CDK4 inhibitors and combinations with CDK2 inhibitors or further agents for use in the treatment of cancer

Abstract

This invention relates to therapies for treating cancers in a subject comprising a cyclin dependent kinase 4 (CDK4) inhibitor, and optionally in combination with a CDK2 or an additional anti-tumor agent, and associated methods of treatment, pharmaceutical compositions, and uses thereof.

Description

[0001] CDK4 INHIBITORS AND COMBINATIONS WITH CDK2 INHIBITORS OR FURTHER AGENTS FOR USE IN THE TREATMENT OF CANCER

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to therapies useful for treating cancers in a subject. In particular, the invention relates to both mono- and combination therapies comprising a cyclin dependent kinase 4 inhibitor. The invention also relates to associated methods of treatment, pharmaceutical compositions, and pharmaceutical uses.

[0004] BACKGROUND

[0005] CDK4 is a cyclin-dependent kinase that plays a crucial role in regulating the cell cycle. It works in conjunction with cyclin D1 (CCND1) to initiate the transition from the G1 to the S phase of the cell cycle. CDK4 inhibitors work by specifically inhibiting the CDK4 / cyclin D complex, blocking the transition from the G1 to the S phase of the cell cycle and potentially inhibiting the growth of cancer cells.

[0006] Ewing sarcoma (ES) is a rare, aggressive malignant pediatric tumor, with most patients harboring a priori micrometastases. ES is characterized by chromosomal tranlocations, giving rise to the oncogene EWS-FLI1 , which triggers the transcription of genes involved in neoplastic transformation including the CCND1 gene. Cancers (Basel), 14, 6 (2022). Approximately 20-25% of ES patients have metastases at the time of initial diagnosis, and these patients are often resistant to intensive treatment. Nat Rev Dis Primers 4, 6 (2018). In addition, the 5-year overall survival (OS) of metastasis patients is only 20-45%, depending on the location of metastasis. Sci Rep \_, 22723 (2021).

[0007] There remains a need for improved therapies for the treatment of cancers. The compounds, compositions and methods of the present invention are believed to have one or more advantages, such as greater efficacy; potential to reduce side effects; potential to reduce drugdrug interactions; potential to enable an improved dosing schedule; or potential to overcome resistance mechanisms, and the like.

[0008] BRIEF SUMMARY OF THE INVENTION

[0009] This present invention relates to methods, combinations, uses, pharmaceutical compositions and kits for treating abnormal cell growth, particularly cancer, comprising a CDK4 inhibitor, optionally in further combination with a CDK2 inhibitor or an additional anti-cancer agent.

[0010] According to an embodiment of the invention, there is provided a method of treating cancer in a subject in need thereof comprising: (1) administering to the subject an amount of a cyclin-dependent kinase 4 (CDK4) inhibitor; or (2) a combination therapy which comprises an amount of a CDK4 inhibitor, and (a) an amount of a CDK2 inhibitor; or (b) an amount of an additional anti-tumor agent; wherein the amount of CDK4 and any optional CDK2 or additional anti-tumor agent is alone or together therapeutically effective in treating cancer.

[0011] Described below are embodiments of the invention, where for convenience Embodiment 1 (E1) is identical to the method of treating a cancer in a subject provided above.

[0012] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 demonstrates the human ewing sarcoma (ES) cell lines are sensitive to PF- 07220060 (“60”).

[0015] FIG. 2 shows dose dependent growth inhibition by palbociclib (“palbo”) and PF-07220060 (“60”) each as a single agent in ewing sarcoma cell line derived multicellular tumor spheroids (MOTS): A673 (FIG. 2A), MHHES-1 (FIG. 2B) and CadoES-1 (FIG. 2C), as average diameter (mm) at concentrations shown.

[0016] FIG. 3 shows dose dependent growth inhibition by palbo, PF-07220060 (“60”) and PF- 07104091 (“91”) each as a single agent, the combination of palbo and PF-07104091 , and the combination of PF-07220060 and PF-07104091 in ES cell line derived MCTS A673, as average diameter (mm) at concentrations shown.

[0017] FIG. 4 shows dose dependent growth inhibition by PF-07220060 (“60”) and PF-07104091 (“91”) each as a single agent, and the combination of PF-07220060 and PF-07104091 in ES cell line derived MCTS MHHES-1 , as average diameter (mm) at concentrations shown.

[0018] FIG. 5 shows dose dependent growth inhibition by palbo, PF-07220060 (“60”) and binimetinib (“bini”) each as a single agent, the combination of palbo and bini, and the combination of PF-07220060 and bini, in MCTS A673, as average diameter (mm) at concentrations shown.

[0019] FIG. 6 shows dose dependent growth inhibition by PF-07220060 (“60”) at different concentration in MCTS A673 (FIG. 6A) and MHHES-1 (FIG. 6B).

[0020] FIG. 7 shows dose dependent growth inhibition by co-treatment of standard of care: SN38 plus TMZ and PF-07220060 (“60”), and SOC alone (i.e. , SN38 + TMZ). The co-treatment of PF- 07220060 partially protects from SN38 / TMZ mediated growth inhibitor of MCTS A673 (FIG. 7A) and MCTS MHHES-1 (FIG. 7B).

[0021] FIG. 8 shows dose dependent growth inhibition by PF-07220060 (“60”) as single agent in both the first treatment (TREAT1) and the second treatment (TREAT2), SOC alone (i.e., SN38 + TMZ) in TREAT1 , co-treatment of PF-07220060 and SOC in TREAT1 followered by PF-07220060 alone in TREAT2, and staggered treatment of SOC in TREAT 1 followered by PF0060 in TREAT2. TREAT 1 was initiated at day 0 and compounds removed at day 5. TREAT2 followed from day 5 to 21. The arrow highlights the switch from TREATIto TREAT2. Sequential treatment of SOC followered by PF-07220060 results in greater growth inhibition of MCTS A673 (FIG. 8A) and MCTS MHHES-1 (FIG. 8B) than co-treatment, or PF-07220060 alone, or SOC alone.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention may be understood more readily by reference to the following detailed description of the embodiments and preferred embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It is further to be understood that unless specifically defined herein, the terminology used herein is to be given its traditional meaning as known in the relevant art.

[0024] E1 A method of treating cancer in a subject in need thereof, as defined above.

[0025] E2. The method of embodiment 1 , wherein the CDK4 inhibitor is a CDK4 selective inhibitor or a CDK4 / 6 inhibitor.

[0026] E3. The method of embodiment 1 , wherein the CDK4 inhibitor is a CDK4 selective inhibitor.

[0027] E4. The method of any one of embodiments 1 to 3, wherein the CDK4 selective inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0028] R1is H, F or Cl;

[0029] R2is C1-C4 alkyl, where said C1-C4 alkyl is optionally substituted by R5;

[0030] R3is H or C1-C4 alkyl, where said C1-C4 alkyl is optionally substituted by R6;

[0031] R4is H or F; and each R5and R6is independently OH, F or C1-C2 alkoxy. E5 The method of any one of embodiments 1 to 4, wherein the compound of Formula (I) is 1,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz- imidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f / 7reo-pentitol.

