Combination therapy for the treatment of breast cancer

Abstract

Provided herein are combination therapies useful for treating breast cancer. The combination comprises an ERa inhibitor and a CDK 4 / 6 inhibitor.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 472,345, filed March 16, 2017, which is incorporated herein by reference. Background Technology

[0003] Breast cancer is the most commonly diagnosed malignant tumor in women today, with nearly 200,000 new cases diagnosed annually in the United States and 1.7 million worldwide. Since approximately 70% of breast tumors are estrogen receptor α (ERα) positive, and ERα is a key oncogenic driver in this subtype of tumor (Spicer DV & Pike MC. Breast cancer prevention through modulation of endogenous hormones. Breast Cancer Res Treat. 1993; 28:179-193), several types of therapeutics have been developed to antagonize ERα function, including: 1) selective estrogen receptor modulators (SERDs), such as fulvestrant; 2) selective estrogen receptor modulators (SERMs), such as tamoxifen; and 3) aromatase inhibitors that lower systemic estrogen levels. These therapeutics have been clinically highly effective in reducing the incidence and progression of ERα+ breast tumors. However, there are specific susceptibility traits associated with these different types of compounds. For example, tamoxifen has been shown to activate signal transduction activity in the endometrium, leading to an increased clinical risk of endometrial cancer (Fisher et al., (1994) J. Natl Cancer Inst. April 6; 86(7):527-37; van Leeuwen et al., (1994) Lancet February 19; 343(8895):448-52). Conversely, because fulvestrant is a pure antagonist, it may lead to bone mineral density loss in postmenopausal women, as ERα activity is crucial for bone formation. In addition to the confirmed side effects, clinical resistance is also beginning to emerge with these types of ERα antagonists, highlighting the need to develop next-generation compounds.

[0004] Several mechanisms of resistance have been identified using in vitro and in vivo models of resistance to various endocrine therapies. These include increased ERa / HER2 "cross-talk" (Shou et al., Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2 / neu cross-talk in ER / HER2-positive breast cancer (2004) J Natl Cancer Inst. June 16; 96(12):926-35), aberrant expression of ERa coactivators / corepressors (Osborne et al., Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2 / neu in tamoxifen resistance in breast cancer (2003) J Natl Cancer Inst. March 5; 95(5):353-61) or complete loss of ERa to allow ER-independent growth (Osborne CK, Schiff R (2011) Annu Rev Med 62:233-47).

[0005] To identify clinically significant mechanisms of resistance, recent efforts have also been made to deeply characterize the genetics of endocrine therapy-resistant metastases isolated from patients. Several independent laboratories have recently published a number of genetic lesions observed in resistant tumors that were not observed in the primary tumors (Li et al. (2013) Cell Rep. Sep 26;4(6): 1116-30; Robinson et al. (2013) Nat Genet. Dec;45(12): 1446-51; Toy et al. (2013) Nat Genet. Dec;45(12): 1439-45). Included are highly recurrent mutations in the ligand-binding domain of ESR1 (the gene encoding the ERa protein) that are significantly enriched in about 30% of resistant tumors relative to endocrine therapy-naive tumors (Jeselsohn et al. (2014) Clin Cancer Res. Apr 1;20(7): 1757-67; Li et al. (2013) Cell Rep. Sep 26;4(6): 1116-30; Toy et al. (2013) Nat Genet. Dec;45(12): 1439-45; Robinson et al. (2013) Nat Genet.December; 45(12): 1446-51; Merenbakh-Lamin et al. (2013) D538G mutation in estrogen receptor-alpha: A novel mechanism for acquired endocrine resistance in breast cancer Cancer Res. December 1; 73(23): 6856-64; Yu et al. (2014) Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility Science 7 July; 345(6193): 216-20; Segal and Dowsett (2014) Estrogen receptor mutations in breast cancer— new focus on an old target Clin Cancer Res 4 April; 20(7): 1724-26; Chandarlapaty et al. (2016) Prevalence of ESR1 Mutations in Cell-Free DNA and Outcomes in Metastatic Breast Cancer: A Secondary Analysis of the BOLERO-2 Clinical Trial. JAMA Oncol. 2:1310-1315), suggesting that these mutations can functionally drive clinical resistance.

[0006] Highly recurrent mutations in the ESR1 ligand binding domain are associated with more aggressive and shorter overall survival disease biology compared to wild type ESR1 (Chandarlapaty et al. (2016)). Furthermore, ERa mutations (ERa MUT ) result in constitutive activation of ERa and confer resistance to existing classes of endocrine therapeutics. Current endocrine therapeutics are only partially effective in the ERa MUT environment and the fact that a substantial proportion of endocrine therapy resistant metastases continue to rely on ERa signaling for growth / survival indicates a continuing need for: 1) development of therapeutics capable of overcoming ERa WT / ERa MUTnext generation ERa therapeutic agents that target 1) the abnormal activity of ERa and / or 2) cellular pathways that can further enhance the efficacy of clinical anti-estrogen therapeutic agents.

[0007] Despite advances in the treatment of breast cancer, particularly ERa positive breast cancer, there remains a need to provide improved methods of treatment for breast cancer. SUMMARY

[0008] Embodiments provide combination therapies comprising an effective amount of Compound 1 and an effective amount of a CDK4 / 6 inhibitor. Other embodiments can provide combination therapies comprising an effective amount of Compound 2 or Compound 3 and an effective amount of a CDK4 / 6 inhibitor. In certain embodiments, the CDK 4 / 6 inhibitor is palbociclib. In other embodiments, the CDK 4 / 6 inhibitor is ribociclib. In other embodiments, the CDK 4 / 6 inhibitor is abemaciclib. The combination therapies provided herein can result in an enhanced reduction in breast cancer cell viability and can result in tumor growth inhibition of breast cancer in a patient in need of treatment. In certain embodiments, the breast cancer cells are ER positive breast cancer cells.

[0009] Embodiments can provide a method of treating breast cancer in a patient in need thereof comprising administering to the patient a combination of (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt of a CDK4 / 6 inhibitor. In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 50 mg-1000 mg. In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 50 mg-500 mg. In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 50 mg-300 mg.

[0010] Embodiments can provide a method of treating breast cancer in a patient in need thereof comprising administering to the patient a combination of (E)-4-((2-(4-((E)-1-(1H- indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide, or a pharmaceutically acceptable salt thereof, and a CDK 4 / 6 inhibitor, or a pharmaceutically acceptable salt thereof. In some embodiments, (E)-4-((2-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide, or a pharmaceutically acceptable salt thereof, is administered at a daily dose of 50 mg - 1000 mg. In some embodiments, (E)-4-((2-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide, or a pharmaceutically acceptable salt thereof, is administered at a daily dose of 50 mg - 500 mg. In some embodiments, (E)-4-((2-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide, or a pharmaceutically acceptable salt thereof, is administered at a daily dose of 50 mg - 300 mg.

[0011] Embodiments can provide a method of treating breast cancer in a patient in need thereof comprising administering to the patient a combination of (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 50 mg - 1000 mg. In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 50 mg - 500 mg. In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 50 mg - 300 mg.

[0012] In some embodiments, the CDK 4 / 6 inhibitor is selected from, for example, 6-acetyl-8- cyclopentyl-5-methyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}pyrido[2,3-d]pyrimidin-7(8H)- one (palbociclib); 7-cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-7H-pyrrolo[2,3- d]pyrimidine-6-carboxylic acid dimethylamide (ribociclib); and N-(5-((4-ethylpiperazin-1- yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6- yl)pyrimidin-2-amine (abemaciclib).

[0013] In some embodiments, the CDK 4 / 6 inhibitor is palbociclib. Palbociclib can be administered, for example, at a dose of 75, 100, or 125 mg per day. Typically, one dose is administered orally as a single capsule for continuous days 21, followed by a 7-day rest period.

[0014] In some embodiments, the CDK 4 / 6 inhibitor is ribociclib. Ribociclib can be administered, for example, at a dose of 200, 400, or 600 mg / day. Typically, ribociclib is administered orally as a 200 mg capsule or tablet for 21 consecutive days followed by a 7 day off period.

[0015] In some embodiments, the CDK 4 / 6 inhibitor is abemaciclib. Abemaciclib can be administered, for example, at a dose of 200, 300, or 400 mg / day. Typically, abemaciclib is administered at a dose of 100, 150, or 200 mg / dose twice daily. Abemaciclib is typically administered for 21 consecutive days or for 28 consecutive days followed by a 7 day off period.

[0016] In some embodiments, the CDK 4 / 6 inhibitor is G1T-38 (2'-((5-(4- isopropylpiperazin-1-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'- pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one dihydrochloride). G1T-38 can be administered, for example, at a dose of 10 mg / kg, 50 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 500 mg / kg, or 10-500 mg / kg or 50-300 mg / kg.

[0017] In some embodiments, the CDK 4 / 6 inhibitor is G1T-28 (2'-((5-(4- methylpiperazin-1-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'- pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one). G1T-28 can be administered, for example, at a dose of 190-200 mg / m 2 .

[0018] In some embodiments, the CDK 4 / 6 inhibitor is AT-7519. AT-7519 can be administered, for example, at a dose of 14.4-32.4 mg / m 2 . AT-7519 can be administered every three weeks, on days 1, 4, 8, and 11. In one embodiment, the dose is 27 mg / m 2 , administered at the above frequency.

[0019] In some embodiments, the CDK 4 / 6 inhibitor is FLX-925. In some embodiments, the CDK 4 / 6 inhibitor is avosentan. Avosentan can be administered, for example, in an amount of 8-122 mg / m 2 . Avosentan can be administered as a 72 hour infusion. The maximum tolerated dose of avosentan has been reported to be 40, 50, or 78 mg / m 2 .

[0020] In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3- fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2- enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof are administered as separate formulations. Typically, administration of each formulation is no more than 12 hours apart. In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3- fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2- enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof are administered as a single formulation. In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3- fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2- enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof are administered sequentially with other treatments. In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3- fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2- enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof are administered concurrently.

[0021] In some embodiments, the administration form of (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4- trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but- 2-enamide is the free base form. In some embodiments, the administration form of (E)-N,N- dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1- yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide is the hydrochloride salt form.

[0022] Other embodiments can provide pharmaceutical formulations comprising (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide is in free base form. In some embodiments, (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide is in hydrochloride form.

[0023] Other embodiments can provide the use of a combination of (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor for the treatment of breast cancer. Other embodiments can provide the use of a combination of (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor for the preparation of a medicament for the treatment of breast cancer.

