Methods of treating CDK4 / 6 inhibitor resistant cancers

Abstract

The present invention discloses a method of treating a CDK4 / 6 inhibitor resistant estrogen receptor alpha positive cancer in a subject having a wild-type estrogen receptor alpha and / or a mutant estrogen receptor alpha, the method comprising administering to the subject a therapeutically effective amount of elastrant, or a pharmaceutically acceptable salt or solvate thereof, wherein the mutated estrogen receptor alpha comprises one or more mutations selected from the group consisting of D538G, Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q, and combinations thereof, wherein: X1 is S, N, or C; and X2 is R or Q. In some embodiments, the CDK4 / 6 inhibitor resistant estrogen receptor alpha positive cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer, and pituitary cancer.

Description

[0001] This application is a divisional application of an invention patent application with an application date of December 6, 2019, application number 201980081052.7 (international application number PCT / US2019 / 065005), and name “Methods for treating CDK4 / 6 inhibitor-resistant cancers”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 776,323 filed on December 6, 2018. The entire contents of the above application, including the drawings, are hereby incorporated by reference in their entirety. Technical Field

[0004] The present invention provides methods of using elacestrant to provide anti-tumor activity in cancer models with ESR1 mutations that are resistant to CDK4 / 6 inhibitors. The present invention also relates to methods of treating estrogen-positive (ER+) cancers with ESR1 mutations that can lead to CDK4 / 6 inhibitor resistance, wherein the cancer is treated with elacestrant. Background Art

[0005] Breast cancer is divided into three subtypes based on the expression of three receptors: estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (Her2). Overexpression of ER is found in many breast cancer patients. ER-positive (ER+) breast cancer accounts for two-thirds of all breast cancers. In addition to breast cancer, estrogen and ER are associated with, for example, ovarian cancer, colon cancer, prostate cancer and endometrial cancer.

[0006] ER can be activated by estrogen and translocate to the nucleus to bind DNA, thereby regulating the activity of various genes. See, for example, Marino et al., "Estrogen Signaling Multiple Pathways to Impact Gene Transcription," Curr. Genomics 7(8): 497-508 (2006); and Heldring et al., "Estrogen Receptors: How Do They Signal and What Are Their Targets," Physiol. Rev. 87(3): 905-931 (2007).

[0007] Agents that inhibit estrogen production, such as aromatase inhibitors (AIs, e.g., letrozole, anastrozole, and exemestane), or agents that directly block ER activity, such as selective estrogen receptor modulators (SERMs, e.g., tamoxifen, toremifene, droloxifene, idoxifene, raloxifene, lasofoxifene, arzoxifene, miproxifene, levormeloxifene, and EM-652 (SCH 57068)) and selective estrogen receptor degraders (SERDs, such as fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 / DPC974, SRN-927, ICI182780, and AZD9496), have been previously used or are being developed for the treatment of ER-positive breast cancer.

[0008] SERM and AI are often used as first-line adjuvant systemic treatments for ER-positive breast cancer. AI inhibits the production of estrogen in peripheral tissues by blocking the activity of aromatase, which converts androgens into estrogens in the body. However, AI cannot prevent the ovaries from producing estrogen. Therefore, AI is mainly used to treat postmenopausal women. In addition, since AI is more effective than SERM tamoxifen and has fewer serious side effects, AI can also be used to treat premenopausal women with suppressed ovarian function. For example, see Francis et al., "Adjuvant Ovarian Suppression in Premenopausal Breast Cancer," the N. Engl. J. Med., 372: 436-446 (2015).

[0009] Resistance to endocrine therapy is a challenging aspect in the management of patients with estrogen receptor-positive (ER+) breast cancer. Recent studies have shown that acquired resistance can develop after treatment with aromatase inhibitors through the emergence of mutations in the estrogen receptor 1 (ESR1) gene. Another mechanism associated with de novo and acquired resistance is the adaptive upregulation of parallel growth factor signaling pathways and crosstalk between these pathways, including the promotion of cyclin D1 expression and cyclin-dependent kinase 4 (CDK4) and CDK6 (CDK4 / 6) activation. Although initial treatment with these agents can be successful, many patients eventually relapse with resistant breast cancer. Mutations affecting the ER have emerged as a possible mechanism for the development of such resistance. See, e.g., Robinson et al., “Activating ESR1 mutations in hormone-resistant metastatic breast cancer,” Nat Genet. 45:1446–51 (2013). Mutations in the ligand binding domain (LBD) of ER were found in 20-40% of metastatic ER-positive breast tumor samples from patients who received at least one endocrine therapy. See Jeselsohn, et al., "ESR1 mutations—a mechanism for acquired endocrine resistance in breast cancer," Nat. Rev. Clin. Oncol., 12: 573-83 (2015).

[0010] Therefore, more durable and effective ER-targeted therapies are still needed to overcome some of the challenges associated with current endocrine therapies and combat the development of CDK4 / 6 resistance. Summary of the invention

[0011] In one aspect, the present invention relates to a method for inhibiting and degrading CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of elastostat or a pharmaceutically acceptable salt or solvate thereof.

[0012] Embodiments of this aspect of the invention may include one or more of the following optional features. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to palbociclib, ribociclib, abemaciclib, or a combination thereof. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to palbociclib. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to ribociclib. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to abemaciclib. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer comprises one or more mutations selected from the group consisting of D538G, Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q, and combinations thereof, wherein: X1 is S, N, or C; and X2 is R or Q. In some embodiments, the mutation is Y537S. In some embodiments, the mutation is D538G. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to a drug selected from the group consisting of antiestrogen drugs, aromatase inhibitors, and combinations thereof. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer, and pituitary cancer. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is advanced or metastatic breast cancer. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is breast cancer. In some embodiments, the subject is a postmenopausal woman. In some embodiments, the subject is a premenopausal woman. In some embodiments, the subject is a postmenopausal woman whose condition has relapsed or progressed after prior treatment with a selective estrogen receptor modulator (SERM) and / or an aromatase inhibitor (AI). In some embodiments, the elastostat is administered to the subject at a dose of about 200 mg / day to about 500 mg / day. In some embodiments, the elastostat is administered to the subject at a dose of about 200 mg / day, about 300 mg / day, about 400 mg / day, or about 500 mg / day. In some embodiments, the elastostat is administered to the subject at a dose of the maximum tolerated dose of the subject.In some embodiments, the method further comprises determining an amount selected from the group consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, H High expression of one or more genes in the group consisting of RAS, IDH1, IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2 and VHL to confirm the subject of the treatment. In some embodiments, the one or more genes are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1 and MTOR. In some embodiments, the ratio of the concentration of elastostat or its salt or solvate in the tumor after administration to the concentration of elastostat or its salt or solvate in plasma (T / P) is at least about 15.

[0013] In another aspect, the present invention relates to a method for treating CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer in a subject having wild-type estrogen receptor α and / or mutant estrogen receptor α, the method comprising administering to the subject a therapeutically effective amount of elastostrat or a pharmaceutically acceptable salt or solvate thereof, wherein the mutant estrogen receptor α comprises one or more mutations selected from the group consisting of D538G, Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q and a combination thereof, wherein: X1 is S, N or C; and X2 is R or Q.

[0014] Embodiments of this aspect of the invention may include one or more of the following optional features. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to palbociclib, ribociclib, abemaciclib, or a combination thereof. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to palbociclib. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to ribociclib. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to abemaciclib. In some embodiments, the cancer is resistant to a drug selected from the group consisting of antiestrogen, aromatase inhibitors, and combinations thereof. In some embodiments, the antiestrogen is selected from the group consisting of tamoxifen, toremifene, and fulvestrant, and the aromatase inhibitor is selected from the group consisting of exemestane, letrozole, and anastrozole. In some embodiments, the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer and pituitary cancer. In some embodiments, the cancer is advanced or metastatic breast cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the subject is a postmenopausal woman. In some embodiments, the subject is a premenopausal woman. In some embodiments, the subject is a postmenopausal woman who has relapsed or progressed after a prior treatment with SERM and / or AI. In some embodiments, the subject expresses at least one mutant estrogen receptor α selected from the group consisting of D538G, Y537S, Y537N, Y537C, E380Q, S463P, L536R, L536Q, P535H, V392I and V534E. In some embodiments, the mutation includes Y537S. In some embodiments, the mutation includes D538G.In some embodiments, the method further comprises determining an amount selected from the group consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, , CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, H High expression of one or more genes in the group consisting of RAS, IDH1, IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2 and VHL to confirm the subject of the treatment. In some embodiments, the one or more genes are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1 and MTOR. In some embodiments, the elastostat is administered to the subject at a dose of about 200 to about 500 mg / day. In some embodiments, the elastostat is administered to the subject at a dose of about 200 mg / day, about 300 mg / day, about 400 mg / day or about 500 mg / day. In some embodiments, the elastostat is administered to the subject at a dose of about 300 mg / day. In some embodiments, the ratio (T / P) of the concentration of elastostat or its salt or solvate in the tumor after administration to the concentration of elastostat or its salt or solvate in plasma is at least about 15.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The present invention describes methods and materials for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0016] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following drawings are provided by way of example and are not intended to limit the scope of the claimed invention.

