WO2020257644A1

WO2020257644A1 - Methods of treating myeloproliferative disorders

Info

Publication number: WO2020257644A1
Application number: PCT/US2020/038727
Authority: WO
Inventors: Adrian SENDEROWICZ; Michael Cooper
Original assignee: Constellation Pharmaceuticals, Inc.
Priority date: 2019-06-21
Filing date: 2020-06-19
Publication date: 2020-12-24

Abstract

The present disclosure relates to the use of 2-((4S)-6-(4-chlorophenyl)-l-methyl-4H- benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide in combination with JAK inhibitors for treating myelofibrosis.

Description

METHODS OF TREATING MYELOPROLIFERATIVE DISORDERS

RELATED APPLICATIONS

[0001] This application claims priority to International Application No.

PCT/US2019/059813, filed November 5, 2019 and International Application No.

PCT/US2019/038506, filed June 21, 2019, the entire contents of each of which are incorporated herein by reference.

BACKGROUND

[0002] Myelofibrosis (MF) is part of a collection of progressive blood cancers known as myeloproliferative neoplasms and is associated with significantly reduced quality of life and shortened survival. As the disease progresses, the bone marrow produces fewer red blood cells. This disease has several important features: severe constitutional symptoms, splenomegaly (enlargement of spleen) and defects in the structure and function of the bone marrow. As part of the defects of the bone marrow, patients have thrombocytopenia, a condition characterized by low platelet counts in the blood. Patients may also have severe anemia (a condition characterized by low red-blood-cell counts) leading to red-blood-cell transfusion requirements. Among other complications, most patients with MF have enlarged spleens as well as many other physical symptoms, including abdominal discomfort, bone pain and extreme fatigue. Although transplantation (HCT) can be curative, it is associated with its own morbidity and mortality, which limit its use to those eligible patients whose prognosis is worse (< 5 years) than the risk of moving forward with the transplant. The remaining treatments are palliative in nature, either due to their mechanism of action (e.g., treatments specifically focused on the anemia that is frequently associated with myelofibrosis) or due to the restricted effects that the treatment can elicit (e.g., the standard of care ruxolitinib). The major cell responsible for MF is the megakaryocyte and preventing megakaryocyte proliferation may lead to improvement in the disease due to decrease in cytokines and decrease in fibrosis.

[0003] There are limited treatment options for patients with MF. Ruxolitinib is the current standard of care and the only approved drug treatment for intermediate- and high-risk MF patients. Ruxolitinib inhibits dysregulated janus kinase 1 and 2, or JAK1/JAK2, signaling that is associated with MF. Ruxolitinib produces spleen reduction and symptom improvement in MF patients. However, its side effects include increased anemia and transfusion dependence. Indeed, in the SIMPLIFY- 1 clinical trial of ruxolitinib in JAK -naive patients, transfusion dependence increased from 24.0% to 40.1% after 24 weeks of treatment with ruxolitinib. See e.g., Journal of Clinical Oncology, 35, No. 34 (December 1, 2017) 3844- 3850. Also, ruxolitinib has not been shown to have a significant effect on bone marrow fibrosis reversion, which has been documented as a primary cause of morbidity and mortality in MF. Unfortunately, many patients (such as those with anemia) cannot initiate ruxolitinib therapy and many other patients may not tolerate treatment with ruxolitinib or will have an insufficient response or no response at all. These disadvantages, coupled with the lack of alternative therapies, provide an unmet medical need for improving the survival rates for subjects with MF.

SUMMARY

[0004] Recently, we identified the Bromodomain and Extra-Terminal (BET) family inhibitor 2-((4S)-6-(4-chlorophenyl)-l-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4- yl)acetamide (herein Compound 1), as an effective second-line treatment for myelofibrosis. See e.g., PCT/US2018/062534, the contents of which are incorporated herein by reference. Unlike ruxolitinib, Compound 1 increased hemoglobin levels in subject with myelofibrosis. See e.g., PCT/US2018/062534. This is particularly important for subjects who are also anemic. Other results showed that transfusion dependence could be reversed with treatment of Compound 1. See e.g., PCT/US2018/062534. Improved bone marrow fibrosis, decreases in spleen size, and positive effects for alleviating uncontrolled thrombocytosis were further realized.

[0005] Interestingly, the benefits resulting from treatment with Compound 1 alone extrapolated through combination therapy arms with ruxolitinib. For example, reductions in spleen size, improvements in platelet counts, depletion of constitutional symptoms, and conversion from transfusion dependence to independence were each noted during myelofibrosis human trial. Based on these profound results, we envision that the benefits achieved from the combination of Compound 1 with ruxolitinib can extend to other j anus kinase inhibitors (JAK).

