EA046068B1 - COMBINED THERAPY FOR THE TREATMENT OF MASTOCYTOSIS - Google Patents

Description

Cross reference to related applications

This application claims benefit from U.S. Patent Application No. 62/624453, filed January 31, 2018, which is incorporated herein by reference in its entirety.

Background of the Invention c-KIT (also known as KIT, CD117 and stem cell growth factor receptor) is a 145 kDa transmembrane protein tyrosine kinase that functions 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 growth factor (SCF, steel factor, kit ligand, mast cell growth factor). The receptor has protein tyrosine kinase activity, and binding of the SCF ligand results in autophosphorylation of c-KIT and its binding to substrates such as phosphatidylinositol 3-kinase (PI3K). Tyrosine phosphorylation by protein tyrosine kinases is particularly important in cell signal transduction and can mediate signaling for essential cellular processes such as proliferation, survival, differentiation, apoptosis, attachment, invasiveness and migration.

The c-KIT receptor tyrosine kinase gene is critical for mast cell growth, maintenance, differentiation, and homeostasis. Activating mutations or overexpression of the c-KIT gene enhance the ability of the c-KIT receptor to stimulate intracellular pathways leading to impaired mast cell proliferation.

Mastocytosis is a rare disorder characterized by abnormal accumulations of mast cells (MC) in the skin, bone marrow, and internal organs (eg, liver, spleen, gastrointestinal tract, and lymph nodes). Cases where the disease begins in adulthood tend to be chronic and involve the bone marrow in addition to the skin, while in childhood the condition is often characterized by cutaneous manifestations without affecting the internal organs and can often disappear during puberty. In most adult patients, mastocytosis is intractable and may progress to more advanced stages in a minority of patients. Mastocytosis can be classified into a specific type depending on the symptoms and general presentation of the patient. Types of mastocytosis include cutaneous mastocytosis (eg, maculopapular cutaneous mastocytosis, mastocytoma, and diffuse cutaneous mastocytosis) and systemic mastocytosis (eg, indolent systemic mastocytosis (ISM), sluggish systemic mastocytosis (SSM), systemic mastocytosis with associated disease lineage-related clonal hematologic cells other than mast cells (SM-AHN), aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL) and mast cell sarcoma). In rare cases, the proliferation of aggressive neoplastic mast cells leads to functional failure of internal organs, with patients having a significantly reduced life expectancy and requiring cytoreductive therapy. Pathological accumulation of neoplastic mast cells due to oncogenic c-KIT mutations has been found to be a cause of systemic mastocytosis. The predominant activating KIT mutation is the substitution of valine for aspartic acid at residue 816 (KIT D816V). Patients with systemic mastocytosis, whose mast cells often harbor the D816V c-KIT activating mutation, may have an indolent to aggressive form of the disease, and they may experience mast cell mediator release-related symptoms. Indolent systemic mastocytosis with recurrent anaphylaxis or vascular collapse in the absence of skin lesions is a specific subtype of indolent systemic mastocytosis, and this disorder of clonal MC activation accounts for a significant portion 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 unclear. Currently, most tyrosine kinase inhibitors have demonstrated only moderate effectiveness in advanced disease and are associated with significant side effects. In addition, some aggressive KIT mutations, including the KIT D816V mutation, are resistant to classical ATP-competitive KIT inhibitors such as imatinib, sunitinib, sorafenib, and regorafenib. Midostaurin, a D816V c-KIT inhibitor, was recently approved for the treatment of SM in 2017.

Although the c-KIT D816V mutation is a primary c-KIT mutation described as a determinant of systemic mastocytosis (SM), secondary c-KIT mutations that confer resistance to some c-KIT inhibitors (secondary c-KIT resistance mutations) , have also been described in patients with mastocytosis, including, for example, the Y269C, Y503_F504insAY, V560D, or K642E point mutation, in-frame deletion or insertion, or missense mutation in the c-KIT gene.

Activating mutations or overexpression of the c-KIT gene are associated with the proliferation of neoplastic mast cells. Given the complex function of c-KIT and the potential utility of c-KIT inhibitors in the treatment of drug-resistant systematic mastocytosis, there is a need for inhibitors and therapeutic treatments with advantageous therapeutic properties.

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Short description

The present invention relates in part to the use of a c-KIT inhibitor, for example 1-[4-bromo5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dighydro-1,6-naphthyrvdin-3-yl] -2-fluorophenyl]-3-phenylurea (compound A), and a MAPKAP pathway inhibitor, for example a MEK inhibitor such as trametinib, or an ERK inhibitor such as ulixertinib, or a RAF inhibitor such as LY3009120, to induce apoptosis of mastocytosis cells.

Also provided by the present invention are methods of 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-oxo1, 2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea (compound A, described herein); and an effective amount of a mitogen-activated protein kinase inhibitor (MEK inhibitor) and/or an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

For example, provided herein is a method of 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 mitogen-activated protein kinase inhibitor (MEK inhibitor) or an ERK inhibitor.

Also provided by the present invention 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 description of graphic materials

In fig. 1A shows graphical representations of cell proliferation following the indicated drug treatment with Compound A compared to vehicle control in the HMC1.1 V560G (left panel) and HMC1.2 V560G/D816V (right panel) cell lines.

In fig. 1B shows graphical representations of cell proliferation following the indicated drug treatment with Compound B compared to vehicle control in the HMC1.1 V560G (left panel) and HMC1.2 V560G/D816V (right panel) cell lines.

In fig. 2A shows a graphical representation of caspase activity after these different treatments with Compound A and trametinib in the HMC1.2 V560G/D816V cell line.

In fig. 2B is a table showing the synergistic effects of the main analytes obtained using the additivity-based method for various Compound A and trametinib treatments in the HMC1.2 V560G/D816V cell line.

In fig. 2C is a graph showing additivity scores for the combination of Compound A with the MEK inhibitor trametinib.

In fig. 3A shows a graphical representation of caspase activity after these different treatments with Compound B and trametinib in the HMC1.2 V560G/D816V cell line.

In fig. 3B is a table showing the synergistic effects of the main analytes obtained using the additivity-based method for various Compound B and trametinib treatments in the HMC1.2 V560G/D816V cell line.

In fig. 3C is a graph showing additivity scores for the combination of Compound B with the MEK inhibitor trametinib.

In fig. 4A shows a graphical representation of caspase activity after these different treatments with Compound A and binimetinib in the HMC1.2 V560G/D816V cell line.

In fig. 4B is a table showing the synergistic effects of the main analytes obtained using the additivity-based method for various Compound A and binimetinib treatments in the HMC1.2 V560G/D816V cell line.

In fig. 4C is a graph showing additivity scores for the combination of Compound A with the MEK inhibitor binimetinib.

In fig. 5A shows a graphical representation of caspase activity after these different treatments with Compound B and binimetinib in the HMC1.2 V560G/D816V cell line.

In fig. 5B is a table showing the synergistic effects of the main analytes obtained using the additivity-based method for various Compound B and binimetinib treatments in the HMC1.2 V560G/D816V cell line.

In fig. 5C is a graph showing additivity scores for the combination of Compound B with the MEK inhibitor binimetinib.

In fig. 6A shows a graphical representation of caspase activity after these different treatments with Compound A and cobimetinib in the HMC1.2 V560G/D816V cell line.

In fig. 6B presents a table with data on the synergistic effects of the main analyzed

- 2,046,068 substances obtained using an additivity-based method for various treatments with Compound A and cobimetinib in the HMC1.2 V560G/D816V cell line.

In fig. 6C is a graph showing additivity scores for the combination of Compound A with the MEK inhibitor cobimetinib.

In fig. 7A shows a graphical representation of caspase activity after these different treatments with Compound B and cobimetinib in the HMC1.2 V560G/D816V cell line.

In fig. 7B is a table showing the synergistic effects of the main analytes obtained using the additivity-based method for various Compound B and cobimetinib treatments in the HMC1.2 V560G/D816V cell line.

In fig. 7C is a graph showing additivity scores for the combination of Compound B with the MEK inhibitor cobimetinib.

In fig. 8A shows a graphical representation of caspase activity following these different treatments with Compound A and the ERK inhibitor ulixertinib in the HMC1.2 V560G/D816V cell line.

In fig. 8B is a table showing the synergistic effects of the main analytes obtained using the additivity-based method for various treatments with Compound A and the ERK inhibitor ulixertinib in the HMC1.2 V560G/D816V cell line.

In fig. 8C is a graph showing additivity scores for the combination of Compound A with the ERK inhibitor ulixertinib.

In fig. 9A shows the inhibition of colony germination due to treatment with Compound A single agent, trametinib single agent, and the combination of Compound A with the MEK inhibitor trametinib in HMC1.2 V560G/D816V cells.

In fig. 9B shows a graphical representation of the inhibition of colony germination due to treatment with the single agent compound A, the single agent trametinib, and the combination of compound A with the MEK inhibitor trametinib in HMC1.2 V560G/D816V cells. Arrows indicate the absence of colony germination.

In fig. 10A shows the inhibition of colony germination due to treatment with Compound B single agent, trametinib single agent, and the combination of Compound B with the MEK inhibitor trametinib in HMC1.2 V560G/D816V cells.

In fig. 10B shows a graphical representation of the inhibition of colony germination due to treatment with the single agent compound B, the single agent trametinib, and the combination of compound B with the MEK inhibitor trametinib in HMC1.2 V560G/D816V cells. Arrows indicate the absence of colony germination.

In fig. 11A shows the inhibition of colony germination due to treatment with Compound A single agent, binimetinib single agent, and the combination of Compound A with the MEK inhibitor binimetinib in HMC1.2 V560G/D816V cells.

In fig. 11B shows a graphical representation of the inhibition of colony germination due to treatment with the single agent compound A, the single agent binimetinib, and the combination of compound A with the MEK inhibitor binimetinib in HMC1.2 V560G/D816V cells. Arrows indicate the absence of colony germination.

In fig. 12A shows inhibition of colony germination by treatment with Compound B single agent, binimetinib single agent, and the combination of Compound B with the MEK inhibitor binimetinib in HMC1.2 V560G/D816V cells.

In fig. 12B shows a graphical representation of the inhibition of colony germination due to treatment with Compound B single agent, binimetinib single agent, and the combination of Compound B with the MEK inhibitor binimetinib in HMC1.2 V560G/D816V cells. Arrows indicate the absence of colony germination.

In fig. 13A shows inhibition of colony germination by treatment with Compound A single agent, cobimetinib single agent, and the combination of Compound A with the MEK inhibitor cobimetinib in HMC1.2 V560G/D816V cells.

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In fig. 13B shows a graphical representation of the inhibition of colony germination due to treatment with Compound A alone, cobimetinib alone, and the combination of Compound A with the MEK inhibitor cobimetinib in HMC1.2 V560G/D816V cells.

In fig. 14A shows inhibition of colony germination by treatment with Compound B single agent, cobimetinib single agent, and the combination of Compound B with the MEK inhibitor cobimetinib in HMC1.2 V560G/D816V cells.

In fig. 14B shows a graphical representation of the inhibition of colony germination due to treatment with Compound B single agent, cobimetinib single agent, and the combination of Compound B with the MEK inhibitor cobimetinib in HMC1.2 V560G/D816V cells.

In fig. 15A shows graphical representations of caspase activity after various treatments with Compound A and trametinib at 24 hours in cells.

HMC1.2 V560G/D816V transfected with empty vector (EV, left panel) or N-ras G12D (right panel).

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

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

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

In fig. 17A shows inhibition of colony germination by treatment with Compound A single agent, trametinib single agent, and the combination of Compound A with the MEK inhibitor trametinib in empty vector (EV)-transfected HMC1.2 V560G/D816V cells. .

In fig. 17B shows a graphical representation of the inhibition of colony germination due to treatment with the single agent compound A, the single agent trametinib, and the combination of compound A with the MEK inhibitor trametinib in HMC1.2 V560G/D816V cells transfected with empty vector ( EV). Arrows indicate the absence of colony germination.

