Methods for treating cancer

Abstract

To provide methods for treating cancer.SOLUTION: Provided is a drug for inhibiting tumor growth or causing tumor regression in a subject with estrogen receptor alpha-positive breast cancer brain metastasis, the subject having one or more estrogen receptor alpha mutations selected from the group consisting of Y537S, Y537C, Y537N, D538G, and S463P. The drug comprises, as an active ingredient, a therapeutically effective amount of RAD1901 having a given structure, or a salt or solvate thereof, which is used in combination with palbociclib.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 154,699, filed April 29, 2015, U.S. Provisional Patent Application No. 62 / 155,451, filed April 30, 2015, U.S. Provisional Patent Application No. 62 / 252,085, filed November 6, 2015, U.S. Provisional Patent Application No. 62 / 265,696, filed December 10, 2015, U.S. Provisional Patent Application No. 62 / 158,469, filed May 7, 2015, U.S. Provisional Patent Application No. 62 / 252,916, filed November 9, 2015, U.S. Provisional Patent Application No. 62 / 265,774, filed December 10, 2015, and U.S. Provisional Patent Application No. 62 / 265,774, filed May 7, 2015. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 192,940, filed July 15, 2015, U.S. Provisional Patent Application No. 62 / 265,658, filed December 10, 2015, U.S. Provisional Patent Application No. 62 / 323,572, filed April 15, 2016, U.S. Provisional Patent Application No. 62 / 192,944, filed July 15, 2015, U.S. Provisional Patent Application No. 62 / 265,663, filed December 10, 2015, and U.S. Provisional Patent Application No. 62 / 323,576, filed April 15, 2016, all of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to methods for treating cancer. [Background technology]

[0003] Breast cancer is classified 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 cancers account for two-thirds of all breast cancers. Estrogen and ER are associated with non-breast cancers, such as ovarian, colon, prostate, and endometrial cancers.

[0004] ER is activated by estrogen and translocates to the cell nucleus where it binds to DNA, thereby regulating the activity of various genes (see, for example, Non-Patent Document 1 and Non-Patent Document 2).

[0005] Drugs that inhibit estrogen production, such as aromatase inhibitors (AIs, e.g., letrozole, anastrozole, and aromasin), or those 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, e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 / DPC974, SRN-927, ICI182782, and AZD9496), are already in use or are being developed in the treatment of ER-positive breast cancer.

[0006] SERMs (e.g., tamoxifen) and AIs are commonly used as first-line adjuvant systemic therapies for ER-positive breast cancer. Tamoxifen is commonly used for ER-positive breast cancer. AIs suppress estrogen production in peripheral tissues by blocking the activity of aromatase, which converts androgens into estrogen in the body. However, AIs cannot stop the ovaries from producing estrogen, so AIs are mainly used to treat postmenopausal women. Furthermore, because AIs have fewer serious side effects and are more effective than tamoxifen, AIs can also be used to treat premenopausal women and suppress ovarian function. See, for example, Non-Patent Document 3.

[0007] Although initial treatment with these agents can be successful, many patients ultimately relapse with drug-resistant breast cancer. Mutations affecting ER have emerged as a potential mechanism for the development of this resistance. See, for example, Non-Patent Document 4. Mutations in the ligand-binding domain (LBD) of ER are found in 21% of metastatic ER-positive breast cancer samples from patients receiving at least one line of endocrine therapy. Non-Patent Document 5.

[0008] Fulvestrant is currently the only SERD approved for the treatment of ER-positive metastatic breast cancer with disease progression after anti-estrogen therapy. Despite its clinical efficacy, fulvestrant's utility is limited by reduced bioavailability due to the amount of drug that can be administered in a single dose. Imaging studies using 18F-fluoroestradiol positron emission tomography (FES-PET) have shown that even at a dose level of 500 mg, some patients may not have complete ER inhibition, suggesting that inadequate dosing may be the reason for treatment failure.

[0009] Another issue associated with estrogen-targeted therapy is that it may have undesirable effects on the uterus, bone, and other tissues. ER directs the transcription of estrogen-responsive genes in a wide variety of tissues and cell types. These effects may be particularly pronounced when endogenous levels of estrogen and other ovarian hormones decline during menopause. For example, tamoxifen acts as a partial agonist in the endometrium, which can cause osteoporosis in premenopausal women and increase the risk of endometrial cancer. In postmenopausal women, AIs can cause more bone loss and fractures than tamoxifen. Due to its mechanism of action, patients treated with fulvestrant may also be at risk of osteoporosis.

[0010] Cell cycle regulators such as cyclins and cyclin-dependent kinases (CDKs) have been reported to have an effect on ER expression. (Non-Patent Document 6) Selective CDK4 / 6 inhibitors (e.g., ribociclib, abemaciclib, and palbociclib) are available for use in tumor types, where CDK4 / 6 plays a crucial role in targeting the cell cycle transition from G1 to S phase, with improved efficacy and fewer side effects compared to normal cells. (Non-Patent Document 7), published online on March 31, 2016 (http: / / www.nature.com / nrclinonc / journal / vaop / ncurrent / full / nrclinonc.2016.26.html). Selective CDK4 / 6 inhibitors showed the best response when tested in combination with endocrine therapy in patients with ER-positive breast cancer.

[0011] Palbociclib in combination with the aromatase inhibitor letrozole (PALoMA-1 / TRIO 18 study) was approved in February 2015 for the treatment of hormone receptor (HR)-positive (HR+), HER2-negative (HER2-) advanced breast cancer as initial endocrine therapy in postmenopausal women. In February 2016, palbociclib in combination with the SERD fulvestrant (PALOMA-3 study) was approved for the treatment of patients with ER+, HER2-negative advanced or metastatic breast cancer who had progressed on prior endocrine therapy. The FDA granted breakthrough therapy designation to the CDK4 / 6 inhibitor abemaciclib (LY2835219) as monotherapy for heavily pretreated patients with refractory HR-positive advanced breast cancer based on data from a phase 1 trial (JPBA trial). Additional combinations of selective CDK4 / 6 inhibitors (eg, ribociclib, abemaciclib, and palbociclib) with endocrine therapy (eg, AIs, SERMs, and SERDs) are currently under development. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Marino et al., “Estrogen Signaling Multiple Pathways to Impact Gene Transcription,” Curr. Genomics 7(8):497-508(2006) [Non-patent document 2] Heldring et al., “Estrogen Receptors: How Do They Signal and What Are Their Targets,” Physiol.Rev.87(3):905-931(2007) [Non-patent document 3] Francis et al., “Adjuvant Ovarian Suppression in Premenopausal Breast Cancer,” N. Engl. J. Med., 372:436-446 (2015) [Non-patent document 4] Robinson et al., “Activating ESR1 mutations in hormone-resistant metastatic breast cancer,” Nat.Genet.45:1446-51 (2013) [Non-Patent Document 5] Jeselsohn, et al., “ESR1 mutations-a mechanism for acquired endocrine resistance in breast cancer,” Nat.Rev.Clin.Oncol.,12:573-83(2015) [Non-patent document 6] Lamb et al., “Cell cycle regulators cyclin D1 and CDK4 / 6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity,” Cell Cycle 12(15):2384-2394(2013) [Non-Patent Document 7] O'Leary et al., “Treating cancer with selective CDK4 / 6 inhibitors,” Nat.Rev.Clin.Oncol.(2016) Summary of the Invention [Problem to be solved by the invention]

[0013] However, CDK4 / 6 inhibitors exhibit toxicities that may require intermittent treatment (O'Leary). Furthermore, there remains a need for more durable and effective ER-targeted therapies that can overcome the challenges associated with current endocrine therapies while providing additional benefits when combined with CDK4 / 6 inhibitors to combat advanced stage and / or prior treatment-resistant cancers. [Means for solving the problem]

[0014] That is, the gist of the present invention relates to the following. Item 1 1. A drug for inhibiting tumor growth or causing tumor regression in a subject with estrogen receptor alpha positive breast cancer brain metastases having one or more estrogen receptor alpha mutations selected from the group consisting of Y537S, Y537C, Y537N, D538G and S463P, comprising, as an active ingredient, a therapeutically effective amount of the structure: [ka] or a salt or solvate thereof, wherein the agent is used in combination with palbociclib. Section 2 Item 1, wherein the mutation is Y537S. Section 3 The agent according to item 1, wherein the ratio (T / P) of the concentration of RAD1901 or a salt or solvate thereof in the tumor to the concentration of RAD1901 or a salt or solvate thereof in the plasma after administration is at least about 15. Section 4 Item 2. The agent according to Item 1, wherein the salt is RAD1901 dihydrochloride. Section 5 Item 2. The drug according to Item 1, wherein the therapeutically effective amount is 150 mg to 2,000 mg. Section 6 1. A medicament for treating breast cancer brain metastases in a subject with estrogen receptor alpha positive breast cancer brain metastases having one or more estrogen receptor alpha mutations selected from the group consisting of Y537S, Y537C, Y537N, D538G and S463P, comprising as an active ingredient a compound having the structure: [ka] or a salt or solvate thereof, wherein the agent is used in combination with a cdk4 / 6 inhibitor. Section 7 7. The method of claim 6, wherein the RAD1901 is administered at a total daily dose of 100 mg to 1,500 mg. Section 8 Item 7. The drug according to Item 6, wherein the cdk4 / cdk6 inhibitor is selected from the group consisting of abemaciclib, ribociclib, and palbociclib. Section 9 Item 9. The agent according to Item 8, wherein the cdk4 / 6 inhibitor is palbociclib. Section 10 Item 10. The agent according to Item 9, wherein the palbociclib is administered at a daily dose of 25 mg to 250 mg. Section 11 Item 9. The agent according to Item 8, wherein the cdk4 / 6 inhibitor is ribociclib. Section 12 Item 12. The agent according to Item 11, wherein the ribociclib is administered at a daily dose of 200 mg to 1,000 mg. Section 13 Item 9. The agent according to Item 8, wherein the cdk4 / 6 inhibitor is abemaciclib. Section 14 Item 14. The method of claim 13, wherein the abemaciclib is administered at a daily dose of 300 mg. [Effects of the Invention]

[0015] The present invention can provide a method for treating cancer. [Brief explanation of the drawings]