[0006] Provided herein, therefore, are methods of using 2-((4S)-6-(4-chlorophenyl)-l- methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide, or a pharmaceutically acceptable salt thereof, and a JAK inhibitor for treating myelofibrosis.

[0007] Also provided herein are pharmaceutical compositions comprising 2-((4S)-6-(4- chlorophenyl)-l-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide, or a

pharmaceutically acceptable salt thereof; and a JAK inhibitor; and optionally a

pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE FIGURES

[0008] FIG. 1 shows the effects of Compound 1 on IL6 and IL10 mRNA transcript levels.

[0009] FIG. 2 depicts histograms of Compound 1 effect on megakaryocyte

differentiation.

[0010] FIG. 3 shows the changes in hemoglobin levels and transfusion requirements in a combination arm of Compound 1 and ruxolitinib.

[0011] FIG. 4 shows the spleen reduction volume and total symptom score (TSS) improvement from a Phase 2 human trial using Compound 1 and ruxolitinib in JAK inhibitor treatment naive myelofibrosis patients.

[0012] FIG. 5A shows the spleen reduction volume improvement from a Phase 2 human trial using Compound 1 monotherapy or as an add-on to ruxolitinib in myelofibrosis patients.

[0013] FIG. 5B shows the total symptom score (TSS) improvement from a Phase 2 human trial using Compound 1 monotherapy or as an add-on to ruxolitinib in myelofibrosis patients.

DETAILED DESCRIPTION

[0014] A. General Description

[0015] In a first embodiment, provided is a method for treating myelofibrosis in a subject comprising administering to the subject an effective amount of 2-((4S)-6-(4-chlorophenyl)-l- methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a JAK inhibitor. Also provided, as part of a first embodiment, is the use of an effective amount of 2-((4S)-6-(4-chlorophenyl)-l-methyl-4H- benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a JAK inhibitor for the manufacture of a medicament for treating myelofibrosis in a subject. Further provided, as part of a first embodiment, is an effective amount of 2-((4S)-6-(4-chlorophenyl)-l-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4- yl (acetamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a JAK inhibitor for treating myelofibrosis in a subject.

[0016] As detailed in the Exemplification section below, Compound 1 was effective in subjects who have myelofibrosis, but are JAK inhibitors naive subjects. Therefore, in a second embodiment, provided herein is a method of treating myelofibrosis in a subject comprising administering to the subject a therapeutically effective amount of 2-((4S)-6-(4- chlorophenyl)-l-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a JAK inhibitor, wherein the subject is janus kinase (JAK) inhibitor naive subject prior to treatment. Also provided is the use of an effective amount of 2-((4S)-6-(4-chlorophenyl)-l-methyl-4H- benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a JAK inhibitor in the manufacture of a medicament for treating myelofibrosis in a subject who is a janus kinase (JAK) inhibitor naive subject prior to treatment. Further provided is an effective amount of 2-((4S)-6-(4-chlorophenyl)-l-methyl- 4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide, or a pharmaceutically acceptable salt thereof, and an effective amount of a JAK inhibitor for treating myelofibrosis in a subject who is a janus kinase (JAK) inhibitor naive subject prior to treatment.

[0017] B. Definitions

[0018] 2-((4S)-6-(4-chlorophenyl)-l-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4- yl)acetamide is exemplified as Compound 144 in Ei.S. Patent No. 8,796,261, the entire contents of which are incorporated herein by reference. 2-((4S)-6-(4-chlorophenyl)-l-methyl- 4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide is used interchangeably herein with Compound 1 and/or CPI-0610, and is represented by the following structural formula:

Crystalline forms of Compound 1 are disclosed in U.S. 9,969,747, the entire contents of which are incorporated by reference herein.

[0019] A j anus kinase inhibitor or JAK inhibitor are agents which inhibit the activity of one or more of the Janus kinase family of enzymes (JAKl, JAK2, JAK3, TYK2) and interfere with the JAK-STAT signaling pathway. JAK inhibitors described herein include biological and chemical agents.

[0020] Pacritinib refers the JAK inhibitor (22Z,23E,8E)-44-(2-(pyrrolidin-l-yl)ethoxy)- 21 ,22-dihydro-6, 11 -dioxa-3 -aza-2(4,2)-pyrimidina- 1 ,4(1 ,3)-dibenzenacyclododecaphan-8- ene, having the following structural formula:

[0021] Momelotinib refers the JAK inhibitor N-(cyanomethyl)-4-(2-((4- morpholinophenyl)amino)pyrimidin-4-yl)benzamide, having the following structural formula:

[0022] Baricitinib refers the JAK inhibitor 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)- lH-pyrazol-l-yl)-l-(ethylsulfonyl)azetidin-3-yl)acetonitrile, having the following structural formula:

[0023] Itacitinib refers to the JAK inhibitor 2-(3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)- lH-pyrazol-l-yl)-l-(l-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3- yl)acetonitrile, having the following structural formula:

[0024] Fedratinib refers the JAK inhibitor N-(l, l-dimethylethyl)-3-[[5-methyl-2-[[4-[2- (l-pyrrolidinyl)ethoxy]phenyl]amino]-4-pyrimidinyl]amino]-benzenesulfonamide, having the following structural formula:

[0025] NS-018 refers the JAK inhibitor (S)-N2-[l-(4-fluorophenyl)ethyl]-4-(l-methyl- lH-pyrazol-4-yl)-N6-(pyrazin-2-yl)pyridine-2, 6-diamine, having the following structural formula:

[0026] The terms“subject” and“patient” may be used interchangeably, and mean a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of treatment.

[0027] The terms“treatment,”“treat,” and“treating” refer to reversing, alleviating, reducing the likelihood of developing, or inhibiting the progress of myelofibrosis, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors), i.e., prophylactic treatment. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. Symptoms specific to

myelofibrosis include, but are not limited to, abdominal discomfort, dyspnea on exertion, early satiety, fatigue, headaches, night sweats, dizziness, fever, chills, insomnia, pruritus, or bone pain.

[0028] A subject who is characterized as progressed/relapsed is one who at one time responded to treatment with a JAK inhibitor, but who no longer responds. A subject who is characterized as refractory/resistant is one who is unresponsive or demonstrates worsening of disease while on treatment with a JAK inhibitor.

[0029] Unless otherwise indicated, the administrations described herein include administering Compound 1 prior to, concurrently with, or after administration of a disclosed JAK inhibitor to treat MF. Thus, simultaneous administration is not necessary for therapeutic purposes. In one aspect, however, Compound 1 is administered concurrently with the JAK inhibitor.

[0030] The term“effective amount” or“therapeutically effective amount” are used interchangeably and include an amount of a compound described herein that will elicit a desired medical response in a subject having myelofibrosis, e.g., reducing the symptoms of and/or slowing the progression of the disease.

[0031] The term“pharmaceutically acceptable carrier” refers to a non-toxic carrier, adjuvant, or vehicle that does not adversely affect the pharmacological activity of the compound with which it is formulated, and which is also safe for human use.

Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances (e.g., microcrystalline cellulose, hydroxypropyl methylcellulose, lactose monohydrate, sodium lauryl sulfate, and crosscarmellose sodium), polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0032] The compounds described herein may be present in the form of pharmaceutically acceptable salts. For use in medicines, the salts of the compounds described herein refer to non-toxic“pharmaceutically acceptable salts.” Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts where possible.

[0033] C. Description of Certain Embodiments

[0034] In a third embodiment, JAK inhibitors of the present methods and compositions are selected from those having the following formulae: 1)

pharmaceutically acceptable salt thereof, wherein, X, R¹, R², R³, and R⁴ are as defined in US 2011/0288065, the contents of which are incorporated herein by reference;

pharmaceutically acceptable salt thereof, wherein R¹,

R², Ar¹, Ar², Z², and L are as defined in WO 2007/058627, the contents of which are incorporated herein by reference;

3)

pharmaceutically acceptable salt thereof, wherein A, Q, G, X, Y, R¹, R², R³, R⁴, R⁵, p, q, and n are as defined in US

2007/0259904, the contents of which are incorporated herein by reference;

4)

pharmaceutically acceptable salt thereof, wherein Q, Z, n, R¹, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are as defined in WO 2008/109943, the contents of which are incorporated herein by reference;

h are incorporated herein by reference; 6)

pharmaceutically acceptable salt thereof, wherein L and R¹ are as defined in WO 2009/144512, the contents of which are incorporated herein by reference;

7)

8)

pharmaceutically acceptable salt thereof, wherein L and R are as defined in WO 2009/144512, the contents of which are incorporated herein by reference; and

10)

pharmaceutically acceptable salt thereof, wherein X,

Y, Z, L, A, n, and R¹ are as defined in WO 2011/112662, the contents of which are incorporated herein by reference

[0035] Alternatively, as part of a third embodiment, the JAK of the present methods and compositions are selected from pacritinib, itacitinib, momelotinib, baricitinib, fedratinib, and NS-018, or a pharmaceutically acceptable salt thereof.

[0036] In a fourth embodiment, the subjects described in the present methods (e.g., as in the first and third embodiments) are characterized as progressed/relapsed to a JAK inhibitor. In a fourth embodiment, the subjects described in the present methods (e.g., as in the first and second embodiments) are characterized is refractory/resistant to a JAK inhibitor.