[0032] E6 The method of any one of embodiments 1 or 2, wherein the CDK4 inhibitor is a CDK4 / 6 inhibitor.

[0033] E7 The method of any one of embodiments 1 or 2, wherein the CDK4 / 6 inhibitor is palbociclib.

[0034] E8 The method of any one of embodiments 1 to 7, wherein the CDK2 inhibitor that is (1 R,3S)-3-[3-({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H-pyrazol-5- yl]cyclopentyl propan-2-yl carbamate.

[0035] E9 The method of any one of embodiments 1 to 7, wherein the additional anti-tumor agent that is selected from the group consisting of binimetinib, SN38, temozolomide, and combinations thereof.

[0036] E10 The method of any one of embodiments 1 to 7, wherein the monotherapy comprises 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7- benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f / 7reo-pentitol , or palbociclib.

[0037] E11 The method of any of embodiments 1 to 9, wherein the combination therapy comprises:

[0038] (a) the CDK4 selective inhibitor that is 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2- hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3- dideoxy-D-f / ireo-pentitol, and the CDK2 inhibitor that is (1 R,3S)-3-[3-({[3- (methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H-pyrazol-5-yl]cyclopentyl propan-2-yl carbamate; or

[0039] (b) the CDK4 selective inhibitor that is 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2- hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3- dideoxy-D-f / ireo-pentitol, and the additional anti-tumor agent that is binimetinib; or

[0040] (c) the CDK4 selective inhibitor 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2- hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3- dideoxy-D-f / ireo-pentitol, and the additional anti-tumor agent that is the combination of SN38 and temozolomide; or

[0041] (d) the CDK4 / 6 inhibitor that is palbociclib, and the CDK2 inhibitor that is (1 R,3S)-3-[3-({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H- pyrazol-5-yl]cyclopentyl propan-2-yl carbamate; or

[0042] (e) the CDK4 / 6 inhibitor that is palbociclib, and the additional anti-tumor agent that is binimetinib; or

[0043] (f) the CDK4 / 6 inhibitor that is palbociclib, and the additional anti-tumor agent that is the combination of SN38 and temozolomide.

[0044] E12 The method any of embodiments 1 to 11 , wherein the administration of the CDK4 inhibitor and the adminstration of the CDK2 inhibitor is concurrent.

[0045] E13 The method any of embodiments 1 to 11 , wherein the administration of the CDK4 inhibitor and the adminstration of the CDK2 inhibitor is sequential.

[0046] E14 The method any of embodiments 1 to 11 , wherein the administration of the CDK4 inhibitor and the adminstration of the additional anti-tumor agent is concurrent.

[0047] E15 The method any of embodiments 1 to 11 , wherein the administration of the CDK4 inhibitor and the adminstration of the additional anti-tumor agent is sequential.

[0048] E16 The method of any of embodiments 1 to 15, wherein the subject is human.

[0049] E17 Use of a CDK4 inhibitor in the manufacture of a medicament, optionally in combination with a) a CDK2 inhibitor, or (b) an additional anti-tumor agent, for treating ewing sarcoma.

[0050] E18 A pharmaceutical combination comprising a CDK4 inhibitor and with a) a CDK2 inhibitor; or b) binimetinib; or c) a further combination of SN38 and temozolomide; and a pharmaceutically acceptable carrier.

[0051] E19 The combination of embodiment 18 wherein the combination is not fixed.

[0052] E20 The combination of embodiment 18 wherein the combination is fixed. Each of the embodiments of the present invention described herein may be combined with one or more other embodiments of the present invention described herein which is not inconsistent with the embodiment(s) with which it is combined. In addition, each of the embodiments below describing the invention envisions within its scope the pharmaceutically acceptable salts of the compound of the invention.

[0053] The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It is further to be understood that unless specifically defined herein, the terminology used herein is to be given its traditional meaning as known in the relevant art.

[0054] As used herein, the singular form "a", "an", and "the" include plural references unless indicated otherwise. For example, "a" substituent includes one or more substituents.

[0055] The invention described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of", and "consisting of' may be replaced with either of the other two terms.

[0056] The term “about” which used to modify a numerically defined parameter means that the parameter may vary by as much as 10% above or below the stated numerical value for that parameter. For example, a dose of about 5mg / kg should be understood to mean that the dose may vary between 4.5mg / kg and 5.5mg / kg.

[0057] Cyclin-dependent kinases (CDKs) and related serine / threonine kinases are important cellular enzymes that perform essential functions in regulating cell division and proliferation. CDK inhibitors include Pan-CDK inhibitors that target a broad spectrum of CDKs or selective CDK inhibitors that target specific CDK(s).

[0058] Cyclin-dependent kinases (CDKs) and related serine / threonine protein kinases are important cellular enzymes that perform essential functions in regulating eukaryotic cell division and proliferation. The CDK catalytic units are activated by regulatory subunits known as cyclins. At least sixteen mammalian cyclins have been identified (Johnson DG, Walker CL. Cyclins and Cell Cycle Checkpoints. Annu. Rev. Pharmacol. Toxicol. (1999) 39:295-312). Cyclin B / CDK1 , cyclin A / CDK2, cyclin E / CDK2, cyclin D / CDK4, cyclin D / CDK6, and likely other heterodynes are important regulators of cell cycle progression. Additional functions of cyclin / CDK heterodynes include regulation of transcription, DNA repair, differentiation and apoptosis (Morgan DO, Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell. Dev. Biol. (1997) 13:261-291).

[0059] CDK inhibitors have been demonstrated to be useful in treating cancer. Increased activity or temporally abnormal activation of cyclin-dependent kinases has been shown to result in the development of human tumors, and human tumor development is commonly associated with alterations in either the CDK proteins themselves or their regulators (Cordon-Cardo C. Mutations of cell cycle regulators: biological and clinical implications for human neoplasia. Am. J. Pathol. (1995) 147:545-560; Karp JE, Broder S. Molecular foundations of cancer: new targets for intervention. Nat. Med. (1995) 1 :309-320; Hall M, Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv. Cancer Res. (1996) 68:67-108).

[0060] CDK4 and CDK6 are important regulators of cell cycle progression at the G1-S checkpoint, which are controlled by D-type cyclins and INK4 endogenous CDK inhibitors, such as p16INK4a(CDKN2A). Dysregulation of the cyclin D-CDK4 / 6-INK4-retinoblastoma (Rb) pathway has been reported to be associated with development of endocrine therapy resistance. Furthermore, CDK4 has been identified as the singular oncogenic driver in many breast cancers and emerging data suggest that cyclin D3-CDK6 inhibition may be linked to hematologic toxicity, suggesting a role for CDK4 selective inhibitors.

[0061] As used herein, a “CDK4 inhibitor” includes a CDK4 selective inhibitor and a CDK4 / 6 inhibitor. “CDK4 selective inhibitor” refers to compounds that inhibit the kinase activity of CDK4 to a greater extent than any other CDKs. “CDK4 / 6 inhibitor” refers to compounds that inhibit the kinase activity of CDK 4 and 6. CDK4 selective inhibitors are disclosed in International Publication No. WO 2019 / 207463. Examples of CDK4 / 6 inhibitors include, but are not limited to, palbociclib.