[0018] Figure 1A For patients with palbociclib sensitivity S ) and palbociclib resistance R ) cell line, the figure shows the development of palbociclib resistance in the ESR1: wild-type LTED cell line.

[0019] Figure 1B About Palbo S and palbo R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[Pabociclib (μM)] for ESR1: wild-type cell lines.

[0020] Figure 1C Control and palbociclib (500 nM) treated palbociclib S and palbo R Figure 3. Colony formation assay of ESR1: wild-type cell line.

[0021] Figure 1D LTED, LTED+palbo, LTED-palbo with ESR1: wild-type gene are shown. R and LTED-palbo R + Western blot of the PALBO model.

[0022] Figure 2A For patients with palbociclib sensitivity S ) and palbociclib resistance R ) cell line, and the figure shows the development of palbociclib resistance in the ESR1:D538G LTED cell line.

[0023] Figure 2B About Palbo S and palbo R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[Pabociclib (μM)] for the ESR1:D538G cell line.

[0024] Figure 2C Control and palbociclib (500 nM) treated palbociclib S and palbo R ESR1:

[0025] Colony formation assay of D538G cell line.

[0026] Figure 2D LTED, LTED+palbo, LTED-palbo with ESR1:D538G mutation are shown R and LTED-palbo R + Western blot of the PALBO model.

[0027] Figure 3A For patients with palbociclib sensitivity S ) and palbociclib resistance R ) cell line, the figure shows the development of palbociclib resistance in the ESR1:Y537S LTED cell line.

[0028] Figure 3B About Palbo S and palbo R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[Pabociclib (μM)] for the ESR1:Y537S cell line.

[0029] Figure 3C Control and palbociclib (500 nM) treated palbociclib S and palbo R ESR1:

[0030] Figure 1. Colony formation assay of Y537S cell line.

[0031] Figure 3D LTED, LTED+palbo, LTED-palbo with ESR1:Y537S mutation are shown R and LTED-palbo R + Western blot of the PALBO model.

[0032] Figure 4A For ribociclib sensitivityS ) and ribociclib resistance R ) cell line, the figure shows the development of resistance of ESR1: wild-type LTED cells to liboxili.

[0033] Figure 4B For abemaciclib sensitivity S ) and abemaciclib resistance (abema R ) cell line, the figure shows the development of abemaciclib resistance in the ESR1: wild-type LTED cell line.

[0034] Figure 4C to ribo S and ribo R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[Riboxil (μM)] for ESR1: wild-type cell lines.

[0035] Figure 4D Control and ribo treated with ribociclib (500 nM) are provided. S and ribo R Figure 3. Colony formation assay of ESR1: wild-type cell line.

[0036] Figure 4E About Abema S and abma R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[abecib (μM)] for ESR1: wild-type cell lines.

[0037] Figure 4F The control and abemaciclib (500 nM) treated abemaciclib cells were provided. S and abema R ESR1:

[0038] Figure 1. Colony formation assay of wild-type cell line.

[0039] Figure 5A For ribociclib sensitivity S ) and resistance to ribociclib R ) cell line, the figure shows the development of resistance of ESR1:D538G LTED cell line to liboxili.

[0040] Figure 5B The development of resistance to abemaciclib divides the ESR1:D538G LTED cell line into abemaciclib-sensitive (abema S ) and abemaciclib resistance (abema R ) cell lines.

[0041] Figure 5C to ribo S and ribo R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[Riboxil (μM)] for the ESR1:D538G cell line.

[0042] Figure 5D Control and ribo treated with ribociclib (500 nM) are provided. S and ribo R ESR1:

[0043] Colony formation assay of D538G cell line.

[0044] Figure 5E About Abema S and abema R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[abecib (μM)] for the ESR1:D538G cell line.

[0045] Fig. 5F The control and abemaciclib (500 nM) treated abemaciclib cells were provided. S and abema R Figure 3. Colony formation assay of ESR1:D538G cell line.

[0046] Fig. 6A For ribociclib sensitivity S ) and ribociclib resistance R ) cell line, the figure shows the development of resistance of ESR1:Y537S LTED cell line to liboxili.

[0047] Figure 6B For abemaciclib sensitivity S ) and abemaciclib resistance (abema R ) cell line, the figure shows the development of abemaciclib resistance in the ESR1:Y537S LTED cell line.

[0048] Figure 6C to ribo S and ribo R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[Riboxil (μM)] for the ESR1:Y537S cell line.

[0049] Fig.6DControl and ribo treated with ribociclib (500 nM) are provided. S and ribo R ESR1:

[0050] Figure 1. Colony formation assay of Y537S cell line.

[0051] Fig. 6E About Abema S and abema R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[abecib (μM)] for the ESR1:Y537S cell line.

[0052] Fig. 6F The control and abemaciclib (500 nM) treated abemaciclib cells were provided. S and abema R Figure 1: Colony formation assay of ESR1:Y537S cell line.

[0053] Fig. 7A EC 50 (nM) values ​​and were sensitive to ESR1: wild-type CDK4 / 6 inhibitors, ESR1: wild-type palbociclib R , ESR1: wild-type lipociclib R and ESR1: wild-type abemaciclib R The percentage of growth inhibition of the cell lines was plotted against Log [erastran (nM)].

[0054] Figure 7B EC 50 (nM) values, and were sensitive to the ESR1:D538G CDK4 / 6 inhibitor, ESR1:

[0055] D538G Palbociclib R 、ESR1:D538G Lipocillin R and ESR1:D538G abemaciclib R The percentage of growth inhibition of the cell lines was plotted against Log [erastran (nM)].

[0056] Figure 7C EC 50 (nM) values ​​and were sensitive to the ESR1:Y537S CDK4 / 6 inhibitor, ESR1:Y537S palbociclib R , ESR1:Y537S lipocili R and ESR1:Y537S abemaciclib R The percentage of growth inhibition of the cell lines was plotted against Log [erastran (nM)].

[0057] Fig. 8A The colony formation assay in the top row shows the control group ESR1: wild-type CDK4 / 6 inhibitor sensitive, ESR1: wild-type palbociclib R , ESR1: wild-type lipociclib R and ESR1: wild-type abemaciclib R The bottom row of images shows the growth of the cell lines, while the ESR1: wild-type CDK4 / 6 inhibitor-sensitive and ESR1: wild-type palbociclib-sensitive R , ESR1: wild-type lipociclib R and ESR1: wild-type abemaciclib R Cell lines were treated with erastatin (300 nM) for cell growth.

[0058] Figure 8B The colony formation assay in the top row shows the control group ESR1:D538G CDK4 / 6 inhibitor sensitive, ESR1:D538G palbociclib R 、ESR1:D538G Lipocillin R and ESR1:D538G abemaciclib R The bottom row of images shows the growth of ESR1:D538G CDK4 / 6 inhibitor-sensitive, ESR1:

[0059] D538G Palbociclib R 、ESR1:D538G Lipocillin R and ESR1:D538G abemaciclib R Cell lines were treated with erastatin (300 nM) for cell growth.

[0060] Figure 8C The colony formation assay in the top row shows the control group ESR1:Y537S CDK4 / 6 inhibitor sensitive, ESR1:Y537S palbociclib R 、ESR1:Y537A Lipocillin R and ESR1:Y537S abemaciclib R The bottom row of images shows the growth of ESR1:Y537S CDK4 / 6 inhibitor-sensitive, ESR1:

[0061] Y537S Palbociclib R 、ESR1:Y537S Lipocillin R and ESR1:Y537S abemaciclib R Cell lines were treated with erastatin (300 nM) for cell growth.

[0062] Fig.9AMean tumor volume over time in athymic nude mice implanted with WHIM43-HIPDX xenografts harboring the ESR1:D538G mutation treated with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg).

[0063] Fig. 9B Quantification of ERα protein levels following treatment with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg) was determined by comparing ERα / vinculin (normalized to control) in the WHIM43-HIPDX xenograft model with the ESR1:D538G mutation.

[0064] Fig. 9C Quantification of E2F1 protein levels in the WHIM43-HIPDX xenograft model with the ESR1:D538G mutation was determined by comparing E2F1 / vinculin (normalized to control) with treatment with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg).

[0065] Fig.9D Quantification of CCNE1 protein levels in the WHIM43-HIPDX xenograft model with the ESR1:D538G mutation was determined by comparing CCNE1 / vinculin (normalized to control) with treatment with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg).

[0066] Fig.9E Quantification of PgRNA levels in the WHIM43-HIPDX xenograft model with the ESR1:D538G mutation was determined by comparing fold change (normalized to control) with treatment with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg).

[0067] Fig.9F Shown are Western blots of treatments with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg) in the WHIM43-HIPDX xenograft model with the ESR1:D538G mutation.

[0068] Fig. 10A In tumor cell models with ESR1: wild-type mutations, palbo S and palbo R Quantification of progesterone receptor (PgR) was determined by comparing PgR mRNA levels (normalized to control) in the treated cell lines.

