Methods for treating cancer

Abstract

Disclosed herein is a method for treating cancer, providing additional benefit by administering to a subject a therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) or a salt thereof and a combination of one or more second therapeutic agents (e.g., everolimus) against advanced cancer and / or cancer resistant to previous treatment. The cancer is an estrogen-dependent cancer, such as breast cancer, ovarian cancer, colon cancer, endometrial cancer, or prostate cancer.

Description

[0001] Divisional application

[0002] This application is a divisional application of the Chinese patent application entitled “Method for Treating Cancer” and application number 201680038907.4 filed on April 29, 2016.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 62 / 154,699 filed on April 29, 2015, U.S. Provisional Application No. 62 / 155,451 filed on April 30, 2015, U.S. Provisional Application No. 62 / 252,085 filed on November 6, 2015, U.S. Provisional Application No. 62 / 265,696 filed on December 10, 2015, U.S. Provisional Application No. 62 / 158,469 filed on May 7, 2015, U.S. Provisional Application No. 62 / 252,916 filed on November 9, 2015, and U.S. Provisional Application No. 62 / 265,696 filed on December 10, 2015. No. 62 / 265,663 filed on December 10, 2015, and U.S. Provisional Application No. 62 / 323,576 filed on April 15, 2016; all of which are incorporated herein by reference in their entirety. Background Art

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

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

[0007] In the treatment of ER-positive breast cancer, agents that inhibit estrogen production, such as aromatase inhibitors (AIs, such as letrozole, anastrozole, and aromoxine), or those that directly block ER activity, such as selective estrogen receptor modulators (SERMs, such as tamoxifen, toremifene, droloxifene, idoxifene, raloxifene, lasofoxifene, arzoxifene, miproxifene, levomeloxifene, and EM-652 (SCH 57068)) and selective estrogen receptor degraders (SERDs, such as fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 / DPC974, SRN-927, ICI182782, and AZD9496) have been used or are being developed.

[0008] SERMs (e.g., tamoxifen) and AIs are often used as first-line adjuvant systemic treatments for ER-positive breast cancer. Tamoxifen is commonly used for ER-positive breast cancer. AIs inhibit estrogen production in peripheral tissues by blocking the activity of aromatase, which converts androgens in the body into estrogens. However, AIs cannot prevent the ovaries from making estrogen. Therefore, AIs are mainly used to treat postmenopausal women. In addition, since AIs are much more effective than tamoxifen and produce fewer serious side effects, AIs can also be used to treat premenopausal women whose ovarian function is suppressed. See, for example, "Adjuvant Ovarian Suppression in Premenopausal Breast Cancer" by Francis et al. (NEngl. J. Med., 372: 436-446 (2015)).

[0009] Although initial treatment with these agents may be successful, many patients eventually relapse with resistant breast cancer. Mutations affecting the ER have emerged as one of the potential mechanisms for the development of this resistance. See, for example, Robinson et al., “Activating ESR1 mutations in hormone-resistant metastatic breast cancer,” Nat. Genet. 45:1446-51 (2013). Mutations in the ER ligand binding domain (LBD) were found in 21% of metastatic ER-positive breast tumor samples from patients who received at least one line of endocrine therapy. Jeselsohn et al., “ESR1 mutations-a mechanism for acquired endocrine resistance in breast cancer,” Nat. Rev. Clin. Oncol., 12:573-83 (2015).

[0010] 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, the utility of fulvestrant has been limited by the dose that can be administered in a single injection and reduced bioavailability. Imaging studies using 18F-fluoroestradiol positron emission tomography (FES-PET) have shown that even at the 500 mg dose level, some patients may not be able to fully suppress ER, and insufficient dosing may be the cause of treatment failure.

[0011] Another challenge associated with estrogen-directed therapies is that they may have undesirable effects on the uterus, bone, and other tissues. ERs direct the transcription of estrogen-responsive genes in several tissues and cell types. These effects may be particularly significant as endogenous levels of estrogen and other ovarian hormones decrease during menopause. For example, tamoxifen can cause osteoporosis in premenopausal women and increase the risk of endometrial cancer due to its partial agonist effect on the endometrium. In postmenopausal women, AIs can cause more bone loss and more fractures than tamoxifen. Due to its mechanism of action, patients treated with fulvestrant may also be at risk for osteoporosis.

[0012] The mammalian target of phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) / mammalian target of rapamycin (mTOR) pathway is an important intracellular signaling pathway in regulating the cell cycle. The frequent activation of the PI3K / AKT / mTOR pathway in cancer and its key role in cell growth and survival pose a challenge to finding the right amount of proliferation-differentiation to exploit this balance in the development of various therapies. See, e.g., Gitto et al., "Recent insights into the pathophysiology of mTOR pathway dysregulation," Res. Rep. Bio., 2: 1-16 (2015).

[0013] Inhibitors of the PI3K pathway show the most promise when administered in combination with other therapies. For example, in 2012, everolimus (an allosteric mTOR inhibitor) was approved for use in combination with the AI ​​exemestane for the treatment of postmenopausal women with advanced hormone receptor-positive (HR+), HER2- breast cancer (BOLERO-2 study). Drugs targeting other components of the PI3K pathway are being developed for the treatment of HR+ cancers, such as ATP-competitive dual inhibitors of PI3K and mTOR (e.g., BEZ235, GDC-0980), pan-PI3K inhibitors that inhibit all four isoforms of class I PI3K (e.g., BKM120, GDC-0941), isoform-specific inhibitors of various PI3K isoforms (e.g., BYL719, GDC-0032), allosteric and catalytic inhibitors of AKT (MK2206, GDC-0068, GSK2110183, GSK2141795, AZD5363), and ATP-competitive inhibitors of mTOR alone (AZD2014, MLN0128, and CC-223). Dienstmann et al., "Picking the point of inhibition: a comparative review of PI3K / AKT / mTOR pathway inhibitors," Mol. Cancer Ther, 13(5): 1021-31 (2014).

[0014] Despite their great potential, adverse side effects associated with mTOR inhibitors have hampered their development as effective cancer therapies. Kaplan et al., "Strategies for the management of adverse events associated with mTOR inhibitors," Transplant Rev (Orlando), 28(3): 126-133 (2014); and Pallet et al., "Adverse events associated with mTOR inhibitors," Expert Opin. Drug Saf. 12(2): 177-186 (2013).

[0015] 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 benefit by combining with a second therapeutic agent (e.g., everolimus and other agents targeting the PI3K / AKT / mTOR pathway) against advanced cancers and / or cancers that are resistant to previous treatments. Summary of the invention

[0016] One aspect of the present invention relates to a method for treating one or more cancers and / or tumors in a subject, comprising administering to the subject a therapeutically effective amount of a combination of RAD1901 or a solvate (e.g., hydrate) or a salt thereof and one or more second therapeutic agents as described herein (e.g., everolimus).

[0017] In some embodiments, the cancer is an estrogen-dependent cancer, such as breast cancer, ovarian cancer, colon cancer, endometrial cancer, or prostate cancer. In some embodiments, the cancer is ER-positive breast cancer.

[0018] RAD1901 or its solvate (e.g., hydrate) or its salt and a second therapeutic agent (e.g., everolimus) are administered in combination to a subject in need. The phrase "combination" refers to the administration of RAD1901 or its solvate (e.g., hydrate) or its salt before, during, or after the administration of the second therapeutic agent (e.g., everolimus). For example, RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent can be administered at the following times: about one week apart, about 6 days apart, about 5 days apart, about 4 days apart, about 3 days apart, about 2 days apart, about 24 hours apart, about 23 hours apart, about 22 hours apart, about 21 hours apart, about 20 hours apart, about 19 hours apart, about 18 hours apart, about 17 hours apart, about 16 hours apart, about 15 hours apart, about 14 hours apart, about 13 hours apart, At intervals of about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes or about 5 minutes. In other embodiments, RAD1901 or its solvate (e.g., hydrate) or its salt and a second therapeutic agent (e.g., everolimus) are administered to the subject simultaneously or substantially simultaneously. In certain embodiments of these embodiments, compound can be administered as a part of a single formulation.

[0019] In some embodiments, RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent are administered in separate preparations. In certain embodiments of these embodiments, the preparation can be a preparation of the same type. For example, both preparations can be designed for oral administration (e.g., by two separate pills) or for injection (e.g., by two separate injectable preparations). In other embodiments, RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus) can be formulated into different types of preparations. For example, a compound can be in a preparation designed for oral administration, and another compound in a preparation designed for injection.

[0020] In other embodiments, RAD1901 or its solvate (e.g., hydrate) or its salt and a second therapeutic agent are administered as part of a single formulation. For example, RAD1901 or its solvate (e.g., hydrate) or its salt and a second therapeutic agent are formulated into a single pill for oral administration or configured into a single injection. Accordingly, in certain embodiments, provided herein are formulations comprising RAD1901 or its solvate (e.g., hydrate) or its salt and one or more second therapeutic agents.

[0021] The routes of administration of RAD1901 or its solvate (e.g., hydrate) or salt thereof and / or the second therapeutic agent include, but are not limited to, topical administration, oral administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intravesical instillation, subcutaneous administration, transdermal administration, and transmucosal administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :RAD1901 inhibits tumor growth (TGI) in several patient-derived xenograft (PDx) models, regardless of the ESR1 status of the tumor and previous endocrine therapy history. It shows the percentage of tumor growth inhibition (TGI) in PDx models treated with RAD1901.

[0023] Figure 2A -C: The combination of RAD1901 and everolimus exhibited tumor growth inhibition and regression in a wild-type (WT) ERα MCF-7 mouse xenograft model (PR+, Her2-). Figure 2A ): Tumor growth in MCF-7 xenograft model treated with vehicle control, everolimus (2.5 mg / kg, oral, once daily), fulvestrant (3 mg / dose, subcutaneous injection, once weekly), the combination of fulvestrant (3 mg / dose, subcutaneous injection, once weekly) and everolimus (2.5 mg / kg, oral, once daily), RAD1901 (60 mg / kg, oral, once daily), and the combination of RAD1901 (60 mg / kg, oral, once daily) and everolimus (2.5 mg / kg, oral, once daily); one-way ANOVA, "ns" is not significant, *p value <0.05, ***p value <0.001; ( Figure 2B): Changes in individual tumor sizes from baseline to the end of the study in the MCF-7 xenograft model treated with vehicle control, everolimus (2.5 mg / kg, oral, once daily), fulvestrant (3 mg / dose, subcutaneous injection, once weekly), the combination of fulvestrant (3 mg / dose, subcutaneous injection, once weekly) and everolimus (2.5 mg / kg, oral, once daily), RAD1901 (60 mg / kg, oral, once daily), and the combination of RAD1901 (60 mg / kg, oral, once daily) and everolimus (2.5 mg / kg, oral, once daily) ; (Figure 2C): Tumor growth in MCF-7 xenograft model treated with vehicle control, everolimus (2.5 mg / kg, oral, once daily), fulvestrant (3 mg / dose, subcutaneous injection, once weekly), the combination of fulvestrant (3 mg / dose, subcutaneous injection, once weekly) and everolimus (2.5 mg / kg, oral, once daily), RAD1901 (30 or 60 mg / kg, oral, once daily), and the combination of RAD1901 (30 or 60 mg / kg, oral, once daily) and everolimus (2.5 mg / kg, oral, once daily).

[0024] Figure 3A -B: In the WT ERα PDx-11 model (PR+, Her2-, previously treated with aromatase inhibitors, fulvestrant, and chemotherapy), the combination of RAD1901 and everolimus demonstrated tumor growth inhibition and regression. ( Figure 3A ): Tumor growth of PDx-11 models treated with vehicle control, fulvestrant (3 mg / dose, subcutaneous injection, once a week), everolimus (2.5 mg / kg, oral, once a day), RAD1901 (60 mg / kg, oral, once a day), and a combination of RAD1901 (60 mg / kg, oral, once a day) and everolimus (2.5 mg / kg, oral, once a day); (Figure 3B): Changes in individual tumor size from baseline to the end of the study in PDx-11 models treated with vehicle control, fulvestrant (3 mg / dose, subcutaneous injection, once a week), RAD1901 (60 mg / kg, oral, once a day), and a combination of RAD1901 (60 mg / kg, oral, once a day) and everolimus (2.5 mg / kg, oral, once a day). n = 8-10 / group.