[0016] [Figure 1] The RAD1901-palbociclib combination demonstrated improved tumor growth inhibition (TGI) compared with RAD1901 monotherapy in various patient-derived xenograft (PDx) models, regardless of ESR1 status and prior endocrine therapy. The percentage of TGI in PDx models treated with RAD1901 alone or in combination with palbociclib is shown. [Figure 2]The combination of RAD1901 and palbociclib demonstrated tumor growth inhibition and regression in wild-type (WT) ERα MCF-7 xenograft models (PR+, HER2-). (A): Vehicle control, palbociclib (45 mg / kg, PO once daily), fulvestrant (3 mg / dose, s.c. once weekly), the combination of fulvestrant (3 mg / dose, s.c. once weekly) and palbociclib (45 mg / kg, PO once daily), RAD1901 (60 mg / kg, PO once daily), and RAD1901 (60 mg / kg, PO once daily) and palbociclib (45 mg / kg, PO once daily). Tumor growth in MCF-7 xenograft models treated with palbociclib (45 mg / kg, once-daily oral administration), fulvestrant (3 mg / dose, once-weekly subcutaneous injection), or fulvestrant (3 mg / dose, once-weekly subcutaneous injection) in combination with palbociclib (45 mg / kg, once-daily oral administration) in a one-way ANOVA; "ns" indicates no significant difference, *p-value < 0.05, and ***p-value < 0.001; (B): Vehicle control, palbociclib (45 mg / kg, once-daily oral administration), fulvestrant (3 mg / dose, once-weekly subcutaneous injection), or fulvestrant (3 mg / dose, once-weekly subcutaneous injection) in combination with palbociclib (45 mg / kg, once-daily oral administration) in a one-way ANOVA; (C): Vehicle control, palbociclib (45 mg / kg, once-daily oral administration), fulvestrant (3 mg / dose, once-weekly subcutaneous injection), or fulvestrant (45 mg / kg, once-daily oral administration) in combination with palbociclib (45 mg / kg, once-daily oral administration) in a one-way ANOVA; "ns" indicates no significant difference, *p-value < 0.05, and ***p-value < 0.001; (B): Vehicle control, palbociclib (45 mg / kg, once-daily oral administration), fulvestrant (3 mg / dose, once-weekly subcutaneous injection), or fulvestrant (45 mg / kg, once-daily oral administration) in combination with palbociclib (45 mg / kg, once-daily oral administration) in a one-way ANOVA; (C): Vehicle control, palbociclib (45 mg / kg, once-daily oral administration), fulvestrant (3 mg (B) Change in individual tumor size from baseline to the end of the study in MCF-7 xenograft models treated with RAD1901 (60 mg / kg, orally once daily), RAD1901 (60 mg / kg, orally once daily), and RAD1901 (60 mg / kg, orally once daily) in combination with palbociclib (45 mg / kg, orally once daily); (C) Change in individual tumor size from baseline to the end of the study in MCF-7 xenograft models treated with RAD1901 (60 mg / kg, orally once daily), RAD1901 (60 mg / kg, orally once daily), and RAD1901 (60 mg / kg, orally once daily) in combination with palbociclib (45 mg / kg, orally once daily); (D) Change in individual tumor size from baseline to the end of the study in MCF-7 xenograft models treated with RAD1901 (60 mg / kg, orally once daily), RAD1901 (60 mg / kg, orally once daily), and RAD1901 (60 mg / kg, orally once daily) in combination with palbociclib (45 mg / kg, orally once daily). Figure 1 shows tumor growth in an MCF-7 xenograft model treated with fulvestrant (3 mg / dose, subcutaneous injection once weekly) and palbociclib (45 mg / kg, oral once daily), a combination of RAD1901 (30 or 60 mg / kg, oral once daily), and a combination of RAD1901 (30 or 60 mg / kg, oral once daily) and palbociclib (45 mg / kg, oral once daily). [Figure 3]The combination of RAD1901 and palbociclib showed tumor growth inhibition and regression in the WT ERα PDx-11 model (PR+, Her2+, previously treated with an aromatase inhibitor, fulvestrant, and chemotherapy). (A): Tumor growth in PDx-11 models treated with vehicle control, fulvestrant (3 mg / dose, subcutaneous injection once weekly), palbociclib (45 mg / kg, oral, once daily), RAD1901 (60 mg / kg, oral, once daily), and the combination of RAD1901 (60 mg / kg, oral, once daily) and palbociclib (45 mg / kg, oral, once daily); (B): Change in individual tumor size from baseline to the end of study in PDx-11 models treated with vehicle control, fulvestrant (3 mg / dose, subcutaneous injection once weekly), RAD1901 (60 mg / kg, oral, once daily), and the combination of RAD1901 (60 mg / kg, oral, once daily) and palbociclib (45 mg / kg, oral, once daily). n=8-10 / group. [Figure 4] The combination of RAD1901 and palbociclib demonstrated inhibition of tumor growth in WT ER+ PDx-2 models (PR+, Her2+, treatment-naive). (A): Tumor growth in PDx-2 models treated with vehicle control, RAD1901 (60 mg / kg, oral, once daily), fulvestrant (3 mg / dose, subcutaneous injection once weekly), and the combination of RAD1901 (60 mg / kg, oral, once daily) and fulvestrant (3 mg / dose, subcutaneous injection once weekly); (B): Tumor growth in PDx-2 models treated with vehicle control, palbociclib (75 mg / kg, oral, once daily), RAD1901 (60 mg / kg, oral, once daily), and the combination of RAD1901 (60 mg / kg, oral, once daily) and palbociclib (75 mg / kg, oral, once daily). n=8~10 / group. [Figure 5] The efficacy of RAD1901 persisted for at least 2 months after terminating treatment with RAD1901, although estradiol treatment continued in the WT ERα PDx-4 model (PR+, Her2+, treatment naive). [Figure 6]The combination of RAD1901 and palbociclib demonstrated inhibition of tumor growth in mutant (Y537S) ERα PDx-5 models (PR+, Her2+, previously treated with an aromatase inhibitor). (A): Tumor growth in PDx-5 models treated with vehicle control, fulvestrant (3 mg / dose, subcutaneous injection once weekly), RAD1901 (60 mg / kg, oral once daily), palbociclib (75 mg / kg, oral once daily), and the combination of RAD1901 (60 mg / kg, oral once daily) and palbociclib (75 mg / kg, oral once daily); (B): Tumor growth in PDx-5 models treated with fulvestrant (3 mg / dose, subcutaneous injection once weekly), palbociclib (75 mg / kg, oral once daily), and RAD1901 (60 mg / kg, oral once daily). (B) Change in individual tumor size from baseline to day 17 in the combination of RAD1901 (60, 120 mg / kg, orally once daily) and palbociclib (75 mg / kg, orally once daily), and (C) change in individual tumor size from baseline to day 56 in the combination of RAD1901 (60 mg / kg, orally once daily) and palbociclib (75 mg / kg, orally once daily). n=8-10 / group. [Figure 7] The combination of RAD1901 and palbociclib showed inhibition of tumor growth in the mutant (Y537S) ERαPDx-5 model (PR+, Her2+, previously treated with an aromatase inhibitor). (A): Tumor growth of PDx-5 models treated with vehicle control, fulvestrant (3 mg / dose, subcutaneous injection once a week), RAD1901 (60 mg / kg, oral once daily), palbociclib (oral once daily), and the combination of RAD1901 (60 mg / kg, oral once daily) and palbociclib (oral once daily); (B): Tumor growth of PDx-5 models treated with vehicle control, fulvestrant (3 mg / dose, subcutaneous injection once a week), RAD1901 (120 mg / kg, oral once daily), palbociclib (oral once daily), and the combination of RAD1901 (120 mg / kg, oral once daily) and palbociclib (oral once daily). [Figure 8] The combination of RAD1901 and palbociclib demonstrated tumor growth inhibition in mutant (Y537S) ERα PDx-5 models (PR+, Her2+, previously treated with an aromatase inhibitor). These models were treated with vehicle control, fulvestrant (3 mg / dose, subcutaneous injection once weekly), RAD1901 (60 mg / kg, oral administration once daily), palbociclib (75 mg, oral administration once daily), the combination of fulvestrant (3 mg / dose, subcutaneous injection once weekly) and palbociclib (75 mg, oral administration once daily), and the combination of RAD1901 (60 mg / kg, oral administration once daily) and palbociclib (75 mg, oral administration once daily). n = 8–10 per group. [Figure 9] Pharmacokinetic analysis of fulvestrant in nude mice. Plasma concentrations of fulvestrant at 1 mg / dose (filled diamonds), 3 mg / dose (filled circles), and 5 mg / dose (filled triangles) are shown. Nude mice received a subcutaneous dose of fulvestrant on day 1 and a second dose on day 8. Plasma concentrations of fulvestrant were monitored at the indicated time points up to 168 hours after the second dose. [Figure 10] Effect of RAD1901 and fulvestrant (Faslodex) on mouse survival in an intracranial MCF-7 tumor model. [Figure 11]Representative images of uterine FES-PET scans from subjects treated with 200 and 500 mg of RAD1901 orally once daily, and changes in ER involvement after RAD1901 treatment. (A): Transverse views of uterine CT scans before (a) and after (c) treatment with 200 mg of RAD1901, and transverse views of uterine FES-PET scans before (b) and after (d) treatment with RAD1901; (B): Sagittal views of uterine CT scans before ((a) upper panel) and after ((a) lower panel) treatment with 500 mg of RAD1901, and uterine FES-PET scans before ((b) upper panel) and after ((b) lower panel) treatment with RAD1901. Sagittal view of ET scan, transverse view of uterine CT scan before ((c) upper panel) and after ((c) lower panel) treatment with RAD1901, transverse view of uterine FES-PET scan before ((d) upper panel) and after ((d) lower panel) treatment with RAD1901; (C): Percent change in ER involvement after treatment with RAD1901 for subjects 1-3 (200 mg) and subjects 4-7 (500 mg) compared to baseline (before RAD1901 treatment). [Figure 12] Representative images of FES-PET scans of the uterus (A) and pituitary gland (B) before (baseline) and after (post-treatment) treatment with RAD1901 (500 mg). (a) Lateral section, (b) longitudinal section, and (c) longitudinal section. [Figure 13] PR and ER expression in MCF-7 xenograft models treated with vehicle control, RAD1901, palbociclib, the combination of RAD1901 and palbociclib, fulvestrant, and the combination of fulvestrant and palbociclib. [Figure 14] RAD1901 treatment resulted in complete ER reduction and inhibited ER signaling in MCF-7 (A) and T47D (B) cell lines in vitro. ER expression was demonstrated in both cell lines treated with RAD1901 and fulvestrant at various concentrations of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM, respectively. ER signaling was demonstrated by three ER target genes tested: PGR, GREB1, and TFF1. [Figure 15]Treatment with RAD1901 resulted in ER degradation and abolition of ER signaling in MCF-7 xenograft models. (A): Western blot showing PR and ER expression in MCF-7 xenograft models treated with vehicle control, 30 and 60 mg / kg RAD1901, and 3 mg / dose of fulvestrant 2 or 8 hours after the last dose; (B): ER protein expression in MCF-7 xenograft models treated with vehicle control, 30 and 60 mg / kg RAD1901, and 3 mg / dose of fulvestrant 2 or 8 hours after the last dose; (C): PR protein expression in MCF-7 xenograft models treated with vehicle control, 30 and 60 mg / kg RAD1901, and 3 mg / dose of fulvestrant 8 hours after the last dose. [Figure 16] Treatment with RAD1901 resulted in a rapid decrease in PR in MCF-7 xenograft models. (A): Western blot showing PR expression in MCF-7 xenograft models treated with vehicle control, 30, 60, and 90 mg / kg RAD1901 8 or 12 hours after a single dose; (B): Western blot showing PR expression in MCF-7 xenograft models treated with vehicle control, 30, 60, and 90 mg / kg RAD1901 4 or 24 hours after the seventh dose; (C): Dose-dependent decrease in PR expression in MCF-7 xenograft models treated with 30, 60, and 90 mg / kg RAD1901. [Figure 17] Treatment with RAD1901 resulted in a sharp decrease in proliferation in MCF-7 xenograft models. (A): Representative photographs of sectioned tumors taken from MCF-7 xenograft models treated with vehicle control and 90 mg / kg RAD1901 8 hours after a single dose and 24 hours after the fourth dose, stained for the proliferation marker Ki-67; (B): Histogram showing the decrease in proliferation marker Ki-67 in MCF-7 xenograft models treated with vehicle control and 90 mg / kg RAD1901 8 hours after a single dose and 24 hours after the fourth dose. [Figure 18]Treatment with 30, 60, and 120 mg / kg RAD1901 resulted in even more significant Ki67 reductions than fulvestrant (1 mg / animal) at the end of study tumor time point of the PDx-4 model at 4 hours on the final day of the 56-day efficacy study. [Figure 19] Treatment with 60 and 120 mg / kg RAD1901 resulted in reduced ER signaling in vivo in the PDx-5 model, concomitant with decreased PR expression. [Figure 20] Effect of RAD1901 on uterine tissue in weaned female Sprague-Dawley rats. (A): Uterine wet weight of rats sacrificed 24 hours after the last dose; (B): Epithelial weight in uterine histological sections; (C): Representative sections of uterine tissue stained with toluidine blue O at 400x magnification; (D): Total RNA extracted from uterine tissue and analyzed by quantitative RT-PCR for levels of complement C3 expression relative to the 18S ribosomal RNA housekeeping gene. [Figure 21] Plasma pharmacokinetic results after administration of 200, 500, 750, and 1000 mg / kg RAD1901 on day 7. [Figure 22] 3ERT(I). [Figure 23] 3ERT(II). [Figure 24] Superposition of ERα LBD-antagonist complexes summarized in Table 10. [Figure 25] Modeling of (A) RAD1901-1R5K and (B) GW5-1R5K. [Figure 26] Modeling of (A) RAD1901-1SJ0 and (B) E4D-1SJ0. [Figure 27] Modeling of (A) RAD1901-2JFA and (B) RAL-2JFA. [Figure 28] Modeling of (A) RAD1901-2BJ4 and (B) OHT-2BJ4. [Figure 29] Modeling of (A) RAD1901-2IOK and (B) IOK-2IOK. [Figure 30]Superposition of the three-dimensional structure of RAD1901 obtained from IFD analysis by 1R5K and 2OUZ. [Figure 31] Superposition of the three-dimensional structure of RAD1901 obtained from IFD analysis using 2BJ4 and 2JFA. [Figure 32] Superposition of the three-dimensional structure of RAD1901 obtained from IFD analysis by 2BJ4, 2JFA, and 1SJ0. [Figure 33] IFD of RAD1901 by 2BJ4. [Figure 34] Protein surface interactions of RAD1901 docked to 2BJ4 by IFD. [Figure 35] IFD of fulvestrant by 2BJ4. [Figure 36] IFD of fulvestrant and RAD1901 by 2BJ4. [Figure 37] Overlay of IFDs of fulvestrant and RAD1901 with 2BJ4. [Figure 38] In vitro binding assay of RAD1901 with WT and LBD mutant ERα constructs. [Figure 39] Locations of exemplary mutations in ERα and their frequencies. [Figure 40] RAD1901 levels in plasma, tumor, and brain of mice implanted with MCF7 cells after 40 days of treatment. [Figure 41] SUVs in uterus, muscle, and bone in human subjects treated with a dose of 200 mg orally once daily for 6 days. [Figure 42] SUVs in uterus, muscle, and bone from human subjects (n=4) treated with a dose of 500 mg orally once daily for 6 days. [Figure 43]Effect of RAD1901 on BMD in ovariectomized rats. Adult female rats underwent either sham surgery or ovariectomy before initiating once-daily treatment with vehicle, E2 (0.01 mg / kg), or RAD1901 (3 mg / kg). (n = 20 per treatment group). BMD was measured by dual-emission X-ray absorptiometry at baseline and 4 weeks after treatment. Data are expressed as mean ± standard deviation. *P < 0.05 vs. matched OVX + Veh controls. BMD, bone mineral density; E2, beta-estradiol; OVX, ovariectomized; Veh, vehicle. [Figure 44] Effect of RAD1901 on femoral microarchitecture in ovariectomized rats. Adult female rats underwent either sham surgery or ovariectomy before initiating once-daily treatment with vehicle, E2 (0.01 mg / kg), or RAD1901 (3 mg / kg). (n = 20 per treatment group). After 4 weeks, bone microarchitecture was assessed using micro-computed tomography. Data are expressed as mean ± standard deviation. *P < 0.05 vs. corresponding OVX + Veh controls. ABD, apparent bone mineral density; BV / TV, bone volume density; ConnD, connective density; E2, beta-estradiol; OVX, ovariectomized; TbN, trabecular number; TbTh, trabecular width; TbSp, trabecular center distance; Veh, vehicle. [Figure 45] Key baseline demographics for a Phase 1 dose-escalation study of RAD1901. [Figure 46] Most frequent (>10%) treatment-related AEs in a Phase 1 dose-escalation study of RAD1901. AEs graded by CTCAE v4.0. Any patient with multiple scenarios of the same preferred term was counted only once to the most severe grade. *>10% of patients in all active groups had any related TEAE. N=number of subjects with at least one treatment-related AE in a given category. [Figure 47] Pharmacokinetic parameters (day 7) in a Phase 1 dose-escalation study of RAD1901. [Figure 48] Frequency of LBD mutations. [Figure 49]Differences in ER-α LBD-antagonist complexes in residual posture versus 3ERT. [Figure 50] Assessment of structural overlap of ER-α LBD-antagonist complexes by RMSD calculations. [Figure 51] Analysis of ligand binding in ER-α LBD-antagonist complexes. [Figure 52] Model evaluation of RAD1901 docking. [Figure 53] Induced fit docking scores of RAD1901 with 1R5K, 1SJ0, 2IFA, 2BJ4, and 2OUZ. DETAILED DESCRIPTION OF THE INVENTION

[0017] As shown in the Examples section below, the combination of RAD1901 and palbociclib (RAD1901-palbociclib combination) (structure below) demonstrated greater tumor growth inhibition than RAD1901 alone in breast cancer xenograft models, regardless of ESR1 status, PR status, and prior endocrine therapy (Example I(A)). Treated xenograft models had tumors expressing wild-type (WT) or mutant (e.g., Y537S) ERα, with or without PR expression, high or low Her2 expression, and with or without prior endocrine therapy (e.g., tamoxifen (tam), AI, fulvestrant), chemotherapy, Her2 inhibitors (Her2i, e.g., trastuzumab, lapatinib), bevacizumab, and / or rituximab (Figure 1). The RAD1901-palbociclib combination demonstrated greater tumor growth inhibition (TGI > 65%) in xenograft models, whereas RAD1901 alone achieved a TGI of 26-64%. The RAD1901-palbociclib combination demonstrated greater tumor growth inhibition (TGI > 65%) in xenograft models, whereas RAD1901 alone achieved a TGI < 25%. The RAD1901-palbociclib combination demonstrated greater tumor regression than RAD1901 alone in xenograft models that were highly responsive to RAD1901 treatment (TGI > 65%), e.g., PDx-11 (Figure 3A-B).

[0018] The ER WT PDx model and the ER mutant PDx model may have different levels of responsiveness to treatment with fulvestrant alone, palbociclib alone, and / or the combination of fulvestrant and palbociclib (fulvestrant-palbociclib combination). However, the RAD1901-palbociclib combination showed improved tumor growth inhibition and / or tumor regression compared to treatment with RAD1901 alone or palbociclib alone, regardless of whether the PDx model was responsive to fulvestrant treatment and / or the fulvestrant-palbociclib combination. In other words, the RAD1901-palbociclib combination may inhibit tumor growth and / or cause tumor regression in fulvestrant-resistant cancers.

[0019] RAD1901-palbociclib combination treatment demonstrated improved tumor growth inhibition and / or tumor regression compared with treatment with fulvestrant alone or the fulvestrant-palbociclib combination. For example, the RAD1901-palbociclib combination produced even more significant tumor regression in WT ER+ xenograft models than treatment with fulvestrant alone, RAD1901 alone, or palbociclib alone, even though these xenograft models have diverse responses to fulvestrant treatment (e.g., the MCF7 cell line xenograft model (Figures 2A-C), the PDx-11 model (Figures 3A-B), and the PDx-2 model (Figures 3A-B), which were the least responsive to fulvestrant treatment). The RAD1901-palbociclib combination also resulted in more significant tumor regression in WT ER+ MCF7 cell line xenograft models and PDx-11 models than treatment with the fulvestrant-palbociclib combination (Figures 2A-C and 3A-B). The RAD1901-palbociclib combination produced similar effects with RAD1901 at doses of 30 mg / kg or 60 mg / kg, but 30 mg / kg RAD1901 alone was not as effective in inhibiting tumor growth as 60 mg / kg RAD1901 alone (Figure 2C). These results suggest that the RAD1901-palbociclib combination using a lower dose of RAD1901 (e.g., 30 mg / kg) was sufficient to maximize the effect of tumor growth inhibition / tumor regression in these xenograft models.

[0020] The RAD1901-palbociclib combination demonstrated tumor regression or improved tumor growth inhibition in mutant ER+ (e.g., Y537S) PDx models that are poorly responsive to fulvestrant treatment. For example, PDx-5 is an ER Y537S mutant PDx model (PR+, Her2+, prior treatment with AI) that is poorly responsive to fulvestrant treatment. The RAD1901-palbociclib combination demonstrated tumor regression in the PDx-5 model, while palbociclib or RAD1901 alone inhibited tumor growth without causing tumor regression (Figures 6A-C and 7A-B). Furthermore, the RAD1901-palbociclib combination produced similar effects to RAD1901 at doses of 60 mg / kg or 120 mg / kg (Figures 7A-B), suggesting that the RAD1901-palbociclib combination using a lower dose of RAD1901 (e.g., 60 mg / kg) was sufficient to maximize tumor growth inhibition / tumor regression in this PDx model. The RAD1901-palbociclib combination produced more significant tumor growth inhibition than RAD1901 alone, palbociclib alone, fulvestrant alone, or the fulvestrant-palbociclib combination in the mutant PDx-5 model (Figure 8). Unexpectedly, the fulvestrant-palbociclib combination did not significantly enhance tumor growth inhibition compared to treatment with palbociclib alone in the PDx-5 model (Figure 8). Therefore, the addition of fulvestrant did not provide benefit in the PDx-5 model when applied in combination with palbociclib, whereas the addition of RAD1901 did. Furthermore, the longer the treatment, the more significant tumor growth inhibition the RAD1901-palbociclib combination achieved compared with RAD1901 or palbociclib treatment alone (Figure 8). Therefore, the RAD1901-palbociclib combination provides a potent antitumor therapy for WT or mutant ER-expressing ER+ breast cancers with or without PR expression, high or low Her2 expression, and resistance to fulvestrant. [ka]

[0021] The results provided herein also demonstrate that RAD1901 can be delivered to the brain (Example II), and that delivery improved mouse survival in an intracranial tumor model expressing wild-type ERα (MCF-7 xenograft model, Example I(B)). Because palbociclib has been reported to cross the blood-brain barrier (O'Leary), the RAD1901-palbociclib combination is likely to be able to cross the blood-brain barrier and treat ER+ tumors in the brain. This represents an additional advantage over the fulvestrant-palbociclib combination for treating ER+ tumors in the brain, as fulvestrant cannot cross the blood-brain barrier (Vergotel et al., "Fulvestrant, a new treatment option for advanced breast cancer: tolerability versus existing agents," Ann. Oncol., 17(2):200-204 (2006)). Combining RAD1901 with other CDK4 / 6 inhibitor(s) that can cross the blood-brain barrier (e.g., abemaciclib (O'Leary)) may also have similar therapeutic effects on ER+ tumors in the brain.

[0022] RAD1901 has demonstrated sustained efficacy in inhibiting tumor growth after treatment is discontinued, although estradiol treatment is continued (e.g., in the PDx-4 model). Therefore, the RAD1901-palbociclib combination is likely to benefit patients by inhibiting tumor growth after treatment is discontinued, especially when CDK4 / 6 inhibitors (e.g., ribociclib, abemaciclib, and palbociclib) can only be administered intermittently due to their side effects (O'Leary).

[0023] The RAD1901-palbociclib combination is likely to have fewer side effects than treatment with palbociclib alone or in combination with other hormonal therapies (e.g., AIs such as letrozole and SERDs such as fulvestrant). For example, both AIs and fulvestrant can cause bone loss in treated patients. RAD1901 is unlikely to have similar side effects. RAD1901 was found to preferentially accumulate in tumors, with a tumor-to-plasma ratio (T / P ratio) of up to about 35 (Example II). Standardized uptake values ​​(SUVs) for uterus, muscle, and bone were calculated for human subjects treated with RAD1901 once daily at doses of about 200 mg up to about 500 mg (Example III(A)). The uterine signal after administration was close to the level from "non-target tissues" (tissues that do not express estrogen receptors), suggesting a complete attenuation of FES-PET uptake after RAD1901 treatment. Little change was observed in pre- versus post-treatment PET scans in tissues that did not significantly express estrogen receptors (e.g., muscle, bone) (Example IIIA). Finally, RAD1901 treatment antagonized estradiol stimulation of uterine tissue in ovariectomized (OVX) rats (Example IV(A)) and largely preserved bone quality in treated subjects. For example, OVX rats treated with RAD1901 exhibited maintained BMD and femoral microarchitecture (Example IV(A)). Therefore, the RAD1901-palbociclib combination may be particularly useful in patients with osteoporosis or at higher risk of osteoporosis.

[0024] Furthermore, RAD1901 was found to degrade wild-type ERα and abolish ER signaling in vivo in MCF7 cell line xenograft models, demonstrating a dose-dependent reduction in PR in these MCF7 cell line xenograft models (Example III(B)). RAD1901 reduced proliferation in MCF7 cell line xenograft models and PDx-4 models, as evidenced by a reduction in the proliferation marker Ki67 in tumors from treated subjects. RAD1901 also reduced ER signaling in vivo in an ER mutant PDx model that was poorly responsive to fulvestrant treatment (Example III(B)).

[0025] The unexpected efficacy of the RAD1901-palbociclib combination in tumors that are poorly responsive to fulvestrant treatment and tumors expressing mutant ERα may be due to a unique interaction between RAD1901 and ERα. Structural models of ERα bound to RAD1901 and compounds that bind to other ERα were analyzed to obtain information about the specific binding interaction (Example V). Computer modeling indicated that the RAD1901-ERα interaction is unlikely to be affected by mutations in the LBD of ERα, such as the Y537X mutant (where X is S, N, or C), D538G, and S463P, which account for approximately 81.7% of LBD mutations found in a recent study of metastatic ER-positive breast tumor samples from patients who had received at least one endocrine therapy (Table 9, Example V). Therefore, a combination of one or more CDK4 and / or CDK6 inhibitors described herein (e.g., ribociclib, abemaciclib, and palbociclib) with RAD1901 or a solvate (e.g., hydrate) or salt thereof is likely to have therapeutic efficacy with relatively low side effects, similar to the RAD1901-palbociclib combination disclosed herein. Computer modeling has led to the identification of specific residues in the C-terminal ligand-binding domain of ERα that are essential for binding, information that can be used to develop compounds that bind to and antagonize not only wild-type ERα, but also certain mutants and variants thereof, and when combined with a CDK4 / 6 inhibitor (e.g., ribociclib, abemaciclib, and palbociclib), may provide a potent antitumor therapy with relatively low side effects, similar to the RAD1901-palbociclib combination disclosed herein.