In fig. 17C shows inhibition of colony germination due to treatment with Compound A single agent, trametinib single agent, and the combination of Compound A with the MEK inhibitor trametinib in HMC1.2 V560G/D816V cells transfected with N-ras G12D.

In fig. 17D shows a graphical representation of the inhibition of colony germination due to treatment with the single agent compound A, the single agent trametinib, and the combination of compound 1 with the MEK inhibitor trametinib in HMC1.2 V560G/D816V cells transfected with N-ras G12D. Arrows indicate the absence of colony germination.

In fig. 18A shows inhibition of colony germination by treatment with Compound A single agent, cobimetinib single agent, and the combination of Compound A with the MEK inhibitor cobimetinib in empty vector (EV)-transfected HMC1.2 V560G/D816V cells. .

In fig. 18B shows a graphical representation of the inhibition of colony germination due to treatment with Compound A single agent, cobimetinib single agent, and the combination of Compound A with the MEK inhibitor cobimetinib in HMC1.2 V560G/D816V cells transfected with empty vector ( EV). Arrows indicate the absence of colony germination.

In fig. 18C shows inhibition of colony germination by treatment with Compound A alone, cobimetinib alone, and the combination of Compound A with the MEK inhibitor cobimetinib in HMC1.2 V560G/D816V cells transfected with N-ras G12D.

In fig. 18D shows a graphical representation of the inhibition of colony germination due to treatment with Compound A single agent, cobimetinib single agent, and the combination of Compound A with the MEK inhibitor cobimetinib in HMC1.2 V560G/D816V cells transfected with N-ras G12D. Arrows indicate the absence of colony germination.

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Detailed description

It has been found that a combination of c-KIT inhibitor, 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 a MAPKAP pathway inhibitor, such as trametinib, which is a MEK inhibitor, or ulixertinib, which is an ERK inhibitor, or LY3009120, which is an RAF inhibitor, unexpectedly has a synergistic effect in inducing apoptosis of mastocytosis cells, demonstrated in the accompanying examples. In addition, the combination therapy methods disclosed herein are cytocidal in addition to cytostatic.

Without wishing to be bound by any particular theory, it is believed that many c-KIT inhibitors only inhibit certain mutant forms of c-KIT and do not effectively inhibit the c-KIT D816V mutation that causes SM. The present invention provides methods for treating cKIT-mediated mastocytosis by inhibiting both c-KIT and MEK using a c-KIT inhibitor in combination with a MAPKAP pathway inhibitor. In specific embodiments, a c-KIT inhibitor is 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. salt, or crenolanib or a pharmaceutically acceptable salt thereof. Surprisingly, a c-KIT inhibitor, such as Compound A and Compound B (and pharmaceutically acceptable salts thereof), has a synergistic effect with a MEK, ERK, or RAF inhibitor in inhibiting the proliferation and inducing apoptosis of mastocytosis cells.

Accordingly, in certain embodiments, the present invention provides methods for inducing cytocidal killing of mast cells, inducing mast cell apoptosis, inhibiting mast cell growth or proliferation, inhibiting mast cell mediator release, reducing the number of accumulated mast cells in a tissue or organ, reducing the volume associated with mastocytosis. a tumor, such as mast cell leukemia or mast cell sarcoma, and/or inhibiting mast cell regrowth by contacting mast cells, eg, mast cells containing a c-KIT mutation, with a c-KIT inhibitor and a MAPKAP pathway inhibitor. In various embodiments, mast cells are contacted in vitro, in vivo, or ex vivo. In specific embodiments, a c-KIT inhibitor is 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. salt or crenolanib or a pharmaceutically acceptable salt thereof; and a MEK inhibitor is 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.

In certain embodiments, the present invention provides methods of treating a subject who has mastocytosis, mast cell leukemia, or acute myeloid leukemia, comprising administering to the subject an effective amount of (i) a KIT inhibitor, such as 1-[4-bromo-5-[1-ethyl -7-(methylamino)-2-oxo-1,2-dihydro-1,6naphthyridin-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, eg trametinib, binimetinib, ulixertinib.

In specific embodiments of any of the methods disclosed herein, mastocytosis is systemic mastocytosis resulting from the D816V mutation in the c-KIT gene in mast cells.

Definitions.

The term compounds A and B as used herein refers to 1-[4-bromo-5-[1-ethyl-7(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridine-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 compounds A and B are also provided in the present invention. The structures of compounds A and B are presented below.

1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3 phenylurea (compound A);

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-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3 phenylurea (compound B).

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

Exemplary methods and materials are described herein. As used in this specification and the accompanying claims, the singular number also includes the plural unless the context clearly indicates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art.

Throughout this disclosure, various patents, patent applications, and publications are incorporated by reference. The disclosures of such patents, patent applications and publications are incorporated herein by reference in their entirety to more fully describe the state of the art known to those skilled in the art as of the date of this disclosure. This disclosure will govern if there is any inconsistency between patents, patent applications and publications and this disclosure.

For convenience, certain terms used in the description, examples and claims are collected in this part. Unless otherwise specified, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one skilled in the art to which the present invention belongs. The original definition provided for a group or term given in this disclosure applies to that group or term throughout this disclosure, alone or as part of another group, unless otherwise indicated.

The term pharmaceutically acceptable carrier, diluent or excipient includes, without limitation, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, coloring agent, flavor enhancer, surfactant, wetting agent, dispersant an agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the United States Food and Drug Administration as acceptable for use in humans or domestic animals.

The term pharmaceutically acceptable salt includes acid addition salts.

The term pharmaceutically acceptable acid addition salt refers to those salts that retain the biological effectiveness and properties of the free bases that are not biologically or otherwise undesirable and that are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, 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- Hydroxyethanphonic acid, ants, fumaric acid, galaxylic acid, hydizinic acid, glucogeptonic acid, glucic acid, glucuronic acid, glutamic acid, glutard acid, 2-oxoglutoic acid, glycerinophosphoric acid, glycolic acid, glycolic acid, gypsum acid, isasyltic acid, and isobilized acid, and isobaric acid, and isobaric acid, and isobilic acid, and amyesyanic acid, and amyesyanic acid, and oilycular acidic acid, and oilycular acid. lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucinic 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, pyruvic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid acid, trifluoroacetic acid, undecylenic acid, etc.

The term pharmaceutical composition refers to a formulation based on a compound described herein (eg, Compound A or a pharmaceutically acceptable salt thereof) and a vehicle generally acceptable in the art for delivering a biologically active compound to mammals, such as humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients intended therefor.

The term MAPKAP pathway inhibitor means an inhibitor of the MAP kinase signaling pathway. Inhibito

- 6 046068 agents in this pathway include RAS inhibitors, RAF inhibitors (eg, vemurafenib, dabrafenib, LY3009120), MEK inhibitors (eg, trametinib, binimetinib, cobimetinib) and ERK inhibitors (eg, ulixertinib).

Subjects or patients in need of treatment with a combination therapy of the present invention, for example, a c-KIT inhibitor in combination with a MAPKAP pathway inhibitor, include subjects with diseases and/or conditions that can be treated with the combination disclosed herein, achieving favorable therapeutic outcome, for example with mastocytosis, mast cell leukemia or acute myeloid leukemia. A positive outcome in the treatment of mastocytosis may include complete response, partial response, clinical improvement, or stable disease as defined by the IWG-MRT-ECNM criterion (Gotlib et al., Blood, 2013, 121:2393-401). In certain embodiments, the subject in need of treatment has cutaneous mastocytosis (eg, maculopapular cutaneous mastocytosis, mastocytoma, and diffuse cutaneous mastocytosis) or systemic mastocytosis (eg, indolent systemic mastocytosis, indolent systemic mastocytosis, systemic mastocytosis with associated lineage-related disease clonal hematologic cells other than mast cells, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma). In specific embodiments, the subjects are mammals, such as humans or other mammals.

The term effective amount when used in connection with a compound or other therapeutic agent disclosed herein refers to an amount of a therapeutic agent, eg, 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 in a combination therapy is the amount of each therapeutic agent that is useful for treating or preventing a disease or disorder when used in a combination therapy, even if the amount of one or both of the therapeutic agents is ineffective in the absence of the other therapeutic agent. to treat or prevent a disease or disorder. In certain embodiments, an effective amount is an amount that results in inducing cytocidal killing of mast cells, inducing mast cell apoptosis, reducing the number of accumulated mast cells in a tissue or organ, reducing the severity of symptoms of mastocytosis, inhibiting mast cell mediator release, inhibiting mast cell growth, and /or inducing regression of mastocytosis, where the mast cells contain activating mutations in c-KIT kinase, including the activating mutation c-KIT D816V. The term effective amount may vary depending on the route of administration, the specific location of the disease or disorder, and the age, body weight, and general health of the subject. The amount of compounds administered will depend on the extent, severity and type of disease or condition, the amount of therapeutic agent required and the release characteristics of the pharmaceutical composition(s). It will also depend on the health status, size, weight, age, gender of the subject and the drug tolerance of the subject. Typically, the compounds are administered for a period of time sufficient to achieve the desired therapeutic effect.

The terms treatment, treat, and treatment delivery mean to include the full range of interventions in the patients being treated, such as patients with mastocytosis or acute myeloid leukemia (AML). Treatment may result in recovery, improvement, or at least partial reduction in the severity of the disorder. In specific embodiments, treatment is administered to induce cytocidal killing of mast cells, induce mast cell apoptosis, reduce the number of accumulated mast cells in a tissue or organ, reduce the severity of symptoms of mastocytosis, inhibit mast cell mediator release, inhibit mast cell growth, and/or induce regression of mastocytosis. in the subject being treated. In certain embodiments, treatment with the combination therapy disclosed herein provides relief, slowing, or reversal of one or more symptoms of mastocytosis and/or induction of regression of mastocytosis, even if the mastocytosis is not actually eliminated. In some embodiments, the treatment includes complete resolution of the disease or disorder, such as mastocytosis or AML. In other embodiments, the treatment results in a complete response, partial response, clinical improvement, or stable disease as determined by the IWG-MRT-ECNM criterion (Gotlib et al., Blood, 2013, 121:2393-401).

The term mast cells as used herein includes mast cells (also called mast cells) and CD34+ mast cell progenitor cells.

The term neoplasm as used herein refers to abnormal tissue that grows through cell proliferation faster than normal. Neoplasms exhibit partial or complete lack of structural organization and functional coordination with normal tissue and usually form a distinct mass of tissue that can be either benign (benign tumor) or malignant (cancer).

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The term tumor as used herein refers to a mass. It is a term that can refer to benign (usually harmless) or malignant (cancerous) growths. Malignant growth can occur in a whole organ or bone marrow. The latter is often called liquid tissue tumors. The term tumors includes mast cell leukemia types and mast cell sarcoma types, collectively referred to herein as mastocytosis tumors.

In certain embodiments, the therapeutic effect of treating mastocytosis in accordance with the methods disclosed herein can be measured using a standard response criterion known in the art. For example, the response criterion term for complete remission (CR), partial remission (PR), clinical improvement (CI), or stable disease (SD), used to quantify the effects of therapy against aggressive systemic mastocytosis, mast cell leukemia, and systemic mastocytosis, associated with a myeloid neoplasm may mean any of those criteria as defined by the IWG-MRT-ECNM (Gotlib et al., Blood, 2013, 121:2393-401). For example, complete remission (CR) requires all 4 criteria and duration of response must be >12 weeks with no compact aggregates of neoplastic mast cells in the BM or other extracutaneous organ biopsied; serum tryptase level <20 ng/ml, calculated peripheral blood remission value defined as ANC >1x10 ⁹ /L with normal differential, Hb level >11 g/dL and platelet count >100x10 ⁹ /L, and complete resolution of palpable hepatosplenomegaly and all biopsy-proven or suspected organ injuries associated with SM (manifestations of CI);

for partial remission (PR)* all 3 criteria are required and duration of response must be >12 weeks in the absence of both CR and progressive disease (PD) with a >50% reduction in the number of neoplastic MCs in the bone marrow and/or other extracutaneous organ at biopsy-demonstrated suitable organ injury associated with SM, reduction in serum tryptase levels by >50% and resolution of 1 or more biopsy-proven or suspected organ injury associated with SM (CI manifestation(s));

clinical improvement (CI) occurs when the duration of response must be >12 weeks, and 1 or more of the criteria for nonhematologic and/or hematologic response must be met in the absence of also CR/PR and disease progression to chronicity or progressive disease ( PD). Stable disease (SD) does not meet criteria for CR, PR, CI or PD.