[0025] Figure 4A -B: In the WT ER+PDx-2 model (PR+, Her2-, treatment naive), the combination of RAD1901 and everolimus showed tumor growth inhibition. ( Figure 4A): Tumor growth of PDx-2 model treated with vehicle control, RAD1901 (60 mg / kg, oral, every other day), fulvestrant (3 mg / dose, subcutaneous injection, once a week), and a combination of RAD1901 (60 mg / kg, oral, every other day) and fulvestrant (3 mg / dose, subcutaneous injection, once a week); ( Figure 4B ): Tumor growth of PDx-2 model treated with vehicle control, everolimus (2.5 mg / kg, orally, once daily), RAD1901 (60 mg / kg, orally, once every other day), and a combination of RAD1901 (60 mg / kg, orally, once daily) and everolimus (2.5 mg / kg, orally, once daily). n=8-10 / group.

[0026] Figure 5 : Estradiol treatment was continued in the WT ERα PDx-4 model (PR+, Her2-, treatment naive) after the end of RAD1901 treatment, and the efficacy of RAD1901 persisted for at least two months.

[0027] Fig. 6A -B: In the mutant (Y537S) ERα PDx-5 model (PR+, Her2+, previously treated with an aromatase inhibitor), the combination of RAD1901 and everolimus demonstrated tumor growth inhibition. ( Fig. 6A ) Tumor growth of PDx-5 models treated with vehicle control, RAD1901 (60 mg / kg, oral, once daily), and fulvestrant (3 mg / dose, subcutaneous injection, once weekly); ( Figure 6B ) Tumor growth of PDx-5 models treated with vehicle control, RAD1901 (60 mg / kg, orally, once daily), everolimus (2.5 mg, orally, once daily), and a combination of RAD1901 (60 mg / kg, orally, once daily) and everolimus (2.5 mg, orally, once daily). n=8-10 / group.

[0028] Figure 7 : Pharmacokinetic analysis of Fulvestrant in nude mice. It shows the plasma concentration of Fulvestrant at 1 mg / dose (solid diamonds), 3 mg / dose (solid circles) and 5 mg / dose (solid triangles). Nude mice were injected subcutaneously with Fulvestrant on day 1 and a second dose was administered on day 8. The plasma concentration of Fulvestrant was monitored at the specified time points for up to 168 hours after the second dose.

[0029] Figure 8 : Effects of RAD1901 and Faslodex on mouse survival in the intracranial MCF-7 tumor model.

[0030] Fig. 9A -C: Representative images of FES-PET scans of the uterus of subjects treated with 200 and 500 mg RAD1901 (oral, once daily) and changes in ER binding after RAD1901 treatment. ( Fig. 9A ): Transverse views of uterine CT scans before (a) and after (c) 200 mg RAD1901 treatment and transverse views of uterine FES-PET scans before (b) and after (d) RAD1901 treatment; ( Fig. 9B ): Sagittal view of uterine CT scan before (upper (a) panel) and after (lower (a) panel) treatment with 500 mg RAD1901, sagittal view of uterine FES-PET scan before (upper (b) panel) and after (lower (b) panel) treatment with RAD1901, transverse view of uterine CT scan before (upper (c) panel) and after (lower (c) panel) treatment with RAD1901, transverse view of uterine FES-PET scan before (upper (d) panel) and after (lower (d) panel) treatment with RAD1901; ( Fig. 9C ): Change in % ER binding after RAD1901 treatment compared to baseline (before RAD1901 treatment) for subjects 1-3 (200 mg) and subjects 4-7 (500 mg).

[0031] Fig. 10A -B: Uterine (before (baseline) and after (after treatment) RAD1901 treatment (500 mg) Fig. 10A ) and pituitary gland ( Fig. 10B Representative images of FES-PET scans of the uterus. (a) lateral section; (b) longitudinal section; and (c) longitudinal section.

[0032] Fig.11 : PR and ER expression in MCF-7 xenograft models treated with vehicle control, RAD1901, everolimus, the combination of RAD1901 and everolimus, fulvestrant, and the combination of fulvestrant and everolimus.

[0033] Fig. 12A -B: In vitro, RAD1901 treatment resulted in the proliferation of MCF-7 cell lines ( Fig. 12A ) and T47D cell lines ( FIG. 12B ). ER expression in two cell lines treated with RAD1901 and Fulvestrant at several concentrations of 0.001 μM, 0.01 μM, 0.1 μM and 1 μM, respectively, is shown. ER signaling is shown by the three ER target genes tested: PGR, GREB1 and TFF1.

[0034] Fig.13A-C: RAD1901 treatment leads to ER degradation and abolishes ER signaling in the MCF-7 xenograft model. Fig.13A ): 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 fulvestrant at 2 hours or 8 hours after the last dose; ( Fig. 13B ): ER protein expression at 2 hours after the last dose in MCF-7 xenograft models treated with vehicle control, 30 and 60 mg / kg RAD1901 and 3 mg / dose fulvestrant; ( Fig. 13C ): PR protein expression at 8 hours after the last dose in MCF-7 xenograft models treated with vehicle control, 30 and 60 mg / kg RAD1901 and 3 mg / dose fulvestrant.

[0035] Fig.14A -C: RAD1901 treatment leads to a rapid decrease in PR in the MCF-7 xenograft model. ( Fig.14A ): Western blot showing PR expression at 8 or 12 hours after a single dose in MCF-7 xenograft models treated with vehicle control and RAD1901 (30, 60 and 90 mg / kg); ( Fig. 14B ): Western blot showing PR expression at 4 hours or 24 hours after the 7th dose in MCF-7 xenograft models treated with vehicle control and RAD1901 (30, 60 and 90 mg / kg); ( Fig. 14C ): Dose-dependent reduction of PR expression in MCF-7 xenograft model treated with 30, 60 and 90 mg / kg RAD1901.

[0036] Fig.15A -B: RAD1901 treatment leads to a rapid decrease in proliferation in the MCF-7 xenograft model. ( Fig.15A ): Representative photographs of tumor sections collected from MCF-7 xenograft models treated with vehicle control and 90 mg / kg RAD1901 at 8 hours after a single dose and 24 hours after the fourth dose, stained for the proliferation marker Ki-67; ( Fig. 15B ): Histogram showing reduction of proliferation marker Ki-67 in MCF-7 xenograft model treated with vehicle control and 90 mg / kg RAD1901 at 8 hours after a single dose and 24 hours after the 4th dose.

[0037] Fig.16: On the last day of the 56-day efficacy study, at 4 hours, 30, 60, and 120 mg / kg RAD1901 treatment significantly reduced Ki67 in end-of-study tumors in the PDx-4 model compared to fulvestrant (1 mg / animal).

[0038] Fig.17 : 60 and 120 mg / kg RAD1901 treatment resulted in reduced in vivo ER signaling in the PDx-5 model, accompanied by decreased PR expression.

[0039] Fig.18A -D: Effects of RAD1901 on uterine tissue of newly weaned female Sprague-Dawley rats. Fig.18A ): uterine wet weight of rats euthanized 24 hours after the last dose; ( Fig.18B ): epithelial height in uterine tissue sections; ( Fig.18C ): Representative sections of uterine tissue stained with toluidine blue O at 400 times magnification, arrows indicate uterine epithelium; ( Fig.18D ): Total RNA extracted from uterine tissue and analyzed by quantitative RT-PCR for the expression level of complement C3 relative to the 18S ribosomal RNA housekeeping gene.

[0040] Fig.19 : Plasma pharmacokinetic results after administration of 200, 500, 750 and 1000 mg / kg RAD1901 on day 7.

[0041] Fig. 20 :3ERT(I).

[0042] Fig.21 :3ERT(II).

[0043] Fig. 22 : Superposition of ERα LBD antagonist complexes summarized in Table 11.

[0044] Fig.23A -B:( Fig.23A )RAD1901-1R5K; and ( Fig. 23B )Modeling of GW5-1R5K.

[0045] Fig.24A -B:( Fig.24A )RAD1901-1SJ0; and ( Fig. 24B )Modeling of E4D-1SJ0.

[0046] Fig.25A -B:( Fig.25A )RAD1901-2JFA; and ( Fig.25B )Modeling of RAL-2JFA.

[0047] Fig.26A -B:( Fig.26A )RAD1901-2BJ4; and ( Fig.26B )Modeling of OHT-2BJ4.

[0048] Fig.27A -B:( Fig.27A )RAD1901-2IOK; and ( Fig.27B )IOK-2IOK modeling.

[0049] Fig.28 : Superposition of RAD1901 conformations obtained from IFD analysis using 1R5K and 2OUZ.

[0050] Fig.29 : Superposition of RAD1901 conformations obtained from IFD analysis using 2BJ4 and 2JFA.

[0051] Fig. 30A -B: Superposition of RAD1901 conformations obtained from IFD analysis using 2BJ4, 2JFA and 1SJ0.

[0052] Fig.31A -C: IFD of RAD1901 using 2BJ4.

[0053] Fig.32A -C: Protein surface interactions of RAD1901 docked in 2BJ4 via IFD.

[0054] Fig.33A - C: IFD of fulvestrant using 2BJ4.

[0055] Fig.34A -B: IFD of fulvestrant and RAD1901 using 2BJ4.

[0056] Fig.35A -B: Superposition of IFDs of fulvestrant and RAD1901 using 2BJ4.

[0057] Fig.36 : RAD1901 in vitro binding assay using WT and LBD mutant ERα constructs.

[0058] Table 1. RAD1901 levels in plasma, tumors and brain of mice implanted with MCF7 cells after 40 days of treatment. BLQ: below the limit of quantification.

[0059] Table 2. SUVs of uterus, muscle and bone in human subjects treated with a 200 mg dose (oral, once daily) for 6 days.

[0060] Table 3. SUV of uterus, muscle and bone in human subjects (n=4) treated with a 500 mg dose (oral, qd) for 6 days.

[0061] Table 4. Effect of RAD1901 on BMD in ovariectomized rats. Adult female rats were subjected to sham or ovariectomy surgery and then started treatment with vehicle, E2 (0.01 mg / kg) or RAD1901 (3 mg / kg) once a day (n=20 / each treatment group). BMD was measured by dual emission x-ray absorptiometry at baseline and after 4 weeks of treatment. Data are expressed as mean+SD. *P<0.05 relative to the corresponding OVX+Veh control. BMD is bone mineral density; E2 is beta estradiol; OVX is ovariectomy; Veh is vehicle.

[0062] Table 5. Effect of RAD1901 on femoral microarchitecture in ovariectomized rats. Adult female rats underwent sham surgery or ovariectomy and then began treatment with vehicle, E2 (0.01 mg / kg) or RAD1901 (3 mg / kg) once a day (n=20 / each treatment group). After 4 weeks, bone microarchitecture was assessed using microcomputed tomography. Data are expressed as mean ± SD. *P < 0.05 relative to the corresponding OVX+Veh control. ABD is apparent bone density; BV / TV is bone volume density; ConnD is connection density; E2 is β-estradiol; OVX is ovariectomy; TbN is trabecular number; TbTh is trabecular thickness; TbSp is trabecular spacing; Veh is vehicle.

[0063] Table 6. Key Baseline Demographics for the Phase 1 Dose-Escalation Study of RAD1901.

[0064] Table 7. Most frequent (>10%) treatment-related AEs in the Phase 1 dose escalation study of RAD1901. AEs were graded according to CTCAE v4.0. Any patient who experienced multiple episodes of the same priority item was scored only once with the most severe grade. *>10% of patients in the total active group with any relevant TEAE. N = number of subjects with at least one treatment-related AE in a given category.

