RU2820478C2 - Methods of treating cdk4/6 inhibitor-resistant cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to treatment of estrogen-positive (ER+) forms of cancer. What is presented is using an elacestrant or a pharmaceutically acceptable salt or solvate thereof for inhibiting and reducing palbociclib-resistant, ribocyclib-resistant or abemacyclib-resistant estrogen receptor-alpha-positive cancer in a subject.

EFFECT: elacestrant has been shown to successfully inhibit growth of cancer cells which are resistant to palbociclib, ribociclib or abemaciclib.

19 cl, 2 ex, 52 dwg

Description

Link to related applications

[0001] This application claims priority under 35 U.S.C. § 119(e) under U.S. Provisional Application No. 62/776,323, filed December 6, 2018. The entire contents of the above application are hereby incorporated by reference in their entirety, including the drawings.

Field of technology to which the present invention relates

[0002] The present invention relates to methods of providing antitumor activity using elacestrant in CDK4/6 inhibitor-resistant cancer models harboring ESR1 mutations. The present invention also provides methods for treating estrogen-positive (ER+) cancers that have ESR1 mutations that may contribute to the development of resistance to CDK4/6 inhibitors if the cancer is treated with elacestrant.

BACKGROUND OF THE INVENTION

[0003] 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). ER overexpression is found in many breast cancer patients. ER-positive (ER+) breast cancer accounts for two-thirds of all breast cancer cases. In addition to breast cancer, estrogen and ER are associated with, for example, ovarian cancer, colon cancer, prostate cancer and endometrial cancer.

[0004] The ER can be activated by estrogen and translocates 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).

[0005] Agents that suppress estrogen production, such as aromatase inhibitors (AI, 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 disruptors (SERDs, e.g. fulvestrant, TTAS-108 (SR 16234), ZK 191703, RU 58668, GDC-0810 (ARN-810), GW5638/DPC974, SRN-927, ICI182780 and AZD9496) have previously been used or are in development for the treatment of ER-positive breast cancer.

[0006] SERMs and AIs are often used as first-line adjuvant systemic therapy for ER-positive breast cancer. AIs suppress estrogen production in peripheral tissues by blocking the activity of aromatase, which converts androgen to estrogen in the body. However, AIs cannot stop the ovaries from producing estrogen. Therefore, AI is mainly used to treat postmenopausal women. Additionally, since AIs are more effective than SERMs such as tamoxifen with fewer serious side effects, AIs can also be used to treat premenopausal women with suppressed ovarian function. See, for example, Francis et al., "Adjuvant Ovarian Suppression in Premenopausal Breast Cancer," the JV. Engl. J. Med., 372:436-446 (2015).

[0007] Resistance to endocrine therapy is a challenging aspect of the management of patients with estrogen receptor positive (ER+) breast cancer. Recent studies have demonstrated that acquired resistance can develop after treatment with aromatase inhibitors due to the occurrence of mutations in the estrogen receptor 1 (ESR1) gene. Another mechanism associated with de novo and acquired resistance is the adaptive activation of parallel growth factor signaling pathways, as well as crosstalk between such pathways, including pathways that promote cyclin D1 expression and activation of cyclin-dependent kinase 4 (CDK4) and CDK6 (CDK4). /6). Although initial treatment with such drugs may be successful, many patients eventually relapse and develop drug-resistant forms of breast cancer. One of the possible mechanisms for the development of such resistance is the emergence of mutations affecting the ER. See, for example, Robinson et al., “Activating ESR1 mutations in hormone-resistant metastatic breast cancer,” Nat Genet. 45:1446-51 (2013). Mutations in the ER ligand binding domain (LBD) are found in 20-40% of metastatic ER-positive breast tumor samples obtained from patients who have received at least one line of endocrine treatment. Jeselsohn, et al., “ESR1 mutations-a mechanism for acquired endocrine resistance in breast cancer,” Nat. Rev. Clin. Oncol., 12:573-83 (2015).

[0008] Therefore, there remains a need for more reliable and effective ER-targeted therapies to overcome some of the shortcomings associated with current endocrine therapies and to combat the development of CDK4/6 resistance.

Brief Disclosure of the Present Invention

[0009] In one aspect, the present invention provides a method of inhibiting and attenuating inhibitor-resistant CDK4/6 estrogen receptor-alpha positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of elacestrant or a pharmaceutically acceptable salt or solvate thereof.

[0010] Embodiments of this aspect of the present invention may include one or more of the following additional 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-alpha positive cancer is characterized by one or more mutations selected from the group consisting of D538G, Y537X ₁ , L536X ₂ , P535H, V534E, S463P, V392I, E380Q and combinations thereof, where: X ₁ represents S, N or C; and X ₂ is R or Q. In some embodiments, the mutation is Y537S. In accordance with some embodiments, the mutation is D538G. In some embodiments, the CDK4/6 inhibitor-resistant estrogen receptor-alpha positive cancer is resistant to a drug selected from the group consisting of antiestrogens, aromatase inhibitors, and combinations thereof. 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. In some embodiments, the CDK4/6 inhibitor-resistant estrogen receptor-alpha positive cancer is an advanced or metastatic breast cancer. In some embodiments, the CDK4/6 inhibitor-resistant estrogen receptor-alpha positive cancer is breast cancer. In accordance with some embodiments, the subject is a postmenopausal woman. In accordance with some embodiments, the subject is a premenopausal woman. In some embodiments, the subject is a postmenopausal woman who has relapsed or progressed following previous treatment with selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs). In accordance with some embodiments, elacestrant is administered to a subject at a dose of from about 200 mg/day to about 500 mg/day. In some embodiments, elacestrant is administered to a subject at a dose of about 200 mg/day, about 300 mg/day, about 400 mg/day, or about 500 mg/day. In accordance with some embodiments, the elacestrant is administered to the subject at a dose that is the maximum tolerated dose for the subject. In accordance with some embodiments, the method further comprises identifying a subject for treatment by measuring the increased expression of one or more genes 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, SEVRA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, ERNB2, 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, RBI, RET , RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2 and VHL. In accordance with some embodiments, one or more genes is selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1, and MTOR. In some embodiments, the ratio of the concentration of elacestrant or a salt or solvate thereof in the tumor to the concentration of elacestrant or a salt or solvate thereof in plasma (T/P) after administration is at least about 15.

[0011] In another aspect, the present invention provides a method of treating estrogen receptor alpha-positive, CDK4/6 inhibitor-resistant cancer in a subject having wild-type and/or mutant estrogen receptor alpha, the method comprising administering subject to a therapeutically effective amount of elacestrant or a pharmaceutically acceptable salt or solvate thereof, wherein the mutant estrogen receptor-alpha contains one or more mutations selected from the group consisting of D538G, Y537X ₁ , L536X ₂ , P535H, V534E, S463P, V392I, E380Q and thereof combinations where: X ₁ represents S, N or C; and X ₂ represents R or Q.

[0012] Embodiments of this aspect of the present invention may include one or more of the following additional 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 accordance with some embodiments, the cancer is resistant to a drug selected from the group consisting of antiestrogens, aromatase inhibitors, and combinations thereof. In accordance with some embodiments, the antiestrogens are selected from the group consisting of tamoxifen, toremifene, and fulvestrant, and the aromatase inhibitors are selected from the group consisting of eczema, letrozole, and anastrozole. 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. In accordance with some embodiments, the cancer is advanced or metastatic breast cancer. In accordance with some embodiments, the cancer is breast cancer. In accordance with some embodiments, the subject is a postmenopausal woman. In accordance with some embodiments, the subject is a premenopausal woman. In accordance with some embodiments, the subject is a postmenopausal woman who has relapsed or progressed after previous treatment with a SERM and/or AI. In some embodiments, 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. In accordance with some embodiments, the mutation includes Y537S. In accordance with some embodiments, the mutation includes D538G. In accordance with some embodiments, the method further comprises identifying a subject for treatment by measuring the increased expression of one or more genes 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, SEVRA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, ERNB2, 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, RBI, RET , RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2 and VHL. In accordance with some embodiments, one or more genes is selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1, and MTOR. In accordance with some embodiments, elacestrant is administered to a subject at a dose of from about 200 to about 500 mg/day. In some embodiments, elacestrant is administered to a subject at a dose of about 200 mg, about 300 mg, about 400 mg, or about 500 mg. In accordance with some embodiments, elacestrant is administered to a subject at a dose of approximately 300 mg/day. In some embodiments, the ratio of the tumor concentration of elacestrant or a salt or solvate thereof to the plasma concentration of elacestrant or a salt or solvate thereof (T/P) after administration is at least about 15.

[0013] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention belongs. Described herein are 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 are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other sources referenced herein are incorporated by reference in their entirety. In the event of any conflict, this description, including definitions, will control.

[0014] Other features and advantages of the present invention will become apparent from the following detailed description and figures, as well as from the claims.