Response assessment guidelines include (1) only disease-related grade 2 organ damage assessed as the primary endpoint in clinical trials;

(2) response scores for CR, PR, SD, PD, and decline or response (LOR) in the trial context should only apply to those presentations of grade >2 organ damage;

(3) disease status at the time of patient removal from the study was separately associated with the updated status of the initial presentation(s) of grade 2 organ injury(s);

(4) diagnosis by exclusion for drug-related toxicity and/or other clinical problems (eg, gastrointestinal bleeding in the case of worsening anemia/transfusion dependence) should be performed before determining the PD or LOR category in a patient with worsening of the initial level of organ damage >2 degrees.

In certain embodiments, the therapeutic effect of treating AML in accordance with the methods disclosed herein can be measured using a standard response criterion known in the art. For example, the term AML treatment response criterion used to quantify the effects of therapy on AML may mean any of those criteria as defined below, including complete remission (CR) without minimal residual disease (CRRMD-), complete remission (CR), CR with incomplete hematologic recovery (CRi), morphologic leukemia free status (MLFS), partial remission (PR), stable disease (SD), or progressive disease (PD) as defined in Blood, 2017 Jan 26, 129(4):424447 and summarized as complete remission without minimal residual disease (CRRMD.). When pretreated with genetic marker-negative CR by RTqPCR or MFC-negative CR, complete remission (CR) is defined as <5% bone marrow blasts; absence of circulating blasts and blasts with Auer bodies; absence of extramedullary disease; absolute neutrophil count (ANC) >1.0x10 ⁹ /l (1000/μl); platelet count >100x10 ⁹ /l (100,000/μl); CR with incomplete hematologic recovery (CRi) meets all criteria for CR except residual neutropenia (<1.0 x 10 ⁹ /L [1000/µL]) or thrombocytopenia (<100 x 10 ⁹ /L [100,000/µL]); morphological state without leukemia (MLFS) provides a blast content in the bone marrow of <5%; absence of blasts with Auer bodies; absence of extramedullary disease; hematological restoration is not required

- 8 046068 xia; partial remission (PR) meets all hematologic criteria for CR; reduction in the percentage of blasts in the bone marrow from 5 to 25%; and a reduction in the percentage of blasts in the bone marrow upon pretreatment by at least 50%.

The term combination therapy means a treatment that involves administering to a patient two or more therapeutic agents, for example, a c-KIT inhibitor (such as Compound A or a pharmaceutically acceptable salt thereof, midostaurin, BLU-285, PLX9486, or crenolanib) and a MAPKAP pathway inhibitor (including but not limited to trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, LY3009120). Two or more therapeutic agents can be delivered simultaneously, for example, in separate pharmaceutical compositions or in the same pharmaceutical composition, or they can be delivered at different points in time. For example, they can be delivered simultaneously or over overlapping periods of time and/or one therapeutic agent can be delivered before or after the other therapeutic agent(s). Treatment with a combination of a KIT inhibitor, such as Compound A, and a MAPKAP pathway inhibitor does not necessarily include treatment with either individual agent, preceded or followed by a period of concurrent treatment with both agents. However, it is assumed that effective amounts of two or more therapeutic agents are present in the patient's body for a period of time.

Methods of treatment.

In one embodiment, the present invention provides methods for treating or preventing mastocytosis, optionally c-KIT-mediated mastocytosis, such as systemic mastocytosis (SM), 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 c-KIT inhibitor. MAPKAP pathway, such as trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, or LY3009120. In one embodiment, the present invention provides methods for treating or preventing mastocytosis, optionally c-KIT-mediated mastocytosis, such as 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, for example trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib or LY3009120. In a related embodiment, the present invention provides methods for treating or preventing a mastocytosis tumor, optionally a c-KIT-mediated mastocytosis tumor, such as mast cell leukemia or mast cell sarcoma, 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, such as trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, or LY3009120. In a related embodiment, the present invention provides methods for treating or preventing a mastocytosis tumor, optionally a c-KIT-mediated mastocytosis tumor, such as 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, for example trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib or LY3009120.

In another example, the present invention 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. Such a MAPKAP pathway inhibitor may be selected from the group consisting of a rapidly progressing fibrosarcoma kinase (RAF) kinase inhibitor, a mitogen-activated protein kinase inhibitor (MEK inhibitor), and an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

In one embodiment of such a disclosed method, mastocytosis is characterized by a c-KIT mutation. In some embodiments, the c-KIT mutation is an activating mutation.

In another embodiment, mastocytosis may involve mast cells containing a primary mutation in exon 17 of the c-KIT gene. In some embodiments, the primary mutation is the c-KIT D816 mutation. In some embodiments, the primary mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and de4l19. In some embodiments, the primary mutation is D816V.

Mastocytosis may also involve mast cells containing a secondary c-KIT mutation. In some embodiments, the secondary c-KIT mutation is in one of exons 9, 11, 13, or 17. In some embodiments, the secondary c-KIT mutation is one of the Y269C, Y503_F504insAY, V560D, or K642E mutations.

The disclosed method may further include determining whether mastocytosis is characterized by a primary c-KIT mutation. For example, the method further includes determining whether mastocytosis is characterized by a secondary c-KIT mutation. In some embodiments, determining whether mastocytosis is characterized by a primary or secondary c-KIT mutation includes identifying

- 9 046068 identification of mutations in DNA isolated from a tumor sample. In yet another embodiment, determining whether mastocytosis is characterized by a primary or secondary c-KIT mutation involves identifying mutations in circulating tumor DNA or identifying mutations in circulating peripheral blood leukocytes.

Mastocytosis may be systemic mastocytosis. In some embodiments, systemic mastocytosis is selected from the group consisting of indolent systemic mastocytosis, indolent systemic mastocytosis, systemic mastocytosis with associated disease associated with a lineage of clonal hematologic cells other than mast cells, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma. mast cells. In some embodiments, the mastocytosis is indolent systemic mastocytosis, optionally systemic mastocytosis with recurrent anaphylaxis or vascular collapse in the absence of skin lesions. In some embodiments, the mastocytosis is an indolent systemic mastocytosis.

In some embodiments, mastocytosis is systemic mastocytosis with an associated disease associated with a lineage of clonal hematologic cells other than mast cells. In some embodiments, the mastocytosis is an 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.

In such a disclosed method, the c-KIT inhibitor may be selected from the group consisting of 1-[4-bromo5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridine- 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 salt.

In addition, the MEK inhibitor may 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 administered substantially simultaneously or sequentially.

The method may also include administering another targeted cancer therapy, a biological targeted cancer therapy, an immune checkpoint inhibitor, or a chemotherapeutic agent.

In addition, two or more weeks of administration of an effective amount of a c-KIT inhibitor, and an effective amount of a mitogen-activated protein kinase inhibitor (MEK inhibitor), and/or an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor), in accordance with the method contemplated may cause the patient to experience at least partial remission.

Also provided is a method of 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-oxo1,2-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 such a disclosed method, the MAPKAP pathway inhibitor is selected from the group consisting of a rapidly progressing fibrosarcoma kinase (RAF) inhibitor, a mitogen-activated protein kinase inhibitor (MEK inhibitor), and an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

In one embodiment, systemic mastocytosis may be characterized by a c-KIT mutation. For example, the mutation may be the 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.

In addition, mastocytosis may be characterized by an additional c-KIT mutation, which is one of the Y269C, Y503_F504insAY, V560D, or K642E mutations.

In such a disclosed method, the MEK inhibitor may 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. The ERK inhibitor may be selected from the group consisting of ulixertinib, SCH772984 and LY3214996.

The present invention further provides a method of treating mastocytosis in a patient,

- 10 046068 in need thereof, comprising administering to the patient an effective amount of a c-KIT inhibitor and an effective amount of a RAF inhibitor.

In such a disclosed method, the RAF inhibitor may be an inhibitor of all isoforms of RAF or B-RAF, including vemurafenib, dabrafenib and LY3009120. Additionally, the c-KIT inhibitor may be 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl] -2fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof.

In specific embodiments of the methods disclosed herein, including methods for treating mastocytosis or a mastocytosis tumor, the methods include inducing cytocidal killing of mast cells, inducing mast cell apoptosis, reducing the number of accumulated mast cells in a tissue or organ, reducing the severity of symptoms of mastocytosis, inhibiting mediator release mast cells, inhibiting mast cell growth and/or inducing regression of mastocytosis, where the mast cells contain one or more activating mutations in c-KIT kinase, such as, for example, the activating KIT mutation D816V. In certain embodiments, the methods include methods for eliminating mastocytosis, such as a mastocytosis tumor, in a subject. In some embodiments, treatment results in a complete response, partial response, clinical improvement, or stable disease as determined by the FWG-MRT-ECNM criterion (Gotlib et al., Blood, 2013, 121:2393-401).

In another related embodiment, the present invention provides methods for 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, for example trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib or LY3009120. In a related embodiment, the present invention provides methods for 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, for example trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib or LY3009120.

When the methods described herein relate to treatment with Compound A or a pharmaceutically acceptable salt thereof or Compound B or a pharmaceutically acceptable salt thereof, it is intended that only one of Compound A or a pharmaceutically acceptable salt thereof or Compound B or a pharmaceutically acceptable salt thereof is required . However, such methods are known to also include 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, wherein Compound A is metabolized in vivo to Compound B, and a mixture of Compound A and Compound B provides effective treatment of the subject in combination with the MAPKAP pathway inhibitor in vivo.

Specific embodiments of the disclosed methods and compositions are practiced using a combination of Compound A or a pharmaceutically acceptable salt thereof and trametinib;

combinations of Compound A or a pharmaceutically acceptable salt thereof and selumetinib;

combinations 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.

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 with c-KIT-mediated mastocytosis. In another embodiment, Compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, eg trametinib or binimetinib, is administered to a subject with c-KIT-mediated mastocytosis.

In a related embodiment, Compound A or a pharmaceutically acceptable salt thereof and a MEK inhibitor, such as trametinib or binimetinib, is administered to a patient with a mastocytosis tumor, including without limitation mast cell leukemia or mast cell sarcoma, where the growth of the mastocytosis tumor or tumor progression is due to a primary activating mutation c-KIT, for example the KIT D816V mutation. In another embodiment, Compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, such as trametinib or binimetinib, is administered to a patient with a mastocytosis tumor, including without limitation mast cell leukemia or mast cell sarcoma, where the growth of the mastocytosis tumor or tumor progression is due to a primary activating mutation c-KIT, for example 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 MEK inhibitor, e.g., trametinib or binimetinib, is administered to a patient with AML, wherein the AML is not necessarily caused by a primary activating c-KIT mutation, e.g., an activating c-KIT mutation in exon 8 or by a c-KIT mutation in exon 17, including but not limited to mutations in D816 or

- 11 046068 in N822 (Journal of Clinical Oncology, 2006, 24:24, 3904-3911).

