Combination therapy for treating mastocytosis

文档序号：975785 发布日期：2020-11-03 浏览：7次中文

阅读说明：本技术 治疗肥大细胞增多症的组合疗法 (Combination therapy for treating mastocytosis ) 是由 D·L·弗林 B·D·史密斯 A·古普塔于 2019-01-31 设计创作，主要内容包括：本公开涉及1-[4-溴-5-[1-乙基-7-(甲基氨基)-2-氧代-1,2-二氢-1,6-萘啶-3-基]-2-氟苯基]-3-苯基脲、或1-(5-(7-氨基-1-乙基-2-氧代-1,2-二氢-1,6-萘啶-3-基)-4-溴-2-氟苯基)-3-苯基脲或它们的药学上可接受的盐与MAPKAP途径抑制剂例如RAS、RAF、MEK或ERK抑制剂组合用于治疗肥大细胞增多症的用途。(The present disclosure relates to the use of 1- [ 4-bromo-5- [ 1-ethyl-7- (methylamino) -2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl ] -2-fluorophenyl ] -3-phenyl urea, or 1- (5- (7-amino-1-ethyl-2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl) -4-bromo-2-fluorophenyl) -3-phenyl urea, or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, such as a RAS, RAF, MEK, or ERK inhibitor, for the treatment of mastocytosis.)

1. A method of treating mastocytosis in a patient in need thereof, the method comprising administering to the patient:

a. an effective amount of an inhibitor of c-KIT; and

b. an effective amount of one or more MAPKAP pathway inhibitors.

2. The method of claim 1, wherein said MAPKAP pathway inhibitor is selected from the group consisting of: mitogen-activated protein kinase (MEK inhibitor), extracellular signal-regulated kinase inhibitor (ERK inhibitor), and rapid-accelerated fibrosarcoma (RAF) kinase inhibitor.

3. The method of claim 1 or 2, wherein the mastocytosis has a c-KIT mutation.

4. The method of any one of claims 1-3, wherein the c-KIT mutation is an activating mutation.

5. The method of any one of claims 1-4, wherein the mastocytosis comprises a mast cell having a primary mutation in exon 17 of the c-KIT gene.

6. The method of claim 5, wherein the primary mutation is a c-KITD816 mutation.

7. The method of claim 5 or 6, wherein the primary mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del 419.

8. The method of any one of claims 5-6, wherein the primary mutation is D816V.

9. The method of any one of claims 1-8, wherein the mastocytosis comprises a mast cell having a secondary c-KIT mutation.

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

11. The method of claim 9 or 10, wherein the secondary c-KIT mutation is one of the Y269C, Y503_ F504insAY, V560D, or K642E mutations.

12. The method of any one of claims 6-11, further comprising determining whether the mastocytosis has the primary mutation of c-KIT.

13. The method of any one of claims 6-11, further comprising determining whether the mastocytosis has the secondary mutation of c-KIT.

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

15. The method of claim 12 or 13, wherein determining whether the mastocytosis has the c-KIT primary or secondary mutation comprises identifying a mutation in circulating tumor DNA or in circulating peripheral blood leukocytes.

16. The method of any one of claims 1-15, wherein the mastocytosis is systemic mastocytosis.

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

18. The method of claim 16, wherein the mastocytosis is indolent systemic mastocytosis, optionally systemic mastocytosis with repeated allergic reactions or vascular collapse in the absence of skin lesions.

19. The method of claim 16, wherein the mastocytosis is systemic smoldering mastocytosis.

20. The method of claim 16, wherein the mastocytosis is systemic mastocytosis with clonal hematologic non-mast cell lineage disease.

21. The method of claim 16, wherein the mastocytosis is an invasive systemic mastocytosis.

22. The method of claim 16, wherein the mastocytosis is a mast cell leukemia or a mast cell sarcoma.

23. The method of any one of claims 1-15, wherein the mastocytosis is cutaneous mastocytosis.

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

25. The method of any one of claims 1-24, wherein the c-KIT inhibitor is selected from the group consisting of: 1- [ 4-bromo-5- [ 1-ethyl-7- (methylamino) -2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl ] -2-fluorophenyl ] -3-phenylurea or a pharmaceutically acceptable salt thereof, midostaurin or a pharmaceutically acceptable salt thereof, imatinib mesylate, sunitinib malate, midostaurin, regorafenib, krynanide, PTX9486, or BLU-285 or a pharmaceutically acceptable salt thereof.

26. The method of any one of claims 2-25, wherein the MEK inhibitor is selected from the group consisting of: trametinib, semetinib, cobitinib and bimitinib.

27. The method of any one of claims 2-26, wherein the MEK inhibitor is bimetinib.

28. The method of any one of claims 2-27, wherein the MEK inhibitor is trametinib.

29. The method of any one of claims 2-25, wherein the ERK inhibitor is selected from the group consisting of: ulitinib, SCH772984 and LY 3214996.

30. The method of any one of claims 2-29, wherein the c-KIT inhibitor and the MEK and/or ERK inhibitor are administered substantially concurrently or sequentially.

31. The method of any one of claims 1-30, further comprising administering another cancer-targeted therapeutic, a cancer-targeted biological immune checkpoint inhibitor, or a chemotherapeutic.

32. The method of any one of claims 1-31, wherein the patient has at least partial remission after two weeks or more of administration.

33. A method of treating systemic mastocytosis in a patient in need thereof, the method comprising administering to the patient:

an effective amount of 1- [ 4-bromo-5- [ 1-ethyl-7- (methylamino) -2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl ] -2-fluorophenyl ] -3-phenylurea or a pharmaceutically acceptable salt thereof; and an effective amount of one or more MAPKAP pathway inhibitors.

34. The method of claim 33, wherein said MAPKAP pathway inhibitor is selected from the group consisting of: mitogen-activated protein kinase (MEK inhibitor) and extracellular signal-regulated kinase inhibitor (ERK inhibitor).

35. The method of claim 33 or 34, wherein the systemic mastocytosis has a c-KIT mutation.

36. The method of claim 33 or 34, wherein the mutation is a c-KITD816 mutation.

37. The method of claim 36, wherein the mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del 419.

38. The method of claim 37, wherein the mutation is one of: A553D, C433Y, D419Y, D572A, D816F, D816H, D816I, D816V, D816Y, D820G, del419, dup (501-) -502), E839K, F522C, I817V, InsFF419, InsV815-I816, K509I, N822I, R815K, T417V, V560G, V559I or Y418Y.

39. The method of any one of claims 34-38, wherein the mutation is D816V.

40. The method of any one of claims 34-38, wherein the mastocytosis has an additional c-KIT mutation, the additional c-KIT mutation being one of the Y269C, Y503_ F504insAY, V560D, or K642E mutations.

41. The method of any one of claims 34-40, wherein the MEK inhibitor is selected from the group consisting of: trametinib, semetinib, cobitinib and bimitinib.

42. The method of any one of claims 34-40, wherein the MEK inhibitor is bimetatinib.

43. The method of any one of claims 34-40, wherein the MEK inhibitor is trametinib.

44. The method of any one of claims 34-42, wherein the ERK inhibitor is selected from the group consisting of: ulitinib, SCH772984 and LY 3214996.

45. A method of treating mastocytosis in a patient in need thereof, the method comprising administering to the patient:

an effective amount of an inhibitor of c-KIT; and

an effective amount of a RAF inhibitor.

46. The method of claim 44, wherein the RAF inhibitor is a pan RAF or B-RAF inhibitor.

47. The method of claim 44 or 45, wherein the c-KIT inhibitor is 1- [ 4-bromo-5- [ 1-ethyl-7- (methylamino) -2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl ] -2-fluorophenyl ] -3-phenylurea or a pharmaceutically acceptable salt thereof.

Background

c-KIT (also known as KIT, CD117 and stem cell factor receptor) is a 145kDa transmembrane tyrosine kinase protein that acts as a type III receptor. The c-KIT proto-oncogene located on chromosome 4q11-21 encodes the c-KIT receptor, the ligand of which is stem cell factor (SCF, steel factor, KIT ligand, mast cell growth factor). The receptor has tyrosine protein kinase activity and binding of ligand SCF results in autophosphorylation of c-KIT and its association with a substrate such as phosphatidylinositol 3-kinase (PI 3K). Tyrosine phosphorylation by protein tyrosine kinases is particularly important in cell signaling and can mediate signals of major cellular processes such as proliferation, survival, differentiation, apoptosis, adhesion, invasiveness and migration.