Brief description of drawings

[0015] The following figures are provided as an example and are not intended to limit the scope of the claimed invention.

[0016] FIG. 1A. Development of palbociclib resistance in ESR1:wild-type LTED cell lines is plotted for palbociclib-sensitive (palbo ^S ) and palbociclib-resistant (palbo ^R ) cell lines.

[0017] FIG. 1B. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[palbociclib(μM)] is plotted for Palbo ^S and Palbo ^R cell lines with ESR1:wild type.

[0018] FIG. 1C. Photographs of colony formation assay results for palbo ^S and palbo ^R cell lines with ESR1:wild type are shown for control and after treatment with palbociclib (500 nM).

[0019] FIG. 1D. Western blot results illustrating the LTED, LTED + palbo, LTED - palbo ^R and LTED - palbo ^R + palbo models having the ESR1:wild type gene.

[0020] FIG. 2A. Development of palbociclib resistance in LTED cell lines with ESR1:D538G is plotted for palbociclib-sensitive (palbo ^S ) and palbociclib-resistant (palbo ^R ) cell lines.

[0021] FIG. 2B. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[palbociclib(μM)] is plotted for palbo ^S and palbo ^R cell lines with ESR1:D538G.

[0022] FIG. 2C. Photographs of colony formation assay results for palbo ^S and palbo ^R cell lines with ESR1:D538G are shown for control and after treatment with palbociclib (500 nM).

[0023] FIG. 2D. Western blot results illustrating the LTED, LTED + palbo, LTED - palbo ^R and LTED - palbo ^R + palbo models having the ESR1:D538G mutation

[0024] FIG. 3A. Development of palbociclib resistance in LTED cell lines with ESR1:Y537S is plotted for palbociclib-sensitive (palbo ^S ) and palbociclib-resistant (palbo ^R ) cell lines.

[0025] FIG. 3B. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[palbociclib(μM)] is plotted for palbo ^S and palbo ^R cell lines with ESR1:Y537S.

[0026] FIG. 3C. Photographs of colony formation assay results for palbo ^S and palbo ^R cell lines with ESR1:Y537S are shown for control and after treatment with palbociclib (500 nM).

[0027] FIG. 3D. Western blot results illustrating the LTED, LTED + palbo, LTED - palbo ^R , and LTED - palbo ^R + palbo models having the ESR1:Y537S mutation.

[0028] FIG. 4A. Development of ribociclib resistance in ESR1:wild-type LTED cell lines is plotted for ribociclib-sensitive (Ribo ^S ) and ribociclib-resistant (Ribo ^R ) cell lines.

[0029] FIG. 4B. Development of abemaciclib resistance in ESR1:wild-type LTED cell lines is plotted for abemaciclib-sensitive (abema ^S ) and abemaciclib-resistant (abema ^R ) cell lines.

[0030] FIG. 4C. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[ribociclib(μM)] is plotted for ESR1:wild-type ribo ^S and ribo ^R cell lines.

[0031] FIG. 4D. Photographs of colony formation assay results for ESR1:wild-type ribo ^S and ribo ^R cell lines are shown for control and after treatment with ribociclib (500 nM).

[0032] FIG. 4E. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[abemaciclib(μM)] is plotted for abema ^S and abema ^R cell lines with ESR1:wild type.

[0033] FIG. 4F. Photographs of colony formation assay results for Abema ^S and Abema ^R cell lines with ESR1:wild type are shown for control and after treatment with abemaciclib (500 nM).

[0034] FIG. 5A. Development of ribociclib resistance in LTED cell lines with ESR1:D538G is plotted for ribociclib-sensitive (Ribo ^S ) and ribociclib-resistant (Ribo ^R ) cell lines.

[0035] FIG. 5V. Development of abemaciclib resistance in LTED cell lines with ESR1:D538G is plotted for abemaciclib-sensitive (abema ^S ) and abemaciclib-resistant (abema ^R ) cell lines.

[0036] FIG. 5C. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[ribociclib(μM)] is plotted for the Ribo ^S and Ribo ^R cell lines with ESR1:D538G

[0037] FIG. 5D. Photographs of colony formation assay results for ribo ^S and ribo ^R cell lines with ESR1:D538G are shown for control and after treatment with ribociclib (500 nM).

[0038] FIG. 5E. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[abemaciclib(μM)] is plotted for the Abema ^S and Abema ^R cell lines with ESR1:D538G.

[0039] FIG. 5F. Photographs of colony formation assay results for Abema ^S and Abema ^R cell lines with ESR1:D538G are shown for control and after treatment with abemaciclib (500 nM).

[0040] FIG. 6A. Development of ribociclib resistance in LTED cell lines with ESR1:Y537S is plotted for ribociclib-sensitive (ribop ^S ) and ribociclib-resistant (ribo ^R ) cell lines.

[0041] FIG. 6B. Development of abemaciclib resistance in LTED cell lines with ESR1:Y537S is plotted for abemaciclib-sensitive (abema ^S ) and abemaciclib-resistant (abema ^R ) cell lines.

[0042] FIG. 6C. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[ribociclib(μM)] is plotted for the Ribo ^S and Ribo ^R cell lines with ESR1:Y537S.

[0043] FIG. 6D. Photographs of colony formation assay results for the Ribo ^S and Ribo ^R cell lines with ESR1:Y537S are shown for control and after treatment with ribociclib (500 nM).

[0044] FIG. 6E. The percentage of growth inhibition (normalized to control, set as 100%) relative to Log[abemaciclib(μM)] is plotted for abema ^S and abema ^R cell lines with ESR1:Y537S.

[0045] FIG. 6F. Photographs of colony formation assay results for Abema ^S and Abema ^R cell lines with ESR1:Y537S are shown for control and after treatment with abemaciclib (500 nM).

[0046] FIG. 7A. EC ₅₀ values (nM) are given and percent growth inhibition is plotted against Log [elacestrant (nM)] for ESR1:wild-type cell line sensitive to CDK4/6 inhibitors, palbociclib ^R cell line with ESR1:wild-type, line ribociclib ^R cell line with ESR1:wild type and abemaciclib ^R cell line with ESR1:wild type.

[0047] FIG. 7B. EC ₅₀ values (nM) are given and percent growth inhibition is plotted against Log [elacestrant (nM)] for CDK4/6 inhibitor-sensitive ESR1:D538G cell line, ESR1:D538G palbociclib ^R cell line, ribociclib cell line ^R with ESR1:D538G and abemaciclib cell lines ^R with ESR1:D538G

[0048] FIG. 7C. EC ₅₀ values (nM) are given and percent growth inhibition is plotted against Log[elacestrant (nM)] for CDK4/6 inhibitor-sensitive ESR1:Y537S cell line, ESR1:Y537S palbociclib ^R cell line, ribociclib cell line ^R with ESR1:Y537S and abemaciclib ^R with ESR1:Y537S cell lines.

[0049] FIG. 8A. Colony formation assay results photographs in the top row visualize the growth of the control ESR1:wild-type cell line sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:wild-type, the ribociclib ^R cell line with ESR1:wild-type, and the abemaciclib cell line ^R with ESR1:wild type, while the photographs in the bottom row visualize cell growth for the control cell line with ESR1:wild type, sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:wild type, the ribociclib ^R cell line with ESR1:wild-type and abemaciclib ^R cell lines with ESR1:wild-type after treatment with elacestrant (300 nM).

[0050] FIG. 8FJ. The photographs of colony formation assay results in the top row visualize the growth of the control cell line with ESR1:D538G, sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESRLD538G, the ribociclib ^R cell line with ESR1:D538G, and the abemaciclib ^R cell line with ESRLD538G, in while the photographs in the bottom row visualize cell growth for the control cell line with ESR1:D538G sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:D538G, the ribociclib ^R cell line with ESRLD538G, and the abemaciclib ^R cell line with ESR1: D538G after treatment with elacestrant (300 nM).

[0051] FIG. 8C. The photographs of colony formation assay results in the top row visualize the growth of the control cell line with ESR1:Y537S, sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:Y537S, the ribociclib ^R cell line with ESR1:Y537A, and the abemaciclib ^R cell line with ESR1 :Y537S, while the photographs in the bottom row visualize cell growth for the control cell line with ESR1:Y537S sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:Y537S, the ribociclib ^R cell line with ESR1:Y537S and the line abemaciclib ^R cells with ESR1:Y537S after treatment with elacestrant (300 nM).

[0052] FIG. 9A. Mean tumor volumes over time in athymic nude mice implanted with a WHIM43-HI PDX xenograft with the ESR1:D538G mutation that were treated with vehicle control, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg) /kg).

[0053] FIG. 9B. ERα protein quantification results were obtained by plotting ERα/vinculin (normalized to control) versus treatment of the WHIM43-HI xenograft PDX model harboring the ESR1:D538G mutation with control medium, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg/kg).