[0062] CDK4 selective inhibitors include, but are not limited to, PF-07220060, BGB-43395 (BeiGene), H RS-6209 (Jiangsu HengRui) and ALI2-94 (Aucentra Therapeutics).

[0063] In an embodiment, CDK4 selective inhibitors of the present invention include “PF- 07220060” which refers to 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1- (propan-2-yl)-1 / 7-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-t / 7reo-pentitol, which has the following chemical structure, including hydrates, salts and polymorphs thereof: PF-07220060 is disclosed in International Publication No. WO 2019 / 207463, U.S. Patent Nos. 10,766,884 and 11 ,220,494, and US Patent Publication US 2022 / 0089580; and International Publication No. WO 2022 / 058871 , the contents of which are incorporated herein by reference in their entirety. Unless indicated otherwise, all references herein to PF-07220060 include references to salts, solvates, hydrates, and complexes thereof, and to solvates, hydrates and complexes of salts thereof, including polymorphs, stereoisomers, and isotopically labelled versions thereof.

[0064] In one embodiment, the CDK4 selective inhibitor having a structure of Formula (I) or a pharmaceucially acceptable salt thereof.

[0065] In one embodiment, the compound of Formula (I) is 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro- 2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3- dideoxy-D-t / ireo-pentitol or a pharmaceucially acceptable salt thereof.

[0066] In one embodiment, 1,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1- (propan-2-yl)-1 / 7-benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-t / 7reo-pentitol is 1,5- anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6- yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-t / 7reo-pentitol monohydrate.

[0067] In one embodiment, the compound of Formula (I) is atirmociclib.

[0068] CDK4 / 6 inhibitors include, but are not limited to, palbociclib, ribociclib, and abemaciclib.

[0069] In an embodiment, a CDK4 / 6 inhibitor of the present invention includes palbociclib.

[0070] Palbociclib, or 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8 / 7- pyrido[2,3-cf]pyrimidin-7-one (also known as “PD-0332991” and referred to herein as “palbo”) is a potent and selective inhibitor of CDK4 and CDK6, having the structure:

[0071] Palbociclib is described 27, No. 2, page 172 (2013). Palbociclib and pharmaceutically acceptable salts thereof are disclosed in International Publication No. WO 2003 / 062236 and U.S. Patent Nos. 6,936,612, 7,456,168 and RE47.739; International Publication No. WO 2005 / 005426 and U.S. Patent Nos. 7,345,171 and 7,863,278; International Publication No. WO 2008 / 032157 and U.S. Patent No. 7,781 ,583; and International Publication No. WO 2014 / 128588. The contents of each of the foregoing references are incorporated herein by reference in their entirety.

[0072] Unless indicated otherwise, all references herein to palbocilib include references to salts, solvates, hydrates, and complexes thereof, and to solvates, hydrates and complexes of salts thereof, including polymorphs, stereoisomers, and isotopically labelled versions thereof.

[0073] CDK2 inhibitors include, but are not limited to, PF-07104091 (Pfizer), BLU-222 (Blueprint Medicines), and those described in W024059010 and W024056019.

[0074] In an embodiment, CDK2 inhibitors of the present invention include “PF-07104091” which refers to (1 R,3S)-3-[3-({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H- pyrazol-5-yl]cyclopentyl propan-2-yl carbamate, which has the following chemical structure, including hydrates, tautomers, salts and polymorphs thereof:

[0075] PF-07104091 is currently in clinical development for the treatment of certain cancers. Preparation of PF-07104091 is disclosed in International Patent Publication No. WO 2020 / 157652 and in United States Patent No. 11 ,014,911 , the contents of each which are incorporated herein by reference in their entirety.

[0076] Unless indicated otherwise, all references herein to PF-07104091 include references to salts, solvates, hydrates, and complexes thereof, and to solvates, hydrates and complexes of salts thereof, including polymorphs, stereoisomers, and isotopically labelled versions thereof.

[0077] It is understood that the above structural formula of PF-07104091 includes all tautomeric forms which may co-exist and be directly interconverted under the appropriate conditions. For example, in some embodiments, PF-07104091 has a structure of formula (la):

[0078] In one embodiment, (1 R,3S)-3-[3-({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5- yl]carbonyl}-amino)-1 H-pyrazol-5-yl]cyclopentyl propan-2-yl carbamate is (1 R,3S)-3-[3-({[3- (methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H-pyrazol-5-yl]cyclopentyl propan-2-yl carbamate monohydrate.

[0079] Another embodiment relates to the pharmaceutically acceptable salts of the compounds described herein. Pharmaceutically acceptable salts of the compounds described herein include the acid addition and base addition salts thereof.

[0080] Another embodiment also relates to the pharmaceutically acceptable acid addition salts of the compounds described herein. Suitable acid addition salts are formed from acids which form non-toxic salts. Non-limiting examples of suitable acid addition salts, i.e., salts containing pharmacologically acceptable anions, include, but are not limited to, the acetate, acid citrate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulphate / sulphate, bitartrate, borate, camsylate, citrate, cyclamate, edisylate, esylate, ethanesulfonate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methanesulfonate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, p-toluenesulfonate, tosylate, trifluoroacetate and xinofoate salts.

[0081] Additional embodiments relate to base addition salts of the compounds described herein. Suitable base addition salts are formed from bases which form non-toxic salts. Non-limiting examples of suitable base salts include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

[0082] The compounds described herein that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds described herein are those that form non-toxic acid addition salts, e.g., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1 ,1’-methylene-bis-(2-hydroxy-3- naphthoate)] salts. The compounds described herein that include a basic moiety, such as an amino group, may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.

[0083] The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of those compounds of the compounds described herein that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N- methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines.

[0084] Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.

[0085] For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Methods for making pharmaceutically acceptable salts of compounds described herein are known to one of skill in the art.

[0086] Administration and Dosing

[0087] "Treat" or "treating" a cancer and / or a cancer-associated disease as used herein means to administer a monotherapy or combination therapy according to the present invention to a subject, participant or patient having a cancer, or diagnosed with a cancer, to achieve at least one positive therapeutic effect, such as, for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastasis or tumor growth, reversing, alleviating, or inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment" or “therapy,” as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cell; inhibiting metastasis or neoplastic cells; shrinking or decreasing the size of tumor; remission of the cancer; decreasing symptoms resulting from the cancer; increasing the quality of life of those suffering from the cancer; decreasing the dose of other medications required to treat the cancer; delaying the progression the cancer; curing the cancer; overcoming one or more resistance mechanisms of the cancer; and I or prolonging survival of patients the cancer. Positive therapeutic effects in cancer may be measured in a number of ways (see, for example, W. A. Weber, J. Nucl. Med. 50:1S-10S (2009)).