[0024] In some embodiments, (E)-4-((2-(4-((E)-l-(lH-indazol-5-yl)-2- phenylbut-l-en-l-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide, or a pharmaceutically acceptable salt thereof, and a CDK 4 / 6 inhibitor, or a pharmaceutically acceptable salt thereof, are administered as separate formulations. Typically, administration of each formulation is no more than 12 hours apart. In some embodiments, (E)-4-((2-(4-((E)-l-(lH-indazol-5-yl)-2- phenylbut-l-en-l-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide, or a pharmaceutically acceptable salt thereof, and a CDK 4 / 6 inhibitor, or a pharmaceutically acceptable salt thereof, are administered as a single formulation. In some embodiments, (E)-4-((2-(4-((E)-l-(lH-indazol-5-yl)-2- phenylbut-l-en-l-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide, or a pharmaceutically acceptable salt thereof, and a CDK 4 / 6 inhibitor, or a pharmaceutically acceptable salt thereof, are administered sequentially with other therapies. In some embodiments, (E)-4-((2-(4-((E)-l-(lH-indazol-5-yl)-2- phenylbut-l-en-l-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide, or a pharmaceutically acceptable salt thereof, and a CDK 4 / 6 inhibitor, or a pharmaceutically acceptable salt thereof, are administered concurrently.

[0025] In some embodiments, the administration form of (E)-4-((2-(4-((E)-l-(lH-indazol-5-yl)-2- phenylbut-l-en-l-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide is the free base form. In some embodiments, the administration form of (E)-4-((2-(4-((E)-l-(lH-indazol-5-yl)-2- phenylbut-l-en-l-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide is the hydrochloride salt form.

[0026] Other embodiments can provide pharmaceutical formulations comprising (E)-4-((2-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, (E)-4-((2-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide is in free base form. In some embodiments, (E)-4-((2-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide is in hydrochloride form.

[0027] Other embodiments can provide the use of a combination of (E)-4-((2-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor for the treatment of breast cancer. Other embodiments can provide the use of a combination of (E)-4-((2-(4-((E)-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)-N,N-dimethylbut-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor for the manufacture of a medicament for the treatment of breast cancer.

[0028] In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3- fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2- enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof are administered as separate formulations. Typically, administration of each formulation is no more than 12 hours apart. In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3- fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2- enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof are administered as a single formulation. In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3- fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2- enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof are administered sequentially with other therapies. In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3- fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2- enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof are administered concurrently.

[0029] In some embodiments, the administration form of (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4- trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2- enamide is the free base form. In some embodiments, the administration form of (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide is the hydrochloride form.

[0030] Other embodiments can provide pharmaceutical formulations comprising (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide is in free base form. In some embodiments, (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide is in hydrochloride salt form.

[0031] Other embodiments can provide the use of a combination of (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor for the treatment of breast cancer. Other embodiments can provide the use of a combination of (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and a CDK 4 / 6 inhibitor for the preparation of a medicament for the treatment of breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Survival of MCF7.6 cells treated with different doses of Compound 1 and palbociclib for 144 hours is shown. Compound 1 and palbociclib synergistically inhibit the growth of MCF7.6 cells in vitro. Inhibition of cell survival was measured using CellTiter-Glo and the Chalice software was used to calculate the inhibition of each Compound 1 and palbociclib dose combination over the Loewe additivity.

[0033] Figure 2 Survival of MCF7.7 cells treated with different doses of Compound 1 and palbociclib for 144 hours is shown. Compound 1 and palbociclib synergistically inhibit the growth of MCF7.7 cells in vitro. Inhibition of cell survival was measured using CellTiter-Glo and the Chalice software was used to calculate the inhibition of each Compound 1 and palbociclib dose combination over the Loewe additivity.

[0034] Figure 3 ST941 cells treated with different doses of Compound 1 and palbociclib for 144 hours. Compound 1 and palbociclib synergistically inhibit the growth of ST941 cells in vitro. Inhibition of cell viability was measured using CellTiter-Glo and Chalice software was used to calculate the inhibition of each Compound 1 and palbociclib dose combination over Loewe additivity.

[0035] Figure 4 MCF7.6 cells treated with different doses of Compound 2 and palbociclib for 144 hours. Compound 2 and palbociclib synergistically inhibit the growth of MCF7.6 cells in vitro. Inhibition of cell viability was measured using CellTiter-Glo and Chalice software was used to calculate the inhibition of each Compound 2 and palbociclib dose combination over Loewe additivity.

[0036] Figure 5 MCF7.7 cells treated with different doses of Compound 2 and palbociclib for 144 hours. Compound 2 and palbociclib synergistically inhibit the growth of MCF7.7 cells in vitro. Inhibition of cell viability was measured using CellTiter-Glo and Chalice software was used to calculate the inhibition of each Compound 2 and palbociclib dose combination over Loewe additivity.

[0037] Figure 6 ST941 cells treated with different doses of Compound 2 and palbociclib for 144 hours. Compound 2 and palbociclib synergistically inhibit the growth of ST941 cells in vitro. Inhibition of cell viability was measured using CellTiter-Glo and Chalice software was used to calculate the inhibition of each Compound 2 and palbociclib dose combination over Loewe additivity.

[0038] Figure 7 MCF7.7 cells treated with different doses of Compound 3 and palbociclib for 144 hours. Compound 3 and palbociclib synergistically inhibit the growth of MCF7.7 cells in vitro. Inhibition of cell viability was measured using CellTiter-Glo and Chalice software was used to calculate the inhibition of each Compound 3 and palbociclib dose combination over Loewe additivity.

[0039] Figure 8 Oral Compound 1 and palbociclib in ERa WT / Y537SAnti-tumor effect (left) and body weight effect (right) in female nude mice bearing subcutaneous ST941 tumor xenografts derived from breast cancer patients carrying ERa

[0040] Figure 9 Anti-tumor effect (left) and body weight effect (right) in female nude mice bearing subcutaneous ST941 tumor xenografts derived from breast cancer patients carrying ERa WT / Y537S Anti-tumor effect (left) and body weight effect (right) in female nude mice bearing subcutaneous ST941 tumor xenografts derived from breast cancer patients carrying ERa DETAILED DESCRIPTION

[0041] Provided herein is a combination therapy of one or more ER-a inhibitors and one or more CDK 4 / 6 inhibitors, which can be used to treat breast cancer. In some embodiments, the breast cancer is ER-a+. In embodiments, the breast cancer expresses an ER-a mutation, which can be L536Q (Robinson et al., Nat Genet. 2013 Dec;45(12)), L536R (Toy et al., Nat Genet. 2013 Dec;45(12):1439-45), Y537S (Toy et al., Nat Genet. 2013 Dec;45(12):1439-45; Robinson et al., Nat Genet. 2013 Dec;45(12); Jeselsohn et al., Clin Cancer Res. 2014 Apr 1;20(7): 1757-67), Y537N (Toy et al., Nat Genet. 2013 Dec;45(12):1439-45; Jeselsohn et al., Clin Cancer Res. 2014 Apr 1;20(7): 1757-67), Y537C (Toy et al., Nat Genet. 2013 Dec;45(12):1439-45; Jeselsohn et al., Clin Cancer Res. 2014 Apr 1;20(7): 1757-67), and D538G (Toy et al., Nat Genet. 2013 Dec;45(12):1439-45; Robinson et al., Nat Genet. 2013 Dec;45(12); Jeselsohn et al., Clin Cancer Res. 2014 Apr 1;20(7): 1757-67; Merenbakh-Lamin et al., Cancer Res. 2013 Dec 1;73(23):6856-64), all of which are incorporated by reference in their entirety for their teachings of ER-a mutations.

[0042] Accordingly, the combinations disclosed herein can also be used to treat other indications and genotypes. ESR1 mutations (Y537C / N) were recently found in 4 out of 373 endometrial cancers (Kandoth et al., Nature 2013 May 2;497(7447):67-73; Robinson et al., Nat Genet. 2013 Dec;45(12)). Since ESR1 mutation Y537C / N has been shown to significantly drive resistance to the current marketed SOC therapeutic, the compounds disclosed herein can be used to treat ERa MUT endometrial cancer.

[0043] The embodiments reported herein relate to the use of an agent that targets cyclin-dependent kinases (CDK) 4 and 6 to inhibit the cell cycle, which has recently emerged as an effective approach to prevent and overcome endocrine therapy resistance in metastatic ER-positive breast cancer (Mancuso and Massarweh, Endocrine therapy and strategies to overcome therapeutic resistance in breast cancer. Curr Probl Cancer. 2016;40:95-105). In contrast to the enrichment of ESR1 mutations observed in therapy-resistant tumors, mutations in other cancer-associated genes could not show such robust enrichment, strongly suggesting the importance of ERa mutations in promoting resistance (Jeselohn et al. (2014) Clin Cancer Res. Apr 1;20(7): 1757-67).

[0044] Compound 1 is a small molecule ERa inhibitor that has the structure shown in Formula I and the chemical name (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide:

[0045]

[0046] Compound 1 and its synthesis are reported in U.S. Patent Application Publication No. US 2016 / 0347717 Al, published December 1, 2016. This document is incorporated herein by reference. Compound 1 can be administered to a patient at any of the following daily doses when used alone or in combination as described herein: 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. Individual doses can be 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. The daily dose can be part of a cycling regimen. In some embodiments, the cycling regimen is a regimen that lasts for 14 days or 21 days. The daily dose can be administered as a single dose or as multiple doses.

[0047] Compound 2 is a small molecule ERa inhibitor having the structure shown in Formula II and the chemical name (E)-4-((2-(4-((E)-l-(lH-indazol-5-yl)-2-phenylbut-l-en-l- yl)phenoxy)ethyl)amino)-NN-dimethylbut-2-enamide:

[0048]

[0049] Compound 2 and its synthesis are reported in U.S. Patent Application Publication No. US 2016 / 0347717 Al, published December 1, 2016. Compound 2 can also be used, alone or in combination as described herein, as a treatment for breast cancer, including ERa+ breast cancer. When used alone or in combination as described herein, Compound 2 can be administered to a patient at any of the following daily doses: 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. Individual doses can be 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. The daily dose can be part of a cycling regimen. In some embodiments, the cycling regimen is a regimen that lasts for 14 days or 21 days. The daily dose can be administered as a single dose or as multiple doses.

[0050] Compound 3 is a small molecule ERa inhibitor having the structure shown in Formula III and the chemical name (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-l-(3-fluoro-lH-indazol-5-yl)-2- phenylbut-l-en-l-yl)phenoxy)ethyl)amino)but-2-enamide:

[0051]

[0052] Compound 3 and its synthesis are reported in U.S. Patent Application Publication No. US 2016 / 0347717 Al, published December 1, 2016. Compound 3 can also be used, alone or in combination as described herein, as a treatment for breast cancer, including ERa+ breast cancer. When used alone or in combination as described herein, Compound 3 can be administered to a patient at any of the following daily doses: 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. Individual doses can be 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. The daily dose can be part of a cycling regimen. In some embodiments, the cycling regimen is a regimen that lasts for 14 days or 21 days. The daily dose can be administered as a single dose or as multiple doses.