[0037] In a sixth embodiment, the subjects described in the present methods (e.g., as in any one of the first through fifth embodiments) are cytopenic. Cytopenic refers to subjects in which the production of one or more blood cell types ceases or is greatly reduced. Types of cytopenia include e.g., anemia (a deficiency of red blood cells), leukopenia or neutropenia (a deficiency of white blood cells), thrombocytopenia (a deficiency in the platelets), and pancytopenia (a deficiency in all three of red blood cells, white blood cells, and platelet counts). [0038] In a seventh embodiment, the subjects described in the present methods (e.g., as in any one of the first through sixth embodiments) are anemic. A subject of the present disclosure (e.g., as in any one of the first through sixth embodiments) is said to be anemic if their hemoglobin value is less than 13.5 g/dL of blood for a male subject or less than 12.0 g/dL of blood for a female subject. In some aspects, a subject (e.g., as in any one of the first through sixth embodiments) is defined herein as being anemic if their hemoglobin value is less than 10.0 g/dL. Subjects treatable by the present methods (e.g., as in any one of the first through sixth embodiments) therefore include those having hemoglobin values less than 13.0 g/dL, less than 12.5 g/dL, less than 12.0 g/dL, less than 11.5 g/dL, less than 11.0 g/dL, less than 10.5 g/dL, less than 10.0 g/dL, less than 9.5 g/dL, less than 9.0 g/dL, or less than 8.5 g/dL for male subjects and less than 11.5 g/dL, less than 11.0 g/dL, less than 10.5 g/dL, less than 10.0 g/dL, less than 9.5 g/dL, less than 9.0 g/dL, or less than 8.5 g/dL for female subjects. In others aspects, a subject (e.g., as in any one of the first through sixth

embodiments) is defined herein as being anemic if their hemoglobin value ranges from 7.5 g/dL of blood to 13.5 g/dL of blood for a male subject or from 7.5 g/dL of blood to 12.0 g/dL of blood for a female subject. In others aspects, a subject (e.g., as in any one of the first through sixth embodiments) is defined herein as being anemic if their hemoglobin value ranges from 7.5 g/dL of blood to 10.5 g/dL of blood for a male subject or from 7.5 g/dL of blood to 10.5 g/dL of blood for a female subject. In others aspects, a subject (e.g., as in any one of the first through sixth embodiments) is defined herein as being anemic if their hemoglobin value ranges from 7.5 g/dL of blood to 10.0 g/dL of blood for a male subject or from 7.5 g/dL of blood to 10.0 g/dL of blood for a female subject. In others aspects, a subject (e.g., as in any one of the first through sixth embodiments) is defined herein as being anemic if their hemoglobin value ranges from 7.7 g/dL of blood to 10.7 g/dL of blood for a male subject or from 7.7 g/dL of blood to 10.5 g/dL of blood for a female subject. In others aspects, a subject (e.g., as in any one of the first through sixth embodiments) is defined herein as being anemic if their hemoglobin value ranges from 7.7 g/dL of blood to 10.0 g/dL of blood for a male subject or from 7.7 g/dL of blood to 10.0 g/dL of blood for a female subject.

[0039] In an eighth embodiment, subjects treated by the methods described herein (e.g., as in any one of the first through seventh embodiments) are thrombocytopenic. A subject of the present disclosure (e.g., as in any one of the first through seventh embodiments) is said to be thrombocytopenic if their platelet count is less than 150,000 platelets/ pL of blood.

Subjects treatable by the present methods (e.g., as in any one of the first through seventh embodiments) therefore include those having platelet levels less than 140,000 platelets/pL, less than 130,000 platelets/pL, less than 120,000 platelets/pL, less than 110,000 platelets/ pL, less than 100,000 platelets/pL, less than 90,000 platelets/pL, less than 80,000 platelets/pL, less than 70,000 platelets/ pL, less than 60,000 platelets/pL or less than 50,000 platelets/ pL, alone or in combination with one or more of the hemoglobin values described above.

[0040] In a ninth embodiment, subjects treated by the methods described herein (e.g., as in any one of the first through seventh embodiments) are thrombocythemic. A subject of the present disclosure subjects treated by the methods described herein (e.g., as in any one of the first through seventh embodiments) is said to be thrombocythemic if their platelet count is more than 400,000 platelets/ pL of blood. Subjects treatable by the present methods (e.g., as in any one of the first through seventh embodiments) therefore include those having platelet levels more than 450,000 platelets/ pL, more than 500,000 platelets/pL, more than 550,000 platelets/pL, or more than 600,000 platelets/ pL, alone or in combination with one or more of the hemoglobin values described above.