[0088] As used herein, the terms, “subject”, “participant” and “patient,” are used interchangeably, to refer to any animal, including mammals. Mammals according to the invention include canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, humans and the like, and encompass mammals in utero. In an embodiment, humans are suitable subjects. Human subjects may be of any gender and at any stage of development. An “amount” for use and for treating a subject refers to an amount that provides, in single or multiple doses, alone, or in combination with one or more other agents, a detectable response of any duration of time (transient, medium or long term), a desired outcome in or an objective or subjective benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for hours, days, months, years, in remission or cured). Such amounts typically are effective to ameliorate a disease, or one, multiple or all adverse effects I symptoms, consequences or complications of the disease, to a measurable extent, although reducing or inhibiting a progression or worsening of the disease, or providing stability (i.e. , not worsening) state of the disease, is considered a satisfactory outcome. The term “therapeutically effective amount” also means an amount of an agent, alone, or in combination with one or more other agents, effective for producing a desired therapeutic effect upon administration to a subject, for example, to stem the growth, or result in the shrinkage, of a cancerous tumor. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis emergence, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth or tumor invasiveness, and / or (4) relieving to some extent (or, preferably, eliminating) one or more signs or symptoms associated with the cancer. Therapeutic or pharmacological effectiveness of the doses and administration regimens may also be characterized as the ability to induce, enhance, maintain or prolong disease control and / or overall survival in patients with these specific tumors, which may be measured as prolongation of the time before disease progression.

[0089] As used herein, “ameliorate” refers to any reduction in the extent, severity, frequency, and / or likelihood of a symptom or clinical sign characteristic of a particular disease. “Symptom” refers to any subjective evidence of disease or of a subject's condition.

[0090] Administration of the compounds of the present invention may 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, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration. Each compound may be administered according to the same or different route of administration.

[0091] In a preferred embodiment, the daily dose of a CDK4 inhibitor or a pharmaceutically acceptable salt thereof, is administered orally.

[0092] A CDK4 inhibitor, or a pharmaceutically acceptable salt, may be present in a pharmaceutical composition which includes a pharmaceutically acceptable excipient. "Pharmaceutically acceptable excipient" refers to a component that may be included in the compositions described herein, is physiologically suitable for pharmaceutical use, and causes no significant adverse effects nor therapeutic effects to a subject. The term ’excipient’ is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0093] The amount of a CDK4 inhibitor, or a pharmaceutically acceptable salt, in the pharmaceutical compositions may be any amounts disclosed herein.

[0094] The compounds of the method, use or combination of the present invention may be formulated prior to administration. The formulation will preferably be adapted to the particular mode of administration. These compounds may be formulated with pharmaceutically acceptable excipients as known in the art and administered in a wide variety of dosage forms as known in the art. Dosage unit forms or pharmaceutical compositions suitable for oral administration include, but are not limited to tablets, capsules, such as gelatin capsules, pills, powders, granules, aqueous and nonaqueous oral solutions and suspensions, packaged in containers adapted for subdivision into individual doses.

[0095] In another embodiment, the dosage of a compound or pharmaceutical composition described herein may vary within the range depending upon the dosage form employed and the route of administration utilized. In another embodiment, an amount of a compound or pharmaceutical composition described herein administered to a subject may be dependent upon factors known to a skilled artisan, including bioactivity and bioavailability of the compound (e.g., half-life and stability of the compound in the body), chemical properties of the compound (e.g., molecular weight, hydrophobility and solubility), route and frequency of administration, and the like. Further, it will be understood that the specific dose of a pharmaceutical composition comprising a compound as disclosed herein may depend on a variety of factors including physical condition of the subject (e.g., age, gender, weight), and medical history of the subject (e.g., medications being taken, health condition other diseases or disorders). The precise dose of a pharmaceutical composition administered to a subject may be determined by methods known to a skilled artisan such as a pharmacologist, or an anesthesiologist.

[0096] In some embodiments, palbociclib, is administered at a daily dosage of about 125 mg once daily, about 100 mg once daily, about 75 mg once daily, about 50 mg daily, or about 25 mg daily. In an embodiment, which is the recommended starting dose, palbociclib is administered at a daily dosage of about 125 mg once a day. For example, palbociclib is administered at a dose of about 100 mg once daily, about 75 mg once daily, or about 50 mg once daily. In an embodiment, palbociclibis administered at a dose of about 100 mg once daily.

[0097] In an embodiment, palbociclib is administered at a dose of about 75 mg once daily. In an embodiment, palbociclib is administered at a dose of about 50 mg once daily.

[0098] In some embodiments, 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1- (propan-2-yl)-1 / 7-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f / 7reo-pentitol, is administered at a daily dosage of from about 10 mg to about 1000 mg per day. In some embodiments, the CDK4 inhibitor is administered at dosages of about: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 275, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475 or 500 mg on a QD, BID, TID or QID schedule.

[0099] In some embodiments, (1 R,3S)-3-[3-({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5- yl]carbonyl}-amino)-1 H-pyrazol-5-yl]cyclopentyl propan-2-yl carbamate is administered at a daily dosage of from about 20 mg to about 1500 mg per day. In some embodiments, the CDK2 inhibitor is administered at a daily dosage from about 150 mg to about 1000 mg per day. In some embodiments, the CDK2 inhibitor is administered at dosages of about: 1 , 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 275, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475 or 500 mg on a QD, BID, TID or QID schedule.

[0100] Repetition of the administration or dosing regimens may be conducted as necessary to achieve the desired reduction or diminution of cancer cells. A “continuous dosing schedule”, as used herein, is an administration or dosing regimen without dose interruptions, e.g., without days off treatment. Repetition of 28-day treatment cycles without dose interruptions between the treatment cycles is an example of a continuous dosing schedule. In an embodiment, the compounds of the combination of the present invention may be administered in a continuous dosing schedule. In an embodiment, the compounds of the combination of the present invention may be administered concurrently in a continuous dosing schedule.

[0101] Method of T reatment

[0102] In one embodiment, the disclosure provides a method of treating a cancer in a subject in need thereof, which includes administering to the subject an amount of a cyclin-dependent kinase 4 (CDK4) inhibitor as described herein.

[0103] "Treat" or "treating" a cancer and / or a cancer-associated disease as used herein means to administer a monotherapy or combination therapy according to the present invention to a subject, participant or patient having a cancer, or diagnosed with a cancer, to achieve at least one positive therapeutic effect, such as, for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastasis or tumor growth, reversing, alleviating, or inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment" or “therapy,” as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cell; inhibiting metastasis or neoplastic cells; shrinking or decreasing the size of tumor; remission of the cancer; decreasing symptoms resulting from the cancer; increasing the quality of life of those suffering from the cancer; decreasing the dose of other medications required to treat the cancer; delaying the progression the cancer; curing the cancer; overcoming one or more resistance mechanisms of the cancer; and I or prolonging survival of patients the cancer. Positive therapeutic effects in cancer may be measured in a number of ways (see, for example, W. A. Weber, J. Nucl. Med. 50:1S-10S (2009)).

[0104] As used herein, the terms, “subject”, “participant” and “patient,” are used interchangeably, to refer to any animal, including mammals. Mammals according to the invention include canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, humans and the like, and encompass mammals in utero. In an embodiment, humans are suitable subjects. Human subjects may be of any gender and at any stage of development.