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

Mastocytosis is a rare disorder characterized by abnormal accumulation of Mast Cells (MC) in the skin, bone marrow, and internal organs such as the liver, spleen, gastrointestinal tract, and lymph nodes. Cases starting during adulthood are often chronic and involve the bone marrow in addition to the skin, however during childhood the condition is usually marked by skin appearance, no internal organs are affected and can usually be alleviated during adolescence. Mastocytosis tends to persist in most adult patients and may progress to a more advanced category in a few patients. Mastocytosis can be classified into a specific type depending on the symptoms and overall performance of the patient. Types of mastocytosis include cutaneous mastocytosis (e.g., maculopapular cutaneous mastocytosis, mastocytoma, and diffuse cutaneous mastocytosis) and systemic mastocytosis (e.g., Indolent Systemic Mastocytosis (ISM), Systemic Smoldering Mastocytosis (SSM), systemic mastocytosis with clonal hematologic non-mast cell lineage disease (SM-AHN), Aggressive Systemic Mastocytosis (ASM), Mast Cell Leukemia (MCL), and mast cell sarcoma). In rare cases, invasive neoplastic mast cell outgrowth leads to end organ failure, whereby patients have a significantly shortened life span and need cytoreductive therapy. Pathological accumulation of neoplastic mast cells caused by oncogenic mutations in c-KIT was found to be the cause of systemic mastocytosis. The major activating KIT mutation is an aspartate to valine substitution at residue 816(KIT D816V). Patients with systemic mastocytosis, whose mast cells often contain an activated D816V c-KIT mutation, may be inert to invasive disease, and they may experience symptoms associated with mast cell mediator release. Indolent systemic mastocytosis with repeated allergic reactions or vascular collapse in the absence of skin lesions is a particular subtype of indolent systemic mastocytosis, and this clonal MC activation disorder represents a large part 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 not yet clear. Currently, most tyrosine kinase inhibitors exhibit only modest efficacy in advanced disease states, with significant side effects. In addition, some invasive KIT mutations (including KIT D816V mutation) are resistant to classical ATP-competitive KIT inhibitors such as imatinib, sunitinib, sorafenib, and regorafenib. The inhibitor midostaurin (midostaurin) of c-KIT D816V was recently approved for the treatment of SM in 2017.

Although the c-KIT D816V mutation is a primary c-KIT mutation reported as a driver of Systemic Mastocytosis (SM), secondary c-KIT mutations that confer resistance to certain c-KIT inhibitors ("secondary resistant c-KIT mutations") have also been reported in mastocytosis patients, including, for example, Y269C, Y503_ F504insAY, V560D, or K642E point mutations, in-frame deletions or insertions, or missense mutations of the c-KIT gene.

Activating mutations or overexpression of the c-KIT gene are associated with neoplastic mast cell proliferation. In view of the complex function of c-KIT and the potential use of c-KIT inhibitors in the treatment of resistant systemic mastocytosis, there is a need for inhibitors and therapeutic treatments with advantageous therapeutic properties.

Disclosure of Invention

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

Also provided in the present disclosure is a method of treating mastocytosis in a patient in need thereof, the method comprising administering to the patient: an effective amount of a 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 as 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 contemplated in the present disclosure is a method of treating mastocytosis in a patient in need thereof, the method comprising administering to the patient: an effective amount of an inhibitor of c-KIT; and an effective amount of a RAF inhibitor.

Drawings

Figure 1A shows a graphical representation of cell proliferation after treatment with the indicated drug of compound a compared to vehicle controls in HMC 1.1V 560G (left panel) and HMC1.2V560G/D816V (right panel) cell lines.

Figure 1B shows a graphical representation of cell proliferation after treatment with the indicated drug for compound B, compared to vehicle controls in HMC 1.1V 560G (left panel) and HMC1.2V560G/D816V (right panel) cell lines.

FIG. 2A shows a graphical representation of caspase activity following various treatments specified with Compound A and trametinib in HMC1.2V560G/D816V cell line.

Figure 2B provides a graph of the synergy matrix based on the combinatorial index approach for various treatments with compound a and trametinib in the HMC1.2V560G/D816V cell line.

Figure 2C provides a combinatorial index of the combination of compound a and the MEK inhibitor trametinib.

Figure 3A shows a graphical representation of caspase activity after various treatments with the designations of compound B and trametinib in HMC1.2V560G/D816V cell line.

Figure 3B provides a graph of the synergy matrix based on the combinatorial indexing method for various treatments with compound B and trametinib in the HMC1.2V560G/D816V cell line.

Figure 3C provides a combinatorial index of the combination of compound B with the MEK inhibitor trametinib.

FIG. 4A shows a graphical representation of caspase activity after various treatments with the designations of Compound A and Bimetinib in an HMC1.2V560G/D816V cell line.

Figure 4B provides a synergy matrix plot based on the combinatorial index approach for various treatments with compound a and bimatinib in HMC1.2V560G/D816V cell line.

Figure 4C provides a combinatorial index of the combination of compound a and the MEK inhibitor bimetinib.

FIG. 5A shows a graphical representation of caspase activity following various treatments with the designations of Compound B and Bimetinib in an HMC1.2V560G/D816V cell line.

Figure 5B provides a synergy matrix plot based on the combinatorial index approach for various treatments with compound B and bimatinib in the HMC1.2V560G/D816V cell line.

Figure 5C provides a combinatorial index of the combination of compound B with the MEK inhibitor bimetinib.

Figure 6A shows a graphical representation of caspase activity after various treatments with the designations of compound a and cobicisinib in HMC1.2V560G/D816V cell line.

Fig. 6B provides a graph of the synergy matrix based on the combinatorial index approach for various treatments with compound a and cobicistinib in the HMC1.2V560G/D816V cell line.

Figure 6C provides a combinatorial index of the combination of compound a and the MEK inhibitor cobicistinib.

Figure 7A shows a graphical representation of caspase activity after various treatments with compound B and cobicisinib as specified in HMC1.2V560G/D816V cell line.

Fig. 7B provides a graph of the synergy matrix based on the combinatorial index approach for various treatments with compound B and cobicistinib in the HMC1.2V560G/D816V cell line.

Figure 7C provides a combinatorial index of the combination of compound B with the MEK inhibitor cobicistinib.

Fig. 8A shows a graphical representation of caspase activity after various treatments with compound a and the ERK inhibitor ulitinib as specified in HMC1.2V560G/D816V cell line.

Fig. 8B provides a graph of the synergy matrix based on the combinatorial index approach for various treatments with compound a and the ERK inhibitor ulitinib in the HMC1.2V560G/D816V cell line.

Figure 8C provides a combinatorial index of the combination of compound a with the ERK inhibitor ulitinib.

Figure 9A shows inhibition of colony growth halo after treatment with single drug compound a, single drug trametinib, and a combination of compound a and the MEK inhibitor trametinib in HMC1.2V560G/D816V cells.

Figure 9B shows a graphical representation of inhibition of colony growth halo after treatment with single drug compound a, single drug trametinib, and a combination of compound a and the MEK inhibitor trametinib in HMC1.2V560G/D816V cells. Arrows indicate no halo of colony growth.

Figure 10A shows inhibition of colony growth halo after treatment with single drug compound B, single drug trametinib, and a combination of compound B and the MEK inhibitor trametinib in HMC1.2V560G/D816V cells.

Figure 10B shows a graphical representation of inhibition of colony growth halo after treatment with single drug compound B, single drug trametinib, and a combination of compound B and the MEK inhibitor trametinib in HMC1.2V560G/D816V cells. Arrows indicate no halo of colony growth.

Figure 11A shows inhibition of colony growth halo after treatment with single drug compound a, single drug bimatinib, and a combination of compound a and MEK inhibitor bimatinib in HMC1.2V560G/D816V cells.