[0069] Fig. 10B In tumor cell models with ESR1: wild-type mutations, palbo S and palbo R Quantification of Trefoil factor 1 (TFF1) was determined by comparing TFF1 mRNA levels (normalized to control) in treated cell lines.

[0070] Fig. 10C In tumor cell models with ESR1: wild-type mutations, palbo S and palbo R Quantification of estrogen-regulated growth (GREB1) was determined by comparing GREB1 mRNA levels (normalized to control) in the treated cell lines.

[0071] Fig.11A In a tumor cell model with ESR1:D538G mutation, palbo S and palbo R Quantification of progesterone receptor (PgR) was determined by comparing PgR mRNA levels in treated cell lines (normalized to control).

[0072] Fig. 11B In a tumor cell model with ESR1:D538G mutation, palbo S and palbo R Quantification of Trefoil factor 1 (TFF1) was determined by comparing TFF1 mRNA levels in treated cell lines (normalized to control).

[0073] Fig. 11C In a tumor cell model with ESR1:D538G mutation, palbo S and palbo R Quantification of estrogen-regulated growth (GREB1) was determined by comparing GREB1 mRNA levels (normalized to control) in treated cell lines.

[0074] Fig. 12A In a tumor cell model with ESR1:Y537S mutation, palbo S and palbo R Quantification of progesterone receptor (PgR) was determined by comparing PgR mRNA levels in treated cell lines (normalized to control).

[0075] Fig. 12B In a tumor cell model with ESR1:Y537S mutation, palbo S and palbo RQuantification of Trefoil factor 1 (TFF1) was determined by comparing TFF1 mRNA levels in treated cell lines (normalized to control).

[0076] Fig. 12C In a tumor cell model with ESR1:Y537S mutation, palbo S and palbo R Quantification of estrogen-regulated growth (GREB1) was determined by comparing GREB1 mRNA levels (normalized to control) in the treated cell lines.

[0077] Fig.13 ST941-HIPDX xenograft model (untreated, treatment- Figure 2. Tumor volume plot of a PDX model previously treated with vehicle or a combination of fulvestrant and palbociclib (data for fulvestrant 3 mg / dose are from a separate study). Tumors from the fulvestrant and palbociclib arm were then reimplanted into another study (ST941-HI treated with palbociclib; first passage) and subsequently treated with vehicle, fulvestrant (3 mg / dose), palbociclib (25 mg / kg), and elastostat (30 mg / kg) to demonstrate that elastostat is still effective in inhibiting tumor growth in a PDX model previously treated with a combination of fulvestrant and palbociclib. DETAILED DESCRIPTION

[0078] As used herein, elastrant or "RAD1901" is an orally bioavailable selective estrogen receptor degrader (SERD) and has the following chemical structure:

[0079]

[0080] Erastolide

[0081] Including its salts, solvates (such as hydrates) and prodrugs. Preclinical data have shown that elastostat effectively inhibits tumor growth in ER+ breast cancer models with wild-type and mutant ESR1. In some embodiments described in the present invention, elastostat is administered in the form of a bishydrochloride (·2HCl) salt having the following chemical structure:

[0082]

[0083] Elacestrant dihydrochloride

[0084] In postmenopausal women, the current standard of care for ER+ cancers such as breast cancer includes inhibition of the ER pathway by: 1) inhibiting the synthesis of estrogen (aromatase inhibitors (AIs)); 2) directly binding to the ER and modulating its activity using SERMs (e.g., tamoxifen); and / or 3) directly binding to the ER and causing receptor degradation using SERDs (e.g., fulvestrant). In premenopausal women, the current standard of care also includes ovarian suppression by oophorectomy or luteinizing hormone-releasing hormone (LHRH) agonists. The addition of cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitors to endocrine agents has been shown in some cases to roughly double progression-free survival (PFS), where these types of results have led to the approval and use of certain CDK4 / 6 inhibitors in combination with either AI in the first-line metastatic setting or in combination with fulvestrant in the second-line metastatic setting. Data showing a trend toward ESR1 mutations and resistance to AIs and / or CDK4 / 6 inhibitors highlight the need for new and improved orally bioavailable endocrine therapies with efficacy against wild-type ESR1 and all ESR1 mutations.

[0085] The addition of cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitors to endocrine agents roughly doubled progression-free survival (PFS), which led to the approval and use of certain CDK4 / 6 inhibitors in combination with either AI in the first-line metastatic setting or in combination with fulvestrant in the first-line or second-line metastatic setting. In the methods described herein, elastostat was shown to induce dose-dependent long-term growth inhibition of CDK4 / 6 inhibitor-resistant cancer cell lines, either with a history of prior treatment or with ESR1 mutations. Erastostat (30 mg / kg) also demonstrated growth inhibition in in vivo PDX models of tumors that were previously treated with palbociclib (>100 days) and / or de novo resistant to palbociclib. Furthermore, elastostat demonstrated in vitro and in vivo growth inhibitory activity in several CDK4 / 6 inhibitor resistance models demonstrating several molecular markers of CDK4 / 6 inhibitor resistance, such as, but not limited to, Rb deficiency, overexpression of cyclin E1, overexpression of E2F1, overexpression of cyclin D1, and overexpression of CDK6.

[0086] Erastolen (30 mg / kg) has been shown to degrade ER, reduce E2F1 expression, and reduce cyclin E1 expression in the WHIM43-HIPDX model (Pabociclib-resistant / Rb-deficient). Characterization of CDK4 / 6 inhibitor resistance demonstrated that ER, ER signaling, and importantly, ER-driven proliferation were preserved in these resistant cell lines. Thus, the present study demonstrates that the effective use of Erastolen to treat CDK4 / 6 inhibitor-resistant cancers with various types of ESR1 mutations is a promising discovery.

[0087] definition

[0088] Unless otherwise stated, the following definitions shall apply to the terms used herein.

[0089] As used herein, the terms "RAD1901" and "erastrant" refer to the same compound and are used interchangeably.

[0090] As used herein, "inhibiting the growth" of an ERα-positive tumor may refer to slowing down the tumor growth rate, or completely stopping the tumor growth.

[0091] "Tumor regression" or "regression" of ERα-positive tumors as used herein may refer to a reduction in the maximum size of the tumor. In certain embodiments, administration of a combination drug as described herein or a solvate (e.g., hydrate) or salt thereof may result in a reduction in tumor size relative to a baseline (i.e., the size before treatment begins), or even eradication or partial eradication of the tumor. Therefore, in certain embodiments, the tumor regression methods provided herein may be considered as methods of reducing tumor size relative to a baseline in this regard.

[0092] As used herein, a "tumor" is a malignant tumor and is used interchangeably with "cancer."

[0093] As used herein, "estrogen receptor alpha" or "ERα" refers to a polypeptide encoded by the gene ESR1 that comprises, consists of, or consists essentially of the wild-type ERα amino acid sequence.

[0094] As used herein, "estrogen receptor alpha positive," "ER+," or "ERα+" tumors refer to tumors in which one or more cells express at least one ERα isoform.

[0095] Treatment

[0096] In some embodiments, the present invention relates to a method for inhibiting and degrading CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of elastostat or a pharmaceutically acceptable salt or solvate thereof.

[0097] In other embodiments, the present invention relates to a method for treating CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer in a subject having wild-type estrogen receptor α and / or mutant estrogen receptor α, the method comprising administering to the subject a therapeutically effective amount of elastostrat or a pharmaceutically acceptable salt or solvate thereof, wherein the mutant estrogen receptor α comprises one or more mutations selected from the group consisting of D538G, Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q and a combination thereof, wherein: X1 is S, N or C; and X2 is R or Q.

[0098] Administration of elastrant

[0099] When administered to a subject, elastrant or a solvate (e.g., hydrate) or salt thereof has a therapeutic effect on one or more cancers or tumors. Tumor growth inhibition or regression may be limited to a single tumor or a group of tumors within a specific tissue or organ, or may be systemic (i.e., affecting tumors in all tissues or organs).

