WO2024124240A1

WO2024124240A1 - Methods of treating myeloproliferative neoplasms

Info

Publication number: WO2024124240A1
Application number: PCT/US2023/083387
Authority: WO
Inventors: Wayne Rothbaum
Original assignee: Kartos Therapeutics
Current assignee: Kartos Therapeutics
Priority date: 2022-12-09
Filing date: 2023-12-11
Publication date: 2024-06-13
Anticipated expiration: 2025-06-09
Also published as: AU2023390908A1; MX2025006677A; KR20250151363A; EP4629993A1; JP2025539944A

Abstract

Therapeutic methods and pharmaceutical compositions for treating a myeloproliferative neoplasm (MPN) with a combination of a MDM2 inhibitor and a JAK inhibitor.

Description

METHODS OF TREATING MYELOPROLIFERATIVE NEOPLASMS

FIELD OF THE INVENTION

Methods of treating a myeloproliferative neoplasm (M PN) using a Mouse double minute 2 homolog (MDM2) inhibitor and a JAK inhibitor.

BACKGROUND OF THE INVENTION p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of numerous genes involved in cell cycle arrest, apoptosis, senescence, and DNA repair. Unlike normal cells, which have infrequent cause for p53 activation, tumor cells are under constant cellular stress from various insults including hypoxia and pro-apoptotic oncogene activation. Thus, there is a strong selective advantage for inactivation of the p53 pathway in tumors, and it has been proposed that eliminating p53 function may be a prerequisite for tumor survival. In support of this notion, three groups of investigators have used mouse models to demonstrate that absence of p53 function is a continuous requirement for the maintenance of established tumors. When the investigators restored p53 function to tumors with inactivated p53, the tumors regressed. p53 is inactivated by mutation and/or loss in 50% of solid tumors and 10% of liquid tumors. Other key members of the p53 pathway are also genetically or epigenetically altered in cancer. MDM2, an oncoprotein, inhibits p53 function, and it is activated by gene amplification at incidence rates that are reported to be as high as 10%. MDM2, in turn, is inhibited by another tumor suppressor, pl4ARF. It has been suggested that alterations downstream of p53 may be responsible for at least partially inactivating the p53 pathway in p53 WT tumors (p53 wild type). In support of this concept, some p53WT tumors appear to exhibit reduced apoptotic capacity, although their capacity to undergo cell cycle arrest remains intact. One cancer treatment strategy involves the use of small molecules that bind MDM2 and neutralize its interaction with p53. MDM2 inhibits p53 activity by three mechanisms: 1) acting as an E3 ubiquitin ligase to promote p53 degradation; 2) binding to and blocking the p53 transcriptional activation domain; and 3) exporting p53 from the nucleus to the cytoplasm. All three of these mechanisms would be blocked by neutralizing the M DM2-p53 interaction. In particular, this therapeutic strategy could be applied to tumors that are p53 WT, and studies with small molecule MDM2 inhibitors have yielded promising reductions in tumor growth both in vitro and in vivo. Further, in patients with p53-inactivated tumors, stabilization of wild type p53 in normal tissues by MDM2 inhibition might allow selective protection of normal tissues from mitotic poisons. As used herein, MDM2 means a human MDM2 protein and p53 means a human p53 protein. It is noted that human MDM2 can also be referred to as HDM2 or hMDM2. Several MDM2 inhibitors are in human clinical trials for the treatment of various cancers.

The myeloproliferative neoplasms (MPN), including but not limited to: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell (HSC) disorders characterized by the clonal proliferation of terminally differentiated myeloid cells. Approximately 1%, 4%, and 20% of ET, PV and PMF patients, respectively, progress to a blast phase (BP) termed MPN-BP over a 10-year period from the time of diagnosis. Cervantes F, et al., Acta Haematol.1991;85(3 '124-127. MPN-BP and de novo acute myeloid leukemia (AML) each have distinct mutational patterns and clinical courses. Rampal R, et al., Proc Natl Acad Sci USA. 2014; lll(50):E5401- 10. Patients with MPN-BP have a particularly dismal prognosis with a median survival of less than 6 months with currently available therapies.

SUMMARY OF THE INVENTION

The present invention relates to a method of suppressing p21 expression levels in a human suffering from a myeloproliferative neoplasm (MPN) comprising administering to the human a therapeutically effective amount of a MDM2 inhibitor in combination with a JAK inhibitor, wherein the administering suppresses p21 expression levels in the human compared to p21 expression levels in MDM2 inhibitor monotherapy.

In one aspect the disclosure provides a method of suppressing p21 expression levels in a human suffering from a myeloproliferative neoplasm (MPN) comprising administering to the human a therapeutically effective amount of a MDM2 inhibitor in combination with a JAK inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the administering suppresses p21 expression levels in the human compared to p21 expression levels in MDM2 inhibitor monotherapy. In an embodiment, the administering suppresses p21 levels by at least 50% compared to MDM2 inhibitor monotherapy, optionally at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In an embodiment, the administering stimulates apoptosis of malignant myeloid cells in the human suffering from the myeloproliferative neoplasm (MPN). In an embodiment, the malignant cells are CD34+ myeloid cells or CD45+ myeloblasts.

In an embodiment, the MPN is polycythemia vera (PV). In an embodiment, the MPN is thrombocythemia. In an embodiment, thrombocythemia is essential thrombocythemia (ET).

In an embodiment, the MPN is myelofibrosis. In an embodiment, the myelofibrosis is selected from primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF).

In an embodiment, the MPN is chronic myelogenous leukemia. In an embodiment, the MPN is systemic mastocystosis (SM). In an embodiment, the MPN is chronic neutrophilic leukemia (CNL). In an embodiment, the MPN is myelodysplastic syndrome (MDS). In an embodiment, the MPN is mast cell disease (SMCD). In an embodiment, the MPN is chronic eosinophilic leukemia. In an embodiment, the MPN is chronic myelomonocytic leukemia (CMML). In an embodiment, the MPN is atypical chronic myeloid leukemia (aCML). In an embodiment, the MPN is juvenile myelomonocytic leukemia (JMML). In an embodiment, the MPN is hypereosinophilic syndromes (HES). In an embodiment, the MDM2 inhibitor is a pharmaceutically acceptable salt of a compound of Formula (I).

In an embodiment, the compound of Formula (I) is administered once daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg. In an embodiment, the compound of Formula (I) is administered twice daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg. In an embodiment, the human is treated with the MDM2 inhibitor for a period selected from the group consisting of about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, and about 56 days. In an embodiment, the compound of Formula (I) is orally administered.

In an embodiment, the JAK inhibitor is selected from the group consisting of AC-410, AT9283, AZ960, AZD-1480, Baricitinib, B MS-911543, CEP-33779, Cerdulatinib, CHZ868, CYT387, Decernotinib, ENMD- 2076, Fetratinib, Filgotinib, Ganetespib, INCB039110, INCB-047986, Itacitinib, JAK3-IN-1, JANEX-1, LFM- A13, LY2784544, NS-018, NSC42834, NVP-BSK805, Oclacitinib, Pacritinib, Peficitinib, Pyridone 6, R348, RGB-286638, Ruxolitinib, Ruxolitinib-S, SAR-20347, SB1317, Solcitinib, TG101209, TG101348, Tofacitinib(3R,4S), Tofacitinib(3S,4R), Tofacitinib(3S,4S), Tofacitinib, TYK2-IN-2, Upadacitinib, WHI-P154, WHI-P97, WP1066, XL019, ZM39923, and pharmaceutically acceptable salts thereof. In an embodiment, the JAK inhibitor is selected from the group consisting of Baricitinib phosphate, CYT387 Mesylate, CYT387 sulfate salt, NS-018 hydrochloride, NS-018 maleate, NVP-BSK805 dihydrochloride, Oclacitinib maleate, Ruxolitinib phosphate, Ruxolitinib sulfate, Tofacitinib citrate, and ZM39923 hydrochloride. In an embodiment, the JAK inhibitor is administered orally.

In an embodiment, the MDM2 inhibitor is administered before administration of the JAK inhibitor. In an embodiment, the MDM2 inhibitor is administered after administration of the JAK inhibitor. In an embodiment, the MDM2 inhibitor is administered concurrently with administration of the JAK inhibitor.

In an embodiment, the therapeutically effective amount of the MDM2 inhibitor is 100 mg.

In an embodiment, the MPN in the human subject has a JAK2V617F mutation. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

FIG. 1 illustrates the effect of Navtemadilin (compound of Formula (I)) and Ruxolitinib on p21 expression ex vivo CD34+ myeloid cells from patients suffering from myelofibrosis. Abbreviations: DMSO, dimethylsulfoxide; pM, micromolar; MF, myelofibrosis; QD, once a day; RUX, ruxolitinib.

FIG. 2A is a graph depicting cytotoxicity of navtemadlin combined with ruxolitinib in UKE-1 cells. FIG. 2B is a graph depicting synergy of navtemadlin and ruxolitinib to drive apoptosis in UKE-1 cells, a JAK2 V617F cell line. Abbreviations: Nvtm, navtemadlin; Rux, ruxolitinib.

FIG. 3 illustrates the effect of navtemadlin and ruxolitinib on apoptosis and p21 protein expression in myelofibrosis patient-derived progenitor cells. Abbreviations: MFI, median fluorescent intensity; NVTM, navtemadlin; Rux, ruxolitinib.

FIG. 4 illustrates the effect of navtemadlin and ruxolitinib on MCL-1 protein expression in myelofibrosis patient-derived progenitor cells. Abbreviations: MFI, median fluorescent intensity; NVTM, navtemadlin; Rux, ruxolitinib.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the invention are shown and described herein, such embodiments are provided by way of example only and are not intended to otherwise limit the scope of the invention.

Various alternatives to the described embodiments of the invention may be employed in practicing the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

The terms "administered in combination with" and "co-administration" as used herein, encompass administration of two or more active pharmaceutical ingredients to a subject so that both agents and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more agents are present. The term "combination" or "pharmaceutical combination" is defined herein to refer to either a fixed combination in one dosage unit form, a non-fixed combination or a kit of parts for the combined administration where the MDM2 and JAK inhibitors may be administered together, independently at the same time or separately within time intervals, which preferably allows that the combination partners show a cooperative, e.g. synergistic effect. Thus, the single compounds of the pharmaceutical combination of the present disclosure could be administered simultaneously or sequentially.