FIG. 11B shows a graphical representation of inhibition of colony growth halo after treatment with the single drug compound A, the single drug bimetinib, and the combination of compound A and the MEK inhibitor bimetinib in HMC1.2V560G/D816V cells. Arrows indicate no halo of colony growth.

Figure 12A shows inhibition of colony growth halo after treatment with single drug compound B, single drug bimatinib, and a combination of compound B and MEK inhibitor bimatinib in HMC1.2V560G/D816V cells.

Figure 12B shows a graphical representation of inhibition of colony growth halo after treatment with single drug compound B, single drug bimatinib, and a combination of compound B and MEK inhibitor bimatinib in HMC1.2V560G/D816V cells. Arrows indicate no halo of colony growth.

Figure 13A shows inhibition of colony growth halo after treatment with single drug compound a, single drug cobicistinib, and a combination of compound a and MEK inhibitor cobicistinib in HMC1.2V560G/D816V cells.

Figure 13B shows a graphical representation of inhibition of colony growth halo following treatment with single drug compound a, single drug cobinib, and a combination of compound a and MEK inhibitor cobinib in HMC1.2V560G/D816V cells.

Figure 14A shows inhibition of colony growth halo after treatment with single drug compound B, single drug cobinib, and a combination of compound B and MEK inhibitor cobinib in HMC1.2V560G/D816V cells.

Figure 14B shows a graphical representation of inhibition of colony growth halo after treatment with single drug compound B, single drug cobinib, and a combination of compound B and MEK inhibitor cobinib in HMC1.2V560G/D816V cells.

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

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

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

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

Figure 17A shows inhibition of colony growth halo after treatment with single drug compound a, single drug trametinib, and a combination of compound a and the MEK inhibitor trametinib in Empty Vector (EV) transfected HMC1.2V560G/D816V cells.

FIG. 17B shows a graphical representation of inhibition of colony growth halo after treatment with single drug Compound A, single drug trametinib, and a combination of Compound A and the MEK inhibitor trametinib in Empty Vector (EV) -transfected HMC1.2V560G/D816V cells. Arrows indicate no halo of colony growth.

FIG. 17C shows inhibition of colony growth halo after treatment with single drug compound A, single drug trametinib, and a combination of compound A and the MEK inhibitor trametinib in N-ras G12D transfected HMC1.2V560G/D816V cells.

FIG. 17D shows a graphical representation of inhibition of colony growth halo after treatment with single drug compound A, single drug trametinib, and a combination of compound 1 and the MEK inhibitor trametinib in N-ras G12D transfected HMC1.2V560G/D816V cells. Arrows indicate no halo of colony growth.

Figure 18A shows inhibition of colony growth halo after treatment with single drug compound a, single drug cobicistinib, and a combination of compound a and MEK inhibitor cobicistinib in Empty Vector (EV) transfected HMC1.2V560G/D816V cells.

FIG. 18B shows a graphical representation of inhibition of colony growth halo after treatment with single drug compound A, single drug cobinib, and a combination of compound A and MEK inhibitor cobinib in Empty Vector (EV) -transfected HMC1.2V560G/D816V cells. Arrows indicate no halo of colony growth.

FIG. 18C shows inhibition of colony growth halo after treatment with single drug compound A, single drug cobinib, and a combination of compound A and MEK inhibitor cobinib in N-ras G12D transfected HMC1.2V560G/D816V cells.

FIG. 18D shows a graphical representation of inhibition of colony growth halo after treatment with single drug compound A, single drug cobinib, and a combination of compound A and MEK inhibitor cobinib in N-ras G12D transfected HMC1.2V560G/D816V cells. Arrows indicate no halo of colony growth.

Detailed Description

It was found that the combination of a c-KIT inhibitor (e.g. 1- [ 4-bromo-5- [ 1-ethyl-7- (methylamino) -2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl ] -2-fluorophenyl ] -3-phenylurea (compound a)) and a MAPKAP pathway inhibitor (e.g. the MEK inhibitor trametinib, or the ERK inhibitor ulitinib, or the RAF inhibitor LY3009120) unexpectedly synergized to induce apoptosis in mastocytosis cells, as demonstrated in the appended examples. In addition, the combination therapy methods disclosed herein have a cytocidal effect, rather than a mere cytostatic effect.

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

Thus, in certain embodiments, the present disclosure provides methods for inducing cytocidal killing of mast cells, inducing apoptosis of mast cells, inhibiting growth or proliferation of mast cells, inhibiting mast cell mediator release, reducing the amount of mast cells accumulated in a tissue or organ, reducing the volume of a mastocytosis-associated tumor (such as a mast cell leukemia or a mast cell sarcoma), and/or inhibiting regrowth of mast cells by contacting mast cells (e.g., mast cells comprising a c-KIT mutation) with a c-KIT inhibitor and a MAPKAP pathway inhibitor. In various embodiments, the mast cell is contacted in vitro, in vivo, or ex vivo. In particular embodiments, the c-KIT inhibitor disclosed herein is compound a or a pharmaceutically acceptable salt thereof, compound B or a pharmaceutically acceptable salt thereof, midostaurin or a pharmaceutically acceptable salt thereof, BLU-285 or a pharmaceutically acceptable salt thereof, PLX9486 or a pharmaceutically acceptable salt thereof, or crenolanib or a pharmaceutically acceptable salt thereof; and the MEK inhibitor disclosed herein is trametinib, cobitinib, semetinib (selumetinib) or bimetinib; ERK inhibitors include, but are not limited to, ulitinib, SCH772984, LY3214996, lavertinib (ravoxertinib), and VX-11 e; and RAF inhibitors include, but are not limited to, LY3009120, vemurafenib or dabrafenib.

In certain embodiments, the present disclosure includes a method for treating a subject having mastocytosis, mast cell leukemia, or acute myeloid leukemia, the method 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, 6-naphthyridin-3-yl ] -2-fluorophenyl ] -3-phenylurea or a pharmaceutically acceptable salt thereof, or 1- (5- (7-amino-1-ethyl-2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl) -4-bromo-2-fluorophenyl) -3-phenylurea or a pharmaceutically acceptable salt thereof; and (ii) MAPKAP kinase inhibitors, such as trametinib, bimetinib, ulitinib. In particular embodiments of any of the methods disclosed herein, the mastocytosis is systemic mastocytosis caused by a D816V mutation of the c-KIT gene in mast cells.

Definition of

As used herein, "Compounds A and B" refer to 1- [ 4-bromo-5- [ 1-ethyl-7- (methylamino) -2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl ] -2-fluorophenyl ] -3-phenylurea, and 1- (5- (7-amino-1-ethyl-2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl) -4-bromo-2-fluorophenyl) -3-phenylurea, respectively. Pharmaceutically acceptable salts, tautomers, hydrates and solvates of compounds a and B are also encompassed by the present disclosure. The structures of compounds a and B are shown 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)

1- (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 8461179B1, the contents of which are incorporated herein by reference.

Illustrative methods and materials are described herein. In the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Throughout this disclosure, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this disclosure in order to more fully describe the state of the art as known to those skilled in the art as of the date of this disclosure. In the event of any inconsistency between a patent, a patent application, and a publication and the present disclosure, the present disclosure shall control.

For convenience, certain terms used in the specification, examples, and claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Unless otherwise indicated, the initial definitions provided for a group or term provided in this disclosure apply to that group or term throughout this disclosure, either alone or as part of another group.

A "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the United States Food and Drug Administration for use in humans or livestock.

"pharmaceutically acceptable salts" include acid addition salts.

"pharmaceutically acceptable acid addition salts" refers to those salts that retain the biological effectiveness and properties of the free base, which are not biologically or otherwise undesirable, and which are formed from 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-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, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, Gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid and the like.

"pharmaceutical composition" refers to a formulation of a compound described herein (e.g., compound a or a pharmaceutically acceptable salt thereof) and a vehicle generally accepted in the art for delivering biologically active compounds to a mammal (e.g., a human). Such media therefore include all pharmaceutically acceptable carriers, diluents or excipients.