[0053] Palbociclib (6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(piperazin-l-yl)pyridin-2-yl]amino}pyrido[2,3- d]pyrimidin-7(8H)-one) is an FDA-approved inhibitor of cyclin-dependent kinases (CDK) 4 and 6. Palbociclib has the following structure:

[0054]

[0055] See U.S. Patent Nos. 6,936,612; 7,208,489; and 7,456,168, which are incorporated herein by reference. Palbociclib in combination with endocrine therapy agents has shown activity in both first- and second-line metastatic disease settings, resulting in a significant improvement in PFS, suggesting that combination therapy can delay the onset of resistance in patients receiving endocrine therapy agents (Finn et al. The cyclin-dependent kinase 4 / 6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1 / TRIO-18): a randomised phase 2 study. Lancet Oncol. (2015) 16(1): 25-35). More recently, the phase III PALOMA-3 trial also showed significant activity of palbociclib in combination with fulvestrant in patients progressing on aromatase inhibitor therapy, suggesting that this combination can also be a viable strategy to overcome resistance to endocrine therapy agents.

[0056] Ribociclib (7-cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-7H-pyrrolo[2,3- d]pyrimidine-6-carboxylic acid dimethylamide) is an FDA-approved inhibitor of cyclin- dependent kinases (CDK) 4 and 6. Ribociclib has the following structure:

[0057]

[0058] See U.S. Patent Application Publication No. US20120115878; PCT Publication No. WO2007140222; PCT Publication No. WO2012061156; PCT Publication No. WO2011130232; PCT Publication No. WO2011101417; and PCT Publication No. WO2010020675, all of which are incorporated herein by reference.

[0059] Ribociclib (7-cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-7H-pyrrolo[2,3- d]pyrimidine-6-carboxylic acid dimethylamide) is an FDA-approved inhibitor of cyclin- dependent kinases (CDK) 4 and 6. Ribociclib has the following structure:

[0060]

[0061] See O'Leary et al., "Treating Cancer with Selective CDK 4 / 6 Inhibitors" Nat. Rev. (published online March 31, 2016); PCT Publication No. WO2016110224, U.S. Patent Application Publication No. 20100160340; and PCT Publication No. WO2016025650, all of which are incorporated herein by reference.

[0062] G1T-38 (also known as GZ-38-1 or G1T38-1) is an inhibitor of CDK 4 / 6 that has been reported. G1T-38 was studied by G1 Therapeutics, Inc. of Research Triangle Park, North Carolina, and reported in Abstract #2824 at the 2016 AACR Annual Meeting, April 16-20 in New Orleans, Louisiana, entitled "G1T38, A Novel, Oral, Potent and Selective CDK 4 / 6 Inhibitor for the Treatment of RB Competent Tumors," by J. Sorrentino, J. Bisi, P. Roberts, and J. Strum, which is incorporated herein by reference. G1T38 has the chemical name 2'-((5-(4- isopropylpiperazin-1-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'- pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one dihydrochloride, and has the structure shown below:

[0063]

[0064] See Bisi et al.,“Preclinical development of G1T38: A novel, potent and selective inhibitor of cyclin dependent kinases 4 / 6 for use as an oral antineoplastic in patients with CDK4 / 6 sensitive tumors,” Oncotarget, Advance Publications 2017 (March 15, 2017); U.S. Patent Application Publication No. US 20140275066 Al; U.S. Patent No. 9,487,530 B2; and PCT International Patent Application Publication No. WO 2014144326, all of which are incorporated herein by reference.

[0065] G1T-28 (also known as trilaciclib) is a CDK 4 / 6 inhibitor with the name 2’-((5-(4- methylpiperazin-1-yl)pyridin-2-yl)amino)-7’,8’-dihydro-6’H-spiro[cyclohexane-1,9’- pyrazino[1’,2’:1,5]pyrrolo[2,3-d]pyrimidin]-6’-one. G1T-28 has the following structure:

[0066]

[0067] See, e.g., Bisi et al.,“Preclinical Characterization of G1T28: A Novel CDK4 / 6 Inhibitor for Reduction of Chemotherapy-induced Myelosuppression” Mol. Cancer Ther.; 15(5) 783-93, May 2016; U.S. Patent Application Publication No. US20160220569; PCT International Patent Application Publication Nos. WO2014144326; WO2014144847; and WO2016040848, all of which are incorporated herein by reference.

[0068] AT-7519 is a CDK 4 / 6 inhibitor with the name N-(4-piperidinyl)-4-(2,6-dichlorobenzoyl amino)-1H- pyrazole-3-carboxamide. AT-7519 has the following structure:

[0069]

[0070] See, e.g., PCT International Patent Application Publication Nos. WO 2005012256; WO 2006077424; WO 2006077426; WO 2008001101; WO 2006077425; WO 2006077428; WO 2008007113; WO 2008007122; and WO 2008009954, all of which are incorporated herein by reference.

[0071] FLX-925 (also known as AMG-925) is a CDK 4 / 6 inhibitor having the name 2-hydroxy-1-[2-[[9-(trans-4-methylcyclohexyl)-9H-pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidin-2-yl]amino]-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]ethanone. FLX-925 has the following structure:

[0072]

[0073] See, e.g., U.S. Patent Application Publication No. 2014163052 and PCT International Patent Application Publication No. WO 2012129344, both of which are incorporated herein by reference.

[0074] Abemaciclib is a CDK 4 / 6 inhibitor having the name 2-(2-chlorophenyl)-5,7-dihydroxy-8-((3S,4R)-3-hydroxy-1-methylpiperidin-4-yl)-4H-chromen-4-one. Abemaciclib has the following structure:

[0075]

[0076] See, e.g., U.S. Patent Application Publication Nos. US2011189175 and US2011189175; PCT International Patent Application Publication Nos. WO 2000044362; WO 2001041747; WO 2001053293; WO 2001053294; WO 2002022133; WO 2007010946, all of which are incorporated herein by reference.

[0077] In some embodiments, the combination therapy comprises administration of a combination of Compound 1 and a CDK 4 / 6 inhibitor. In certain embodiments, the CDK 4 / 6 inhibitor is palbociclib. In other embodiments, the CDK 4 / 6 inhibitor is ribociclib. In yet other embodiments, the CDK 4 / 6 inhibitor is abemaciclib. In yet other embodiments, the CDK 4 / 6 inhibitor is G1T-38. In other embodiments, the CDK 4 / 6 inhibitor is relacimib. In yet other embodiments, the CDK 4 / 6 inhibitor is AT-7519. In other embodiments, the CDK 4 / 6 inhibitor is FLX-925. In other embodiments, the CDK 4 / 6 inhibitor is alvocidib.

[0078] Provided herein are combinations of therapeutic agents and methods of administering the combinations of therapeutic agents to treat breast cancer. As used herein, "combination of therapeutic agents" and like terms refer to a combination of two types of therapeutic agents: (1) Compound 1 and / or a pharmacologically active salt thereof and (2) a CDK 4 / 6 inhibitor and / or a pharmacologically active salt thereof. As used herein, "combination" (including the term "combination of therapeutic agents") refers to these types of therapeutic agents being co-formulated in a single dosage form, separately formulated and co-administered, or separately formulated and administered sequentially.

[0079] Compound 1 is a small molecule ERa inhibitor having the structure shown in Formula I and the chemical name (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide:

[0080]

[0081] Compound 1 and its synthesis are reported in U.S. Patent Application Publication No. US 2016 / 0347717 Al, published December 1, 2016. This document is incorporated herein by reference. Compound 1 can also be used, alone or in combination as described herein, for the treatment of breast cancer, including ERa+ breast cancer. When used alone or in combination as described herein, Compound 1 can be administered to a patient at any of the following daily doses: 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. Individual doses can be 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. The daily dose can be part of a cycling regimen. In some embodiments, the cycling regimen is a regimen that lasts for 14 days or 21 days. The daily dose can be administered as a single dose or as multiple doses.

[0082] Compound 2 is a small molecule ERa inhibitor that has the structure shown in Formula II and the chemical name (E)-4-((2-(4-((E)-l-(lH-indazol-5-yl)-2-phenylbut-l-en-l- yl)phenoxy)ethyl)amino)-NN-dimethylbut-2-enamide:

[0083]

[0084] Compound 2 and its synthesis are reported in U.S. Patent Application Publication No. US 2016 / 0347717 Al, published December 1, 2016. Compound 2 can also be used, alone or in combination as described herein, for the treatment of breast cancer, including ERa+ breast cancer. When used alone or in combination as described herein, Compound 2 can be administered to a patient at any of the following daily doses: 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. Individual doses can be 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. The daily dose can be part of a cycling regimen. In some embodiments, the cycling regimen is a regimen that lasts for 14 days or 21 days. The daily dose can be administered as a single dose or as multiple doses.

[0085] Compound 3 is a small molecule ERa inhibitor having the structure shown in Formula III and the chemical name (E)-N,N-dimethyl-4-((2-(4-((E)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenoxy)ethyl)amino)but-2-enamide:

[0086]

[0087] Compound 3 and its synthesis are reported in U.S. Patent Application Publication No. US 2016 / 0347717 Al, published December 1, 2016. This document is incorporated herein by reference. Compound 3 can also be used, alone or in combination as described herein, as a treatment for breast cancer, including ERa+ breast cancer. When used alone or in combination as described herein, Compound 3 can be administered to a patient at any of the following daily doses: 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. Individual doses can be 50 mg - 1000 mg; 50 mg - 500 mg; 50 mg - 300 mg; 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1000 mg. The daily dose can be part of a cycling regimen. In some embodiments, the cycling regimen is a regimen that lasts for 14 days or 21 days. The daily dose can be administered as a single dose or as multiple doses.

[0088] CDK 4 / 6 inhibitors suitable for use herein can include, for example, ribociclib, palbociclib, and abemaciclib, G1T-38, relaciclib, AT-7519, FLX-925, and alvocidib, as well as pharmaceutically acceptable salts and hydrates thereof.

[0089] Administration of a therapeutic agent combination includes administration of the individual therapeutic agents of the combination in a single formulation or unit dosage form, administration of the individual therapeutic agents of the combination simultaneously but separately, or administration of the individual therapeutic agents of the combination sequentially by any appropriate route. The dosages of the individual therapeutic agents of the combination can require that one agent be administered more frequently than the other agent(s) in the combination. Thus, to allow for appropriate administration, a packaged pharmaceutical product can contain one or more dosage forms containing the combination of agents, and one or more dosage forms containing one agent of the combination of agents but not the other agent(s) of the combination.