[0100] Since it is known that elastrant preferentially binds to ERα relative to estrogen receptor β (ERβ), unless otherwise indicated, estrogen receptor, estrogen receptor α, ERα, ER and wild-type ERα are used interchangeably in the present invention. In certain embodiments, ER+ cells overexpress ERα. In certain embodiments, the patient has one or more cells expressing one or more forms of ERβ in the tumor. In certain embodiments, ERα-positive tumors and / or cancers are associated with breast cancer, uterine cancer, ovarian cancer or pituitary cancer. In some of these embodiments, the patient has a tumor located in breast, uterine, ovarian or pituitary tissue. In those embodiments in which the patient has a tumor located in the breast, the tumor may be associated with luminal breast cancer that may or may not be HER2-positive, and for HER2+ tumors, the tumor may be highly expressed or lowly expressed HER2. In other embodiments, the patient has a tumor located in another tissue or organ (e.g., bone, muscle, brain), but is still associated with breast cancer, uterine cancer, ovarian cancer or pituitary cancer (e.g., tumors caused by migration or metastasis from breast cancer, uterine cancer, ovarian cancer or pituitary cancer). Therefore, in certain embodiments of the tumor growth inhibition or tumor regression methods provided by the present invention, the targeted tumor is a metastatic tumor and / or a tumor with ER overexpression in other organs (e.g., bones and / or muscles). In certain embodiments, the targeted tumor is a brain tumor and / or brain cancer.In certain embodiments, the targeted tumor is more sensitive to treatment with elastostat than to other treatments using another SERD (e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 / DPC974, SRN-927, and AZD9496), a Her2 inhibitor (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine and / or pertuzumab), chemotherapy drugs (e.g., albumin-bound paclitaxel (abraxane), adriamycin, carboplatin, cyclophosphamide (cytoxan), daunorubicin, liposomal doxorubicin hydrochloride (doxil), epirubicin (ellence), fluorouracil (fluorouracil), gemzar, helaven, ixabepilone (lxempra), methotrexate, silk The invention relates to the treatment of leukemia with a selective estrogen receptor modulator (e.g., tamoxifen, raloxifene, lasofoxifene and / or toremifene), angiogenesis inhibitors (e.g., bevacizumab), and / or rituximab.

[0101] In addition to demonstrating the ability of elastostat to inhibit tumor growth in tumors expressing wild-type ERα, elastostat also exhibited the ability to inhibit the growth of tumors expressing a mutant form of ERα, namely, Y537S ERα. Computer modeling evaluation of examples of ERα mutations showed that none of these mutations would be expected to affect the LBD or specifically block elastostat binding, such as ERα with one or more mutations selected from ERα with Y537X (where X is S, N, or C), ERα with D538G mutation, and Based on these results, the present invention provides a method for inhibiting the growth of ERα-positive tumors having one or more mutations in the ligand binding domain (LBD) or causing regression thereof by administering a therapeutically effective amount of elastostat or a solvate (e.g., hydrate) or salt thereof to a cancer patient, wherein the mutation is selected from the group consisting of Y537X1, D538G, L536X2, P535H, V534E, S463P, V392I, E380Q, and in particular Y537S ERα, wherein X1 is S, N or C, wherein X2 is R or Q. As used herein, "mutant ERα" refers to ERα comprising one or more substitutions or deletions and variants thereof, which comprises, consists of, or essentially consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to the amino acid sequence of ERα.

[0102] In addition to inhibiting breast cancer tumor growth in animal xenograft models, elastostat shows significant accumulation in tumor cells and is able to penetrate the blood-brain barrier. The survival of brain metastasis xenograft models was significantly prolonged by elastostat administration, confirming its ability to penetrate the blood-brain barrier. Therefore, in certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, the targeted ERα-positive tumor is located in the brain or other locations in the central nervous system. In some of these embodiments, ERα-positive tumors are mainly related to brain cancer. In other embodiments, ERα-positive tumors are metastatic tumors, which are mainly related to another type of cancer, such as breast cancer, uterine cancer, ovarian cancer or pituitary cancer, or tumors migrated from another tissue or organ. In some of these embodiments, the tumor is a brain metastasis, such as breast cancer brain metastasis (BCBM). In certain embodiments of the methods disclosed in the present invention, elastostat or its solvate (e.g., hydrate) or salt accumulates in one or more cells in the target tumor.

[0103] In certain embodiments of the methods disclosed herein, elastostat or a solvate (e.g., hydrate) or salt thereof preferably accumulates in the tumor with a T / P ratio (elastostat concentration in tumor / elastostat concentration in plasma) of about 15 or more, about 18 or more, about 19 or more, about 20 or more, about 25 or more, about 28 or more, about 30 or more, about 33 or more, about 35 or more, or about 40 or more.

[0104] dose

[0105] The therapeutically effective amount of the combination of elastrant or its solvate (e.g., hydrate) or salt used in the method of the present invention is an amount that results in achieving one or more treatment benchmarks (e.g., slowing or stopping tumor growth, causing tumor regression, symptom cessation, etc.) when administered within a specific time interval. The combination used in the method of the present invention can be administered to the subject once or multiple times. In those embodiments in which the compound is administered multiple times, they can be administered at set intervals, such as every day, every other day, every week, or every month. Alternatively, they can be administered at irregular intervals, such as based on symptoms, patient health conditions, etc., as needed. The combination of a therapeutically effective amount can be administered once a day (qd), continuously administered for 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 15 days. Optionally, the state of cancer or the regression of the tumor is detected during or after treatment, such as by monitoring by FES-PET scanning of the subject. Depending on the state of cancer or the regression of the detected tumor, the dose of the combination administered to the subject can be increased or decreased.

[0106] Ideally, the therapeutically effective amount does not exceed the maximum tolerated dose that causes 50% or more of the treated subjects to experience nausea or other toxic side effects that prevent further administration. The therapeutically effective amount for the subject may vary according to various factors, including the type and degree of symptoms, sex, age, weight or general health of the subject, mode of administration and salt or solvate type, changes in sensitivity to the drug, the specific type of disease, etc.

[0107] Examples of dosages of therapeutically effective amounts of elastrant or a solvate (e.g., hydrate) or salt thereof for use in the methods disclosed herein include, but are not limited to, about 150 to about 1,500 mg, about 200 to about 1,500 mg, about 250 to about 1,500 mg, or about 300 to about 1,500 mg per day for subjects with resistant ER-driven tumors or cancers; about 150 to about 1,500 mg, about 200 to about 1,000 mg, or about 250 to about 1,500 mg per day for subjects with both wild-type ER-driven tumors and / or cancers and resistant tumors and / or cancers. About 1,000 mg or about 300 to about 1,000 mg; and for subjects with primarily wild-type ER-driven tumors and / or cancers, about 300 to about 500 mg, about 300 to about 550 mg, about 300 to about 600 mg, about 250 to about 500 mg, about 250 to about 550 mg, about 250 to about 600 mg, about 200 to about 500 mg, about 200 to about 550 mg, about 200 to about 600 mg, about 150 to about 500 mg, about 150 to about 550 mg, or about 150 to about 600 mg per day. In certain embodiments, in the methods provided herein, the dosage of a compound of Formula I (e.g., elastrant) or a salt or solvate thereof for an adult subject is typically about 200 mg, 400 mg, 30 mg to 2,000 mg, 100 mg to 1,500 mg, or 150 mg to 1,500 mg orally per day. The daily dose may be achieved by single administration or by multiple administrations.

[0108] The dosage of elastrant for treating breast cancer including those with drug resistance and expressing mutant receptors is in the range of 100 mg to 1,000 mg per day. For example, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 mg of elastrant can be administered daily. In particular, 200 mg, 400 mg, 500 mg, 600 mg, 800 mg and 1,000 mg per day. The surprisingly long half-life of elastrant in humans after oral (PO) administration makes this option particularly feasible. Therefore, the drug can be administered at 200 mg BID (400 mg per day), 250 mg BID (500 mg per day), 300 mg BID (600 mg per day), 400 mg BID (800 mg per day) or 500 mg BID (1,000 mg per day). In some embodiments, the administration is oral.

[0109] In certain embodiments of the methods disclosed herein, elastrant or a solvate (e.g., hydrate) or salt thereof preferably accumulates in the tumor with a T / P ratio (elastrant concentration in tumor / elastrant concentration in plasma) of about 15 or more, about 18 or more, about 19 or more, about 20 or more, about 25 or more, about 28 or more, about 30 or more, about 33 or more, about 35 or more, or about 40 or more.

[0110] Elastranostran or its solvate (e.g., hydrate) or salt can be administered to the subject once or multiple times. In those embodiments where the compound is administered multiple times, the drug can be administered at set intervals, such as daily, every other day, weekly, or monthly. Alternatively, the drug can be administered at irregular intervals, such as as needed based on symptoms, patient health, etc.

[0111] preparation

[0112] In some embodiments, elastrant or a solvate (e.g., hydrate) or salt thereof is administered as part of a single formulation. For example, elastrant or a solvate (e.g., hydrate) or salt thereof is formulated in a single pill for oral administration or in a single amount for injection. In certain embodiments, administering the compound in a single formulation improves patient compliance.

[0113] In some embodiments, the formulation comprising elastrant or a solvate (eg, hydrate) or salt thereof may further comprise one or more pharmaceutical excipients, carriers, adjuvants and / or preservatives.

[0114] The elastrant or its solvate (e.g., hydrate) or salt used in the method disclosed in the present invention can be formulated into a unit dosage form, which refers to a physically discrete unit suitable as a unit dose for a subject to be treated, wherein each unit contains a predetermined amount of active substance calculated to produce an expected therapeutic effect, optionally in combination with a suitable pharmaceutical carrier. The unit dosage form can be one of a single daily dose or multiple daily doses (e.g., about 1 to 4 times or more per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose. In certain embodiments, the compound can be formulated into a controlled release formulation.