Furthermore, the pharmaceutical combination of the present disclosure may be in the form of a fixed combination or in the form of a non-fixed combination.

The term "effective amount" or "therapeutically effective amount" refers to that amount of an active pharmaceutical ingredient or combination of active pharmaceutical ingredients as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, and other factors which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, (e.g., the reduction of platelet adhesion and/or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

The term "fixed combination" means that the MDM2 and JAK inhibitors, e.g., the single compounds of the combination, are in the form of a single entity or dosage form.

"MPN-BP" refers to blast phase (BP) of the myeloproliferative neoplasms (MPN) described in this disclosure.

The term "non-fixed combination" means that the MDM2 and JAK inhibitors, e.g., the single compounds of the combination, are administered to a patient as separate entities or dosage forms either simultaneously or sequentially with no specific time limits, wherein preferably such administration provides therapeutically effective levels of the two JAK inhibitors in the body of the subject, e.g., a mammal or human in need thereof. "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, and absorption delaying agents. The use of such media and agents for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional media or agent is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingredients can also be incorporated into the described compositions. Unless otherwise specified, or clearly indicated by the text, reference to MDM2 and JAK inhibitors useful in the pharmaceutical combination of the present disclosure includes both the free acid or free base of the compounds, and all pharmaceutically acceptable salts of the compounds.

The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counter ions known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In selected embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts. The term "cocrystal" refers to a molecular complex derived from a number of cocrystal formers known in the art. Unlike a salt, a cocrystal typically does not involve proton transfer between the cocrystal and the drug, and instead involves intermolecular interactions, such as hydrogen bonding, aromatic ring stacking, or dispersive forces, between the cocrystal former and the drug in the crystal structure.

A "therapeutic effect" as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

When ranges are used herein to describe, for example, physical or chemical properties such as molecular weight or chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. Use of the term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes those embodiments such as, for example, an embodiment of any composition of matter, method or process that "consist of" or "consist essentially of" the described features.

The invention encompasses a method of regulating p21 expression in CD34+ myeloid cells using a combination of a MDM2 inhibitor and a JAK inhibitor. p21, also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1, is a cyclin-dependent kinase inhibitor (CKI) that is capable of inhibiting all cyclin/CDK complexes, though it is primarily associated with inhibition of CDK2. p21 represents a major target of p53 activity and thus is associated with linking DNA damage to cell cycle arrest. This protein is encoded by the CDKN1A gene located on chromosome 6 (6p21.2) in humans. MDM2/p53 inhibition axes upregulate p21 expression which functions to put a damaged cell into cell cycle arrest. MDM2 inhibitors must overcome this checkpoint to drive apoptosis. JAK inhibitors (i.e. Ruxolitinib) do not upregulate p21 which is not surprising as it would not be expected that Ruxolitinib could biologically modulate p21. However, the combination treatment with an MDM2 inhibitor and a JAK inhibitor of CD34+ myeloid cells from patients with myelofibrosis results in supression of the p21 levels (i.e. they still do not increase). Thus, the invention is based on the surprising finding that p21 expression would have still been upregulated in the presence of the MDM2 inhibitor, which does not occur when combined with a JAK inhibitor. Because it is no longer necessary to overcome this p21 check point, the apoptotic threshold is much lower and more apoptosis occurs in the presence of the MDM2 inhibitor. This effect enhances the activity of the MDM2 inhibitor, making it more effective in the treatment of MPN. Co-administration of compounds

The present invention relates to pharmaceutical combinations or pharmaceutical compositions that are particularly useful as a medicine. Specifically, the combinations or compositions of the present disclosure can be applied in the treatment of a cancer. In an embodiment, the cancer is a MPN. The present invention also relates to use of pharmaceutical combinations or pharmaceutical compositions of the present disclosure for the preparation of a medicament for the treatment of a cancer, in particular a MPN, and to a method for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination according to the present disclosure, or the pharmaceutical composition according to the present disclosure.

In an embodiment, the MPN is selected from the group consisting of polycythemia vera (PV), myelofibrosis, primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), idiopathic myelofibrosis, systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), and systemic mast cell disease (SMCD).

In an embodiment, the myelofibrosis is selected from the group consisting of primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF).

In an embodiment, the primary myelofibrosis (PMF) is selected from the group consisting of prefibrotic/early stage PMF and overt fibrotic stage PMF.

In an embodiment, the MPN is selected from the group consisting of chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia, chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML), hypereosinophilic syndromes (HES), and myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T).

An embodiment of the invention is a composition, such as a pharmaceutical composition comprising a combination comprising a MDM2 inhibitor in combination with a JAK inhibitor. Another embodiment is a kit containing both components formulated into separate pharmaceutical compositions, which are formulated for co-administration. Another embodiment of the invention is a method of for treating a myeloproliferative neoplasm (MPN), wherein the MPN is selected from the group consisting of polycythemia vera (PV), myelofibrosis, thrombocythemia, idiopathic myelofibrosis, chronic myelogenous leukemia, systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), and systemic mast cell disease (SMCD) in a subject, comprising co-administering to the subject in need thereof a therapeutically effective amount of a combination comprising a MDM2 inhibitor in combination with a JAK inhibitor. The pharmaceutical composition comprising the combination, and the kit, are both for use in treating such disease or condition.

In an embodiment, the MDM2 inhibitor is a compound of Formula (I) or Formula (II).

In an embodiment, the MDM2 inhibitor is selected from the group consisting of a compound of Formula (I), Formula (II), RG7388, Triptolide, HDM201, RG7112, CGM097A, CGM0970B, SJ-172550, SAR405838, MI-773, MX69, YH239-EE, RO8994, Nutlin-3, Nutlin-3a, Nutlin-3b, Serdemetan, NSC59984, CHEMBL2386350, MK-8242, DS-3032, DS-3032B, RO6839921, APG-115, MI-1601, and pharmaceutically acceptable salts thereof.

In an embodiment, the MDM2 inhibitor is selected from the group consisting of a compound of Formula (I), Formula (II), RG7388, HDM201, RG7112, CGM097A, CGM0970B, SAR405838, MK-8242, DS-3032B, RO6839921, APG-115, MI-1601, and pharmaceutically acceptable salts thereof.

In an embodiment, the JAK inhibitor is a JAK1 inhibitor.

In an embodiment, the JAK inhibitor is a JAK2 inhibitor.

In an embodiment, the JAK inhibitor is a JAK3 inhibitor.

In an embodiment, the JAK inhibitor is a selective JAK inhibitor.

In an embodiment, the JAK inhibitor is a pan JAK inhibitor.

In an embodiment, the JAK inhibitor is selected from the group consisting of AC-410, AT9283, AZ960, AZD-1480, Baricitinib, BMS-911543, CEP-33779, Cerdulatinib, CHZ868, CYT387, Decernotinib, ENMD- 2076, fedratinib, Filgotinib, Ganetespib, INCB039110, INCB-047986, Itacitinib, JAK3-IN-1, JANEX-1, LFM- A13, LY2784544, NS-018, NSC42834, NVP-BSK805, Oclacitinib, Pacritinib, Peficitinib, Pyridone 6, R348, RGB-286638, Ruxolitinib, Ruxolitinib-S, SAR-20347, SB1317, Solcitinib, TG101209, TG101348, Tofacitinib(3R,4S), Tofacitinib(3S,4R), Tofacitin ib(3S,4S), Tofacitinib, TYK2-IN-2, Upadacitinib, WHI-P154, WHI-P97, WP1066, XL019, ZM39923, and pharmaceutically acceptable salts thereof.

In an embodiment, the JAK inhibitor is selected from the group consisting of Baricitinib phosphate, CYT387 Mesylate, CYT387 sulfate salt, NS-018 hydrochloride, NS-018 maleate, NVP-BSK805 dihydrochloride, Oclacitinib maleate, Ruxolitinib phosphate, Ruxolitinib sulfate, Tofacitinib citrate, and ZM39923 hydrochloride.

The combination may be administered by any route known in the art. In an exemplary embodiment, the MDM2 inhibitor and JAK inhibitor are independently administered by oral, intravenous, intramuscular, intraperitoneal, subcutaneous or transdermal means. In one embodiment, the MDM2 inhibitor is administered orally.

In an exemplary embodiment, the MDM2 inhibitor is in the form of a pharmaceutically acceptable salt.

In an exemplary embodiment, the MDM2 inhibitor is administered to the subject before administration of the JAK inhibitor.

In an exemplary embodiment, the MDM2 inhibitor is administered to the subject after administration of the JAK inhibitor.

In an exemplary embodiment, the MDM2 inhibitor is administered to the subject concurrently with administration of the JAK inhibitor.

In an embodiment, the disclosure provides a method of for treating a blast phase myeloproliferative neoplasm (MPN-BP) in a subject, comprising co-administering to the subject in need thereof a therapeutically effective amount of a combination comprising a MDM2 inhibitor in combination with a JAK inhibitor. The pharmaceutical composition comprising the combination, and the kit, are both for use in treating such disease or condition. In an embodiment, the MPN-BP is selected from the group consisting of blast phase polycythemia vera (BP-PV), blast phase myelofibrosis, blast phase primary myelofibrosis, blast phase thrombocythemia, blast phase essential thrombocythemia (BP-ET), blast phase idiopathic myelofibrosis, blast phase systemic mastocystosis (BP-SM), blast phase chronic neutrophilic leukemia (BP-CNL), blast phase myelodysplastic syndrome (BP-MDS), and blast phase systemic mast cell disease (BP-SMCD). In an embodiment, the blast phase myelofibrosis is selected from the group consisting of blast phase primary myelofibrosis (BP-PMF), blast phase post-polycythemia vera myelofibrosis (BP-post-PV MF), and blast phase post-essential thrombocythemia myelofibrosis (BP-post- ET MF). In an embodiment, the blast phase primary myelofibrosis (BP-PMF) is selected from the group consisting of blast phase prefibrotic/early stage PMF and blast phase overt fibrotic stage PMF. In an embodiment, the MPN-BP is selected from the group consisting of blast phase chronic neutrophilic leukemia (BP-CNL), blast phase chronic eosinophilic leukemia, blast phase chronic myelomonocytic leukemia (BP-CMML), blast phase atypical chronic myeloid leukemia (BP-aCML), blast phase juvenile myelomonocytic leukemia (BP-JMML), blast phase hypereosinophilic syndromes (BP-HES), and blast phase myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (BP- MDS/MPN-RS-T). In an embodiment, the MDM2 inhibitor is a compound of Formula (I) or Formula (II). In an embodiment, the MDM2 inhibitor is selected from the group consisting of a compound of Formula (I), Formula (II), RG7388, Triptolide, HDM201, RG7112, CGM097A, CGM0970B, SJ-172550, SAR405838, MI-773, MX69, YH239-EE, RO8994, Nutlin-3, Nutlin-3a, Nutlin-3b, Serdemetan, NSC59984, CHEMBL2386350, MK-8242, DS-3032, DS-3032B, RO6839921, APG-115, MI-1601, and pharmaceutically acceptable salts thereof. In an embodiment, the MDM2 inhibitor is selected from the group consisting of a compound of Formula (I), Formula (II), RG7388, HDM201, RG7112, CGM097A, CGM0970B, SAR405838, MK-8242, DS-3032B, RO6839921, APG-115, MI-1601, and pharmaceutically acceptable salts thereof.