Specific embodiments of the disclosed methods and compositions are practiced using a combination of Compound A or a pharmaceutically acceptable salt thereof and ulixertinib;

combinations of compound A or a pharmaceutically acceptable salt thereof and SCH772984;

combinations of compound A or a pharmaceutically acceptable salt thereof and LY3214996;

combinations of compound A or a pharmaceutically acceptable salt thereof and ravoxertinib; or a combination of Compound A or a pharmaceutically acceptable salt thereof and and VX-11.

In one embodiment, Compound A or a pharmaceutically acceptable salt thereof and an ERK inhibitor, eg ulixertinib, is administered to a subject with c-KIT-mediated mastocytosis. In another embodiment, Compound B or a pharmaceutically acceptable salt thereof and an ERK inhibitor, eg ulixertinib, is administered to a subject with c-KIT-mediated mastocytosis.

In a related embodiment, Compound A or a pharmaceutically acceptable salt thereof and an ERK inhibitor, for example ulixertinib, is administered to a patient with a mastocytosis tumor, including without limitation mast cell leukemia or mast cell sarcoma, where the growth of the mastocytosis tumor or tumor progression is due to a primary activating mutation c- KIT, for example the KIT D816V mutation. In another embodiment, Compound B or a pharmaceutically acceptable salt thereof and an ERK inhibitor, such as ulixertinib, is administered to a patient with a mastocytosis tumor, including without limitation mast cell leukemia or mast cell sarcoma, where the growth of the mastocytosis tumor or tumor progression is due to a primary activating mutation c- KIT, for example 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 an ERK inhibitor, e.g., ulixertinib, is administered to a patient with AML, wherein the AML is not necessarily caused by a primary activating mutation of c-KIT, e.g., an activating mutation of c-KIT in an exon 8 or a c-KIT mutation in exon 17, including but not limited to mutations in D816 or N822 (Journal of Clinical Oncology, 2006, 24:24, 3904-3911).

Specific embodiments of the disclosed methods and compositions are practiced using a combination of Compound A or a pharmaceutically acceptable salt thereof and LY3009120;

combinations of Compound A or a pharmaceutically acceptable salt thereof and dabrafenib; or a combination of Compound A or a pharmaceutically acceptable salt thereof and vemurafenib.

In one embodiment, Compound A or a pharmaceutically acceptable salt thereof and a RAF inhibitor, eg LY3009120, dabrafenib or vemurafenib, is administered to a subject with c-KIT-mediated mastocytosis. In another embodiment, Compound B or a pharmaceutically acceptable salt thereof and a RAF inhibitor, eg LY3009120, dabrafenib or vemurafenib, is administered to a subject with 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, is administered to a patient with a mastocytous tumor, including, without limitation, mast cell leukemia or mast cell sarcoma, where the growth of the mastocytous tumor or progression of the tumor is due to a primary an activating mutation of c-KIT, for example 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, is administered to a patient with a mastocytous tumor, including, without limitation, mast cell leukemia or mast cell sarcoma, where the growth of the mastocytous tumor or progression of the tumor is due to a primary an activating mutation of c-KIT, for example 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, e.g., LY3009120, dabrafenib, or vemurafenib, is administered to a patient with AML, wherein the AML is not necessarily caused by a primary activating cKIT mutation, e.g., an activating c-KIT mutation in exon 8 or a c-KIT mutation in exon 17, including but not limited to mutations in D816 or N822 (Journal of Clinical Oncology, 2006, 24:24, 3904-3911).

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 may 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.

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. tramethin

- 12 046068 bom, binimetinib, ulixertinib or LY3009120, covers the administration of Compound A or a pharmaceutically acceptable salt thereof or Compound B or a pharmaceutically acceptable salt thereof before, after, concurrently with, or during a period of time overlapping with the administration of a 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, such as trametinib, binimetinib, ulixertinib, or LY3009120, may differ when used in combinations disclosed herein, as opposed to when any of these agents are used by themselves for the same purpose, for example, to treat or prevent mastocytosis or a mast cell tumor, such as mast cell leukemia or AML. In specific embodiments, the effective amount of Compound A or a pharmaceutically acceptable salt thereof or Compound B or a pharmaceutically acceptable salt thereof is a lower amount when administered as a combination therapy with a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, compared to when administered as monotherapy, for example to treat or prevent mastocytosis or mast cell tumor. In specific embodiments, the effective amount of a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, is a lower amount when administered as part of a combination therapy with Compound A or a pharmaceutically acceptable salt thereof or when administered as part of a combination therapy with Compound B or a pharmaceutically acceptable salt thereof. an acceptable salt, for example, for the treatment or prevention of mastocytosis or mast cell tumor.

Any of the methods disclosed herein may further include determining whether the treated mastocytosis cells, mast cell tumors, or AML contain one or more mutations in the c-KIT gene. Such determination can be made by conventional methods for determining the presence of a mutation in a gene in a biological sample, such as a bone marrow sample, a tissue sample, a peripheral blood sample, or a blood plasma sample obtained from a subject. In addition, such determination can be made by reviewing the results of studies conducted to determine the presence of one or more mutations in the c-KIT gene in a biological sample obtained from the patient. In certain embodiments of any of the methods disclosed herein, the methods are performed on subjects in whom mastocytosis, mast cell tumor, or AML has been identified as containing one or more mutations in the c-KIT gene. Mutations in the c-KIT gene include, without limitation, any of those specifically described herein. In certain embodiments of any of the methods disclosed herein, the methods are not performed on subjects in whom mastocytosis, mast cell tumor, or AML has been identified as not containing one or more mutations in the c-KIT gene.

In various aspects 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 cytocidal killing of obese cells, induces mast cell apoptosis, reduces the number of accumulated mast cells in a tissue or organ, reduces the severity of symptoms of mastocytosis, inhibits mast cell mediator release, inhibits mast cell growth, and/or induces regression of mastocytosis where mast cells contain activating mutations in c-KIT kinase, including the activating mutation KIT D816V. Methods for measuring or quantifying mast cell apoptosis, mast cell killing, inhibition of mast cell growth and proliferation, inhibition of mast cell mediator release, mutant KIT allelic load in peripheral blood, elimination of mastocytosis and mastocytous tumors, complete response, partial response, clinical improvement, or stable disease are known in the art and include any of the methods described herein.

In specific 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, and a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, results in an increase in the rate of apoptosis of mastocytosis cells or mast cells compared to a quantitative measure of apoptosis of either untreated or treated with a MAPKAP pathway inhibitor alone or a c-KIT inhibitor alone, eg Compound A or Compound B, mastocytosis cells or mast cells of the same type. For example, the apoptosis rate may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, according to at least 80%, at least 90%, at least twice, at least three times, at least four times, at least five times, at least 10 times or at least 20 times. In certain embodiments, apoptosis is quantified 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.

- 13 046068

In specific embodiments, treatment with a combination of a c-KIT inhibitor, such as Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, and a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, results in decreased accumulation of mast cells in the skin or internal organ, such as the liver, spleen, bone marrow and/or small intestine, compared to a quantitative measure of mast cell accumulation in the same or untreated or treated only with a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib or LY3009120, or with a c-inhibitor alone. KIT, for example midostaurin, BLU-285, compound A or compound B, organ. For example, the number 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%.

In specific embodiments, treatment with a combination of a c-KIT inhibitor, such as Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, and a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, results in a complete response, as determined by IWG-MRT-ECNM criterion (Gotlib et al., Blood, 2013, 121:2393-401).

In specific embodiments, treatment with a combination of a c-KIT inhibitor, such as Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, and a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, results in a partial response, as determined by IWG-MRT-ECNM criterion (Gotlib et al., Blood, 2013, 121:2393-401).

In specific embodiments, treatment with a combination of a c-KIT inhibitor, such as Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, and a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, results in clinical improvement, as determined by IWG-MRT-ECNM criterion (Gotlib et al., Blood, 2013, 121:2393-401).

In specific embodiments, treatment with a combination of a c-KIT inhibitor, such as Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, and a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, results in stable disease, as determined by IWG-MRT-ECNM criterion (Gotlib et al., Blood, 2013, 121:2393-401).

In specific 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, and a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, induces apoptosis or growth inhibition of resistant mastocytosis cells. containing a secondary KIT mutation in addition to the primary KIT D816V mutation, where the secondary KIT mutation includes, but is not limited to, a Y269C, Y503_F504insAY, V560D, or K642E point mutation, an in-frame deletion or insertion, or a missense mutation in the c-KIT gene (Lasho et al., Br. J. Haematol., 173, 153-156).

In specific 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, and a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, induces apoptosis or growth inhibition of resistant mastocytosis cells. containing mutations, such as resistance mutations, in other genes, including any of those disclosed herein. In certain embodiments, such mutations in 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). Other mutations in epigenetic or transcriptional regulators in SM have been identified, including DNMT3A, ASXL1, and CBL mutations in 12%, 12%, and 4% of patients, respectively (PloS one. 2012, 7:e43090). In addition, some patients with mastocytosis also have mutations in the spliceosome mechanism. Spliceosomes ensure the correct linear order of the exons spliced onto the mRNA. Hansens et al. reported mutation rates in a group of 72 patients with mastocytosis of 23.6% for SRSF2, 5.6% for SF3B1, and 2.7% for U2AF1 (Haematologica, 2014, 99:830-35). Treatment disclosed herein with a combination of a c-KIT inhibitor and a MEK inhibitor, compared with no treatment or treatment with a MEK inhibitor alone, such as trametinib, or a c-KIT inhibitor alone, such as Compound A or Compound B, may have a beneficial effect on such mastocytosis. , characterized by complex genomic initiators. For example, the apoptosis rate may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, according to at least 80%, at least 90%, at least twice, at least three times, at least four times, at least five times, at least 10 times or at least 20 times. For example, a quantitative indicator of growth or number

- 14 046068 lo resistant mastocytosis cells may be limited to 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%.

In specific embodiments, the resistant mastocytosis cells are resistant to apoptosis-mediated cell death or cytotoxic 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 death cells or cytotoxic activity through treatment with a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib or LY3009120, when used as monotherapy.

In specific embodiments, treatment with a combination of a c-KIT inhibitor, such as 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, such as trametinib, binimetinib, ulixertinib, or LY3009120, results in the elimination of mastocytosis. such as mastocytosis tumor. In certain embodiments, resolution of mastocytosis means that the patient no longer has detectable mastocytosis. In specific embodiments, the patient will no longer have detectable mastocytosis for at least twelve weeks, at least twenty-four weeks, at least one year, at least two years, or at least 5 years after initiation of combination therapy treatment for mastocytosis, disclosed in this document. Resolution of mastocytosis can be determined by a complete response criterion, as defined by the IWG-MRT-ECNM criterion (Gotlib et al., Blood, 2013, 121:2393-401).

The present invention describes combination therapies that comprise the administration of a c-KIT inhibitor, for example, either Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof and a MAPKAP pathway inhibitor, for example trametinib, binimetinib, ulixertinib or LY3009120. The combination therapies described herein can be used alone or in combination with one or more additional therapeutic agents. For example, either Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof and a MAPKAP pathway inhibitor may be administered together with a targeted cancer therapy therapeutic agent, a targeted cancer therapy biological agent, an immune checkpoint inhibitor, or a chemotherapeutic agent. In another embodiment, Compound A or Compound B and a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, are administered without any other therapeutic agent. The therapeutic agents can be administered sequentially or together with another therapeutic agent described herein as part of a combination therapy.

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 covered by combination therapy. Although two or more agents in combination therapy can be administered simultaneously, this is not required. For example, administration of the first agent (or combination of agents) may precede administration of the second agent (or combination of agents) by minutes, hours, days or weeks. Thus, two or more agents can be administered with an interval of several minutes between administrations, or with an interval of 1, 2, 3, 6, 9, 12, 15, 18 or 24 hours between administrations, or with an interval of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days between injections, or with an interval of 2, 3, 4, 5, 6, 7, 8, 9 weeks between injections. In some cases, even longer intervals are possible. Although many cases require that two or more agents used in combination therapy be present in the patient's body at the same time, this is not necessary.