[0026] Based on these results, provided herein are methods for inhibiting the growth of or causing regression of an ERα-positive tumor in a subject in need thereof by administering to the subject a therapeutically effective amount of a combination of RAD1901, or a solvate (e.g., hydrate) or salt thereof, and one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib). In certain embodiments, administration of RAD1901, or a solvate (e.g., hydrate) or salt thereof, has additional therapeutic benefits in addition to tumor growth, including, for example, inhibiting cancer cell proliferation or inhibiting ERα activity (e.g., by inhibiting estradiol binding or by degrading ERα). In certain embodiments, the method does not produce negative effects on muscle, bone, breast, and / or uterus.

[0027] In certain embodiments, RAD1901, or a solvate (eg, hydrate) or salt thereof, modulates and / or degrades ERα and mutant ERα.

[0028] In certain embodiments of the methods of tumor growth inhibition or tumor regression provided herein, provided herein are methods for inhibiting the growth of or causing the regression of an ERα-positive tumor in a subject in need thereof by administering to the subject a therapeutically effective amount of a combination of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901, or a solvate (e.g., hydrate) or salt thereof. In certain of these embodiments, the salt has the structure: [ka] RAD1901 dihydrochloride has the formula:

[0029] CDK4 and / or CDK6 inhibitors In certain embodiments, CDK4 and / or CDK6 inhibitors include, but are not limited to, palbociclib, abemaciclib, ribociclib, AMG925, compounds of formula II, compounds of formula III, and compounds of formula IV, solvates thereof, salts thereof, and combinations thereof, as disclosed below. [ka] [ka] [ka] [ka]

[0030] Compounds of Formula II have the structure of Formula II, including pharmaceutically acceptable solvates (e.g., hydrates) thereof, and pharmaceutically acceptable salts thereof: [ka] During the ceremony, each X is independently a heteroatom (e.g., O, S, and N); Each R1 is independently selected from the group consisting of hydrogen, lower alkyl, carboxy lower alkyl, oxygen, and cycloalkyl.

[0031] Unless otherwise specified, a lower alkyl as used herein is an alkyl having 1, 2, 3, 4, 5, or 6 carbons.

[0032] Compounds of formula III have the structure of formula III, including pharmaceutically acceptable solvates (e.g., hydrates) thereof, and pharmaceutically acceptable salts thereof: [ka] During the ceremony, R2 is selected from the group consisting of hydrogen, halogen atoms, NH2, NHR2, NHCOR2, NO2, CN, CH2NH2CH2NHR2, phenyl and heteroaromatic groups, wherein the phenyl or heteroaromatic group is optionally substituted with further substituents selected from the group consisting of lower alkyl, carboxy lower alkyl, oxygen, and cycloalkyl groups; Ar is a phenyl or heteroaromatic group, wherein the phenyl or heteroaromatic group is optionally substituted with further substituents selected from the group consisting of lower alkyl, carboxy lower alkyl (—(C═O)-lower alkyl), oxygen (═O), or a cycloalkyl group; n is 0, 1, 2, or 3.

[0033] The compound of formula IV has the structure of formula IV, as disclosed in EP1295878B1 (herein incorporated by reference), and further includes pharmaceutically acceptable solvates (e.g., hydrates) thereof, and pharmaceutically acceptable salts thereof: [ka] During the ceremony, Ar2 is [ka] and Ar is [ka] or Ar2 is the same as above, and Ar is [ka] is.

[0034] Combination therapy (1) A combination of RAD1901 or a solvate (e.g., hydrate) or salt thereof with one or more CDK4 and / or CDK6 inhibitor(s). When administered alone to a subject, RAD1901 or a solvate (e.g., hydrate) or salt thereof and a CDK4 and / or CDK6 inhibitor(s) both have a therapeutic effect on one or more cancers or tumors (Examples I(A) and I(B)). When administered in combination to a subject, RAD1901 or a solvate (e.g., hydrate) or salt thereof and a CDK4 and / or CDK6 inhibitor(s) have a significantly improved effect on cancers / tumors (Examples I(A) and I(B)).

[0035] As used herein, "inhibiting the growth" of an ERα-positive tumor can refer to slowing the rate of tumor growth or stopping tumor growth altogether.

[0036] As used herein, "tumor regression" or "regression" of an ERα-positive tumor may refer to a reduction in the maximum size of the tumor. In certain embodiments, administration of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) in combination with RAD1901 or a solvate (e.g., hydrate) or salt thereof may result in a reduction in tumor size relative to baseline (i.e., size before treatment begins), or even the eradication or partial eradication of the tumor. Thus, in certain embodiments, the methods of tumor regression provided herein may alternatively be characterized as methods of reducing tumor size relative to baseline.

[0037] As used herein, a "tumor" is a malignant tumor and is used synonymously with "cancer."

[0038] Tumor growth inhibition or regression can be localized to a single tumor or a series of tumors in a particular tissue or organ, or can be systemic (ie, affecting tumors in all tissues or organs).

[0039] Because RAD1901 is known to preferentially bind to ERα versus estrogen receptor beta (ERβ), unless otherwise specified, estrogen receptor, estrogen receptor alpha, ERα, ER, wild-type ERα, and ESR1 are used interchangeably herein. As used herein, "estrogen receptor alpha" or "ERα" refers to a polypeptide comprising, consisting of, or consisting essentially of the wild-type ERα amino acid sequence encoded by the gene ESR1. As used herein, a tumor that is "estrogen receptor alpha positive," "ERα positive," "ER+," or "ERα+" refers to a tumor in which one or more cells express at least one isoform of ERα. In certain embodiments, these cells overexpress ERα. In certain embodiments, a patient has one or more cells in the tumor that express one or more forms of ERβ. In certain embodiments, the ERα-positive tumor and / or cancer is associated with breast, uterine, ovarian, or pituitary cancer. In certain 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, which may or may not be positive for HER2 and HER2+ tumors, and the tumor may express high or low HER2 (e.g., Figure 1). In other embodiments, the patient has a tumor located in another tissue or organ (e.g., bone, muscle, brain), but is nevertheless not associated with breast, uterine, ovarian, or pituitary cancer (e.g., a tumor resulting from migration or metastasis of breast, uterine, ovarian, or pituitary cancer). Thus, in certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, the targeted tumor is a metastatic tumor and / or the tumor has overexpression of ER in other organs (e.g., bone and / or muscle). In certain embodiments, the targeted tumor is a brain tumor and / or carcinoma.In certain embodiments, the targeted tumor is treated with another SERD (e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 / DPC974, SRN-927, ICI182782, and AZD9496), a Her2 inhibitor (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine, and / or pertuzumab), chemotherapy (e.g., Abraxane, Adriamycin, Carboplatin, Cytoxan, Daunorubicin, Doxil, Elence, Fluorouracil, Gemzar, Heraben, Ixempra, and more sensitive to treatment with RAD1901 and the CDK4 and / or CDK6 inhibitors disclosed herein than to treatment with methotrexate, mitomycin, micoxantrone, navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda), aromatase inhibitors (e.g., anastrozole, exemestane, and letrozole), selective estrogen receptor modulators (e.g., tamoxifen, raloxifene, lasofoxifene, and / or toremifene), angiogenesis inhibitors (e.g., bevacizumab), and / or rituximab.

[0040] In certain embodiments of the methods of tumor growth inhibition or tumor regression provided herein, the methods further comprise determining whether the patient has a tumor that expresses ERa prior to administering a combination of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901, or a solvate (e.g., hydrate) or salt thereof. In certain embodiments of the methods of tumor growth inhibition or tumor regression provided herein, the methods further comprise determining whether the patient has a tumor that expresses mutant ERa prior to administering a combination of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901, or a solvate (e.g., hydrate) or salt thereof. In certain embodiments of the methods of tumor growth inhibition or tumor regression provided herein, the methods further comprise determining whether the patient has an ERα-expressing tumor that will or will not respond to fulvestrant treatment prior to administering a combination of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901 or a solvate (e.g., hydrate) or salt thereof. These determinations can be made using any method of detecting expression known in the art and can be made in vitro using a tumor or tissue sample removed from the subject.

[0041] In addition to demonstrating the ability of RAD1901 to inhibit tumor growth in tumors expressing wild-type ERα, the results provided herein demonstrate the unexpected ability of RAD1901 to inhibit the growth of tumors expressing a mutant form of ERα, i.e., Y537S ERα (Example I(A)). Computer modeling evaluation of exemplary ERα mutations, such as ERα with one or more mutations selected from the group consisting of ERα with a Y537X mutation (wherein X is S, N, or C), ERα with a D538G mutation, and ERα with a S463P mutation, indicated that none of these mutations were expected to affect the LBD or specifically interfere with RAD1901 binding (Example V(A)). Based on these results, provided herein are methods for inhibiting the growth of or causing regression of tumors positive for ERα having one or more mutations in the ligand binding domain (LBD) selected from the group consisting of Y537X1 (wherein X1 is S, N, or C), D538G, L536X2 (wherein X2 is R or Q), P535H, V534E, S463P, V392I, E380Q, particularly Y537S ERα, by administering to the subject a therapeutically effective amount of a combination of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901, or a solvate (e.g., hydrate) or salt thereof. In certain embodiments, the combination is RAD1901, or a solvate (e.g., hydrate) or salt thereof. As used herein, "mutant ERα" refers to ERα and variants thereof that contain one or more substitutions or deletions and comprise, consist of, or consist essentially of an amino acid sequence that has 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% identity to the amino acid sequence of ERα.

[0042] In addition to inhibiting breast cancer tumor growth in animal xenograft models, the results disclosed herein demonstrate that RAD1901 exhibits significant accumulation within tumor cells and is capable of crossing the blood-brain barrier (Example II). The ability to cross the blood-brain barrier was confirmed by the fact that administration of RAD1901 significantly prolonged survival in a brain metastasis xenograft model (Example I(B)). Thus, 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 elsewhere in the central nervous system. In certain of these embodiments, the ERα-positive tumor is primarily associated with brain cancer. In other embodiments, the ERα-positive tumor is primarily a metastatic tumor associated with another type of cancer, such as breast, uterine, ovarian, or pituitary cancer, that has migrated from another tissue or organ. In certain of these embodiments, the tumor is a brain metastasis, such as a breast cancer brain metastasis (BCBM). In certain embodiments of the methods disclosed herein, RAD1901, or a solvate (eg, hydrate) or salt thereof, accumulates in one or more cells within the target tumor.

[0043] In certain embodiments of the methods disclosed herein, RAD1901 or a solvate (e.g., hydrate) or salt thereof preferably accumulates in the tumor at a T / P (RAD1901 concentration in tumor / RAD1901 concentration in plasma) ratio 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.

[0044] The results provided herein demonstrate that RAD1901 administration prevents bone loss in ovariectomized rats (Example IV(A)). Thus, in certain embodiments of the methods of tumor growth inhibition or tumor regression provided herein, the combination of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) with RAD1901 or a solvate (e.g., hydrate) or salt thereof does not have undesirable effects on bone, including, for example, undesirable effects on bone volume density, bone surface density, bone mineral density, trabecular number, trabecular width, trabecular central distance, connectivity density, and / or apparent bone density of a treated subject. Because tamoxifen may be associated with bone loss in premenopausal women, and fulvestrant may harm bone structure through its mechanism of action, a combination of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) with RAD1901 or a solvate (e.g., hydrate) or salt thereof may be particularly useful in premenopausal women, tumors resistant to tamoxifen or anti-estrogen therapy, and patients with osteoporosis and / or a high risk of osteoporosis.

[0045] The results provided herein demonstrate that RAD1901 antagonizes estradiol stimulation of uterine tissue in ovariectomized rats (Example IV(A)). Furthermore, in human subjects treated once daily with RAD1901 at doses of 200 mg or up to 500 mg, standardized uptake values ​​(SUVs) for uterine, muscle, and bone tissues, which do not significantly express ER, showed little change in pre- and post-treatment signals (Example III(A)). Thus, in certain embodiments, such administration also does not result in undesirable effects on other tissues, including, for example, uterine, muscle, or breast tissue.

[0046] RAD1901 or a solvate (e.g., hydrate) or salt thereof and a CDK4 and / or CDK6 inhibitor (e.g., ribociclib, abemaciclib, and palbociclib) disclosed herein are co-administered to a subject in need thereof. The term "co-administered" means that RAD1901 or a solvate (e.g., hydrate) or salt thereof can be administered before, during, or after the administration of the CDK4 and / or CDK6 inhibitor. For example, RAD1901 or a solvate (e.g., hydrate) or salt thereof and CDK4 and / or CDK6 inhibitors (e.g., ribociclib, abemaciclib, and palbociclib) disclosed herein can be administered at least once a week, at least once every 6 days, at least once every 5 days, at least once every 4 days, at least once every 3 days, at least once every 2 days, at least once every 24 hours, at least once every 23 hours, at least once every 22 hours, at least once every 21 hours, at least once every 20 hours, at least once every 19 hours, at least once every 18 hours, at least once every 17 hours, at least once every 16 hours, or at least once every 28 hours. about every 15 hours, about every 14 hours, about every 13 hours, about every 12 hours, about every 11 hours, about every 10 hours, about every 9 hours, about every 8 hours, about every 7 hours, about every 6 hours, about every 5 hours, about every 4 hours, about every 3 hours, about every 2 hours, about every 1 hour, about every 55 minutes, about every 50 minutes, about every 45 minutes, about every 40 minutes, about every 35 minutes, about every 30 minutes, about every 25 minutes, about every 20 minutes, about every 15 minutes, about every 10 minutes, or about every 5 minutes. In other embodiments, RAD1901 or a solvate (e.g., hydrate) or salt thereof and a CDK4 and / or CDK6 inhibitor (e.g., ribociclib, abemaciclib, and palbociclib) disclosed herein are administered to a subject simultaneously or substantially simultaneously. In certain of these embodiments, RAD1901 or a solvate (e.g., hydrate) or salt thereof and a CDK4 and / or CDK6 inhibitor (e.g., ribociclib, abemaciclib, and palbociclib) disclosed herein may be administered as part of a single formulation.

[0047] In some embodiments, a combination of RAD1901 or a solvate (e.g., hydrate) or salt thereof and a single CDK4 and / or CDK6 inhibitor is administered to a subject. In other embodiments, a combination of RAD1901 or a solvate (e.g., hydrate) or salt thereof and more than one CDK4 and / or CDK6 inhibitor is administered to a subject. For example, RAD1901 or a solvate (e.g., hydrate) or salt thereof can be mixed with two or more CDK4 and / or CDK6 inhibitors to treat cancer / tumor.

[0048] (2) Dosage A therapeutically effective amount of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901, or a solvate (e.g., hydrate) or salt thereof, for use in the methods disclosed herein, is an amount that, when administered over a specified time interval, results in the achievement of one or more therapeutic criteria (e.g., slowing or halting tumor growth, causing tumor regression, cessation of symptoms, etc.). Combinations for use in the presently disclosed methods may be administered to a subject one or more times. In those embodiments in which the compounds are administered multiple times, they may be administered at a series of intervals, e.g., daily, every other day, weekly, or monthly. Alternatively, they may be administered at irregular intervals as needed, e.g., based on symptoms, the patient's health, etc. A therapeutically effective amount of the combination may be administered once daily, for one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least ten days, or at least 15 days. Optionally, the cancer status or tumor regression is monitored during or after treatment, for example, by FES-PET scan of the subject. The dosage of the combination administered to the subject can be increased or decreased according to the detected cancer status or tumor regression.

[0049] Ideally, a therapeutically effective amount will not exceed the maximum tolerated dose at which 50% or more of treated subjects experience nausea or other toxic reactions that prevent further drug administration. The therapeutically effective amount may vary in subjects depending on various factors, including various symptoms, sex, age, weight range, or general health of the subject, mode of administration and salt or solvate type, varying sensitivity to the drug, the particular type of disease, etc.

[0050] Examples of therapeutically effective amounts of RAD1901 or a solvate (e.g., hydrate) or salt thereof for use in the methods disclosed herein include doses of 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 once daily in subjects with resistant ER-driven tumors or cancers; and doses of about 150 to about 1,500 mg, about 200 to about 1,000 mg, about 250 to about 1,500 mg, or about 300 to about 1,500 mg once daily in subjects with both wild-type ER-driven tumors and / or cancers and resistant tumors and / or cancers. In subjects with primarily wild-type ER-driven tumors and / or cancers, doses include, but are not limited to, 50 to about 1,000 mg, or about 300 to about 1,000 mg, and, primarily, once daily doses of 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. In certain embodiments, the dosage of a compound of Formula I (e.g., RAD1901) or a salt or solvate thereof for use in the presently disclosed methods in an adult subject can be 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 administered once daily. This daily dosage can be achieved via a single dose or multiple doses.

[0051] Therapeutically effective amounts or dosages of the CDK4 and / or CDK6 inhibitors described herein (e.g., ribociclib, abemaciclib, and palbociclib) vary depending on their specific type. Generally, the daily dosage of the CDK4 and / or CDK6 inhibitors described herein (e.g., ribociclib, abemaciclib, and palbociclib) ranges from about 1 mg to about 1,500 mg, about 1 mg to about 1,200 mg, about 1 mg to about 1,000 mg, about 1 mg to about 800 mg, about 1 mg to about 600 mg, about 1 mg to about 500 mg, about 1 mg to about 200 mg, about The ranges range from 1 mg to about 100 mg, from about 1 mg to about 50 mg, from about 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg, from about 50 mg to about 1,500 mg, from about 100 mg to about 1,200 mg, from about 150 mg to about 1,000 mg, from about 200 mg to about 800 mg, from about 300 mg to about 600 mg, and from about 350 mg to about 500 mg.

[0052] Abemaciclib Administration of RAD1901 with abemaciclib can be achieved at 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mg of RAD1901 per day. Doses of 200 mg, 400 mg, 500 mg, 600 mg, 800 mg, and 1,000 mg per day are particularly well-known. Under certain circumstances, a twice-daily administration schedule is preferred. The surprisingly long half-life of RAD1901 in humans after oral administration makes this option particularly viable. Thus, the drug can be administered at 200 mg twice daily (400 mg total per day), 250 mg twice daily (500 mg total per day), 300 mg twice daily (600 mg total per day), 400 mg twice daily (800 mg total per day), or 500 mg twice daily (1,000 mg total per day). Preferably, administration is oral. The dose of abemaciclib can be 50 mg to 500 mg daily or 150 mg to 450 mg daily, and this administration can be every day in a 28-day cycle or for less than 28 days per 28-day cycle, such as 21 days per 28-day cycle, 14 days per 28-day cycle, or 7 days per 28-day cycle. In some embodiments, abemaciclib is administered once daily, or preferably twice daily, where administration is oral. When administered twice daily, the doses can be 4 hours, 8 hours, or 12 hours apart. In certain embodiments, abemaciclib is administered orally at 150 mg twice daily, with doses recommended 12 hours apart.