An "inhibitor of the MAPKAP pathway" is an inhibitor of the MAP kinase signaling pathway. Inhibitors of this pathway include RAS inhibitors, RAF inhibitors (e.g., vemurafenib, dabrafenib, LY3009120), MEK inhibitors (e.g., trametinib, bimetinib, cobitinib), and ERK inhibitors (e.g., ulitinib).

A subject or patient "in need of treatment with a combination therapy of the present disclosure (e.g., a combination of a c-KIT inhibitor and a MAPKAP pathway inhibitor)" includes a subject having a disease and/or disorder (e.g., mastocytosis, mast cell leukemia, or acute myeloid leukemia) that can be treated with a combination disclosed herein to achieve a beneficial therapeutic outcome. Beneficial outcomes for treating mastocytosis may include complete response, partial response, clinical improvement, or stable disease, as defined by the IWG-MRT-ECNM standard (Gotlib et al, Blood 2013; 121: 2393-. In certain embodiments, a subject in need of treatment suffers from cutaneous mastocytosis (e.g., maculopapular cutaneous mastocytosis, mastocytoma, and diffuse cutaneous mastocytosis) as well as systemic mastocytosis (e.g., indolent systemic mastocytosis, systemic smoldering mastocytosis, systemic mastocytosis with clonal hematologic non-mast cell lineage disease, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma). In particular embodiments, the subject is a mammal, e.g., a human or other mammal.

The term "effective amount" when used in conjunction with a compound disclosed herein or other therapeutic agent, refers to an amount of the therapeutic agent (e.g., compound a or a MAPKAP pathway inhibitor), alone or in combination, that is useful for treating or preventing a disease or disorder. An effective amount of a therapeutic agent for use in combination therapy is the amount of each therapeutic agent that is useful for treating or preventing a disease or disorder when used in combination therapy, even though the amount of one or both of the therapeutic agents is not effective for treating or preventing the disease or disorder in the absence of the other therapeutic agent. In certain embodiments, an effective amount is one that results in inducing cytocidal mast cell killing, inducing apoptosis of mast cells, reducing the amount of mast cells accumulated in a tissue or organ, alleviating a symptom of mastocytosis, inhibiting mast cell mediator release, inhibiting growth of mast cells, and/or inducing regression of mastocytosis, wherein mast cells possess an activating mutation in c-KIT kinase, including an activating c-KITD816V mutation. The "effective amount" may vary depending on the mode of administration, the particular site of the disease or disorder, and the age, weight, and general health of the subject. The amount of compound administered will depend on the extent, severity and type of the disease or condition, the amount of therapy desired and the release profile of the one or more pharmaceutical formulations. It will also depend on the health, size, weight, age, sex and tolerance to drugs of the subject. Typically, the compound is administered for a sufficient period of time to achieve the desired therapeutic effect.

The terms "treatment" and "treating" are intended to include the full range of interventions on a patient being treated, for example a patient with mastocytosis or Acute Myeloid Leukemia (AML). Treatment may be to cure, ameliorate, or at least partially alleviate the disorder. In particular embodiments, treatment is intended to induce cytocidal mast cell killing, induce apoptosis of mast cells, reduce the amount of mast cells accumulated in a tissue or organ, alleviate symptoms of mastocytosis, inhibit mast cell mediator release, inhibit growth of mast cells, and/or induce regression of mastocytosis in a subject being treated. In certain embodiments, treatment with the combination therapies disclosed herein reduces, slows, or reverses one or more symptoms of mastocytosis and/or induces regression of mastocytosis, even if mastocytosis is not actually eliminated. In some embodiments, treatment includes complete elimination of the disease or disorder, e.g., mastocytosis or AML. In other embodiments, treatment results in a complete response, partial response, clinical improvement, or disease stabilization, as defined by the IWG-MRT-ECNM standard (Gotlib et al, Blood 2013; 121: 2393-.

As used herein, "mast cells" include mast cells (also known as mast cells) and CD34+ mast cell precursors.

As used herein, "neoplasm" refers to abnormal tissue that grows faster than normal by cell proliferation. Neoplasms exhibit a partial or complete lack of structural tissue and functional coordination with normal tissue, and often form significant tissue masses, which may be benign (benign tumor) or malignant (cancer).

As used herein, "tumor" refers to a lump. This is a term that may refer to benign (generally harmless) or malignant (cancerous) growth. Malignant growth may originate from a solid organ or bone marrow. The latter is commonly referred to as a liquid tumor. "tumor" encompasses mast cell leukemia and mast cell sarcoma, collectively referred to herein as "mastocytosis tumor".

In certain embodiments, the therapeutic effect of treating mastocytosis according to the methods disclosed herein can be measured using standard reaction criteria known in the art. For example, for quantitative therapy on invasive systemic fertilizers"Complete Remission (CR), Partial Remission (PR), Clinical Improvement (CI) or disease Stabilization (SD) response criteria" for the effects of macrocytosis, mast cell leukemia, and systemic mastocytosis associated with bone marrow neoplasms may be any of those defined by IWG-MRT-ECNM (Gotlib et al, Blood 2013; 121: 2393-. For example, Complete Remission (CR) requires all 4 criteria and the duration of the reaction must be ≧ 12 weeks, with the absence of dense aggregates of neoplastic mast cells in the dermal outer organs of BM or other biopsies. Serum tryptase levels<20ng/mL

Peripheral blood count remission is defined as ANC ≧ 1 × 10 by ordinary differentiation⁹Hb levels ≥ 11g/dL and platelet counts ≥ 100X 10⁹L, and complete resolution of palpable hepatosplenomegaly and all biopsy confirmed or suspected SM-associated organ damage (CI findings); in the absence of both CR and Progressive Disease (PD), Partial Remission (PR) requires that all 3 criteria be met and the duration of the reaction must be ≧ 12 weeks, where: a reduction of neoplastic MC in bone marrow and/or other dermal outer organs by more than or equal to 50% at the time of biopsy indicates a qualified SM associated organ damage, a reduction of serum tryptase levels by more than or equal to 50%; and resolving 1 or more biopsy confirmed or suspected SM-associated organ lesions (one or more CI findings); clinical Improvement (CI) is the need to meet one or more non-hematological and/or hematological response criteria in the absence of both CR/PR and assigned or Progressive Disease (PD) where the duration of the response must be ≧ 12 weeks (see Table 3). Disease Stability (SD) does not meet the criteria for CR, PR, CI or PD.

Criteria for response determination include (1) only disease-related grade 2 or more organ damage can be assessed as a primary endpoint in clinical trials. (2) Response decisions for CR, PR, SD, PD, and Loss Or Response (LOR) should only apply to these class 2 organ injury findings in the experimental context. (3) The disease state at the time the patient was removed from the study was only associated with an updated state of one or more initial stage 2 organ injury findings. (4) In patients with exacerbations with baseline grade 2 or greater organ damage, elimination of drug-related toxicity and/or other clinical problems (e.g., gastrointestinal bleeding in the case of anemia/transfusion-dependent exacerbations) should be performed prior to assigning the designation PD or LOR.

In certain embodiments, the therapeutic effect of treating AML according to the methods disclosed herein can be measured using standard response criteria known in the art. For example, a "response criteria to AML treatment" for quantifying the effect of a therapy on AML can be any of those criteria defined below, including Complete Remission (CR) without minimal residual disease (CR)_MRD-) Complete Remission (CR), CR with incomplete blood recovery (CR)_i) A morphologically leukemia-free state (MLFS), Partial Remission (PR), disease Stabilization (SD), or Progressive Disease (PD), as described by blood.2017jan 26; 129(4) 424 and 447, and the sum is: complete remission without minimal residual disease (CR)_MRD-): CR has a negative for the genetic marker as detected by RT-qPCR or CR has a negative as detected by MFC if prior study treatment; complete Remission (CR): bone marrow primary cells<5 percent; absence of circulating primary cells and primary cells with Auer bodies; absence of bulbar disease; absolute Neutrophil Count (ANC) ≥ 1.0 × 10⁹L (1000/. mu.L); platelet count is not less than 100X 10⁹L (100000/μ L); CR (CR) with incomplete hematological recovery_i): removal of residual neutropenia (<1.0×10⁹/L[1000/μL]) Or thrombocytopenia (<100×10⁹/L[100 000/μL]) All CR standards except; status of Morphic Leukemia (MLFS): bone marrow primary cells<5 percent; absence of primary cells with Auer bodies; absence of bulbar disease; no desired hematological recovery; partial Remission (PR): all hematological criteria for CR; the percentage of bone marrow primary cells is reduced to 5% to 25%; and the percentage of bone marrow primary cells decreased by at least 50% before treatment.