Combination therapy may also include two or more administrations of one or more agents used in combination using a different sequence of agent components. For example, if agent X and agent Y are used in combination, they may be administered sequentially in any combination one or more times, for example in the order X-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc. In addition, administration of two or more combination agents may occur before or after the end of dosing intervals during which at least one of the combination agents was withdrawn from treatment.

Additional therapeutic agents that can be administered in accordance with the present invention, for example, for the treatment of mastocytosis or mastocytosis tumor include, but are not limited to, agents selected from the group consisting of STAT5 inhibitors, including, without limitation, ruxolitinib, tofacitinib, or fedratinib;

BTK inhibitors, including without limitation ibrutinib, PCI 29732, acalabrutinib or AVL-292;

PI3 kinase inhibitors including, without limitation, idelalisib, dactolisib, pictilisib, LY294002, buparlisib, pilaralisib, duvelisib, PF-04691502, voxtalisib, omipalisib, gedatolisib, apitolisib, or wortmannin;

- 15 046068 AKT kinase inhibitors, including without limitation MK-2206, perifosine, GSK690693, GSK2141795, ipatasertib, AZD5363, afuresertib or AT7867;

DNA methylation inhibitors, including without limitation 5-azacytidine or 5-aza-2'deoxycytidine;

proteasomal inhibitors including, but not limited to, bortezomib, carfilzomib, MLN9708, ONX 0912;

interferon-alpha (IFN-α);

cladribine and lysosomotropic agents, including but not limited to chloroquine, hydroxychloroquine or quinocrine.

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

In specific embodiments, treatment of a subject in need thereof is carried out with a combination of Compound A or a pharmaceutically acceptable salt thereof, one or two of a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib or LY3009120, and 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.

In specific embodiments, treatment of a subject in need thereof is carried out with a combination of Compound A or a pharmaceutically acceptable salt thereof, a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib or LY3009120, and 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.

In specific embodiments, treatment of a subject in need thereof is carried out with a combination of Compound A or a pharmaceutically acceptable salt thereof, a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib or LY3009120, and 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 can be administered in accordance with the present invention include, but are not limited to, arsenic trioxide, cyclophosphamide, cytarabine, daunorubicin, doxorubicin, enasidenib, idarubicin, quizartinib, mitoxantrone, thioguanine, or vincristine. In specific embodiments, treatment of a subject who has AML is performed with a combination of Compound A or a pharmaceutically acceptable salt thereof and arsenic trioxide, cyclophosphamide, cytarabine, daunorubicin, doxorubicin, enasidenib, idarubicin, quizartinib, mitoxantrone, thioguanine, or vincristine.

Pharmaceutical compositions.

Aspects of the present invention are directed to methods of treatment comprising administering a combination of compounds disclosed herein, or one or more pharmaceutical compositions containing such compounds and a pharmaceutically acceptable diluent, excipient or carrier. In specific embodiments, the methods disclosed herein comprise administering a first pharmaceutical composition comprising a c-KIT inhibitor, such as 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 , for example trametinib, binimetinib, ulixertinib or LY3009120, and a pharmaceutically acceptable diluent, excipient or carrier. In specific embodiments, the methods disclosed herein include 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 containing a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib or LY3009120, and a pharmaceutically acceptable diluent, excipient or carrier. In specific embodiments, the methods disclosed herein include administering a pharmaceutical composition comprising a c-KIT inhibitor, such as Compound A or a pharmaceutically acceptable salt thereof, a MAPKAP pathway inhibitor, such as trametinib, binimetinib, ulixertinib, or LY3009120, and a pharmaceutically acceptable diluent, excipient substance or carrier. In specific embodiments, the methods disclosed herein include administering a pharmaceutical composition comprising Compound B or a pharmaceutically acceptable salt thereof,

- 16 046068 MAPKAP pathway inhibitor, for example trametinib, binimetinib, ulixertinib or LY3009120, and a pharmaceutically acceptable diluent, excipient or carrier.

When using pharmaceutical compositions based on the compounds described herein, pharmaceutically acceptable carriers can be either solid or liquid. Solid forms include powders, tablets, dispersible granules, capsules, starch capsules and suppositories. Powders and tablets may contain from about 5% to about 95% active ingredient. Suitable solid carriers are known in the art, for example magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, starches and capsules can be used in solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods for preparing various compositions can be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th edition, (1990), Mack Publishing Co., Easton, Pa., which is incorporated herein by reference in its entirety. completeness.

Preparations in liquid form include solutions, suspensions and emulsions. For example, aqueous solutions or solutions based on water and propylene glycol for parenteral injection or the addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Preparations in liquid form may also include solutions for intranasal administration.

Liquid, in particular injectable, compositions can, for example, be prepared by dissolving, dispersing, etc. For example, the disclosed compound is mixed with or dissolved in a pharmaceutically acceptable solvent, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, thereby forming an isotonic injection solution or suspension.

Proteins such as albumin, chylomicron particles or whey proteins can be used to solubilize the disclosed compounds.

Parenteral administration by injection is typically used for subcutaneous, intramuscular or intravenous injections and infusions. Injectable preparations can be prepared in conventional forms, either as liquid solutions or suspensions, or as solid forms suitable for dissolution in liquid prior to injection.

You can also use aerosol preparations suitable for inhalation. These preparations may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as an inert pressurized gas such as nitrogen.

It also contemplates the use of solid form preparations that are intended to be converted shortly before use into liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

Dosage.

In some embodiments, wherein Compound A or Compound B (or pharmaceutically acceptable salts thereof) is used in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, for a treatment protocol, wherein the two therapeutic agents may be administered together or in dual mode, where the two therapeutic agents are dosed and administered separately. When Compound A or B (or pharmaceutically acceptable salts thereof) and a MAPKAP pathway inhibitor are dosed separately, the typical dosage of Compound A or Compound B (or pharmaceutically acceptable salts thereof) administered to a subject in need of treatment is typically from about 5 mg per day to about 5000 mg per day, and in other embodiments from about 50 mg per day to about 1000 mg per day. Other dosages may range from about 10 mmol to about 250 mmol per day, from about 20 mmol to about 70 mmol per day, or even from about 30 mmol to about 60 mmol per day. Amounts representing an effective dosage of the disclosed compounds, when used to provide the indicated effects, range from about 0.5 mg to about 5000 mg of the disclosed compound as needed to treat the condition. Compositions for in vivo or in vitro use may contain about 0.5, 5, 20, 50, 75, 100, 150, 250, 500, 750, 1000, 1250, 2500, 3500 or 5000 mg of the disclosed compound, or an amount ranging from one quantity to another quantity in the list of doses. The usual recommended daily dosage for an oral administration regimen may range from about 1 mg/day to about 500 mg/day, or from 1 to 200 mg/day in a single dose or in two to four divided doses. In one embodiment, the typical daily dose for an oral administration regimen is 150 mg.

In certain embodiments, the dosage of the MAPKAP pathway inhibitors corresponds to previously disclosed dosages and/or dosages approved for use by the Food and Drug Administration. In other embodiments, the dosage of the MAPKAP pathway inhibitor is less than previously approved dosages, such as about 20%, about 50%, or about 80% of the approved dosage. In certain embodiments, the dosage of trametinib is from about 5 to 20 mg per day orally, such as about 1 mg per day or about 2 mg per day. In certain variants

- 17 046068 The dosage of cobimetinib is from about 10 to 200 mg per day, for example about 30 mg or about 60 mg per day. In certain embodiments, the dosage of binimetinib is from about 10 mg to about 200 mg twice daily, such as about 25 mg or about 45 mg twice daily. In certain embodiments, the dosage of selumetinib is from about 10 mg to about 200 mg per day, or about 30 mg, or about 75 mg twice daily.

The amount and frequency of administration of the compounds described herein, and/or their pharmaceutically acceptable salts, and other therapeutic agents will be adjusted in accordance with the judgment of the attending physician, taking into account such factors as the age, condition and weight of the patient, as well as the severity of symptoms, treatment which are carried out.

The compounds of the present invention (eg, 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 (eg, with food) in capsules, suspensions, tablets, pills, dragees, liquids, gels, syrups, slurries, and the like. Methods for encapsulating compositions (such as coating with hard gelatin or cyclodextran) are known in the art (Baker et al., Controlled Release of Biological Active Agents, John Wiley and Sons, 1986, which is incorporated herein by reference in its entirety). The compounds can be administered to a subject in combination with an acceptable pharmaceutical carrier as part of a pharmaceutical composition. The composition of the pharmaceutical composition will vary according to the chosen route of administration. Suitable pharmaceutical carriers may contain inert ingredients that do not react with the compound. The carriers are biologically compatible, i.e. non-toxic, non-inflammatory, non-immunogenic and have no other adverse reactions at the injection site.

Exemplary pharmaceutical compositions are tablets and gelatin capsules containing a compound described herein, for example, Compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, such as

a) a diluent, for example purified water, triglyceride oils such as hydrogenated or partially hydrogenated vegetable oil or mixtures thereof, corn oil, olive oil, sunflower oil, safflower oil, fish oil such as EPA or DHA or their esters or triglycerides, or mixtures thereof, omega-3 fatty acids or derivatives thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose and/or glycine;

b) a lubricant, for example silicon, talc, stearic acid, its magnesium or calcium salt, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and/or polyethylene glycol; also for tablets;

c) a binder, for example magnesium aluminosilicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or beta-lactose, corn sugar, natural and synthetic gums such as acacia, tragacanth or sodium alginate, waxes and/or polyvinylpyrrolidone if necessary;

d) disintegrant, for example starches, agar, methylcellulose, bentonite, xanthan gum, alginic acid or its sodium salt or effervescent mixtures;

e) absorbent, coloring, flavoring and sweetening agent;

f) an emulsifier or dispersant such as Tween 80, Labrazole, HPMC, DOSS, Caproyl 909, Labrafac, Labrafil, Peceol, Transcutol, Capmule MCM, Capmule PG-12, Captex 355, Gelucir, Vitamin E TGPS or other suitable emulsifier; and/or

g) an agent that enhances the absorption of the compound, such as cyclodextrin, hydroxypropylcyclodextrin, PEG400, PEG200.

When formulated as a fixed dose, such combination products employ the compounds described herein within the dosage range described herein or known to those skilled in the art.

Because the compounds described herein (e.g., compounds A and B and MAPKAP pathway inhibitors) are intended for use in pharmaceutical compositions, those skilled in the art will appreciate that they can be obtained in substantially pure forms, e.g., at least 60% % pure, at least 75% pure, at least 85% pure and at least 98% pure (w/w). The pharmaceutical preparation may be in a unit dosage form. In such form, the preparation is divided into unit doses of suitable size containing suitable amounts of compounds A or B, for example an effective amount to achieve the desired purpose described herein.

Examples

Treatment with a combination of either Compound A or a pharmaceutically acceptable salt thereof in combination with a MAPKAP pathway inhibitor was found to unexpectedly and synergistically induce apoptosis of mutant c-KIT-controlled mast cells, which are the cause of systemic mastocytosis. In addition, this combination therapy provides inhibition of mastocytosis cell proliferation. In addition, the combination therapy disclosed herein was found to have a cytotoxic effect on mastocytosis, as opposed to a purely cytostatic effect, as determined by caspase activation induced by the combination treatment. Characterization of this unexpected discovery has been undertaken in biochemical assays and cellular assays, including those described herein.

Accordingly, the present invention is further illustrated by the following examples, which should not be construed as limiting the present invention in the scope or spirit of the specific procedures described herein. It should be understood that the examples are provided to illustrate certain embodiments and that the scope of the present invention is not intended to be limited thereby. It should also be understood that various other embodiments, modifications and equivalents thereof may be contemplated by those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

Example 1 Treatment of KIT mutant mast cell lines with Compound A or Compound B inhibits cell proliferation and KIT phosphorylation of mastocytosis cell lines.