[0053] As discovered and described herein, significant synergy between RAD1901 and cdk4 / 6 inhibitors appears to exist, and therefore, dose reductions of RAD1901 and / or abemaciclib from the normally recommended or approved doses are contemplated and described herein. For example, RAD1901 may be recommended for monotherapy at doses of 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mg per day, or more specifically, at doses of 200 mg, 400 mg, 500 mg, 600 mg, 800 mg, and 1,000 mg per day. In combination, a given dose reduction by a given percentage means that a dose of 25% to 75% below the usual dose is possible. By way of non-limiting example, a recommended dose of 400 mg of RAD1901 per day can be reduced to a final dose of 100 mg to 300 mg per day, or by 100 mg per day, 200 mg per day, or 300 mg per day. When the dose of RAD1901 is reduced as described, the same percentage reduction generally applies whether the administration is twice daily or once daily. For example, a 50% reduction in twice-daily dose of 400 mg can be administered on a 200 mg twice-daily schedule. In some exceptions, a reduction in the recommended twice-daily dose may be sufficient to allow the total daily dose to be administered as a single daily dose. For example, a typical twice-daily dose of 300 mg given in combination with abemaciclib can be reduced by 50%. Thus, the dose can be administered as 150 mg twice-daily or 300 mg once-daily.

[0054] Similarly, the normally recommended dose of abemaciclib may be reduced when used in combination with RAD1901. If the reduced dose is 25% to 75% lower than the normally recommended dose, as illustrated directly above, the abemaciclib dose may be reduced and combined with the normally recommended monotherapy dose of RAD1901 or a reduced RAD1901 dose. For example, a recommended dose of 150 mg twice daily may be administered 25% to 75% less twice daily than the 150 mg twice daily dose. For example, a 150 mg twice daily dose of abemaciclib may be reduced to a twice daily dose of 37.5 mg to 112.5 mg (75 mg to 225 mg total per day). Alternatively, it may be desirable to reduce the frequency of abemaciclib from the recommended 28-day cycle to a somewhat lower dose. For example, the dosing frequency can be reduced to 22 to 27 days in a 28-day cycle or 21 days in a 28-day cycle, or the dosing frequency can be reduced to 15 to 20 days in a 28-day cycle or 14 days in a 28-day cycle, or the dosing frequency can be reduced to 8 to 13 days in a 28-day cycle or as few as 7 days in a 28-day cycle. The number of days administered can be sequential or combined, as needed under the circumstances. In one embodiment, the total amount over the dosing interval is reduced by 25% to 75% of the recommended dose, and the reduction can occur as a result of less frequent dosing, a dose reduction, or a combination thereof. For example, a recommended 28-day dosing cycle of abemaciclib at a dose of 150 mg twice daily (300 mg total per day) provides a total dose of 8,400 mg over 28 days (28 days x 300 mg total per day). This amount can be reduced from 2,100 mg per 28 days to 6,300 mg per 28 days.

[0055] Ribociclib Administration of RAD1901 with ribociclib can be achieved at 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mg of RAD1901 per day. Doses of 200 mg, 400 mg, 500 mg, 600 mg, 800 mg, and 1,000 mg per day are particularly well-known. Under certain circumstances, a twice-daily administration schedule is preferred. The surprisingly long half-life of RAD1901 in humans after oral administration makes this option particularly viable. Thus, the drug may be administered at 200 mg twice daily (400 mg total per day), 250 mg twice daily (500 mg total per day), 300 mg twice daily (600 mg total per day), 400 mg twice daily (800 mg total per day), or 500 mg twice daily (1,000 mg total per day). Preferably, administration is oral. The dose of ribociclib may be 200 mg to 1,000 mg daily or 250 mg to 750 mg daily, and this administration may be every day in a 28-day cycle or for less than 28 days per 28-day cycle, such as 21 days per 28-day cycle, 14 days per 28-day cycle, or 7 days per 28-day cycle. In some embodiments, ribociclib is administered once daily, where administration is oral. In one particular embodiment, the dose of ribociclib used in combination with RAD1901 is 600 mg once daily, with a dosing interval of 21 days within a 28-day cycle.

[0056] As discovered and described herein, significant synergy between RAD1901 and cdk4 / 6 inhibitors appears to exist, and therefore dose reductions of RAD1901 and / or ribociclib from the normally recommended or approved doses are contemplated and described herein. For example, RAD1901 may be recommended for monotherapy at doses of 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mg per day, or more specifically, at doses of 200 mg, 400 mg, 500 mg, 600 mg, 800 mg, and 1,000 mg per day. In combination, a given dose reduction by a given percentage means that a dose of 25% to 75% below the usual dose is possible. By way of non-limiting example, a recommended dose of 400 mg of RAD1901 per day can be reduced to a final dose of 100 mg to 300 mg per day, or by 100 mg per day, 200 mg per day, or 300 mg per day. When the dose of RAD1901 is reduced as described, the same percentage reduction generally applies whether the administration is twice daily or once daily. For example, a 50% reduction in twice-daily dose of 400 mg can be administered on a 200 mg twice-daily schedule. In some cases, a reduction in the recommended twice-daily dose may be sufficient to allow the total daily dose to be administered as a single daily dose. For example, a typical twice-daily dose of 300 mg given in combination with ribociclib can be reduced by 50%. Thus, the dose can be administered as 150 mg twice-daily or 300 mg once-daily.

[0057] Similarly, the normally recommended dose of ribociclib may be reduced when used in combination with RAD1901. If the reduced dose is 25% to 75% lower than the normally recommended dose, as illustrated directly above, the ribociclib dose may be reduced and combined with the normally recommended monotherapy dose of RAD1901 or a reduced RAD1901 dose. For example, a recommended dose of 600 mg of ribociclib once daily may be administered 25% to 75% less once daily than a 600 mg once daily dose. For example, a recommended dose of 600 mg of ribociclib may be reduced to a dose of 150 mg to 450 mg. Alternatively, it may be desirable to reduce the frequency of ribociclib from the recommended 21 days out of a 28-day cycle to some amount less. For example, the dosing frequency can be reduced to 15 to 20 days in a 28-day cycle or to 14 days in a 28-day cycle, or the dosing frequency can be reduced to 8 to 13 days in a 28-day cycle or to 7 days in a 28-day cycle. The number of days administered can be sequential or combined, as needed under the circumstances. In one embodiment, the total amount over the dosing interval is reduced by 25% to 75% of the recommended dose, and the reduction can occur as a result of less frequent administration, a dose reduction, or a combination thereof. For example, a recommended 28-day dosing cycle of ribociclib (21 days at a 600 mg dose once daily) provides a total dose of 12,600 mg over 28 days (21 dosing days x 600 mg total per day). This amount can be reduced from 3,150 mg per 28-day cycle to 9,450 mg per 28-day cycle.

[0058] Palbociclib RAD1901 administration with palbociclib can be achieved at 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mg of RAD1901 per day. Doses of 200 mg, 400 mg, 500 mg, 600 mg, 800 mg, and 1,000 mg per day are particularly well-known. Under certain circumstances, a twice-daily administration schedule is preferred. The surprisingly long half-life of RAD1901 in humans after oral administration makes this option particularly viable. Thus, the drug may be administered at 200 mg twice daily (400 mg total per day), 250 mg twice daily (500 mg total per day), 300 mg twice daily (600 mg total per day), 400 mg twice daily (800 mg total per day), or 500 mg twice daily (1,000 mg total per day). Preferably, administration is oral. The dose of palbociclib may be 25 mg to 250 mg daily, 50 mg to 125 mg daily, 75 mg to 125 mg daily, 75 mg daily, 100 mg daily, or 125 mg daily. Administration may be daily in a 28-day cycle, or for less than 28 days per 28-day cycle, such as 21 days per 28-day cycle, 14 days per 28-day cycle, or 7 days per 28-day cycle. In some embodiments, palbociclib is administered orally once a day.In certain embodiments, the dose of palbociclib used in combination with RAD1901 is 125 mg once a day, with an administration interval of 21 days in a 28-day cycle, or 100 mg once a day, with an administration interval of 21 days in a 28-day cycle, or 75 mg, with an administration interval of 21 days in a 28-day cycle.

[0059] As discovered and described herein, significant synergy between RAD1901 and palbociclib appears to exist, and therefore, dose reductions of RAD1901 and / or palbociclib from the normally recommended or approved dosing are contemplated and described herein. For example, RAD1901 may be recommended for monotherapy at doses of 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mg per day, or more specifically, at doses of 200 mg, 400 mg, 500 mg, 600 mg, 800 mg, and 1,000 mg per day. In combination, a given dose reduction by a given percentage means that a dose of 25% to 75% below the usual dose is possible. By way of non-limiting example, a recommended dose of 400 mg of RAD1901 per day can be reduced to a final dose of 100 mg to 300 mg per day, or by 100 mg per day, 200 mg per day, or 300 mg per day. When the dose of RAD1901 is reduced as described, the same percentage reduction generally applies whether the administration is twice daily or once daily. For example, a 50% reduction in twice-daily dose of 400 mg can be administered on a 200 mg twice-daily schedule. In some cases, a reduction in the recommended twice-daily dose may be sufficient to allow the total daily dose to be administered as a single daily dose. For example, a typical twice-daily dose of 300 mg administered in combination with palbociclib can be reduced by 50%. Thus, the dose can be administered as 150 mg twice-daily or 300 mg once-daily.

[0060] Similarly, the normally recommended dose of palbociclib may be reduced when used in combination with RAD1901. If the reduced dose is 25% to 75% less than the normally recommended dose, as illustrated directly above, the palbociclib dose may be reduced and combined with the normally recommended monotherapy dose of RAD1901 or the reduced RAD1901 dose. For example, a recommended dose of 125 mg palbociclib once daily may be administered 25% to 75% less once daily than the 125 mg once daily dose. For example, a recommended dose of 125 mg palbociclib may be reduced to a dose of 31.25 mg to 93.75 mg. In some embodiments, specific pre-specified dose reductions of 125 mg to 100 mg daily or 125 mg to 75 mg daily may be used. Alternatively, it may be desirable to reduce the frequency of palbociclib from the recommended 21 days out of a 28-day cycle to some amount less. For example, the dosing frequency can be reduced to 15 to 20 days in a 28-day cycle or to 14 days in a 28-day cycle, or the dosing frequency can be reduced to 8 to 13 days in a 28-day cycle or to 7 days in a 28-day cycle. The number of days administered can be sequential or combined, as needed under the circumstances. In one embodiment, the total amount over the dosing interval is reduced by 25% to 75% of the recommended dose, and the reduction can occur as a result of less frequent administration, a dose reduction, or a combination thereof. For example, a recommended 28-day dosing cycle of palbociclib (21 days at a 125 mg dose once daily) provides a total dose of 2,625 mg over 28 days (21 dosing days x 125 mg total per day). This amount can be reduced from 656.25 mg per 28-day cycle to 1,968.75 mg per 28-day cycle. In another embodiment, the recommended 28 day cycle dose of 2,625 mg total is reduced to 2,100 mg per 28 day cycle.

[0061] In certain embodiments, a therapeutically effective amount of the combination may utilize a therapeutically effective amount of either compound administered alone. In other embodiments, due to the significantly improved synergistic therapeutic effect achieved by the combination, the therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) or salt thereof and the CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) when administered in combination may be less than the therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) or salt thereof and the CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) required when administered alone, and one or both compounds may be administered at a lower dosage than would normally be administered if administered separately. Without being bound by any particular theory, the combination therapy achieves significantly improved efficacy by reducing the dosage of at least one or all of RAD1901 or a solvate (e.g., hydrate) or salt thereof and the CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib), thereby eliminating or reducing undesirable toxic side effects.

[0062] In some embodiments, a therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) or salt thereof when administered as part of a combination is about 30% to about 200%, about 40% to about 200%, about 50% to about 200%, about 60% to about 200%, about 70% to about 200%, about 80% to about 200%, about 90% to about 200%, about 100% to about 200%, 30% to about 150%, about 40% to about 150%, or about 50% to about 150% of the therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) or salt thereof when administered alone. , about 60% to about 150%, about 70% to about 150%, about 80% to about 150%, about 90% to about 150%, about 100% to about 150%, about 30% to about 120%, about 40% to about 120%, about 50% to about 120%, about 60% to about 120%, about 70% to about 120%, about 80% to about 120%, about 90% to about 120%, about 100% to about 120%, 30% to about 110%, about 40% to about 110%, about 50% to about 110%, about 60% to about 110%, about 70% to about 110%, about 80% to about 110%, about 90% to about 110%, or about 100% to about 110%.In some embodiments, when administered as part of a combination, a therapeutically effective amount of a CDK4 and / or CDK6 inhibitor described herein (e.g., ribociclib, abemaciclib, and palbociclib) exhibits about 30% to about 200%, about 40% to about 200%, about 50% to about 200%, about 60% to about 200%, about 70% to about 200%, about 80% to about 200%, about 90% to about 200%, about 100% to about 200%, 30% to about 15% of the CDK4 and / or CDK6 inhibitors described herein (e.g., ribociclib, abemaciclib, and palbociclib) when administered alone. 0%, approx. 40% to approx. 150%, approx. 50% to approx. 150%, approx. 60% to approx. 150%, approx. 70% to approx. 150%, approx. 80% to approx. 150%, approx. 90% to approx. 150%, approx. 100% to approx. 150%, approx. 30% to approx. 120%, approx. 40% to approx. 120%, approx. 50% to approx. 120%, approx. 60% to approx. 120%, approx. 70% to approx. 20%, about 80% to about 120%, about 90% to about 120%, about 100% to about 120%, 30% to about 110%, about 40% to about 110%, about 50% to about 110%, about 60% to about 110%, about 70% to about 110%, about 80% to about 110%, about 90% to about 110%, or about 100% to about 110%.

[0063] In certain embodiments, the cancer or tumor is a resistant ER-driven cancer or tumor (e.g., a mutant ER binding domain (e.g., an ER-driven cancer or tumor) containing one or more mutations, including, but not limited to, Y537X1 (wherein X1 is S, N, or C), D538G, L536X2 (wherein X2 is R or Q), P535H, V534E, S463P, V392I, E380Q, and combinations thereof). α)), and overexpression of ER or tumor and / or cancer growth becomes ligand-independent or is mediated by another SERD (e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 / DPC974, SRN-927, ICI182782, and AZD9496), Her2 inhibitors (e.g., trastuzumab, and / or pertuzumab), chemotherapy (e.g., abraxane, adriamycin, carboplatin, cytoxan, daunorubicin, doxil, erence, fluorouracil, gemzar, heraben, lxempra, methotrexate, mitomycin, micoxantrone, navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda), aromatase inhibitors (e.g., anastrozole, exemestane, and letrozole), selective estrogen receptor modulators (e.g., tamoxifen, raloxifene, lasofoxifene, and / or toremifene), angiogenesis inhibitors (e.g., bevacizumab), and / or rituximab.

[0064] In certain embodiments, the dosage of RAD1901 or a solvate (e.g., hydrate) or salt thereof in combination with a CDK4 and / or CDK6 inhibitor described herein (e.g., ribociclib, abemaciclib, and palbociclib) for general use in the presently disclosed methods in adult subjects can be about 30 mg to 2,000 mg, 100 mg to 1,500 mg, or 150 mg to 1,500 mg, administered orally once daily. This daily dosage can be achieved via a single dose or multiple doses.

[0065] The combination of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901, or a solvate (e.g., hydrate) or salt thereof, can be administered to a subject one or more times. In those embodiments in which the compounds are administered multiple times, they can be administered at a series of intervals, e.g., daily, every other day, weekly, or monthly. Alternatively, they can be administered at irregular intervals as needed, e.g., based on symptoms, the patient's health, etc.

[0066] (3) Preparation In some embodiments, RAD1901 or a solvate (e.g., hydrate) or salt thereof and the CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) are administered in separate formulations. In certain of these embodiments, the formulations may be of the same type. For example, both formulations may be designed for oral administration (e.g., via two separate pills) or for injection (e.g., via two injectable formulations). In other embodiments, RAD1901 or a solvate (e.g., hydrate) or salt thereof and the CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) may be formulated in different types of formulations. For example, one compound may be a formulation designed for oral administration, while the other may be a formulation designed for injection.

[0067] In other embodiments, RAD1901 or a solvate (e.g., hydrate) or salt thereof and one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) are administered as part of a single formulation. For example, RAD1901 or a solvate (e.g., hydrate) or salt thereof and one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) are formulated in a single pill for oral administration or a single dose for injection. In certain embodiments, provided herein are combination formulations comprising RAD1901 or a solvate (e.g., hydrate) or salt thereof and one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib). In certain embodiments, administering the compounds in a single formulation improves patient compliance.

[0068] When administered in combination, the therapeutically effective amount of each compound may be lower than the therapeutically effective amount of each compound when administered alone.

[0069] In some embodiments, formulations comprising RAD1901 or a solvate (e.g., hydrate) or salt thereof, one or more CDK4 and / or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib, and palbociclib), or both RAD1901 or a solvate (e.g., hydrate) or salt thereof and one or more CDK4 and / or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib, and palbociclib) may further comprise one or more pharmaceutical excipients, carriers, adjuvants, and / or preservatives.

[0070] RAD1901 or a solvate (e.g., hydrate) or salt thereof and CDK4 and / or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib, and palbociclib) for use in the presently disclosed methods can be formulated into a unit dosage form, meaning physically discrete units suitable as a single administration for a subject undergoing treatment, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be one of a single daily dose or multiple daily doses (e.g., once daily, about 1 to 4 times daily, or more). When multiple daily doses are used, the unit dosage form can be the same or different for each dose. In certain embodiments, the compounds can be formulated for controlled release.