[0065] Table 8. Pharmacokinetic parameters in the Phase 1 dose escalation study of RAD1901 (Day 7).

[0066] Table 9. Frequency of LBD mutations.

[0067] Table 10. Differences in residue configurations of ER-α LBD-antagonist complexes and 3ERT.

[0068] Table 11. Structural overlap of ER-α LBD-antagonist complexes assessed by RMSD calculations.

[0069] Table 12. Analysis of ligand binding in ER-α LBD-antagonist complexes.

[0070] Table 13. Model evaluation of RAD1901 docking.

[0071] Table 14. Induced fit docking scores of RAD1901 with 1R5K, 1SJ0, 2IFA, 2BJ4 and 2OUZ. DETAILED DESCRIPTION

[0072] As described in the Examples section below, the combination of RAD1901 and everolimus (RAD1901-everolimus combination) (structures shown below) exhibited greater tumor growth inhibition than RAD1901 alone in several breast cancer xenograft models, including: the wild-type (WT) ERα MCF-7 xenograft model ( Figure 2A -C), WT ERα PDx-2 ( Figure 4A -B) and PDx-11 models ( Figure 3A -B), and mutant (e.g., Y537S) ERα PDx-5 models ( Fig. 6A -B), regardless of the ESR1 status of the tumor and the history of previous endocrine therapy as described in Example 1. PDx-2, PDx-5, and PDx-11 models have models expressing WT or mutant (e.g., Y537S) ERα, with PR expression, with high or low Her2 expression, and with or without previous endocrine therapy (e.g., AI, fulvestrant), and / or chemotherapy (chemo) ( Figure 1 ) tumors. RAD1901 alone also inhibited Figure 1 Tumor growth in all other PDx models listed in , i.e., tumors expressing WT or mutant (e.g., Y537S) ERα, with PR expression, with high or low Her2 expression, and with or without prior endocrine therapy (e.g., tamoxifen (tam), AI, fulvestrant), chemotherapy (chemo), Her2 inhibitors (Her2i, such as trastuzumab, lapatinib), bevacizumab, and / or rituximab.

[0073] ER WT PDx models and ER mutant PDx models can have different response levels for treatment with fulvestrant alone, everolimus alone, and / or a combination of fulvestrant and everolimus (fulvestrant-everolimus combination). However, the RAD1901-everolimus combination showed improved tumor growth inhibition and / or tumor regression compared to treatment with RAD1901 alone or everolimus alone, regardless of whether the PDx model responded to fulvestrant treatment and / or fulvestrant-everolimus combination treatment. In other words, the RAD1901-everolimus combination can inhibit tumor growth and / or produce tumor regression in fulvestrant-resistant cancers.

[0074] Compared to treatment with fulvestrant alone or with the fulvestrant-everolimus combination, RAD1901-everolimus combination treatment exhibited improved tumor growth inhibition and / or tumor regression. For example, the RAD1901-everolimus combination produced more significant tumor regression in more WT ER+ xenograft models than treatment with fulvestrant alone, RAD1901 alone, or everolimus alone, even though these xenograft models had different responsiveness to fulvestrant treatment (e.g., MCF7 cell line xenograft models that responded to fulvestrant treatment (Figure 2); PDx-11 models that responded to fulvestrant treatment (Figure 3); and PDx-2 models that were least responsive to fulvestrant treatment (Figure 4)). The RAD1901-everolimus combination also produced more significant tumor regressions in more WT ER+MCF7 cell line xenograft models and PDx-11 models than treatment with the fulvestrant-everolimus combination (Figures 2 and 3). The RAD1901-everolimus combination provided similar effects to 30 mg / kg or 60 mg / kg doses of RAD1901, but RAD1901 alone at 30 mg / kg was not as effective as RAD1901 alone at 60 mg / kg in inhibiting tumor growth (Figures 2 and 3). Figure 2C ). The results indicate that a RAD1901-everolimus combination with a lower dose of RAD1901 (eg, 30 mg / kg) is sufficient to maximize the tumor growth inhibition / tumor regression effect in the xenograft model.

[0075] The RAD1901-everolimus combination demonstrated tumor regression or improved tumor growth inhibition in mutant ER+ (e.g., Y537S) PDx models that had little response to fulvestrant treatment ( Fig. 6A). For example, PDx-5 is in an ER Y537S mutant PDx model (PR+, Her2+, with prior AI treatment) that has little response to fulvestrant treatment. The RAD1901-everolimus combination showed tumor regression in the PDx-5 model, while everolimus alone or RAD1901 alone only inhibited tumor growth without causing tumor regression ( Figure 6B The RAD1901-everolimus combination produced more significant tumor growth inhibition in the mutant PDx-5 model compared with RAD1901 alone, everolimus alone, or fulvestrant alone ( Figure 6B ). Therefore, when administered in combination with RAD1901, the addition of everolimus benefits the PDx-5 model. Therefore, the RAD1901-everolimus combination provides a potent anti-tumor therapy for ER+ breast cancer expressing WT or mutant ER, with PR expression, with high or low Her2 expression, and with or without resistance to Fulvestrant.

[0076]

[0077] The results provided herein also show that RAD1901 can be delivered to the brain (Example II), and that the delivery improves mouse survival in an intracranial tumor model expressing wild-type ERα (MCF-7 xenograft model, Example I (B)). Everolimus has been approved for the treatment of subependymal giant cell astrocytoma (SEGA), a brain tumor seen in tuberous sclerosis (TS). Thus, both components of the RAD1901-everolimus combination may be able to cross the brain-blood barrier and treat ER+ tumors in the brain. This represents an additional advantage in treating ER+ tumors in the brain compared to the fulvestrant-everolimus combination, because 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)). The combination of RAD1901, which is able to cross the blood-brain barrier, with other second treatments (e.g., mTOR inhibitors such as rapamycin analogs (Geoerger et al., "Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor / medu1loblastoma models as single agent and in combination chemotherapy," Cancer Res. 61: 1527-1532 (2001)) may also have similar therapeutic effects on ER+ tumors in the brain.

[0078] RAD1901 showed sustained efficacy in inhibiting tumor growth when estradiol treatment was continued after RAD1901 treatment was completed (e.g., PDx-4 models). Therefore, the RAD1901-everolimus combination may benefit patients by inhibiting tumor growth after treatment is completed, especially when the second treatment may be discontinued (e.g., 29% for everolimus) or reduced or delayed (70% for everolimus-treated patients) due to adverse reactions. http: / / www.fda.gov / Drugs / InformationOnDrugs / ApprovedDrugs / ucm488028.htm.

[0079] Compared with everolimus alone or with everolimus combined with other hormonal therapies (eg, AIs (eg, letrozole) and SERDs Compared with treatment with a combination of (ri, such as fulvestrant), the RAD1901-everolimus combination may have lower and / or less severe side effects. For example, both AI and fulvestrant may cause bone loss in treated patients. RAD1901 is unlikely to have similar side effects. It has been found that RAD1901 preferentially accumulates in tumors, with tumor RAD1901 levels: plasma RAD1901 levels (T / P ratio) as high as about 35 (Example II). The standard uptake values ​​(SUV) of the uterus, muscle, and bone of human subjects treated with RAD1901 at a dose of about 200 mg to up to about 500 mg (once a day) were calculated (Example III (A)). The uterine signal after administration is close to the level from "non-target tissues" (tissues that do not express estrogen receptors), which shows that FES-PET uptake after RAD1901 treatment is completely weakened. In tissues that do not significantly express estrogen receptors (such as muscle, bone), almost no changes were observed in PET scans before and after treatment (Example III (A)). Finally, RAD1901 treatment antagonizes estradiol stimulation of uterine tissue in ovariectomized (OVX) rats (Example IV (A)), and substantially preserves bone mass in treated subjects. For example, OVX rats treated with RAD1901 showed maintained BMD and femoral microarchitecture (Example IV (A)). Therefore, the RAD1901-everolimus combination may be particularly useful for patients with osteoporosis or at a higher risk of osteoporosis.

[0080] In addition, gene expression analysis has been reported to be effective in identifying patients who respond to everolimus treatment. Yoon et al., "Gene expression profiling identifies responsive patients with cancer of unknown primary treated with carboplatin, paclitaxel, and everolimus: NCCTG N0871 (alliance)," Ann. Oncol., 27(2): 339-44 (2016). Study NCT00805129 found that everolimus was more effective in patients with somatic mutations in TSC1 because the mutations led to increased relapse and increased response time to everolimus. Therefore, the method disclosed herein may further include performing genetic analysis on the subject to be treated to identify subjects with greater response and / or longer response time.

[0081] In addition, it has been found that RAD1901 degrades wild-type ERα and eliminates ER signaling in vivo in MCF-7 cell line xenograft models, and shows a dose-dependent reduction in PR in these MCF-7 cell line xenograft models (Example III (B)). RAD1901 reduces proliferation in MCF-7 cell line xenograft models and PDx-4 models, as confirmed by a decrease in the proliferation marker Ki67 in tumors collected from treated subjects. In a mutant ER PDx model that has little response to fulvestrant treatment, RAD1901 also reduces ER signaling in vivo (Example III (B)).

[0082] The unexpected efficacy of the RAD1901-everolimus combination in tumors that have little response to fulvestrant treatment and tumors expressing mutant ERα may be due to the unique interaction between RAD1901 and ERα. Structural models of ERα bound to RAD1901 and other ERα-binding compounds were analyzed to obtain information about specific binding interactions (Example V). Computer modeling showed that the RAD1901-ERα interaction is unlikely to be affected by LBD mutants of ERα, such as Y537X mutants, 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 receiving at least one line of endocrine therapy (Table 9, Example V). Therefore, the combination of one or more second therapeutic agents (e.g., everolimus) and RAD1901 or a salt thereof or a solvate thereof (e.g., hydrate) may have a therapeutic effect similar to that of RAD1901-everolimus disclosed herein, while having relatively low side effects. Computer modeling enables the identification of specific residues in the C-terminal ligand binding domain of ERα that are critical for binding, and this information can be used to develop compounds that not only bind to and antagonize wild-type ERα but also bind to and antagonize certain mutants and variants thereof, which, when combined with a second therapeutic agent (e.g., everolimus), can provide a strong anti-tumor therapy similar to that of the RAD1901-everolimus combination disclosed herein, while having relatively low side effects.

[0083] Based on these results, provided herein are methods for inhibiting the growth or causing regression of ERα-positive tumors in a subject in need thereof by administering to the subject a therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) or salt thereof in combination with one or more second therapeutic agents described herein (e.g., everolimus).

[0084] In certain embodiments, in addition to inhibiting tumor growth (including, for example, inhibiting cancer cell proliferation or inhibiting ERα activity (e.g., by inhibiting estradiol binding or by downregulating ERα)), administration of RAD1901 or a salt thereof or a solvate thereof (e.g., hydrate) has additional therapeutic benefits. In certain embodiments, the method does not negatively affect muscle, bone, breast, and uterus.

[0085] In certain embodiments, RAD1901 or a solvate (eg, hydrate) or a salt thereof modulates and / or degrades ERa and mutant ERa.

[0086] In certain embodiments of the tumor growth inhibition or tumor regression methods provided herein, a method of inhibiting ERα-positive tumor growth or causing regression in a subject in need thereof is provided by administering to the subject a therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) thereof or a salt thereof in combination with one or more second therapeutic agents described herein. In certain embodiments of these embodiments, its salt is a RAD1901 dihydrochloride salt having the following structure:

[0087]

[0088] Second therapeutic agent

[0089] The second therapeutic agent used in the method provided herein can be a chemotherapeutic agent or an AKT inhibitor, androgen receptor, angiogenesis inhibitor, aromatase, Aurora kinase A, BCL2, EGFR, estrogen pathway, estrogen signaling pathway, estrogen receptor, HER2, HER3, heat shock protein 90 (Hsp90), Hedgehog (Hh) signaling pathway, histone deacetylase (HDAC), KIT pathway, mTOR (e.g., TORC1 and / or TORC2), microtubules, MYC, nucleoside metabolism, PARP, pan-PI3K, PI3K, protein kinase CK2, RAS pathway, steroid sulfatase (STS), TK, Top2A, tyrosine kinase, VEGF receptor tyrosine kinase, or any combination thereof. The second therapeutic agent can also be an antibody, such as an anti-TGF β antibody, an anti-type 1 insulin-like growth factor receptor antibody, an anti-TROP-2 antigen antibody, an anti-HER3 antibody, an anti-PD1 antibody, or a drug conjugate thereof.