[0105] An “amount” for use and for treating a subject refers to an amount that provides, in single or multiple doses, alone, or in combination with one or more other agents, a detectable response of any duration of time (transient, medium or long term), a desired outcome in or an objective or subjective benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for hours, days, months, years, in remission or cured). Such amounts typically are effective to ameliorate a disease, or one, multiple or all adverse effects I symptoms, consequences or complications of the disease, to a measurable extent, although reducing or inhibiting a progression or worsening of the disease, or providing stability (i.e. , not worsening) state of the disease, is considered a satisfactory outcome. The term “therapeutically effective amount” also means an amount of an agent, alone, or in combination with one or more other agents, effective for producing a desired therapeutic effect upon administration to a subject, for example, to stem the growth, or result in the shrinkage, of a cancerous tumor. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis emergence, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth or tumor invasiveness, and / or (4) relieving to some extent (or, preferably, eliminating) one or more signs or symptoms associated with the cancer. Therapeutic or pharmacological effectiveness of the doses and administration regimens may also be characterized as the ability to induce, enhance, maintain or prolong disease control and / or overall survival in patients with these specific tumors, which may be measured as prolongation of the time before disease progression. As used herein, “ameliorate” refers to any reduction in the extent, severity, frequency, and / or likelihood of a symptom or clinical sign characteristic of a particular disease. “Symptom” refers to any subjective evidence of disease or of a subject's condition.

[0106] Administration of the compounds of the present invention may 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, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration. Each compound may be administered according to the same or different route of administration.

[0107] As used herein, an “effective dosage”, “effective amount” or “therapeutically effective amount” of a compound or pharmaceutical composition is the amount that, when used as indicated (which may be alone if used as a single agent or together with other agents if used in combination) is sufficient to affect one or more beneficial or desired outcomes, including preventing, ameliorating or treating the biochemical, histological or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presenting during development of the disease. For prophylactic use, beneficial or desired outcomes may include: eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease. For therapeutic use, beneficial or desired outcomes may include: reducing the incidence or ameliorating one or more symptoms of the disease, reducing the dose of another medication used to treat the disease, enhancing the efficacy or safety of another medication used to treat the disease, or delaying the time to disease progression.

[0108] In reference to the treatment of cancer, beneficial or desired outcomes provided by the invention may include: (1) reducing the size of the tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth or tumor invasiveness, (4) reducing the incidence or ameliorating (that is, reducing to some extent, preferably, eliminating) one or more signs or symptoms associated with the cancer, (5) decreasing the dosage of another medication required to treat the cancer, (6) enhancing the efficacy or safety of another medication used to treat the cancer, and / or (7) delaying the time to progression of the cancer.

[0109] One of ordinary skill in the art would be able to determine such amounts based on such factors as the patient’s size, the severity of the patient’s symptoms, and the particular combination, composition or route of administration selected. The patient or subject may be a human or non-human mammal in need of treatment. In one embodiment, the patient is human.

[0110] As used herein, the term “combination,” unless otherwise indicated, means a fixed-dose combination or a combination of agents that is administered concurrently (co-treatment) or sequentially (staggered), according to the same or different route of administration and according to the same or different dosage schedules. In some embodiments, the combination therapy using a CDK4 inhibitor combined with an additional anti-tumor agent in a staggered dosing schedule.

[0111] In some embodiments, the CDK4 inhibitor (i.e., CDK4 selective inhibitor, or CDK4 / 6 inhibitor) and the additional anti-tumor agent are administered to the subject using a staggered dosing schedule.

[0112] In some embodiments, the combination comprises CDK4 / 6 inhibitor that is palbociclib, and an additional anti-tumor agent that is the combination of SN38 and temozolomide, wherein the use of such combination is effective in treating ewing sarcoma.

[0113] In some embodiments, the combination therapy methods provided herein, using a staggered dosing schedule, can improve efficacy observed in human subjects relative to alternative combination co-treatment methods not using a staggered dosing schedule.

[0114] In some embodiments of each of the combinations and uses described herein, the combination of (i) a CDK4 inhibitor and (ii) either a CDK2 inhibitor or an additional anti-tumor agent, is synergistic, and the invention provides the synergistic combination, or use of the synergistic combination, as described. In some embodiments of the combinations and uses described herein, the combination of a CDK4 inhibitor and a CDK2 inhibitor is synergistic and the invention provides the synergistic combination, or use of the synergistic combination, as described. In some embodiments of the combinations and uses described herein, the combination of a CDK4 inhibitor and an additional anti-tumor agent is synergistic and the invention provides the synergistic combination, or use of the synergistic combination, as described.

[0115] In some embodiments, the combination comprises a CDK4 inhibitor that is 1 ,5-anhydro- 3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6- yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f / 7reo-pentitol, and a CDK2 inhibitor that is (1 R,3S)-3-[3- ({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H-pyrazol-5-yl]cyclopentyl propan-2-yl carbamate, wherein the use of such combination is effective in treating ewing sarcoma.

[0116] In some embodiments, the combination comprises a CDK4 inhibitor that is 1 ,5-anhydro- 3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6- yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f / 7reo-pentitol, and an additional anti-tumor agent that is binimetinib, wherein the use of such combination is effective in treating ewing sarcoma.

[0117] In some embodiments, the combination comprises a CDK4 inhibitor that is 1 ,5-anhydro- 3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6- yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f / 7reo-pentitol, and an additional anti-tumor agent that is the combination of SN38 and temozolomide, wherein the use of such combination is effective in treating ewing sarcoma. In some embodiments, the combination comprises CDK4 / 6 inhibitor that is palbociclib, and a CDK2 inhibitor that is (1 R,3S)-3-[3-({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5- yl]carbonyl}-amino)-1 H-pyrazol-5-yl]cyclopentyl propan-2-yl carbamate, wherein the use of such combination is effective in treating ewing sarcoma.

[0118] In some embodiments, the combination comprises CDK4 / 6 inhibitor that is palbociclib, and an additional anti-tumor agent that is binimetinib, wherein the use of such combination is effective in treating ewing sarcoma.

[0119] In some embodiments, the combination comprises CDK4 / 6 inhibitor that is palbociclib, and an additional anti-tumor agent that is the combination of SN38 and temozolomide, wherein the use of such combination is effective in treating ewing sarcoma.

[0120] The term “additive” is used to mean that the result of the combination of two compounds, components or targeted agents is no greater than the sum of each compound, component or targeted agent individually.

[0121] The term “synergy” or “synergistic” are used to mean that the result of the combination of two or more compounds, components or targeted agents is greater than the sum of each compound, component or targeted agent individually. This improvement in the disease, condition or disorder being treated is a “synergistic” effect and combinations providing a synergistic effect may be referred to as synergistic combinations. A “synergistic amount” is an amount of the combination of the two compounds, components or targeted agents that results in a synergistic effect, as “synergistic” is defined herein.

[0122] Determining a synergistic interaction between one or two components, the optimum range for the effect and absolute dose ranges of each component for the effect may be definitively measured by administration of the components over different dose ranges, or dose ratios to patients in need of treatment. The observation of synergy in in vitro models or in vivo models can be predictive of the effect in humans and other species to measure a synergistic effect. The results of such studies can also be used to predict effective dose and plasma concentration ratio ranges and the absolute doses and plasma concentrations required in humans and other species such as by the application of pharmacokinetic or pharmacodynamics methods.