A study was conducted which demonstrated that treatment with Compound A or Compound B inhibited cell proliferation of KIT mutant mast cell lines HMC1.1 KIT V560G and HMC1.2 KIT V560G/D816V. Assays were performed in 96-well plates seeded at 10,000 cells per well. Cells were treated with vehicle control, Compound A, or Compound B at various concentrations, allowed to grow for 72 hours, and then cell proliferation was assessed.

In fig. 1A is a graphical representation showing the relative percentage of cell proliferation determined for various concentrations of Compound A. Treatment with Compound A provided inhibition of increased cell proliferation in the HMC1.1 V560G and HMC1.2 V560G/D816V mast cell cell lines with IC50 values being 2.6 and 97 nM, respectively.

In fig. 1B is a graphical representation showing the relative percentage of cell proliferation determined for various concentrations of Compound B. Treatment with Compound B inhibited the increase in cell proliferation in the HMC1.1 V560G and HMC1.2 V560G/D816V cell lines with IC50 values of 2 .3 and 61 nM, respectively.

Example 2 Combination treatment with Compound A and trametinib induces apoptosis in mastocytosis cell lines.

A study was conducted to demonstrate that combination treatment with Compound A and the MEK inhibitor trametinib induced apoptosis in the V560G/D816V HMC1.2 mastocytosis cell lines. Assays were performed in 96-well plates seeded at 10,000 cells per well. Cells were treated with vehicle control, Compound A, trametinib, or combinations thereof at various concentrations and allowed to grow for 24 hours. Apoptosis was assessed by measuring caspase 3/7 activity.

In fig. 2A is a graphical representation showing the relative percentage of cell apoptosis determined for various treatments. In fig. 2B is a table showing the synergistic effects of the main analytes obtained using the additivity index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). CI<1 indicates synergism, CI=1 indicates additive effect, and CI>1 indicates antagonism. Combination treatments with Compound A and the MEK inhibitor trametinib unexpectedly showed strong synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. In fig. 2C is a graph based on the CI additivity score demonstrating the strong synergy for the combination of Compound A with trametinib to induce apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 3 Combination treatment with Compound B and trametinib induced apoptosis in mastocytosis cell lines.

A study was also conducted to demonstrate that combination treatment with Compound B and trametinib induced apoptosis in the V560G/D816V HMC1.2 mastocytosis cell line. Assays were performed as explained in Example 2. Apoptosis was assessed by measuring caspase 3/7 activity.

In fig. 3A is a graphical representation showing the relative percentage of cell apoptosis determined for various treatments. In fig. 3B is a table showing the synergistic effects of the main analytes obtained using the additivity index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). CI<1 indicates si

- 19 046068 nergism, CI=1 indicates an additive effect, and CI>1 indicates antagonism. Combination treatments with Compound B and the MEK inhibitor trametinib unexpectedly showed strong synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. In fig. 3C is a graph based on the CI additivity score demonstrating the strong synergy for the combination of Compound B with trametinib to induce apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 4 Combination treatment with Compound A and binimetinib induces apoptosis in mastocytosis cell lines.

Combination treatment with Compound A and binimetinib also induced apoptosis in HMC1.2 KIT V560G/D816V mastocytosis cell lines. Assays were performed as explained in Example 2. Apoptosis was assessed by measuring caspase 3/7 activity.

In fig. 4A is a graphical representation showing the relative percentage of cell apoptosis determined for various treatments. In fig. 4B is a table showing the synergistic effects of the main analytes obtained using the additivity index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). Combination treatments with Compound A and the MEK inhibitor binimetinib unexpectedly showed strong synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. In fig. 4C is a graph based on the CI additivity score demonstrating the strong synergy for the combination of Compound A with binimetinib to induce apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 5 Combination treatment with Compound B and binimetinib induces apoptosis in mastocytosis cell lines.

In fig. 5A is a graphical representation showing the relative percentage of cell apoptosis determined for various treatments with Compound B and binimetinib. In fig. 5B is a table showing the synergistic effects of the main analytes obtained using the additivity index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). CI<1 indicates synergism, CI=1 indicates additive effect, and CI>1 indicates antagonism. Combination treatments with Compound B and the MEK inhibitor binimetinib unexpectedly showed strong synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. In fig. 5C is a graph based on the CI additivity score demonstrating the strong synergy for the combination of Compound B with binimetinib to induce apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 6 Combination treatment with Compound A and cobimetinib induces apoptosis in mastocytosis cell lines.

In fig. 6A is a graphical representation showing the relative percentage of cell apoptosis determined for various Compound A and cobimetinib treatments. In fig. 6B is a table showing the synergistic effects of the main analytes obtained using the additivity index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). Combination treatments with Compound A and the MEK inhibitor cobimetinib unexpectedly showed strong synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. In fig. 6C is a graph based on the CI additivity score demonstrating the strong synergy for the combination of Compound A with cobimetinib to induce apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 7 Combination treatment with Compound B and cobimetinib induces apoptosis in mastocytosis cell lines.

In fig. 7A is a graphical representation showing the relative percentage of cell apoptosis determined for various Compound B and cobimetinib treatments. In fig. 7B is a table showing the synergistic effects of the main analytes obtained using the additivity index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). CI<1 indicates synergism, CI=1 indicates additive effect, and CI>1 indicates antagonism. Combination treatments with Compound B and the MEK inhibitor cobimetinib unexpectedly showed strong synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. In fig. 7C is a CI additivity graph showing strong synergy for the combination of Compound B with cobimetinib to induce apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 8 Combination treatment with Compound A and ulixertinib induces apoptosis in mastocytosis cell lines.

In fig. 8A is a graphical representation demonstrating the relative

- 20 046068 percentage of cell apoptosis determined for various treatments with Compound A and the ERK inhibitor ulixertinib. In fig. 8B is a table showing the synergistic effects of the main analytes obtained using the additivity index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). Combination treatments with Compound A and ulixertinib unexpectedly showed strong synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. In fig. 8C is a plot based on the CI additivity score demonstrating the strong synergy for the combination of Compound A with ulixertinib to induce apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 9 Combination treatment with Compound B and ulixertinib induces apoptosis in mastocytosis cell lines.

Combination treatment with Compound B and the ERK inhibitor ulixertinib can be assessed for the induction of apoptosis in HMC1.2 KIT V560G/D816V mast cells. A table containing data on the synergistic effects of the main analytes obtained using the additivity index (CI) method can be created as described by Chou and Talalay (1984) and using the computer software of Chou and Martin (2005). Combination treatments with Compound B and ulixertinib can be used to demonstrate synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. A plot based on the CI additivity score can be used to demonstrate synergy for the combination of Compound B and ulixertinib in inducing apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 10 Combination treatment with Compound A and SCH772984 induces apoptosis in mastocytosis cell lines.

Combination treatment with Compound A and SCH772984, an ERK inhibitor, can be assessed for the induction of apoptosis in HMC1.2 KIT V560G/D816V mast cells. A table containing data on the synergistic effects of the main analytes obtained using the additivity index (CI) method can be created as described by Chou and Talalay (1984) and using the computer software of Chou and Martin (2005). Combination treatments with Compound A and SCH772984 can be used to demonstrate synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. A plot based on the CI additivity score can be used to demonstrate the synergism for the combination of Compound A and SCH772984 in inducing apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 11 Combined treatment with Compound B and SCH772984 induces apoptosis in mastocytosis cell lines.

Combination treatment with Compound B and SCH772984, an ERK inhibitor, can be assessed for the induction of apoptosis in HMC1.2 KIT V560G/D816V mast cells. A table containing data on the synergistic effects of the main analytes obtained using the additivity index (CI) method can be created as described by Chou and Talalay (1984) and using the computer software of Chou and Martin (2005). Combination treatments with Compound A and SCH772984 can be used to demonstrate synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. A graph based on the CI additivity score can be used to demonstrate the synergy for the combination of Compound B and SCH772984 in inducing apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 12 Combined treatment with Compound A and LY3009120 induces apoptosis in mastocytosis cell lines.

Combination treatment with Compound A and the RAF inhibitor LY3009120 can be assessed for the induction of apoptosis in HMC1.2 KIT V560G/D816V mast cells. A table containing data on the synergistic effects of the main analytes obtained using the additivity index (CI) method can be created as described by Chou and Talalay (1984) and using the computer software of Chou and Martin (2005). Combination treatments with Compound A and LY3009120 can be used to demonstrate synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. A plot based on the CI additivity score can be used to demonstrate the strong synergy for the combination of Compound A and LY3009120 in inducing apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 13 Combined treatment with Compound B and LY3009120 induces apoptosis in mastocytosis cell lines.

Combination treatment with Compound B and the RAF inhibitor LY3009120 can be assessed for the induction of apoptosis in HMC1.2 KIT V560G/D816V mast cells. Table with data on the synergistic effects of the main analytes obtained using

- 21 046068 However, a method based on determining the additivity index (CI) can be created as described by Chou and Talalay (1984) and using computer software by Chou and Martin (2005). Combination treatments with Compound B and LY3009120 can be used to demonstrate synergy in inducing apoptosis in HMC1.2 KIT V560G/D816V cells. A CI additivity graph can be used to demonstrate synergism for the combination of Compound B and LY3009120 in inducing apoptosis in HMC1.2 KIT V560G/D816V mast cells.

Example 14 Combination treatment with Compound A and trametinib results in a synergistic reduction in colony germination of the HMC1.2 KIT D816V mast cell line.

A study was conducted to demonstrate that combination treatment with Compound A and the MEK inhibitor trametinib resulted in reduced HMC1.2 colony growth compared to treatment with either agent alone. 10,000 HMC1.2 cells were grown in soft agar and incubated with various concentrations of Compound A, trametinib, or a combination of Compound A and trametinib for 10 days. Treatment drugs were removed and viable cell colony germination was monitored for 5 additional days.

In fig. 9A is an illustrative depiction of HMC1.2 mast cell colony germination following treatment with trametinib (0, 10 or 25 nM), Compound A (0, 25 or 50 nM) as single agents or as a matrix combination of various concentrations of Compound A with various concentrations of trametinib . While treatment with the single agent Compound A or trametinib resulted in colony germination at all concentrations 5 days after drug removal, the combination of Compound A with trametinib resulted in less colony germination 5 days after drug removal. The combination of compound A (25 nM) with trametinib (25 nM) resulted in complete cessation of colony germination 5 days after drug removal. The combination of Compound A (50 nM) with trametinib (10 or 25 nM) unexpectedly resulted in a complete arrest of viable HMC1.2 cell germination to the limit of detection, as determined by microscopy imaging using a 5X objective 5 days after drug removal, at This discontinuation was not observed following treatment with the single agent Compound A or the single agent trametinib. By further increasing colony germination for 8 additional days (13 days total) after drug removal, the cessation of colony germination was still maintained to the limit of detection by the combination of Compound A (50 nM) and trametinib (10 and 25 nM). However, ~15-20 colonies germinated after an additional 8 days of incubation with the combination of Compound A (25 nM) and trametinib (25 nM). (Fig. 9A right panel).

In fig. 9B is a graphical representation of the quantification of HMC1.2 mast cell colony germination after the various treatments shown in FIG. 9A. Combination treatment with Compound A at 50 nM and trametinib at either 10 nM or 25 nM unexpectedly resulted in cessation of colony germination to the limit of detection as determined by microscopy imaging using a 5X objective (see arrows in FIG. 9B). Combination treatment with Compound A at 25 nM and trametinib at 25 nM unexpectedly resulted in cessation of colony germination to the limit of detection as determined by microscopy imaging using a 5X objective (see arrows in Fig. 9B), with no cessation observed after treatment a separate agent, which is either Compound A or trametinib.

Example 15 Combination treatment with Compound B and trametinib results in a synergistic reduction in colony germination of the HMC1.2 KIT D816V mast cell line.