[0090] The combinations reported herein can include embodiments in which one or more of Compound 1 and the CDK 4 / 6 inhibitor are administered as a pharmaceutically acceptable salt or free base. There is no requirement that both compounds be administered as the same pharmaceutically acceptable salt, but they can be. In particular embodiments, the combination comprises the free base form of Compound 1 and the free base form of the CDK 4 / 6 inhibitor. In other embodiments, the combination comprises the HC1 form of Compound 1 and the HC1 form of the CDK 4 / 6 inhibitor. In some embodiments, the CDK 4 / 6 inhibitor can be the free base. In some embodiments, the CDK 4 / 6 inhibitor can be a pharmaceutically acceptable salt. In some embodiments, the CDK 4 / 6 inhibitor can be a hydrate.

[0091] As used herein, "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt of a compound in the disclosure. A pharmaceutically acceptable salt is any salt of the parent compound that retains its activity and does not impart any undesired or adverse effects in its intended environment of administration to its intended individual. Pharmaceutically acceptable salts include, but are not limited to, metal complexes and salts of inorganic and carboxylic acids. Pharmaceutically acceptable salts also include metal salts, such as aluminum, calcium, iron, magnesium, manganese, and complex salts. In addition, pharmaceutically acceptable salts include, but are not limited to, acid salts, such as acetate, aspartate, alkylsulfonate, arylsulfonate, axetil, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, butyrate, calcium edetate, camphorsulfonate, carbonate, chlorobenzoate, citrate, edetate, edisylate, estolic, esyl, esylic, formate, fumarate, gluceptic, gluconate, glutamate, glycolate, glycolylarsanilic, hexamic, hexylresorcinoic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoate, isethionic, lactic, lactobionic, maleic, malic, malonic, mandelic, mesylic, methylnitrate, methylsulfate, mucic, muconic, napsylic, nitric, oxalic, p-nitromethanesulfonic, pamoic, pantothenic, phosphoric, monohydrogen, dihydrogen, phthalic, polygalacturonic, propionic, salicylic, stearic, succinic, sulfamic, sulfanilic, sulfonic, sulfuric, tannic, tartaric, teoclic, toluenesulfonic, and the like.

[0092] The embodiment can be a hydrochloride salt. Pharmaceutically acceptable salts can be derived from amino acids, including but not limited to cysteine. Methods of making salt forms of compounds are known to those skilled in the art (e.g., see Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J. Pharm. Sci. 66: 1, 1977).

[0093] An“effective amount” of a therapeutic agent combination (e.g., Compound 1 and a CDK 4 / 6 inhibitor) is an amount sufficient to provide an observable therapeutic benefit compared to untreated breast cancer in an individual or patient.

[0094] The active agents described herein can be combined with a pharmaceutically acceptable carrier to provide pharmaceutical formulations thereof. The specific choice of carrier and formulation depends on the particular route of administration intended for the composition.

[0095] As used herein, a“pharmaceutically acceptable carrier” refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of this application include, but are not limited to, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, sodium phosphate dibasic, potassium phosphate monobasic, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene glycol, and lanolin.

[0096] The compositions of the present application can be suitable for parenteral, oral, inhalation spray, topical, rectal, nasal, buccal, vaginal, or implant reservoir administration, among others. In some embodiments, the formulation comprises ingredients from natural or non-natural sources. In some embodiments, the formulation or carrier can be provided in sterile form. Non-limiting examples of sterile carriers include endotoxin-free water or pyrogen-free water.

[0097] The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In particular embodiments, the compounds are administered by intravenous, oral, subcutaneous, or intramuscular administration. Sterile injectable forms of the compositions of the present application can be aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0098] For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids, such as those of olive oil, have also been described for use in injection formulations. These oil solutions or suspensions also can contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers which are commonly used in manufacturing pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.

[0099] For oral administration, the compounds or salts can be provided in an acceptable oral dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, can also be added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient can be combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added. In addition, preserving agents can be added. Suitable examples of pharmaceutically acceptable preservatives include, but are not limited to, various antibacterial and antifungal agents, such as solvents, e.g., ethyl alcohol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (e.g., methyl paraben, ethyl paraben, propyl paraben, etc.).

[0100] "Immediate release" is intended to include conventional release, in which the drug begins to release immediately after administration. The term "immediate release" as used herein includes dosage forms that allow dissolution of the drug in the gastrointestinal contents without the intent to delay or prolong dissolution or absorption of the drug. The goal is rapid release of the drug after administration, e.g., at least 80% of the drug can be released within about 30 minutes after the onset of dissolution in a dissolution test.

[0101] "Continuous release" or "extended release" includes dosage forms whose drug release time course and / or location characteristics are selected to achieve a therapeutic or convenience objective that would not be provided by conventional dosage forms such as solutions or immediate-release dosage forms.

[0102] The term "steady state" refers to the blood plasma levels of a given active agent or combination of active agents that have reached a level that is equal to or above the minimum effective therapeutic level of a given active agent and below the minimum toxic plasma level of a given active agent, and subsequent doses of the active agent(s) keep it at that level.

[0103] The term "single formulation" as used herein refers to a single carrier or vehicle that is formulated to deliver an effective amount of both therapeutic agents to a patient. The single vehicle is designed to deliver an effective amount of each agent as well as any pharmaceutically acceptable carriers or excipients. In some embodiments, the vehicle is a tablet, capsule, pill, or patch.

[0104] The term "unit dose" is used herein to refer to the simultaneous administration of both agents to a patient being treated in one dosage form. In some embodiments, the unit dose is a single formulation. In certain embodiments, the unit dose includes one or more vehicles such that each vehicle contains an effective amount of at least one agent (Compound 1 or a CDK 4 / 6 inhibitor) as well as pharmaceutically acceptable carriers and excipients. In some embodiments, the unit dose is one or more tablets, capsules, pills, or patches that are administered to a patient simultaneously. When agents are "administered simultaneously," they can be administered as a single unit dose or as separate doses within close proximity in time; in one non-limiting example, the two agents can be administered separately within five minutes of each other.

[0105] The term "dose range" as used herein refers to the upper and lower limits of acceptable variation of a specified agent dose. Typically, any amount of an agent dose within a specified range can be administered to a patient being treated.

[0106] The term "treat" is used herein to refer to alleviating, abating or ameliorating at least one symptom of a disease in a subject. For example, with respect to breast cancer, the term "treat" can refer to halting, delaying onset (i.e., the period prior to clinical manifestation of a disease or disease symptoms), and / or reducing the risk of developing or worsening of disease symptoms. The term "protect" is used herein to refer to preventing, delaying or treating (or all, as the case can be) the development or persistence or worsening of a symptom of a disease in a subject.

[0107] The term "subject" or "patient" is intended to include an animal that can have or be afflicted with breast cancer. Examples of subjects or patients include mammals such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human having breast cancer, at risk of having breast cancer, or potentially capable of having breast cancer.

[0108] The term "about" or "approximately," refers generally to within 20%, more preferably within 10%, and most preferably still within 5%, of a given value or range. Alternatively, especially in biological systems, the term "about" means within an order of magnitude, i.e., within a factor of about 10, of a given value.

[0109] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0110] Exemplary cell proliferative disorders that can be treated using one or more of the compounds disclosed herein include, but are not limited to, breast cancer, a precancerous or precancer condition of the breast, a benign growth or lesion of the breast, and a malignant growth or lesion of the breast, as well as metastatic lesions in tissues and organs other than the breast. Cell proliferative disorders of the breast can include hyperplasia, metaplasia, and dysplasia of the breast.

[0111] The breast cancer to be treated can occur in a male or female individual. The breast cancer to be treated can occur in a premenopausal female individual or a postmenopausal female individual. The breast cancer to be treated can occur in an individual who is 30 years of age or older or an individual who is less than 30 years of age. The breast cancer to be treated can occur in an individual who is 50 years of age or older or an individual who is less than 50 years of age. The breast cancer to be treated can occur in an individual who is 70 years of age or older or an individual who is less than 70 years of age.

[0112] The compounds disclosed herein, or pharmaceutically acceptable salts thereof, can be used to treat or prevent a cell proliferative disorder of the breast, or to treat or prevent breast cancer in an individual at increased risk of developing breast cancer relative to the majority of the population, or to identify suitable candidates for these purposes. An individual at increased risk of developing breast cancer relative to the majority of the population is a female individual with a family or personal history of breast cancer. An individual at increased risk of developing breast cancer relative to the majority of the population is a female who is greater than 30 years of age, greater than 40 years of age, greater than 50 years of age, greater than 60 years of age, greater than 70 years of age, greater than 80 years of age, or greater than 90 years of age.

[0113] The term "enhanced effect" as used herein refers to an effect that is stronger or better provided by the action of two agents administered together as compared to the administration of the individual agents alone without co-administration of the other agent. An enhanced effect can be provided by the administration of the agents together, either simultaneously or sequentially. Sequential administration of the agents includes administration that is separated by seconds, minutes, hours, or days. An enhanced effect can be provided by the administration of the agents together when the agents are administered as part of a single formulation or in separate formulations. Examples of agents that can be administered together include Compound 1 and a CDK4 / 6 inhibitor. Other examples of agents that can be administered together include: i) Compound 1 and ribociclib; ii) Compound 1 and palbociclib; and iii) Compound 1 and abemaciclib.

[0114] The stronger or better effect of the enhanced effect can include, for example, one or more of: i) an improved quality of tumor response, ii) an improved speed of tumor response, and iii) a tumor response that is greater than the sum of the responses that would have been achieved by the individual agents administered alone. An example of an improved quality of tumor response can include complete remission (CR) instead of partial remission (PR), stable disease (SD), or progressive disease (PD). Another example of an improved quality of tumor response can include partial remission (PR) instead of stable disease (SD) or progressive disease (PD). Another example of an improved quality of tumor response can include stable disease (SD) instead of progressive disease (PD). For example, control studies can be performed in mice, rats, dogs, monkeys, or other animals to determine whether administration of the agents together achieves an enhanced effect of tumor response such that the tumor response is greater than the sum of the corresponding responses that would have been achieved by the individual agents administered separately. Such control studies can assess, for example, tumor volume or metastasis or other status produced. Likewise, control studies can be used to determine an enhanced effect to achieve a faster tumor response.

[0115] Therapeutic methods

[0116] Provided herein are combination therapies useful for the treatment of breast cancer. As described below, the combinations provided herein can have a number of advantages.

[0117] One advantage of the combinations disclosed herein is the unexpected enhanced effect of the combination of Compound 1 and a CDK 4 / 6 inhibitor on tumor growth inhibition and treatment of breast cancer.

[0118] In some embodiments, provided herein are single pharmaceutical formulations containing a combination of Compound 1 and a CDK 4 / 6 inhibitor. An advantage provided herein is that the therapeutic effect on breast cancer is enhanced compared to treatment with either agent in a single dose. The "pill burden" on the breast cancer patient is not increased when the agents are provided in a single unit dose or single formulation.