In an embodiment, the MPN in the human subject is characterized by a CALR mutation (calreticulin, located on chromosome 19pl3.2), as described in Massie, N. Engl. J. Med. (2013) 25: 2379-2390.

In an embodiment, the MPN in the human subject is characterized by an MPL mutation (myeloproliferative leukemia virus oncogene; located on chromosome lp34), as described in Pikman, Pios Med. (2006) 3(7): e270.

In an embodiment, the MPN in the human subject is characterized by a JAK2V617F mutation. The JAK2V617F mutation is a functional mutation promoting cytokine-independent growth of myeloid cells and accounts for a majority of myeloproliferative neoplasms (MPN), as described in Nakatake, Oncogene (2012) 31, 1323-1333.

In an embodiment, the MPN in the human subject is characterized by having one or more mutations selected from the group consisting of JAK2V617F, MPL, CALR, and combinations thereof.

In an exemplary embodiment, the subject is a mammal, such as a human. MDM2 inhibitors

The compound of Formula (I) has is known as 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-l-((S)- l-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl) acetic acid.

In an embodiment, the MDM2 inhibitor is a compound of Formula (II) known as 4-(2-((3R,5R,6S)-l-((S)-2- (tert-butylsulfonyl)-l-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)-5-(3-chlorophenyl)-3-methyl-2- oxopiperidin-3-yl)acetamido)-2-methoxybenzoic acid.

In an embodiment, the MDM2 inhibitor is RG7388. RG7388 is known as 4-[[(2R,3S,4R,5S)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-(2,2-dimethylpropyl)pyrrolidine-2- carbonyl]amino]-3-methoxybenzoic acid.

In an embodiment, the MDM2 inhibitor is triptolide. Triptolide is known as (5bS,6aS,7aS,8R,8aR,9aS,9bS,10aS,10bS)-8-hydroxy-8a-isopropyl-10b-methyl- 2,5,5b,6,6a,8,8a,9a,9b,10b-decahydrotris(oxireno) [2',3':4b,5;2",3":6,7;2"',3"':8a,9] phenanthro[l,2- c]furan-3(lH)-one.

In an embodiment, the MDM2 inhibitor is Nutlin-3a. Nutlin-3a is known as 4-[(4S,5R)-4,5-bis(4- chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-l-carbonyl]piperazin-2-one.

In an embodiment, the MDM2 inhibitor is HDM201. HDM201 is known as (4S)-5-(5-chloro-l-methyl-2- oxopyridin-3-yl)-4-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-3-propan-2-yl-4H-pyrrolo[3,4- d]imidazol-6-one.

In an embodiment, the MDM2 inhibitor is RG7112. RG7112 is known as [(4S,5R)-2-(4-tert-butyl-2- ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethylimidazol-l-yl]-[4-(3-methylsulfonylpropyl)piperazin- l-yl]methanone.

In an embodiment, the MDM2 inhibitor is CGM097A. CGM097A is known as (lS)-l-(4-chlorophenyl)-6- methoxy-2-[4-[methyl-[[4-(4-methyl-3-oxopiperazin-l-yl)cyclohexyl]methyl]amino]phenyl]-7-propan-2- yloxy-l,4-dihydroisoquinolin-3-one.

In an embodiment, the MDM2 inhibitor is nutlin-3. Nutlin-3 is known as 4-[4,5-bis(4-chlorophenyl)-2-(4- methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-l-carbonyl]piperazin-2-one. In an embodiment, the MDM2 inhibitor is SJ-172550. SJ-172550 is known as methyl 2-[2-chloro-6- ethoxy-4-[(3-methyl-5-oxo-l-phenylpyrazol-4-ylidene)methyl]phenoxy]acetate.

In an embodiment, the MDM2 inhibitor is SAR405838. SAR405838 is known as (2'R,3R,3'S,5'S)-6-chloro- 3'-(3-chloro-2-fluorophenyl)-5'-(2,2-dimethylpropyl)-N-(4-hydroxycyclohexyl)-2-oxospiro[lH-indole-3,4'- pyrrolidine]-2'-carboxamide.

In an embodiment, the MDM2 inhibitor is MI-773. MI-773 is known as (2'R,3S,3'S,5'R)-6-chloro-3'-(3- chloro-2-fluorophenyl)-5'-(2,2-dimethylpropyl)-N-(4-hydroxycyclohexyl)-2-oxospiro[lH-indole-3,4'- pyrrolidine]-2'-carboxamide.

In an embodiment, the MDM2 inhibitor is MX69. MX69 is known as 4-[8-[(3,4- dimethylphenyl)sulfamoyl]-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-4-yl] benzoic acid.

In an embodiment, the MDM2 inhibitor is YH239-EE. YH239-EE is known as ethyl 3-[2-(tert-butylamino)-

1-[(4-chlorophenyl)methyl-formylamino]-2-oxoethyl]-6-chloro-lH-indole-2-carboxylate.

In an embodiment, the MDM2 inhibitor is RO8994. RO8994 is known as (2'R,3R,3'S,5'S)-N-(4- carbamoyl-2-methoxyphenyl)-6-chloro-3'-(3-chloro-2-fluorophenyl)-5'-(2,2-dimethylpropyl)-2- oxospiro[lH-indole-3,4'-pyrrolidine]-2'-carboxamide.

In an embodiment, the MDM2 inhibitor is nutlin-3b. Nutlin-3b is known as 4-[(4R,5S)-4,5-bis(4- chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-l-carbonyl]piperazin-2-one.

In an embodiment, the MDM2 inhibitor is Serdemetan. Serdemetan is known as l-N-[2-(lH-indol-3- yl)ethyl]-4-N-pyridin-4-ylbenzene-l,4-diamine.

In an embodiment, the MDM2 inhibitor is NSC59984. NSC59984 is known as (E)-l-(4-methylpiperazin-l- yl)-3-(5-nitrofuran-2-yl)prop-2-en-l-one.

In an embodiment, the MDM2 inhibitor is CHEMBL2386350. CHEMBL2386350 is known as 2-[4-[(4S,5R)-

2-(4-tert-butyl-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethylimidazole-l-carbonyl]piperazin-l- yl]-l-morpholin-4-ylethanone.

In an embodiment, the MDM2 inhibitor is CGM0970B. CGM0970B is known as (lR)-l-(4-chlorophenyl)- 6-methoxy-2-[4-[methyl-[[4-(4-methyl-3-oxopiperazin-l-yl)cyclohexyl]methyl]amino]phenyl]-7-propan- 2-yloxy-l,4-dihydroisoquinolin-3-one. In an embodiment, the MDM2 inhibitor is MK-8242. MK-8242 is known as 4-amino-l-[(2R,3S,4S,5R)-3,4- dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one.

In an embodiment, the MDM2 inhibitor is DS-3032. DS-3032 is known as (3'R,4'S,5'R)-N-((3R,6S)-6- carbamoyltetrahydro-2H-pyran-3-yl)-6"-chloro-4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2"- oxodispiro[cyclohexane-l,2'-pyrrolidine-3',3"-indoline]-5'-carboxamide.

In an embodiment, the MDM2 inhibitor is DS-3032B. DS-3032B is known as (3'R,4'S,5'R)-N-((3R,6S)-6- carbamoyltetrahydro-2H-pyran-3-yl)-6"-chloro-4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2"- oxodispiro[cyclohexane-l,2'-pyrrolidine-3',3"-indoline]-5'-carboxamide 4-methylbenzenesulfonate.

In an embodiment, the MDM2 inhibitor is APG-115. APG-115 is known as 4-((3'R,4'S,5'R)-6"-Chloro-4'- (3-chloro-2-fluorophenyl)-l'-ethyl-2"-oxodispiro[cyclohexane-l,2'-pyrrolidine-3',3"-indoline]-5'- carboxamido)bicyclo[2.2.2]octane-l-carboxylic acid.

In an embodiment, the MDM2 inhibitor is APG-115. APG-115 is known as 4-((3'R,4'S,5'R)-6"-chloro-4'- (3-chloro-2-fluorophenyl)-2"-oxodispiro[cyclohexane-l,2'-pyrrolidine-3',3"-indoline]-5'- carboxamido)benzoic acid.

JAK Inhibitors

In an embodiment, the JAK inhibitor is Ruxolitinib (available from Incyte Corp, and Novartis AG).

Ruxolitinib is known as (R)-3-(4-(7H-pyrrolo[2,3-c/]pyrimidin-4-yl)-lH-pyrazol-l-yl)-3- cyclopentylpropanenitrile.

In an embodiment, the JAK inhibitor is Ruxolitinib phosphate (available from Incyte Corp, and Novartis AG). In an embodiment, the JAK inhibitor is the phosphate salt of (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin- 4-yl)-lH-pyrazol-l-yl)-3-cyclopentylpropanenitrile.

In an embodiment, the JAK inhibitor is Baricitinib (available from Incyte Corp, and Eli Lilly & Co.).

Baricitinib is known as 2-(3-(4-(7/-/-pyrrolo[2,3-d] pyrimidin-4-yl)-l/-/-pyrazol-l-yl)-l- (ethylsulfonyl)azetidin-3-yl)acetonitrile. In an embodiment, the JAK inhibitor is Momelotinib (Gilead Sciences). Momelotinib is also known as CYT-387. Momelotinib is known as /V-(cyanomethyl)-4-(2-((4-morpholinophenyl)amino)pyrimidin-4- yl)benzamide.