[0054] FIG. 9C. E2F1 protein quantification results were obtained by plotting E2E1/vinculin (normalized to control) versus treatment of the WHIM43-HI xenograft PDX model harboring the ESR1:D538G mutation with control medium, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg/kg).

[0055] FIG. 9D. CCNE1 protein level quantification results were obtained by plotting CCNE1/vinculin (normalized to control) versus treatment of the WHIM43-HI xenograft PDX model harboring the ESR1:D538G mutation with control medium, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg/kg).

[0056] FIG. 9E. PgR mRNA levels were determined by plotting the fold change (normalized to control) against treatment of the WHIM43-HI xenograft PDX model harboring the ESR1:D538G mutation with control medium, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg/dose). kg).

[0057] FIG. 9F. Western blot results are presented illustrating a WHIM43-HI xenograft PDX model harboring the ESR1:D538G mutation treated with vehicle control, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg/kg).

[0058] FIG. 10A. Results from progesterone receptor (PgR) quantification in ESR1:wild-type mutant tumor cell models were obtained by plotting PgR mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0059] FIG. 10V. Results from quantification of trefoil factor 1 (TFF1) in ESR1:wild-type mutant tumor cell models were obtained by plotting TFF1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0060] FIG. 10C. Results from quantification of estrogen-regulated growth (GREB1) in ESR1:wild-type mutant tumor cell models were obtained by plotting GREB1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0061] FIG. 11. Results from progesterone receptor (PgR) quantification in tumor cell models harboring the ESR1:D538G mutation were obtained by plotting PgR mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0062] FIG. 11B. Results from quantification of trefoil factor 1 (TFF1) in tumor cell models harboring the ESR1:D538G mutation were obtained by plotting TFF1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0063] FIG. 11C. Results from quantification of estrogen-regulated growth (GREB1) in tumor cell models harboring the ESR1:D538G mutation were obtained by plotting GREB1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0064] FIG. 12A. Results from progesterone receptor (PgR) quantification in tumor cell models harboring the ESR1:Y537S mutation were obtained by plotting PgR mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0065] FIG. 12V. Results from quantification of trefoil factor 1 (TFF1) in tumor cell models harboring the ESR1:Y537S mutation were obtained by plotting TFF1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0066] FIG. 12C. Results from quantification of estrogen-regulated growth (GREB1) in tumor cell models harboring the ESR1:Y537S mutation were obtained by plotting GREB1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines.

[0067] FIG. 13. The graph shows tumor volume of the WHIM43-HI xenograft PDX model (previously untreated) as a function of treatment day when the model was treated with vehicle or the combination of fulvestrant and palbociclib (data for fulvestrant 3 mg/dose are from a separate study). Tumors from the fulvestrant and palbociclib group were then re-implanted in another study (with palbociclib treatment ST941-HI; first passage) and then treated with vehicle, fulvestrant (3 mg/dose), palbociclib (25 mg/kg) and elacestrant (30 mg /kg) to demonstrate that elacestrant was still effective in inhibiting tumor growth in a PDX model previously treated with the combination of fulvestrant and palbociclib.

Detailed Disclosure of the Present Invention

[0068] As used herein, elacestrant or "RAD1901" is an orally bioavailable selective estrogen receptor disruptor (SERD) and has the following chemical structure:

including its salts, solvates (eg hydrate) and prodrugs. From preclinical data, elacestrant was seen to be effective in inhibiting tumor growth in both wild-type and ESR1 mutant ER+ breast cancer models. In some embodiments, the elacestrant described herein is administered as a bis-hydrochloride salt (⋅2HCl) having the following chemical structure:

[0069] In postmenopausal women, the current standard of care for ER+ cancers, such as breast cancer, involves inhibition of the ER pathway through: 1) inhibition of estrogen synthesis (aromatase inhibitors (AI)); 2) direct binding to the ER and modulation of its activity by SERMs (eg, tamoxifen); and/or 3) direct binding to the ER and induction of receptor destruction by SERD (eg, fulvestrant). In premenopausal women, the current standard of care additionally involves ovarian suppression through oophorectomy or a luteinizing hormone-releasing hormone (LHRH) agonist. The addition of a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor to an endocrine agent has been shown to approximately double progression-free survival (PFS) in some cases, with these types of results leading to the approval and use of certain CDK4 inhibitors/ 6 in combination either with AI when prescribing treatment of metastatic forms with first-line therapy, or in combination with fulvestrant when prescribing treatment of metastatic forms with second-line therapy. Data showing a tendency for ESR1 to mutate and become resistant to AI and/or CDK4/6 inhibitors highlight the need for new and improved orally bioavailable endocrine therapies that are effective against wild-type ESR1 and all mutant forms of ESR1.

[0070] The addition of a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor to an endocrine agent approximately doubles progression-free survival (PFS) and has led to the approval and use of certain CDK4/6 inhibitors in combination with either an AI when prescribing treatment of metastatic forms with first-line therapy, or in combination with fulvestrant when prescribing treatment of metastatic forms with either first-line or second-line therapy. The methods described herein show that elacestrant induces dose-dependent, long-term growth inhibition in cancer cell lines resistant to CDK4/6 inhibitors, regardless of previous treatment or ESR1 mutant status. In vivo tumor growth inhibition was also observed with elacestrant (30 mg/kg) in PDX models that had previously been treated with palbociclib (>100 days) and/or had developed de novo resistance to palbociclib. In addition, using elacestrant, inhibitory activity was observed in vitro and in vivo in several CDK4/6 inhibitor-resistant models in which several molecular markers of CDK4/6 inhibitor resistance were observed, such as, but not limited to, Rb loss, cyclin E1 overexpression, E2F1 overexpression , overexpression of cyclin D1 and overexpression of CDK6.

[0071] Elacestrant (30 mg/kg) has been demonstrated to degrade the ER, reduce E2F1 expression, and reduce cyclin E1 expression in the WHIM43-HI (palbociclib-resistant/no-Rb) PDX model. Characteristics of resistance to CDK4/6 inhibitors revealed that these resistant cell lines retained the ER, ER-mediated signaling, and, importantly, ER-driven proliferation. Therefore, the results of the studies presented herein demonstrating the effective use of elacestrant as a treatment for CDK4/6 inhibitor-resistant cancers harboring various types of ESR1 mutations are promising.

Definitions

[0072] Unless otherwise specified, the following definitions apply for the purposes of this document.

[0073] As used herein, the terms “RAD1901” and “elacestrant” refer to the same chemical compound and are used interchangeably.

[0074] “Growth inhibition” of an ERα-positive tumor as used herein may refer to slowing the rate of tumor growth or stopping tumor growth entirely.

[0075] "Tumor regression" or "regression" of an ERα-positive tumor as used herein may refer to a reduction in the maximum size of the tumor. In certain embodiments, administration of a combination described herein or its solvates (eg, hydrate) or salts may result in tumor size being reduced from baseline (i.e., pre-treatment size) or even eradicating or partial destruction of the tumor. Therefore, in accordance with certain embodiments, methods of tumor regression presented herein can alternatively be characterized as methods of reducing the size of a tumor compared to a baseline.

[0076] "Tumor" as used herein means a malignant tumor and is used synonymously with the term "cancer".

[0077] "Estrogen receptor alpha" or "ERα" as used herein refers to a polypeptide containing, consisting of, or substantially consisting of the wild-type ERα amino acid sequence that is encoded by the ESR1 gene.

[0078] A tumor that is “estrogen receptor alpha positive,” “ERα positive,” “ER+,” or “ERα+” as used herein refers to a tumor in which one or more cells express at least one isoform ERα.

Treatment methods

[0079] In accordance with some embodiments, the present invention provides a method of inhibiting and attenuating inhibitor-resistant CDK4/6 estrogen receptor-alpha positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of elacestrant or a pharmaceutically acceptable salt or solvate thereof.

[0080] In accordance with other embodiments, the present invention provides a method of treating estrogen receptor-alpha-positive, CDK4/6 inhibitor-resistant cancer in a subject having wild-type and/or mutant estrogen receptor-alpha, wherein The method involves administering to a subject a therapeutically effective amount of elacestrant or a pharmaceutically acceptable salt or solvate thereof, wherein the mutant estrogen receptor alpha contains one or more mutations selected from the group consisting of D538G, Y537X ₁ , L536X ₂ , P535H, V534E, S463P, V392I, E380Q and combinations thereof, where: X ₁ represents S, N or C; and X ₂ represents R or Q.

Introduction of elacestrant

[0081] Elacestrant or its solvates (eg, hydrate) or salts, when administered to a subject, provide a therapeutic effect on one or more forms of cancer or tumors. Inhibition or regression of tumor growth may be localized to a single tumor or group of tumors in a specific tissue or organ, or may be systemic (ie, affecting tumors in all tissues or organs).