[0090] Further examples of second therapeutic agents include, but are not limited to, abiraterone acetate, ADI-PEG 20, ado-trastuzumab emtansine, afatinib, alisertib, anastrozole, paclitaxel and paclitaxel derivatives (e.g., ANG1005, paclitaxel polymer micelles), ARN-810, azacitidine, AZD2014, AZD5363, bevacizumab, BP-C1, buparlisib (BKM120), BYL719, capecitabine, carboplatin, cedritinib maleate, cetuximab, cisplatin / AC4-CDDP4, CR1447, CX-4945, dasatinib, denosumab, docetaxel, doxorubicin, eniluracil, entinostat, enzalutamide, epirubicin, eribulin, exemestane, everolimus, fluorouracil, fulvestrant, fresolimumab, ganetespib, ganitumab, GDC-0032, GDC-0941, gemcitabine, glembatumumab Vedotin, GnRH agonists (e.g., goserelin acetate), GRN1005, GSK 2141795, ibandronate, IMMU-132, irinotecan, irosustat, epothilones (e.g., ixabepilone), lapatinib, sonidegib (LDE225), letrozole, LGK974, LJM716, lucitanib, methotrexate, MK-2206, MK-3475, MLN0128, MM-302, neratinib, niraparib, olaparib, antiandrogens (e.g., orteronel), oxaliplatin, pazopanib, pertuzumab, PF-05280014, PM01183, progesterone, pyrotinib, romidepsin, ruxolitinib, sorafenib, sunitinib, talazoparib, tamoxifen, taxanes, T-DM1, telapristone (CDB-4124), temozolomide, temsirolimus, tetrathiomolybdate, tesetaxel, TLR7 agonists, TPI 287, trametinib, trastuzumab, TRC105, trebananib (AMG 386), triptorelin, veliparib, vinflunine, vinorelbine, vorinostat, noradex and zoledronic acid, including solvates (e.g., hydrates) and salts thereof.

[0091] In certain embodiments, the second therapeutic agent is selected from ado-trastuzumab emtansine, an Aurora kinase A inhibitor (e.g., alisertib), an AI (e.g., anastrozole, exemestane, letrozole), ARN-810, an mTOR inhibitor (e.g., everolimus, AZD2014, BEZ235, GDC-0980, CC-223, MLN0128), an AKT inhibitor (e.g., AZD5363, GDC-0068, GSK2110183, GSK2141795, GSK690693, MK2206), a PI3K inhibitor (e.g., BKM120, BYL719, GDC-0032, GDC-0941), a selective histone deacetylase (HDAC) inhibitor (e.g., entinostat), GnRH agonists (e.g., goserelin acetate), GRN1005 and its combination with trastuzumab, lapatinib, tyrosine kinase inhibitors (e.g., lucitanib, neratinib), antiandrogens (e.g., orteronel), pertuzumab, temozolomide, and antibodies (e.g., keytruda and BYM338).

[0092] In certain embodiments, the second therapeutic agent can be an AI (e.g., anastrozole, aromasin, and letrozole), another SERM (e.g., arzoxifene, droloxifene, EM-652 (SCH 57068), indoxifen, lasofoxifene, levomeloxifen, miproxifene, raloxifene, tamoxifen, and toremifene), or another SERD (e.g., fulvestrant, GDC-0810 (ARN-810), GW5638 / DPC974, ICI182782, RU58668, SRN-927, TAS-108 (SR16234), and ZK191703), including solvates (e.g., hydrates) and salts thereof.

[0093] Other examples of second therapeutic agents include, but are not limited to, abraxane, AMG 386, cabazitaxel, kalei, capecitabine, docetaxel, eribulin, gemcitabine, Herceptin, neratinib, pazopanib (GW786034), rapalog (rapamycin and its analogs), paclitaxel (including analogs / alternative preparations), TDM1, temozolamide, tadalafil, veliparib (ABT-888) and vinorelbine, including solvates (e.g., hydrates) and salts thereof.

[0094] The second therapeutic agent targets the PI3K / AKT / mTOR pathway

[0095] In some embodiments, the second therapeutic agent targets the PI3K / AKT / mTOR pathway and can be an mTOR inhibitor, an mTOR dual inhibitor, a PI3K / mTOR inhibitor. In some embodiments, the second therapeutic agent is a rapamycin derivative (aka rapalog), such as rapamycin (sirolimus or rapamycin, Pfizer), everolimus (Afinitor or RAD001, Novartis), ridaforolimus (AP23573 or MK-8669, Merck and ARIAD Pharmaceuticals), temsirolimus (Torisel or CCI779, Pfizer), including solvates thereof (e.g., hydrates) and salts thereof. In some embodiments, the second therapeutic agent is an mTOR dual inhibitor that inhibits both mTORC1 and mTORC2, such as MLN0128 (castration-resistant prostate cancer (CRPC), Memorial Sloan-Kettering Cancer Center), CC115 and CC223 (Celgene), OSI-027 (OSI Pharmaceuticals), and AZD8055 and AZD2014 (AstraZeneca), including solvates (e.g., hydrates) and salts thereof. In some embodiments, the second therapeutic agent is a PI3K / mTOR inhibitor, such as GDC-0980, SAR245409 (XL765), LY3023414 (Eli Lilly), NVP-BEZ235 (Novartis), NVP-BGT226 (Novartis), SF1126, and PKI-587 (Pfizer), including solvates (e.g., hydrates) and salts thereof.

[0096] In certain embodiments, more than one second therapeutic agent disclosed above can be used together with RAD1901 or a solvate thereof (e.g., hydrate) or a salt thereof. For example, an mTOR inhibitor can be used together with another mTOR inhibitor or with a PI3K / mTOR inhibitor. Moreover, it is known in the art that the above-mentioned second therapeutic agent including an mTOR inhibitor, an mTOR dual inhibitor, and a PI3K / mTOR inhibitor can be administered together with other active agents to enhance the therapeutic effect. For example, an mTOR inhibitor can be used in combination with a JAK2 inhibitor (Bogani et al., PLOS One, 8(1): e54826 (2013)), a chemotherapeutic agent (Yardley, Breast Cancer (Auckl) 7: 7-22 (2013)), or an endocrine therapy (e.g., tamoxifen or exemestane (Vinayak et al., "mTOR inhibitors in the treatment of breast cancer," Oncology, published January 15, 2013 (http: / / www.cancernetwork.com / breast-cancer / mtor-inhibitors-treatment-breast-cancer)). Accordingly, the second therapeutic agent also includes these auxiliary active agents.

[0097] Combination therapy

[0098] Combination of RAD1901 or a solvate (eg, hydrate) or a salt thereof with one or more second therapeutic agents

[0099] When administered alone to a subject, RAD1901 or a solvate thereof (e.g., a hydrate) or a salt thereof and a second therapeutic agent (e.g., everolimus) have a therapeutic effect on one or more cancers or tumors (Examples 1(A) and 1(B)). Unexpectedly, it has been found that when administered in combination to a subject, RAD1901 or a solvate thereof (e.g., a hydrate) or a salt thereof and a second therapeutic agent (e.g., everolimus) have a significant ameliorative effect on cancer / tumors (Examples 1(A) and 1(B)).

[0100] As used herein, "inhibiting the growth" of an ERa-positive tumor may refer to slowing down the rate of tumor growth, or completely stopping tumor growth.

[0101] As used herein, "tumor regression" or "regression" of ERα-positive tumors may refer to a reduction in the maximum size of a tumor. In certain embodiments, administration of one or more second therapeutic agents described herein (e.g., everolimus) (e.g., ribociclib, abemaciclib, and everolimus) in combination with RAD1901 or a solvate thereof (e.g., hydrate) or a salt thereof may result in a reduction in tumor size relative to a baseline (i.e., the size before treatment was started) or even eradication or partial eradication of the tumor. Accordingly, in certain embodiments, the tumor regression methods provided herein may alternatively be characterized as methods for reducing tumor size relative to a baseline.

[0102] As used herein, a "tumor" is a malignant tumor and is used interchangeably with "cancer."

[0103] Tumor growth inhibition or regression may be localized to a single tumor or group of tumors within a specific tissue or organ, or may be systemic (ie, affecting tumors in all tissues or organs).

[0104] Since RAD1901 is known to preferentially bind to ERα rather than estrogen receptor β (ERβ), unless otherwise indicated, estrogen receptor, estrogen receptor α, ERα, ER, wild-type ERα and ESR1 are used interchangeably herein. As used herein, "estrogen receptor α" or "ERα" refers to a polypeptide comprising a wild-type ERα amino acid sequence encoded by the gene ESR1, a polypeptide consisting of a wild-type ERα amino acid sequence encoded by the gene ESR1, or a polypeptide consisting essentially of a wild-type ERα amino acid sequence encoded by the gene ESR1. As used herein, "estrogen receptor α-positive", "ERα-positive", "ER+" or "ERα+" tumors refer to tumors in which one or more cells express at least one ERα isoform. In certain embodiments, these cells overexpress ERα. In certain embodiments, the patient has one or more cells expressing one or more forms of estrogen receptor β within the tumor. In certain embodiments, ERα-positive tumors and / or cancers are associated with breast cancer, uterine cancer, ovarian cancer, or pituitary cancer. In certain embodiments of these embodiments, the patient has a tumor located in the breast, uterus, ovary, 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 for a HER2+ tumor, the tumor may express high HER2 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 still associated with breast cancer, uterine cancer, ovarian cancer, or pituitary cancer (e.g., a tumor that is derived from the migration or metastasis of breast cancer, uterine cancer, ovarian cancer, or pituitary cancer). Accordingly, in certain embodiments of the tumor growth inhibition or regression methods provided herein, the targeted tumor is a metastatic tumor and / or the tumor has ER overexpression in other organs (e.g., bone and / or muscle). In certain embodiments, the targeted tumor is a brain tumor and / or cancer. In certain embodiments, the targeted tumor is more sensitive to treatment with RAD1901 and a second therapeutic agent disclosed herein than with treatment with 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, lapatinib, trastuzumab et al., and / or pertuzumab), chemotherapy (e.g., abraxane, doxorubicin, carboplatin, cyclophosphamide, daunorubicin, doxil, ellence, fluorouracil, gemcitabine, helaven, ixabepilone, methotrexate, mitomycin, micoxantrone, norvinblastine, paclitaxel, 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.

[0105] In certain embodiments of the tumor growth inhibition or regression methods provided herein, the method further comprises a step of determining whether the patient has a tumor expressing ERα before administering a combination of RAD1901 or its solvate (e.g., hydrate) or its salt and one or more second therapeutic agents (e.g., everolimus). In certain embodiments of the tumor growth inhibition or regression methods provided herein, the method further comprises a step of determining whether the patient has a tumor expressing mutant ERα before administering a combination of RAD1901 or its solvate (e.g., hydrate) or its salt and one or more second therapeutic agents (e.g., everolimus). In certain embodiments of the tumor growth inhibition or regression methods provided herein, the method further comprises a step of determining whether the patient has a tumor expressing ERα that responds or does not respond to fulvestrant treatment before administering a combination of RAD1901 or its solvate (e.g., hydrate) or its salt and one or more second therapeutic agents (e.g., everolimus). These determinations can be performed using any expression detection method known in the art, and can be performed in vitro using a tumor or tissue sample removed from a subject.