In an embodiment, the JAK inhibitor is Ganetespib. Ganetespib is known as 5-(2,4-dihydroxy-5- isopropylphenyl)-4-(l-methyl-lH-indol-5-yl)-2,4-dihydro-3H-l,2,4-triazol-3-one.

In an embodiment, the JAK inhibitor is NS-O18. NS-018 is known as (S)-/V²-(l-(4-fluorophenyl)ethyl)-6-(l- methyl-lH-pyrazol-4-yl)-A/⁴-(pyrazin-2-yl)pyrimidine-2,4-diamine.

In an embodiment, the JAK inhibitor is BMS-911543. BMS-911543 is known as A/,/\/-dicyclopropyl-4- ((l,5-dimethyl-l/-/-pyrazol-3-yl)amino)-6-ethyl-l-methyl-l,6-dihydroimidazo[4,5-d]pyrrolo[2,3- b]pyridine-7-carboxamide.

In an embodiment, the JAK inhibitor is Gandotinib. Gandotinib is known as 3-(4-chloro-2-fluorobenzyl)- 2-methyl-A/-(5-methyl-lH-pyrazol-3-yl)-8-(morpholinomethyl)imidazo[l,2-h]pyridazin-6-amine.

In an embodiment, the JAK inhibitor is ENMD-2076. ENMD-2076 is known as (Ej-/V-(5-methyl-lH- pyrazol-3-yl)-6-(4-methylpiperazin-l-yl)-2-styrylpyrimidin-4-amine.

In an embodiment, the JAK inhibitor is AT-9283. AT-9283 is known as l-cyclopropyl-3-(3-(5-

In an embodiment, the JAK inhibitor is Pacritinib. Pacritinib is known as ll-(2-pyrrolidin-l-yl-ethoxy)-

14,19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).l(8,12)]heptacosa- l(25),2(26),3,5,8,10,12(27),16,21,23-decaene.

In an embodiment, the JAK inhibitor is AC -410 (available from Ambit Biosciences). AC -410 is known as (S)-(4-fluorophenyl)(4-((5-methyl-lH-pyrazol-3-yl)amino)quinazolin-2-yl)methanol.

In an embodiment, the JAK inhibitor is AZD-1480. AZD-1480 is known as (S)-5-chloro-N²-(l-(5- fluoropyrimidin-2-yl)ethyl)-N⁴-(5-methyl-lH-pyrazol-3-yl)pyrimidine-2,4-diamine.

In an embodiment, the JAK inhibitor is CYT387. CYT387 is known as N-(cyanomethyl)-4-(2-(4- morpholinophenylamino)pyrimidin-4-yl)benzamide.

In an embodiment, the JAK inhibitor is TYK2-IN-2. TYK2-IN-2 is known as 6-((3,5-dimethylphenyl)amino)- 8-(methylamino)imidazo[l,2-b]pyridazine-3-carboxamide. In an embodiment, the JAK inhibitor is SAR-20347. SAR-20347 is known as 2-(2-chloro-6-fluorophenyl)- 5-[4-(morpholine-4-carbonyl)anilino]-l,3-oxazole-4-carboxamide.

In an embodiment, the JAK inhibitor is Upadacitinib (ABT-494). Upadacitinib is known as (3S,4R)-3-ethyl- 4-(3H-imidazo[l,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-l-carboxamide.

In an embodiment, the JAK inhibitor is WP1066. WP1066 is known as (E)-3-(6-bromopyridin-2-yl)-2- cyano-N-[(lS)-l-phenylethyl]prop-2-enamide.

In an embodiment, the JAK inhibitor is GLPG0634 (Filgotinib). GLPG0634 is known as N-[5-[4-[(l,l- dioxo-l,4-thiazinan-4-yl)methyl]phenyl]-[l,2,4]triazolo[l,5-a]pyridin-2-yl]cyclopropanecarboxamide.

In an embodiment, the JAK inhibitor is TG101348 (Fedratinib; SAR 302503). TG101348 is known as N- tert-butyl-3-[[5-methyl-2-[4-(2-pyrrolidin-l-ylethoxy)anilino]pyrimidin-4-yl]amino]benzenesulfonamide.

In an embodiment, the JAK inhibitor is Cerdulatinib (PRT062070; PRT2070). Cerdulatinib is known as 4- (cyclopropylamino)-2-[4-(4-ethylsulfonylpiperazin-l-yl)anilino]pyrimidine-5-carboxamide.

In an embodiment, the JAK inhibitor is Tofacitinib. Tofacitinib is known as 3-[(3R,4R)-4-methyl-3- [methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-l-yl]-3-oxopropanenitrile.

In an embodiment, the JAK inhibitor is Itacitinib. Itacitinib is known as 2-[l-[l-[3-fluoro-2- (trifluoromethyl)pyridine-4-carbonyl]piperidin-4-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-l- yl]azetidin-3-yl]acetonitrile.

In an embodiment, the JAK inhibitor is Decernotinib. Decernotinib is known as (2R)-2-methyl-2-[[2-(lH- pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl]amino]-N-(2,2,2-trifluoroethyl)butanamide.

In an embodiment, the JAK inhibitor is CHZ868. CHZ868 is known as N-[4-[2-(2,4-difluoroanilino)-l,4- dimethylbenzimidazol-5-yl]oxypyridin-2-yl]acetamide.

In an embodiment, the JAK inhibitor is SB1317. SB1317 is known as (E)-6-methyl-12-oxa-3,6-diaza- 2(4,2)-pyrimidina-l,4(l,3)-dibenzenacyclododecaphan-8-ene.

In an embodiment, the JAK inhibitor is Solcitinib. Solcitinib is known as N-[5-[4-(3,3-dimethylazetidine-l- carbonyl)phenyl]-[l,2,4]triazolo[l,5-a]pyridin-2-yl]cyclopropanecarboxamide. In an embodiment, the JAK inhibitor is Peficitinib. Peficitinib is known as 4-[[(lR,3S)-5-hydroxy-2- adamantyl]amino]-lH-pyrrolo[2,3-b]pyridine-5-carboxamide.

In an embodiment, the JAK inhibitor is CEP-33779. CEP-33779 is known as N-[3-(4-methylpiperazin-l- yl)phenyl]-8-(4-methylsulfonylphenyl)-[l,2,4]triazolo[l,5-a]pyridin-2-amine.

In an embodiment, the JAK inhibitor is Pyridone 6. Pyridone 6 is known as 2-(tert-butyl)-9-fluoro-3H- benzo[h]imidazo[4,5-f]isoquinolin-7-ol.

In an embodiment, the JAK inhibitor is LFM-A13. LFM-A13 is known as (Z)-2-cyano-N-(2,5- dibromophenyl)-3-hydroxybut-2-enamide.

In an embodiment, the JAK inhibitor is BMS-911543. BMS-911543 is known as (Z)-N,N-dicyclopropyl-4- ((l,5-dimethyl-l,2-dihydro-3H-pyrazol-3-ylidene)amino)-6-ethyl-l-methyl-l,6-dihydroimidazo[4,5- d]pyrrolo[2,3-b]pyridine-7-carboxamide.

In an embodiment, the JAK inhibitor is NS-018. NS-018 is known as 6-N-[( lS)-l-(4-f luorophenyl )ethyl]- 4-(l-methylpyrazol-4-yl)-2-N-pyrazin-2-ylpyridine-2,6-diamine.

In an embodiment, the JAK inhibitor is JANEX-1. JAN EX-1 is known as 4-[(6,7-dimethoxyquinazolin-4- yl)amino]phenol.

In an embodiment, the JAK inhibitor is TG101209. TG101209 is known as N-tert-butyl-3-[[5-methyl-2- [4-(4-methylpiperazin-l-yl)anilino]pyrimidin-4-yl]amino]benzenesulfonamide.

In an embodiment, the JAK inhibitor is WHI-P154. WHI-P154 is known as 2-bromo-4-[(6,7- dimethoxyquinazolin-4-yl)amino] phenol.

In an embodiment, the JAK inhibitor is NVP-BSK805. NVP-BSK805 is known as 4-[[2,6-dif luoro-4-[3-( 1- piperidin-4-ylpyrazol-4-yl)quinoxalin-5-yl]phenyl]methyl]morpholine.

In an embodiment, the JAK inhibitor is ZM39923. ZM39923 is known as 3-[benzyl(propan-2-yl)amino]-l- naphthalen-2-ylpropan-l-one.

In an embodiment, the JAK inhibitor is Ruxolitinib-S. Ruxolitinib-S is known as (3S)-3-cyclopentyl-3-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-l-yl]propanenitrile. In an embodiment, the JAK inhibitor is XL019. XL019 is known as (2S)-N-[4-[2-(4-morpholin-4- ylanilino)pyrimidin-4-yl]phenyl]pyrrolidine-2-carboxamide.

In an embodiment, the JAK inhibitor is AZ960. AZ960 is known as 5-fluoro-2-[[(lS)-l-(4- fluorophenyl)ethyl]amino]-6-[(5-methyl-lH-pyrazol-3-yl)amino]pyridine-3-carbonitrile.

In an embodiment, the JAK inhibitor is JAK3-IN-1. JAK3-IN-1 is known as N-[3-[[[5-chloro-2-[2-methoxy- 4-(4-methylpiperazin-l-yl)anilino]pyrimidin-4-yl]amino]methyl]phenyl]prop-2-enamide.

In an embodiment, the JAK inhibitor is WHI-P97. WHI-P97 is known as 2,6-dibromo-4-[(6,7- dimethoxyquinazolin-4-yl)amino] phenol.

In an embodiment, the JAK inhibitor is RGB-286638. RGB-286638 is known as l-[3-[4-[[4-(2- methoxyethyl)piperazin-l-yl]methyl]phenyl]-4-oxo-lH-indeno[l,2-c]pyrazol-5-yl]-3-morpholin-4- ylurea;dihydrochloride.

In an embodiment, the JAK inhibitor is Tofacitinib(3R,4S). Tofacitinib(3R,4S) is known as 3-[(3R,4S)-4- methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-l-yl]-3-oxopropanenitrile.

In an embodiment, the JAK inhibitor is NSC42834. NSC42834 is known as 2-methyl-l-phenyl-4-pyridin-2- yl-2-(2-pyridin-2-ylethyl)butan-l-one.