[0041] In a tenth embodiment, subjects treated by the methods described herein (e.g., as in any one of the first through ninth embodiments) are leukopenic. A subject (e.g., as in any one of the first through ninth embodiments) is said to be leukopenic if their white blood cell (WBC) count is less than 4,000 WBCs/pL of blood. In certain aspects, subjects treatable by the present methods (e.g., as in any one of the first through ninth embodiments) include those having WBC counts of less than 3,500 WBCs/pL, 3,200 WBCs/pL, 3,000 WBCs/pL, or 2,500 WBCs/pL, alone or in combination with one or more of the hemoglobin and/or platelet values described above.

[0042] In an eleventh embodiment, subjects treated by the methods described herein is (e.g., as in any one of the first through tenth embodiments) are neutropenic. In one aspect, a subject of the present disclosure (e.g., as in any one of the first through tenth embodiments) is said to be neutropenic if their neutrophil count is less than 1500 neutrophil s/pL of blood. In certain aspects, subjects treatable by the present methods (e.g., as in any one of the first through tenth embodiments) include those having neutrophil counts of less than 1250 neutrophil s/pL, 1000 neutrophil s/pL, 750 neutrophil s/pL, or 500 neutrophil s/pL, alone or in combination with one or more of the hemoglobin, platelet, and/or WBC values described above. [0043] In a twelfth embodiment, subjects treatable by the present methods (e.g., as in any one of the first through eleventh embodiments) include those having an enlarged spleen or liver.

[0044] In a thirteenth embodiment, subjects treatable by the present methods (e.g., as in any one of the first through twelfth embodiments) may also be experiencing one or more additional symptoms. These symptoms include, but are not limited to, abdominal discomfort, dyspnea on exertion, early satiety, fatigue, headaches, night sweats, dizziness, insomnia, pruritus, or bone pain.

[0045] In a fourteenth embodiment, subjects treated by the present methods (e.g., as in any one of the first through thirteenth embodiments) are transfusion dependent prior to treatment with Compound 1. In some aspects,“transfusion dependent” means that a subject requires red blood cell (RBC) transfusions in order to maintain an acceptable level of hemoglobin. An acceptable level of hemoglobin is determined by those skill in the art and can range from e.g., from 13.5 to 17.5 g/dL of blood for men and from 12.0 to 15.5 g/dL of blood in women. It will be understood that subjects undergoing treatment with a JAK inhibitor may have lower hemoglobin levels than those described above and still be deemed an“acceptable” level in order for treatment to continue.

[0046] In a fifteenth embodiment, subjects treated by the present methods (e.g., as in any one of the first through fourteenth embodiments) experience a reduction in spleen size. In one aspect, the reduction comprises a 10% or more (e.g., a 15% or more, a 20% or more, a 25% or more, a 30% or more, a 35% or more, a 40% or more, a 45% or more, a 50% or more, a 55% or more, a 60% or more, or a 65% or more reduction in spleen volume from baseline. In another aspect, the reduction comprises from a 10% to a 65% reduction in spleen volume from baseline.

[0047] In a sixteenth embodiment, subjects treated by the present methods (e.g., as in any one of the first through seventeenth embodiments) experience a reduction in headaches.

[0048] In a ninteenth embodiment, subjects treated by the present methods (e.g., as in any one of the first through eighteenth embodiments) have a reduction in the number of blood transfusion.

[0049] In a twentieth embodiment, subjects treated by the present methods (e.g., as in any one of the first through ninteenth embodiments) experience a normalization of platelets.

[0050] In a twenty-first embodiment, subjects treated by the present methods (e.g., as in any one of the first through twentieth embodiments) experience an increase in hemoglobin values. [0051] In a twenty-second embodiment, provided is a pharmaceutically acceptable composition comprising Compound 1, or a pharmaceutically acceptable salt thereof; and a JAK inhibitor (e.g., as in the third embodiment); and optionally a pharmaceutically acceptable carrier.

[0052] The compounds of the methods described herein can be formulated as

pharmaceutical compositions and administered to a subject, such as a human, in a variety of forms adapted to the chosen route of administration. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, buccal, transdermal, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrathecal, intrasternal injection or infusion techniques. Methods of formulating pharmaceutical compositions are well known in the art, for example, as disclosed in“Remington: The Science and Practice of Pharmacy,” University of the Sciences in Philadelphia, ed., 21st edition, 2005, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0053] Pharmaceutical compositions of the invention can be prepared by combining a compound of the methods described herein with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Thus, the present compounds of the methods described herein may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable excipient such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0054] A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound described herein in the composition will also depend upon the particular compound in the composition. In one aspect, however, Compound 1, or a pharmaceutically acceptable salt thereof, may be formulated at a dose of from 50 mg to 500 mg for e.g., administration once, twice, or three times daily. For example, in combination therapies, Compound 1 may be administered at a dosage of from 100 mg to 300 mg/day, from 100 mg to 200 mg/day, or at 100 mg/day, 125 mg/day, 150 mg/day, 175 mg/day, or 200 mg/day.