[0123] A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively. Ma & Motsinger-Reif, Current Method for Quantifying Drug Synergism, Proteom. Bioinform (2019) 1(2):43-48; Tang et al., What is Synergy? The Saariselka Agreement Revisited, Front Pharmacol. (2015) Article 181 , 6: 1-5.

[0124] These and other embodiments of the invention, including the exemplary specific embodiments listed below, will be apparent from the teachings contained herein.

[0125] EXAMPLES

[0126] Materials and Methods

[0127] Cell Culture

[0128] A673 cells were obtained from ATCC and cultured in DM EM supplemented with 10% FBS. MHHES1 cells were obtained from DSMZ cultured in RPMI1640 supplemented with 10% FBS. All cells were maintained in a humidified incubator at 37°C with 5% CO2.

[0129] Animals

[0130] Female NSG mice were obtained from Jackson Laboratories at 6-8 weeks of age and were allowed to acclimate for at least 72 hours before animal manipulation. Mice were housed 5 / cage and were allowed food and water ad libitum with a 12-hour light / dark cycle.

[0131] Compound Formulation

[0132] PF07220060 and palbociclib were formulated as a suspension in 0.5% methylcellulose A4M / water.

[0133] Temozolomide (TMZ) was formulated as a suspension in 0.2% Tween 80 in 0.5% methylcellulose A4M / water

[0134] Irinotecan was formulated in 0.9% Saline (sodium chloride USP).

[0135] In vivo efficacy evaluation and statistical analysis

[0136] Tumor volume and body weights were measured twice a week. Tumor volume was calculated using the [(Length x Width x Width) / 2)] formula. TGI was calculated as 100*(1-AT / AC). The AC (AT) was obtained by subtracting the mean tumor burden in the vehicle (treated) group on the first day of treatment (Day 0) from the mean tumor burden in vehicle (treated) group on the assessment day. Statistical analysis was performed using ANCOVA.

[0137] Example 1 - In Vitro Screen in Ewing Sarcoma Cells

[0138] This example demonstrates that the human ewing sarcoma (ES) cell lines are sensitive to PF-07220060.

[0139] Seven cell lines of Human ewing sarcoma cell lines were evaluated. Human ewing sarcoma cell lines, MHH-ES1 , RD-ES, A-673, SK-NEP-1 , SK-ES-1 , CADO- ES1 and SK-N-MC were obtained from the American Type Culture Collection, ATCC (Manassas, Virginia, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum and penicillin-streptomycin. All cells were maintained in a humidified incubator at 37°C with 5% CO2. 1000 cells per well were seeded into 96 well plates and allowed to incubate overnight. All cell lines were cultured under 2D conditions.

[0140] This example demonstrates that the human ewing sarcoma (ES) cell lines are sensitive to PF-07220060 was tested against various commercially available human ewing sarcoma cell lines over a 6-day treatment period. This example demonstrates that the human ewing sarcoma (ES) cell lines are sensitive to PF-07220060 was checked with a range of concentrations to determine the IC50 of the molecule in actively growing cell cultures.

[0141] Sensitivity coincided with the presence of RB1 wild-type (RB1 WT) across the cell line panel (MHH-ES1 , RD-ES, A-673, SK-NEP-1 , SK-ES-1 , CADO-ES1). RB1 loss-of-function (RB1 LOF) plausibly accounts for resistance to PF-07220060 seen in SK-N-MC cells, which is consistent with the role of RB1 as mediator of CDK4 function and repressor of the endogenous CDK4 inhibitor. Oncogene (2014) 33, 1890-1903.

[0142] The IC50 values are shown in FIG.1 and Table 1 . Six out of seven commercially available ES cell lines are sensitive to This example demonstrates that the human ewing sarcoma (ES) cell lines are sensitive to PF-07220060 (I05o<0.19 pM).

[0143] Table 1 . IC50 values of This example demonstrates that the human ewing sarcoma (ES) cell lines are sensitive to PF-07220060 in various ewing sarcoma cell lines

[0144] Example 2: In-Vitro Study: Effects of Treatment of PF-07220060 alone, in combination with binimetinib, and in combination with PF-07104091 on Human Ewing Sarcoma (ES) Multicellular Tumor Spheroid (MCTS) cell lines This example evaluates the efficacy of PF-07220060 alone, in combination with binimetinib, and in combination with a CDK2 inhibitor in ewing sarcoma MCTS derived from ES cell lines A-673, MHHES-1 and CADO-ES1.

[0145] Spheroid assays were performed in 96 well ultralow attachment plates (ULA-96U) from Nexcelom & Thermo Fisher Scientific. One hundred twenty (120) ES MCTS cells were dispensed in 200 pL of complete growth medium per well (n = 10 to 12 wells per treatment group) of each ultralow attachment plate to allow formation of one spheroid per well with a diameter between 200 and 250 pm before the start of treatment (cell seeding numbers were previously optimized so that formed spheroids possessed this desired dimension). To aid spheroid formation, dispensed cells were centrifuged at 220 x g for 6 minutes in the ultralow attachment plates and allowed to form compact spheroids for 4 days prior to the initiation of treatment. After spheroids were formed, 150 pL of medium was aspirated from each well without disturbing the spheroid, and fresh RPMI medium of the same volume was added containing single agent compounds: palbociclib (‘palbo’), binimetinib (‘bini’), PF-07220060 (‘60’), or PF-07104091 (‘91’), or selected combinations thereof. Final concentrations of each compound in the wells were: 30 or 100 nM for palbociclib; 30 nM binimetinib; 100, 300, 500 or 1000 nM for PF-07220060; and 250, 500 or 1000 nM for PF-07104091. DMSO (0.01 %) was used as the vehicle control. DMSO and all compounds were diluted in cell medium. Medium and compounds were replenished twice per week, with 3 and 4-day intervals. Replenishment was executed by aspirating 150 pL of medium per well without disturbing the spheroid and then adding the same volume of premixed medium / compound solution to spheroids. In some cases, an extended phase of treatment was followed by a ‘recovery’ phase where medium was replenished without addition of compounds. Spheroid average diameter was quantified with a Celigo image cytometer (Celigo 200-BFFL-S, Nexcelcom) twice a week (on every 3rdor 4thday) throughout the duration of the assay, and the spheroid growth curves were obtained (shown in FIGS. 2A-C, FIG. 3, FIG. 4 and FIG. 5 described below).

[0146] Growth of these MCTS was monitored over time to assess the duration of response to PF-07220060 and combination treatments while on treatment. All of treatments of spheroids were initiated at Day 0 (X-axis).

[0147] Treatment of PF-07220060 alone:

[0148] Ewing Sarcoma (ES) cell line derived MCTS (A673, MHHES-1 , and CADO-ES1) were treated with vehicle control (0.01% DMSO), increasing concentrations of 100, 300, or 1000 nM PF-07220060 (‘60’) and compared to palbociclib (‘palbo’) at the concentration of 30 or 100 nM for 3-4 weeks. PF-07220060 and palbo are equipotent at inhibiting growth of ES MCTS at 100 nM but increased PF-07220060 concentrations allow for greater spheroid growth inhibition. PF- 07220060 showed dose-dependent inhibition of ewing sarcoma spheroid growth. (FIGS. 2A, 2B and 2C).