A "combination therapy" is a treatment that includes administering to a patient two or more of the following therapeutic agents: for example, c-KIT inhibitors (such as compound a or a pharmaceutically acceptable salt thereof, midostaurin, BLU-285, PLX9486 or crenolanib) and MAPKAP pathway inhibitors (including but not limited to trametinib, cobitinib, semetinib, bimetinib, ulitinib, LY 3009120). The two or more therapeutic agents may be delivered simultaneously, e.g., in separate pharmaceutical compositions or in the same pharmaceutical composition, or they may be delivered at different times. For example, they may be delivered concurrently or over overlapping time periods, and/or one therapeutic agent may be delivered before or after the other therapeutic agent or agents. Treatment with a combination of a KIT inhibitor (such as compound a) and a MAPKAP pathway inhibitor optionally includes treatment with either agent before or after a concurrent treatment period with both agents. However, it is contemplated that an effective amount of two or more therapeutic agents may be present in a patient over a period of time.

Method of treatment

In one embodiment, the present disclosure provides a method of treating or preventing mastocytosis (optionally c-KIT mediated mastocytosis, e.g., Systemic Mastocytosis (SM)), the method comprising providing or administering to a subject in need thereof an effective amount of a c-KIT inhibitor in combination with an effective amount of a MAPKAP pathway inhibitor (e.g., trametinib, cobitinib, semetinib, bimetinib, ulitinib, or LY 3009120). In one embodiment, the present disclosure provides a method of treating or preventing mastocytosis (optionally c-KIT mediated mastocytosis, e.g., Systemic Mastocytosis (SM)), comprising providing or administering to a subject in need thereof an effective amount of compound a (or a pharmaceutically acceptable salt thereof) or compound B (or a pharmaceutically acceptable salt thereof) in combination with an effective amount of a MAPKAP pathway inhibitor (e.g., trametinib, cobitinib, semetinib, bimetinib, ulitinib, or LY 3009120). In related embodiments, the present disclosure provides methods of treating or preventing a mastocytosis tumor (optionally a c-KIT mediated mastocytosis tumor, such as a mast cell leukemia or a mast cell sarcoma), the method comprising providing or administering to a subject in need thereof an effective amount of a c-KIT inhibitor in combination with an effective amount of a MAPKAP pathway inhibitor (e.g., trametinib, cobitinib, semetinib, bimetinib, ulitinib, or LY 3009120). In related embodiments, the present disclosure provides a method of treating or preventing a mastocytosis tumor (optionally a c-KIT mediated mastocytosis tumor, such as a mast cell leukemia or a mast cell sarcoma), the method comprising providing or administering to a subject in need thereof an effective amount of compound a (or a pharmaceutically acceptable salt thereof) or compound B (or a pharmaceutically acceptable salt thereof) in combination with an effective amount of a MAPKAP pathway inhibitor (e.g., trametinib, cobitinib, semetinib, bimetinib, ulitinib, or LY 3009120).

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

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

In another embodiment, mastocytosis may comprise a mast cell having a primary mutation in exon 17 of the c-KIT gene. In some embodiments, the primary mutation is a c-KITD816 mutation. In some embodiments, the primary mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del 419. In some embodiments, the primary mutation is D816V.

Mastocytosis may also comprise mast cells with 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 methods may also include determining whether mastocytosis has a primary mutation of c-KIT. For example, the method further comprises determining whether mastocytosis has a secondary mutation of c-KIT. In some embodiments, determining whether mastocytosis has a c-KIT primary or secondary mutation comprises identifying a mutation in DNA extracted from a tumor sample. In yet another embodiment, determining whether mastocytosis has a c-KIT primary or secondary mutation comprises identifying a mutation in circulating tumor DNA or identifying a mutation in circulating peripheral blood leukocytes.

The mastocytosis may be systemic mastocytosis. In some embodiments, the systemic mastocytosis is selected from the group consisting of: indolent systemic mastocytosis, systemic smoldering mastocytosis, systemic mastocytosis with clonal hematologic non-mast cell lineage disease, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma. In some embodiments, the mastocytosis is an indolent systemic mastocytosis, optionally with repeated allergic reactions or vascular collapse in the absence of skin lesions. In some embodiments, the mastocytosis is systemic smoldering mastocytosis.

In some embodiments, the mastocytosis is a systemic mastocytosis with clonal hematologic non-mast cell lineage disease. In some embodiments, the mastocytosis is an invasive systemic mastocytosis. In some embodiments, the mastocytosis is a mast cell leukemia or a mast cell sarcoma. In some embodiments, the mastocytosis is a cutaneous mastocytosis. In some embodiments, the mastocytosis is selected from the group consisting of: maculopapular cutaneous mastocytosis, mastocytoma or diffuse cutaneous mastocytosis.

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

Further, the MEK inhibitor may be selected from the group consisting of: trametinib, semetinib, cobitinib and bimitinib. In some embodiments, the MEK inhibitor is bimatoinib. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the ERK inhibitor is selected from the group consisting of: ulitinib, SCH772984 and LY 3214996. In some embodiments, the c-KIT inhibitor and the MEK and/or ERK inhibitor are administered substantially concurrently or sequentially.

The method may further comprise administering another cancer-targeted therapeutic agent, a cancer-targeted biological immune checkpoint inhibitor, or a chemotherapeutic agent.

Additionally, an effective amount of a c-KIT inhibitor for two weeks or more according to the intended method; 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) may result in the patient having at least partial remission.

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

In one embodiment, systemic mastocytosis may have a c-KIT mutation. For example, the mutation may be a c-KITD816 mutation. In some embodiments, the mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del 419. 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 have an additional c-KIT mutation that is one of the Y269C, Y503_ F504insAY, V560D, or K642E mutations.

In this disclosed method, the MEK inhibitor may be selected from the group consisting of: trametinib, semetinib, cobitinib and bimitinib. In some embodiments, the MEK inhibitor is bimatoinib. In some embodiments, the MEK inhibitor is trametinib. The ERK inhibitor may be selected from the group consisting of: ulitinib, SCH772984 and LY 3214996.

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

In the disclosed methods, the RAF inhibitor can be a pan-RAF or B-RAF inhibitor, including vemurafenib, dabrafenib, and LY 3009120. Further, the c-KIT inhibitor may be 1- [ 4-bromo-5- [ 1-ethyl-7- (methylamino) -2-oxo-1, 2-dihydro-1, 6-naphthyridin-3-yl ] -2-fluorophenyl ] -3-phenylurea or a pharmaceutically acceptable salt thereof.

In particular embodiments of the methods disclosed herein, there is included a method of treating mastocytosis or a mastocytosis tumor, the method comprising: inducing cytocidal mast cell killing, inducing apoptosis of mast cells possessing one or more activating mutations in c-KIT kinase, e.g., an activated c-KIT D816V mutation, reducing the amount of mast cells accumulated in a tissue or organ, alleviating a symptom of mastocytosis, inhibiting mast cell mediator release, inhibiting growth of mast cells, and/or inducing regression of mastocytosis. In certain embodiments, the methods encompass methods for ablating mastocytosis (e.g., a mastocytosis tumor) in a subject. In some embodiments, treatment results in a complete response, partial response, clinical improvement, or disease stabilization, as defined by the IWG-MRT-ECNM standard (Gotlib et al, Blood 2013; 121: 2393-.

In another related embodiment, the present disclosure provides a method of treating or preventing Acute Myeloid Leukemia (AML) (optionally c-KIT mediated (AML)), the method comprising administering to a subject in need thereof an effective amount of a c-KIT inhibitor in combination with an effective amount of a MAPKAP pathway inhibitor (e.g., trametinib, cobitinib, semetinib, bimetinib, ulitinib, or LY 3009120). In related embodiments, the present disclosure provides a method of treating or preventing Acute Myeloid Leukemia (AML) (optionally c-KIT mediated (AML)), the method comprising administering to a subject in need thereof an effective amount of compound a (or a pharmaceutically acceptable salt thereof) or compound B (or a pharmaceutically acceptable salt thereof) in combination with an effective amount of a MAPKAP pathway inhibitor (e.g., trametinib, cobicistinib, semetinib, bimetinib, ulitinib, or LY 3009120).