[0119] As described above, in one aspect, provided herein is a pharmaceutical combination useful for treating, preventing, arresting, delaying onset of, and / or reducing the risk of developing, or reversing breast cancer in a mammal, comprising administering to the mammal a combination therapy comprising an effective amount of Compound 1 and an effective amount of a CDK 4 / 6 inhibitor.

[0120] In some embodiments, the individual (e.g., patient) to be treated is determined to be non-responsive or resistant to one or more breast cancer therapeutic agents (e.g., Compound 1). In other embodiments, the individual to be treated is responsive to a Compound 1 therapeutic agent, but administration of a CDK 4 / 6 inhibitor can improve the treatment. For example, a patient is administered Compound 1 (e.g., 50-600 mg per day, 200-400 mg per day, or 300 mg per day, for a period of time, e.g., more than one day, more than two days, more than three days, more than one week, for 21 days, more than one month, etc.). Thereafter, a CDK 4 / 6 inhibitor can be administered to the patient in combination with Compound 1.

[0121] The amount of CDK 4 / 6 inhibitor can vary depending on the CDK 4 / 6 inhibitor used. For example, palbociclib can be administered at a dose of, e.g., 75, 100, or 125 mg per day; ribociclib can be administered at a dose of, e.g., 200, 400, or 600 mg per day. Typically, one dose is administered orally as a single capsule for 21 consecutive days, followed by a 7-day rest period.

[0122] The daily dose can be part of a cycle regimen that lasts for 14 to 21 days or more. The daily dose can be administered as a single dose or multiple doses.

[0123] One skilled in the art appreciates that the effective dose of an active drug can be less than the actual amount administered. Thus, provided herein are the amounts necessary to achieve a therapeutic dose.

[0124] In various embodiments, provided herein are methods of treating breast cancer by administering to an individual having breast cancer an effective amount of Compound 1 and a CDK 4 / 6 inhibitor. The amounts of the combination of agents are effective to treat breast cancer. In one embodiment, the combination of agents has a synergistic effect. In one embodiment, even though one or more of the agents is effective when administered alone at a particular dose, the treatment is more effective when administered in combination at the same dose of each agent. For example, in one embodiment, the combination of Compound 1 and palbociclib is more effective than either agent administered alone. In another embodiment, the combination of Compound 1 and ribociclib is more effective than either agent administered alone.

[0125] Dose

[0126] The optimal dosage of the pharmaceutical combination for the treatment of breast cancer can be empirically determined for each individual using known methods, and will depend on a variety of factors, including the activity of the pharmaceuticals; the age, body weight, general health, sex and diet of the individual; the time and route of administration; and the other medications the individual is taking. The optimal dosage can be determined using routine testing and procedures well known in the art.

[0127] For the combination therapy of the present application, the daily dosage of Compound 1 is in the range of 50 mg - 1000 mg. In some embodiments, the daily dosage of Compound 1 is up to 1000 mg. In certain embodiments, the daily dosage of Compound 1 is up to 500 mg. In various embodiments, the daily dosage of Compound 1 is up to 300 mg. In certain embodiments, the daily dosage of Compound 1 is 50 mg. In one embodiment, the daily dosage is 300 mg.

[0128] For the combination therapy of the present application, the daily dosage of Compound 2 is in the range of 50 mg - 1000 mg. In some embodiments, the daily dosage of Compound 2 is up to 1000 mg. In certain embodiments, the daily dosage of Compound 2 is up to 500 mg. In various embodiments, the daily dosage of Compound 2 is up to 300 mg. In certain embodiments, the daily dosage of Compound 1 is 50 mg. In one embodiment, the daily dosage is 300 mg.

[0129] For the combination therapy of the present application, the daily dosage of Compound 3 is in the range of 50 mg - 1000 mg. In some embodiments, the daily dosage of Compound 3 is up to 1000 mg. In certain embodiments, the daily dosage of Compound 3 is up to 500 mg. In various embodiments, the daily dosage of Compound 3 is up to 300 mg. In certain embodiments, the daily dosage of Compound 3 is 50 mg. In one embodiment, the daily dosage is 300 mg.

[0130] The time of administration can be chosen such that both drugs are administered simultaneously, separately or sequentially in the morning or in the evening. Alternatively, one drug can be administered in the morning and the other drug in the evening. In certain embodiments, both drugs can be administered once a day as a single tablet, capsule, pill, patch or jelly formulation, in the morning or in the evening.

[0131] The amount of the pharmaceutical combination that can be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration. In some embodiments, a unit dosage form containing the pharmaceutical combination described herein will contain an amount of each pharmaceutical in the combination which is generally administered at a time.

[0132] Pharmaceutical formulations and routes of administration

[0133] Provided herein are pharmaceutical formulations comprising a combination of agents for the treatment of breast cancer. The pharmaceutical formulations can additionally comprise a carrier or excipient, a stabilizer, a flavoring agent, and / or a coloring agent.

[0134] The combination of agents can be administered using a variety of routes of administration known to those of skill in the art. Routes of administration include oral administration. In certain embodiments, the pharmaceutical formulation comprising the combination of agents can be administered orally in the form of a liquid, syrup, tablet, capsule, powder, spray, chewable tablet, or dissolvable wafer. Alternatively, the pharmaceutical formulations of the present application can be administered intravenously or transdermally. Other routes of administration are known to those of skill in the art (see, e.g., Remington's Pharmaceutical Sciences, Gennaro A.R., ed., 20th ed., Mack Publishing Co., Easton, Pa.).

[0135] In some embodiments, Compound 1 and a CDK 4 / 6 inhibitor are formulated as a paste, jelly, or suspension. For example, the drug is dissolved, entrapped, or suspended in a gelatinous solution or semi-solid in the form of drug particles, microencapsulated particles, or drug-polymer particles. One advantage of oral jelly formulations is that it is easier to administer the drug to patients who have difficulty swallowing tablets, capsules, or pills. In certain embodiments, the two agents are thoroughly mixed and suspended in a suitable medium to form a paste or gel. Other agents can optionally be mixed to provide flavor during oral administration. Peanut butter or alginic acid flavored with raspberries and sweeteners are examples of many suitable taste masking agents. In various embodiments, the paste or jelly can also be formulated with suitable binders or excipients known in the art for topical administration.

[0136] Methods of preparing sustained release formulations in the form of tablets, capsules, or pills are known in the art. In some embodiments, sustained release formulations are prepared by coating the active ingredients of the drug with a polymer, preferably a water-insoluble polymer. For example, water-insoluble polymers used in the pharmaceutical arts as sustained release coating agents, enteric coating agents, or gastric coating agents. The water-insoluble polymers can include, for example, ethyl cellulose, purified shellac, white shellac, aminoalkyl methacrylate copolymer RS, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, methacrylic acid copolymer L, methacrylic acid copolymer LD, methacrylic acid copolymer S, aminoalkyl methacrylate copolymer E, or polyvinyl acetal diethylaminoacetate.

[0137] The type, degree of substitution, and molecular weight of the water-insoluble polymer can depend on the solubility of the active ingredient in water or alcohol, the desired sustained release level, etc. The water-insoluble polymer can be used alone or in combination. Hydrogenated oil, stearic acid, or cetyl alcohol can also be incorporated as a coating aid, and medium-chain triglyceride, triacetin, or cetyl alcohol as a plasticizer.

[0138] In some embodiments, the sustained release formulation is a matrix-type tablet or granule. The active ingredient can be coated with up to three different types of polymers. The three different types of polymers can include: 1) a water-insoluble polymer, such as ethyl cellulose; 2) a pH-independent gelling polymer, such as hydroxypropyl methyl cellulose; and 3) a pH-dependent gelling polymer, such as sodium alginate. The three different types of polymers can be used together to reduce the release rate of the drug.

[0139] Dosage form: Release property

[0140] The sustained release formulation can achieve a certain degree of sustained effect. However, the exposure and / or bioavailability of the active ingredient can vary based on a variety of factors, such as the absorption window, the carrier or excipient used in the formulation, the delivery mode of the formulation, and / or the transit time of the active ingredient through the gastrointestinal tract of the patient.

[0141] The combination therapy can contain at least one sustained release part for performing a sustained release function and one immediate release part for performing an immediate release function. In certain embodiments, when the combination therapy is a single dosage form, it can be in the form of a tablet formed from a mixture of sustained release granules constituting the sustained release part and immediate release granules constituting the immediate release part, a capsule formulation obtained by filling a capsule with sustained release granules and immediate release granules, or a compressed coated tablet in which an outer layer constituting the immediate release part is formed on an inner core constituting the sustained release part. However, there is no limitation to the above-described embodiments.

[0142] Furthermore, there is no particular limitation to the state of containment of each drug in the composition or the immediate release part or the sustained release part; Compound 1 can be uniformly dispersed in the composition, the immediate release part, or the sustained release part, or can be contained only in a portion of the composition, the immediate release part, or the sustained release part, or can be contained in a manner having a concentration gradient.

[0143] The sustained release part in the composition according to the present application can contain at least one pH-independent polymer substance or pH-dependent polymer substance for controlling the release of the drug.

[0144] The polymer substance independent of pH used herein can include a polymer substance whose charge state is hardly changed under the pH conditions usually found in the gastrointestinal tract, particularly pH 1 to pH 8. This means, for example, that the polymer substance does not have a functional group whose charge state changes with pH, such as a basic functional group such as an amino group or an acidic functional group such as a carboxylic acid group. Note that the polymer substance independent of pH can be included for the purpose of imparting sustained release to the composition according to the present application, but can also be included for another purpose. Furthermore, the polymer substance independent of pH used in the present application can be water-insoluble, or can swell in water or dissolve in water to form a gel.

[0145] Examples of the water-insoluble pH-independent polymeric substances include, but are not limited to, cellulose ethers, cellulose esters, and methacrylic acid-acrylic acid copolymers (trade name Eudragit, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany). Examples include, but are not limited to, cellulose alkyl ethers such as ethyl cellulose (trade name Ethocel, manufactured by Dow Chemical Company, USA), ethyl methyl cellulose, ethyl propyl cellulose, or isopropyl cellulose, and butyl cellulose; cellulose arylalkyl ethers such as benzyl cellulose; cellulose cyanoalkyl ethers such as cyanoethyl cellulose; cellulose organic acid esters such as cellulose acetate butyrate, cellulose acetate, cellulose propionate, or cellulose butyrate, and cellulose acetate propionate; ethyl acrylate-methyl methacrylate copolymer (trade name Eudragit NE, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), and methacrylic acid aminoalkyl ester copolymer RS (trade names Eudragit RL, Eudragit RS). The average particle diameter of the water-insoluble polymer used in the present application is not particularly limited, but generally the smaller the average particle diameter, the better the performance, and the average particle diameter is preferably 0.1 to 100 μm, more preferably 1 to 50 μm, particularly preferably 3 to 15 μm, and most preferably 5 to 15 μm. Furthermore, examples of the water-soluble or water-swellable pH-independent polymeric substances include, but are not limited to, polyethylene oxide (trade name Polyox, manufactured by Dow Chemical Company, molecular weight: 100,000 to 7,000,000), low-substituted hydroxypropyl cellulose (trade name L-HPC, manufactured by Shin-Etsu Chemical, Japan), hydroxypropyl cellulose (trade name HPC, manufactured by Nippon Soda, Co., Ltd, Japan), hydroxypropyl methyl cellulose (trade names Metolose 60SH, 65SH, 90SH, manufactured by Shin-Etsu Chemical, Japan), and methyl cellulose (trade name Metolose SM, manufactured by Shin-Etsu Chemical, Japan).