[0106] In addition to demonstrating the ability of RAD1901 to inhibit tumor growth in tumors expressing wild-type ERα, the results provided herein also show that RAD1901 exhibits an unexpected ability to inhibit the growth of tumors expressing mutant forms of ERα (i.e., Y537SERα) (Example I (A)). Computer simulation evaluation of ERα mutant examples showed that none of these mutations were expected to affect the LBD and would not specifically hinder RAD1901 binding (Example V (A)), such as ERα with one or more mutants selected from the following: ERα with a Y537X mutant, wherein X is S, N or C; ERα with a D538G mutant; and ERα with a S463P mutant. Based on these results, provided herein is a method for inhibiting tumor growth or causing regression in a subject with cancer by administering to the subject a therapeutically effective amount of a combination of one or more second therapeutic agents (e.g., everolimus) and RAD1901 or a solvate (e.g., hydrate) or a salt thereof, wherein the tumor is positive for ERα having one or more mutants within the ligand binding domain (LBD), wherein the mutant is selected from Y537X1, wherein X1 is S, N or C; D538G; L536X2, wherein X2 is R or Q; P535H; V534E; S463P; V392I; E380Q; and in particular Y537SERα. As used herein, "mutant ERα" refers to ERα and variants thereof comprising one or more substitutions or deletions, which comprise, consist of, or consist essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of ERα.

[0107] In addition to inhibiting breast cancer tumor growth in animal xenograft models, the results disclosed herein also show that RAD1901 shows significant accumulation in tumor cells and is able to penetrate the blood-brain barrier (Example II). The ability to penetrate the blood-brain barrier was confirmed by showing that RAD1901 administration significantly prolongs survival in brain metastasis xenograft models (Example I (B)). Accordingly, in certain embodiments of the tumor growth inhibition or regression method provided herein, the targeted ERα-positive tumor is located in the brain or elsewhere in the central nervous system. In certain embodiments of these embodiments, ERα-positive tumors are mainly associated with brain cancer. In other embodiments, ERα-positive tumors are metastatic tumors mainly associated with another type of cancer (e.g., breast cancer, uterine cancer, ovarian cancer, or pituitary cancer) or tumors that have been transferred from another tissue or organ. In certain embodiments of these embodiments, the tumor is a brain metastasis, such as breast cancer brain metastasis (BCBM). In certain embodiments of the method disclosed herein, RAD1901 or its salt or its solvate accumulates in one or more cells in the target tumor.

[0108] In certain embodiments of the methods disclosed herein, RAD1901 or a solvate (e.g., hydrate) thereof or a 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.

[0109] The results provided herein show that RAD1901 administration prevents bone loss in ovariectomized rats (Example IV (A)). Accordingly, in certain embodiments of the tumor growth inhibition or regression method provided herein, the combination of one or more second therapeutic agents (e.g., everolimus) and RAD1901 or its solvate (e.g., hydrate) or its salt is administered without adverse effects on bone, including, for example, the adverse effects of bone volume density, bone surface density, bone mineral density, trabecular number, trabecular thickness, trabecular spacing, connection density and / or apparent bone density of the treated subject. Tamoxifen may be associated with bone loss in premenopausal women, and fulvestrant may damage bone structure due to its mechanism of action. The combination of one or more second therapeutic agents (e.g., everolimus) and RAD1901 or its solvate (e.g., hydrate) or its salt can be particularly useful for premenopausal women, tumors resistant to tamoxifen or anti-estrogen therapy, and patients with osteoporosis and / or high osteoporosis risk.

[0110] The results provided herein show that RAD1901 antagonizes the stimulation of uterine tissue by estradiol in ovariectomized rats (Example IV (A)). In addition, in human subjects treated with RAD1901 at a dose of 200 mg or up to 500 mg (once daily), the standard uptake values ​​(SUV) of uterine, muscle, and bone tissues that do not significantly express ER showed almost no signal changes before and after treatment (Example III (A)). Accordingly, in certain embodiments, such administration will not cause adverse effects on other tissues (including, for example, uterine, muscle, or breast tissue).

[0111] RAD1901 or its solvate (e.g., hydrate) or its salt and a second therapeutic agent (e.g., everolimus) are administered in combination to a subject in need. The phrase "combination" refers to the administration of RAD1901 or its solvate (e.g., hydrate) or its salt before, during, or after the administration of the second therapeutic agent (e.g., everolimus). For example, RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus) can be administered at the following times: about one week apart, about 6 days apart, about 5 days apart, about 4 days apart, about 3 days apart, about 2 days apart, about 24 hours apart, about 23 hours apart, about 22 hours apart, about 21 hours apart, about 20 hours apart, about 19 hours apart, about 18 hours apart, about 17 hours apart, about 16 hours apart, about 15 hours apart, about 14 hours apart, about 1 In some embodiments, RAD1901 or its solvate (such as hydrate) or its salt and the second therapeutic agent (such as everolimus) are administered to the subject at the same time or substantially at the same time. In some embodiments of these embodiments, RAD1901 or its solvate (such as hydrate) or its salt and the second therapeutic agent (such as everolimus) can be administered as a part of a single preparation.

[0112] In some embodiments, a combination of RAD1901 or a solvate thereof (e.g., a hydrate) or a salt thereof and a single second therapeutic agent (e.g., everolimus) is administered to a subject. In other embodiments, a combination of RAD1901 or a solvate thereof (e.g., a hydrate) or a salt thereof and more than one second therapeutic agent (e.g., everolimus) is administered to a subject. For example, RAD1901 or a solvate thereof (e.g., a hydrate) or a salt thereof can be combined with two or more second therapeutic agents (e.g., everolimus) for the treatment of cancer / tumors.

[0113] (2) Dosage

[0114] The therapeutically effective amount of the combination of RAD1901 or its solvate (e.g., hydrate) or its salt and one or more second therapeutic agents (e.g., everolimus) for the methods disclosed herein is as follows: when applied within a specific time interval, the amount results in achieving one or more treatment benchmarks (e.g., slowing or stopping tumor growth, causing tumor regression, symptom cessation, etc.). The combination for the methods disclosed herein can be applied to the subject once or multiple times. In those embodiments where the compound is applied multiple times, the compound can be applied at set intervals (e.g., every day, every other day, every week, or every month). Alternatively, the compound can be applied at irregular intervals, such as based on symptoms, patient health, etc., as needed. The combination of a therapeutically effective amount can be applied once a day for 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 15 days. Optionally, during or after treatment, the state of cancer or the regression of the tumor is monitored, such as by FES-PET scanning of the subject. The dosage of the combination applied to the subject can be increased or decreased according to the detected cancer state or tumor regression.

[0115] Ideally, the therapeutically effective amount does not exceed the maximum tolerated dose, i.e., the dose at which 50% or more of the treated subjects experience nausea or other toxic reactions (which prevent further drug administration). The therapeutically effective amount for a subject may vary according to a variety of factors, including the type and extent of the subject's symptoms, sex, age, weight or general health, mode of administration and type of salt or solvate, changes in sensitivity to the drug, specific disease type, etc.

[0116] Examples of therapeutically effective amounts of RAD1901 or a solvate (e.g., hydrate) or a salt thereof for use in the methods disclosed herein include, but are not limited to, about 150 to about 1500 mg, about 200 to about 1500 mg, about 250 to about 1500 mg, or about 300 to about 1500 mg daily for subjects with drug-resistant ER-driven tumors or cancers; about 150 to about 1500 mg, about 200 to about 1000 mg, or about 250 to about 1000 mg, or about 300 to about 1000 mg daily for subjects with both wild-type ER-driven tumors and / or cancers and drug-resistant tumors and / or cancers; and 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 10 ... 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 daily dose.

[0117] The therapeutically effective amount or dosage of the second therapeutic agent (e.g., everolimus) depends on its specific type. Typically, the scope of the daily dose of the second therapeutic agent (e.g., everolimus) is from about 1 mg to about 1500 mg, from about 1 mg to about 1200 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 800 mg, from about 1 mg to about 600 mg, from about 1 mg to about 500 mg, from about 1 mg to about 200 mg, from about 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 1500 mg, from about 100 mg to about 1200 mg, from about 150 mg to about 1000 mg, from about 200 mg to about 800 mg, from about 300 mg to about 600 mg, from about 350 mg to about 500 mg. The daily dose of the second therapeutic agent (e.g., everolimus) can be about 1 to about 100 mg / kg, about 1 to about 75 mg / kg, about 1 to about 50 mg / kg, about 1 to about 45 mg / kg, about 1 to about 40 mg / kg, about 1 to about 30 mg / kg, about 1 to about 20 mg / kg, about 1 to about 10 mg / kg, about 2 to about 100 mg / kg, about 2 to about 75 mg / kg, about 2 to about 50 mg / kg, about 2 to about 45 mg / kg g, about 2 to about 40 mg / kg, about 2 to about 30 mg / kg, about 2 to about 20 mg / kg, about 2 to about 10 mg / kg, about 2.5 to about 100 mg / kg, about 2.5 to about 75 mg / kg, about 2.5 to about 50 mg / kg, about 2.5 to about 45 mg / kg, about 2.5 to about 40 mg / kg, about 2.5 to about 30 mg / kg, about 2.5 to about 20 mg / kg, or about 2.5 to about 10 mg / kg.

[0118] In certain embodiments, a therapeutically effective amount of a combination can utilize a therapeutically effective amount of any compound administered alone. In other embodiments, since the combination achieves a significantly improved synergistic therapeutic effect, the therapeutically effective amount of RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus) administered in the combination can be less than the therapeutically effective amount of RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus) required for separate administration; and one or both compounds can be administered at a dose lower than the dose usually administered when administered separately. Without being limited by any particular theory, by reducing at least one or all of the doses of RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus), the combination therapy significantly improves the effect, thereby eliminating or mitigating undesirable toxic side effects.

[0119] In some embodiments, when administered as part of a combination, the therapeutically effective amount of RAD1901, or a solvate (e.g., hydrate), or a salt thereof, 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%, about 50% to about 150%, about 60% to about 150%, about 70% to about 150%, about 80% to about 200%, about 90% to about 200%, about 100% to about 200%, 30% to about 150%, about 40% to about 150%, about 50% to about 150%, about 60% to about 150%, about 70% to about 150%, about 80% to about 150%, about From about 90% to about 150%, from about 100% to about 150%, from about 30% to about 120%, from about 40% to about 120%, from about 50% to about 120%, from about 60% to about 120%, from about 70% to about 120%, from about 80% to about 120%, from about 90% to about 120%, from about 100% to about 120%, from 30% to about 110%, from about 40% to about 110%, from about 50% to about 110%, from about 60% to about 110%, from about 70% to about 110%, from about 80% to about 110%, from about 90% to about 110%, or from about 100% to about 110%. In some embodiments, when administered as part of a combination, the therapeutically effective amount of the second therapeutic agent (e.g., everolimus) 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%, about 30% to about 150%, about 40% to about 150%, about 50% to about 150%, about 60% to about 150%, about 70% to about 200%, about 80% to about 200%, about 90% to about 200%, about 100% to about 200%, about 30% to about 150%, about 40% to about 150%, about 50% to about 150%, about 60% to about 150%, about 70% to about 150%, about From about 80% to about 150%, from about 90% to about 150%, from about 100% to about 150%, from about 30% to about 120%, from about 40% to about 120%, from about 50% to about 120%, from about 60% to about 120%, from about 70% to about 120%, from about 80% to about 120%, from about 90% to about 120%, from about 100% to about 120%, from 30% to about 110%, from about 40% to about 110%, from about 50% to about 110%, from about 60% to about 110%, from about 70% to about 110%, from about 80% to about 110%, from about 90% to about 110%, or from about 100% to about 110%.