[0149] Treatment of PF-07220060 in combination with PF-07104091 :

[0150] ES cell line derived MOTS (A673) were treated with vehicle control (0.01 % DMSO), 300 nM PF-07220060, 1000 nM PF-07104091 , combination of 30 nM palbociclib and 1000 nM PF- 07104091 , or combination of 300 nM PF-07220060 and 1000 nM PF-07104091 for 5-6 weeks. Cotreatment of PF-07220060 and PF-07104091 leads MCTS growth stasis over time. (FIG. 3).

[0151] ES cell line derived MHHHES1 were treated with vehicle control (0.01% DMSO), 500 nM PF-07220060, 250 or 500 nM PF-07104091 , combination of 500 nM PF-07220060 and 250 nM PF-07104091 , or combination of 500 nM PF-07220060 and 500 nM PF-07104091 for 21 days. Cotreatment of PF-07220060 and PF-07104091 did not show as significant growth stasis in MHHES1 compared to the previous experiment conducted with A673 cells. (FIG. 4).

[0152] Treatment of PF-07220060 in combination with binimetinib:

[0153] ES cell line derived MCTS (A673) were treated with vehicle control (0.01 % DMSO), 300 nM PF-07220060, 30 nM binimetinib (bini), combination of 30 nM palbo and 30 nM binimetinib, or combination of 300 nM PF-07220060 and 30 nM binimetinib for 2-6 weeks. Cotreatment of PF- 07220060 and binimetinib induced only mild combinatorial benefit. (FIG. 5).

[0154] Conclusion:

[0155] PF-07220060 demonstrates superior efficacy against ewing sarcoma derived multicellular tumor spheroids (MCTS) versus palbociclib at clinically relevant concentrations.

[0156] Example 3 - In-Vitro Study: Effects of Treatment of PF-07220060 Alone, Co-treatment of PF-07220060 with Standard of Care Agents SN38 (an Active Metabolite of Irinotecan) plus Temozolomide (‘TMZ’), and Sequential Treatment of SN38 plus TMZ followed by PF- 07220060 on Human ES MCTS Cell Lines

[0157] This example evaluates the efficacy of PF-07220060 alone, in simultaneous and sequential combination with SN38 plus TMZ in ewing sarcoma MCTS derived from ES cell lines A-673 and MHHES-1. Particularly, this example evaluates whether the addition of PF-07220060 to the SN38 plus TMZ regimen would enhance the efficacy of this SN38 plus TMZ chemotherapeutic ES treatment option.

[0158] The average diameter of each spheroid was quantified with an Incucyte S3 imager every 12 hours.

[0159] All treatments of spheroids were initiated at Day 0 (X-axis). Treatment of PF-07220060 alone:

[0160] ES cell line derived MCTS (A-673, MHHES-1) were treated with vehicle control (0.01% DMSO), 300 or 1000 nM of PF-07220060 for three weeks in ES cell line A-673 (FIG. 6A) and ES cell line MHHES-1 (FIG. 6B). Medium change (and drug replacement) was perfomed every 3-4 days.

[0161] The spheroid growth curves in response to PF-07220060 single agent treatment are highly reproducible when recorded with different instruments, i.e., Celigo Image Cytometer (FIGS. 2A-C) versus Incucyte S3 (FIGS. 3A-B), where the latter allows for more frequent spheroid diameter readings (every 12 hours) and increased sample number analysis through utilization of 384 well plates.

[0162] Co-Treatment of PF-07220060 and SN38 plus TMZ:

[0163] Spheroids were treated with vehicle (DMSO) or the indicated treatment combinations for 5 days (TREAT) in ES cell line A-673 (FIG. 7A) and ES cell line MHHES-1 (FIG. 7B); drugs were then removed, and medium changes continued every 3-4 days. SN38 (7-Ethyl-10- hydroxycamptothecin) is an active metabolite of Irinotecan.

[0164] Co-treatment of PF-07220060 with SN38 plus TMZ accelerated regrowth of A-673 and MHHES-1 spheroids when compared to treatment with SN38 plus TMZ alone, thus partially negating the effects of cytotoxic chemotherapy.

[0165] Sequential Treatment of PF-07220060 with SN38 plus TMZ:

[0166] Spheroids were treated with vehicle (DMSO), or PF-07220060 alone, or SN38 plus TMZ alone, or spheroids were co-treated with PF-07220060 and SN38 plus TMZ for 5 days then immediately followed by PF-07220060 alone, or spheroids were first treated with SN38 plus TMZ alone for 5 days then immediately followed by PF-07220060 alone. All treatments were evaluated in ES cell line A-673 (FIG. 8A) and ES cell line MHHES-1 (FIG. 8B).

[0167] The first treatment (TREAT1) was initiated at day 0 and drugs were removed at day 5. The second treatment (TREAT2) followed from day 5 to 21 . The arrow highlights the switch from first treatment to second treatment. Corresponding treatment sequences of the individual compounds or combinations are indicated in the panel. The treatment switch again indicated by the arrow.

[0168] As shown in both FIG. 8A and FIG. 8B, when the 5-day treatment with only SN38 plus TMZ was immediately followed by PF-07220060, spheroid growth stasis was extended from 1 to 3 weeks in the A-673 ES model, and 1 to 2 weeks in the MHHES-1 ES model (3 weeks spanning the entire course of the spheroid growth assay). Conclusion:

[0169] Sequential treatment of SN38 plus TMZ followed by PF-07220060 results in greater growth inhibition than co-treatment, or PF-07220060 alone, or SN38 plusTMZ alone.

[0170] Example 4 - Efficacy evaluation of triple agent combination with PF-07220060 with SN38 plus TMZ, or palbociclib with SN38 plus TMZ in Ewing Sarcoma Cell-Derived Xenograft Models (CDX)

[0171] In vivo studies were performed in two clinically relevant, cell-derived xenograft (CDX) models of ewing sarcoma to determine whether PF-07220060 or palbociclib provided additional anti-tumor efficacy benefit when used in combination with standard of Care agents Irinotecan (SN38) plus Temozolomide (TMZ).

[0172] A673 cells were obtained from ATCC and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MHHES1 cells were obtained from DSMZ cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS. All cells were maintained in a humidified incubator at 37°C with 5% CO2.

[0173] Female NSG mice were obtained from Jackson Laboratories at 6-8 weeks of age and were allowed to acclimate for at least 72 hours before animal manipulation. Mice were housed 5 / cage and were allowed food and water ad libitum with a 12-hour light / dark cycle.

[0174] PF07220060 and palbociclib were formulated as a suspension in 0.5% methylcellulose A4M / water. Temozolomide (TMZ) was formulated as a suspension in 0.2% Tween 80 in 0.5% methylcellulose (A4M) / water Irinotecan was formulated in 0.9% Saline (Sodium Chloride USP).