EXEMPLIFICATION

[0055] Compound 1 can be obtained following the procedures describe in U.S. Patent No. 8,796,261 and WO 2015/195862, both of which are incorporated herein by reference.

Inhibitory Effect on Cytokine Release In Vitro

[0056] Compound 1 was assessed for its ability to suppress the expression of NF-KB target genes in two experiments. In one experiment, THP-1 acute leukemia cell lines were exposed to lipopoly saccharide treatment and then Compound 1 for 16 hours. IL6 release from the THP-1 acute leukemia cells was inhibited, with an ICso of 0.069 mM. In the other experiment, the ability of Compound 1 to suppress both IL6 and IL10 expression in TMD8 ABC-DLBCL cells was investigated (data on file). TMD8 cells were incubated with DMSO or 1.6 pM Compound 1 for 6 or 24 hours. RNA was then extracted from the cells and quantified using qRT-PCR. As shown in FIG. 1, Compound 1 substantially suppressed mRNA transcription of both IL6 and IL10 after 6 and 24 hours of treatment.

Effect of Compound 1 as a Single Agent on Megakaryocyte Differentiation

[0057] The effects of Compound 1 on megakaryocyte differentiation and proliferation were evaluated using CD34+ cells isolated from healthy donor bone marrow (data on file). The CD34+ cells were grown in megakaryocyte differentiation serum-free stem cell differentiation base medium with a megakaryocyte-driving cytokine cocktail for 14 days with DMSO or Compound 1 at concentrations ranging from 3 nM to 500 nM. The cells were then stained for CD34 (progenitor marker), CD45 (leukocyte marker) and CD41a (mature megakaryocyte marker) and assessed by FACS for viability and marker expression. CD41a expression and cell size were used as markers of megakaryocyte differentiation. Compound 1 reduced the number of cells with high CD41a expression in a concentration-dependent manner. The shift from high to low CD41a expression began at approximately 50 nM, with pronounced effects observed at 200 to 500 nM, as shown in FIG. 2. The loss of CD41a-high- expressing cells suggests impaired megakaryocyte differentiation and loss of mature megakaryocytes. Clinical Data

[0058] Patient 245, a 66 year-old female was diagnosed with myelofibrosis in May 2014, remained treatment naive until January 2016 when she initiated treatment with the JAK inhibitor ruxolitinib 15 mg twice daily (BID). Panobinostat was added in February 2016 and discontinued in March 2017, due to the development of anemia. From March 2017, while on ruxolitinib alone, the patient became resistant to ruxolitinib, with her spleen increasing 25% in size.

[0059] At entry into the study with Compound 1, her spleen volume by MRI was 1404 cc and was 12 cm by palpation. The patient presented with early satiety, night sweats, and dyspnea at the start of the study. Within 4 months of treatment with Compound 1, 125 mg QD and ruxolitinib 15 mg BID, the patient had resolution of early satiety; her spleen was 5 cm by palpation and her liver was no longer palpable. The lowest spleen volume by MRI was 1144 cc, a 19% reduction, at the 6-month MRI. The dose of ruxolitinib was reduced on Cycle 10 to 7.5 mg BID to address decreasing platelet count. Her platelets counts gradually improved following the dose reduction of ruxolitinib and remained below the protocol- specified criteria of 100 x 10⁹/L for two treatment cycles to permit a dose increase in Compound 1.

[0060] Patient 246 is a 53 year-old female who was diagnosed with myelofibrosis in 2009. During 2002 and 2006, the patient cycled between epoetin alfa, lenalidomide and thalidomide then received lenalidomide for 7 years until 2013. She required RBC

transfusions during 2013 and initiated interferon in 2014, which allowed her to become transfusion independent. Interferon was discontinued almost a year later due to fatigue. The patient remained transfusion independent and without further treatment until late 2016 when they once again became transfusion dependent. Ruxolitinib 5 mg BID was started in January 2017. Ruxolitinib was increased to 10 mg BID in April 2017, but the patient remained transfusion dependent and symptomatic. She was considered ruxolitinib resistant due an increasing spleen size and exacerbated symptoms (extreme fatigue, shortness of breath, distress on exertion, occasional nausea and night sweats) while on ruxolitinib therapy.

[0061] When Patient 246 initiated combination treatment with Compound 1 125 mg QD and ruxolitinib 10 mg BID her spleen volume was 607 cc by MRI and 2 cm by palpation and she required regular transfusions (2 units of RBC every 3-4 weeks). The dose of Compound 1 was titrated up to 175 mg QD after five treatment cycles and within 7 months of combination therapy, the patient had become transfusion independent (defined as >12 weeks without a transfusion and hemoglobin > 8 g/dL; see FIG. 3), which has been maintained for >30 weeks (most recent hemoglobin measurement was 10.9g/dL). She has also experienced a clinically meaningful improvement in her associated constitutional symptoms (fatigue and dyspnea) and has had an incremental decrease in spleen volume, achieving a 37% reduction in spleen volume by Cycle 12 (380 cc).