[0120] In certain embodiments, the cancer or tumor is a drug-resistant ER-induced cancer or tumor (e.g., a cancer or tumor with a mutant ER binding domain (e.g., an ERα comprising 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), an overexpressor of ER or tumor and / or cancer proliferation becomes ligand-independent, or a tumor and / or cancer that progresses in the presence of treatment with a 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, lapatinib, trastuzumab et al., and / or pertuzumab), chemotherapy (e.g., abraxane, doxorubicin, carboplatin, cyclophosphamide, daunorubicin, doxil, ellence , fluorouracil, gemcitabine, helaven, ixabepilone, methotrexate, mitomycin, micoxantrone, norvinblastine, paclitaxel, 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.

[0121] In certain embodiments, the dosage of RAD1901 or its solvate (e.g., hydrate) or its salt in combination with the second therapeutic agent (e.g., everolimus) (e.g., ribociclib, abemaciclib and everolimus) of the disclosed methods for adult subjects can be about 30 μg to 2000 mg, 100 μg to 1500 mg, or 150 mg to 1500 mg (oral, once daily). The daily dose can be achieved by single administration or multiple administration.

[0122] The combination of one or more second therapeutic agents (e.g., everolimus) and RAD1901 or a solvate thereof (e.g., hydrate) or a salt thereof can be administered to a subject once or multiple times. In those embodiments where the compound is administered multiple times, the compound can be administered at set intervals (e.g., daily, every other day, weekly, or monthly). Alternatively, the compound can be administered at irregular intervals, such as as needed based on symptoms, patient health, etc.

[0123] (3) Preparation

[0124] In some embodiments, RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus) are administered in separate preparations. In certain embodiments of these embodiments, the preparation can be a preparation of the same type. For example, both preparations can be designed for oral administration (e.g., by two separate pills) or for injection (e.g., by two separate injectable preparations). In other embodiments, RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus) can be formulated into different types of preparations. For example, one compound can be in a preparation designed for oral administration, while another compound is in a preparation designed for injection.

[0125] In other embodiments, RAD1901 or its solvate (e.g., hydrate) or its salt and a second therapeutic agent (e.g., everolimus) are administered as part of a single formulation. For example, RAD1901 or its solvate (e.g., hydrate) or its salt and a second therapeutic agent (e.g., everolimus) are formulated into a single pill or a single injection for oral administration. In certain embodiments, provided herein is a combination formulation comprising RAD1901 or its solvate (e.g., hydrate) or its salt and one or more second therapeutic agents (e.g., everolimus). In certain embodiments, administration of the compound with a single formulation improves patient compliance.

[0126] When administered in combination, the therapeutically effective amount of each compound may be lower than the therapeutically effective amount of each compound administered alone.

[0127] In some embodiments, the formulation comprising RAD1901 or a solvate (e.g., hydrate) or a salt thereof, one or more second therapeutic agents (e.g., everolimus), or both RAD1901 or a solvate (e.g., hydrate) or a salt thereof and one or more second therapeutic agents (e.g., everolimus) may further comprise one or more pharmaceutical excipients, carriers, adjuvants and / or preservatives.

[0128] RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus) used in the disclosed method can be formulated into a unit dosage form, i.e., a physically separated unit suitable as a unit dosage of a subject undergoing treatment, wherein each unit contains a predetermined amount of active substance calculated to produce a desired therapeutic effect, which is optionally combined with a suitable pharmaceutical carrier. The unit dosage form can be used for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 times or more per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose. In certain embodiments, the compound can be formulated into controlled release.

[0129] RAD1901 or its solvate (e.g., hydrate) or its salt and the second therapeutic agent (e.g., everolimus) for the disclosed method can be formulated according to any available conventional method. Examples of preferred dosage forms include tablets, powders, microgranules, granules, coated tablets, capsules, syrups, lozenges, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nasal drops, ear drops, papuam, lotions, etc. In the preparation, commonly used additives such as diluents, adhesives, disintegrants, lubricants, colorants, flavorings, and stabilizers, emulsifiers, absorption promoters, surfactants, pH regulators, preservatives, antioxidants, etc., can be applied. In addition, according to conventional methods, preparations are also carried out by combining compositions commonly used as raw materials for pharmaceutical preparations. Examples of these compositions include, for example: (1) oils such as soybean oil, tallow and synthetic glycerides; (2) hydrocarbons such as liquid paraffin, squalane and paraffin wax; (3) ester oils such as octyldodecyl myristic acid and isopropyl myristic acid; (4) higher alcohols such as cetearyl alcohol and behenyl alcohol; (5) Silicone resin; (6) Silicone oil; (7) Surfactants, such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, solid polyoxyethylene castor oil and polyoxyethylene polyoxypropylene block copolymers; (8) Water-soluble polymers, such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone and methyl cellulose; (9) Lower alcohols, such as ethanol and isopropanol; (10) Polyols, such as glycerol, propylene glycol, dipropylene glycol and sorbitol; (11) Sugars, such as glucose and sucrose; (12) Inorganic powders, such as anhydrous silicic acid, magnesium aluminum silicate and aluminum silicate; (13) Purified water, etc. Additives for the above-mentioned preparations may include, for example: 1) lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose and silicon dioxide as diluents; 2) polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth gum, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, polypropylene glycol-polyoxyethylene block copolymer, meglumine, calcium citrate, dextrin, pectin, etc. as binders; 3) disintegrants such as cellulose, dextrin, and pectin; 4) disintegrants such as cellulose, cellulose acetate, cellulose ether, methyl cellulose ... Starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, carboxymethyl cellulose / calcium, etc. as disintegrators; 4) magnesium stearate, talc, polyethylene glycol, silicon dioxide, concentrated vegetable oil, etc. as lubricants; 5) any pharmaceutically acceptable colorant can be added as a colorant; 6) cocoa powder, menthol, aromatics, peppermint oil, cinnamon powder as flavoring agents; 7) pharmaceutically acceptable antioxidants such as ascorbic acid or α-phenol.

[0130] RAD1901 or its solvate (e.g., hydrate) or its salt and one or more second therapeutic agents (e.g., everolimus) used in the disclosed method can be formulated into a pharmaceutical composition as any one or more active compounds described herein and a physiologically acceptable carrier (also referred to as a pharmaceutically acceptable carrier or solution or diluent). Such carriers and solutions include pharmaceutically acceptable salts and solvates of the compounds used in the methods of the present invention, and mixtures comprising two or more such compounds, pharmaceutically acceptable salts of the compounds, and pharmaceutically acceptable solvates of the compounds. Such compositions are prepared according to acceptable pharmaceutical methods, such as described in Remington's Pharmaceutical Sciences (18th edition. Alfonso R. Gennaro, Mack Publishing Company, Eaton, Pa. (1990)), which are incorporated herein by reference.

[0131] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic reactions or other adverse reactions in the patient to which it is applied and is compatible with other ingredients in the preparation. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers that are suitably selected and conform to conventional pharmaceutical practice according to the expected form of administration. 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., polymethyl acrylate), calcium carbonate, magnesium oxide, talc or mixtures thereof. Pharmaceutically acceptable carriers may further include a small amount of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, which improve the shelf life or effectiveness of the therapeutic agent.

[0132] The free form of one or more second therapeutic agents (e.g., everolimus) and RAD1901 or its solvate (e.g., hydrate) or its salt can be converted into a salt by conventional methods. The term "salt" used herein is not limited as long as the salt is formed using RAD1901 or its solvate (e.g., hydrate) or its salt and is pharmaceutically acceptable; preferred examples of salts 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., aspartate, glutamate, etc.), quaternary ammonium salts, alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (magnesium salts, calcium salts, etc.), etc. In addition, hydrochloride, sulfate, methanesulfonate, acetate and the like are preferred as the "pharmaceutically acceptable salt" of the compound according to the present invention.

[0133] Isomers (e.g., geometric isomers, optical isomers, rotational isomers, tautomers, etc.) of RAD1901 or its solvate (e.g., hydrate) or its salt and / or the second therapeutic agent (e.g., everolimus) can be purified into single isomers using general separation means, including, for example, recrystallization, optical resolution such as diastereoisomeric salt method, enzyme fractionation, various chromatography methods (e.g., thin layer chromatography, column chromatography, glass chromatography, etc.). The term "single isomer" herein includes not only isomers with a purity of 100%, but also includes isomers that are present even after conventional purification operations, except for the target. For RAD1901 or its solvate (e.g., hydrate) or its salt and / or the second therapeutic agent (e.g., everolimus), crystalline polymorphs sometimes exist, and all of its crystalline polymorphs are included in the present invention. Crystalline polymorphs are sometimes single and sometimes mixtures, both of which are included in this article.

[0134] In certain embodiments, RAD1901 or its solvate (e.g., hydrate) or its salt and / or the second therapeutic agent (e.g., everolimus) can be in the form of a prodrug, which means that it must undergo some changes (e.g., oxidation or hydrolysis) to achieve its active form. Alternatively, RAD1901 or its solvate (e.g., hydrate) or its salt and / or the second therapeutic agent (e.g., everolimus) can be a compound produced by changing a parent prodrug to its active form.

[0135] (4) Route of administration

[0136] The administration routes of RAD1901 or its solvate (e.g., hydrate) or salt thereof and / or the second therapeutic agent (e.g., everolimus) disclosed herein include, but are not limited to, topical administration, oral administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intravesical instillation, subcutaneous administration, transdermal administration, and transmucosal administration.

[0137] (5) Genetic analysis

[0138] In certain embodiments, the tumor growth inhibition methods provided herein further comprise performing a genetic analysis on the subject, wherein the gene to be analyzed is one or more genes selected from the group consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, 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.

[0139] In some embodiments, the second agent is everolimus and the subject has a somatic mutation in TSC1.

[0140] In some embodiments, the present invention provides the following methods: treating a subpopulation of breast cancer patients, wherein the subpopulation has increased expression of one or more of the following genes; and treating the subpopulation with an effective dose of a combination of RAD1901 or a solvate (e.g., hydrate) or a salt thereof and one or more second therapeutic agents (e.g., everolimus) according to the dosing embodiments described in the present disclosure.

[0141] (6) Dosage adjustment

[0142] In addition to determining the ability of RAD1901 to inhibit tumor growth, the results provided herein show that RAD1901 inhibits estradiol from binding to ER in the uterus and pituitary (Example III (A)). In these experiments, estradiol was assessed for binding to ER in uterine and pituitary tissues by FES-PET imaging. After treatment with RAD1901, the observed ER binding levels were at or below background levels. These results confirm that the antagonism of RAD1901 to ER activity can be assessed by real-time scanning. Based on these results, provided herein is a method for monitoring the therapeutic efficacy of RAD1901 or its solvate or salt in the combination therapy disclosed herein by measuring estradiol-ER in one or more target tissues, wherein the combination is reduced or disappears indicating that efficacy has been produced.

[0143] Also provided is a method for adjusting the dosage of RAD1901 or its solvate or its salt in combination based on estradiol-ER binding. In certain embodiments of these methods, the binding is measured at a certain time point after one or more administrations of the first dose of the compound. If estradiol-ER binding is not affected or shows a reduction below a predetermined threshold (e.g., compared to baseline levels, the binding reduction is less than 5%, less than 10%, less than 20%, less than 30% or less than 50%), the first dose is considered to be too low. In certain embodiments, these methods include an additional step of administering an increased second dose of the compound. These steps can be repeated, and the dose is repeatedly increased until the desired estradiol-ER binding reduction is achieved. In certain embodiments, these steps can be incorporated into the methods for inhibiting tumor growth provided herein. In these methods, estradiol-ER can be combined as an alternative indicator for tumor growth inhibition, or a supplementary means for assessing growth inhibition. In other embodiments, these methods can be used in combination with the administration of RAD1901 or its solvate (e.g., hydrate) or its salt for purposes other than inhibiting tumor growth, including, for example, inhibiting cancer cell proliferation.