In an embodiment, the JAK inhibitor is PF-06651600. PF-06651600 is known as benzyl 2- (hydroxymethyl)-5-[(2-methylpropan-2-yl)oxycarbonylamino]piperidine-l-carboxylate.

In an embodiment, the JAK inhibitor is Tofacitinib(3S,4S). Tofacitinib(3S,4S) is known as 3-[(3S,4S)-4- methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-l-yl]-3-oxopropanenitrile.

In an embodiment, the JAK inhibitor is Tofacitinib(3S,4R). Tofacitinib(3S,4R) is known as 3-[(3S,4R)-4- methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-l-yl]-3-oxopropanenitrile.

In an embodiment, the JAK inhibitor is AEG3482. AEG3482 is known as 6-phenylimidazo[2,l- b][l,3,4]thiadiazole-2-sulfonamide.

In an embodiment, the JAK inhibitor is Lestaurtinib (CEP-701). Lestaurtinib is known as (5R,7S,8S)-7- hydroxy-7-(hydroxymethyl)-8-methyl-5,6,7,8,13,14-hexahydro-15H-16-oxa-4b,8a,14-triaza-5,8- methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacen-15-one. In an embodiment, the JAK inhibitor is Oclacitinib. Oclacitinib is known as N-methyl-l-[4-[methyl(7H- pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclohexyl]methanesulfonamide.

In an embodiment, the JAK inhibitor is (F)-4-(2-(pyrrolidin-l-yl)ethoxy)-6,ll-dioxa-3-aza-2(4,2)- pyrimidina-l(2,5)-furana-4(l,3)-benzenacyclododecaphan-8-ene.

In an embodiment, the JAK inhibitor is (9E)-15-(2-(pyrrolidin-l-yl)ethoxy)-7,12,25-trioxa-19,21,24-triaza- tetracyclo[18.3.1.1(2,5).l(14,18)]hexacosa-l(24),2,4,9,14(26),15,17,20,22-nonaene.

In an embodiment, the JAK inhibitor is (R)-(4-fluorophenyl)(4-((5-methyl-lH-pyrazol-3- yl)amino)quinazolin-2-yl)methanol, which is also known in the art to be active as a JAK inhibitor. In an embodiment, the JAK inhibitor is racemic (4-fluorophenyl)(4-((5-methyl-l/-/-pyrazol-3- yl)amino)quinazolin-2-yl)methanol, which is also known in the art to be active as a JAK inhibitor.

In an embodiment, the JAK inhibitor is (S)-5-fluoro-2-((l-(4-fluorophenyl)ethyl)amino)-6-((5-methyl-lH- pyrazol-3-yl)amino)nicotinonitrile.

In an embodiment, the JAK inhibitor is ((/?)-7-(2-aminopyrimidin-5-yl)-l-((l-cyclopropyl-2,2,2- trifluoroethyl)amino)-5H-pyrido[4,3-b]indole-4-carboxamide, which is also named 7-(2-aminopyrimidin- 5-yl)-l-{[(lR)-l-cyclopropyl-2,2,2-trifluoroethyl]amino}-5H-pyrido[4,3-b]indole-4-carboxamide.

Pharmaceutical Compositions

In some embodiments, the invention provides pharmaceutical compositions comprising a combination comprising a MDM2 inhibitor and a JAK inhibitor. In an embodiment, the MPN is selected from the group consisting of polycythemia vera (PV), myelofibrosis, primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), idiopathic myelofibrosis, systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), and systemic mast cell disease (SMCD). In an embodiment, the myelofibrosis is selected from the group consisting of primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF). In an embodiment, the primary myelofibrosis (PMF) is selected from the group consisting of prefibrotic/early stage PMF and overt fibrotic stage PMF. In an embodiment, the MPN is selected from the group consisting of chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia, chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML), hypereosinophilic syndromes (HES), and myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN- RS-T).

In an embodiment, the MDM2 inhibitor is a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof.

In an embodiment, thrombocythemia is essential thrombocythemia (ET).

In an embodiment, myelofibrosis is selected from primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF).

Polycythemia Vera In some embodiments, the invention provides pharmaceutical compositions comprising a combination of the compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof and a JAK inhibitor; wherein the JAK inhibitor is selected from the group consisting of AC -410, AT9283, AZ960, AZD-1480, Baricitinib, B MS-911543, CEP-33779, Cerdulatinib, CHZ868, CYT387, Decernotinib, ENMD- 2076, fedratinib, Filgotinib, Ganetespib, INCB039110, INCB-047986, Itacitinib, JAK3-IN-1, JANEX-1, LFM- A13, LY2784544, NS-018, NSC42834, NVP-BSK805, Oclacitinib, Pacritinib, Peficitinib, Pyridone 6, R348, RGB-286638, Ruxolitinib, Ruxolitinib-S, SAR-20347, SB1317, Solcitinib, TG101209, TG101348, Tofacitinib(3R,4S), Tofacitinib(3S,4R), Tofacitinib(3S,4S), Tofacitinib, TYK2-IN-2, Upadacitinib, WHI-P154, WHI-P97, WP1066, XL019, ZM39923, and pharmaceutically acceptable salts thereof.

Essential Thrombocythemia (ET)

In some embodiments, the invention provides pharmaceutical compositions comprising a combination of the compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof and a JAK inhibitor; wherein the JAK inhibitor is selected from the group consisting of AC -410, AT9283, AZ960, AZD-1480, Baricitinib, BMS-911543, CEP-33779, Cerdulatinib, CHZ868, CYT387, Decernotinib, ENMD- 2076, Filgotinib, Ganetespib, INCB039110, INCB-047986, Itacitinib, JAK3-IN-1, JANEX-1, LFM-A13, LY2784544, NS-018, NSC42834, NVP-BSK805, Oclacitinib, Pacritinib, Peficitinib, Pyridone 6, R348, RGB- 286638, Ruxolitinib, Ruxolitinib-S, SAR-20347, SB1317, Solcitinib, TG101209, TG101348, Tofacitinib(3R,4S), Tofacitinib(3S,4R), Tofacitinib(3S,4S), Tofacitinib, TYK2-IN-2, Upadacitinib, WHI-P154, WHI-P97, WP1066, XL019, ZM39923, and pharmaceutically acceptable salts thereof.

Myelofibrosis

In some embodiments, the invention provides pharmaceutical compositions comprising a combination of the compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof and a JAK inhibitor; wherein the JAK inhibitor is selected from the group consisting of AC -410, AT9283, AZ960, AZD-1480, Baricitinib, BMS-911543, CEP-33779, Cerdulatinib, CHZ868, CYT387, Decernotinib, ENMD- 2076, Filgotinib, Ganetespib, INCB039110, INCB-047986, Itacitinib, JAK3-IN-1, JANEX-1, LFM-A13, LY2784544, NS-018, NSC42834, NVP-BSK805, Oclacitinib, Pacritinib, Peficitinib, Pyridone 6, R348, RGB- 286638, Ruxolitinib, Ruxolitinib-S, SAR-20347, SB1317, Solcitinib, TG101209, TG101348, Tofacitinib(3R,4S), Tofacitinib(3S,4R), Tofacitinib(3S,4S), Tofacitinib, TYK2-IN-2, Upadacitinib, WHI-P154, WHI-P97, WP1066, XL019, ZM39923, and pharmaceutically acceptable salts thereof. In an embodiment, myelofibrosis is selected from primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF).

The pharmaceutical compositions are typically formulated to provide a therapeutically effective amount of a MDM2 inhibitor and a JAK inhibitor. Where desired, the pharmaceutical compositions contain a pharmaceutically acceptable salt and/or coordination complex thereof, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.

In selected embodiments, the concentration of a MDM2 inhibitor and a JAK inhibitor provided in the pharmaceutical compositions of the invention is independently less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In selected embodiments, the concentration of a MDM2 inhibitor and a JAK inhibitor provided in the pharmaceutical compositions of the invention is independently greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v.

In selected embodiments, the concentration of a MDM2 inhibitor and a JAK inhibitor is independently in the range from approximately 0.0001% to approximately 50%, approximately 0.001% to approximately 40%, approximately 0.01% to approximately 30%, approximately 0.02% to approximately 29%, approximately 0.03% to approximately 28%, approximately 0.04% to approximately 27%, approximately 0.05% to approximately 26%, approximately 0.06% to approximately 25%, approximately 0.07% to approximately 24%, approximately 0.08% to approximately 23%, approximately 0.09% to approximately 22%, approximately 0.1% to approximately 21%, approximately 0.2% to approximately 20%, approximately 0.3% to approximately 19%, approximately 0.4% to approximately 18%, approximately 0.5% to approximately 17%, approximately 0.6% to approximately 16%, approximately 0.7% to approximately 15%, approximately 0.8% to approximately 14%, approximately 0.9% to approximately 12% or approximately 1% to approximately 10% w/w, w/v or v/v.

In selected embodiments, the concentration of a MDM2 inhibitor and a JAK inhibitor is independently in the range from approximately 0.001% to approximately 10%, approximately 0.01% to approximately 5%, approximately 0.02% to approximately 4.5%, approximately 0.03% to approximately 4%, approximately 0.04% to approximately 3.5%, approximately 0.05% to approximately 3%, approximately 0.06% to approximately 2.5%, approximately 0.07% to approximately 2%, approximately 0.08% to approximately 1.5%, approximately 0.09% to approximately 1%, approximately 0.1% to approximately 0.9% w/w, w/v or v/v.

In selected embodiments, the amount of a MDM2 inhibitor and a JAK inhibitor is independently equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In selected embodiments, the amount of a MDM2 inhibitor and a JAK inhibitor is independently more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

A MDM2 inhibitor is effective over a wide dosage range. For example, in the treatment of adult humans, dosages independently ranging from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.

Pharmaceutical Compositions for Oral Administration

In selected embodiments, the invention provides a pharmaceutical composition for oral administration a combination comprising a MDM2 inhibitor and a JAK inhibitor, and a pharmaceutical excipient suitable for oral administration.

In selected embodiments, the invention provides a solid pharmaceutical composition for oral administration containing: (i) a combination comprising an effective amount of a MDM2 inhibitor and a JAK inhibitor, in combination with (ii) a pharmaceutical excipient suitable for oral administration. In selected embodiments, the composition further contains (iii) an effective amount of at least one additional active ingredient.