[0115] The elastrant or solvate (e.g., hydrate) or its salt and salt or solvate used in the method disclosed by the present invention can be prepared according to any available conventional method. Preferred dosage form examples include tablets, powders, fine particles, coated tablets, capsules, syrups, lozenges, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nose drops, ear drops, pastes, lotions, etc. In the preparation, commonly used additives such as diluents, adhesives, disintegrants, lubricants, colorants, flavoring agents, and if necessary, stabilizers, emulsifiers, absorption promoters, surfactants, pH regulators, preservatives, antioxidants, etc. can be used. In addition, the composition commonly used as a raw material for pharmaceutical preparations can also be mixed and prepared according to conventional methods. Examples of these compositions include, for example, (1) oils such as soybean oil, tallow and synthetic glycerides; (2) hydrocarbons such as liquid paraffin, squalane and paraffin wax; (3) ester oils such as octyldodecyl myristicacid and isopropyl myristic acid; (4) higher alcohols such as cetostearyl alcohol and behenyl alcohol; (5) silicone resins; (6) silicone oils; (7) surfactants such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, solid polyoxyethylene castor oil and polyoxyethylene polyoxypropylene block copolymers. co-polymer); (8) water-soluble polymers, such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone and methyl cellulose; (9) lower alcohols, such as ethanol and isopropanol; (10) polyvalent alcohols, such as glycerol, propylene glycol, dipropylene glycol and sorbitol; (11) sugars, such as glucose and sucrose; (12) inorganic powders, such as anhydrous silicic acid, magnesium aluminum silicate and aluminum silicate; (13) purified water, etc.The additives used in the above-mentioned preparations may include, for example, 1) lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose and silicon dioxide as diluents; 2) polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, gum tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, polypropylene glycol-poly oxyethylene-block copolymers; co-polymer), meglumine, calcium citrate, dextrin, pectin, etc. as binders; 3) starch, agar, gum powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, carboxymethyl cellulose / calcium, etc. as disintegrants; 4) magnesium stearate, talc, polyethylene glycol, silicon dioxide, concentrated vegetable oil, etc. as lubricants; 5) any colorant that is pharmaceutically acceptable is suitable for addition as a colorant; 6) cocoa powder, menthol, aromatics, peppermint oil, cinnamon powder as flavorings; and 7) a pharmaceutically acceptable antioxidant, such as ascorbic acid or tocopherol (α-tophenol).

[0116] The elastrant or its solvate (e.g., hydrate) or salt used in the method disclosed by the present invention can be used as any one or more of the active compounds described in the present invention and a physiologically acceptable carrier (also referred to as a pharmaceutically acceptable carrier or solution or diluent) to prepare a pharmaceutical composition. These carriers and solutions include pharmaceutically acceptable salts and solvates of the compounds used in the method of the present invention, and mixtures containing two or more of these compounds, pharmaceutically acceptable salts of these compounds, and pharmaceutically acceptable solvates of these compounds. These compositions are prepared according to acceptable pharmaceutical methods, such as those described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Eaton, Pa. (1985), which is incorporated herein by reference.

[0117] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic reactions or other adverse reactions in patients to whom the drug is administered and is compatible with other ingredients in the formulation. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers that are appropriately selected according to the intended form of administration and consistent with conventional pharmaceutical practice. For example, solid carriers / diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., crystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc or mixtures thereof. Pharmaceutically acceptable carriers may further contain a small amount of auxiliary substances that increase the shelf life or effectiveness of the therapeutic agent, such as a wetting agent or emulsifier, a preservative or a buffer.

[0118] Free form of elastrant or its solvate (e.g., hydrate) or salt can be converted into salt by conventional methods. The term "salt" used in the present invention is not limited as long as the salt is formed by elastrant or its solvate (e.g., hydrate) or salt and is pharmacologically acceptable; preferred examples of salts include hydrohalide salts (e.g., hydrochloride, hydrobromide, hydroiodide, etc.), inorganic acid salts (e.g., sulfate, nitrate, perchlorate, phosphate, carbonate, bicarbonate, etc.), organic carboxylate salts (e.g., acetate, maleate, tartrate, fumarate, citrate, etc.), organic sulfonate salts (e.g., methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, camphorsulfonate, etc.), amino acid salts (e.g., aspartate, glutamate, etc.), quaternary ammonium salts, alkali metal salts (e.g., sodium salt, potassium salt, etc.), alkaline earth metal salts (magnesium salt, calcium salt, etc.), etc. In addition, hydrochloride, sulfate, methanesulfonate, acetate, etc. are preferred as the "pharmacologically acceptable salt" of the compound of the present invention.

[0119] Isomers (e.g., geometric isomers, optical isomers, rotational isomers, tautomers, etc.) of elastostat or its solvates (e.g., hydrates) or salts can be purified into single isomers using general separation methods, including, for example, recrystallization, optical resolution such as diastereomeric salt method, enzyme fractionation, various chromatography methods (e.g., thin layer chromatography, column chromatography, glass chromatography, etc.). The term "single isomer" herein includes not only isomers with a purity of 100%, but also isomers that exist in conventional purification operations other than the target substance. Erastostat or its solvates (e.g., hydrates) or salts and / or fulvestrant sometimes have crystalline polymorphs, and all of their crystalline polymorphs are included in the present invention. Crystalline polymorphs are sometimes single and sometimes mixed, and both are included in the present invention.

[0120] In certain embodiments, elastrant or a solvate (e.g., hydrate) or salt thereof can be in the form of a prodrug, meaning that it must undergo some change (e.g., oxidation or hydrolysis) to obtain its active form. Alternatively, elastrant or a solvate (e.g., hydrate) or salt thereof can be a compound produced by changing a parent prodrug to its active form.

[0121] Route of administration

[0122] The administration route of elastrant or its solvate (e.g., hydrate) or salt includes, but is not limited to, topical administration, oral administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intravesical infusion, subcutaneous administration, transdermal administration, and transmucosal administration. In some embodiments, the administration route is oral.

[0123] Genetic profiling

[0124] In certain embodiments, the method for tumor growth inhibition or tumor regression provided by the present invention further comprises performing a gene profile analysis on the subject, wherein the gene to be analyzed is selected from ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR 2. One or more genes from the group consisting of FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1, IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2, and VHL. In other embodiments, the gene to be analyzed is one or more genes selected from the group consisting of AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1 and MTOR.

[0125] In some embodiments, the present invention provides a method for treating a subpopulation of breast cancer patients, wherein the subpopulation has high expression of one or more genes disclosed above, and treating the subpopulation with an effective dose of elastostat or a solvate (e.g., hydrate) or salt thereof according to the administration embodiments described in the present invention.

[0126] Dosage Considerations

[0127] In addition to the discovery of elastostat's ability to inhibit tumor growth, elastostat also inhibits estradiol from binding to ER in the uterus and pituitary. In these experiments, the binding of estradiol to ER in uterine and pituitary tissues was evaluated by FES-PET imaging. After treatment with elastostat, the observed ER binding levels were at or below background levels. These results confirm that the antagonistic effect of elastostat on ER activity can be evaluated by real-time scanning. Based on these results, the present invention provides a method for monitoring the efficacy of elastostat or its solvate (e.g., hydrate) or salt in the combined treatment disclosed by the present invention by measuring the binding of estradiol-ER in one or more target tissues, wherein the reduction or disappearance of the binding indicates effectiveness.

[0128] Further provided are methods for adjusting the dosage of elastostat or its solvate (e.g., hydrate) or salt in the combination therapy disclosed in the present invention based on estradiol-ER binding. In certain embodiments of these methods, binding is measured at a certain time point after one or more administrations of the first dose of the compound. If estradiol-ER binding is not affected or shows a decrease below a predetermined threshold (e.g., a decrease in binding relative to baseline of less than 5%, less than 10%, less than 20%, less than 30%, or less than 50%), the first dose is considered too low. In certain embodiments, these methods include an additional step of administering the compound at an increased second dose. These steps can be repeated, and the dose is repeatedly increased until the desired amount of reduction in estradiol-ER binding is reached. In certain embodiments, these steps can be incorporated into the methods for inhibiting tumor growth provided by the present invention. In these methods, estradiol-ER binding can be used as an alternative mode of tumor growth inhibition, or as a supplementary means of evaluating growth inhibition. In other embodiments, these methods can be used together with the administration of elastostat or its solvate (e.g., hydrate) or salt for purposes other than inhibiting tumor growth, including, for example, inhibiting cancer cell proliferation.

[0129] In certain embodiments, the method provided herein for adjusting the dosage of elastrant or a salt or solvate (eg, hydrate) thereof in combination therapy comprises:

[0130] (1) administering a first dose of elastrant or a salt or solvate (e.g., hydrate) thereof (e.g., about 350 to about 500 or about 200 to about 600 mg / day) for 3, 4, 5, 6, or 7 days;

[0131] (2) detecting estradiol-ER binding activity; wherein:

[0132] (i) if ER binding activity is undetectable or below a predetermined threshold level, continuing administration of the first dose (i.e., maintaining the dose level); or

[0133] (ii) if the ER binding activity is detectable or above a predetermined threshold level, administering a second dose greater than the first dose (e.g., the first dose plus about 50 to about 200 mg) for 3, 4, 5, 6 or 7 days, and then performing step (3);

[0134] (3) detecting estradiol-ER binding activity; wherein

[0135] (i) if ER binding activity is undetectable or below a predetermined threshold level, continuing with the second dose (i.e., maintaining the dose level); or

[0136] (ii) if the ER binding activity is detectable or above a predetermined threshold level, administering a third dose greater than the second dose (e.g., the second dose plus about 50 to about 200 mg) for 3, 4, 5, 6 or 7 days, and then performing step (4);

[0137] (4) Repeat the above steps to the fourth dose, the fifth dose, and so on, until no ER binding activity is detected.