[0144] In certain embodiments, the methods provided herein for adjusting the dosage of RAD1901 or a salt thereof or a solvate thereof (eg, hydrate) in combination therapy include:

[0145] (1) administering a first dose of RAD1901 or a solvate (e.g., hydrate) or a salt thereof (e.g., about 350 to about 500 mg, or about 200 to about 600 mg / day) for 3, 4, 5, 6, or 7 days;

[0146] (2) detecting estradiol-ER binding activity, for example using FES-PET imaging as disclosed herein; wherein:

[0147] (i) if the ER binding activity is undetectable or below a predetermined threshold level, continuing to administer the first dose (ie, the maintenance dose level); or

[0148] (ii) if the ER binding activity is detectable or above a predetermined threshold level, administering a second dose greater than the first dose (e.g., the first dose plus about 50 to about 200 mg) for 3, 4, 5, 6, or 7 days, and then performing step (3);

[0149] (3) detecting estradiol-ER binding activity, for example using FES-PET imaging disclosed herein; wherein,

[0150] (i) if the ER binding activity is undetectable or below a predetermined threshold level, continuing with a second dose (ie, a maintenance dose level); or

[0151] (ii) if the ER binding activity is detectable or above a predetermined threshold level, administering a third dose that is greater than the second dose (e.g., the second dose plus about 50 to about 200 mg) for 3, 4, 5, 6, or 7 days, and then performing step (4);

[0152] (4) Repeat the above steps with the fourth dose, the fifth dose, and so on, until no ER binding activity can be detected.

[0153] In certain embodiments, the invention encompasses the use of PET imaging to detect and / or administer drugs to ER-sensitive or ER-resistant cancers.

[0154] (7) Combinations for use in the methods disclosed herein

[0155] Another aspect of the invention relates to a pharmaceutical composition for use in the combination methods described herein, comprising a therapeutically effective amount of RAD1901 or a solvate (eg, hydrate) or a salt thereof as disclosed herein and / or a second therapeutic agent (eg, everolimus).

[0156] RAD1901-ERα interaction

[0157] (1) Mutant ERα in ER-positive breast tumor samples from patients who received at least one line of endocrine therapy

[0158] In five studies reported in the past two years, a total of 187 metastatic ER-positive breast 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). The 6 most frequent LBD mutations in the 39 patients are shown in Scheme 1 (adapted from Jeselsohn).

[0159]

[0160] The frequencies of all LBD mutations are summarized in Table 9.

[0161] Table 9

[0162] LBD mutation frequency

[0163] frequency(%) D538G 29.5 Y537S 25.0 Y537N 13.6 Y537C 9.1 E380Q 6.8 S463P 4.5 L536R 2.3 L536Q 2.3 P535H 2.3 V392I 2.3 V534E 2.3

[0164] Computer modeling showed that the RAD1901-ERα interaction is unlikely to be affected by LBD mutants of ERα, such as the Y537X mutant, where X is S, N, or C; D538G; and S463P, which account for approximately 81.7% of the LBD mutations found in a recent study of metastatic ER-positive breast tumor samples from patients who received at least one line of endocrine therapy (Table 10, Example V).

[0165] Provided herein are complexes and crystals of RAD1901 that bind to ERα and / or mutant ERα, wherein the 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.

[0166] 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. The term "and / or" as used herein includes both "and" and "or" situations.

[0167] In certain embodiments, provided herein are methods for treating a disorder associated with ERα and / or mutant ERα activity or expression in a subject in need thereof, comprising administering to the subject one or more second therapeutic agents (e.g., everolimus) and one or more compounds capable of binding to ERα and / or mutant ERα via LBD. In certain embodiments, the subject is a mammal, and in certain embodiments of these embodiments, the subject is a human. In certain embodiments, the disorder is a tumor and / or cancer, including but not limited to ER-positive tumors and / or cancers as disclosed herein.

[0168] In certain embodiments of the compounds and 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.

[0169] In certain embodiments of the compounds and methods provided herein, the compound capable of binding to ERα and / or mutant ERα via LBD is a selective estrogen receptor degrader (SERD) or a selective estrogen receptor modulator (SERM). In certain embodiments, the compound capable of binding to ERα and / or mutant ERα via LBD completes binding via one or more interactions selected from the following: hydrogen bond interactions with residues E353, D351, R349 and / or L536 and π-interactions with residue F404 of ERα and / or mutant ERα. An example of such a compound is RAD1901.

[0170] In certain embodiments, provided herein are methods for treating a disorder associated with the activity or expression of a mutant ERα, the mutant ERα comprising 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, wherein the method comprises administering to the subject one or more second therapeutic agents (e.g., everolimus) and one or more compounds capable of binding to ERα via LBD. In certain embodiments, the disorder is cancer, including but not limited to ER-positive cancer, breast cancer, ER-positive breast cancer, and metastatic breast cancer, and in certain embodiments, the compound is RAD1901 or a pharmaceutically acceptable solvate (e.g., hydrate) thereof or a pharmaceutically acceptable salt thereof.

[0171] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. With regard to the scope of the specific materials mentioned, this is merely for illustrative purposes and is not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without exerting creative ability and without departing from the scope of the invention. It will be understood that many changes can be made to the procedures described herein and still within the scope of the invention. The inventor intends to include such changes within the scope of the invention.

[0172] Example

[0173] Materials and methods

[0174] Test compound

[0175] RAD1901 used in the following examples is (6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-2-ol dihydrochloride manufactured by IRIX Pharmaceuticals (Florence, SC). RAD1901 is stored as a dry powder, prepared as a uniform suspension of 0.5% (w / v) methylcellulose in deionized water for use, and orally administered to an animal model. Tamoxifen, raloxifene, and estradiol (E2) were obtained from Sigma-Aldrich (St. Louis, MO) and administered by subcutaneous injection. Fulvestrant was obtained from Tocris Biosciences (Minneapolis, MN) and administered by subcutaneous injection. Unless otherwise noted, other laboratory reagents were purchased from Sigma-Aldrich.

[0176] Cell lines

[0177] MCF-7 cells (human breast metastatic adenocarcinoma) were purchased from the American Type Culture Collection (Rockville, MD) and routinely maintained in phenol red-free minimum essential medium (MEM) containing 2 mM L-glutamine and Earle's BSS, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate supplemented with 0.01 mg / ml bovine insulin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA) in 5% CO2.

[0178] T47D cells were cultured to approximately 75% confluence in 10 cm dishes in a 5% CO2 incubator in RPMI growth medium supplemented with 10% FBS and 5 μg / mL human insulin.

[0179] In vivo xenograft models

[0180] All mice were housed in pathogen-free housing (with sterile and dust-free supplies) in individually ventilated cages with free access to sterile food and water, under a dark-light cycle (12-14 h circadian cycle of artificial light) and controlled room temperature and humidity. Tumors were measured twice a week with a vernier caliper, and the volume was calculated using the following formula: (L×W 2 )×0.52.

[0181] PDx Models

[0182] Some examples of patient-derived xenograft models (PDx models) are shown in Figure 1 Patient-derived PDx models of breast cancer tumors were established 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. For each experiment, the pre-study tumor volume was recorded approximately one week before its estimated start date. When the tumor reached the appropriate tumor volume initiation (TVI) range (150-250mm 3 ), animals were randomized into treatment and control groups and dosing began (Day 0, 8-10 subjects per group); all animals in the study were individually dosed for each experiment. Initial dosing began on Day 0; animals in all groups were dosed by weight (0.01 mL / g; 10 ml / kg). Vehicle (control, oral, once daily to endpoint), tamoxifen (1 mg / subject, subcutaneous, once every other day to endpoint), fulvestrant ( 1 mg / subject or 3 mg / subject, subcutaneous injection, weekly × 5, extended if necessary), or RAD1901 (30, 60 or 120 mg / kg subject, oral, once daily to endpoint) were treated in each group starting on day 0 as indicated. Depending on the model, the treatment period lasted 56-60 days. 17β-estradiol was supplemented in the drinking water of these PDx models.

[0183] Drug efficacy

[0184] For all studies, tumor size was measured by digital caliper starting from day 0, and data including individual and mean estimated tumor volumes (mean TV ± SEM) were recorded for each group; 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 × (length) × 0.52. Once the mean tumor volume of the group evaluated reaches the tumor volume (TV) endpoint (time endpoint is 60 days; volume endpoint is group mean 2 cm 3 ), each group or study was terminated; tumors with a volume of 2 cm were removed from the study. 3 or more individual mice and included the final measurement in the group average until the group average reached the volume endpoint or the study reached the time endpoint.

[0185] Power calculation and statistical analysis

[0186] % 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) - control (C) using the initial (i) and final (f) tumor measurements by the following 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.

[0187] statistics

[0188] TGI study - one-way ANOVA + Dunnett's multiple comparison test (Corbett TH et al.).

[0189] Sample collection

[0190] Tumors were removed at endpoint. One fragment was snap frozen, while the other was placed in 10% NBF for at least 24 hours and formalin fixed paraffin embedded (FFPE). Snap frozen samples were stored at -80°C; FFPE blocks were stored at room temperature.

[0191] Western blotting

[0192] Cells were harvested and analyzed for protein expression using standard procedures. Tumors were collected at designated time points after the last day of dosing and homogenized in RIPA buffer with protease and phosphatase inhibitors using Tissuelyser (Qiagen). Equal amounts of protein were separated by MW, transferred to nitrocellulose membranes, and blotted using the following antibodies using standard procedures:

[0193] Estrogen receptor (SantaCruz (HC-20); sc-543)

[0194] Progesterone receptor (Cell Signaling Technologies; 3153)

[0195] Vinculin (Sigma-Aldrich, v9131)

[0196] qPCR analysis was performed as follows: cells were harvested, mRNA was extracted, and equal amounts were used for cDNA analysis and qPCR using primers specific for progesterone receptor, GREB1 and TFF1 (LifeTech). Bands were quantified using ID Quant software (GE).

[0197] Immunohistochemistry

[0198] Tumors were collected, fixed with formalin and embedded in paraffin. Embedded tumors were sectioned (6 μM) and stained with ER, PR and Her2 specific antibodies. Quantification was performed as follows: five areas were counted for positive cells (0-100%) and staining intensity (0-3+). H scores (0-300) were calculated using the following formula: % Positive × Intensity.

[0199] Example I. RAD1901-everolimus combination enhances tumor growth inhibition in tumors and / or cancers expressing WT ER or mutant ER (eg, Y537S) in the presence of different prior endocrine therapies.

[0200] I(A). Effectiveness of RAD1901 in animal xenograft models

[0201] I(A)(i)RAD1901 inhibits tumor growth in PDx models (PDx-1 to PDx-12) regardless of the ER status and previous endocrine therapy history of the tumor.

[0202] Figure 1 Tumor growth inhibition in several PDx models was shown for mice treated with RAD1901 alone. 12 patient-derived xenograft models were screened to test RAD1901 response in several genetic backgrounds with different ER, PR, and Her2 levels. Full efficacy studies were conducted on PDx models marked with "*" (PDx-1 to PDx-4 and PDx-12), where n=8-10. Screening studies (where n=3) were conducted for other PDx models (PDx-5 to PDx-11), treated with vehicle (negative control) or RAD1901 at a dose of 60 mg / kg (oral, once daily) for 60 days. As Figure 1 As shown, PDx models in which growth is driven by ER and additional drivers (e.g., PR+ and / or Her2+) derive benefit from RAD1901 treatment. RAD1901 effectively inhibits tumor growth in models with ER mutations and / or high Her2 expression levels (PDx), regardless of the tumor's prior treatment history, i.e., treatment naive or treatment with aromatase inhibitors, tamoxifen (tam), chemotherapy (chemo), Her2 inhibitors (Her2i, such as trastuzumab, lapatinib), bevacizumab, fulvestrant and / or rituximab.

[0203] I(A)(ii) The RAD1901-everolimus combination induces more regressions than RAD1901 alone in a xenograft model expressing WT ER

[0204] I(A)(ii)(1) RAD1901-everolimus induces more regressions than RAD1901 alone in MCF-7 xenografts responding to fulvestrant treatment.