[0175] Cell Implant and Compound Treatment

[0176] To generate the A673 xenograft model, cells were subcutaneously implanted (1 x 106) into the right upper flank of female NSG mice. When the TV reached a range of 115-170 mm3, animals were matched by tumor size and assigned into treatment groups (n=10 per group) and subsequently treated with: 1) Vehicle (0.5% (w / v) methylcellulose (A4M) in deionized water); 2) SN38 at 3mg / kg (IP) plus TMZ at 35 mg / kg (PO) QDx5 every 21 days; 3) Palbociclib at 10 mg / kg BID; 4) PF-07220060 at 30 mg / kg BID; 5) PF-07220060 at 60 mg / kg BID; 6) PF-07220060 at 30 mg / kg BID plus SN38 +TMZ; 7) PF-07220060 at 60 mg / kg BID plus SN38 +TMZ; 8) Palbociclib at 10 mg / kg BID plus SN38 +TMZ. TGI was assessed on day 28.

[0177] To generate the MHHE-S1 xenograft model, cells were subcutaneously implanted (1 x 106) into the right upper flank of female NSG mice. When the TV reached a range of 121-167 mm3, animals were matched by tumor size and assigned into treatment groups (n=10 per group) and subsequently treated with: 1) vehicle (0.5% (w / v) methylcellulose (A4M) in deionized water); 2) SN38 at 3mg / kg (IP) plus TMZ at 35 mg / kg (PO) QDx5 every 21 days ; 3) Palbociclib at 10 mg / kg BID; 4) PF-07220060 at 60 mg / kg BID; 5) PF-07220060 at 60 mg / kg BID plus IRI+TMZ;

[0178] 6) Palbociclib at 10 mg / kg BID plus SN38 +TMZ; 7) Palbociclib at 10 mg / kg BID plus SN38 +TMZ (co treatment). TGI was assessed on day 35.

[0179] In vivo efficacy evaluation and statistical analysis

[0180] Tumor volume and body weights were measured twice a week. Tumor volume was calculated using the [(Length x Width x Width) / 2)] formula. TGI was calculated as 100*(1-AT / AC). The AC (AT) was obtained by subtracting the mean tumor burden in the vehicle (treated) group on the first day of treatment (Day 0) from the mean tumor burden in vehicle (treated) group on the assessment day. Statistical analysis was performed using ANCOVA.

[0181] Table 2: Efficacy evaluation of PF-07220060 single or combination treatment in the ewing sarcoma A-673 and MHH-ES1 PDX models

[0182] Statistical analysis was performed in GraphPad Prism with an unpaired t test, followed by Mann-Whitney test.a: indicates p <0.05 vs. SM38 plus TMZ;b: indicates p<0.05 vs PF- 07220060;c: indicates p<0.05 vs palbociclib;d: indicates p<0.05 vs palbociclib + SM38 plus TMZ;e: indicates p<0.05 vs palbociclib + SM38 plus TMZ (co-treatment).

[0183] All publications and patent applications cited in the specification are herein incorporated by reference in their entirety. Although the foregoing invention has been described in some detail by way of illustration and example, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMS1. A method of treating cancer in a subject in need thereof comprising: (1) administering to the subject an amount of a cyclin-dependent kinase 4 (CDK4) inhibitor; or (2) a combination therapy which comprises an amount of a CDK4 inhibitor, and (a) an amount of a CDK2 inhibitor; or (b) an amount of an additional anti-tumor agent; wherein the amount of CDK4 and any optional CDK2 or additional anti-tumor agent is alone or together therapeutically effective in treating cancer.

2. The method of claim 1 , wherein the CDK4 inhibitor is a CDK4 selective inhibitor or a CDK4 / 6 inhibitor.

3. The method of claim 1 , wherein the CDK4 inhibitor is a CDK4 selective inhibitor.

4. The method of any one of claims 1 to 3, wherein the CDK4 selective inhibitor is a compound of Formula (I):or a pharmaceutically acceptable salt thereof, wherein:R1is H, F or Cl;R2is C1-C4 alkyl, where said C1-C4 alkyl is optionally substituted by R5;R3is H or C1-C4 alkyl, where said C1-C4 alkyl is optionally substituted by R6;R4is H or F; and each R5and R6is independently OH, F or C1-C2 alkoxy.

5. The method of any one of claims 1 to 4, wherein the compound of Formula (I) is 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz- imidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f / 7reo-pentitol.

6. The method of any one of claims 1 or 2, wherein the CDK4 inhibitor is a CDK4 / 6 inhibitor.

7. The method of any one of claims 1 or 2, wherein the CDK4 / 6 inhibitor is palbociclib.

8. The method of any one of claims 1 to 7, wherein the CDK2 inhibitor that is (1 R,3S)- 3-[3-({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H-pyrazol-5- yl]cyclopentyl propan-2-yl carbamate.

9. The method of any one of claims 1 to 7, wherein the additional anti-tumor agent that is selected from the group consisting of binimetinib, SN38, temozolomide, and combinations thereof.

10. The method of any one of claims 1 to 7, wherein the monotherapy comprises 1 ,5- anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6- yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f / 7reo-pentitol.11 . The method of any of claims 1 to 9, wherein the combination therapy comprises:(a) the CDK4 selective inhibitor that is 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2- hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3- dideoxy-D-f / ireo-pentitol, and the CDK2 inhibitor that is (1 R,3S)-3-[3-({[3- (methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H-pyrazol-5-yl]cyclopentyl propan-2-yl carbamate; or(b) the CDK4 selective inhibitor that is 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2- hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3- dideoxy-D-f / ireo-pentitol, and the additional anti-tumor agent that is binimetinib; or(c) the CDK4 selective inhibitor 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2- hydroxypropan-2-yl)-1-(propan-2-yl)-1 / 7-benz-imidazol-6-yl]pyrimidin-2-yl}amino)-2,3- dideoxy-D-f / ireo-pentitol, and the additional anti-tumor agent that is the combination of SN38 and temozolomide; or(d) the CDK4 / 6 inhibitor that is palbociclib, and the CDK2 inhibitor that is (1 R,3S)-3-[3-({[3-(methoxymethyl)-1-methyl-1 H-pyrazol-5-yl]carbonyl}-amino)-1 H- pyrazol-5-yl]cyclopentyl propan-2-yl carbamate; or(e) the CDK4 / 6 inhibitor that is palbociclib, and the additional anti-tumor agent that is binimetinib; or(f) the CDK4 / 6 inhibitor that is palbociclib, and the additional anti-tumor agent that is the combination of SN38 and temozolomide.

12. The method any of claims 1 to 11, wherein the administration of the CDK4 inhibitor and the adminstration of the additional anti-tumor agent is concurrent.

13. The method any of claims 1 to 11 , wherein the administration of the CDK4 inhibitor and the adminstration of the additional anti-tumor agent is sequential.

14. The method of any of claims 1 to 13, wherein the subject is human.

15. Use of a CDK4 inhibitor in the manufacture of a medicament, optionally in combination with a) a CDK2 inhibitor, or (b) an additional anti-tumor agent, for treating ewing sarcoma.

16. A pharmaceutical combination comprising a CDK4 inhibitor and with a) a CDK2 inhibitor; or b) binimetinib; or c) a further combination of SN38 and temozolomide; and a pharmaceutically acceptable carrier.

17. The combination of claim 16 wherein the combination is not fixed.

18. The combination of claim 16 wherein the combination is fixed.

Images