Expanded Clinical Data Set

A. Phase 2 Study of Compound 1 and Ruxolitinib in JAK Inhibitor Treatment Naive Myelofibrosis Patients

[0062] A global, multicenter, open label Phase 2 study of Compound 1 in combination with the JAK inhibitor ruxolitinib was conducted. Key eligibility criteria of included JAKi naive myelofibrosis (MF) patients (pts) with Dynamic International Prognostic Scoring (DIPSS) score int-1 or higher, ECOG performance status <2, platelet counts >100 x 109/L, peripheral blood blast count <10%, anemia (hemoglobin < lOg/dL), >5 cm palpable spleen,

>2 symptoms measurable (score >3) or a total symptom score (TSS) of >10 using the MFSAF v4.0. Primary endpoint: spleen volume response (SVR); key secondary endpoints: change in TSS, safety and PK; additional endpoints: changes in proinflammatory Ck levels, BM morphology and mutant allele burden.

[0063] A total of 11 patients were treated with 4 patients on treatment for > 4 cycles (> 12 weeks). Demographics and results are as follows: Baseline median age: 71 years (52-76), gender: 8 male (72.7%), ECOG <l : 10 (90.9%) patients, primary MF: 8 (72.7%) patients, DIPSS score: int-1 /int-2/high: 2/7/2 patients, median platelet: 368 x 109/L (112-951), 9 (81.8%) patients with hemoglobin <10 g/dL, median spleen volume: 1379 cc (580-2807), median TSS: 11.8 (4.1-17), driver mutations: 11 (100%) with >1 JAK2IMPLICALR mutations, HMR (high molecular risk) mutations: 6 (56%) patients, and >3 mutations: 4 (36%) patients. All 4 (100%) patients on treatment for > 12 weeks achieved >35% spleen volume reduction (median: -52.4%, [range -68.7%, -42.7%]) and all 4 patients (100%) achieved >50% improvement in TSS (median best change: -79.35% [range -90.2%, - 70.1%]). See FIG. 4, where * is data post cut off-date. Suppression of proinflammatory Ck, including IL-8, IL-18 and CRP, was also observed. Safety data from the first 6 patients who received treatment for at least 1 cycle were reviewed: no DLTs or grade >3 thrombocytopenia was observed. The most common treatment-emergent adverse events (TEAE) observed in > 2 patients include anemia (1 grade 3), fatigue (all < grade 2), and non-cumulative reversible thrombocytopenia (all < grade 2). [0064] Overall, the combination of Compound 1 and ruxolitinib was generally well- tolerated demonstrating that the safety of this combination is acceptable in JAKi naive MF patients with anemia. Early clinical activity was observed with the combination: all 4 evaluable patients achieved both >35% SVR and >50% improvement in TSS as early as 3 months after treatment. Available data in JAKi naive anemic MF patients, a population with poor prognosis, along-with additional information on reduction in pro-inflammatory Ck and BM fibrosis improvement in Compound 1 treated patients in ruxolitinib refractory MF, collectively indicate that addition of Compound 1 to ruxolitinib could have disease modifying effects in JAKi naive MF patients.

B. Phase 2 Study of Compound 1 as Monotherapy or Add-On to Ruxolitinib in Patients with Refractory or Intolerant Advanced Myelofibrosis

[0065] A global, multicenter, open label Phase 2 study of Compound 1 monotherapy in myelofibrosis (MF) patients (pts) who were refractory/intolerant to ruxolitinib (Arm 1) or Compound 1 and ruxolitinib in MF patients who had suboptimal response to ruxolitinib (Arm 2) was conducted. Patients were further stratified based on transfusion dependence status [transfusion dependent (TD), defined as an average of >2 units per month over 12 wks, or non-TD cohorts]. The starting dose of Compound 1 was 125 mg daily on days 1-14 of a 21 -day cycle in both arms. Primary endpoint: spleen volume response (SVR) for non-TD cohorts or TD to transfusion independence (TI, no transfusion for consecutive 12 wks) conversion for TD cohorts; secondary endpoints: change in total symptom score (TSS) per MFSAF v4.0, patient global impression of change (PGIC), safety and PK; additional endpoints: changes in proinflammatory Ck levels, BM morphology and mutant allele burden.