[0082] Because elacestrant is known to preferentially bind ERα rather than estrogen receptor beta (ERβ), unless otherwise noted, estrogen receptor, estrogen receptor alpha, ERα, ER, and wild-type ERα are used interchangeably herein. In accordance with certain embodiments, ER+ cells overexpress ERα. In accordance with certain embodiments, the patient has one or more cells in the tumor expressing one or more forms of ERβ. In certain embodiments, the ERα-positive tumor and/or ERα-positive cancer is associated with breast, uterine, ovarian, or pituitary cancer. In accordance with some such embodiments, the patient has a tumor located in breast, uterine, ovarian, or pituitary tissue. In such embodiments, if a patient has a tumor located in the breast, the tumor may be associated with ductal breast cancer, which may or may not be HER2 positive, and in the case of HER2+ tumors, the tumors may be high or low. HER2 expression. In other embodiments, the patient has a tumor located in another tissue or organ (eg, bone, muscle, brain) but is nonetheless associated with breast, uterine, ovarian, or pituitary cancer (for example, tumors resulting from migration or metastasis of breast, uterine, ovarian or pituitary cancer). Therefore, in certain embodiments of the methods presented herein for inhibiting tumor growth or tumor regression, the targeted tumor is a metastatic tumor, and/or the tumor overexpresses ER in other organs (eg, bone and/or muscle). In certain embodiments, the targeted tumor is a tumor and/or cancer of the brain. In certain embodiments, the target tumor may be more sensitive to treatment with elacestrant than treatment with another SERD (eg, fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 /DPC974, SRN-927 and AZD9496), Her2 inhibitors (eg, trastuzumab, lapatinib, ado-trastuzumab, emtansine and/or pertuzumab), chemotherapy (eg, Abraxane, Adriamycin, Carboplatin, Cytoxan, daunorubicin, Doxil, Ellens, fluorouracil , gemzar, chelaven, lxempra, methotrexate, mitomycin, mycoxantrone, navelbine, taxol, taxotere, thiotepa, vincristine and xeloda), aromatase inhibitor (for example, anastrozole, exemestane and letrozole), selective estrogen receptor modulators (for example, tamoxifen, raloxifene, oxyphene and/or toremifene), an angiogenesis inhibitor (eg, bevacizumab) and/or rituximab.

[0083] In addition to demonstrating the ability of elacestrant to inhibit tumor growth in tumors expressing wild-type ERα, elacestrant is characterized by the ability to inhibit the growth of tumors expressing a mutant form of ERα, namely Y537S ERα. From the computer modeling evaluation of examples of ERα mutations, it was clear that none of these mutations would be expected to affect the LBD or, in particular, to impede the binding of elacestrant to, for example, ERα having one or more of the selected mutations from the group consisting of ERα with the Y537X mutation, where X is S, N or C, ERα with the D538G mutation, and ERα with the S463P mutation. Based on these results, methods are provided herein for inhibiting the growth or achieving regression of a tumor that is positive for ERα having one or more mutations in the ligand binding domain (LBD) selected from the group consisting of Y537X1, where X1 is S , N or C, D538G, L536X2, where X ₂ is R or Q, P535H, V534E, S463P, V392I, E380Q, especially Y537S ERα, in a subject with cancer by administering to the subject a therapeutically effective amount of elacestrant or solvates thereof (eg, hydrate ) or salt. "Mutant ERα" as used herein refers to an ERα containing one or more substitutions or deletions, and variants thereof comprising, consisting of, or substantially consisting of an amino acid sequence of at least 80%, at least 85%, at least with 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of ERα.

[0084] In addition to inhibiting tumor growth in breast cancer in a xenograft animal model, elacestrant exhibits significant accumulation in tumor cells and is able to cross the blood-brain barrier. The ability to cross the blood-brain barrier was confirmed by demonstrating that administration of elacestrant significantly prolonged survival in a xenograft model of brain metastases. Therefore, in certain embodiments of the methods presented herein for inhibiting tumor growth or tumor regression, the targeted ERα-positive tumor is located in the brain or elsewhere in the central nervous system. In accordance with some such embodiments, the ERα-positive tumor is predominantly associated with brain cancer. In other embodiments, an ERα-positive tumor is a metastatic tumor that is predominantly associated with another type of cancer, such as breast, uterine, ovarian, or pituitary cancer, or a tumor that has migrated from another tissue or organ. In accordance with some such embodiments, the tumor is a brain metastasis, such as breast cancer brain metastases (BCBM). In accordance with certain embodiments of the methods disclosed herein, elacestrant or its solvates (eg, hydrate) or salts accumulate in one or more cells in the target tumor.

[0085] In accordance with certain embodiments of the methods disclosed herein, elacetrant or its solvates (e.g., hydrate) or salts preferably accumulate in the tumor at a TUR ratio (tumor elacetrant concentration/plasma elacetrant concentration) of about 15 or greater than about 18 or higher, approximately 19 or higher, approximately 20 or higher, approximately 25 or higher, approximately 28 or higher, approximately 30 or higher, approximately 33 or higher, approximately 35 or higher, or approximately 40 or higher.

Dose

[0086] A therapeutically effective amount of a combination of elacestrant or its solvates (e.g., hydrate) or salts for use in the methods disclosed herein is an amount that, when administered over a specified period of time, results in the achievement of one or more therapeutic endpoints (e.g., slowing or stopping tumor growth, resulting in tumor regression, disappearance of symptoms, etc.). The combination for use in the methods disclosed herein can be administered to a subject once or multiple times. According to such embodiments, when the compounds are administered multiple times, they can be administered at a predetermined interval, for example, once a day, every other day, once a week, or once a month. Alternatively, they may be administered at irregular intervals, for example, as needed based on symptoms, the patient's health condition, and the like. A therapeutically effective amount of the combination may be administered once daily for one 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, cancer status or tumor regression is monitored during or after treatment, for example, using a FES-PET scan of the subject. The dose of the combination administered to the subject may be increased or decreased depending on the cancer status or regression of the detected tumor.

[0087] Ideally, the therapeutically effective amount does not exceed the maximum tolerated dose at which 50% or more of the treated subjects experience nausea or other toxicity that prevents further administration of the drug. The therapeutically effective amount may vary for a subject depending on a number of factors, including the variety and severity of symptoms, sex, age, body weight or general health of the subject, route of administration and type of salt or solvate, changes in sensitivity to the drug, the particular type diseases, etc.

[0088] Examples of therapeutically effective amounts of elacestrant or solvates (e.g., hydrate) or salts thereof for use in the methods disclosed herein include, but are not limited to, about 150 to about 1500 mg, about 200 to about 1500 mg, about 250 to about 1500 mg or about 300 to about 1500 mg dose once daily for subjects with resistant ER-dependent tumors or cancers; about 150 to about 1500 mg, about 200 to about 1000 mg, or about 250 to about 1000 mg, or about 300 to about 1000 mg once daily doses for subjects having both wild-type ER-dependent tumors and/or forms cancer, as well as resistant tumors and/or forms of cancer; and from about 300 to about 500 mg, from about 300 to about 550 mg, from about 300 to about 600 mg, from about 250 to about 500 mg, from about 250 to about 550 mg, from about 250 to about 600 mg, from 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 once daily dosage for subjects having predominantly wild-type ER-dependent tumors and/or cancers. In certain embodiments, the dosage of a compound of Formula I (e.g., elacestrant) or a salt or solvate thereof for use in the general adult subject methods disclosed herein may be about 200 mg, 400 mg, 30 mg to 2000 mg. , 100 to 1500 mg or 150 to 1500 mg orally per day. This daily dose can be achieved by a single administration or multiple administrations.

[0089] The dose of elacestrant for the treatment of breast cancer, including resistant forms, as well as in cases expressing one or more mutant receptors, ranges from 100 mg to 1000 mg per day. For example, elacestrant can be taken in doses of 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg per day. In particular, 200 mg, 400 mg, 500 mg, 600 mg, 800 mg and 1000 mg per day should be noted. The unusually long half-life of elacestrant in humans following oral dosing makes this option particularly noteworthy. Accordingly, the drug can be administered in an amount of 200 mg twice daily (total 400 mg per day), 250 mg twice daily (total 500 mg per day), 300 mg twice daily (total 600 mg per day), 400 mg twice daily (800 mg per day) or 500 mg twice daily (total 1000 mg per day). In accordance with some embodiments, the dosage is administered orally.

[0090] In accordance with certain embodiments of the methods disclosed herein, elacetrant or a solvate (e.g., hydrate) or salt thereof preferably accumulates in a tumor at a T/P ratio (tumor elacetrant concentration/plasma elacetrant concentration) of approximately 15 or greater , about 18 or higher, about 19 or higher, about 20 or higher, about 25 or higher, about 28 or higher, about 30 or higher, about 33 or higher, about 35 or higher, or about 40 or higher.