Where the methods described herein involve treatment with compound a or a pharmaceutically acceptable salt thereof, or with compound B or a pharmaceutically acceptable salt thereof, it is meant that only one of compound a or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, is required. However, it is to be understood that these methods also encompass administering to the 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 encompass administering compound a and a MAPKAP pathway inhibitor to a subject, whereupon compound a is metabolized in vivo to compound B, and the in vivo mixture of compound a and compound B in combination with the MAPKAP pathway inhibitor effectively treats the subject.

Particular embodiments of the disclosed methods and compositions are implemented using the following compounds: a combination of compound a or a pharmaceutically acceptable salt thereof and trametinib; a combination of compound a or a pharmaceutically acceptable salt thereof and sematinib; a combination of compound a or a pharmaceutically acceptable salt thereof and cobicistinib; or a combination of compound a or a pharmaceutically acceptable salt thereof and bimatonil.

In one embodiment, compound a or a pharmaceutically acceptable salt thereof and a MEK inhibitor (e.g., trametinib or bimatinib) are administered to a subject suffering from c-KIT mediated mastocytosis. In another embodiment, compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor (e.g., trametinib or bimatinib) are administered to a subject suffering from c-KIT mediated mastocytosis.

In related embodiments, compound a or a pharmaceutically acceptable salt thereof and a MEK inhibitor (e.g., trametinib or bimatinib) are administered to a patient suffering from a mastocytosis tumor, including but not limited to mast cell leukemia or mast cell sarcoma, wherein tumor growth or tumor progression in mastocytosis is caused by a primary activating c-KIT mutation (e.g., KIT D816V mutation). In another embodiment, compound B, or a pharmaceutically acceptable salt thereof, and a MEK inhibitor (e.g., trametinib or bimetinib) are administered to a patient suffering from a mastocytosis tumor, including but not limited to mast cell leukemia or mast cell sarcoma, wherein tumor growth or tumor progression in mastocytosis is caused by a primary activating c-KIT mutation (e.g., 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 bimetinib) are administered to a patient with AML, optionally wherein the AML is caused by a primary activating c-KIT mutation (e.g., a c-KIT exon 8 activating mutation, or a c-KIT exon 17 mutation, including but not limited to a mutation at D816 or at N822 (Journal of clinical Oncology 200624:24, 3904-.

Particular embodiments of the disclosed methods and compositions are implemented using the following compounds: a combination of compound a or a pharmaceutically acceptable salt thereof and ulitinib; a combination of compound a or a pharmaceutically acceptable salt thereof and SCH 772984; a combination of compound a or a pharmaceutically acceptable salt thereof and LY 3214996; a combination of compound a or a pharmaceutically acceptable salt thereof and lavendustinib; or a combination of compound a or a pharmaceutically acceptable salt thereof and VX-11.

In one embodiment, compound a or a pharmaceutically acceptable salt thereof and an ERK inhibitor (e.g., ulitinib) are administered to a subject having c-KIT mediated mastocytosis. In another embodiment, compound B or a pharmaceutically acceptable salt thereof and an ERK inhibitor (e.g., ulitinib) are administered to a subject suffering from c-KIT mediated mastocytosis.

In a related embodiment, compound a or a pharmaceutically acceptable salt thereof and an ERK inhibitor (e.g., ulitinib) are administered to a patient having a mastocytosis tumor, including but not limited to mast cell leukemia or mast cell sarcoma, wherein tumor growth or tumor progression in mastocytosis is caused by a primary activating c-KIT mutation (e.g., KIT D816V mutation). In another embodiment, compound B or a pharmaceutically acceptable salt thereof and an ERK inhibitor (e.g., ulitinib) are administered to a patient having a mastocytosis tumor, including but not limited to mast cell leukemia or mast cell sarcoma, wherein tumor growth or tumor progression in mastocytosis is caused by a primary activating c-KIT mutation (e.g., 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., ulitinib) are administered to a patient with AML, optionally wherein the AML is caused by a primary activating c-KIT mutation (e.g., a c-KIT exon 8 activating mutation, or a c-KIT exon 17 mutation, including but not limited to a mutation at D816 or at N822 (journal of clinical Oncology 200624:24, 3904-.

In one embodiment, compound a or a pharmaceutically acceptable salt thereof and a RAF inhibitor (e.g., LY3009120, dabrafenib, or vemurafenib) are administered to a subject having c-KIT mediated mastocytosis. In another embodiment, compound B or a pharmaceutically acceptable salt thereof and a RAF inhibitor (e.g., LY3009120, dabrafenib, or vemurafenib) are administered to a subject having c-KIT mediated mastocytosis.

In related embodiments, compound a or a pharmaceutically acceptable salt thereof and a RAF inhibitor (e.g., LY3009120, dabrafenib, or vemurafenib) are administered to a patient having a mastocytosis tumor, including but not limited to mast cell leukemia or mast cell sarcoma, wherein tumor growth or tumor progression of mastocytosis caused by a primary activating c-KIT mutation (e.g., KIT D816V mutation). In another embodiment, compound B or a pharmaceutically acceptable salt thereof and a RAF inhibitor (e.g., LY3009120, dabrafenib, or vemurafenib) are administered to a patient having a mastocytosis tumor, including but not limited to mast cell leukemia or mast cell sarcoma, wherein tumor growth or tumor progression of mastocytosis caused by a primary activating c-KIT mutation (e.g., 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) are administered to a patient with AML, optionally wherein the AML is caused by a primary activating c-KIT mutation (e.g., a c-KIT exon 8 activating mutation, or a c-KIT exon 17 mutation, including but not limited to mutations at D816 or N822 (journal of clinical Oncology 200624:24, 3904-.

Illustrative c-KIT inhibitors that may 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. Illustrative MEK inhibitors that may be used in accordance with the disclosed methods and compositions include, but are not limited to, trametinib, sematinib, cobitinib, and bimatinib. Illustrative ERK inhibitors that may be used in accordance with the disclosed methods and compositions include, but are not limited to, ulitinib, SCH772984, LY3214996, lavendib (ravoxerttinib), and VX-11 e. Illustrative 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., trametinib, bimetinib, ulitinib, or LY3009120) encompasses administration of compound a or a pharmaceutically acceptable salt thereof or compound B or a pharmaceutically acceptable salt thereof, before, after, simultaneously with, or overlapping with administration of the MAPKAP pathway inhibitor. It is to be understood that the effective amount of any of compound a or a pharmaceutically acceptable salt thereof, compound B or a pharmaceutically acceptable salt thereof, another c-KIT inhibitor or a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120) when used in a combination disclosed herein can be different than when either of these agents is used alone for the same purpose (e.g., for treating or preventing mastocytosis or a mast cell tumor, such as mast cell leukemia or AML). In particular embodiments, the effective amount of compound a or a pharmaceutically acceptable salt thereof or the effective amount of compound B or a pharmaceutically acceptable salt thereof is a lower amount when administered as a combination therapy with a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120) than when administered as a monotherapy (e.g., for treating or preventing mastocytosis or mast cell tumors). In particular embodiments, the effective amount of a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120) is a lower amount when administered as a combination therapy with compound a or a pharmaceutically acceptable salt thereof, or when administered as a combination therapy with compound B or a pharmaceutically acceptable salt thereof (e.g., for treating or preventing mastocytosis or mast cell tumors).

Any of the methods disclosed herein can further comprise determining that the mastocytosis cell, mast cell tumor, or AML being treated has one or more c-KIT gene mutations. Such determination can be made by conventional methods for determining the presence of a gene mutation in a biological sample (e.g., a bone marrow sample, a tissue sample, a peripheral blood sample, or a plasma sample) obtained from a subject. In addition, such determination may be made by reviewing the results of tests performed to determine the presence of one or more c-KIT gene mutations in a biological sample obtained from the patient. In certain embodiments of any of the methods disclosed herein, the method is performed on a subject in which mastocytosis, mast cell tumor, or AML has been identified as having one or more c-KIT gene mutations. c-KIT gene mutations include, but are not limited to, any of those specifically described herein. In certain embodiments of any of the methods disclosed herein, the method is not performed on a subject in which mastocytosis, mast cell tumor, or AML has been identified as having one or more c-KIT gene mutations.