[0138] In certain embodiments, the invention encompasses the use of PET imaging to detect and / or quantify ER-sensitive or ER-resistant cancers.

[0139] The following examples are used to better illustrate the claimed invention, and should not be construed as limiting the scope of the present invention. With regard to the specific materials mentioned, they are only used for illustrative purposes, rather than to limit the present invention. Those skilled in the art can exploit equivalent means or reactants without departing from the scope of the present invention, without exerting creative ability. It is to be understood that many changes can be made in the process described here, and still remain within the scope of the present invention. The inventor's intention is that such changes are included within the scope of the present invention.

[0140] Example

[0141] Materials and methods

[0142] Test compound

[0143] The elastostat used in the following examples is (6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-2-ol dihydrochloride, such as manufactured by IRIX Pharmaceuticals, Inc. (Florence, SC). Erastostat is stored as a dry powder and is formulated into a uniform suspension in deionized water containing 0.5% (w / v) methylcellulose and administered orally to an animal model. Tamoxifen, raloxifene, and estradiol (E2) were obtained from Sigma-Aldrich (St. Louis, MO) and administered by subcutaneous injection. Fulvestrant was obtained from Tocris Biosciences (Minneapolis, MN) and was administered by subcutaneous injection. Other laboratory reagents were purchased from Sigma-Aldrich unless otherwise stated.

[0144] Development of resistance to CDK4 / 6 inhibitors

[0145] In vitro testing

[0146] Currently Palbo R and Ribo R Cells were maintained at 500 nM palbociclib and riboxiclib, respectively, Abema R HCC1428-LTED-CDK4 / 6 cells maintained at 250 nM abemaciclib R Cells, MCF7-Y537S-CDK4 / 6 R and MCF7-D538G-CDK4 / 6 R Generated by exposing HCC1428-LTED cells to increasing concentrations of the appropriate CDK4 / 6i.

[0147] In vivo testing

[0148] Xenograft fragments derived from ST941-HI patients were implanted into athymic nude mice. Tumors were measured twice / week with a vernier caliper; volume was calculated using the formula: (L*W2)*0.5. The tumors were treated with vehicle, Fulvestrant (3 mg / dose / week) + Palbociclib (25 mg / kg daily), and RAD1901 (30 mg / kg daily). Tumors grown in the presence of Fulvestrant (3 mg / dose / week) + Palbociclib (25 mg / kg daily) were grown to >1500 mm 3, and then harvested and re-implanted into a new cohort of mice considered for passage (P1).

[0149] PDX Models

[0150] Tumors were passaged as fragments into athymic nude mice (Nu(NCR)-Foxn1Nu). Xenograft fragments derived from WHIM43 patients were implanted into ovariectomized mice without estradiol supplementation (in view). All mice were housed in pathogen-free feeding rooms in individually ventilated cages with sterile and dust-free bedding cobs, with ad libitum access to sterile food and water under a light-dark cycle (12-14 hour circadian cycle of artificial light) and controlled room temperature and humidity. Tumors were measured twice / week with a vernier caliper; volume was calculated using the following formula: (L*W2)*0.52. Elastroc and palbociclib were administered orally daily during the study. Fulvestrant was administered subcutaneously once / week.

[0151] Quantitative real-time PCR ( RT-qPCR )

[0152] In vivo xenograft models

[0153] Study ends with cryoPREP TM Tumors were crushed with an impactor (Covaris), and total RNA was extracted using the RNeasy mini kit (Qiagen). Taqman Fast Virus 1-Step Master Mix and TaqMan TM qPCR was performed using the 2-ΔΔCT method and GAPDH as an internal reference to analyze Ct values ​​to assess the relative changes in PgR (progesterone receptor) mRNA expression.

[0154] In vitro xenograft models

[0155] At the end of the treatment, cells were lysed with lysis buffer from the 1-step Cells-to-Ct kit and the lysates were processed according to the manufacturer's instructions. TM qPCR was performed using the 2-ΔΔCT method with GAPDH as an internal reference to analyze Ct values ​​to assess the relative changes in the expression of PgR (progesterone receptor) mRNA, tff1 (trefoil factor 1) mRNA, and GREB1 (estrogen-regulated growth) mRNA.

[0156] Proliferation assay

[0157] Cells were plated at a density of 5000 cells / well and treated with the corresponding treatment the next day. Viability was measured after 7 days of incubation with the drug, and the data were normalized relative to the control value as 100%. Data were plotted as % growth inhibition relative to the control at day 7.

[0158] Colony formation assay

[0159] Plate at a density of 1000-10000 cells / well and grow for 2-5 weeks depending on the cell line. Treatments were performed in triplicate; media and compounds were changed weekly. At the end of treatment, cells were fixed in paraformaldehyde and visualized with crystal violet staining.

[0160] Western blot analysis

[0161] Cells or tumors were harvested after dosing) and the following protein expressions were analyzed using standard protocols and antibodies: ERa, PR, E2F1, CCNE1, CCNE2, CCND1, Rb, pRb, CDK2, CDK4, CDK6 (Cell Signaling Technologies, Cat#13258; #3153; #3742, #4129, #4132, #2978,

[0162] The results were as follows: (a) 10 μg / ml (Abcam: ab168458, ab6545), (b) 10 μg / ml (Abcam: ab168458, ab6545), and (c) 10 μg / ml (Abcam: ab168458, ab6545), and (d ...

[0163] Example

[0164] Reference Figures 1A-1D , provide the generation and characterization of palbociclib resistance models in wild-type and mutant ESR1 backgrounds. Figure 1A Figure 3 shows the generation of ESR1:palbociclib-sensitive wild-type LTED cell line. S ) and palbociclib resistance R ) cell lines are resistant to palbociclib. Figure 1B In the palbo S and palbo RThe percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[Pabociclib (μM)] for ESR1: wild-type cell lines. Figure 1C The results for the control and palbociclib (500 nM) treatment are provided. S and palbo R Figure 1: Colony formation assay of ESR1 wild-type cell line. Figure 1D In the figure, LTED, LTED+palbo, LTED-palbo with ESR1: wild-type gene are shown. R and LTED-palbo R + Western blot of the PALBO model.

[0165] Reference Figures 2A-2D , provide the generation and characterization of palbociclib resistance models in wild-type and mutant ESR1 backgrounds. Figure 2A The palbociclib sensitivity of the generated ESR1:D538G LTED cell line was plotted. S ) and palbociclib resistance R ) cell lines are resistant to palbociclib. Figure 2B In the palbo S and palbo R The percentage of growth inhibition (normalized to 100% relative to the control) was plotted against Log[Pabociclib (μM)] for the ESR1:D538G cell line. Figure 2C The results for the control and palbociclib (500 nM) treatment are provided. S and palbo R Figure 1 shows the colony formation assay of the ESR1:D538G cell line. Figure 2D Figure 2 shows LTED, LTED+palbo, LTED-palbo with ESR1:D538G mutation. R and LTED-palbo R + Western blot of the PALBO model.

[0166] Reference Figures 3A-3D , provide the generation and characterization of palbociclib resistance models in wild-type and mutant ESR1 backgrounds. Figure 3A The palbociclib sensitivity of the generated ESR1:Y537S LTED cell line was plotted. S ) and palbociclib resistance R ) cell lines are resistant to palbociclib. Figure 3B In the palbo Sand palbo R The percentage of growth inhibition (normalized to 100% relative to the control) was plotted against Log[Pabociclib (μM)] for the ESR1:Y537S cell line. Figure 3C The results for the control and palbociclib (500 nM) treatment are provided. S and palbo R Figure 1: Colony formation assay of ESR1:Y537S cell line. Figure 3D Figure 2 shows LTED, LTED+palbo, LTED-palbo with ESR1:Y537S mutation. R and LTED-palbo R + Western blot of the PALBO model.

[0167] Now refer to Figures 4A-4F , provide the generation and characterization of ribociclib and abemaciclib resistance models in wild-type and mutant ESR1 backgrounds. Figure 4A The resulting ESR1:ribosome sensitivity in wild-type LTED cell lines is plotted. S ) and ribociclib resistance R ) cell lines are resistant to lipociclib. Figure 4B The resulting ESR1:abema sensitivity in wild-type LTED cell lines is plotted. S ) and abemaciclib resistance (abema R ) cell lines are resistant to abemaciclib. Figure 4C In the case of ribo S and ribo R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[Riboxil (μM)] for ESR1: wild-type cell lines. Figure 4D The results for the control and ribociclib (500 nM) treatment are provided. S and ribo R Figure 1: Colony formation assay of ESR1 wild-type cell line. Figure 4E In, for abema S and abema R The percentage of growth inhibition (normalized to 100% relative to control) was plotted against Log[abecib (μM)] for ESR1: wild-type cell lines. Figure 4F The results of the control and abemaciclib (500 nM) treatments are provided in S and abema R Figure 3. Colony formation assay of ESR1: wild-type cell line.