[0091] The elacestrant or its solvates (eg, hydrate) or salts can be administered to a subject once or multiple times. According to such embodiments, when the compounds are administered multiple times, they can be administered at a predetermined interval, for example, once a day, every other day, once a week, or once a month. Alternatively, they may be administered at irregular intervals, for example, as needed based on symptoms, the patient's health condition, and the like.

Compound

[0092] In accordance with some embodiments, the elacestrant or its solvates (eg, hydrate) or salts are administered as part of a single formulation. For example, elacestrant or its solvates (eg, hydrate) or salts are formulated as a single tablet for oral administration or as a single dose for injection. In accordance with certain embodiments, administering the compounds as a single formulation improves patient compliance with the treatment regimen.

[0093] In accordance with some embodiments, the composition containing elacetrant or its solvates (eg, hydrate) or salts may further contain one or more pharmaceutical excipients, carriers, excipients and/or preservatives.

[0094] Elacestrant or its solvates (e.g., hydrate) or salts for use in the methods disclosed herein can be formulated in unit dosage forms, which means physically discrete units suitable as a unit dose for subjects undergoing treatment, each unit contains a predetermined amount of active material calculated to obtain the desired therapeutic effect, optionally in combination with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of several doses comprising a daily dose (eg, about 1 to 4 or more times per day). When multiple doses are used to make up a daily dose, the unit dosage form may be the same or different for each dose. In accordance with certain embodiments, the compounds can be formulated for controlled release.

[0095] The elacestrant or its solvates (eg, hydrate) or salts and salts or solvates for use in the methods disclosed herein can be formulated in accordance with any available conventional method. Examples of preferred dosage forms include tablet, powder, fine granule, granule, coated tablet, capsule, syrup, lozenge, inhalant, suppository, injectable, ointment, eye ointment, eye drops, nasal drops, ear drops, hot compress, lotion, etc. The composition can include commonly used additives such as a diluent, binder, disintegrant, lubricant, colorant, flavor and, if necessary, stabilizer, emulsifier, absorption enhancer, surfactant, pH regulator, antiseptic , antioxidant, etc. In addition, the preparation of the composition is also carried out in accordance with conventional methods by combining compositions that are usually used as raw materials for the preparation of a pharmaceutical composition. Examples of these compositions include, for example, (1) an oil such as soybean oil, beef tallow, and synthetic glyceride; (2) hydrocarbon such as liquid paraffin, squalane and solid paraffin; (3) ester oil such as octyldodecylmyristic acid and isopropylmyristic acid; (4) higher alcohol such as cetostearyl alcohol and behenyl alcohol; (5) silicone resin; (6) silicone oil; (7) a surfactant such as polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerol fatty acid ester, polyoxyethylene sorbitan fatty acid ester, solid polyoxyethylene castor oil and polyoxyethylene-polyoxypropylene block copolymer; (8) a water-soluble macromolecule such as hydroxyethylcellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinylpyrrolidone and methylcellulose; (9) lower alcohol such as ethanol and isopropanol; (10) polyvalent alcohol such as glycerin, propylene glycol, dipropylene glycol and sorbitol; (11) sugar such as glucose and cane sugar; (12) inorganic powder such as anhydrous silicic acid, magnesium aluminum silicate and aluminum silicate; and (13) purified water, etc. Additives for use in the above formulations may include, for example, 1) lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose and silica as a diluent; 2) polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum acacia, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polypropylene glycol-polyoxyethylene block copolymer, meglumine, calcium citrate, dextrin, pectin and the like as a binder; 3) starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectic acid, carboxymethylcellulose/calcium and the like as a disintegrant; 4) magnesium stearate, talc, polyethylene glycol, silicon dioxide, condensed vegetable oil and the like as a lubricant; 5) any dyes, the addition of which as a dye is acceptable from a pharmaceutical point of view; 6) cocoa powder, menthol, flavoring, peppermint oil, cinnamon powder as flavoring; and 7) antioxidants whose addition is pharmaceutically acceptable, such as ascorbic acid or alpha-tophenol.

[0096] The elacestrant or solvates (e.g., hydrate) or salts thereof for use in the presently disclosed methods may be included in the pharmaceutical composition as any one or more of the active compounds described herein, and may also be included in a physiologically acceptable carrier (also referred to as a pharmaceutically acceptable carrier). carrier, or solution, or diluent). Such carriers and solutions include pharmaceutically acceptable salts and solvates of the compounds used in the methods of the present invention, and mixtures containing two or more such compounds, pharmaceutically acceptable salts of such compounds, and pharmaceutically acceptable solvates of such compounds. Such compositions are prepared in accordance with acceptable pharmaceutical procedures, 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.

[0097] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other adverse effect in patients to whom it is administered and is compatible with the other ingredients in the formulation. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers, preferably selected based on the intended form of administration and consistent with generally accepted pharmaceutical practices. For example, solid carriers/diluents include, but are not limited to, gum, starch (e.g., corn starch, gelatinized starch), sugar (e.g., lactose, mannitol, sucrose, dextrose), cellulosic material (e.g., microcrystalline cellulose), acrylate (e.g., polymethyl acrylate ), calcium carbonate, magnesium oxide, talc or mixtures thereof. Pharmaceutically acceptable carriers may further contain small amounts of auxiliary agents, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness of the therapeutic agent.

[0098] Elacestrant or its solvates (eg, hydrate) or salts in free form can be converted to a salt using conventional methods. The term “salt” as used herein is not limited so long as the salt is formed with the elacetrant or its solvates (eg, hydrate) or salts and is pharmacologically acceptable; Preferable examples of salts include hydrohalide salt (for example, hydrochloride, hydrobromide, hydroiodide and the like), inorganic acid salt (for example, sulfate, nitrate, perchlorate, phosphate, carbonate, bicarbonate and the like), organic carboxylate salt (for example , acetate salt, maleate salt, tartrate salt, fumarate salt, citrate salt, etc.), organic sulfonate salt (for example, methanesulfonate salt, ethanesulfonate salt, benzenesulfonate salt, toluenesulfonate salt, camphorsulfonate salt, etc.), salt amino acids (for example, aspartate salt, glutamate salt, etc.), quaternary ammonium salt, alkali metal salt (for example, sodium salt, potassium salt, etc.), alkaline earth metal salt (magnesium salt, calcium salt, etc.) etc.) etc. Moreover, as the “pharmacologically acceptable salt” of the compounds of the present invention, hydrochloride salt, sulfate salt, methanesulfonate salt, acetate salt and the like are preferred.

[0099] Isomers of elacestrate or its solvates (e.g., hydrate) or salts (e.g., geometric isomers, optical isomers, rotamers, tautomers, etc.) can be purified by common means of separation, including, for example, recrystallization, optical separation methods such as diastereomeric salt method, enzymatic fractionation method, various chromatography (eg thin layer chromatography, column chromatography, glass column chromatography, etc.) to obtain a separate isomer. The term “distinct isomer” as used herein includes not only an isomer having 100% purity, but also an isomer containing a different isomer from the target that is present even after a conventional purification operation. A crystalline polymorph sometimes exists for elacestrant or its solvates (eg, hydrate) or salts and/or fulvestrant, and all crystalline polymorphs thereof are included in the present invention. The crystalline polymorph is sometimes single and sometimes present as a mixture, and both the former and the latter are included herein.

[00100] In certain embodiments, the elacestrant or its solvates (eg, hydrate) or salts may be in prodrug form, meaning that it must undergo some change (eg, oxidation or hydrolysis) to achieve its active form. Alternatively, the elacestrant or its solvates (eg, hydrate) or salts may be a compound obtained by converting the parent prodrug to its active form.

Route of administration

[00101] Routes of administration of elacestrant or its solvates (eg, hydrate) or salts include, but are 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 accordance with some embodiments, the route of administration is oral.

Gene profiling

[00102] In accordance with certain embodiments, methods of inhibiting tumor growth or tumor regression provided herein further comprise gene profiling of a subject, wherein the gene to be profiled is one or more genes selected from the group consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, VAR, VAR1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, SEVRA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, ERNB2, 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, NOTCH 1 , NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RBI, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2 and VHL. In other embodiments, the gene to be profiled is one or more genes selected from the group consisting of AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1, and MTOR.

[00103] In accordance with some embodiments, the present invention provides a method of treating a subpopulation of patients with breast cancer, wherein said subpopulation is characterized by increased expression of one or more genes disclosed above, and treating said subpopulation with an effective dose of elacestrant or solvates thereof (e.g., hydrate) or salts in accordance with the dosing embodiments described herein.

Dose adjustment

[00104] In addition to establishing the ability of elacestrant to inhibit tumor growth, elacestrant inhibits the binding of estradiol to the ER in the uterus and pituitary gland. In these experiments, estradiol binding to ER in uterine and pituitary tissue was assessed using FES-PET imaging. Following elacestrant treatment, the observed level of ER binding was at or below background levels. Such results indicate that the antagonistic effect of elacestrant on ER activity can be assessed using real-time scanning. Based on such results, the present invention provides methods for monitoring the effectiveness of treatment with elacestrant or its solvates (e.g., hydrate) or salts in a combination therapy disclosed herein by measuring the binding of estradiol to ER in one or more target tissues, with a decrease or disappearance of binding indicating for efficiency.