In selected embodiments, the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption. Pharmaceutical compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such dosage forms can be prepared by any of the methods, but all methods include the step of bringing the active ingredient(s) into association with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The invention further encompasses anhydrous pharmaceutical compositions and dosage forms since water can facilitate the degradation of some compounds. For example, water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms of the invention which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.

A combination of a MDM2 inhibitor with a JAK inhibitor can be further combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and combinations thereof.

Examples of suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and combinations thereof.

Disintegrants may be used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which disintegrate in the bottle. Too little may be insufficient for disintegration to occur, thus altering the rate and extent of release of the active ingredients from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegrant, or about 1 to about 5 weight percent of disintegrant, may be used in the pharmaceutical composition. Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or combinations thereof.

Lubricants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, or combinations thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or combinations thereof. A lubricant can optionally be added, in an amount of less than about 1 weight percent of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Surfactants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and combinations thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.

A suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10. An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of non-ionic amphiphilic compounds is the hydrophilic-lipophilic balance ("HLB" value). Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions. Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable. Similarly, lipophilic (/.e., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10. However, HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrial, pharmaceutical and cosmetic emulsions.

Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and combinations thereof. Within the aforementioned group, ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and combinations thereof.

Ionic surfactants may be the ionized forms of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP-phosphatidylethanolamine, lactylic esters of fatty acids, stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides, mono/diacetylated tartaric acid esters of mono/diglycerides, citric acid esters of mono/diglycerides, cholylsarcosine, caproate, caprylate, caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate, lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, and salts and combinations thereof.

Hydrophilic non-ionic surfactants may include, but not limited to, alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and analogues thereof; polyoxyethylated vitamins and derivatives thereof; polyoxyethylenepolyoxypropylene block copolymers; and combinations thereof; polyethylene glycol sorbitan fatty acid esters and hydrophilic transesterification products of a polyol with at least one member of the group consisting of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol may be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide.

Other hydrophilic-non-ionic surfactants include, without limitation, PEG-10 laurate, PEG-12 laurate, PEG- 20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32 distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6 caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides, polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30 soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG-100 succinate, PEG-24 cholesterol, polyglyceryl-10 oleate, Tween 40, Tween 60, sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG 15- 100 octyl phenol series, and poloxamers.

Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil-soluble vitamins/vitamin derivatives; and combinations thereof. Within this group, preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and combinations thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides.

In an embodiment, the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present invention and to minimize precipitation of the compound of the present invention. This can be especially important for compositions for non-oral use, such as for compositions for injection. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

Examples of suitable solubilizers include, but are not limited to, the following: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetra hydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG; amides and other nitrogen-containing compounds such as 2-pyrrolidone, 2- piperidone, E-caprolactam, W-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, /V-alkylpiperidone, N- alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esters such as ethyl propionate, tributylcitrate, acetyl triethylcitrate, acetyl tributyl citrate, triethylcitrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, epsilon- caprolactone and isomers thereof, 6-valerolactone and isomers thereof, (3-butyrolactone and isomers thereof; and other solubilizers known in the art, such as dimethyl acetamide, dimethyl isosorbide, N- methyl pyrrolidones, monooctanoin, diethylene glycol monoethyl ether, and water.

Mixtures of solubilizers may also be used. Examples include, but not limited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol.

The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art. In some circumstances, it may be advantageous to include amounts of solubilizers far in excess of bioacceptable amounts, for example to maximize the concentration of the drug, with excess solubilizer removed prior to providing the composition to a patient using conventional techniques, such as distillation or evaporation. Thus, if present, the solubilizer can be in a weight ratio of 10%, 25%, 50%, 100%, or up to about 200% by weight, based on the combined weight of the drug, and other excipients. If desired, very small amounts of solubilizer may also be used, such as 5%, 2%, 1% or even less.

Typically, the solubilizer may be present in an amount of about 1% to about 100%, more typically about 5% to about 25% by weight.

The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and combinations thereof.

In addition, an acid or a base may be incorporated into the composition to facilitate processing, to enhance stability, or for other reasons. Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable are bases that are salts of a pharmaceutically acceptable acid, such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, and the like. Salts of polyprotic acids, such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used. When the base is a salt, the cation can be any convenient and pharmaceutically acceptable cation, such as ammonium, alkali metals and alkaline earth metals. Examples may include, but are not limited to, sodium, potassium, lithium, magnesium, calcium and ammonium.

Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, and the like. Examples of suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, parabromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid and uric acid. Pharmaceutical Compositions for Injection

In selected embodiments, the invention provides a pharmaceutical composition for injection comprising a combination comprising a MDM2 inhibitor and a JAK inhibitor, and a pharmaceutical excipient suitable for injection.

The forms in which the compositions of the present invention may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (and suitable combinations thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and thimerosal.

Sterile injectable solutions are prepared by incorporating a MDM2 inhibitor and a JAK inhibitor in the required amounts in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, certain desirable methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Administration of a combination comprising a MDM2 inhibitor and a JAK inhibitor can be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, intra-arterial, subcutaneous, intramuscular, intravascular, or infusion), topical (e.g., transdermal application), via local delivery by catheter or stent. Exemplary parenteral administration forms include solutions or suspensions of active compound in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.

The invention also provides kits. The kits include a MDM2 inhibitor and a JAK inhibitor, either alone or in combination in suitable packaging, and written material that can include instructions for use, discussion of clinical studies and listing of side effects. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. The kit may further contain another active pharmaceutical ingredient. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in selected embodiments, be marketed directly to the consumer. In an embodiment, the invention provides a kit comprising a combination comprising a MDM2 inhibitor and a JAK inhibitor for use in the treatment of a MPN. In an embodiment, the MPN is selected from the group consisting of polycythemia vera (PV), myelofibrosis, primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), idiopathic myelofibrosis, systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), and systemic mast cell disease (SMCD). In an embodiment, the myelofibrosis is selected from the group consisting of primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF). In an embodiment, the primary myelofibrosis (PMF) is selected from the group consisting of prefibrotic/early stage PMF and overt fibrotic stage PMF. In an embodiment, the MPN is selected from the group consisting of chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia, chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML), hypereosinophilic syndromes (HES), and myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN- RS-T). Dosages and Dosing Regimens

The amount of a MDM2 inhibitor and a JAK inhibitor administered will be independently dependent on the human being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compounds and the discretion of the prescribing physician. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, such as about 0.05 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect by dividing such larger doses into several small doses for administration throughout the day.

In some embodiments, a MDM2 inhibitor and JAK inhibitor are independently administered in a single dose. Typically, such administration will be by injection, e.g., intravenous injection, in order to introduce the agents quickly. However, other routes may be used as appropriate. A single dose of a MDM2 inhibitor and a JAK inhibitor may also be used for treatment of an acute condition.

In some embodiments, a MDM2 inhibitor and a JAK inhibitor are independently administered in multiple doses for treating a MPN. In an embodiment, a MDM2 inhibitor and a JAK inhibitor are independently administered in multiple doses orally. In an embodiment, dosing may be once, twice, three times, four times, five times, six times, or more than six times per day. In an embodiment, dosing may be selected from the group consisting of once a day, twice a day, three times a day, four times a day, five times a day, six times a day, once every other day, once weekly, twice weekly, three times weekly, four times weekly, biweekly, and monthly. In some embodiments a MDM2 inhibitor and a JAK inhibitor are independently administered three times a week, including every Monday, Wednesday, and Friday.

Administration of a MDM2 inhibitor and a JAK inhibitor may independently continue as long as necessary. In some embodiments, the MDM2 inhibitor and the JAK inhibitor are independently administered for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 1 , 28, 29, 30, 31 or more days. In some embodiments, the MDM2 inhibitor and the JAK inhibitor are independently administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the MDM2 inhibitor and the JAK inhibitor are independently administered for about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days. In some embodiments, the MDM2 inhibitor and the JAK inhibitor are independently administered chronically on an ongoing basis for the treatment of chronic effects. In another embodiment, the administration of the MDM2 inhibitor and the JAK inhibitor independently continues for less than about 7 days. In yet another embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or one year. In some embodiments, the administration continues for more than about one year, two years, three years, four years, or five years. In some embodiments, continuous dosing is achieved and maintained as long as necessary.

In some embodiments, an effective dosage of the MDM2 inhibitor and the JAK inhibitor is independently in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 10 mg to about 200 mg, about 20 mg to about 150 mg, about 30 mg to about 120 mg, about 10 mg to about 90 mg, about 20 mg to about 80 mg, about 30 mg to about 70 mg, about 40 mg to about 60 mg, about 45 mg to about 55 mg, about 48 mg to about 52 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 202 mg. In some embodiments, an effective dosage of the MDM2 inhibitor and the JAK inhibitor is about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, or about 500 mg. In some embodiments, an effective dosage of a MDM2 inhibitor or a JAK inhibitor is 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, or 500 mg.

In some embodiments, an effective dosage of the MDM2 inhibitor or the JAK inhibitor is independently in the range of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg. In some embodiments, an effective dosage of a MDM2 inhibitor or a JAK inhibitor is about 0.35 mg/kg, about 0.7 mg/kg, about 1 mg/kg, about 1.4 mg/kg, about 1.8 mg/kg, about 2.1 mg/kg, about 2.5 mg/kg, about 2.85 mg/kg, about 3.2 mg/kg, or about 3.6 mg/kg.

In some embodiments, a MDM2 inhibitor or a pharmaceutically acceptable salt thereof is administered at a dosage of 10 to 500 mg BID, including a dosage of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg BID.

In some embodiments, a MDM2 inhibitor or a pharmaceutically acceptable salt thereof is administered at a dosage of 10 to 500 mg QD, including a dosage of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg QD.

An effective amount of the MDM2 inhibitor or the JAK inhibitor may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including buccal, sublingual, and transdermal routes, by intra-arterial injection, intravenously, parenterally, intramuscularly, subcutaneously or orally.

In some embodiments, the MDM2 inhibitor and the JAK inhibitor are independently administered to a subject intermittently, known as intermittent administration. By "intermittent administration", it is meant a period of administration of a therapeutically effective dose of a MDM2 inhibitor and/or the JAK inhibitor, followed by a time period of discontinuance, which is then followed by another administration period and so on. In each administration period, the dosing frequency can be independently select from three times daily, twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly or monthly. In an embodiment, the MDM2 inhibitor is the compound of Formula (I) or Formula (II). In an embodiment, the JAK inhibitor is selected from the group consisting of Ruxolitinib, Ruxolitinib-S, and Fedratinib.