Compound B and the MEK inhibitor trametinib were also tested to evaluate the reduction of colony germination in HMC1.2 cells as described in Example 14.

In fig. 10A is an illustrative depiction of HMC1.2 mast cell colony germination following treatment with trametinib (0, 10 or 25 nM), Compound B (0, 25 or 50 nM) as single agents or as a matrix combination of various concentrations of Compound B with various concentrations of trametinib . While single agent treatment with Compound B or trametinib resulted in colony germination at all concentrations 5 days after drug removal, the combination of Compound B with trametinib resulted in less colony germination 5 days after drug removal. The combination of Compound B (25 nM) with trametinib (25 nM) resulted in a significant reduction in viable HMC1.2 cell germination 5 days after drug removal. The combination of Compound B (50 nM) with trametinib (10 or 25 nM) unexpectedly resulted in complete inhibition of viable HMC1.2 cell germination to the limit of detection, as determined by microscopy imaging using a 5X objective 5 days after drug removal, at This cessation was not observed following treatment with the single agent Compound B or the single agent trametinib. With additional

- 22 046068 No increase in colony germination for 8 additional days (13 days total) after drug removal still maintained cessation of colony germination to the limit of detection by the combination of Compound B (50 nM) and trametinib (25 nM).

In fig. 10B is a graphical representation of the quantification of HMC1.2 mast cell colony germination after various treatments shown in FIG. 10A. Combination treatment with Compound B at 50 nM and trametinib at either 10 nM or 25 nM unexpectedly resulted in cessation of colony germination to the limit of detection as determined by microscopy imaging using a 5X objective, with no cessation observed following treatment with the individual agent representing being either Compound B or trametinib (see arrows in FIG. 10B).

Example 16 Combination treatment with Compound A and binimetinib results in a synergistic reduction in colony germination of the HMC1.2 KIT D816V mast cell line.

In fig. 11A is an illustrative depiction of HMC1.2 mast cell colony germination following treatment with binimetinib (0, 250 or 500 nM), Compound A (0, 25 or 50 nM) as single agents or as a matrix combination of various concentrations of Compound A with various concentrations of binimetinib . While treatment with the single agent Compound A or binimetinib resulted in colony germination at all concentrations 5 days after drug removal, the combination of Compound A with binimetinib resulted in less colony germination 5 days after drug removal. The combination of compound A (25 nM) with binimetinib (250 nM) resulted in a significant reduction in viable HMC1.2 cell germination 5 days after drug removal. The combination of Compound A (50 nM) with binimetinib (250 or 500 nM) unexpectedly resulted in complete inhibition of viable HMC1.2 cell germination to the limit of detection, as determined by microscopy imaging using a 5X objective 5 days after drug removal, at This cessation was not observed following treatment with the single agent Compound A or the single agent binimetinib. By further increasing colony germination for 8 additional days (13 days total) after drug removal, the cessation of colony germination was still maintained to the limit of detection by the combination of Compound A (50 nM) and binimetinib (500 nM). However, ~10-15 colonies germinated when the combination of Compound A (50 nM) and binimetinib (250 nM) was used.

In fig. 11B is a graphical representation of the quantification of HMC1.2 mast cell colony germination after various treatments shown in FIG. 11A. Combination treatment with Compound A at 50 nM and binimetinib at 250 or 500 nM unexpectedly resulted in cessation of colony germination to the limit of detection as determined by microscopy imaging using a 5X objective, with no cessation observed following treatment with either compound alone. A, or binimetinib (see arrows in Fig. 11B).

Example 17 Combination treatment with Compound B and binimetinib results in a synergistic reduction in colony germination of the HMC1.2 KIT D816V mast cell line.

In fig. 12A is an illustrative depiction of HMC1.2 mast cell colony germination following treatment with binimetinib (0, 250 or 500 nM), Compound B (0, 25 or 50 nM) as single agents or as a matrix combination of various concentrations of Compound B with various concentrations of binimetinib . While treatment with the single agent Compound B or binimetinib resulted in colony germination at all concentrations 5 days after drug removal, the combination of Compound B with binimetinib resulted in less colony germination 5 days after drug removal. The combination of Compound B (25 nM) with binimetinib (250 nM) resulted in a significant reduction in viable HMC1.2 cell germination 5 days after drug removal. The combination of Compound B (25 nM) with binimetinib (500 nM) resulted in complete inhibition of viable HMC1.2 cell germination to the limit of detection 5 days after drug removal, as determined by microscopy imaging using a 5X objective. The combination of Compound B (50 nM) with binimetinib (250 or 500 nM) unexpectedly resulted in complete inhibition of viable HMC1.2 cell germination to the limit of detection as determined by microscopy imaging using a 5X objective 5 days after drug removal, at This cessation was not observed following treatment with the single agent Compound B or the single agent binimetinib. By further increasing colony germination for 8 additional days (13 days total) after drug removal, the cessation of colony germination was still maintained to the limit of detection by the combination of Compound B (50 nM) and binimetinib (250 and 500 nM) and Compound B (25 nM ) and binimetinib (500 nM).

In fig. 12B is a graphical representation of the quantification of HMC1.2 mast cell colony germination after various treatments shown in FIG. 12A. Combini

- 23 046068 treatment with Compound B at 25 nM and binimetinib at 500 nM and combined treatment with Compound B at 50 nM and binimetinib at either 250 nM or 500 nM unexpectedly resulted in cessation of colony germination to the limit of detection as determined by imaging microscopy using a 5X objective, and no termination was observed following treatment with either Compound B or binimetinib as a single agent (see arrows in FIG. 12B).

Example 18 Combination treatment with Compound A and cobimetinib results in a synergistic reduction in colony germination of the HMC1.2 KIT D816V mast cell line.

In fig. 13A is an illustrative depiction of HMC1.2 mast cell colony growth following treatment with cobimetinib (0, 25 or 50 nM), Compound A (0, 25 or 50 nM) as single agents or as a matrix combination of various concentrations of Compound A with various concentrations of cobimetinib . While treatment with Compound A or cobimetinib alone resulted in colony germination at all concentrations 5 days after drug removal, the combination of Compound A with cobimetinib resulted in less colony germination 5 days after drug removal compared to treatment by any single means. The combination of Compound A (50 nM) with cobimetinib (25 or 50 nM) resulted in a significant reduction in viable HMC1.2 cell germination 5 days after drug removal.

In fig. 13B is a graphical representation of the quantification of HMC1.2 mast cell colony germination after various treatments shown in FIG. 13A. Combination treatment with Compound A at 50 nM and cobimetinib at either 25 nM or 50 nM resulted in a significant reduction in colony germination compared to treatment with either Compound A or cobimetinib alone.

Example 19 Combination treatment with Compound B and cobimetinib results in a synergistic reduction in colony germination of the HMC1.2 KIT D816V mast cell line.

In fig. 14A is an illustrative depiction of HMC1.2 mast cell colony growth following treatment with cobimetinib (0, 25 or 50 nM), Compound B (0, 25 or 50 nM) as single agents or as a matrix combination of various concentrations of Compound B with various concentrations of cobimetinib . However, treatment with Compound B or cobimetinib alone resulted in colony germination at all concentrations 5 days after drug removal, while the combination of Compound B with cobimetinib resulted in less colony germination 5 days after drug removal compared with treatment with any separate means. The combination of Compound B (50 nM) with cobimetinib (25 or 50 nM) resulted in a significant reduction in viable HMC1.2 cell germination 5 days after drug removal.

In fig. 14B is a graphical representation of the quantification of HMC1.2 mast cell colony germination after various treatments shown in FIG. 14A. Combination treatment with Compound B at 50 nM and cobimetinib at either 25 nM or 50 nM resulted in a significant reduction in colony germination compared to treatment with either Compound B or cobimetinib alone.

Example 20 Combination treatment with Compound A and the ERK inhibitor ulixertinib resulted in reduced colony germination of the HMC1.2 KIT D816V mast cell line.

Combination treatment with Compound A and ulixertinib can be assessed for inhibition of colony germination of the HMC1.2 KIT D816V mast cell line according to the method of Example 14.

Example 21 Combination treatment with Compound B and the ERK inhibitor ulixertinib resulted in decreased colony germination of the HMC1.2 KIT D816V mast cell line.

Combination treatment with Compound B and ulixertinib can be assessed for inhibition of colony germination of the HMC1.2 KIT D816V mast cell line according to the method of Example 14.

Example 22 Combined treatment with Compound A and the RAF inhibitor LY3009120 resulted in decreased colony germination of the HMC1.2 KIT D816V mast cell line.

Combination treatment with Compound A and the RAF inhibitor LY3009120 can be evaluated for inhibition of colony germination of the HMC1.2 KIT D816V mast cell line according to the method of Example 14.

Example 23. Combined treatment with compound B and LY3009120 leads to a decrease in colony germination of the mast cell line HMC1.2 KIT D816V.

Combination treatment with Compound B and the RAF inhibitor LY3009120 can be assessed for inhibition of colony germination of the HMC1.2 KIT D816V mast cell line according to the method of Example 14.

Example 24 Combination treatment with Compound A and the MEK inhibitor trametinib induces apoptosis in the HMC1.2 KIT mastocytosis mutant cell line

- 24 046068

V560G/D816V transfected with N-ras G12D.

A study was conducted to demonstrate that combination treatment with Compound A and trametinib induced apoptosis in HMC1.2 V560G/D816V mutant cells transfected with N-ras G12D. Assays were performed in 96-well plates seeded at 10,000 cells per well. Cells were treated with vehicle control, Compound A, trametinib, or combinations thereof at various concentrations and allowed to grow for 24 and 48 hours. Apoptosis was assessed by measuring caspase 3/7 activity.

In fig. 15A is a graphical representation showing the relative percentage of caspase activity determined for different 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 HMC1.2 V560G/D816V mutant cells transfected with N-ras G12D (right panel). Combination of Compound A with trametinib results in a synergistic increase in apoptosis in EV-transfected and N-ras G12D-transfected HMC1.2 cells.

In fig. 15B is a graphical representation showing the relative percentage of caspase activity determined for different 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 HMC1.2 V560G/D816V mutant cells transfected with N-ras G12D (right panel). Combination of Compound A with trametinib results in a synergistic increase in apoptosis in EV-transfected and N-ras G12D-transfected HMC1.2 cells. Apoptosis was higher in cells transfected with N-ras than in cells transfected with EVs.

Example 25 Combination treatment with Compound A and the MEK inhibitor cobimetinib induces apoptosis in the HMC1.2 KIT V560G/D816V mastocytosis mutant cell line transfected with N-ras G12D.

In fig. 16A is a graphical representation showing the relative percentage of caspase activity determined for different 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 HMC1.2 V560G/D816V mutant cells transfected with N-ras G12D (right panel). Combination of Compound A with cobimetinib results in a synergistic increase in apoptosis in EV-transfected and N-ras G12D-transfected HMC1.2 cells. Apoptosis was higher in cells transfected with N-ras than in cells transfected with EVs.

In fig. 16B is a graphical representation showing the relative percentage of caspase activity determined for different treatments at 48 hours. Combined 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 HMC1.2 V560G/D816V mutant cells transfected with N-ras G12D (right panel). Combination of Compound A with cobimetinib results in a synergistic increase in apoptosis in EV-transfected and N-ras G12D-transfected HMC1.2 cells. Apoptosis was higher in cells transfected with N-ras than in cells transfected with EVs.

Example 26 Combination treatment with Compound A and the MEK inhibitor trametinib resulted in a synergistic reduction in colony germination of the HMC1.2 KIT D816V mast cell line transfected with N-ras G12D or empty vector.

A study was conducted to demonstrate that combination treatment with Compound A and trametinib resulted in reduced germination of an empty vector (EV)-transfected HMC1.2 colony or N-ras G12D-transfected colony compared to treatment with either agent alone. HMC1.2 cells were incubated with various concentrations of Compound A, trametinib, or a combination of Compound A and trametinib for 10 days. Treatment drugs were removed and viable cell colony germination was monitored for 5 or 13 additional days.