[0071] RAD1901 or its solvates (e.g., hydrates) or salts and salts or solvates and CDK4 and / or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib, and palbociclib) for use in the presently disclosed methods can be formulated according to any available conventional method. Examples of preferred dosage forms include tablets, powders, fine granules, granules, coated tablets, capsules, syrups, lozenges, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nasal drops, ear drops, poultices, lotions, and the like. Commonly used additives such as diluents, binders, disintegrants, lubricants, colorants, flavoring agents, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, and antioxidants can be used in the formulation. Additionally, formulation can also be carried out by mixing ingredients commonly used as raw materials for pharmaceutical preparations according to conventional methods. Examples of these compositions include, for example, (1) oils such as soybean oil, beef tallow, and synthetic glycerides; (2) hydrocarbons such as liquid paraffin, squalane, and solid paraffin; (3) ester oils such as octyldodecyl myristate and isopropyl myristate; (4) higher alcohols such as cetostearyl alcohol and behenyl alcohol; (5) silicone resins; (6) silicone oils; (7) polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, solid polyoxyethylene castor oil, and polyoxyethylene polyethylene fatty acid esters. (8) surfactants such as propylene glycol block copolymers, (9) water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymers, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose, (10) lower alcohols such as ethanol and isopropanol, (11) polyhydric alcohols such as glycerol, propylene glycol, dipropylene glycol, and sorbitol, (12) sugars such as glucose and sucrose, (13) inorganic powders such as silicic anhydride, magnesium aluminum silicate, and aluminum silicate, and (14) purified water.Examples of additives for use in the above formulations include: 1) diluents such as lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide; 2) binders such as polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polypropylene glycol-polyoxyethylene-block copolymer, meglumine, calcium citrate, dextrin, and pectin; and 3) binders such as polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polypropylene glycol-polyoxyethylene-block copolymer, meglumine, calcium citrate, dextrin, and pectin. ) disintegrants such as starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, carboxymethylcellulose / calcium, etc.; 4) lubricants such as magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, etc.; 5) any coloring agent sufficient to serve as a pharmaceutically acceptable coloring agent when added; 6) flavoring agents such as cocoa powder, menthol, aromatizer, peppermint oil, and cinnamon powder; and 7) pharmaceutically acceptable antioxidants such as ascorbic acid or alpha-tophenol when added.

[0072] One or more CDK4 and / or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901 or solvates (e.g., hydrates) or salts thereof described herein for use in the presently disclosed methods can be formulated into pharmaceutical compositions containing any one or more of the active compounds described herein and a physiologically acceptable carrier (also referred to as a pharmaceutically acceptable carrier, solution, or diluent). Such carriers and solutions include pharmaceutically acceptable salts and solvates of the compounds used in the methods of the invention, as well as mixtures containing two or more of such compounds, pharmaceutically acceptable salts of the compounds, and pharmaceutically acceptable solvates of the compounds. Such compositions are prepared in accordance with accepted pharmaceutical procedures, such as those described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Eaton, Pa. (1985) (incorporated herein by reference).

[0073] The term "pharmaceutically acceptable carrier" refers to a carrier that can be administered to a patient without causing an allergic or other untoward reaction in the patient and that is compatible with other ingredients in the formulation. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients, or carriers appropriately selected for 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., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers that enhance the shelf life or effectiveness of the therapeutic agent.

[0074] One or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) and RAD1901 or solvates (e.g., hydrates) or salts thereof, in free form, can be converted to salts by conventional methods. The term "salt" as used herein is not limited, so long as the salt is formed with RAD1901 or a solvate (e.g., hydrate) or salt thereof and is pharmacologically acceptable. Preferred examples of the salt include hydrohalides (e.g., hydrochlorides, hydrobromides, hydroiodides, etc.), inorganic acid salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, bicarbonates, etc.), organic carboxylates (e.g., acetates, maleates, tartrates, fumarates, citrates, etc.), organic sulfonates (e.g., methanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, camphorsulfonates, etc.), amino acid salts (e.g., aspartates, glutamates, etc.), quaternary ammonium salts, alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (magnesium salts, calcium salts, etc.), and the like. Additionally, hydrochlorides, sulfates, methanesulfonates, acetates, and the like are preferred as "pharmacologically acceptable salts" of compounds according to the present invention.

[0075] Isomers (e.g., geometric isomers, optical isomers, rotamers, tautomers, etc.) of RAD1901 or a solvate (e.g., hydrate) or salt thereof and / or the CDK4 and / or CDK6 inhibitor(s) disclosed herein (e.g., ribociclib, abemaciclib, and palbociclib) can be purified into single isomers using conventional separation methods, including, for example, recrystallization, optical resolution such as diastereomeric salt methodology, enzymatic fractionation, and various chromatographic methods (e.g., thin-layer chromatography, column chromatography, glass chromatography, etc.). As used herein, the term "single isomer" includes not only isomers having 100% purity, but also isomers containing isomers other than the target one, which may be present even through conventional purification procedures. Crystalline polymorphs sometimes exist for RAD1901 or its solvates (e.g., hydrates) or salts and / or CDK4 and / or CDK6 inhibitors (e.g., ribociclib, abemaciclib, and palbociclib), all of which are included in the present invention. Crystalline polymorphs are sometimes single and sometimes mixtures, both of which are included herein.

[0076] In certain embodiments, RAD1901 or a solvate (e.g., hydrate) or salt thereof and / or a CDK4 and / or CDK6 inhibitor (e.g., ribociclib, abemaciclib, and palbociclib) may be in a prodrug form, meaning that some transformation (e.g., oxidation or hydrolysis) must be performed to achieve its active form. Alternatively, RAD1901 or a solvate (e.g., hydrate) or salt thereof and / or a CDK4 and / or CDK6 inhibitor (e.g., ribociclib, abemaciclib, and palbociclib) may be a compound produced by transformation of a parent prodrug into its active form.

[0077] (4) Administration route Routes of administration of RAD1901 or a solvate (e.g., hydrate) or salt thereof and / or the CDK4 and / or CDK6 inhibitor(s) disclosed herein (e.g., ribociclib, abemaciclib, and palbociclib) include, but are not limited to, topical, oral, intradermal, intramuscular, intraperitoneal, intravenous, intravesical, subcutaneous, transdermal, and transmucosal administration.

[0078] (5) Gene profiling In certain embodiments, the methods of tumor growth inhibition or tumor regression provided herein further comprise genetic profiling of the subject, wherein the profiled genes include 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, FGF5, FGF6, FGF7, FGF8, FGF9 ... The gene is one or more selected from GFR1, 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.

[0079] In some embodiments, the present invention provides methods of treating a subpopulation of breast cancer patients, wherein the subpopulation has increased expression of one or more of the genes disclosed above, and methods of treating the subpopulation with an effective dose of one or more CDK4 and / or CDK6 inhibitor(s) described herein (e.g., ribociclib, abemaciclib, and palbociclib) in combination with RAD1901 or a solvate (e.g., hydrate) or salt thereof, according to the administration embodiments described in this disclosure.

[0080] (6) Dose adjustment In addition to demonstrating RAD1901's ability to inhibit tumor growth, the results provided herein also demonstrate that RAD1901 inhibits estradiol binding to ER in the uterus and pituitary gland (Example III(A)). In these experiments, estradiol binding to ER in uterine and pituitary tissues was assessed by FES-PET imaging. After treatment with RAD1901, the observed levels of ER binding were at or below background levels. These results suggest that the antagonistic effect of RAD1901 on ER activity can be assessed using real-time scanning. Based on these results, provided herein are methods for monitoring the effectiveness of treatment with RAD1901, or a solvate (e.g., hydrate) or salt thereof, in the combination therapies disclosed herein, by measuring estradiol-ER binding in one or more target tissues, where reduced or absent binding indicates efficacy.

[0081] Further provided are methods for adjusting the dosage of RAD1901 or a solvate (e.g., hydrate) or salt thereof in the combination therapy disclosed herein based on estradiol-ER binding. In certain embodiments of these methods, binding is measured at several time points after administration of one or more of the first doses of the compounds. If estradiol-ER binding is unaffected 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 the further step of administering an increased second dose of the compound. These steps can be repeated to iteratively increase the dosage until the desired decrease in estradiol-ER binding is achieved. In certain embodiments, these steps can be incorporated into the methods for inhibiting tumor growth provided herein. In these methods, estradiol-ER binding can serve as a surrogate for tumor growth inhibition or an auxiliary means for assessing growth inhibition. In other embodiments, these methods can be used in combination with administration of RAD1901 or a solvate (e.g., hydrate) or salt thereof for purposes other than inhibiting tumor growth, including, for example, inhibiting cancer cell proliferation.

[0082] In certain embodiments, the methods provided herein for adjusting the dosage of RAD1901, or a salt or solvate (e.g., hydrate) thereof, in combination therapy include: (1) administering a first dose of RAD1901 or a salt or solvate (e.g., a 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; (2) detecting estradiol-ER binding activity, for example, using FES-PET imaging as disclosed herein, (i) continuing to administer the first dose (i.e., maintaining the dose level) if the ER binding activity is undetectable or below a predetermined threshold level; or (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 proceeding to step (3); (3) detecting estradiol-ER binding activity, for example, using FES-PET imaging as disclosed herein, (i) if the ER binding activity is undetectable or below a predetermined threshold level, continue to administer a second dose (i.e., maintain the dose level); or (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 proceeding to step (4); (4) Repeating the above steps through a fourth dose, a fifth dose, etc., until no ER binding activity is detectable.

[0083] In certain embodiments, the present invention involves the use of PET imaging to detect and / or treat ER-sensitive or ER-resistant cancers.

[0084] (7) Combinations for the methods disclosed herein Another aspect of the present invention relates to a pharmaceutical composition comprising RAD1901 or a solvate (e.g., hydrate) or salt thereof and / or a CDK4 and / or CDK6 inhibitor(s) disclosed herein (e.g., ribociclib, abemaciclib, and palbociclib) in a therapeutically effective amount as disclosed herein for the combination methods described herein.

[0085] RAD1901-ERα interaction (1) Mutant ERα in ER-positive breast cancer tumor samples from patients receiving at least one line of endocrine therapy In five studies reported over the past two years, a total of 187 metastatic ER-positive breast cancer tumor samples from patients receiving at least one line of endocrine therapy were sequenced, and ER LBD mutations were identified in 39 patients (21%) (Jeselsohn). Among the 39 patients, the six most frequent LBD mutations are shown in Figure 39, adapted from Jeselsohn.

[0086] The frequencies of all LBD mutations are summarized in Table 9.

[0087] Computer modeling indicated that the RAD1901-ERα interaction was unlikely to be affected by mutations in the ERα LBD, such as the Y537X mutation (where X is S, N, or C), D538G, and S463P, which accounted for approximately 81.7% of LBD mutations found in a recent study of metastatic ER-positive breast tumor samples from patients who had received at least one of the endocrine therapies (Table 10, Example V).

[0088] Provided herein are complexes and crystals of RAD1901 binding to ERα and / or mutant ERα, wherein the mutant ERα contains one or more mutations, including, but not limited to, Y537X1 (wherein X1 is S, N, or C), D538G, L536X2 (wherein X2 is R or Q), P535H, V534E, S463P, V392I, E380Q, and combinations thereof.

[0089] In certain embodiments of the methods provided herein, the LBD of ERα and mutant ERα comprises AF-2. In other embodiments, the LBD comprises, consists of, or consists essentially of amino acids 299-554 of ERα. In certain embodiments, the LBD of mutant ERα comprises one or more mutations, including, but not limited to, Y537X1 (wherein X1 is S, N, or C), D538G, L536X2 (wherein X2 is R or Q), P535H, V534E, S463P, V392I, E380Q, and combinations thereof. As used herein, the term "and / or" includes both "and" and "or."

[0090] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, it is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art will develop equivalent means or reactions without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein while still remaining within the scope of the invention. It is the intention of the inventors that such variations be included within the scope of the invention. [Example]

[0091] Materials and Methods Test Compound RAD1901 used in the following examples was (6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-2-ol dihydrochloride, manufactured by IRIX Pharmaceuticals, Inc. (Florence, SC). RAD1901 was stored as a dry powder, formulated for use as a uniform suspension in 0.5% (w / v) methylcellulose in deionized water, and administered orally in animal models. 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 administered by subcutaneous injection. Other experimental reagents were purchased from Sigma-Aldrich unless otherwise noted.

[0092] cell line MCF-7 cells (human breast metastatic adenocarcinoma) were purchased from the American Type Culture Collection (Rockville, MD) and were routinely maintained in phenol red-free minimum essential medium (MEM) containing 2 mM L-glutamine and Earle's BSS, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate, supplemented with 0.01 mg / ml bovine insulin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA), at 5% CO.

[0093] T47D cells were cultured in 10 cm dishes in a 5% CO 2 incubator in RPMI growth medium supplemented with 10% FBS and 5 μg / mL human insulin until approximately 75% confluent.

[0094] In vivo xenograft model All mice were housed in sterile housing in individually ventilated cages with sterile, dust-free bedding cobs under a light-dark cycle (12-14 hour circadian cycle of artificial light) and controlled room temperature and humidity, with ad libitum access to sterile food and water. Tumors were measured twice weekly with calipers and volume was calculated using the formula: (L*W 2 )*0.52.

[0095] PDx Model Some examples of patient-derived xenograft models (PDx models) are shown in Figure 1. PDx models with patient-derived breast cancer tumors were constructed from viable human tumor tissue or fluid that had been serially passaged a limited number of times in animals (athymic nude mice (Nu(NCF)-Foxn1nu)) to maintain tumor heterogeneity. Pre-test tumor volumes were recorded for each experiment starting approximately 1 week before its estimated start date. Tumors were grown to approximately the tumor volume onset (TVI) range (150-250 mm). 3 When the animals reached the age of 18, they were randomized into treatment and control groups, and dosing began (Day 0, 8–10 animals in each group). All animals in each study were followed individually for each experiment. Dosing began on Day 0, and animals in all groups were dosed by body weight (0.01 mL per gram, 10 ml / kg). Starting on Day 0, each group was treated with vehicle (control, orally administered once daily until endpoint), tamoxifen (1 mg / subject, subcutaneous injection, daily until endpoint), fulvestrant (Faslodex®; 1 mg / subject or 3 mg / subject, subcutaneous injection once weekly x 5, extended as needed), or RAD1901 (30, 60, or 120 mg / kg, orally administered once daily until endpoint), as specified. The treatment period lasted 56–60 days, depending on the model. Drinking water for these PDx models was supplemented with 17β-estradiol.

[0096] Drug effectiveness In all studies, starting on day 0, tumor dimensions were measured by digital calipers with individual and average estimated tumor volumes (mean TV ± standard error) recorded for each group, and tumor volume was calculated using the formula (Yasui et al. Invasion Metastasis 17:259-269 (1997), which is incorporated herein by reference): TV = width 2 Each group or study was terminated as soon as the tumor volume (TV) endpoint was reached (time endpoint was 60 days, and volume endpoint was a group mean of 2 cm). 3 ), 2cm 3 Individual mice reaching tumor volumes above this level were removed from the study and final measurements were included in the group average until the average reached the volume endpoint or the study reached the time endpoint.

[0097] Efficacy calculations and statistical analysis Tumor growth inhibition (%) (TGI(%)) values ​​were calculated at a single time point (when the control group reached tumor volume or time endpoint) and reported for each treatment group (T) versus control (C) using the initial (i) and final (f) tumor measurements according to the formula (Corbett TH et al. In vivo methods for screening and preclinical testing. In: Teicher B, ed., Anticancer Drug Development Guide. Totowa, NJ: Humana. 2004:99-123.): TGI(%)=1-Tf-Ti / Cf-Ci.

[0098] statistics TGI test - one-way analysis of variance + Dunnett multiple comparison test (Corbett TH et al.).

[0099] Sample collection At endpoint, tumors were removed. One section was snap-frozen, while another was placed in 10% NBF for at least 24 hours and formalin-fixed, paraffin-embedded (FFPE). The snap-frozen samples were stored at -80°C, and the FFPE blocks were stored at room temperature.

[0100] Western blot Cells were harvested and protein expression was analyzed using standard techniques. Tumors were harvested at the indicated time points after the last day of dosing and homogenized in RIPA buffer with protease and phosphatase inhibitors using a Tissuelyser (Qiagen). Equal amounts of protein were separated by MW, transferred to nitrocellulose membranes, and blotted with the following antibodies using standard techniques: Estrogen receptor (SantaCruz (HC-20); sc-543) Progesterone receptor (Cell Signaling Technologies; 3153) Vinculin (Sigma-Aldrich, v9131)

[0101] qPCR analysis was performed as follows: cells were harvested, mRNA was extracted, and equal amounts were used for cDNA synthesis and qPCR with primers specific for the progesterone receptor, GREB1, and TFF1 (LifeTech). Bands were quantified using 1D Quant software (GE).

[0102] immunohistochemistry Tumors were harvested, fixed in formalin, and embedded in paraffin. Embedded tumors were sectioned (6 μM) and stained with antibodies specific for ER, PR, and Her2. Quantification was performed as follows: five fields were counted for positive cells (0-100%) and staining intensity (0-3+). H-scores (0-300) were calculated using the following formula: positivity (%) * intensity.

[0103] Example I. The RAD1901-palbociclib combination provided enhanced tumor growth inhibition in tumors and / or cancers expressing WT ER or mutant ER (e.g., Y537S) with different prior endocrine therapies.

[0104] I(A). Efficacy of RAD1901 in animal xenograft models I(A)(i) The RAD1901-palbociclib combination demonstrated improved tumor growth inhibition in PDx models (PDx-1 to PDx-12) despite ER status and prior endocrine therapy.

[0105] Figure 1 demonstrates tumor growth inhibition in various PDx models in mice treated with RAD1901 alone and / or the RAD1901-palbociclib combination. Twelve patient-derived xenograft models were screened to test RAD1901 response in various genetic backgrounds with varying levels of ER, PR, and Her2. Full efficacy studies were performed on PDx models marked with an "*" (PDx-1 through PDx-4 and PDx-12) with n=8-10. These PDx models were treated with vehicle (negative control), RAD1901 administered orally once daily at a dose of 60 mg / kg, or the RAD1901-palbociclib combination (60 mg / kg RAD1901 and palbociclib) administered orally once daily for 60 days. Screening studies were conducted on other PDx models (PDx-5 to PDx-11) with n=3 treated with vehicle (negative control) or RAD1901 at a dose of 90 mg / kg, administered orally once daily for 60 days. As shown in Figure 1, RAD1901 treatment was effective in PDx models with growth-driven ER and additional drivers (e.g., PR+ and / or Her2+). RAD1901 was effective in inhibiting tumor growth in models with ER mutations and / or high levels of Her2 expression (PDx) despite prior treatment, naive (Rx-negative), or treatment with aromatase inhibitors, tamoxifen (TAM), chemotherapy, Her2 inhibitors (Her2i, e.g., trastuzumab, lapatinib), bevacizumab, fulvestrant, and / or rituximab.

[0106] The RAD1901-palbociclib combination demonstrated enhanced tumor growth inhibition in PDx models (PDx-2, PDx-5, PDx-7, PDx-8, PDx-9, and PDx-10) in which single-agent RAD1901 treatment achieved a TGI of 64% or less. These PDx models included naive models (PDx-2, ER++, PR++, and Her2+) and models previously treated with aromatase inhibitors (AIs, tamoxifen, chemotherapy, Her2 inhibitors (Her2i, e.g., trastuzumab, lapatinib), bevacizumab, fulvestrant, and / or rituximab (PDx-5, PDx-7, PDx-8, PDx-9, and PDx-10). The PDx-5 model expressed mutant ESR1, whereas the other PDx models expressed wild-type ESR1. The PDx-2 and PDx-5 models were PR+ and Her2+, while the other PDx models were PR- and HER2+. Because RAD1901 single-agent treatment provided a TGI of 65% or more in the PDx-6 and PDx-11 models, the difference between RAD1901 treatment with and without palbociclib could not be shown in Figure 1. See, for example, Example (I)(A)(ii) and Figure 3B, which show that the combination of RAD1901 and palbociclib resulted in more significant tumor regression than RAD1901 alone in the PDx11 model.