By "period of discontinuance" or "discontinuance period" or "rest period", it is meant to the length of time when discontinuing of the administration of the MDM2 inhibitor and/or the JAK inhibitor. The time period of discontinuance may be longer or shorter than the administration period or the same as the administration period. For example, where the administration period comprises three times daily, twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly or monthly dosing, the discontinuance period may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, one month, two months, three months, four months or more days. During the discontinuance period, JAK inhibitors other than a MDM2 inhibitor and the JAK inhibitor may be administered.

In an embodiment, the MDM2 inhibitor and the JAK inhibitor are independently administered to a human subject in need thereof for treating a myeloproliferative neoplasm (MPN) for a first administration period, then followed by a discontinuance period, then followed by a second administration period, and so on. In an embodiment, the MPN is selected from the group consisting of polycythemia vera (PV), myelofibrosis, primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), idiopathic myelofibrosis, systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), and systemic mast cell disease (SMCD). In an embodiment, the myelofibrosis is selected from the group consisting of primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post-ET MF). In an embodiment, the primary myelofibrosis (PMF) is selected from the group consisting of prefibrotic/early stage PMF and overt fibrotic stage PMF. In an embodiment, the MPN is selected from the group consisting of chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia, chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML), hypereosinophilic syndromes (HES), and myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T). The first administration period, the second administration period, and the discontinuance period are independently selected from the group consisting of more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, one month, two months, three months, four months and more days, in which the MDM2 inhibitor and the JAK inhibitor are independently administered to a subject three times daily, twice daily, daily, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly or monthly. In an embodiment, the first administration period is at same length as the second administration period. In an embodiment, the first administration period is shorter than the second administration period. In an embodiment, the first administration period is longer than the second administration period. In an embodiment, the first administration period and the second administration period are about three weeks, in which the MDM2 inhibitor and the JAK inhibitor are independently administered to a subject daily; and the discontinuance is about two weeks. In an embodiment, the first administration period and the second administration period are about three weeks, in which the MDM2 inhibitor and the JAK inhibitor are independently administered to a subject weekly; and the discontinuance is about two weeks. In an embodiment, the first administration period and the second administration period are about four weeks, in which the MDM2 inhibitor and the JAK inhibitor are independently administered to a subject daily; and the discontinuance is about two weeks. In an embodiment, the first administration period and the second administration period are about four weeks, in which the MDM2 inhibitor and the JAK inhibitor are independently administered to a subject weekly; and the discontinuance is about two weeks. In an embodiment, the MDM2 inhibitor is the compound of Formula (I) or Formula (II). In an embodiment, the JAK inhibitor is selected from the group consisting of Ruxolitinib, Ruxolitinib-S and fedratinib.

In an embodiment, the MDM2 inhibitor is administered to a human intermittently, while the JAK inhibitor is administered to a human non-intermittently. In an embodiment, the JAK inhibitor is administered to a human intermittently, while the MDM2 inhibitor is administered to a human non- intermittently. In an embodiment, both the MDM2 inhibitor and the JAK inhibitor are administered to a human intermittently. In an embodiment, both the MDM2 inhibitor and the JAK inhibitor are administered to a human non-intermittently.

Methods of Treating a Myeloproliferative Neoplasm (MPN)

In an embodiment, the invention encompasses a method of suppressing p21 expression levels in a human suffering from a myeloproliferative neoplasm (MPN) comprising administering to the human a therapeutically effective amount of a MDM2 inhibitor in combination with a JAK inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the administering suppresses p21 expression levels in the human compared to p21 expression levels in MDM2 inhibitor monotherapy. In an embodiment, the invention relates to a method of treating a MPN in a human that comprises the step of administering to said human a therapeutically effective amount of a MDM2 inhibitor and a JAK inhibitor in a dosage independently selected from the group consisting of 15 mg QD, 25 mg QD, 30 mg QD, 50 mg QD, 60 mg QD, 75 mg QD, 100 mg QD, 120 mg QD, 150 mg QD, 175 mg QD, 200 mg QD, 225 mg QD, 240 mg QD, 250 mg QD, 275 mg QD, 300 mg QD, 325 mg QD, 350 mg QD, 360 mg QD, 375 mg QD, 480 mg QD, 15 mg BID, 25 mg BID, 30 mg BID, 50 mg BID, 60 mg BID, 75 mg BID, 100 mg BID, 120 mg BID, 150 mg BID, 175 mg BID, 200 mg BID, 225 mg BID, 240 mg BID, 250 mg BID, 275 mg BID, 300 mg BID, 325 mg BID, 350 mg BID, 360 mg BID, 375 mg BID, and 480 mg BID. In an embodiment, the MPN is selected from the group consisting of polycythemia vera (PV), myelofibrosis, primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), idiopathic myelofibrosis, systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), and systemic mast cell disease (SMCD). In an embodiment, the myelofibrosis is selected from the group consisting of primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and postessential thrombocythemia myelofibrosis (post-ET MF). In an embodiment, the primary myelofibrosis (PMF) is selected from the group consisting of prefibrotic/early stage PMF and overt fibrotic stage PMF. In an embodiment, the MPN is selected from the group consisting of chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia, chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML), hypereosinophilic syndromes (HES), and myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T). In an embodiment, the MDM2 inhibitor is the compound of Formula (I) or Formula (II). In an embodiment, the JAK inhibitor is selected from the group consisting of Ruxolitinib, Ruxolitinib- S and fedratinib.

In an embodiment, the invention relates to a combination of a therapeutically effective amount of a MDM2 inhibitor and a JAK inhibitor for use of treating a MPN in a human, wherein the MDM2 inhibitor and the JAK inhibitor are administered in a dosage independently selected from the group consisting of 15 mg QD, 25 mg QD, 30 mg QD, 50 mg QD, 60 mg QD, 75 mg QD, 100 mg QD, 120 mg QD, 150 mg QD, 175 mg QD, 200 mg QD, 225 mg QD, 240 mg QD, 250 mg QD, 275 mg QD, 300 mg QD, 325 mg QD, 350 mg QD, 360 mg QD, 375 mg QD, 480 mg QD, 15 mg BID, 25 mg BID, 30 mg BID, 50 mg BID, 60 mg BID, 75 mg BID, 100 mg BID, 120 mg BID, 150 mg BID, 175 mg BID, 200 mg BID, 225 mg BID, 240 mg BID, 250 mg BID, 275 mg BID, 300 mg BID, 325 mg BID, 350 mg BID, 360 mg BID, 375 mg BID, and 480 mg BID. In an embodiment, the MPN is selected from the group consisting of polycythemia vera (PV), myelofibrosis, primary myelofibrosis, thrombocythemia, essential thrombocythemia (ET), idiopathic myelofibrosis, systemic mastocystosis (SM), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), and systemic mast cell disease (SMCD). In an embodiment, the myelofibrosis is selected from the group consisting of primary myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF), and postessential thrombocythemia myelofibrosis (post-ET MF). In an embodiment, the primary myelofibrosis (PMF) is selected from the group consisting of prefibrotic/early stage PMF and overt fibrotic stage PMF. In an embodiment, the MPN is selected from the group consisting of chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia, chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML), hypereosinophilic syndromes (HES), and myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T). In an embodiment, the MDM2 inhibitor is the compound of Formula (I) or Formula (II). In an embodiment, Ruxolitinib, Ruxolitinib-S and fedratinib.

The methods described above may be used as first-line cancer therapy, or after treatment with conventional chemotherapic active pharmaceutical ingredients, including cyclophosphamide, fludarabine (FC chemotherapy), and chlorambucil.

The combination of the MDM2 inhibitor and the JAK inhibitor may also be used in combination with radiation therapy, hormone therapy, surgery and immunotherapy, which therapies are well known to those skilled in the art.

EXAMPLES

The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

Example 1: Effects of the combination of the compound of Formula (I) with a JAK inhibitor on CD34+ myeloid cells from patients with myelofibrosis

The procedure of testing the effects of the combination of the compound of Formula (I) with a JAK inhibitor on CD34+ myeloid cells follows that described in Lu, Blood (2012) 120(15); 3098-3105. The following describes the procedure briefly.

Peripheral blood is be obtained from myelofibrosis (MF) patients. The peripheral blood samples are layered onto Ficoll-Hypaque (1.077 g/mL; GE Healthcare) and low-density mononuclear cells are separated via centrifugation. The CD34+ cells are isolated using a human CD34+ cell selection kit (StemCell Technologies) according to the manufacturer's instructions. The purity of the CD34+ cell population is analyzed using a FACSCalibur flow cytometer (BD Biosciences); and is required for at least 85% for all experiments. Fresh normal human bone marrow CD34+ cells from ALLCELLS are utilized as a control.

The effects of the compound of Formula (I) in combination with Ruxolitinib on MF patients can be assessed by the HPC assays, described in Lu, Blood (2012) 3098-3105. In brief, CD34+ cells are cultured in serum free medium (StemCell Technologies) containing 50 ng/mL stem cell factor (SCF), 50 ng/mL thrombopoietin (TPO), 50 ng/mL fms-like tyrosine kinase 3 (Flt-3) ligand, and 50 ng/mL IL-3, and are treated with various doses of the compound of Formula (I) and/or Ruxolitinib for 4 days. After 4 days of treatment, CD34+ cells are assayed in semisolid media as described in Bruno, Blood (2006) 3128-3134, the entirety of which is incorporated by reference. Briefly, 5 x 10² CD34+ cells are plated per dish in duplicate cultures containing 1 mL IMDM with 1.1% methylcellulose and 20% FBS, to which SCF, TPO, Flt-3 ligand, IL-3, and GM-CSF at each 50 ng/mL, and 2 U/mL erythropoietin (EPO) are added. Colonies are enumerated after 14 days of incubation, and individual colonies isolated and genotyped for JAK2V617F.

Genomic DNA is isolated from randomized plucked colonies using the Extract-N-Amp Blood PCR Kits (Sigma-Aldrich). JAK2V617F is detected by using a nested allele-specific PCR as described in Bruno, Blood (2006) 3128-3134. The final PCR products is analyzed on 2.0% agarose gels. A 279-bp product indicates allele-specific JAK2V617F-positive, whereas a 229-bp product indicates JAK2V617F-negative. Colonies are classified as homozygous for JAK2V617F if they contained only the 279-bp band, whereas heterozygous colonies are identified based on the presence of both the 279-bp and 229-bp bands.