In various aspects of any of the methods disclosed herein, treatment with compound a or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120) induces cytocidal mast cell killing, induces apoptosis of mast cells, reduces the amount of mast cells accumulated in a tissue or organ, reduces symptoms of mastocytosis, inhibits mast cell mediator release, inhibits growth of mast cells, and/or induces regression of mastocytosis, wherein mast cells possess an activating mutation in c-KIT kinase, including an activating c-KIT D816V mutation. Measuring or determining mast cell apoptosis, amount of mast cell killing, inhibition of mast cell growth and proliferation, inhibition of mast cell mediator release, peripheral blood mutant KIT allele burden, mastocytosis, and eradication of mastocytosis tumors; methods of complete response, partial response, clinical improvement, or disease stabilization are known in the art and include any of the methods described herein.

In particular embodiments, treatment is performed with a combination of: c-KIT inhibitors (e.g., compound a or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof); and MAPKAP pathway inhibitors (e.g., trametinib, bimetinib, ulitinib, or LY3009120), result in an increase in the amount of apoptosis in mastocytosis cells or mast cells. For example, apoptosis 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%, at least 80%, at least 90%, at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least 10-fold, or at least 20-fold. In certain embodiments, the amount of apoptosis is determined by measuring caspase activity of KIT mutant mast cells or mast cell lines, including HMC1.2 mast cell lines possessing the KIT D816V mutation.

In particular embodiments, treatment is performed with a combination of: c-KIT inhibitors (e.g., compound a or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof); and MAPKAP pathway inhibitors (e.g., trametinib, bimetinib, ulitinib, or LY3009120), result in a decrease in accumulation of mast cells in the skin or internal organs (e.g., liver, spleen, bone marrow, and/or small intestine). For example, the amount or number of mast cells accumulated within 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 particular embodiments, the treatment is performed with a combination of: c-KIT inhibitors (e.g., compound a or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof); and MAPKAP pathway inhibitors (e.g., trametinib, bimetinib, ulitinib, or LY3009120) induce apoptosis or inhibition of growth of resistant mastocytosis cells containing mutations (e.g., resistance mutations) in other genes, including any of those disclosed herein. In certain embodiments, such other genetic mutations may include NRas gain-of-function mutations (Haematologica 2011; 96(03):459-463.doi:10.3324/haematol.2010.031690) or TET2 loss-of-function mutations (leukamia 2009; 23: 900-04; Blood, 2012, 12/6/2012; 120(24): 4846-49). The presence of other epigenetic or transcriptional regulator mutations has been detected in SM, including DNMT3A, ASXL1, and CBL mutations in 12%, and 4% of patients, respectively (Plosone.2012; 7: e 43090). In addition, some mastocytosis patients also exhibit mutations in the splice body machinery. Spliceosomes ensure the correct linear order of spliced exons in the mRNA. Hanssens et al reported that in a group of 72 mastocytosis patients, the incidence of mutations in SRSF2 was 23.6%, SF3B1 was 5.6%, and U2AF1 was 2.7% (Haematologica 2014; 99: 830-35). Such mastocytosis with complex genomic drivers may benefit from treatment with a combination of a c-KIT inhibitor and a MEK inhibitor as disclosed herein, compared to untreated or treatment with a MEK inhibitor only (e.g., trametinib) or c-KIT inhibitor only (e.g., compound a or compound B). For example, apoptosis 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%, at least 80%, at least 90%, at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least 10-fold, or at least 20-fold. For example, the amount or number of growth of resistant mastocytosis cells may be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

In particular embodiments, when used as monotherapy, the resistant mastocytosis cells are resistant to apoptosis-mediated cell death or cytocidal activity by treatment with a c-KIT inhibitor (e.g., compound a, compound B, midostaurin, BLU-285, PLX9486, or crenolanib) and/or resistant to apoptosis-mediated cell death or cytocidal activity by treatment with a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY 3009120).

The present disclosure describes combination therapies involving the administration of a c-KIT inhibitor (e.g., compound a or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof), and a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY 3009120). The combination therapies described herein may be used alone as such or in combination with one or more additional therapeutic agents. For example, compound a or a pharmaceutically acceptable salt thereof or compound B or a pharmaceutically acceptable salt thereof and a MAPKAP pathway inhibitor may be administered with a cancer-targeted therapeutic agent, a cancer-targeted biological immune checkpoint inhibitor, or a chemotherapeutic agent. In another embodiment, compound a or compound B and the MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120) are administered in the absence of any other therapeutic agent. A therapeutic agent may be administered in combination therapy with another therapeutic agent described herein or sequentially therewith.

Combination therapy can be achieved by: administering two or more therapeutic agents, each formulated and administered separately; or two or more therapeutic agents are applied in a single formulation. Other combinations are also contemplated by combination therapy. While two or more agents in a combination therapy may be administered simultaneously, they need not be so administered. For example, the administration of the first agent (or combination of agents) can be minutes, hours, days, or weeks before the administration of the second agent (or combination of agents). Thus, the two or more agents may be administered within minutes of each other, or within 1,2, 3, 6, 9, 12, 15, 18, or 24 hours of each other, or within 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other, or within 2, 3, 4, 5, 6, 7, 8, 9, or weeks of each other. In some cases, even longer intervals are possible. While in many cases it is desirable that two or more agents used in combination therapy be present in the patient at the same time, this is not essential.

Combination therapy may also include the use of two or more administrations in a different order of the component agents of one or more of the agents used in the combination. For example, if agent X and agent Y are used in combination, one may administer them sequentially in any combination, once or more, for example, in the order of X-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc. Furthermore, the administration of two or more agents in combination may precede or follow an administration dosage interval during which at least one combined agent is omitted from treatment.

Additional therapeutic agents that may be administered according to the present disclosure, for example, to treat mastocytosis or a mastocytosis tumor, include, but are not limited to, agents selected from the group consisting of: STAT5 inhibitors including, but not limited to, ruxolitinib, tofacitinib, or fedratinib; BTK inhibitors, including but not limited to ibrutinib, PCI 29732, acarabtinib (acalaburtinib), or AVL-292; PI3 kinase inhibitors including, but not limited to, idelalisib, dactylisib, pictiliib, LY294002, buparlisib, pilalaisib, duvelisib, PF-04691502, voxtalisib, omiplasib, gedatolisib, apitolisib or wortmannin (wortmannin); AKT kinase inhibitors including, but not limited to, MK-2206, perifosine (perifosine), GSK690693, GSK2141795, iptastartib, AZD5363, affrescertib, or AT 7867; inhibitors of DNA methylation including, but not limited to, 5-azacytidine or 5-aza-2' -deoxycytidine; proteosome inhibitors, including but not limited to bortezomib, carfilzomib, MLN9708, ONX 0912; interferon alpha (IFN-); cladribine (cladribine); and lysosomotropic agents including, but not limited to, chloroquine, hydroxychloroquine, or quinacrine (quinacrine).

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

In certain embodiments, a subject in need thereof is treated with a combination of one or two of compound a or a pharmaceutically acceptable salt thereof, a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120), and 5-azacytidine. In certain embodiments, the MAPKAP pathway inhibitor is trametinib. In certain embodiments, the MAPKAP pathway inhibitor is bimatinib. In certain embodiments, the MAPKAP pathway inhibitor is ulitinib. In certain embodiments, the MAPKAP pathway inhibitor is LY3009120, dabrafenib, or vemurafenib.