[0107] I(A)(ii) The RAD1901-palbociclib combination caused more regression than RAD1901 alone in a xenograft model expressing WT ER.

[0108] I(A)(ii)(1) RAD1901-palbociclib caused more regression than RAD1901 alone in MCF-7 xenografts that responded to fulvestrant treatment.

[0109] MCF7 xenograft model Two days before cell transplantation, Balb / C nude mice were incubated with 17β-estradiol pellets at a release of 0.18 / 90 days. MCF7 cells (PR+, Her2-) were harvested and 1 × 10 7 Cells were implanted subcutaneously into the right flank of Balb / C-nude mice. Tumors were 200 mm 3 When the mean tumor volume reached 100, mice were randomized into treatment groups according to tumor volume and treated with test compounds. Each group was treated with vehicle (control, orally administered once daily until endpoint), fulvestrant (Faslodex®; 3 mg / subject, subcutaneously administered once weekly x 5, extended as needed), RAD1901 (30 mg / kg or 60 mg / kg of subject, orally administered once daily until endpoint), palbociclib (45 mg / kg, 75 mg / kg, or 100 mg / kg, orally administered once daily until endpoint), or a RAD1901-palbociclib combination, as specified from day 0. The treatment period lasted 28 days.

[0110] Figures 2A-C show that in an MCF7 xenograft model, the RAD1901-palbociclib combination, with RAD1901 administered orally at 60 mg / kg once daily and palbociclib administered orally at 45 mg / kg once daily, significantly reduced tumor size by approximately 50% compared with RAD1901 (60 mg / kg administered orally once daily) at day 14. RAD1901 and palbociclib showed efficacy in inhibiting tumor growth when administered alone.

[0111] To confirm these results, MCF7 xenograft mice were treated with vehicle (negative control), RAD1901 (30 or 60 mg / kg, orally once daily), palbociclib (45 mg / kg, orally once daily), a combination of RAD1901 (30 or 60 mg / kg, orally once daily) and palbociclib (45 mg / kg, orally once daily), fulvestrant (3 mg / dose, subcutaneously once weekly), or a combination of fulvestrant (3 mg / dose, subcutaneously once weekly) and palbociclib (45 mg / kg, orally once daily). Tumor size was measured at various time points for 27 days.

[0112] The results are shown in Figures 2A-B. Treatment with the combination of RAD1901 (60 mg / kg) and palbociclib (45 mg / kg) again resulted in significant tumor regression and was superior to treatment with RAD1901, palbociclib, or fulvestrant alone, or in combination with fulvestrant and palbociclib (Figures 2A-B).

[0113] Figure 2C shows that the RAD1901-palbociclib combination produced similar effects with RAD1901 at doses of 30 mg / kg or 60 mg / kg, but 30 mg / kg of RAD1901 alone was not as effective at inhibiting tumor growth as 60 mg / kg of RAD1901 alone. These results suggest that the RAD1901-palbociclib combination using a lower dose of RAD1901 (e.g., 30 mg / kg) was sufficient to maximize the effects of tumor growth inhibition / tumor regression in this xenograft model.

[0114] Treatment with the combination of RAD1901 and palbociclib was also more effective at reducing ER and PR expression in vivo in an MCF-7 xenograft model than treatment with RAD1901, palbociclib, or fulvestrant alone, or in combination with fulvestrant and everolimus (Figure 13; tumors harvested 2 hours after the last dose).

[0115] I(A)(ii)(2) RAD1901-palbociclib induced greater tumor regression than RAD1901 alone in PDx-11 and PDx-2 models that responded to fulvestrant treatment.

[0116] The ER WT PDx models PDx-2 (PR+, Her2-, treatment-naive) and PDx-11 (PR+, Her2+, treated with AI, fulvestrant, and chemotherapy) showed different sensitivities to fulvestrant (3 mg / dose, subcutaneous injection once weekly). PDx-2 and PDx-11 models were treated with the combination of RAD1901 (60 mg / kg, oral once daily) and palbociclib (75 mg / kg, oral once daily), RAD1901 alone (60 mg / kg, oral once daily), palbociclib alone (75 mg / kg, oral once daily), or fulvestrant alone (3 mg / dose, subcutaneous injection once weekly). The PDx-11 model was also treated with a combination of palbociclib (75 mg / kg, orally, once daily) and fulvestrant (3 mg / dose, subcutaneously once weekly).

[0117] In the PDx-11 model, fulvestrant or palbociclib alone significantly inhibited tumor growth, with mice treated with fulvestrant showing better tumor growth inhibition. The combination of fulvestrant and palbociclib showed slight tumor regression. Surprisingly, administration of RAD1901 alone or in combination with palbociclib resulted in significant tumor regression, and this combination achieved a more significant tumor regression effect than in the wild-type ESR1 PDx model (Figure 3B).

[0118] In the PDx-2 model, oral administration of RAD1901 alone achieved a better tumor growth inhibition effect compared with injection of fulvestrant alone (Figure 4A). Furthermore, administration of RAD1901 or palbociclib alone significantly inhibited tumor growth. Unexpectedly, administration of RAD1901 in combination with palbociclib produced an even more potent effect in inhibiting tumor growth (Figure 4B).

[0119] Furthermore, in PDx-4 models that responded to fulvestrant treatment (1 mg / dose, subcutaneous injection once weekly), RAD1901-mediated inhibition of tumor growth was maintained in the absence of treatment for at least 2 months after the end of the RAD1901 treatment (30 mg / kg, oral administration once daily) period (Figure 5).

[0120] Therefore, the RAD1901-palbociclib combination is likely to benefit patients by inhibiting tumor growth after treatment is terminated, especially when CDK4 / 6 inhibitors (e.g., ribociclib, abemaciclib, and palbociclib) can only be administered intermittently due to their side effects (O'Leary).

[0121] I(A)(iii) RAD1901-palbociclib caused more regression than RAD1901 alone in a xenograft model expressing mutant ER (ERα Y537S).

[0122] I(A)(iii)(1) RAD1901-palbociclib induced regressions more than RAD1901 alone in a PDx-5 model that was poorly responsive to fulvestrant.

[0123] The PDx-5 model was prepared following the same protocol as described above for the PDx model: Tumor size in each treatment group was measured twice weekly with a caliper, and the volume was calculated using the formula (L*W2)*0.52.

[0124] In the mutant ER PDx-5 model (a PDx model with breast cancer tumors derived from patients with the Y537S estrogen receptor mutation, PR+, Her2+, and prior aromatase inhibitor treatment), tumor growth inhibition by RAD1901 (60 mg / kg, orally once daily) in combination with palbociclib (100 mg / kg, reduced to an interim 75 mg / kg dose due to resistance issues), palbociclib (100 mg / kg, orally once daily, reduced to an interim 75 mg / kg dose due to resistance issues), and RAD1901 (60 mg / kg, orally once daily) was evaluated using the methods described above. For tumors expressing certain ERα mutations (e.g., Y537S), combined treatment with RAD1901 and palbociclib was more effective at inhibiting tumor growth than treatment with either agent alone (Figure 6A). These PDx models were not as sensitive as treatment with fulvestrant (3 mg / dose) (Figure 6A). Combination treatment with RAD1901 and palbociclib was more effective than treatment with either agent alone in causing tumor regression in the RDx-5 model (Figure 6B-C), which was shown as the change in individual tumor size from baseline at days 17 and 56, respectively.

[0125] PDx-5 models were treated with vehicle (negative control), fulvestrant (faslodex 3 mg / dose, subcutaneous injection once weekly), RAD1901 (60 mg / kg or 120 mg / kg, administered orally once daily), palbociclib (100 mg / kg, administered orally once daily, reduced to an interim dose of 75 mg / kg due to resistance issues), or RAD1901 (60 or 120 mg / kg, administered orally once daily) and palbociclib (100 mg / kg, administered orally once daily, reduced to an interim dose of 75 mg / kg due to resistance issues). The measured tumor sizes are shown in Figure 7A-B. Although the PDx-5 model was resistant to fulvestrant treatment, RAD1901 and palbociclib, either alone or in combination, inhibited tumor growth. At a dose of 60 mg / kg, RAD1901 alone had virtually identical efficacy to palbociclib alone at 100 mg / kg (Figure 7A), whereas at a dose of 120 mg / kg, RAD1901 alone had improved efficacy compared to palbociclib alone at 100 mg / kg (Figure 7B). Oral administration of 60 mg / kg RAD1901 achieved significant inhibition of tumor growth (Figure 8). Furthermore, palbociclib alone or in combination with fulvestrant significantly inhibited tumor growth in the mutant PDx model, but the combination did not further enhance the inhibition (Figure 8).

[0126] Surprisingly, RAD1901 alone or in combination with palbociclib achieved even more significant tumor inhibition; this combination almost completely inhibited tumor growth in the mutant PDx model, and the RAD1901-palbociclib combination using a lower dose of RAD1901 (e.g., 60 mg / kg) was sufficient to maximize tumor growth inhibition / tumor regression in the PDx-5 model (Figure 7A-B).

[0127] Therefore, these results demonstrated that RAD1901 was an effective endocrine scaffold that promoted targeted drug inhibition of tumor growth. Furthermore, RAD1901 demonstrated potent antitumor activity in PDx models derived from patients who had received multiple prior endocrine therapies, including those who were non-responsive to fulvestrant.

[0128] I(A)(iv) Pharmacokinetic evaluation of fulvestrant treatment on non-tumor-bearing mice.

[0129] Various doses of fulvestrant were administered to mice, which demonstrated significant dose exposure to the target (Figure 9).

[0130] Fulvestrant was administered subcutaneously at 1, 3, or 5 mg / dose to nude mice on days 1 (D1 Rx) and 8 (D8 Rx, n=4 / dose level). Blood was collected at the indicated time points up to 168 hours after the second dose, centrifuged, and plasma was analyzed by liquid chromatography-mass spectrometry.

[0131] I(B)RAD1901 promoted survival in a mouse xenograft model of brain metastasis (MCF-7 intracranial model).

[0132] The potential of RAD1901 to cross the blood-brain barrier and inhibit tumor growth was further evaluated using an MCF-7 intracranial tumor xenograft model.

[0133] Female athymic nude mice (Crl:NU(NCr)-Foxn1nu) were used for tumor xenograft testing. Three days before tumor cell implantation, estrogen pellets (0.36 mg E2, 60-day release; Innovative Research of America, Sarasota, FL) were implanted subcutaneously between the shoulder blades of all test animals using a sterile trocar. MCF-7 human breast adenocarcinoma cells were cultured to mid-log phase in RPMI-1640 medium containing 10% fetal bovine serum, 100 units / mL penicillin G, 100 μg / mL streptomycin sulfate, 2 mM glutamine, 10 mM HEPES, 0.075% sodium bicarbonate, and 25 μg / mL gentamicin. On the day of tumor cell implantation, cells were trypsinized, pelleted, and cultured at 5 × 10 7 The cells were resuspended in phosphate-buffered saline at a concentration of 1 × 10 cells / mL. 6 MCF-7 cells were implanted intracranially.

[0134] Five days after tumor cell implantation (represented as day 1 of the study), mice were randomized into three groups of 12 animals each and treated with vehicle, fulvestrant (0.5 mg / animal once daily), or RAD1901 (120 mg / kg once daily) as described above.

[0135] The endpoint was defined as mortality or a three-fold increase in survival rate compared with the control group, whichever occurred first. Treatment tolerance was assessed by weight measurement and frequent observation for clinical signs of treatment-related side effects. Animals with a weight loss of more than 30% at one measurement or more than 25% at three measurements were humanely sacrificed and classified as treatment-related deaths. Acceptable toxicity was defined as a group mean weight loss of less than 20% over the study period and at most one treatment-related death among 10 treated animals, or 10%. At the end of the study, animals were sacrificed by terminal cardiac puncture under isoflurane anesthesia. RAD1901 and fulvestrant concentrations in plasma and tumors were determined using LC-MS / MS.

[0136] Kaplan-Meier survival analysis showed that RAD1901 significantly prolonged survival compared with fulvestrant (P<0.0001; Figure 10). No animals in the control or fulvestrant groups survived beyond days 20 and 34, respectively, whereas 41% (5 / 12) of the RAD1901-treated animals survived to the end of the study on day 54.

[0137] The concentration of RAD1901 in plasma was 738±471 ng / mL and in intracranial tumors was 462±105 ng / g, supporting the hypothesis that RAD1901 can effectively cross the blood-brain barrier. In contrast, the concentration of fulvestrant was substantially lower in plasma (21±10 ng / mL) and intracranial tumors (8.3±0.8 ng / g).

[0138] Example II. RAD1901 preferably accumulates in tumors and can be delivered to the brain.

[0139] As described in Example I(A)(i), MCF-7 xenografts were further evaluated for RAD1901 concentrations in plasma and tumors using LC-MS / MS. At the end of the study, the RAD1901 concentration in plasma was 344±117 ng / mL, and in tumors it was 11,118±3,801 ng / mL for the 60 mg / kg dose level. Similar tumor-to-plasma ratios were also observed at lower dose levels, where tumor concentrations were approximately 20-30 times higher than in plasma. RAD1901 levels in plasma, tumors, and brains of mice treated for 40 days are summarized in Table 1. Significant amounts of RAD1901 were delivered to the brains of treated mice (see, e.g., the B / P ratio (RAD1901 concentration in brain / RAD1901 concentration in plasma)), indicating that RAD1901 was able to cross the blood-brain barrier (BBB). Surprisingly, RAD1901 accumulated favorably in tumors, see for example the T / P (RAD1901 concentration in tumor / RAD1901 concentration in plasma) ratios shown in Table 1.

[0140] Example III. RAD1901 inhibited the ER pathway and degraded ER.

[0141] III(A). RAD1901 reduced ER engagement in the uterus and pituitary gland of healthy postmenopausal female human subjects.

[0142] Subjects were amenorrheic for at least 12 months and had serum FSH levels consistent with menopause. 2 Subjects were aged 40 to 75 years, with a BMI of 0.01 or 0.02 and an intact uterus. Patients were excluded if they had evidence of clinically relevant pathology, had a history of stroke or a previous venous thromboembolic event at increased risk, or were taking concomitant medications (paracetamol was allowed up to 3 days prior) within 14 days of enrolling at the clinical research center.

[0143] FES-PET was performed at baseline and 6 days after exposure to RAD1901 to assess ER involvement in utero. RAD1901 resulted in 83% and 92% ER involvement in utero at the 200 mg (7 subjects) and 500 mg (6 subjects) dose levels, respectively.

[0144] FES-PET images showed a significant decrease in the binding of labeled estradiol to both the uterus and pituitary gland after treatment with 200 mg or 500 mg of RAD1901 (oral administration once daily for 6 days).

[0145] Due to high ER expression, the uterus showed a strong FES-PET signal at baseline before RAD1901 treatment (Figure 11A, transverse view of baseline uterine FES-PET scan of subject 3 treated at the 200 mg dose level; Figure 11B, sagittal and transverse views, respectively, of baseline uterine FES-PET scan of subject 7 treated at the 500 mg dose level). However, when scanned 4 hours after dosing on day 6 during the study, the uterus was barely visible (at or near the background FES-PET signal (Figure 11A, transverse view of day 6 uterine scan of subject 3; and Figure 11B, sagittal and transverse views, respectively, of day 6 uterine scan of subject 7). Such data are consistent with ER degradation and / or competition for receptor binding. Figures 11A-B also include CT scans of the uterus scanned by FES-PET, showing the presence of the uterus before and after RAD1901 treatment.

[0146] The FES-PET uterine scan results were further quantified to show the change in ER binding from baseline to post-dose for seven subjects, shown as subjects 1-3 and subjects 4-7 in the 200 mg and 500 mg dose groups, respectively (Figure 11(C)). RAD1901 demonstrated robust ER engagement at the low dose (200 mg).

[0147] Figure 12 shows representative images of FES-PET scans of the uterus (A) and pituitary gland (B) before (baseline) and after (post-treatment) treatment with 500 mg of RAD1901 orally once daily for 6 days. Figure 12A shows FES-PET scans of the uterus in (a) lateral, (b) longitudinal, and (c) longitudinal views.

[0148] Post-treatment FES-PET scans of the uterus and pituitary gland showed no significant signal for ER binding in the uterus (FIG. 12A, post-treatment) and pituitary gland (FIG. 12B, post-treatment), respectively.

[0149] Therefore, the results showed that RAD1901 effectively engaged the ER in humans when administered orally once daily for 6 days at doses of 200 and 500 mg.

[0150] Standardized uptake values ​​(SUVs) for uterus, muscle, and bone were calculated and are summarized in Tables 2 and 3 for treatment with RAD1901 administered orally once daily at 200 mg and 500 mg, respectively. Post-treatment uterine signal was very close to levels from "non-target tissues," suggesting complete attenuation of FES-PET uptake after RAD1901 treatment. Little change was observed for pre- versus post-treatment PET scans in tissues that did not significantly express estrogen receptors.

[0151] Therefore, RAD1901 or a salt or solvate (e.g., hydrate) thereof can be used in treating cancer and / or tumor cells (e.g., breast cancer, uterine cancer, and ovarian cancer) with ER overexpression without adverse effects on other organs (e.g., bone, muscle). RAD1901 or a salt or solvate (e.g., hydrate) thereof may be particularly useful in treating metastatic cancers and / or tumors with ER overexpression in other organs, such as original breast cancer, uterine cancer, and / or ovarian cancer that migrate to other organs (e.g., bone, muscle), without adverse effects on the organs, for treating breast cancer, uterine cancer, and / or ovarian cancer lesions in the other organs (e.g., bone, muscle).

[0152] III(B). RAD1901 reduced ER expression and inhibited the ER pathway.

[0153] III(B)(i)(1) The RAD1901-palbociclib combination reduced ER and PR expression in an MCF7 xenograft model and was more effective than treatment with RAD1901, palbociclib, or fulvestrant alone or in combination with fulvestrant-palbociclib.

[0154] Treatment with the combination of RAD1901 and palbociclib was also more effective in reducing ER and PR expression in vivo in an MCF7 xenograft model (as described in Example I(A)(ii)) than treatment with RAD1901, palbociclib, or fulvestrant alone, or the combination of fulvestrant and palbociclib (Figure 13, tumors harvested 2 hours after the final dose).