Treated cells are collected and washed with PBS for staining with annexin-V (BD Biosciences); the staining procedures are performed according to the protocols provided by the manufacturer. Data is acquired on a FACSCalibur flow cytometer (BD Biosciences), and at least 10,000 live cells are acquired for each analysis (BD FACS Diva software; BD Biosciences).

CD34+ cells are purified from the peripheral blood of patients with MF and cultured in serum-free medium contained with SCF, FL-3 ligand, IL-3, and TPO. The cells are treated with various dose of the compound of Formula (I) for 4 hours. Cells are harvested and the whole cells protein extracts are prepared with RIPA lysis buffer (Boston BioProducts) for Western blotting.

To prepare the cytoplasmic and nuclear protein fractions of cells from patients with MF, CD34+ cells are expanded in serum-free media containing SCF, FL-3 ligand, and IL-3 for 10 days. CD34+ cells are then repurified and treated with various doses of the compound of Formula (I) and/or Ruxolitinib for 48 hours in the presence of SCF, FL-3 ligand, IL-3, and TPO. The protein extracts are prepared using the NEPER nuclear and cytoplasmic extraction reagent (Thermo Scientific) according to the manufacturer's instructions.

Before Western blotting, all the samples are denatured with Laemmli SDS-sample buffer (Boston BioProducts) by heating at 95°C for 5 minutes; each sample will be separated on SDS-PAGE gels and transferred to polyvinyldif luoridine membranes (Bio-Rad). Phospho-p53, p53, MDM2, p21, p-STATl, PUMA, and Bak were visualized using antibodies (Cell Signaling Technologies) and ECL Western blotting reagents (Denville Scientific).

MDM2/p53 inhibition axes upregulate p21 expression which function is to put a damaged cell into cell cycle arrest. MDM2 inhibitors must overcome this checkpoint to drive apoptosis. JAK inhibitors (i.e. Ruxolitinib) do not upregulate p21 expression which is not surprising as it would not be expected that Ruxolitinib could biologically modulate p21 expression. However, the combination treatment of CD34+ cells from patients with myelofibrosis with Ruxolitinib and the compound of formula (I) results in p21 expression levels which still do not increase, despite the inclusion of treatment with the MDM2 inhibitor (compound of formula (I)). It would have been expected that p21 expression would have still been upregulated in the presence of the MDM2 inhibtor, which it is not. Because it is no longer necessary to overcome this p21 check point, the apoptotic threshold is much lower and more ex vivo apoptosis is observed (see Fig. 1). As shown in Fig. 1, monotherapy navtemadlin treatment increased p21 levels, a pre-cursor apoptotic checkpoint, while adding ruxolitinib to navtemadlin inhibited p21-mediated cell cycle arrest.

Example 2: Cyctotoxicity of the combination of the compound of Formula (I) with a JAK inhibitor in UKE- 1 cell line.

Cell viability was assessed following the procedure described in Canon et aL, Mol Cancer Ther (2015) 14 (3): 649-658. The following describes the procedure briefly.

UKE-1 cells were plated in 96- or 384-well plates at optimum initial seeding densities to ensure that cells did not reach confluency by the end of the assay. The cells were treated with DMSO control or navtemadlin and ruxolitinib combination at various concentrations for a 24 hour incubation. A commercial cell viability assay kit was used to determine the numbers of viable cells. Growth inhibition (Gl) was calculated on a 200-point scale according to the following equations, where V₂₄ was luminescence of DMSO control at 24 hours and T₂₄ was luminescence of the compound-treated sample: if T₂₄ > Vo, then Gl = 100 x (1 - ((T₂₄ - V₀)/(V₂₄ - V_o))); if T₂₄ < V_o, then Gl = 100 x (1 - ((T₂₄ - V_o)/V_o)). Gl values of 0, 100, and 200 represent uninhibited cell growth (i.e., DMSO control), cell stasis, and complete cell killing, respectively. The results are shown in FIG. 2A.

These data were analyzed for synergy using the HSA model (Combenefit software) and the results are shown in FIG. 2B. The y-axis shows percentage of baseline cell proliferation under control conditions (i.e., with no navtemadlin and no ruxolitinib). Decreased y-axis values correspond to increased apoptosis. Dark blue/blue colors show drug combinations eliciting synergistic apoptosis, which exceeded the sum of cell death produced by the drugs when presented alone.

Example 3: Apoptosis and protein expression after 24 hour and 72 hour exposure to navtemadlin and ruxolitinib.

The effect of navtemadlin combined with ruxolitinib on apoptosis and protein expression in myelofibrosis subject-derived progenitor cells was assessed. As shown in FIG. 3, navtemadlin combined with ruxolitinib enhances apoptosis in the myelofibrosis subject-derived progenitor cells and complete suppression of p21 was observed with the addition of ruxolitinib. Live cells were defined as CD45+mid, SSCIow, CD14-, cPARP-. As shown in FIG. 4, navtemadlin combined with ruxolitinib reduced pro-survival MCL-1 levels.

The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

Claims

1. A method of suppressing p21 expression levels in a human suffering from a myeloproliferative neoplasm (MPN) comprising administering to the human a therapeutically effective amount of a MDM2 inhibitor in combination with a JAK inhibitor, wherein the MDM2 inhibitor is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein the administering suppresses p21 expression levels in the human compared to p21 expression levels in MDM2 inhibitor monotherapy.

2. The method of claim 1, wherein the administering suppresses p21 levels by at least 50% compared to MDM2 inhibitor monotherapy, optionally at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

3. The method of claim 1 wherein the administering stimulates apoptosis of malignant myeloid cells in the human suffering from the myeloproliferative neoplasm (MPN).

4. The method of claim 3, wherein the malignant cells are CD34+ myeloid cells or CD45+ myeloblasts.

5. The method of any one of claims 1 to 4, wherein the MPN is polycythemia vera (PV).

6. The method of any one of claims 1 to 4, wherein the MPN is thrombocythemia.

7. The method of claim 6, wherein thrombocythemia is essential thrombocythemia (ET).

8. The method of any one of claims 1 to 4, wherein the MPN is myelofibrosis.

9. The method of claim 8, wherein the myelofibrosis is selected from primary myelofibrosis (PMF), postpolycythemia vera myelofibrosis (post-PV MF), and post-essential thrombocythemia myelofibrosis (post- ET MF).

10. The method of any one of claims 1 to 4, wherein the MPN is chronic myelogenous leukemia.

11. The method of any one of claims 1 to 4, wherein the MPN is systemic mastocystosis (SM).

12. The method of any one of claims 1 to 4, wherein the MPN is chronic neutrophilic leukemia (CNL).

13. The method of any one of claims 1 to 4, wherein the MPN is myelodysplastic syndrome (MDS).

14. The method of any one of claims 1 to 4, wherein the MPN is mast cell disease (SMCD).

15. The method of any one of claims 1 to 4, wherein the MPN is chronic eosinophilic leukemia.

16. The method of any one of claims 1 to 4, wherein the MPN is chronic myelomonocytic leukemia (CMML).

17. The method of any one of claims 1 to 4, wherein the MPN is atypical chronic myeloid leukemia (aCML).

18. The method of any one of claims 1 to 4, wherein the MPN is juvenile myelomonocytic leukemia (JMML).

19. The method of any one of claims 1 to 4, wherein the MPN is hypereosinophilic syndromes (HES).

20. The method of any one of claims 1 to 19, wherein the MDM2 inhibitor is a pharmaceutically acceptable salt of a compound of Formula (I).

21. The method of any one of claims 1 to 20, wherein the compound of Formula (I) is administered once daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.

22. The method of any one of claims 1 to 20, wherein the compound of Formula (I) is administered twice daily at a dose selected from the group consisting of 15 mg, 25 mg, 30 mg, 50 mg, 60 mg, 75 mg, 100 mg, 120 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 360 mg, 375 mg, and 480 mg.

23. The method of any one of claims 1 to 22, wherein the human is treated with the MDM2 inhibitor for a period selected from the group consisting of about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, and about 56 days.

24. The method of any one of claims 1 to 23, wherein the compound of Formula (I) is orally administered.

25. The method of any one of claims 1 to 24, wherein the JAK inhibitor is selected from the group consisting of AC-410, AT9283, AZ960, AZD-1480, Baricitinib, BMS-911543, CEP-33779, Cerdulatinib, CHZ868, CYT387, Decernotinib, ENMD-2076, Fetratinib, Filgotinib, Ganetespib, INCB039110, INCB- 047986, Itacitinib, JAK3-IN-1, JANEX-1, LFM-A13, LY2784544, NS-018, NSC42834, NVP-BSK805, Oclacitinib, Pacritinib, Peficitinib, Pyridone 6, R348, RGB-286638, Ruxolitinib, Ruxolitinib-S, SAR-20347, SB1317, Solcitinib, TG101209, TG101348, Tofacitinib(3R,4S), Tofacitinib(3S,4R), Tofacitinib(3S,4S), Tofacitinib, TYK2-IN-2, Upadacitinib, WHI-P154, WHI-P97, WP1066, XL019, ZM39923, and pharmaceutically acceptable salts thereof.

26. The method of any one of claims 1 to 24, the JAK inhibitor is selected from the group consisting of Baricitinib phosphate, CYT387 Mesylate, CYT387 sulfate salt, NS-018 hydrochloride, NS-018 maleate, NVP-BSK805 dihydrochloride, Oclacitinib maleate, Ruxolitinib phosphate, Ruxolitinib sulfate, Tofacitinib citrate, and ZM39923 hydrochloride.

27. The method of claim 25 or 26, wherein the JAK inhibitor is administered orally.

28. The method of any one of claims 1 to 4, wherein the MDM2 inhibitor is administered before administration of the JAK inhibitor.

29. The method of any one of claims 1 to 4, wherein the MDM2 inhibitor is administered after administration of the JAK inhibitor.

30. The method of any one of claims 1 to 4, wherein the MDM2 inhibitor is administered concurrently with administration of the JAK inhibitor.

31. The method of any one of claims 1 to 30, wherein the therapeutically effective amount of the MDM2 inhibitor is 100 mg.

32. The method of any one of claims 1 to 31, wherein the MPN in the human subject has a JAK2V617F mutation.