In certain embodiments, a subject in need thereof is treated with a combination of compound a or a pharmaceutically acceptable salt thereof, a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120), and cladribine. In certain embodiments, the MAPKAP pathway inhibitor is trametinib. In certain embodiments, the MAPKAP pathway inhibitor is bimatinib. In certain embodiments, the MAPKAP pathway inhibitor is ulitinib. In certain embodiments, the MAPKAP pathway inhibitor is LY3009120, dabrafenib, or vemurafenib. Additional therapeutic agents that may be administered according to the present disclosure include, but are not limited to, arsenic trioxide, cyclophosphamide, cytarabine, daunorubicin, doxorubicin, enzidipine (enasidib), idarubicin, quinazatinib (quizartinib), mitoxantrone, thioguanine, or vincristine. In certain embodiments, a subject having AML is treated with a combination of compound a, or a pharmaceutically acceptable salt thereof, and arsenic trioxide, cyclophosphamide, cytarabine, daunorubicin, doxorubicin, enzipine, idarubicin, quinatinib, mitoxantrone, thioguanine, or vincristine.

Pharmaceutical composition

Aspects of the present disclosure relate to methods of treatment involving administering a combination of compounds disclosed herein, or one or more pharmaceutical compositions comprising such compounds and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein relate to administering a first pharmaceutical composition comprising a c-KIT inhibitor (e.g., compound a or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, excipient, or carrier) and a second pharmaceutical composition comprising a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120), and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein involve administering a first pharmaceutical composition comprising compound B or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, excipient, or carrier, and a second pharmaceutical composition comprising a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120), and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein relate to administering a pharmaceutical composition comprising a ciKIT inhibitor (e.g., compound a or a pharmaceutically acceptable salt thereof), a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120), and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein relate to administering a pharmaceutical composition comprising compound B or a pharmaceutically acceptable salt thereof, a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120), and a pharmaceutically acceptable diluent, excipient, or carrier.

In using the pharmaceutical compositions of the compounds described herein, the pharmaceutically acceptable carrier can be a solid or a liquid. Solid forms include powders, tablets, dispersible granules, capsules, cachets, and suppositories. Powders and tablets may contain from about 5% to about 95% of the active ingredient. Suitable solid carriers are known in the art, for example, magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods of making various compositions can be found in a.gennaro (ed.), Remington's pharmaceutical Sciences, 18 th edition, (1990), Mack Publishing co.

Liquid form preparations include solutions, suspensions, and emulsions. For example, water or water-propylene glycol solutions are used for parenteral injection, or sweeteners and opacifiers are added for oral solutions, suspensions and emulsions. Liquid form formulations may also include solutions for intranasal administration.

Liquid, in particular injectable, compositions may be prepared, for example, by dissolution, dispersion, or the like. For example, the disclosed compounds are dissolved in or mixed with a pharmaceutically acceptable solvent (e.g., water, saline, aqueous dextrose, glycerol, ethanol, and the like), thereby forming an injectable isotonic solution or suspension. Proteins such as albumin, chylomicron, or serum proteins may be used to solubilize the disclosed compounds.

Parenteral injection administration is commonly used for subcutaneous, intramuscular or intravenous injection and infusion. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, or in solid form suitable for dissolution in liquid prior to injection.

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

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

Dosage form

In some embodiments where compound a or compound B (or a pharmaceutically acceptable salt thereof) is used in combination with a MAPKAP pathway inhibitor (e.g., trametinib, bimetinib, ulitinib, or LY3009120) for a treatment regimen, the two therapeutic agents may be administered together or in a "dual regimen" in which the two therapeutic agents are administered separately and administered. When compound a or B (or a pharmaceutically acceptable salt thereof) and the MAPKAP pathway inhibitor are administered alone, typical dosages of compound a or compound B (or a pharmaceutically acceptable salt thereof) administered to a subject in need of treatment will generally range from about 5mg per day to about 5000mg per day, and in other embodiments, from about 50mg per day to about 1000mg per day. Other doses may be from about 10mmol per day to about 250mmol per day, from about 20mmol per day to about 70mmol per day, or even from about 30mmol per day to about 60mmol per day. When used for the indicated effects, an effective dose of the disclosed compounds is in an amount ranging from about 0.5mg to about 5000mg of the disclosed compounds as required 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 5000mg of the disclosed compound, or, alternatively, in a range from one amount to another in a dosage list. Typical recommended daily dosage regimens for oral administration may range from about 1 mg/day to about 500 mg/day or 1 mg/day to 200 mg/day in a single dose or in two to four divided doses. In one embodiment, a typical daily oral dosage regimen is 150 mg.

In certain embodiments, the dosage of the MAPKAP pathway inhibitor is consistent with a previously disclosed dose and/or a dose approved for use by the Food and Drug Administration. In other embodiments, the dose of the MAPKAP pathway inhibitor is less than a previously approved dose, e.g., about 20%, about 50%, or about 80% of an approved dose. In certain embodiments, the dose of trametinib is about.5 mg to 20mg orally daily, e.g., about 1mg daily or about 2mg daily. In certain embodiments, the dose of cobicistinib is from about 10mg to 200mg per day, for example about 30mg or about 60mg per day. In certain embodiments, the dose of bimatinib is from about 10mg to about 200mg twice daily, for example about 25mg or about 45mg twice daily. In certain embodiments, the dose of sematinib is from about 10mg to about 200mg daily, or about 30mg or about 75mg twice daily.

The amount and frequency of administration of the compounds described herein and/or pharmaceutically acceptable salts thereof and other therapeutic agents will also be adjusted at the discretion of the attending clinician, taking into account factors such as age, condition and size of the patient and the severity of the symptoms being treated.

The compounds of the present disclosure (e.g., compound a or compound B (and pharmaceutically acceptable salts thereof), MAPKAP pathway inhibitors, and other therapeutic agents) can be administered by any suitable route. The compounds can be administered orally (e.g., via the diet) in the form of capsules, suspensions, tablets, pills, dragees, liquids, gels, syrups, slurries and the like. Methods of encapsulating compositions, such as in coatings of hard gelatin or cyclodextrins, are known in the art (Baker et al, "Controlled release of biological Active Agents", John Wiley and Sons,1986, which is hereby incorporated 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 formulation of the pharmaceutical composition will vary depending on the route of administration selected. Suitable pharmaceutical carriers may contain inert ingredients that do not interact with the compound. The carrier is biocompatible, i.e., non-toxic, non-inflammatory, non-immunogenic, and has no other adverse reactions at the site of administration.

Illustrative pharmaceutical compositions are tablets and gelatin capsules comprising a compound described herein (e.g., compound a or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier such as: a) diluents, for example purified water, triglyceride oils (such as hydrogenated or partially hydrogenated vegetable oils, or mixtures thereof), corn oil, olive oil, sunflower oil, safflower oil, fish oils (such as EPA or DHA, or esters or triglycerides thereof, or mixtures thereof), omega-3 fatty acids or derivatives thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose and/or glycine; b) lubricants, for example silica, 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) binders, for example magnesium aluminium silicate, starch paste, gelatin, gum tragacanth, methyl cellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums (such as gum acacia), gum tragacanth or sodium alginate, waxes and/or polyvinylpyrrolidone, if desired; d) disintegrating agents, such as starch, agar, methylcellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or effervescent mixtures; e) absorbents, colorants, flavors, and sweeteners; f) emulsifying or dispersing agents such as Tween 80, Labrasol, HPMC, DOSS, capryl 909, labrafac, labrafil, peceol, transcutol, capmulMCM, capmul PG-12, captex 355, gelucire, vitamin E TGPS or other acceptable emulsifying agents; and/or g) agents that enhance absorption of the compound, such as cyclodextrin, hydroxypropyl-cyclodextrin, PEG400, PEG 200.

If formulated as a fixed dose, such combination products employ the compounds described herein within the dosage ranges described herein or as known to those of skill in the art.

Since the compounds described herein (e.g., compounds a and B and MAPKAP pathway inhibitors) are intended for use in pharmaceutical compositions, the skilled artisan will appreciate that they can be provided in substantially pure form (e.g., at least 60% pure, at least 75% pure, at least 85% pure, and at least 98% pure (w/w)). The pharmaceutical preparation may be in unit dosage form. In this form, the formulation is subdivided into unit doses of appropriate size containing an appropriate amount of compound a or B, e.g. an effective amount to achieve the desired purpose as described herein.

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Combination therapy for treating mastocytosis

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