[0155] III(B)(i)(2) Comparison of RAD1901 and Fulvestrant in MCF7 and T47D Cell Lines

[0156] The effects of RAD1901 and fulvestrant were compared at various concentrations of 0.01 μM, 0.1 μM, and 1 μM using MCF7 and T47D cell lines (both human breast cancer cell lines) (FIG. 14A for the MCF7 cell line assay and FIG. 14B for the T47D cell line assay). Three ER target genes, progesterone receptor (PgR), estrogen growth regulation in breast cancer 1 (GREB1), and trefoil factor 1 (TFF1), were used as markers. RAD1901 caused ER degradation and inhibited ER signaling (FIG. 14). Surprisingly, as disclosed above in Example I(A) and Example I(B), RAD1901 was as effective as or more effective than fulvestrant in inhibiting tumor growth and causing tumor regression.

[0157] III(B)(i)(3) RAD1901 treatment resulted in ER degradation and abolition of ER signaling in the MCF7 xenograft model, as described above in Example I(A)(ii)(1).

[0158] RAD1901 treatment led to ER degradation in vivo (Figures 15A-C, Student's t-test: *p-value < 0.05, **p-value < 0.01) and inhibited ER signaling in vivo (Figures 15A and 15C, Student's t-test: *p-value < 0.05, **p-value < 0.01).

[0159] Tumors harvested from MCF-7 xenografts 2 hours after the final dose of RAD1901 (30 mg / kg, 60 mg / kg, oral administration once daily) or fulvestrant (3 mg / dose, subcutaneous injection once weekly) showed significantly decreased expression of ER and PR (Figures 15A-B). Tumors harvested from MCF-7 xenografts 8 hours after the final dose of fulvestrant treatment showed altered expression of PR and ER. However, tumors harvested from MCF-7 xenografts 8 hours after the final dose of RAD1901 treatment showed decreased expression of PR and ER (Figures 15A and 15C).

[0160] Tumors harvested from MCF-7 xenografts 8 or 12 hours after a single dose of RAD1901 (30 mg / kg, 60 mg / kg, or 90 mg / kg, orally administered once daily) showed a rapid decrease in PR expression (Figure 16A). Tumors harvested from MCF-7 xenografts 4 or 24 hours after the seventh dose of RAD1901 (30 mg / kg, 60 mg / kg, orally administered once daily) showed consistent and stable inhibition of ER signaling (Figure 16B). Quantification of Western blot analysis of tumors harvested from MCF-7 xenografts at various time points during RAD1901 treatment (30 mg / kg, 60 mg / kg, orally administered once daily) demonstrated a dose-dependent decrease in PR expression (Figure 16C).

[0161] RAD1901 treatment caused a rapid decrease in proliferation in the MCF-7 xenograft model. For example, tumors harvested from the MCF-7 xenograft model were sectioned and stained 8 hours after a single dose of RAD1901 (90 mg / kg, PO, once daily) and 24 hours after the fourth dose of RAD1901 (90 mg / kg, PO, once daily), and showed a rapid decrease in the proliferation marker Ki67 (Figures 17A-B).

[0162] These results suggest that RAD1901 treatment leads to ER degradation and inhibition of ER signaling in vivo in ER WT xenografts.

[0163] III(B)(i)(4) RAD1901 treatment resulted in ER degradation and abolition of ER signaling in the PDx-4 model, as described above in Example I(A)(ii).

[0164] RAD1901 treatment caused a rapid decrease in proliferation in the PDx-4 model. For example, tumors harvested from PDx-4 models treated with RAD1901 (30, 60, or 120 mg / kg, orally once daily) or fulvestrant (1 mg / animal, once weekly) were sectioned 4 hours after the final dose on the final day of the 56-day efficacy study and showed a rapid decrease in the proliferation marker Ki67 compared to PDx-4 models treated with fulvestrant (Figure 18).

[0165] These results suggest that RAD1901 treatment leads to ER degradation and inhibition of ER signaling in vivo in ER WT xenografts.

[0166] III(B)(ii) RAD1901 treatment resulted in decreased ER signaling in the mutant ER xenograft PDx-5 model, as described above in Example I(A)(iii)(1).

[0167] Tumors were harvested at the indicated time points after the last day of treatment (unless otherwise specified) and homogenized in RIPA buffer with protease and phosphatase inhibitors using a Tissuelyser (Qiagen). Equal amounts of protein were separated by MW, transferred to nitrocellulose membranes, and blotted with the following antibodies as described in Materials and Methods: progesterone receptor (PR, Cell Signaling Technologies; 3153).

[0168] Bands were quantified using 1D Quant software (GE), and PR IHC Allred scores obtained from the PDx-5 model, as described in Example I(A)(iii)(1), are shown in Figure 19. Fulvestrant had little effect on PR expression, whereas RAD1901 showed efficacy at both the 60 mg / kg and 120 mg / kg doses (administered orally once daily, III(B)(ii)—Figure 1).

[0169] These results indicate that for tumors expressing certain ERα mutations (e.g., Y537S), RAD1901 was even more effective than fulvestrant in inhibiting tumor growth, and was particularly effective in inhibiting the growth of tumors that were poorly responsive to fulvestrant treatment (e.g., 3 mg / dose, subcutaneous injection once weekly; for PDx-5, Figure 6A). Furthermore, for tumors that were poorly responsive to fulvestrant treatment (e.g., PDx-5), RAD1901 was effective, but fulvestrant was not, in reducing PR expression in vivo (Figure 19).

[0170] Example IV Effect of RAD1901 Treatment on Uterine Tissue and / or BMD IV(A(1)): RAD1901 antagonized estradiol stimulation of uterine tissue.

[0171] The uterotropic effects of RAD1901 were investigated in immature rats by assessing changes in uterine weight, histology, and C3 gene expression. Results from a representative study are shown in Figures 20A-D.

[0172] Evaluation of uterotropic activity Sprague-Dawley rat pups were weaned at 19 days of age and randomized into groups (n = 4). They were administered vehicle (aqueous methylcellulose), E2 (0.01 mg / kg), raloxifene (3 mg / kg), tamoxifen (1 mg / kg), RAD1901 alone (0.3–100 mg / kg), or RAD1901 in combination with E2 (0.01 mg / kg) (0.01–10 mg / kg) either subcutaneously or orally (see Reagents above) as needed, once daily for three consecutive days. Twenty-four hours after the final administration, all animals were sacrificed by carbon dioxide inhalation. Body weight and wet uterine weight were recorded for each animal. Similar assays were also performed with RAD1901 (0.03–100 mg / kg) in rats and mice (Charles River Laboratories, Montreal, QC).

[0173] Fresh uterine tissue from each rat was fixed in 4% paraformaldehyde, dehydrated in ethanol, and embedded in JB4 plastic resin. Sections were cut at 8 μm and stained with 0.1% toluidine blue O. Endometrial epithelial tissue thickness was measured using a Zeiss Axioskop 40 microscope and the Spot Advanced program, and the mean of nine measurements per specimen was calculated.

[0174] Uterine component 3 (C3) gene expression To determine the relative expression levels of C3 in the treated uterine tissues, RNA was extracted from the remaining tissues using a Micro to Midi Total RNA Purification Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA was quantified, and equal amounts were reverse transcribed using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA).

[0175] Quantitative PCR was performed using an ABI Prism 7300 System (Applied Biosystems). PCR was performed using Taqman Universal Master Mix with probe sets for C3 and 18S ribosomal RNA as a reference gene. Thermal cycling conditions included an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

[0176] Relative gene expression was determined by normalizing each sample to an endogenous control (18S) and comparing with the calibrator (vehicle). Relative gene expression was determined using the equation: 2-ΔΔCt, where Ct = cycle threshold or the cycle number at which PCR product was first detected, ΔCt = normalized sample value, and ΔΔCt = normalized difference between treated subjects and vehicle. Five replicate gene expression determinations were performed for each treatment within each study.

[0177] Treatment with E2 (0.01 mg / kg), raloxifene (RAL, 3 mg / kg), or tamoxifen (TAM, 1 mg / kg) resulted in a significant increase in uterine wet weight compared with vehicle alone, whereas RAD1901 treatment at doses ranging from 0.3 to 100 mg / kg had no significant effect on uterine wet weight (Figure 20A). Data shown (Figure 20A) are means (± mean error), n = 4 rats per group. P vs. vehicle: *<0.05; vs. E2: ‡<0.05. Furthermore, when coadministered with E2 (0.01 mg / kg), RAD1901 antagonized E2-mediated uterine stimulation in a dose-dependent manner, demonstrating significant inhibition of uterotropic activity at doses of 0.1 mg / kg and above and complete inhibition at 3 mg / kg. The EC of RAD1901 50 The dose of RAD1901 in the uterine wet weight and epithelial thickness was approximately 0.3 mg / kg. Similar results were obtained in mice with doses of 0.03 to 100 mg / kg, which did not affect uterine wet weight or epithelial thickness (data not shown).

[0178] Treatment-dependent changes in uterine tissue were further investigated by quantitative microscopic histology. There was a statistically significant increase in endometrial epithelial thickness after treatment with E2 at both 0.01 and 0.3 mg / kg (Figure 20B). A significant increase in epithelial thickness was also observed after treatment with tamoxifen (1 mg / kg) or raloxifene (3 mg / kg). In contrast, RAD1901 treatment did not increase endometrial epithelial thickness up to the highest evaluated dose of 100 mg / kg. Representative images of endometrial epithelium are shown in Figure 20C.

[0179] Consistent with the changes in both uterine weight and endometrial epithelial thickness, E2, tamoxifen, and raloxifene all significantly increased the expression of the estrogen-regulated component gene C3 (Figure 20D). In contrast, RAD1901 did not increase C3 gene expression at any of the doses tested (0.3–100 mg / kg). Furthermore, RAD1901 at 1, 3, and 10 mg / kg significantly suppressed E2-stimulated C3 gene expression.

[0180] RAD1901 did not stimulate the uterus in immature female rats Immature female rats were orally treated once daily for three consecutive days with vehicle (VEH), estradiol (E2), raloxifene (RAL), tamoxifen (TAM), RAD1901, or RAD1901 + E2. Uterine wet weights were measured. Data shown (Figure 20A) are means (± mean error), n = 4 rats per group. P vs. vehicle: *<0.05; vs. E2: ‡<0.05.

[0181] Example II(A)(2). Treatment with RAD1901 prevented bone loss in ovariectomized rats The bone-specific effects of RAD1901 were tested in ovariectomized rats.

[0182] Ovariectomy was performed in anesthetized female Sprague-Dawley rats as a model of postmenopausal bone loss, with sham-operated controls. After surgery, ovariectomized rats, with 20 animals per group, were treated once daily for 4 weeks with vehicle, E2 (0.01 mg / kg), or RAD1901 (0.1, 0.3, 1, or 3 mg / kg), administered as described above. Animals in the sham-operated group were treated with vehicle. All animals were sacrificed by carbon dioxide inhalation 24 hours after the final administration. Bone mineral density was assessed at baseline and after 4 weeks of treatment using PIXImus dual-emission X-ray absorptiometry.

[0183] At necropsy, the left femur of each animal was removed, dissected free of soft tissue, and stored in 70% ethanol before analysis. Detailed qualitative and quantitative 3D evaluation was performed using a micro-CT40 system (Scanco Systems, Wayne, PA). For each specimen, 250 images of the distal femoral metaphysis were required. Morphological parameters were determined using a direct 3D approach in preselected analysis regions. Trabecular parameters determined included bone volume density, bone surface density, trabecular number, trabecular width, trabecular center distance, connectivity density, and apparent bone mineral density.

[0184] After ovariectomy, untreated (vehicle control) rats had decreased bone mineral density in both the complete femur and lumbar vertebrae compared to baseline (Table 5). Treatment with E2 was associated with prevention of bone loss in both the femur and vertebrae. Treatment with RAD1901 resulted in a dose-dependent and statistically significant inhibition of ovariectomy-induced bone loss (data shown for the 3 mg / kg treatment group). At doses of 0.1 mg / kg to 3 mg / kg, bone mineral density in RAD1901-treated rats was intact and not statistically significantly different from the E2-treated group.

[0185] MicroCT analysis of the distal femur (Table 6) showed that ovariectomy induced significant changes in numerous key microarchitectural parameters compared to sham-operated animals. These changes were consistent with bone loss and included decreased bone volume, decreased trabecular number, thickness, and density, and increased trabecular spacing. Consistent with the preservation of bone mineral density observed after treatment with RAD1901, significant preservation of trabecular architecture was observed in the key microarchitectural parameters (Table 6).

[0186] Example IV(B): Phase 1 Dose-Escalation Study of RAD101 in Healthy Postmenopausal Women In a phase 1 study, safety, tolerability, and pharmacokinetics were evaluated in 44 healthy postmenopausal women. No dose-limiting toxicities (DLTs) were observed, and no maximum tolerated dose (MTD) was established. Plasma exposure increased more than dose-proportionally across the dose range tested.

[0187] subject Forty-four healthy postmenopausal women were enrolled in this phase 1 study. Subjects had been amenorrheic for at least 12 months and had serum FSH levels consistent with menopause. Subjects were 18.0-30 kg / m 2 Patients were aged 40 to 75 years, with a BMI of 0.01 or lower. Patients were excluded if they had evidence of clinically relevant pathology, had an increased risk of stroke or previous venous thromboembolic events, or were using concomitant medications (paracetamol was allowed up to 3 days prior) within 14 days of enrolling at the clinical research center.

[0188] Administration Subjects were treated with placebo or at least one oral dose once daily for 7 days after a light breakfast at dose levels of 200 mg, 500 mg, 750 mg, and 1000 mg, respectively. Key baseline demographics of the 44 postmenopausal women enrolled in the Phase 1 study are summarized in Table 7.

[0189] Treatment-emergent adverse events (TEAEs) TEAEs were recorded, and the most frequent (>10% of patients in all active groups had any relevant TEAE) adverse events (AEs) are summarized in Table 8, where "n" is the number of subjects with at least one treatment-related AE in a given category. AEs were graded as per Common Terminology Criteria for Adverse Events (CTCAE) v4.0; any patient with multiple scenarios of the same preferred term was counted only once, up to the most severe grade. No dose-limiting toxicities were observed, and no maximum tolerated dose (MTD) was established.

[0190] Pharmacokinetic evaluation Serial blood samples were drawn during the study for analysis of RAD1901 in plasma. Five-mL blood samples were drawn either via an indwelling IV catheter or by direct venipuncture into tubes containing K3-EDTA as an anticoagulant. Steady state was achieved on the fifth day of treatment. The geometric mean (Ge-Mean) plasma concentration-time profile of RAD1901 was evaluated. The plasma pharmacokinetic results for the groups (N=35) treated with RAD1901 (200, 500, 750, or 1,000 mg) on ​​day 7 of the study are provided as examples in Table 8 and Figure 21. Median t 1 / 2 The median time to onset was 37.5 to 42.3 hours (Table 8). max was 3 to 4 hours after administration.

[0191] Example V(A)-1. Modeling RAD1901-ERα binding using selected published ER structures. Unless otherwise stated, when structures are shown as their stick models, each end of a bond is colored the same color as the atom to which it is attached: gray is carbon, red is oxygen, blue is nitrogen, and white is hydrogen.

[0192] Fourteen published structures (i.e., models) of the ERα ligand-binding domain (LBD) complexed with various ER ligands were carefully selected from 96 published models. One of these 14 models was 3ERT (human ERα LBD (OHT) bound to 4-hydroxytamoxifen). OHT is the active metabolite of tamoxifen, a first-generation SERM that acts as an antagonist in breast tissue.

[0193] In 3ERT (Figures 21 and 22), the ERα binding site introduces a three-layer "helical sandwich" that forms a hydrophobic poem, including helix 3 (H3), helix 5 (H5), and helix 11 (H11) (Figure 21). The dotted box in Figure 22 represents the binding site and residues within the binding site that are critical or achieved by OHT binding. OHT acts as an antagonist by substituting H12 into the site that binds the LXXLL coactivator(s). OHT occupies the space normally filled by L540 and modifies the conformation of four residues (G521, H524, L525, and M528) on the C-terminus of helix 11. OHT also forms a salt bridge with D351, resulting in charge neutralization.

[0194] Thirteen other ERα LBD-ER ligand models were compared with 3ERT, and their residue structural differences are summarized in Table 10. Superposition of the ERα structures of the 14 models (Figure 23) shows that these structures differed significantly at residues E380, M421, G521, M522, H524, Y526, S527, M528, P535, Y537, L540, and various combinations of these.

[0195] Calculations of the standard deviation (RMSD) of any pair of the 14 models are summarized in Table 11. Structures were considered to be superimposed if the RMSD was less than 2 Å. Table 11 shows that all 14 models had an RMSD of less than 1.5 Å. Use of format analysis suggested that 1R5K and 3UUC were not very similar to other models (analysis not shown). Therefore, 1R5K and 3UUC were considered to be unique, separate structural clusters tested.

[0196] The ERα residues bound by the ligand in the 14 models are summarized in Table 12. Table 12 also shows the EC 50 Of the 14 models, 13 models showed H-bond interactions between the ligand and E353, 12 models showed π interactions between the ligand and F404, 5 models showed H-bond interactions between the ligand and D351, 6 models showed H-bond interactions between the ligand and H524, 4 models showed H-bond interactions between the ligand and R394, and 1 model (3UUC) showed an interaction between the ligand and T347.

[0197] Each of the 14 models was used to dock a random library of 1,000 compounds in addition to the ligand, and the model was published with known antagonists to determine whether it could identify and prioritize known antagonists. If the model could identify known antagonists, it was determined that the model itself could predict the structure of the published ligand. EF 50 The strength of the model was quantified to see how much better it was than random selection by calculating the EC. RAD1901 was docked into the selected model (e.g., Figures 24-28). The docking scores of the published ligands and RAD1901 in the model were determined. EC 50The docking scores were also determined. Visual inspection of RAD1901 showed that it "followed" the interactions shown with the published ligands in 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. No spatial clashes were observed. In certain embodiments, for example, in 1R5k and 2BJ4, RAD1901 had higher docking scores than the published ligands.

[0198] The evaluation results of nine models (1ERR, 3ERT, 3UCC, 2IOK, 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ) are summarized in Table 13.

[0199] 1ERR and 3ERT could not predict the exact structure of their crystalline ligands. RAD1901 could not be docked to 3UCC. In 2IOK-RAD1901, tetrahydronaphthalene was bound in a non-traditional manner.

[0200] The major difference between models 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ was the residues at the C-terminus of helix 11 (G521-M528).

[0201] Figure 24 shows modeling of RAD1901-1R5K (a) and GW5-1R5K (b). RAD1901 bound through H-bond interactions with E353, R394, and L536, and p-interactions with F404.

[0202] Figure 25 shows modeling of RAD1901-1SJ0 (a) and E4D-1SJ0 (b). RAD1901 bound through H-bond interactions with E353 and D351 and p-interactions with F404.