[0168] Now refer to Figures 5A-5F , provide the generation and characterization of ribociclib and abemaciclib resistance models in wild-type and mutant ESR1 backgrounds. Figure 5A The ribociclib sensitivity of the generated ESR1:D538G LTED cell line was plotted. S ) and ribociclib resistance R ) cell lines are resistant to lipociclib. Figure 5B In the figure, the abemaciclib sensitivity of the generated ESR1:D538G LTED cell line was plotted. S ) and abemaciclib resistance (abema R ) cell lines are resistant to abemaciclib. Figure 5C In the case of ribo S and ribo R ESR1:

[0169] The D538G cell line was plotted as a graph of percent growth inhibition relative to Log[Riboxil (μM)] (normalized to 100% relative to the control). Figure 5D The results for the control and ribo treated with ribociclib (500 nM) are provided. S and ribo R Figure 1 shows the colony formation assay of the ESR1:D538G cell line. Figure 5E In, for abema S and abema R The percentage of growth inhibition (normalized to 100% relative to the control) was plotted against Log[abecib (μM)] for the ESR1:D538G cell line. Fig. 5F The results for the control and abemaciclib (500 nM) treatment are provided in S and abema R Figure 3. Colony formation assay of ESR1:D538G cell line.

[0170] Now refer to Figures 6A-6F , provide the generation and characterization of ribociclib and abemaciclib resistance models in wild-type and mutant ESR1 backgrounds. Fig. 6A The ribociclib sensitivity in the resulting ESR1:Y537S LTED cell line was plotted. S ) and ribociclib resistance R ) cell lines are resistant to lipociclib. Figure 6B In the figure, the abemaciclib sensitivity of the generated ESR1:Y537S LTED cell line was plotted.S ) and abemaciclib resistance (abema R ) cell lines are resistant to abemaciclib. Figure 6C In the case of ribo S and ribo R ESR1:

[0171] Y537S cell line was plotted as the percentage of growth inhibition relative to Log[Riboxil (μM)] (normalized to 100% relative to control). Fig.6D The results for the control and ribo treated with ribociclib (500 nM) are provided. S and ribo R Figure 1: Colony formation assay of ESR1:Y537S cell line. Fig. 6E In, for abema S and abema R The percentage of growth inhibition (normalized to 100% relative to the control) was plotted against Log[abecib (μM)] for the ESR1:Y537S cell line. Fig. 6F The results for the control and abemaciclib (500 nM) treatment are provided in S and abema R Figure 1: Colony formation assay of ESR1:Y537S cell line.

[0172] Now refer to Figures 7A-7C Regardless of prior treatment history or ESR1 status, elastrant demonstrated dose-dependent tumor growth inhibition and tumor regression. Fig. 7A EC 50 (nM) values ​​were plotted against Log[erastatre (nM)] to show ESR1: wild-type CDK4 / 6 inhibitor sensitivity, ESR1: wild-type palbociclib sensitivity R , ESR1: wild-type lipociclib R and ESR1: wild-type abemaciclib R The percentage of cell line growth inhibition curve is shown in Figure 2. Figure 7B EC 50 (nM) values ​​were plotted relative to Log[erastatre (nM)] for ESR1:D538G CDK4 / 6 inhibitor sensitivity and ESR1:D538G palbociclib R 、ESR1:D538G Lipocillin R and ESR1:D538G abemaciclib R The percentage of cell line growth inhibition curve is shown in Figure 2. Figure 7C EC 50(nM) values ​​were plotted against Log[erastatre (nM)] for ESR1:Y537S CDK4 / 6 inhibitor sensitivity and ESR1:Y537S palbociclib R 、ESR1:Y537S Lipocillin R and ESR1:Y537S abemaciclib R The percentage of cell line growth inhibition is shown in Figure 3.

[0173] Reference Figures 8A-8C In this study, elastatin showed long-term growth inhibition in cell lines resistant to CDK4 / 6 inhibitors. Fig. 8A The top row of colony formation assays shows the control group ESR1: wild-type CDK4 / 6 inhibitor sensitive, ESR1: wild-type palbociclib R , ESR1: wild-type lipociclib R and ESR1: wild-type abemaciclib R The bottom row of images shows the growth of cell lines treated with elastostat (300 nM), ESR1: wild-type CDK4 / 6 inhibitor sensitive, ESR1: wild-type palbociclib R , ESR1: wild-type lipociclib R and ESR1: wild-type abemaciclib R Cell growth of cell lines. Figure 8B The colony formation assay in the top row shows the control group ESR1:D538G CDK4 / 6 inhibitor sensitive, ESR1:D538G palbociclib R 、ESR1:D538G Lipocillin R and ESR1:D538G abemaciclib R The bottom row of images shows the growth of ESR1:D538G CDK4 / 6 inhibitor-sensitive, ESR1:D538G palbociclib-sensitive cells after treatment with elastostat (300 nM). R 、ESR1:D538G Lipocillin R and ESR1:D538G abemaciclib R Cell growth of cell lines. Figure 8C In the colony formation assay in the top row, the control group ESR1:Y537S CDK4 / 6 inhibitor sensitive, ESR1:Y537S palbociclib R 、ESR1:Y537S Lipocillin R and ESR1:Y537S abemaciclib R The bottom row of images shows the growth of ESR1:Y537S CDK4 / 6 inhibitor-sensitive, ESR1:Y537S palbociclib-sensitive cells after treatment with elastostat (300 nM). R、ESR1:Y537S Lipocillin R and ESR1:Y537S abemaciclib R Cell growth of the cell lines.

[0174] Now refer to Figures 9A-9F In this study, elastostat showed growth inhibition in a PDX model that was insensitive to palbociclib. Fig.9A In , mean tumor volumes over time in athymic nude mice implanted with WHIM43 PDX xenografts harboring the ESR1:D538G mutation treated with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg) are shown. Fig. 9B Quantification of ERα protein levels was determined by comparing ERα / vinculin (normalized to control) versus treatment of a WHIM43 PDX xenograft model with an ESR1:D538G mutation with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg) as shown in the figure. Fig. 9C Quantification of E2F1 protein levels was determined by comparing E2F1 / vinculin (normalized to control) versus treatment of the WHIM43 PDX xenograft model with the ESR1:D538G mutation with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg) as shown in the figure. Fig.9D Quantification of CCNE1 protein (or cyclin E1) levels was determined by comparing PgR mRNA levels (normalized to control) shown in the figure with vehicle control, palbociclib, fulvestrant (3 mg / dose) and elastostat (30 and 60 mg / kg) treatment of the WHIM43 PDX xenograft model with the ESR1:D538G mutation. Fig.9E In the Figure, PgR mRNA levels were determined by comparing the PgR mRNA levels shown in the figure (normalized to control) with vehicle control, palbociclib, fulvestrant (3 mg / dose) and elastostat (30 and 60 mg / kg) in the WHIM43 PDX xenograft model with the ESR1:D538G mutation. Fig.9F , Western blot images of a WHIM43PDX xenograft model with an ESR1:D538G mutation treated with vehicle control, palbociclib, fulvestrant (3 mg / dose), and elastostat (30 and 60 mg / kg) are shown. .

[0175] Now refer to Figures 10A-10C , elastatin showed inhibition of ER signaling in a CDK4 / 6 inhibitor resistance model. Fig. 10AIn the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R Progesterone receptor (PgR) quantification in tumor models with ESR1: wild-type mutations was determined by comparing PgR mRNA levels (normalized to controls) in cell lines. Fig. 10B In the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R TFF1 mRNA levels (normalized to controls) of cell lines were compared to determine trefoil factor 1 (TFF1) quantification in tumor models with ESR1: wild-type mutations. Fig. 10C In the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R GREB1 mRNA levels (normalized to controls) of the cell lines were compared to determine the quantification of estrogen-regulated growth (GREB1) in a tumor model with an ESR1: wild-type mutation.

[0176] Now refer to Figures 11A-11C , elastatin showed inhibition of ER signaling in a CDK4 / 6 inhibitor resistance model. Fig.11A In the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R Progesterone receptor (PgR) quantification in a tumor model with the ESR1:D538G mutation was determined by comparing PgR mRNA levels (normalized to controls) in the cell lines. Fig. 11B In the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R TFF1 mRNA levels (normalized to controls) of cell lines were compared to determine trefoil factor 1 (TFF1) quantification in tumor models harboring the ESR1:D538G mutation. Fig. 11C In the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R GREB1 mRNA levels (normalized to controls) of the cell lines were compared to determine estrogen-regulated growth (GREB1) quantification in a tumor model with the ESR1:D538G mutation.