[00105] In addition, the present invention provides methods for adjusting the dose of elacestrant or its solvates (eg, hydrate) or salts in a combination therapy disclosed herein based on the binding of estradiol to the ER. In accordance with certain embodiments of such methods, binding is measured at the same time point after one or more administrations of the first dose of the compound. If estradiol binding to the ER is intact or is reduced below a predetermined threshold (eg, a decrease in binding from baseline that is 5% less, 10% less, 20% less, 30% less, or 50% less ), the first dose is considered too low. In accordance with certain embodiments, such methods include the additional step of administering an increased second dose of the compound. Such steps can be repeated, periodically increasing the dose until the desired reduction in estradiol binding to the ER is achieved. In accordance with certain embodiments, such methods may be included in methods of inhibiting tumor growth provided herein. In such methods, estradiol binding to the ER may serve as an indicator of tumor growth inhibition or as an additional means of assessing growth inhibition. In other embodiments, such methods may be used in combination with the administration of elacestrant or solvates (eg, hydrate) or salts thereof for a purpose other than inhibiting tumor growth, including, for example, inhibiting the proliferation of cancer cells.

[00106] In certain embodiments, methods provided herein for adjusting the dose of elacestrant or a salt or solvate thereof (e.g., hydrate) in combination therapy include:

(1) administering a first dose of elacestrant or a salt or solvate (eg, hydrate) thereof (eg, about 350 to about 500 or about 200 to about 600 mg/day) for 3, 4, 5, 6, or 7 days;

(2) detection of estradiol binding activity to the ER; and:

(i) if ER binding activity is undetectable or below a predetermined threshold level, continuing with the first dose (ie, maintaining the dose level); or

(ii) if ER binding activity is detectable or exceeds a predetermined threshold level, administering a second dose that is greater than the first dose (eg, the first dose plus about 50 to about 200 mg) over 3, 4, 5, 6, or 7 days, then move to stage (3);

(3) detection of estradiol binding activity to the ER; and:

(i) if ER binding activity is undetectable or below a predetermined threshold level, continuing with a second dose (ie, maintaining the dose level); or

(ii) if ER binding activity is detectable or exceeds a predetermined threshold level, administering a third dose that is greater than the second dose (eg, a second dose plus about 50 to about 200 mg) over 3, 4, 5, 6, or 7 days, then transition to stage (4);

(4) repeating the above steps until the fourth dose, fifth dose, etc. until no ER binding activity is detected.

[00107] In accordance with certain embodiments, the present invention relates to the use of PET imaging for the detection and/or dose treatment of ER-sensitive or ER-resistant forms of cancer.

[00108] The following examples are provided to better illustrate the claimed invention and should not be interpreted as limiting the scope of the present invention. Where specific materials are mentioned, they are mentioned for purposes of illustration only and are not intended to limit the present invention. One skilled in the art will be able to develop equivalent agents or reagents without resorting to inventive approaches and without departing from the scope of the present invention. It will be understood that many changes can be made to the procedures described herein while remaining within the scope of the present invention. It is intended by the inventors of the present invention that all such variations are included within the scope of the present invention.

Examples

Materials and methods

Tested connections

[00109] The elacestrant used in the examples below was (6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5 ,6,7,8-tetrahydronaphthalene-2-ol dihydrochloride, available for example from IRIX Pharmaceuticals, Inc. (Florence, South Carolina). Elacestrant was stored as a dry powder formulated for use as a homogeneous suspension in 0.5% (w/v) methyl cellulose in deionized water and was administered orally in animal models. Tamoxifene, raloxifene, and estradiol (E2) were purchased from Sigma-Aldrich (St. Louis, MO) and administered by subcutaneous injection. Fulvestrant was purchased from Tocris Biosciences (Minneapolis, MN) and administered by subcutaneous injection. Unless otherwise stated, other laboratory reagents were purchased from Sigma-Aldrich.

Development of resistance to CDK4/6 inhibitors

In vitro

[00110] HCC1428-LTED-CDK4/ ^6R , MCF7-Y537S-CDK4/ ^6R , and MCF7-D538G-CDK4/ ^6R cells were generated by exposing HCC1428-LTED cells to increasing concentrations of the respective CDK4/6i while maintaining the cells in conditions of 500 nM palbociclib and ribociclib in the case of Palbo ^R and Ribo ^R cells, respectively, and 250 nM abemaciclib in the case of Abema ^R cells.

In vivo

[00111] Xenograft fragments obtained from patient ST941-HI were implanted into athymic nude mice. Tumors were measured twice weekly with a vernier caliper; volumes were calculated using the formula: (L*W2)*0.5. Tumors were treated with medium, fulvestrant (3 mg/dose per week) + palbociclib (25 mg/kg per day) and RAD1901 (30 mg/kg per day). Tumors growing in the presence of fulvestrant (3 mg/dose per week) + palbociclib (25 mg/kg per day) were allowed to grow to a size >1500 mm ³ and then collected and re-transplanted into a new cohort of mice, which was considered passage (P1 ).

PDX models

[00112] Tumors were transplanted as fragments into athymic nude mice (Nu(NCR)-Foxnlnu). Xenograft fragments obtained from patient WHIM43 were implanted into ovariectomized mice without the addition of estradiol (Horizon). All mice were housed in a sterile facility, individually ventilated cages with sterilized and dust-free corn bedding, with unlimited access to sterilized food and water, under a light/dark cycle (12–14 h circadian light cycle), and at controlled room temperature. and humidity. Tumors were measured twice weekly with a vernier caliper; volumes were calculated using the formula: (L*W2)*0.52. Elacestrant and palbociclib were administered orally every day throughout the study period. Fulvestrant was administered subcutaneously once a week.

Real-time quantitative PCR (RT-qPCR)

In vivo xenograft models

[00113] Tumors at the end of the study were crushed using a cryoPREP™ impactor (Covaris) and total RNA was extracted using an RNeasy mini kit (Qiagen). qPCR was performed using Taqman Fast Virus 1-Step Master Mix and TaqMan™ probes (Applied Biosystems). Ct values using the 2-ΔΔCT method were analyzed to evaluate the relative changes in PgR (progesterone receptor) mRNA expression with GAPDH as an internal control.

In vitro xenograft models

[00114] At the end of the treatment, cells were lysed with lysis buffer from the Cells-to-Ct 1-Step Kit and lysates were processed according to the manufacturer's instructions. qPCR was performed using 1-step master mix and TaqMan™ probes (Applied Biosystems). Ct values using the 2-DDST method were analyzed to assess the relative changes in the expression of PgR (progesterone receptor) mRNA, tff1 (trefoil factor 1) mRNA and GREB1 (estrogen-regulated growth) mRNA with GAPDH as an internal control.

Proliferation assays

[00115] Cells were seeded at a density of 5000 cells/well and treated with the appropriate treatment regimens the next day. Viability was measured after 7 days of incubation with the drug, and the data were normalized to control values, taken as 100%. The data are presented as a graph of % growth inhibition compared to control on the 7th day.

Colony formation assay

[00116] Cells were seeded at a density of 1000-10000 cells/well and allowed to grow for 2-5 weeks depending on the cell line. The procedures were carried out in triplicate; The medium and connection were replaced weekly. At the end of the treatment cycle, cells were fixed in paraformaldehyde and stained with crystal violet for visualization.

Western blotting

Cell Signaling Technologies, cat. No. 13258; No. 3742, No. 4129, No. 2978, No. 9309, No. 8516, No. 2546, No. 12790, No. 13331), p107, p130 (Abeam: ab 168458, ab6545) Vinculin: Sigma-Aldrich, no. v9131. Protein expression was quantified using AzureSpot software and normalized to vinculin expression. The differential effects of palbociclib on susceptible and resistant lines were examined by treating both lines with 500 nM palbociclib for 24 hours and comparing them to their relative controls. The in vivo effect of elacestrant, fulvestrant, and palbociclib was examined at the end of the study on tumors that were collected 4 hours after the last dose.

Examples

[00118] Referring now to FIG. 1A-1D, which depict the generation and characterization of palbociclib-resistant models in wild-type and ESR1 mutant backgrounds. In fig. 1A Palbociclib resistance in engineered ESR1:wild-type LTED cell lines is plotted for palbociclib-sensitive (palbo ^S ) and palbociclib-resistant (palbo ^R ) cell lines. In fig. 1B, the percentage of growth inhibition (normalized to control set as 100%) relative to Log[palbociclib(μM)] is plotted for palbo ^S and palbo ^R cell lines with ESR1:wild type. In fig. 1C photographs of colony formation assay results for palbo ^S and palbo ^R cell lines with ESR1:wild type are shown for control and after treatment with palbociclib (500 nM). In fig. 1D Western blot results are illustrated for LTED, LTED + palbo, LTED - palbo ^R and LTED - palbo ^R + palbo models having the ESR1:wild type gene.