[0146] In some embodiments, a single pH-independent polymeric substance can be contained in the composition, or a plurality of pH-independent polymeric substances can be contained. If used in the embodiments described herein, the pH-independent polymeric substance can be a water-insoluble polymeric substance, more preferably ethyl cellulose, ethyl acrylate-methyl methacrylate copolymer (trade name Eudragit NE), or aminoalkyl methacrylate copolymer RS (trade name Eudragit RL, Eudragit RS). At least one of ethyl cellulose and aminoalkyl methacrylate copolymer RS is particularly preferred. Ethyl cellulose is most preferred. The amount of the pH-independent polymeric substance contained in the composition is not particularly limited; the amount can be appropriately adjusted depending on the purpose, for example, to control sustained release of the drug.

[0147] The pH-dependent polymeric substance that can be used in the embodiments described herein can be a polymeric substance whose charge state changes under the pH conditions generally found in the gastrointestinal tract, particularly pH 1 to pH 8. This means, for example, that the polymeric substance has a functional group whose charge state changes with pH, such as a basic functional group such as an amino group or an acidic functional group such as a carboxylic acid group. The pH-dependent functional group of the pH-dependent polymeric substance is preferably an acidic functional group, and the pH-dependent polymeric substance most preferably has a carboxylic acid group.

[0148] The pH-dependent polymeric substance used in the present application can be water-insoluble, or can swell in water or dissolve in water to form a gel. Examples of the pH-dependent polymeric substance used in the present application include, but are not limited to, enteric polymeric substances. Examples of the enteric polymeric substance include, but are not limited to, methacrylic acid-methyl methacrylate copolymer (Eudragit L100, Eudragit S100, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), methacrylic acid-ethyl acrylate copolymer (Eudragit L100-55, Eudragit L30D-55, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), hydroxypropyl methylcellulose phthalate (HP-55, HP-50, manufactured by Shin-Etsu Chemical, Japan), hydroxypropyl methylcellulose acetate succinate (AQOAT, manufactured by Shin-Etsu Chemical, Japan), carboxymethyl ethyl cellulose (CMEC, manufactured by Freund Corporation, Japan), and cellulose acetate phthalate.

[0149] Examples of the pH-dependent polymer substance which swells in water or dissolves in water to form a gel include, but are not limited to, alginic acid, pectin, carboxyvinyl polymer, and carboxymethyl cellulose. In the present application, a single pH-dependent polymer substance can be contained in the composition, or a plurality of pH-dependent polymer substances can be contained. The pH-dependent polymer substance used in the present application is preferably an enteric polymer substance, more preferably a methacrylic acid-ethyl acrylate copolymer, a methacrylic acid-methyl methacrylate copolymer, hydroxypropyl methyl cellulose phthalate, or hydroxypropyl methyl cellulose acetate succinate, and particularly preferably a methacrylic acid-ethyl acrylate copolymer.

[0150] When the pH-dependent polymer substance is used in the production process of the composition according to the present application, a commercially available product in powder or granular form can be used as it is, or a suspension-type product in which the pH-dependent polymer substance has been previously dispersed in a solvent, or such a commercially available product can be dispersed in water or an organic solvent for use. The smaller the particle diameter of the pH-dependent polymer substance, the better the performance, and the pH-dependent polymer substance is preferably in powder form. In the case of a methacrylic acid-ethyl acrylate copolymer, one example is Eudragit L100-55. The average particle diameter of the pH-dependent polymer substance used in the present application is not particularly limited, but the average particle diameter is preferably 0.05 to 100 μm, more preferably 0.05 to 70 μm, and most preferably 0.05 to 50 μm. Furthermore, the amount of the pH-dependent polymer substance is not particularly limited, for example, in the case of an enteric polymer substance, the amount is usually 0.1 to 90 parts by weight, preferably 1 to 70 parts by weight, more preferably 5 to 60 parts by weight, and particularly preferably 10 to 50 parts by weight, based on 100 parts by weight of the composition.

[0151] The combination therapy according to the embodiments described herein can also contain any of various additives as needed, such as any of various pharmacologically acceptable carriers, such as diluents, lubricants, binders, and disintegrants, as well as preservatives, colorants, sweeteners, plasticizers, film-coating agents, and the like. Examples of diluents include, but are not limited to, lactose, mannitol, calcium hydrogen phosphate, starch, pregelatinized starch, crystalline cellulose, light silicic anhydride, synthetic aluminum silicate, magnesium aluminum metasilicate, and the like. Examples of lubricants include, but are not limited to, magnesium stearate, calcium stearate, talc, sodium stearyl fumarate, and the like. Examples of binders include, but are not limited to, hydroxypropyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, and the like. Examples of disintegrants include, but are not limited to, carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, sodium carboxymethyl starch, low-substitution hydroxypropyl cellulose, and the like. Examples of preservatives include, but are not limited to, p-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenyl ethanol, dehydroacetic acid, sorbic acid, and the like. Preferred examples of colorants include, but are not limited to, water-insoluble lake pigments, natural pigments (e.g., β-carotene, chlorophyll, iron oxide red), iron oxide yellow, iron oxide red, iron oxide black, and the like. Preferred examples of sweeteners include, but are not limited to, sodium saccharin, dipotassium glycyrrhizinate, aspartame, stevia, and the like. Examples of plasticizers include, but are not limited to, glycerol fatty acid esters, triethyl citrate, propylene glycol, polyethylene glycol, and the like. Examples of film-coating agents include, but are not limited to, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, and the like.

[0152] Manufacturing method

[0153] For the manufacture of the embodiments described herein, a single conventional method or a combination of conventional methods can be used. For example, when manufacturing drug-containing granules as sustained-release portions or immediate-release portions, granulation is the main operation, but this operation can be combined with other operations, such as mixing, drying, sieving, and sorting. As a granulation method, a wet granulation method in which a binder and a solvent are added to a powder and granulation is performed, a dry granulation method in which a powder is compressed and granulation is performed, a melt granulation method in which a heated and molten binder is added and heating and granulation are performed, and the like can be used.

[0154] Further, according to the granulation method, a mixing granulation method using a planetary mixer, a screw mixer, or the like, a high-speed mixing granulation method using a Henschel mixer, a Super mixer, or the like, an extrusion granulation method using a cylinder-type granulator, a rotary granulator, a screw extrusion granulator, a granulator mill-type granulator, or the like, a wet-type high-shear granulation method, a fluidized bed granulation method, a compression granulation method, a pulverization granulation method, or a spray granulation method can be used. After granulation, drying can be performed using a dryer, a fluidized bed, or the like, and pulverization and sieving can be performed to obtain granules or fine granules for use. Further, when the composition according to the present application is prepared, a granulation solvent can be used. The granulation solvent is not particularly limited, and can be any of water or various organic solvents, such as water, a lower alcohol such as methanol or ethanol, a ketone such as acetone or methyl ethyl ketone, dichloromethane, or a mixture thereof.

[0155] For the sustained-release granules contained in the embodiments, at least one drug and at least one polymer selected from the group consisting of a polymer substance independent of pH and a polymer substance dependent on pH are mixed together, a diluent and a binder are added as necessary, and granulation is performed to obtain a granular substance. The obtained granular substance is dried using a compartment dryer, a fluidized bed dryer, or the like, and sieving is performed using a grinder or a shaker, whereby sustained-release granules can be obtained. Alternatively, as a method for manufacturing sustained-release granules in the present application, a dry compactor such as a roller compactor or a slug tabletting machine can be used to add at least one drug, at least one polymer selected from the group consisting of a polymer substance independent of pH and a polymer substance dependent on pH, and a diluent and a binder as necessary, and compression molding is performed while mixing, and then granulation is performed by pulverization to an appropriate size. The granular substance prepared using this granulation mechanism can be used as it is as a granule or fine granule according to the present application, or can be further pulverized using a dynamic mill, a roller granulator, a rotor speed mill, or the like, and sieved to obtain sustained-release granules. Note that immediate-release granules can also be manufactured as sustained-release granules.

[0156] The compression molded product can be manufactured using a single conventional method or a combination of conventional methods into a drug-containing sustained release portion or an immediate release portion, or the composition described herein. For example, using at least one drug, at least one polymer material selected from the group consisting of a polymer material independent of pH and a polymer material dependent on pH, a diluent such as mannitol or lactose, a binder such as polyvinylpyrrolidone or crystalline cellulose, a disintegrant such as sodium carboxymethyl cellulose or crospovidone, and a lubricant such as magnesium stearate or talc, and using ordinary methods, a compression molded product can be obtained by tabletting. In this case, tabletting is the main operation in the method of manufacturing a compression molded product, but this operation can be combined with other operations, such as mixing, drying, sugar coating, and coating.

[0157] Examples of the tabletting method include, but are not limited to, direct compression molding, in which at least one drug and a pharmacologically acceptable additive are mixed together, and then the mixture is directly compression molded into a tablet using a tabletting machine, and dry granule compression or wet granule compression, in which the sustained release granules or the immediate release granules according to the present application are compression molded after adding a lubricant or a disintegrant as needed. There is no particular limitation on the tabletting machine used for compression molding; for example, a single punch tabletting machine, a rotary tabletting machine, or a press-coated tabletting machine can be used.

[0158] The drug-containing sustained release granules or the immediate release granules according to the embodiments herein, or the compression molded product, can be used as a composition in the form of granules or tablets, but can also be further processed to manufacture a composition. For example, the compression molded product or the granules can be film-coated using a film base material such as ethyl cellulose, casein, methyl cellulose, hydroxypropyl methyl cellulose, methacrylic acid copolymer L, cellulose acetate phthalate, shellac, etc., or sugar-coated using a sugar coating liquid containing sucrose, sugar alcohol, gum arabic powder, talc, etc., thereby manufacturing a film-coated tablet or a sugar-coated tablet. One of the solvents in this coating technique can be pure water, but an organic solvent such as an alcohol, a ketone, an ether, or a chlorinated hydrocarbon, or a mixture thereof can also be used. For example, ethanol, acetone, dichloromethane, etc. can be used as the organic solvent. Furthermore, as the coating equipment, equipment commonly used in coating techniques for manufacturing drugs can be used, examples of which include a spray coating equipment in which coating is performed by spraying a coating liquid, etc., and a rotor fluidized bed granulator for layering.