[0177] Now refer to Figures 12A-12C , elastatin showed inhibition of ER signaling in a CDK4 / 6 inhibitor resistance model. Fig. 12A In the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R Progesterone receptor (PgR) quantification in a tumor model with the ESR1:Y537S mutation was determined by comparing PgR mRNA levels (normalized to controls) in the cell lines. Fig. 11B In the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R TFF1 mRNA levels (normalized to controls) of cell lines were compared to determine trefoil factor 1 (TFF1) quantification in tumor models harboring the ESR1:Y537S mutation. Fig. 11C In the figure, palbo treated with control or elastostat (100 nM and 1000 nM) as shown S and palbo R GREB1 mRNA levels (normalized to controls) of the cell lines were compared to determine estrogen-regulated growth (GREB1) quantification in a tumor model with the ESR1:Y537S mutation.

[0178] Now refer to Fig.13 In this study, elastrant was shown to inhibit tumor growth in PDX models previously treated with a combination of fulvestrant and palbociclib. Fig.13 In the ST941-HIPDX xenograft model (untreated) treated with vehicle or fulvestrant and palbociclib combination (data for fulvestrant 3 mg / dose are from a separate study) tumor volume is plotted against treatment day. Tumors from the fulvestrant and palbociclib arm were then reimplanted in another study (ST941-HI palbociclib treatment; first passage) and then treated with vehicle, fulvestrant (3 mg / dose), palbociclib (25 mg / kg), and elastostat (30 mg / kg) to demonstrate that elastostat is still effective in inhibiting tumor growth in a PDX model previously treated with fulvestrant and palbociclib combination.

[0179] Other Implementations

[0180] All publications and patents mentioned in the present invention are incorporated by reference into the present invention to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference into the present invention. If the meaning of the term in any patent or publication incorporated by reference conflicts with the meaning of the term used in the present invention, the meaning of the term in the present invention shall prevail. In addition, the foregoing only discloses and describes exemplary embodiments of the present invention. Those skilled in the art will easily recognize from these contents and the accompanying drawings and claims that various changes, modifications and variations can be made therein without departing from the spirit and scope of the present invention as defined in the appended claims.

[0181] The present invention also relates to the following embodiments:

[0182] Item 1. A method for inhibiting and degrading CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of elastostrat or a pharmaceutically acceptable salt or solvate thereof.

[0183] Item 2. The method according to Item 1, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to palbociclib, ribociclib, abemaciclib or a combination thereof.

[0184] Item 3. The method according to Item 1, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to palbociclib.

[0185] Item 4. The method according to Item 1, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to ribociclib.

[0186] Item 5. The method according to Item 1, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to abemaciclib.

[0187] Item 6. A method according to any one of Items 1-5, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer contains one or more mutations selected from the group consisting of D538G, Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q and combinations thereof, wherein: X1 is S, N or C; and X2 is R or Q.

[0188] Item 7. The method according to Item 6, wherein the mutation is Y537S.

[0189] Item 8. The method according to Item 6, wherein the mutation is D538G.

[0190] Item 9. The method according to any one of Items 1 to 8, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is also resistant to a drug selected from the group consisting of anti-estrogen drugs, aromatase inhibitors, and combinations thereof.

[0191] Item 10. The method according to any one of Items 1 to 9, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer and pituitary cancer.

[0192] Item 11. The method according to any one of Items 1-10, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is advanced or metastatic breast cancer.

[0193] Item 12. The method according to any one of Items 1-10, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is breast cancer.

[0194] Item 13. A method according to any one of Items 1-12, wherein the subject is a postmenopausal female.

[0195] Item 14. A method according to any one of Items 1-12, wherein the subject is a premenopausal female.

[0196] Item 15. A method according to any one of Items 1-12, wherein the subject is a postmenopausal woman whose disease has recurred or progressed after prior treatment with a selective estrogen receptor modulator (SERM) and / or an aromatase inhibitor (AI).

[0197] Item 16. The method according to any one of Items 1-15, wherein the elastrant is administered to the subject at a dose of about 200 mg / day to about 500 mg / day.

[0198] Item 17. The method according to any one of Items 1-16, wherein the elastrant is administered to the subject at a dose of about 200 mg / day, about 300 mg / day, about 400 mg / day, or about 500 mg / day.

[0199] Item 18. The method according to any one of Items 1-15, wherein the elastrant is administered to the subject at a dose that is the maximum tolerated dose of the subject.

[0200] Item 19. The method according to any one of Items 1 to 18, further comprising:

[0201] By measuring ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1 , IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2 and VHL to confirm the subject for treatment.

[0202] Item 20. A method according to Item 19, wherein the one or more genes are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1 and MTOR.

[0203] Item 21. A method according to any one of Items 1-20, wherein the ratio (T / P) of the concentration of elastostat or its salt or solvate in the tumor to the concentration of elastostat or its salt or solvate in plasma after administration is at least about 15.

[0204] Item 22. A method for treating CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer in a subject having wild-type estrogen receptor α and / or mutant estrogen receptor α, the method comprising:

[0205] administering to the subject a therapeutically effective amount of elastrant or a pharmaceutically acceptable salt or solvate thereof,

[0206] The mutant estrogen receptor α comprises one or more mutations selected from the group consisting of D538G, Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q and a combination thereof, wherein: X1 is S, N or C; and X2 is R or Q.

[0207] Item 23. A method according to Item 22, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to palbociclib, ribociclib, abemaciclib or a combination thereof.

[0208] Item 24. The method according to Item 22, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to palbociclib.

[0209] Item 25. A method according to Item 22, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to ribociclib.

[0210] Item 26. A method according to Item 22, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to abemaciclib.

[0211] Item 27. The method according to any one of Items 22-26, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is also resistant to a drug selected from the group consisting of anti-estrogen drugs, aromatase inhibitors, and combinations thereof.

[0212] Item 28. The method according to Item 27, wherein the anti-estrogen is selected from the group consisting of tamoxifen, toremifene and fulvestrant, and the aromatase inhibitor is selected from the group consisting of exemestane, letrozole and anastrozole.

[0213] Item 29. The method according to any one of Items 22-28, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer and pituitary cancer.

[0214] Item 30. A method according to any one of Items 22-29, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is advanced or metastatic breast cancer.

[0215] Item 31. A method according to any one of Items 22-29, wherein the CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is breast cancer.

[0216] Item 32. A method according to any one of Items 22-31, wherein the subject is a postmenopausal female.

[0217] Item 33. A method according to any one of Items 22-31, wherein the subject is a premenopausal female.

[0218] Item 34. A method according to any one of Items 22-31, wherein the subject is a postmenopausal woman whose disease has recurred or progressed after prior treatment with SERMs and / or AIs.

[0219] Item 35. A method according to any one of Items 22-34, wherein the subject expresses at least one mutant estrogen receptor alpha selected from the group consisting of D538G, Y537S, Y537N, Y537C, E380Q, S463P, L536R, L536Q, P535H, V392I and V534E.

[0220] Item 36. A method according to Item 35, wherein the mutation includes Y537S.

[0221] Item 37. A method according to Item 35, wherein the mutation includes D538G.

[0222] Item 38. The method according to any one of items 22 to 37, further comprising:

[0223] By measuring ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8,, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1 , IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2 and VHL to confirm the subject for treatment.

[0224] Item 39. A method according to Item 38, wherein the one or more genes are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1 and MTOR.

[0225] Item 40. The method according to any one of Items 22-39, wherein the elastrant is administered to the subject at a dose of about 200 to about 500 mg / day.

[0226] Item 41. The method according to any one of Items 22-40, wherein the elastrant is administered to the subject at a dose of about 200 mg / day, about 300 mg / day, about 400 mg / day or about 500 mg / day.

[0227] Item 42. A method according to any one of Items 22-41, wherein the ratio (T / P) of the concentration of elastostat or its salt or solvate in the tumor to the concentration of elastostat or its salt or solvate in plasma after administration is at least about 15.

Claims

1. A method for inhibiting and degrading CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of elastostrat or a pharmaceutically acceptable salt or solvate thereof.

2. The method according to claim 1, wherein: The CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is resistant to palbociclib, ribociclib, abemaciclib, or a combination thereof.

3. The method according to claim 1, wherein: The CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to palbociclib.

4. The method according to claim 1, wherein: The CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to ribociclib.

5. The method according to claim 1, wherein: The CDK4 / 6 inhibitor-resistant estrogen receptor alpha-positive cancer is resistant to abemaciclib.

6. The method according to any one of claims 1 to 5, wherein: The CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer comprises one or more mutations selected from the group consisting of D538G, Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q and a combination thereof, wherein: X1 is S, N or C; and X2 is R or Q.

7. The method according to claim 6, wherein: The mutation is Y537S.

8. The method according to claim 6, wherein: The mutation is D538G.

9. The method according to any one of claims 1 to 8, wherein: The CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is also resistant to a drug selected from the group consisting of anti-estrogen drugs, aromatase inhibitors, and a combination thereof.

10. The method according to any one of claims 1 to 9, wherein: The CDK4 / 6 inhibitor-resistant estrogen receptor α-positive cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer and pituitary cancer.

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