[00119] Referring now to FIG. 2A-2D, which depict the generation and characterization of palbociclib-resistant models in wild-type and ESR1 mutant backgrounds. In fig. 2A, the development of palbociclib resistance in established LTED cell lines with ESR1:D538G is plotted for palbociclib-sensitive (palbo ^S ) and palbociclib resistant (palbo ^R ) cell lines. In fig. 2B, the percentage of growth inhibition (normalized to control, set as 100%) relative to Log[palbociclib(μM)] is plotted for Palbo ^S and Palbo ^R cell lines with ESR1:D538G. In fig. 2C photographs of colony formation assay results for palbo ^S and palbo ^R cell lines with ESR1:D538G are shown for control and after treatment with palbociclib (500 nM). In fig. 2D Western blot results are illustrated for the LTED, LTED + palbo, LTED - palbo ^R and LTED - palbo ^R + palbo ^R models having the ESR1:D538G mutation.

[00120] Referring now to FIG. 3A-3D, which depict the generation and characterization of palbociclib-resistant models in wild-type and ESR1 mutant backgrounds. In fig. 3A, the development of palbociclib resistance in established LTED cell lines with ESR1:Y537S is plotted for palbociclib-sensitive ( ^palboR ) and palbociclib-resistant ( ^palboR ) cell lines. In fig. 3B, the percentage of growth inhibition (normalized to control set as 100%) versus Log[palbociclib(μM)] is plotted for Palbo ^S and Palbo ^R cell lines with ESR1:Y537S. In fig. 3C shows photographs of colony formation assays of the palbo ^S and palbo ^R cell lines with ESR1:Y537S for control and after treatment with palbociclib (500 nM). In Fig. 3D, Western blot results are illustrated for the LTED, LTED + palbo, LTED - palbo ^R and LTED - palbo ^R + palbo models having the ESR1:Y537S mutation.

[00121] Referring now to FIG. 4A-4F, which depict the generation and characterization of ribociclib- and abemaciclib-resistant models in wild-type and ESR1 mutant ESR1 backgrounds. In fig. 4A, ribociclib resistance in engineered ESR1:wild-type LTED cell lines is plotted for ribociclib-sensitive (Ribo ^S ) and ribociclib-resistant (Ribo ^R ) cell lines. In fig. 4B, abemaciclib resistance in engineered ESR1:wild-type LTED cell lines is plotted for abemaciclib-sensitive (abema ^S ) and abemaciclib-resistant (abema ^R ) cell lines. In fig. 4C, the percentage of growth inhibition (normalized to control set as 100%) versus Log[ribociclib(μM)] is plotted for ESR1:wild-type ribo ^S and ribo ^R cell lines. In fig. 4D photographs of colony formation assay results for ESR1:wild-type ribo ^S and ribo ^R cell lines are shown for control and after treatment with ribociclib (500 nM). In fig. 4E percent growth inhibition (normalized to control, set to 100%) versus Log[abemaciclib(μM)] is plotted for abema ^S and abema ^R cell lines with ESR1:wild type. In fig. 4F photographs of colony formation assay results for Abema ^S and Abema ^R cell lines with ESR1:wild type are shown for control and after treatment with abemaciclib (500 nM).

[00122] Referring now to FIG. 5A-5F, which depict the generation and characterization of ribociclib- and abemaciclib-resistant models in wild-type and ESR1 mutant backgrounds. In fig. In Figure 5A, ribociclib resistance in established LTED cell lines with ESR1:D538G is plotted for ribociclib-sensitive (pn6o ^S ) and ribociclib-resistant (ribo ^R ) cell lines. In fig. 5B, abemaciclib resistance in established LTED cell lines with ESR1:D538G is plotted for abemaciclib-sensitive (abema ^R ) and abemaciclib-resistant (abema ^R ) cell lines. In fig. 5C, the percentage of growth inhibition (normalized to control set as 100%) versus Log[ribociclib(μM)] is plotted for Ribo ^S and Ribo ^R cell lines with ESR1:D538G. FIG. 5D photographs of colony formation assay results for Ribo ^S and Ribo ^R cell lines with ESR1:D538G are shown for control and after treatment with ribociclib (500 nM). In fig. 5E percent growth inhibition (normalized to control set to 100%) versus Log[abemaciclib(μM)] is plotted for Abema ^S and Abema ^R cell lines with ESR1:D538G. FIG. 5F photographs of colony formation assay results for Abema ^S and Abema ^R cell lines with ESR1:D538G are shown for control and after treatment with abemaciclib (500 nM).

[00123] Referring now to FIG. 6A-6F, which depict the generation and characterization of ribociclib- and abemaciclib-resistant models in wild-type and ESR1 mutant ESR1 backgrounds. In fig. In Figure 6A, ribociclib resistance in established LTED cell lines with ESR1:Y537S is plotted for ribociclib-sensitive (pn6o ^S ) and ribociclib-resistant (ribo ^R ) cell lines. In fig. 6B, abemaciclib resistance in established LTED cell lines with ESR1:Y537S is plotted for abemaciclib-sensitive (abema ^S ) and abemaciclib-resistant (abema ^R ) cell lines. In fig. 6C, the percentage of growth inhibition (normalized to control, set as 100%) versus Log[ribociclib(μM)] is plotted for the Ribo ^S and Ribo ^R cell lines with ESR1:Y537S. In fig. 6D photographs of colony formation assay results for ribo ^S and ribo ^R cell lines with ESR1:Y537S are shown for control and after treatment with ribociclib (500 nM). In fig. 6E percent growth inhibition (normalized to control, set to 100%) versus Log[abemaciclib(μM)] is plotted for Abema ^S and Abema ^R cell lines with ESR1:Y537S. In fig. 6F photographs of colony formation assay results for Abema ^S and Abema ^R cell lines with ESR1:Y537S are shown for control and after treatment with abemaciclib (500 nM).

[00124] Referring now to FIG. 7A-7C, dose-dependent tumor growth inhibition and tumor regression were observed for elacestrant, regardless of previous treatment or ESR1 mutant status. In fig. 7A shows the EC ₅₀ (nM) values and the percentage of growth inhibition is plotted against Log[elacestrant (nM)] for the ESR1:wild-type cell line sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:wild-type, ribociclib ^R cell line with ESR1:wild type and abemaciclib ^R cell line with ESR1:wild type. In fig. 7B shows the EC ₅₀ (nM) values and the percentage of growth inhibition is plotted against Log[elacestrant (nM)] for the ESR1:D538G cell line sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line ESR1:D538G, the ribociclib cell line ^R with ESR1:D538G and abemaciclib cell lines ^R with ESR1:D538G FIG. 7C shows the EC ₅₀ values (nM) and the percentage of growth inhibition is plotted against Log [elacestrant (nM)] for the ESR1:Y537S cell line sensitive to CDK4/6 inhibitors, palbociclib ^R cell line with ESR1:Y537S cell line ribociclib ^R with ESR1:Y537S and abemaciclib ^R cell lines with ESR1:Y537S.

[00125] Referring now to FIG. 8A-8C, long-term growth inhibition of cell lines resistant to CDK4/6 inhibitors was observed for elacestrant. In fig. 8A shows photographs of colony formation assays showing, in the top row, the growth of a control ESR1:wild-type cell line sensitive to CDK4/6 inhibitors, a palbociclib ^R cell line with ESR1:wild-type, a ribociclib ^R cell line with ESR1:wild-type, and abemaciclib ^R cell line with ESR1:wild type, while the photographs in the bottom row visualize cell growth for the control cell line with ESR1:wild type, sensitive to CDK4/6 inhibitors, palbociclib ^R cell line with ESR1:wild type, line ribociclib ^R cell line with ESR1:wild type and abemaciclib ^R cell line with ESR1:wild type after treatment with elacestrant (300 nM). In fig. 8B shows photographs of colony formation assays, in which the top row shows the growth of the control cell line with ESR1:D538G, sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:D538G, the ribociclib ^R cell line with ESR1:D538G, and the abemaciclib cell line ^R with ESR1:D538G, while the photographs in the bottom row visualize cell growth for the control cell line with ESR1:D538G, sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:D538G, the ribociclib ^R cell line with ESR1: D538G and abemaciclib ^R cell lines with ESR1:D538G after treatment with elacestrant (300 nM). In fig. 8C shows photographs of colony formation assays, in which the top row shows the growth of the control cell line with ESR1:Y537S, sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:Y537S, the ribociclib ^R cell line with ESR1:Y537A, and the abemaciclib cell line ^R with ESR1:Y537S, while the photographs in the bottom row visualize cell growth for the control cell line with ESR1:Y537S, sensitive to CDK4/6 inhibitors, the palbociclib ^R cell line with ESR1:Y537S, the ribociclib ^R cell line with ESR1: Y537S and abemaciclib ^R cell lines with ESR1:Y537S after treatment with elacestrant (300 nM).