[0203] Figure 26 shows modeling of RAD1901-2JFA (a) and RAL-2JFA (b). RAD1901 bound via a p interaction with F404.

[0204] Figure 27 shows modeling of RAD1901-2BJ4 (a) and OHT-2BJ4 (b). RAD1901 bound through H-bond interactions with E353 and R394 and p-interaction with F404.

[0205] Figure 28 shows modeling of RAD1901-2IOK (a) and IOK-2IOK (b). RAD1901 bound through H-bond interactions with E353, R394, and D351, and p-interactions with F404.

[0206] The ligand published in the model has the following structure: [ka] [ka] [ka] [ka]

[0207] Example V(A)-2. Induced Fit Docking (IFD) of ERα with RAD1901 and Fulvestrant In ERα, the binding conformation of RAD1901 was further optimized by IFD analysis of five ERα crystal structures: 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. IFD analysis revealed receptor flexibility (upon ligand binding) to adapt its precise binding conformation.

[0208] A library of different conformations for each ligand (e.g., RAD1901 and fulvestrant) was generated by searching for local minima as a function of rotation about the rotatable bond. The library for RAD1901 had 25 different conformations.

[0209] Five ERα crystal structures were prepared and minimized, and the corresponding ligands in published X-ray structures were used to define the ERα binding pocket.

[0210] The RAD1901 conformation was docked onto the prepared ERα structure, allowing for the induction of side-chain or skeletal movements to residues located in the binding pocket. These movements allowed ERα to change its binding site, thereby more closely matching the shape and binding mode of the RAD1901 conformation. In some cases, small skeletal relaxations and significant side-chain conformational changes in the receptor structure were possible during IFD analysis.

[0211] An empirical scoring function was used to estimate the ligand binding free energy to provide a docking score or G score. The G score, also known as the GlideScore, may be used synonymously in this example. The docking score was an estimate of binding affinity. Thus, the lower the docking score, the "better" the ligand bound to its receptor. A docking score of -13 to -14 corresponded to a very good binding interaction.

[0212] The RAD1901 conformations obtained from IFD analysis of 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ were overlaid to show their differences (Figures 29-31, shown in stick models). All bonds in each RAD1901 conformation are shown in the same color in Figures 29, 30, and 31(a).

[0213] The conformation of RAD1901 obtained from IFD analysis with 1R5K (blue) and 2OUZ (yellow) had the N-benzyl-N-ethylaniline group of RAD1901 on the front side (Figure 29). The conformation of RAD1901 obtained from IFD analysis with 2BJ4 (green) and 2JFA (pink) had the N-benzyl-N-ethylaniline group of RAD1901 on the back side (Figure 30). The conformations of RAD1901 obtained from IFD analysis with 2BJ4 (green), 2JFA (pink), and 1SJ0 (brown) were nearly similar, as shown by their superposition (Figures 31(a) and (b)). The IFD docking scores of RAD1901 are summarized in V(A) - Table 5.

[0214] The IFD of RAD1901 by 2BJ4 showed hydrogen-bond interactions with E353 and D351 and π-interactions with F404 (Figures 32(a)-(c)). Figure 32(a) shows the regions within the binding site suitable for H-bond acceptor groups (red), H-bond donor groups (blue), and hydrophobic groups (yellow). In Figures 32(a)-(b), light blue indicates carbons of RAD1901. Figures 33(a)-(c) show the protein surface interactions of the IFD of RAD1901 and 2BJ4. V(A) - Figures 33(a) and (b) are front views, and Figure 33(c) is a side view. The molecular surface of RAD1901 is blue in Figure 33(a) and green in Figure 33(c). Figures 33(b)-(c) show the electrostatics of the solvent-accessible surface of ERα, with red indicating negative and blue indicating positive.

[0215] A similar IFD analysis was performed on fulvestrant with 2BJ4, as described above. The IFD of fulvestrant-2BJ4 yielded a G score of -14.945, indicating hydrogen-bond interactions with E353, Y526, and H524, as well as π interactions with F404 (Figures 34(a)-(c)). Figure 34(a) shows regions within the binding site suitable for H-bond acceptor groups (red), H-bond donor groups (blue), and hydrophobic groups (yellow). In Figure 34(a), light blue indicates carbon in RAD1901.

[0216] Figures 35(a)-(b) show that RAD1901 and fulvestrant docked to 2BJ4 by IFD had both π interactions with F404 and hydrogen-bonding interactions with E353. Furthermore, RAD1901 had hydrogen-bonding interactions with D351 (blue represents the RAD1901 molecular surface, Figure 35(b)), whereas fulvestrant had hydrogen-bonding interactions with Y526 and H524 (green represents the fulvestrant molecular surface, Figure 35(c)). The superposition of 2BJ4 docked with RAD1901 and fulvestrant is shown in Figures 36(a)-(b). In Figure 36(a), green represents the fulvestrant molecular surface, and blue represents the RAD1901 molecular surface. In Figure 36(b), the brown structure is fulvestrant, and the blue structure is RAD1901.

[0217] Example V(A)-3. Modeling evaluation of selected ERα mutations. The effects of various ERα mutations on the C-terminal ligand-binding domain were evaluated. The specific ERα mutations evaluated were the Y537X mutant (where X is S, N, or C), D538G, and S463P.

[0218] The Y537 residue in helix 12, once phosphorylated, may regulate ligand binding, homodimerization, and DNA binding, allowing ERα to escape phosphorylation-mediated controls and potentially provide cells with a selective tumorigenic advantage. Additionally, it may alter the conformation that renders the receptor constitutively active.

[0219] The Y537S mutation favors a closed pocket structure that is transcriptionally active, regardless of whether it is occupied by a ligand. A closed, but unoccupied, pocket may explain the constitutive activity of ERα (Carlson et al., Biochemistry 36:14897-14905 (1997)). Ser537 forms a hydrogen-bonding interaction with Asp351, resulting in an altered conformation of the helix 11-12 loop and burying Leu536 in a solvent-inaccessible position. This may contribute to the constitutive activity of the Y537S mutant protein. The Y537S surface mutation does not affect the surface of the LBD pocket.

[0220] Y537N is frequently found in ERα-negative metastatic breast cancer. Mutations at this site may allow ERα to escape phosphorylation-mediated control, potentially providing cells with a selective tumorigenic advantage. Specifically, the Y537N substitution induces a conformational change in ERα that may mimic hormone binding, conferring constitutive transactivation function to the receptor without affecting its ability to dimerize (Zhang et al. Cancer Res 57:1244-1249 (1997)).

[0221] Y537C has the same effect as Y537N.

[0222] D538G, although more preferably the active conformation, may alter the overall energy landscape by stabilizing both active and inactive conformations, which may result in constitutive activity of this mutant in the absence of hormone, as observed in hormone-resistant breast cancer (Huang et al., "A newfound cancer-activating mutation reshapes the energy landscape of estrogen-binding domain," J. Chem. Theory Comput. 10:2897-2900 (2014)).

[0223] None of these mutations are expected to affect the ligand-binding domain or specifically prevent RAD1901 binding. Y537 and D538 may alter conformation that results in constitutive receptor activation independent of ligand binding.

[0224] Example V(B). In vitro binding assay of wild-type and LBD mutant ERα constructs with RAD1901 and other compounds In vitro binding assays of wild-type (WT) and LBD mutant ERα constructs with RAD1901 showed that RAD1901 bound to the mutant ERα with similar affinity to WT ERα.

[0225] WT and LBD mutant ERα constructs were prepared by expressing and purifying the corresponding LBD residues 302–552 with N-terminal thioredoxin and a 6xHis tag cleaved by TEV protease.

[0226] Fluorescence polarization (FP) was used to determine the binding of test compounds (RAD1901, fulvestrant, bazedoxifene, raloxifene, tamoxifen, and AZD9496) to ERα using 2 nM fluoromone and 100 nM WT or LBD mutant ERα constructs as per the manufacturer's instructions (Polar Screen, Invitrogen). Each set was performed in duplicate, and IC values ​​for different ERα constructs were calculated. 50 Certain test compounds were tested to determine the binding activity of the RAD1901 binding compounds (Figure 38 for RAD1901 binding assay).

[0227] As stated above, the foregoing is intended to merely illustrate various embodiments of the present invention. The specific modifications discussed above should not be construed as limitations on the scope of the invention. Since various equivalents, changes, and modifications can be made without departing from the scope of the invention, it should also be understood that such equivalent embodiments are intended to be included herein, as will be apparent to those skilled in the art. All references cited herein are incorporated by reference as if fully set forth herein.

[0228] The present invention includes the following aspects. [1] A method of inhibiting tumor growth or causing tumor regression in a subject with drug-resistant estrogen receptor alpha-positive cancer, comprising administering to the subject a therapeutically effective amount of palbociclib and a compound of the structure: [ka] or a salt or solvate thereof. [2] A method of inhibiting tumor growth or causing tumor regression in a subject having a mutant estrogen receptor alpha-positive cancer, comprising administering to the subject a therapeutically effective amount of palbociclib and a compound of the structure: [ka] or a salt or solvate thereof. [3] The method according to [1] or [2], wherein the cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer, and pituitary cancer. [4] The method according to [1] or [2], wherein the cancer is metastatic cancer. [5] The cancer is positive for mutant estrogen receptor alpha containing one or more mutations selected from the group consisting of Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q, and combinations thereof, wherein: The method according to [1] or [2], wherein X1 is S, N, C, or D538G, and X2 is R or Q. [6] The method according to [5], wherein the mutation is Y537S. [7] The method according to [1] or [2], wherein the ratio (T / P) of the concentration of RAD1901 or a salt or solvate thereof in the tumor to the concentration of RAD1901 or a salt or solvate thereof in the plasma after administration is at least about 15. [8] The method according to [1] or [2], wherein the subject has osteoporosis or is at higher risk of osteoporosis. [9] The method according to [1] or [2], wherein the subject is a premenopausal woman.

[10] The method according to [1] or [2], wherein the subject is a postmenopausal woman who has experienced recurrence or progression of disease after previous treatment with a SERM and / or AI.

[11] The method according to [1] or [2], wherein the therapeutically effective amount is about 150 to about 1,500 mg once a day.

[12] The method according to [1] or [2], wherein the salt is RAD1901 dihydrochloride.

[13] The method according to [1] or [2], wherein the tumor exhibits resistance to a drug selected from the group consisting of antiestrogen, an aromatase inhibitor, and a combination thereof.

[14] The method according to

[13] , wherein the anti-estrogen is tamoxifen or fulvestrant.

[15] The method according to

[13] , wherein the aromatase inhibitor is aromasin.

[16] The method according to [1] or [2], wherein the therapeutically effective amount is 150 mg to 2,000 mg.

[17] The method according to

[16] , wherein the therapeutically effective amount is 200 mg, 400 mg, or 500 mg.

[18] A pharmaceutical composition comprising palbociclib and RAD1901, or a salt or solvate thereof.

[19] A method of treating breast cancer in a subject having drug-resistant estrogen receptor alpha-positive cancer, comprising administering to the subject a therapeutically effective amount of a cdk4 / 6 inhibitor and a compound having the structure: [ka] or a salt or solvate thereof.

[20] The method of

[19] , wherein the drug-resistant breast cancer is resistant to one or more anti-estrogen or aromatase inhibitor therapies.

[21] The method of

[20] , wherein the one or more antiestrogens are selected from the group consisting of tamoxifen, toremifene, and fulvestrant, and the one or more aromatase inhibitors are selected from the group consisting of aromasin, letrozole, and anastrozole.

[22] The method of any one of

[19] to

[21] , wherein the female 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.

[23] The method of

[22] , wherein the mutant estrogen receptor alpha is selected from the group consisting of Y537S, Y537N, Y537C, D538G, L536R, S463P, and E380Q.

[24] The method according to

[22] or

[23] , wherein the mutant receptor alpha is Y537S.

[25] The method according to any one of

[19] to

[24] , wherein the RAD1901 is administered at a total daily dose of 100 mg to 1,500 mg.

[26] The method of

[25] , wherein the RAD1901 is administered at a total daily dose of 100 mg to 1,000 mg.

[27] The method of

[26] , wherein the RAD1901 is administered at a total daily dose of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1,000 mg.

[28] The method according to any one of

[25] to

[27] , wherein the daily dose is delivered in two separate doses.

[29] The method of

[28] , wherein the separate doses are equal doses.

[30] The method of

[29] , wherein the equivalent doses are 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg, respectively.

[31] The method according to any one of

[25] to

[30] , wherein the dose is delivered by oral route.

[32] The method according to any one of

[19] to

[31] , wherein the woman is a postmenopausal woman.

[33] The woman first had a diagnosis 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, 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.

[34] The method according to

[33] , wherein the one or more genes are selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1, and MTOR.

[35] The method according to any one of

[19] to

[34] , wherein the cdk4 / cdk6 inhibitor is selected from the group consisting of abemaciclib, ribociclib, and palbociclib.

[36] The method according to

[35] , wherein the cdk4 / 6 inhibitor is palbociclib.

[37] The method according to

[36] , wherein the palbociclib is administered at a daily dose of 25 mg to 250 mg.

[38] The method according to

[37] , wherein the palbociclib is administered at a daily dose of 50 to 125 mg.

[39] The method according to

[37] , wherein the palbociclib is administered at a daily dose of 75 mg to 125 mg.

[40] The method of

[37] , wherein the palbociclib is administered at 75 mg, 100 mg, or 125 mg daily.

[41] The method according to

[37] , wherein the palbociclib is administered at a daily dose of 31.25 mg to 93.75 mg.

[42] The method according to any one of

[36] to

[41] , wherein the palbociclib is administered for 21 days in a 28-day cycle.

[43] The method according to any one of

[36] to

[41] , wherein the palbociclib is administered for less than 21 days in a 28-day cycle.

[44] The method according to any one of

[36] to

[41] , wherein the palbociclib is administered for 7 to 20 days in a 28-day cycle.

[45] The method according to any one of

[36] to

[41] , wherein the palbociclib is administered on 7 or 14 days in a 28-day cycle.

[46] The method according to

[36] , wherein the palbociclib is administered in a total amount of 2,625 mg every 28 days.

[47] The method according to

[36] , wherein the palbociclib is administered in a total amount of 656.25 mg to 1,968.75 mg every 28 days.

[48] ​​The method according to

[35] , wherein the cdk4 / 6 inhibitor is ribociclib.

[49] The method of

[48] , wherein the ribociclib is administered at a daily dose of 200 mg to 1,000 mg.

[50] The method according to

[49] , wherein the ribociclib is administered at a daily dose of 250 to 750 mg.

[51] The method of

[50] , wherein the ribociclib is administered at a daily dose of 600 mg.

[52] The method of

[48] , wherein the ribociclib is administered at a daily dose of 150 mg to 450 mg.

[53] The method according to any one of

[48] to

[52] , wherein the ribociclib is administered for 21 days in a 28-day cycle.

[54] The method according to any one of

[48] to

[52] , wherein the ribociclib is administered for less than 21 days in a 28-day cycle.

[55] The method according to

[54] , wherein the ribociclib is administered for 7 to 20 days in a 28-day cycle.

[56] The method of

[55] , wherein the ribociclib is administered on 7 or 14 days in a 28-day cycle.

[57] The method of

[48] , wherein the ribociclib is administered in a total amount of 12,600 mg every 28 days.

[58] The method according to

[48] , wherein the ribociclib is administered in a total amount of 3,150 mg to 9,450 mg every 28 days.

[59] The method according to any one of

[35] to

[58] , wherein the ribociclib or the palbociclib is administered orally.

[60] The method of

[59] , wherein the ribociclib and the palbociclib are orally administered once daily.

[61] The method according to

[34] , wherein the cdk4 / 6 inhibitor is abemaciclib.

[62] The method of

[61] , wherein the abemaciclib is administered at a daily dose of 300 mg.

[63] The method of

[61] , wherein the abemaciclib is administered at a daily dose of 150 mg twice daily.

[64] The method according to any one of

[61] to

[63] , wherein the abemaciclib is administered for 28 days in a 28-day cycle.

[65] The method according to any one of

[61] to

[63] , wherein the abemaciclib is administered for less than 28 days in a 28-day cycle.

[66] The method according to

[65] , wherein the abemaciclib is administered on days 21 to 27 of a 28-day cycle.

[67] The method of

[65] , wherein the abemaciclib is administered on 7, 14, or 21 days in a 28-day cycle.

[68] The method according to

[61] , wherein the abemaciclib is administered in a total dose of 8,400 mg every 28 days.

[69] The method according to

[61] , wherein the abemaciclib is administered in a total dose of 2,100 mg to 6,300 mg every 28 days.

[70] The method according to any one of

[61] to

[69] , wherein the abemaciclib is administered orally.

Claims

1. 1. A drug for inhibiting tumor growth or causing tumor regression in a subject with estrogen receptor alpha positive breast cancer brain metastases having one or more estrogen receptor alpha mutations selected from the group consisting of Y537S, Y537C, Y537N, D538G and S463P, comprising, as an active ingredient, a therapeutically effective amount of the structure: 【Chemistry 1】 or a salt or solvate thereof, wherein the agent is used in combination with palbociclib.

2. The agent of claim 1, wherein the mutation is Y537S.

3. The method of claim 1, wherein the ratio (T / P) of the concentration of RAD1901 or its salt or solvate in the tumor to the concentration of RAD1901 or its salt or solvate in the plasma after administration is at least about 15.

4. 2. The method of claim 1, wherein the salt is RAD1901 dihydrochloride.

5. The method of claim 1, wherein the therapeutically effective amount is 150 mg to 2,000 mg.

6. 1. A medicament for treating breast cancer brain metastases in a subject with estrogen receptor alpha positive breast cancer brain metastases having one or more estrogen receptor alpha mutations selected from the group consisting of Y537S, Y537C, Y537N, D538G and S463P, comprising as an active ingredient a compound of the structure: 【Chemistry 2】 or a salt or solvate thereof, wherein the agent is used in combination with a cdk4 / 6 inhibitor.

7. 7. The method of claim 6, wherein the RAD1901 is administered in a total daily dose of 100 mg to 1,500 mg.

8. The method of claim 6, wherein the cdk4 / cdk6 inhibitor is selected from the group consisting of abemaciclib, ribociclib, and palbociclib.

9. The method of claim 8, wherein the cdk4 / 6 inhibitor is palbociclib.

10. 10. The method of claim 9, wherein the palbociclib is administered in a daily dose of 25 mg to 250 mg.

11. The method of claim 8, wherein the cdk4 / 6 inhibitor is ribociclib.

12. 12. The method of claim 11, wherein the ribociclib is administered in a daily dose of 200 mg to 1,000 mg.

13. The drug of claim 8, wherein the cdk4 / 6 inhibitor is abemaciclib.

14. 14. The method of claim 13, wherein the abemaciclib is administered at a daily dose of 300 mg.

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