[0066] A total of 48 patients were enrolled with 12 being treated with Compound 1 monotherapy (Arm 1) and 36 with Compound 1 and ruxolitinib (Arm 2). Demographics and results are as follows: At baseline, median age: 69 years (41-88), gender: 28 (58%) male, ECOG <l : 45 (94%) patients, primary MF: 33 (69%) patients, DIPSS score high: 10 (21%) patients, median platelet: 199 x 109/L (77-895), 34 (71%) patients with Hgb <10 g/dL, median spleen volume: 2183 cc (123-3909), median TSS: 17.6 (1.4-56), 46 (96%) patients had >1 JAK2IMPLICALR mutations, and 34 (71%) patients had HMR (high molecular risk) mutations. 33 (69%) patients on treatment for > 12 wks, 4 on treatment for > 18 months.

[0067] Spleen volume reduction observed in 29 of 31 (94%) patients (median best change: -17% [range: -50.7, 10.2]) (FIG. 5A). TSS improvement was reported in 26 of 28 (93%) patients (median best change: -46.4% [range: -95.3%, 27%]), 11 (39%) patients with >50% TSS improvement (FIG. 5B). PGIC improvement score in 28 of 33 (85%) patients; 21 (64%) reported much or very much improved scores. Increase in hemoglobin by 1.5 mg/dL post-baseline observed with both Compound 1 monotherapy (4 of 8, 50%) and Compound 1 with ruxolitinib (4 of 25, 16%)). Improvement in BM fibrosis and/or reticulin by >1 Gr reported in 7 of 12 (58%) evaluable patients with baseline and 1 post-baseline biopsy and as early as 6-months of Compound 1 treatment. 4 TD patients in Arm 2 treated with Compound 1 with ruxolitinib converted to TI- 2 of whom are TI for >36 wks, no longer anemic, and showed spleen volume reduction, improvement in symptom and BM fibrosis; 12 additional patients are being monitored for potential TI conversion. 41 patients remain active on treatment and 7 patients discontinued, including 1 patient, initially transplant ineligible, underwent stem cell transplantation after 6 cycles of Compound 1 with ruxolitinib treatment. Most common (>20%) treatment-emergent adverse events (TEAE) of any Gr include diarrhea, nausea, cough and upper respiratory tract infection. Most common (>5%) >3 Gr TEAE include anemia (8.3%) and thrombocytopenia (8.3%, asymptomatic, non-cumulative and generally reversible).

[0068] This data indicates that Compound 1 alone or“add-on” to ruxolitinib is generally well-tolerated and provides clinical benefits in MF patients with inadequate responses or who are refractory to ruxolitinib. Improvement in BM fibrosis and anemia responses indicate the potential for meaningful disease modification.

[0069] While have described a number of embodiments of this, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this disclosure. Therefore, it will be appreciated that the scope of this disclosure is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

[0070] The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

Claims

Listing of Claims:

1. A method of treating myelofibrosis in a subject comprising administering to the subject a therapeutically effective amount of 2-((4S)-6-(4-chlorophenyl)-l-methyl-4H- benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide and a therapeutically effective amount of a janus kinase (JAK) inhibitor selected from pacritinib, itacitinib, momelotinib, baricitinib, fedratinib, and NS-018, or a pharmaceutically acceptable salt of any of the foregoing.

2. The method of Claim 1, wherein the subject is a janus kinase (JAK) inhibitor naive subject prior to treatment.

3. The method of Claim 1 or 2, wherein the subject is cytopenic.

4. The method of any one of Claims 1 to 3, wherein the subject is anemic.

5. The method of any one of Claims 1 to 4, wherein the subject has a hemoglobin count of less than 10 g/dL.

6. The method of any one of Claims 1 to 5, wherein the subject is thrombocytopenic.

7. The method of any one of Claims 1 to 6, wherein the subject’s platelet count is less than 120,000 platelets/pL.

8. The method of any one of Claims 1 to 5, wherein the subject is thrombocythemic.

9. The method of any one of Claims 1 to 5 and 8, wherein the subject’s platelet count is more than 400,000 platelets/ pL.

10. The method of any one of Claims 1 to 9, wherein the subject is neutropenic.

11. The method of any one of Claims 1 to 10, wherein the subject’s absolute neutrophil count is less than 1000 neutrophil s/pL of blood.

12. The method of any one of Claims 1 to 11, wherein the subject has an enlarged spleen or liver.

13. The method of any one of Claims 1 to 12, wherein the subject is suffering from abdominal discomfort, dyspnea on exertion, early satiety, fatigue, headaches, night sweats, dizziness, insomnia, pruritus, or bone pain.

14. The method of any one of Claims 1 to 13, wherein the subject is transfusion dependent.

15. A pharmaceutical composition comprising an effective amount of 2-((4S)-6-(4- chlorophenyl)-l-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide and an effective amount of a j anus kinase (JAK) inhibitor selected from pacritinib, momelotinib, baricitinib, fedratinib, and NS-018; or a pharmaceutically acceptable salt of any of the foregoing; and optionally a pharmaceutically acceptable carrier.