[00126] Referring now to FIG. 9A-9F, elacestrant was observed to inhibit the growth of a palbociclib-insensitive PDX model. In fig. Figure 9A shows mean tumor volumes over time in athymic nude mice implanted with a WHIM43 PDX xenograft with the ESR1:D538G mutation that were treated with vehicle control, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg). /kg). In fig. 9B shows ERα protein quantification results obtained by plotting ERα/vinculin (normalized to control) versus treatment of a WHIM43 xenograft PDX model harboring the ESR1:D538G mutation with vehicle control, palbociclib, fulvestrant (3 mg/dose). and elacestrant (30 and 60 mg/kg). In fig. Figure 9C shows the results of quantification of E2F1 protein levels, which were obtained by plotting E2E1/vinculin (normalized to control) versus treatment of a WHIM43 xenograft PDX model harboring the ESR1:D538G mutation with control medium, palbociclib, fulvestrant (3 mg/dose). and elacestrant (30 and 60 mg/kg). In fig. 9D shows the results of quantification of CCNE1 (or cyclin E1) protein levels, which were obtained by plotting PgR mRNA levels (normalized to control) relative to treatment of a WHIM43 xenograft PDX model harboring the ESR1:D538G mutation with control medium, palbociclib, fulvestrant ( 3 mg/dose) and elacestrant (30 and 60 mg/kg). In fig. 9E shows PgR mRNA levels, which were determined by plotting PgR mRNA levels (normalized to control) against treatment of the WHIM43 xenograft PDX model harboring the ESR1:D538G mutation with control medium, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg/kg). In fig. 9F shows Western blot results illustrating a WHIM43-HI xenograft PDX model harboring the ESR1:D538G mutation treated with vehicle control, palbociclib, fulvestrant (3 mg/dose), and elacestrant (30 and 60 mg/kg).

[00127] Referring now to FIG. 10A-10C, which demonstrate that elacestrant inhibits ER signaling in CDK4/6 inhibitor-resistant models. In fig. 10A shows the results of progesterone receptor (PgR) quantification in ESR1:wild-type mutant tumor cell models, which were obtained by plotting PgR mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines with control and elacestrant ( 100 nM and 1000 nM). In fig. 10B shows the results of quantification of trefoil factor 1 (TFF1) in ESR1:wild-type mutant tumor cell models, which were obtained by plotting TFF1 mRNA levels (normalized to control) relative to treatment of Palbo ^S and Palbo ^R control cell lines. and elacestrant (100 nM and 1000 nM). In fig. 10C shows the results of quantification of estrogen-regulated growth (GREB1) in ESR1:wild-type mutant tumor cell models, which were obtained by plotting GREB1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines with control and elacestrant (100 nM and 1000 nM).

[00128] Referring now to FIG. 11A-11C, which demonstrate that elacestrant inhibits ER signaling in CDK4/6 inhibitor-resistant models. In fig. 11A shows the results of progesterone receptor (PgR) quantification in tumor cell models harboring the ESR1:D538G mutation, which were obtained by plotting PgR mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines with control and elacestrant (100 nM and 1000 nM). In fig. 11B shows the results of quantification of trefoil factor 1 (TFF1) in tumor cell models harboring the ESR1:D538G mutation, which were obtained by plotting TFF1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines with control and elacestrant (100 nM and 1000 nM). In fig. 11C shows the results of quantification of estrogen-regulated growth (GREB1) in tumor cell models harboring the ESR1:D538G mutation, which were obtained by plotting GREB1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines with control and elacestrant ( 100 nM and 1000 nM).

[00129] Referring now to FIG. 12A-12C, which demonstrate that elacestrant inhibits ER signaling in CDK4/6 inhibitor-resistant models. In fig. 12A shows the results of progesterone receptor (PgR) quantification in tumor cell models harboring the ESR1:Y537S mutation, which were obtained by plotting PgR mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines with control and elacestrant (100 nM and 1000 nM). In fig. 11B shows the results of quantification of trefoil factor 1 (TFF1) in tumor cell models harboring the ESR1:Y537S mutation, which were obtained by plotting TFF1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines with control and elacestrant (100 nM and 1000 nM). In fig. 11C shows the results of quantification of estrogen-regulated growth (GREB1) in tumor cell models harboring the ESR1:Y537S mutation, which were obtained by plotting GREB1 mRNA levels (normalized to control) relative to treatment of palbo ^S and palbo ^R cell lines with control and elacestrant ( 100 nM and 1000 nM).

[00130] Referring now to FIG. 13, which demonstrated that elacestrant inhibited tumor growth in a PDX model previously treated with a combination of fulvestrant and palbociclib. In fig. 13 graph shows tumor volume of the WHIM43-HI PDX xenograft model (previously untreated) as a function of treatment day when the model was treated with vehicle or the combination of fulvestrant and palbociclib (data for fulvestrant 3 mg/dose are from a separate study). Tumors from the fulvestrant and palbociclib group were then re-implanted in another study (with palbociclib treatment ST941-HI; first passage) and then treated with vehicle, fulvestrant (3 mg/dose), palbociclib (25 mg/kg) and elacestrant (30 mg /kg) to demonstrate that elacestrant was still effective in inhibiting tumor growth in a PDX model previously treated with the combination of fulvestrant and palbociclib.

Other embodiments

[00131] All publications and patents mentioned in this disclosure are incorporated herein by reference to the same extent as if each individual publication or patent application had been specifically and individually identified as being incorporated by reference. If the meaning of terms in any of the patents or publications incorporated by reference conflicts with the meaning of terms used in this disclosure, the meaning of the terms in this disclosure is intended to control. Moreover, in the foregoing discussion only exemplary embodiments of the present invention have been disclosed and described. One skilled in the art will readily understand from such consideration and from the accompanying drawings and claims that various changes, modifications and variations may be made therein without departing from the spirit and scope of the present invention as defined in the following claims.

Claims (19)

1. The use of elacestrant or a pharmaceutically acceptable salt or solvate thereof for the inhibition and attenuation of palbociclib-resistant, ribociclib-resistant or abemaciclib-resistant estrogen receptor-alpha positive cancer in a subject. 2. Use according to claim 1, wherein the resistant estrogen receptor-alpha positive cancer is resistant to palbociclib. 3. Use according to claim 1, wherein the stable estrogen receptor-alpha positive cancer is resistant to ribociclib. 4. Use according to claim 1, wherein the resistant estrogen receptor-alpha positive cancer is resistant to abemaciclib. 5. Application according to any one of paragraphs. 1-4, wherein the stable estrogen receptor-alpha positive cancer is characterized by one or more mutations selected from the group consisting of D538G, Y537X ₁ , L536X ₂ , P535H, V534E, S463P, V392I, E380Q and combinations thereof, where: X ₁ is S, N or C; and X ₂ represents R or Q. 6. Use according to claim 5, wherein the mutation is Y537S. 7. Use according to claim 5, wherein the mutation is D538G. 8. Application according to any one of paragraphs. 1-7, wherein the persistent estrogen receptor-alpha positive cancer is additionally resistant to or progresses with respect to a drug selected from the group consisting of antiestrogens, aromatase inhibitors, and combinations thereof. 9. Application according to any one of paragraphs. 1-8, wherein the stable estrogen receptor-alpha positive cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer, breast cancer brain metastasis, and pituitary cancer. 10. Application according to any one of paragraphs. 1-9, wherein the persistent estrogen receptor-alpha positive cancer is an advanced stage or metastatic breast cancer. 11. Application according to any one of paragraphs. 1-9, wherein the persistent estrogen receptor-alpha positive cancer is breast cancer. 12. Application according to any one of paragraphs. 1-11, wherein the subject is a postmenopausal woman. 13. Application according to any one of paragraphs. 1-11, wherein the subject is a premenopausal woman. 14. Application according to any one of paragraphs. 1-11, wherein the subject is a postmenopausal woman who has relapsed or progressed after previous treatment with selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs). 15. Application according to any one of paragraphs. 1-14, wherein elacestrant is administered to the subject at a dose of from about 200 mg/day to about 500 mg/day. 16. Application according to any one of paragraphs. 1-15, wherein elacestrant 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. 17. Application according to any one of paragraphs. 1-14, wherein the elacestrant is administered to the subject at a dose that is the maximum tolerated dose for the subject. 18. Application according to any one of paragraphs. 1-17, wherein the subject is identified for treatment by measuring the increased expression of one or more genes 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, 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. 19. Use according to claim 18, wherein one or more genes are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1 and MTOR.