[0159] In the case of manufacturing a capsule preparation, the capsule preparation can be manufactured by filling the sustained release granules or immediate release granules as described above or microtablets into a hard gelatin capsule or an HPMC capsule using an automatic capsule filling machine. Alternatively, in the case of a tube administration preparation or a dry syrup for use with water or the like at the time of taking, the sustained release granules or immediate release granules as described above can be mixed with a thickening agent or a dispersing agent to disperse the granules, and then the mixture is made into granules or tablets. In addition, a liquid or jelly can be made using water and a substance selected from a dispersing agent, an emulsifying agent, a thickening agent, a preservative, a pH adjusting agent, a sweetening agent, a flavoring agent, an aromatic agent, and the like. However, there is no limitation on the above with respect to other manufacturing methods.

[0160] In order to more fully understand the embodiments described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

[0161] Examples

[0162] Overall, the data below indicate that the combination with palbociclib, a CDK4 / 6 inhibitor, can significantly enhance the anti-proliferative and anti-tumor effects of compounds 1, 2 and 3 in ERa WT and / or ERa Y537S positive cell lines and compound 1 in patient-derived xenograft models representing ERa WT / Y537S positive breast cancer.

[0163] Materials and Methods

[0164] Cell lines tested

[0165] MCF7 BUS cells (Coser et al., (2003) PNAS 100(24): 13994-13999) were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 4 mM L-glutamine and lx non-essential amino acids. Lenti-X 293T cells (Clontech, Cat# 632180) were routinely cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS. Cells were engineered to overexpress ERa WT (MCF7.6) or ERa Y537SThe MCF7 cell line (MCF7.7) was derived from MCF7 BUS cells of H3 Biomedicine, Inc. The ST941 cell line was derived from a patient-derived breast cancer xenograft (PDX) model (ST941) positive for endogenous Y537S hotspot mutations in ERα. HEK293T cells used for virus generation were derived from Clontech. All cell lines were confirmed to be free of Mycobacterium contamination, and their identity was confirmed by analysis of short tandem repeats of nine labeled cells.

[0166] Cell line maintenance and study conditions

[0167] The culture medium conditions for the growth of engineered MCF7 cell lines include supplementation with 10% v / v fetal bovine serum (FBS). 30-2021 TM ), 2.0 mM L-glutamine ( 30-2214 TM Dalberg modified Eagle medium (DMEM) containing 1.0% non-essential amino acids (ThermoFisher#11140050) and 5 μg / mL blast fungicide (ThermoFisher#A1113903) 30-2002 TM The culture medium conditions for ST941 cell growth included supplementation with 20% v / v fetal bovine serum (FBS). 30-2021 TM Dalberg Modified Eagle Medium (DMEM) 30-2002 TM All cells were maintained at 37°C, 5% CO2, and 95% relative humidity before and during experiments. Cells were passaged 2–3 times per week, with the number of passages limited to 6–20. During in vitro experiments, cells were seeded at an appropriate density to provide logarithmic growth during and for at least 24 hours after the duration of exposure to the target compound.

[0168] Site-directed mutagenesis and cell line engineering

[0169] The Y537S mutation was generated in exon 8 of ERα using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, catalog number 200523). Wild-type ESR1 cDNA (GeneCopoeia Inc., catalog number GC-A0322, accession number NM 000125) was used as a template, and the following mutagenic primers were used (underlined nucleotides represent site mutations): Y537S: F-AAG AAC GTG GTG CCC CTC TC T GAC CTG CTG CTG GAG ATG (SEQ ID NO: 1), R-CAT CTC CAG CAG CAG GTC A G A GAG GGG CAC CAC GTT CTT (SEQ ID NO: 2). Wild-type and mutant ESR1 cDNAs were cloned into the indicated lentiviral vector pLenti6.3 / V5-Dest (Invitrogen, Cat. No. V533-06). To prepare lentiviral particles, DNA (wild-type and mutant ESR1) was co-transfected with packaging plasmids into HEK293T cells using lipofectamine 2000. Forty-eight hours post-transfection, the virus-containing media was filtered and added to MCF7 cells in the presence of 8 μΜ polybrene overnight. The next day, cells were placed under selection with 6 μΜ blasticidin for 2 weeks to stabilize expression.

[0170] Compound preparation and cell presentation

[0171] These preparation methods involved the following Examples 1-3. Compounds for testing were prepared as stock solutions in 90% dimethyl sulfoxide (DMSO), assessed for purity by LC / MS, and serially diluted in DMSO in an 11-point half-log serial dilution to generate the master dose response (MDR) source for all testing using low volume liquid handlers (VIAFLO ASSIST and VIAFLO II electronic 16-channel pipettes, 0.5-12.5 μΐ^).

[0172] Compound transfer from the MDR source plate to the cell plate was accomplished directly by low energy acoustic transfer (ATS100, EDC Biosystems) using custom combination-specific transfer tracks (Transfer Track, BioSero). After compound transfer to the assay plate, cells were subjected to a dose response range typically 2.5 μΜ - 25 pM (5 logs), with a final DMSO concentration of 0.1% consistent across the assay. Each assay plate was self-anchored containing a duplicate dilution series of each compound as a single agent, a repeated 11 x 11 combination matrix, vehicle / DMSO negative controls, a cidal positive controls (0.5 μΜ bortezomib + 0.5 μΜ staurosporine), and static control reagent cycloheximide (3 μΜ).

[0173] Determination of anti-proliferative activity of treated cells

[0174] Cell proliferation and viability assays were performed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat. No. G7571) 144 hours post-treatment CellTiter-Glo® Luminescent Cell Viability Assay (Promega), according to manufacturer's instructions (Product G7570, G7571, G7572, G7573 CellTiter-Glo® Luminescent Cell Viability Assay (Promega), according to manufacturer's instructions (Product G7570, G7571, G7572, G7573

[0175] Cell proliferation was assessed using the time zero (T0) signal as a positive control and the in-plate vehicle well (DMSO) as a negative control. Data were converted to percent inhibition and ranged from 0% to 100% growth, where 0% equals the signal at T0 and 100% equals no inhibition or maximum growth. Cell growth of 0% or near 0% was considered a static response.

[0176] Cell viability was assessed using the response data of the in-plate lethal control compound (0.5 mM bortezomib / 0.5 mM staurosporine) and the T0 signal as a negative control. Data were converted to percent inhibition and ranged from -100% to 0%. Cell growth of -100% or near -100% was considered a lethal response.

[0177] Determination of in vitro compound synergy

[0178] Compounds 1, 2 and 3 and the CDK4 / 6 inhibitor palbociclib were tested as single agents and in combination against the MCF7.6, MCF7.7 and ST941 breast cancer cell line models. Relative percent inhibition data were calculated by the in-house data analysis software (ECABIA, H3 Biomedicine) described and then converted to Chalice software (Horizon Discovery) format compatible for further analysis (i.e. -100%, 0% and 100% converted to 200% (lethal), 100% (static) and 0% (no effect) respectively).

[0179] The combination effect was then assessed using Chalice software, comparing the combination response to its matched single agent effects using the Loewe additivity model (Lehar J et al., 2009 and Zimmermann GR et al., 2006). The range of drug concentrations where synergistic effects occur can be observed in Chalice by comparing the full dose matrix plot to the Loewe additivity model plot and by directly observing the Excess Response Chart. Similar quantitative assessments can be made in an anchor study in one study and also by the area and intensity of the combination response provided by the Chalice synergy score. The cross-over experiment and other tests containing only the additive combination serve as baseline controls.

[0180] Generation of xenografts, administration, and determination of anti-tumor activity

[0181] To generate a representative ERa WT / Y537S patient-derived xenograft (PDX) of breast cancer, solid tumor tissue from the ST941 xenograft model (ST941) carrying ERa WT / Y537S was cut into 70 mg pieces, mixed with Matrigel (Corning, 354234) and subcutaneously implanted into the right flank of female athymic nude (Crl:NU(NCr)-Foxn1nu) mice supplied with drinking water containing estradiol (Sigma-Aldrich, E1024-25G). When the tumor volume (TV) reached 125-250 mm 3 , 72 animals were selected according to TV and randomly divided into 9 treatment groups of 8 animals each. From three days before treatment and for the remainder of the study, no exogenous estradiol was supplied in the drinking water. Treatment was performed with oral (PO) administration of Compound 1 (3 mg / kg and 10 mg / kg) and palbociclib (25 mg / kg and 75 mg / kg) or vehicle once a day (QD). The PO administration volume was calculated according to body weight (BW) before compound administration (0.1 mL / 10 g of body weight). Body weight and tumor volume measurements were recorded twice a week.

[0182] TV (mm 3 ) was calculated according to the following formula:

[0183] TV = length x width x 0.5 2

[0184] Length: maximum diameter of the tumor (mm)

[0185] Width: diameter perpendicular to the length (mm)

[0186] Tumor growth inhibition % (TGI) was calculated according to the following formula:

[0187]

[0188] where day X is any day of the measurements.

[0189] The antitumor effect of treatment, stable disease (SD) and progressive disease (PD) was defined by the xenograft model response criteria (see below). Mice with a body weight loss of more than 20% compared to the body weight of day 0, or mice with a tumor mass of more than 10% of the animal body weight were euthanized to prevent the animals from suffering any pain and distress. All studies were performed under the guidelines established by the START IACUC and defined in the START Animal Care and Use Protocol (Protocol 09-001).

[0190] ​Statistical analysis

[0191] For TV and BW, data are presented as mean ± SEM. Differences in tumor volume between vehicle and Compound 1 or palbociclib treatment groups on day 38 were analyzed by two-way ANOVA followed by Tukey’s test. Relative body weight changes were analyzed by two-way ANOVA followed by Tukey’s test. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software, La Jolla, CA).

[0192] Xenograft model response criteria

[0193] Progressive disease (PD): 3 consecutive measurements greater than 120% of the initial volume, or an increase of 3 consecutive measurements from best response; stable disease (SD): 3 consecutive measurements greater than 50% and less than 120% of the initial volume.

[0194] Formulations of Compound 1 and palbociclib for in vivo xenograft studies

[0195] In the four in vivo xenograft examples reported below, Compound 1 and palbociclib were formulated as follows. This type of formulation is exemplary and not essential in particular embodiments of the application. In these examples, palbociclib was provided as the free base. Compound 1 was formulated in 10% 2-hydroxypropyl- -cyclodextrin (HPpCD) in 5% dextrose, vortexed and sonicated until clear.