In fig. 17A is an illustrative depiction of colony germination of HMC1.2 mast cells transfected with EVs following treatment with trametinib (0, 1, 10 or 25 nM), Compound A (0, 25 or 50 nM) as single agents or as a matrix combination of various concentrations compound A with different concentrations of trametinib. While treatment with the single agent Compound A or trametinib resulted in colony germination at all concentrations 5 days after drug removal, the combination of Compound A with trametinib resulted in less colony germination 5 days after drug removal. The combination of Compound A (50 nM) with trametinib (1 nM) resulted in a significant reduction in colony germination in combination with 1 nM trametinib compared to treatment with Compound A alone or treatment with trametinib alone. The combination of compound A (50 nM) with trametinib (10 or 25 nM) unexpectedly led to

- 25 046068 complete cessation of germination of viable HMC1.2 cells to the limit of detection, as determined by microscopy imaging using a 5X objective 5 days after drug removal, where cessation was not observed after treatment with a separate compound A agent, or a separate agent, trametinib. By further increasing colony germination for 8 additional days after drug removal (a total of 13 days after drug removal), colony germination was still maintained to the limit of detection by the combination of Compound A (50 nM) and trametinib (25 nM).

In fig. 17B is a graphical representation of the quantification of colony germination of EV-transfected HMC1.2 mast cells after the various treatments shown in FIG. 17A. Combination treatment with Compound A at 50 nM and trametinib at either 10 nM or 25 nM unexpectedly resulted in cessation of colony germination to the limit of detection as determined by microscopy imaging using a 5X objective, while cessation was not observed after treatment with the individual agent representing is either Compound A or trametinib (see arrows in FIG. 17B).

In fig. 17C is an illustrative image of colony germination of N-ras G12D transfected HMC1.2 cells following treatment with trametinib (0, 1, 10 or 25 nM), Compound A (0, 25 or 50 nM) as single agents or as a matrix combination different concentrations of Compound A with different concentrations of trametinib. While treatment with the single agent Compound A or trametinib resulted in colony germination at all concentrations 5 days after drug removal, the combination of Compound A with trametinib resulted in less colony germination 5 days after drug removal. The combination of Compound A (25 nM) with trametinib (25 nM) resulted in a significant reduction in viable HMC1.2 cell germination 5 days after drug removal, and the combination of Compound A (50 nM) with trametinib (10 or 25 nM) unexpectedly resulted in complete cessation of germination of viable N-ras G12D-transfected HMC1.2 cells to the limit of detection as determined by microscopy imaging using a 5X objective 5 days after drug removal, with no cessation observed following treatment with a separate drug representing either compound A or trametinib. By further increasing colony germination for 8 additional days after drug removal (a total of 13 days after drug removal), the cessation of colony germination was still maintained by the combination of Compound A (50 nM) and trametinib (25 nM) (Figure 17C).

In fig. 17D is a graphical representation of the quantification of colony germination after various treatments shown in FIG. 17C. Combination treatments with Compound A and trametinib resulted in superior colony germination blocking compared to either treatment alone. Combination treatment with Compound A at 50 nM and trametinib at either 10 nM or 25 nM unexpectedly resulted in cessation of colony germination to the detection limit as determined by microscopy imaging using a 5X objective 5 days after drug removal, but no cessation. was observed after treatment with either Compound A or trametinib as a single agent (see arrows in FIG. 17D).

Example 27 Combination treatment with Compound A and cobimetinib results in a synergistic reduction in colony germination of the HMC1.2 KIT D816V mast cell line transfected with N-ras G12D or empty vector.

A study was conducted to demonstrate that combination treatment with Compound A and cobimetinib resulted in reduced germination of an empty vector (EV)-transfected HMC1.2 colony or N-ras G12D-transfected colony compared to 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. Treatment drugs were removed and viable cell colony germination was monitored for 5 or 10 additional days.

In fig. 18A is an illustrative depiction of colony germination of HMC1.2 mast cells transfected with EVs following treatment with cobimetinib (25 or 50 nM), Compound A (0, 25 or 50 nM) as single agents or as a matrix combination of various concentrations of Compound A with various cobimetinib concentrations. While treatment with the single agent Compound A or cobimetinib resulted in colony germination at all concentrations 5 days after drug removal, the combination of Compound A with cobimetinib resulted in less colony germination 5 days after drug treatment was removed. The combination of Compound A (25 nM) with cobimetinib (25 and 50 nM) resulted in a significant reduction in viable HMC1.2 cell germination 5 days after removal of drug treatment. The combination of compound A (50 nM) with cobimetinib (50 nM) is unexpected

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Claims (26)

resulted in complete cessation of germination of viable HMC1.2 cells to the limit of detection as determined by microscopy imaging using a 5X objective 5 days after removal of drug treatment, and cessation was not observed after treatment with a separate compound A drug, or a separate agent, cobimetinib. By further increasing colony germination for 5 additional days after drug treatment was removed (for a total of 10 days after drug removal), colony germination was still maintained to the limit of detection by the combination of Compound A (50 nM) and cobimetinib (50 nM). In fig. 18B is a graphical representation of the quantification of colony germination of EV-transfected HMC1.2 mast cells after the various treatments shown in FIG. 18A. Combination treatment with Compound A at 50 nM and cobimetinib at 50 nM unexpectedly resulted in cessation of colony germination to the limit of detection as determined by microscopy imaging using a 5X objective, with no cessation observed following treatment with either A or A alone. cobimetinib (see arrows in Fig. 18B). In fig. 18C is an illustrative image of the germination of a colony of HMC1.2 mast cells transfected with N-ras G12D following treatment with cobimetinib (0, 25 or 50 nM), Compound A (0, 25 or 50 nM) as individual agents or as a matrix combination of various concentrations of compound A with different concentrations of cobimetinib. While single agent treatment with Compound A or cobimetinib resulted in colony germination at all concentrations 5 days after drug treatment was removed, the combination of Compound A with cobimetinib resulted in less colony germination 5 days after drug treatment was removed. The combination of Compound A (25 nM) with cobimetinib (25 and 50 nM) resulted in a significant reduction in viable HMC1.2 cell germination 5 days after drug removal. The combination of Compound A (50 nM) with cobimetinib (25 or 50 nM) unexpectedly resulted in complete inhibition of viable HMC1.2 cell germination to the limit of detection, as determined by microscopy imaging using a 5X objective 5 days after removal of drug treatment. however, discontinuation was not observed following treatment with the single agent Compound A or the single agent cobimetinib. By further increasing colony germination for 10 additional days after removal of drug treatment, colony germination was still maintained to the limit of detection by the combination of Compound A (50 nM) and cobimetinib (25 or 50 nM). In fig. 18D is a graphical representation of the quantification of colony germination after various treatments shown in FIG. 18C. Combination treatments with Compound A and cobimetinib resulted in superior colony germination blocking compared to either treatment alone. Combination treatment with Compound A at 50 nM and cobimetinib at 25 or 50 nM unexpectedly resulted in cessation of colony germination to the limit of detection as determined by microscopy imaging using a 5X objective 5 days after drug removal, with no cessation observed after treatment a separate agent, which is either compound A or cobimetinib (see arrows in Fig. 18D). Equivalents. One skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments specifically described in the present invention. Such equivalents are intended to be included within the scope of the following claims. CLAIM 1. A method of treating mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of a c-KIT inhibitor represented by Compound A (Compound A), or a pharmaceutically acceptable salt thereof; and an effective amount of one or more MAPKAP pathway inhibitors selected from the group consisting of a mitogen-activated protein kinase (MEK inhibitor), selected from the group consisting of trametinib, selumetinib, cobimetinib and binimetinib, or an extracellular signal-regulated kinase inhibitor (ERK inhibitor) , selected from the group consisting of ulixertinib, - 27 046068 SCH772984 and LY3214996, and a rapidly progressing fibrosarcoma (RAF) kinase inhibitor selected from the group consisting of vemurafenib, dabrafenib and LY3009120. 2. The method according to claim 1, wherein mastocytosis is characterized by a c-KIT mutation. 3. The method of claim 2, wherein the c-KIT mutation is an activating mutation. 4. The method of claim 3, wherein the c-KIT mutation is a c-KIT D816 mutation. 5. The method according to any one of claims 2 to 4, wherein the c-KIT D816V mutation is a primary c-KIT mutation. 6. The method according to any one of claims 1 to 5, wherein the mastocytosis involves mast cells containing a secondary c-KIT mutation. 7. The method of claim 6, wherein the secondary c-KIT mutation is one of the Y269C, Y503_F504insAY, V560D, or K642E mutations. 8. The method according to any one of claims 5 to 7, further comprising determining whether mastocytosis is characterized by a primary c-KIT mutation. 9. The method according to any one of claims 5 to 7, further comprising determining whether mastocytosis is characterized by a secondary c-KIT mutation. 10. The method of claim 8 or 9, wherein determining whether mastocytosis is characterized by a primary or secondary c-KIT mutation includes identifying mutations in DNA isolated from the tumor sample. 11. The method of claim 8 or 9, wherein determining whether mastocytosis is characterized by a primary or secondary c-KIT mutation includes identifying mutations in circulating tumor DNA or in circulating peripheral blood leukocytes. 12. The method according to any one of claims 1 to 11, wherein the mastocytosis is systemic mastocytosis. 13. The method of claim 12, wherein systemic mastocytosis is selected from the group consisting of indolent systemic mastocytosis, indolent systemic mastocytosis, systemic mastocytosis with associated disease associated with a lineage of clonal hematological cells other than mast cells, aggressive systemic mastocytosis, leukemia from mast cell and mast cell sarcoma. 14. The method of claim 12, wherein the mastocytosis is indolent systemic mastocytosis, optionally systemic mastocytosis with recurrent anaphylaxis or vascular collapse in the absence of skin lesions. 15. The method of claim 12, wherein the mastocytosis is mast cell leukemia or mast cell sarcoma. 16. Method according to any one of claims 1 to 11, wherein the mastocytosis is cutaneous mastocytosis. 17. The method of claim 16, wherein the mastocytosis is selected from the group consisting of maculopapular cutaneous mastocytosis, mastocytoma, or diffuse cutaneous mastocytosis. 18. The method according to any one of claims 1 to 17, wherein the MAPKAP pathway inhibitor is a MEK inhibitor selected from the group consisting of trametinib, selumetinib, cobimetinib and binimetinib. 19. The method according to any one of claims 1 to 17, wherein the MAPKAP pathway inhibitor is an ERK inhibitor selected from the group consisting of ulixertinib, SCH772984 and LY3214996. 20. The method according to any one of claims 1 to 19, wherein the c-KIT inhibitor and the MEK and/or ERK inhibitor are administered substantially simultaneously or sequentially. 21. The method according to any one of claims 1 to 20, further comprising administering another targeted anticancer therapy agent, a biological targeted anticancer therapy agent, an immune checkpoint inhibitor, or a chemotherapeutic agent. 22. A method of treating systemic mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of Compound A or a pharmaceutically acceptable salt thereof; and an effective amount of one or more MAPKAP pathway inhibitors selected from the group consisting of a mitogen-activated protein kinase (MEK inhibitor), selected from the group consisting of trametinib, selumetinib, cobimetinib and binimetinib, an extracellular signal-regulated kinase inhibitor (ERK inhibitor), selected from the group consisting of ulixertinib, SCH772984 and LY3214996. 23. The method according to claim 22, wherein systemic mastocytosis is characterized by a c-KIT mutation. 24. The method of claim 23, wherein the mutation is a c-KIT D816 mutation. 25. The method according to claim 23 or 24, where the mutation is D816V. 26. The method according to any one of claims 22-25, where mastocytosis is characterized by an additional mutation -