[0196] Palbociclib was formulated in 50 mM sodium lactate at pH 4.0. The compound was stable in this formulation for 7 days.

[0197] Example 1 - Compound 1 and palbociclib

[0198] In Examples 1-4, Compound 1 was present as the HC1 salt.

[0199] Figure 1 , 2 Figures 1-3 show that Compound 1 and palbociclib synergistically inhibit the growth of breast cancer cell models in vitro. MCF7.6 cells were treated with different doses of Compound 1 and palbociclib for 144 hours, see Figure 1 Figures 1-3 show that Compound 1 and palbociclib synergistically inhibit the growth of breast cancer cell models in vitro. MCF7.6 cells were treated with different doses of Compound 1 and palbociclib for 144 hours, see Figure 2 Figures 1-3 show that Compound 1 and palbociclib synergistically inhibit the growth of breast cancer cell models in vitro. MCF7.6 cells were treated with different doses of Compound 1 and palbociclib for 144 hours, see Figure 3CellTiter-Glo to measure inhibition of cell viability and Chalice software to calculate the excess inhibition over Loewe additivity for each compound 1 and palbociclib dose combination. At the highest doses, 1.0 μΜ compound 1 and 2.5 μΜ palbociclib did not completely cause cell stasis as single agents, but the combination of 1.0 μΜ compound 1 and 2.5 μΜ palbociclib resulted in complete cell stasis (i.e., 100% effect) for all cell models tested. In addition, at lower dose ranges, cell proliferation was reduced to a greater extent when compound 1 and palbociclib were combined compared to the corresponding single agent doses. Excess inhibition over additivity was calculated using the Loewe additivity model and synergistic values were observed starting at 0.010 μΜ compound 1 and 0.025 μΜ palbociclib.

[0200] Example 2 - Compound 2 and palbociclib

[0201] Figure 4 、 5 Figures 5, 6, and 7 show that compound 2 and palbociclib synergistically inhibit the growth of breast cancer cell models in vitro. MCF7.6 cells were treated with different doses of compound 2 and palbociclib for 144 hours, see Figure 4 Figures 5, 6, and 7 show that compound 2 and palbociclib synergistically inhibit the growth of breast cancer cell models in vitro. MCF7.6 cells were treated with different doses of compound 2 and palbociclib for 144 hours, see Figure 5 Figures 5, 6, and 7 show that compound 2 and palbociclib synergistically inhibit the growth of breast cancer cell models in vitro. MCF7.6 cells were treated with different doses of compound 2 and palbociclib for 144 hours, see Figure 6 CellTiter-Glo to measure inhibition of cell viability and Chalice software to calculate the excess inhibition over Loewe additivity for each compound 2 and palbociclib dose combination. At the highest doses, 1.0 μΜ compound 2 and 2.5 μΜ palbociclib did not completely cause cell stasis as single agents, but the combination of 1.0 μΜ compound 2 and 2.5 μΜ palbociclib resulted in complete cell stasis (i.e., 100% effect) for all cell models tested. In addition, at lower dose ranges, cell proliferation was reduced to a greater extent when compound 2 and palbociclib were combined compared to the corresponding single agent doses. Excess inhibition over additivity was calculated using the Loewe additivity model and synergistic values were observed starting at 0.010 μΜ compound 2 and 0.025 μΜ palbociclib.

[0202] Example 3 - Compound 3 and palbociclib

[0203] Figure 7Compound 3 and palbociclib were shown to synergistically inhibit the growth of MCF7.7 breast cancer cell model in vitro. MCF7.7 cells were treated with different doses of Compound 3 and palbociclib for 144 hours. Cell viability inhibition was measured using CellTiter-Glo and excess inhibition over Loewe additivity was calculated using Chalice software for each Compound 3 and palbociclib dose combination. As single agents, the highest doses of 1.0 mM Compound 3 and 2.5 mM palbociclib did not completely cause cell stasis, but the combination of 1.0 mM Compound 3 and 2.5 mM palbociclib caused complete cell stasis (i.e., 100% effect). In addition, at lower dose ranges, cell proliferation was reduced to a greater extent when Compound 3 and palbociclib were combined compared to the corresponding single agent doses. Excess inhibition over additivity was calculated using the Loewe additivity model and synergistic values were observed starting from 0.010 mM Compound 3 and 0.025 mM palbociclib.

[0204] Example 4 - Oral Compound 1 and palbociclib in ERa WT / Y537S carrying subcutaneous Anti-tumor effect and body weight effect in female nude mice of tumor xenografts.

[0205] Figure 8 and Figure 9 Compound 1 was shown to inhibit the growth of ST941 PDX model in female nude mice with ERa WT / Y537S . The anti-tumor effect (left) and body weight effect (right) of 3 mg / kg and 10 mg / kg Compound 1 as single agent or 3 mg / kg and 10 mg / kg Compound 1 in combination with 25 mg / kg and 75 mg / kg palbociclib, administered orally daily. On Day 38, palbociclib at 25 mg / kg as single agent did not significantly inhibit tumor growth with a TGI of 13% while 75 mg / kg significantly inhibited tumor growth with a TGI of 69% (P < 0.0001, Figure 8 and Figure 9 ) without causing SD. On Day 38, 3 mg / kg and 10 mg / kg of Compound 1 as single agent resulted in significant inhibition of tumor growth with a TGI of 50% and 71% (P < 0.0001 ( Figure 8 and Figure 9 ) and induced SD in 1 / 8 mice.

[0206] On Day 38, the combination of 3 mg / kg Compound 1 and 25 mg / kg or 75 mg / kg palbociclib resulted in a significant enhancement of tumor growth inhibition relative to vehicle control and induced SD in 4 / 8 and 8 / 8 mice, respectively (TGI of 80% and 88%, P < 0.0001 ( Figure 8 ).

[0207] On day 38, the combination of 10 mg / kg compound 1 and 25 mg / kg or 75 mg / kg palbociclib also resulted in significantly enhanced tumor growth inhibition relative to the mediator control, inducing SD in 6 / 8 and 8 / 8 mice, respectively (TGI 86% and 91%, P < 0.0001). Figure 9 Furthermore, the combination of compound 1 at 3 mg / kg or 10 mg / kg and palbociclib at 75 mg / kg improved the durability of the response, and regression was observed with longer treatment durations.

[0208] sequence list <110> Eisai R&D Management Co., Ltd. C. Carl Natalie Riu Manaf Kopo Peter Smith <120> Combination therapy for treating breast cancer <130> 0080171-000414 <150> US 62 / 472,345 <151> 2017-03-16 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 39 <212> DNA <213> Homo sapiens <400> 1 aagaacgtgg tgcccctctc tgacctgctg ctggagatg 39 <210> 2 <211> 39 <212> DNA <213> Homo sapiens <400> 2 catctccagc agcaggtcag agaggggcac cacgttctt 39

Claims

1. ( E )- N,N -dimethyl-4-((2-((5-(( Z )-4,4,4-trifluoro-1-(3-fluoro-1 H -Indazole-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and 6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}pyrido[2,3- d Pyrimidine-7(8) H Use of β-ketone (palbociclib) or a pharmaceutically acceptable combination of its salts in the preparation of a medicament for the treatment of estrogen receptor-positive (ER-positive) breast cancer in patients of need. The drug contains an effective amount of ( E )- N,N -dimethyl-4-((2-((5-(( Z )-4,4,4-trifluoro-1-(3-fluoro-1 H -Indazole-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and an effective amount of palbociclib or a pharmaceutically acceptable salt thereof, The effective amount of ( E )- N,N -dimethyl-4-((2-((5-(( Z )-4,4,4-trifluoro-1-(3-fluoro-1 H -Indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof, and said effective amount of palbociclib or a pharmaceutically acceptable salt thereof, synergistically inhibit the growth of estrogen receptor-positive (ER-positive) breast cancer cells.

2. The use according to claim 1, wherein (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 50 mg to 1000 mg.

3. The use according to claim 1, wherein (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof is administered at a daily dose of 50 mg to 300 mg.

4. The use according to claim 1, wherein the palbociclib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 75 mg.

5. The use according to claim 1, wherein the palbociclib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 100 mg.

6. The use according to claim 1, wherein the palbociclib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 125 mg.

7. The use according to any one of claims 1-6, wherein (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and said palbociclib or a pharmaceutically acceptable salt thereof are administered as a single formulation.

8. The use according to any one of claims 1-6, wherein (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and said palbociclib or a pharmaceutically acceptable salt thereof are administered as a single formulation.

9. The use according to any one of claims 1-6, wherein (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and said palbociclib or a pharmaceutically acceptable salt thereof are administered sequentially.

10. The use according to any one of claims 1-6, wherein (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and said palbociclib or a pharmaceutically acceptable salt thereof are administered simultaneously.

11. The use according to any one of claims 1-6, wherein (E)-N,N-dimethyl-4-((2-((5-(((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide is the free base form of (E)-N,N-dimethyl-4-((2-((5-(((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide.

12. The use according to any one of claims 1-6, wherein the pharmaceutically acceptable salt of (E)-N,N-dimethyl-4-((2-((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide is a hydrochloride salt.

13. A pharmaceutical preparation for treating estrogen receptor-positive (ER-positive) breast cancer, comprising an effective amount of ( E )- N,N -dimethyl-4-((2-((5-(( Z )-4,4,4-trifluoro-1-(3-fluoro-1 H -Indazole-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and an effective amount of 6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}pyrido[2,3- d Pyrimidine-7(8) H )-ketone (palbociclib) or a pharmaceutically acceptable salt thereof. The effective amount of ( E )- N,N -dimethyl-4-((2-((5-(( Z )-4,4,4-trifluoro-1-(3-fluoro-1 H -Indazole-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide or a pharmaceutically acceptable salt thereof and said effective amount of palbociclib or a pharmaceutically acceptable salt thereof synergistically inhibit the growth of estrogen receptor-positive (ER-positive) breast cancer cells.

14. Effective amount ( E )- N,N -dimethyl-4-((2-((5-(( Z )-4,4,4-trifluoro-1-(3-fluoro-1 H -Indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)ethyl)amino)but-2-enamide hydrochloride and effective amounts of 6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}pyrido[2,3- d Pyrimidine-7(8) H Use of α-ketone (palbociclib) or a combination thereof, comprising pharmaceutically acceptable salts, in the preparation of a medicament for the treatment of estrogen receptor-positive (ER-positive) breast cancer. The effective amount of ( E )- N,N -dimethyl-4-((2-((5-(( Z )-4,4,4-trifluoro-1-(3-fluoro-1 H The hydrochloride salt of 5-indazole-2-phenylbut-1-en-1-ylpyridin-2-yloxyethylaminobut-2-enamide and the effective amount of palbociclib or a pharmaceutically acceptable salt thereof synergistically inhibit the growth of ER-positive breast cancer cells.

Images