[0205] MCF-7 xenograft model

[0206] Two days before cell implantation, Balb / C-Nude mice were inoculated with 0.18 / 90 day-release 17β-estradiol pellets. MCF-7 cells (PR+, Her2-) were harvested and 1×10 7 The cells were implanted subcutaneously in the right axilla of Balb / C-Nude mice. When the tumors reached an average of 200 mm 3 At 4 hr, mice were randomly divided into treatment groups according to tumor volume and treated with the test compound. Each group was treated with RAD1901 (30 mg / kg or 60 mg / kg subject, orally, once daily, to endpoint), everolimus (2.5 mg / kg, orally, once daily, to endpoint) or the specified dose of RAD1901-everolimus combination starting on day 0. The treatment period lasted 28 days.

[0207] MCF7 xenograft mice were treated with vehicle (negative control), RAD1901 (60 mg / kg, oral, once daily), everolimus (2.5 mg / kg, oral), a combination of RAD1901 (30 mg / kg or 60 mg / kg, oral, once daily) and everolimus (2.5 mg / kg, oral), fulvestrant (3 mg / dose, subcutaneous injection, once weekly), or a combination of fulvestrant (3 mg / dose, subcutaneous injection, once weekly) and everolimus (2.5 mg / kg, oral). Tumor size was measured at several time points for 27 days.

[0208] The results are shown in Figure 2A -B. Combination treatment with RAD1901 (60 mg / kg) and everolimus (2.5 mg / kg) again resulted in significant tumor regression, which was superior to treatment with RAD1901, everolimus or fulvestrant alone, or with the combination of fulvestrant and everolimus ( Figure 2B ).

[0209] Figure 2C It was shown that the RAD1901-everolimus combination with 30 mg / kg or 60 mg / kg RAD1901 provided similar effects, but RAD1901 alone at 30 mg / kg was not as effective as RAD1901 alone at 60 mg / kg in inhibiting tumor growth. The results indicate that the RAD1901-everolimus combination with a lower dose of RAD1901 (e.g., 30 mg / kg) is sufficient to maximize the tumor growth inhibition / tumor regression effect in the xenograft model.

[0210] Treatment with the combination of RAD1901 and everolimus was also more effective in reducing ER and PR expression in the MCF7 xenograft model in vivo compared with treatment with RAD1901, everolimus or fulvestrant alone, or the combination of fulvestrant and everolimus ( Fig.11 ); tumors were collected two hours after the last dose).

[0211] I(A(ii)(2) In PDx-11 and PDx-2 models that responded to fulvestrant, RAD1901-everolimus caused more tumor regressions than RAD1901 alone.

[0212] 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, sc, once weekly). PDx-2 and PDx-11 models were treated with a combination of RAD1901 (60 mg / kg, orally, once daily) and everolimus (2.5 mg / kg, orally), RAD1901 (60 mg / kg, orally, once daily), everolimus (2.5 mg / kg, orally) alone, or fulvestrant (3 mg / dose, sc, once weekly) alone.

[0213] In the PDx-11 model, administration of either fulvestrant or everolimus alone significantly inhibited tumor growth, while mice treated with fulvestrant showed better results in terms of tumor growth inhibition. Fulvestrant treatment showed mild tumor regression (Figure 3B). Unexpectedly, administration of RAD1901 alone or in combination with everolimus resulted in significant tumor regression, and the combination achieved even more significant tumor regression in the wild-type ESR1 PDx model (Figure 3B). Figure 3B ).

[0214] In the PDx-2 model, oral administration of RAD1901 alone resulted in superior inhibition of tumor growth compared with fulvestrant alone ( Figure 4A ). In addition, administration of RAD1901 or everolimus alone significantly inhibited tumor growth. Unexpectedly, administration of RAD1901 in combination with everolimus resulted in an even stronger tumor growth inhibitory effect ( Figure 4B ).

[0215] Furthermore, in a PDx-4 model that responded to fulvestrant treatment (1 mg / dose, subcutaneously, once weekly), RAD1901-mediated tumor growth inhibition was maintained for at least two months without treatment when estradiol treatment was continued after the RAD1901 treatment (30 mg / kg, orally, once daily) period. Figure 5 ).

[0216] Therefore, the combination of one or more second therapeutic agents with RAD1901 may be beneficial to patients in inhibiting tumor growth after treatment is completed, especially when the one or more second therapeutic agents (eg, everolimus) may be reduced or delayed due to adverse effects.

[0217] I(A)(iii) In a xenograft model expressing mutant ER (ERαY537S), RAD1901-everolimus induced more regressions than RAD1901 alone

[0218] I(A)(iii)(1) In a PDx-5 model that had little response to fulvestrant treatment, RAD1901-everolimus induced more regressions than RAD1901 alone.

[0219] The PDx-5 model was prepared following a similar protocol as described above for the PDx model. The tumor size of the dosing group was measured twice a week with a vernier caliper, and the volume was calculated using the formula (L×W2)×0.52.

[0220] RAD1901 (60 mg / kg, orally, once daily), everolimus (2.5 mg / kg, orally), and the combination of RAD1901 (60 mg / kg, orally, once daily) and everolimus (2.5 mg / kg) were evaluated for tumor growth inhibition in the mutant ERPDx-5 model (a PDx model with breast cancer tumors from patients with Y537S estrogen receptor mutations, PR+, Her2+, with prior treatment with aromatase inhibitors) using the methods described herein. For tumors expressing certain ERα mutations (e.g., Y537S), combination treatment with RAD1901 and everolimus was more effective in inhibiting tumor growth than treatment with either agent alone ( Figure 6B These PDx models were insensitive to treatment with fulvestrant (3 mg / dose). Combination treatment with RAD1901 and everolimus was more effective in causing tumor regression in the RDX-5 model than treatment with either agent alone ( Figure 6B ).

[0221] Thus, the results show that RAD1901 is an effective endocrine scaffold to enhance tumor growth inhibition by targeted drugs. In addition, RAD1901 showed potent antitumor activity in PDx models derived from patients who had received multiple prior endocrine therapies, including those insensitive to fulvestrant.

[0222] I(A)(iv) Pharmacokinetic evaluation of fulvestrant treatment in non-tumor bearing mice.

[0223] Several doses of fulvestrant were administered to mice, which showed significant dose exposure to the subjects ( Figure 7 ).

[0224] Fulvestrant was administered subcutaneously to nude mice at 1, 3, or 5 mg / dose on Day 1 (D1 Rx) and Day 8 (D8 Rx, n=4 / dose level). Blood was collected at designated time points up to 168 hours after the second dose, centrifuged, and plasma analyzed by liquid chromatography-mass spectrometry.

[0225] I(B) RAD1901 promotes survival in a mouse xenograft model of brain metastasis (MCF-7 intracranial model).

[0226] The potential ability of RAD1901 to cross the blood-brain barrier and inhibit tumor growth was further evaluated using the MCF-7 intracranial tumor xenograft model.

[0227] Female athymic nude mice (Cr1: NU (NCr) -Foxn1nu) were used for tumor xenograft studies. Three days before tumor cell implantation, estrogen pellets (0.36 mg E2, 60-day release (Innovative Research of America, Sarasota, Florida)) were subcutaneously implanted between the shoulder blades of all test animals using a sterile trocar. MCF-7 human breast cancer cells were cultured to mid-logarithmic growth 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 g / mL gentamicin. On the day of tumor cell implantation, cells were trypsinized, pelleted, and 5×10 7个 The cells were resuspended in phosphate-buffered saline at a concentration of 1×10 cells / mL. 6 MCF-7 cells.

[0228] On day 5 after tumor cell implantation (designated study day 1), 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.

[0229] The endpoint was defined as mortality or 3× survival rate in the control group, whichever came first. Treatment tolerance was assessed by body weight measurement and frequent observation for clinical signs of treatment-related adverse reactions. Animals with a weight loss of more than 30% measured once or more than 25% measured three times were humanely euthanized and classified as treatment-related deaths. Acceptable toxicity was defined as a group mean weight loss of less than 20% during the study and no more than one treatment-related death (or 10%) among 10 treated animals. At the end of the study, animals were euthanized by terminal cardiac puncture under isoflurane anesthesia. RAD1901 and fulvestrant concentrations in plasma and tumors were determined by LC-MS / MS.

[0230] Kaplan Meier survival analysis showed that RAD1901 significantly prolonged survival compared with fulvestrant (P < 0.0001; Figure 8 ). No animals in the control or fulvestrant groups survived beyond day 20 and day 34, respectively, whereas 41% (5 / 12) of RAD1901-treated animals survived until the end of the study on day 54.

[0231] The concentration of RAD1901 in plasma was 738±471 ng / mL and in intracranial tumors was 462±105 ng / g, which supports the hypothesis that RAD1901 can effectively cross the blood-brain barrier. In contrast, the concentration of fulvestrant in plasma (21±10 ng / mL) and intracranial tumors (8.3+0.8 ng / g) was significantly lower.

[0232] I(C). Phase 1 study of RAD1901 in the treatment of ER+ advanced breast cancer.

[0233] In a Phase 1 study, safety, tolerability, and pharmacokinetics were evaluated in 44 healthy postmenopausal women. No dose-limiting toxicity was observed, and the maximum tolerated dose (MTD) was not determined. Within the dose range tested, plasma exposure increased proportionally with increasing dose.

[0234] Subjects

[0235] Eight postmenopausal women with advanced breast cancer (ER+ tumors with no less than 1% IHC staining, HER2-negative tumors with an ECOG performance status of 0 or 1) were recruited as subjects for this Phase 1 study. Subjects must have received the following prior treatments:

[0236] No more than 2 prior chemotherapy regimens in the advanced / metastatic setting

[0237] 6 months of prior endocrine therapy and has progressed on prior endocrine therapy

[0238] Patients with untreated or symptomatic CNS metastases or who received prior anticancer therapy within the following time windows were excluded:

[0239] Tamoxifen <14 days before first dose of study treatment

[0240] Fulvestrant < 90 days before first dose of study treatment

[0241] Chemotherapy < 28 days before first dose of study treatment

[0242] LHRH analogue < 12 months before first dose of study treatment

[0243] DLT Standards

[0244] Any grade ≥ 3 non-hematologic toxicity (excluding alopecia and nausea, vomiting, or diarrhea not treated with optimal medication)

[0245] Any grade of hematologic toxicity not less than 3

[0246] Any grade toxicity leading to study drug interruption for >7 days

[0247] The dose-limiting toxicity observation period is from day 1 to day 28 of cycle 1.

[0248] Treatment-emergent adverse events (TEAEs)

[0249] TEAEs were recorded throughout the study. Preliminary data are summarized in Table 12. "n" is the number of subjects with at least one treatment-related AE in a given category (AEs are graded according to Common Criteria for Adverse Events (CTCAE) v4.0), and any patient experiencing multiple situations of the same priority project is only calculated once with the most severe grade. No death or dose-limiting toxicity was observed, and the maximum tolerated dose (MTD) was not determined. Most AEs were Grade 1 or 2. The most common treatment-related AEs were dyspepsia (5 / 8 patients) and nausea (3 / 8 patients). Two severe AEs (SAEs) were observed, one was Grade 3 treatment-related constipation, and the other was shortness of breath (pleural effusion), which was unrelated to treatment.

[0250] Subjects who were heavily pretreated in this Phase 1 study included subjects who had previously been treated with a variety of endocrine and targeted agents (e.g., CDK4 / 6, PI3K, and mTOR inhibitors). After treatment with RAD1901 for up to 6 months at a dose of 200 mg (oral, once daily) and up to 2 months at a dose of 400 mg (oral, once daily), no dose-limiting toxicity was observed. Therefore, RAD1901 shows the potential for treating ER+ advanced breast cancer, especially in subjects who had previously been treated with endocrine and / or targeted agents such as CDK4 / 6, PI3K, and mTOR inhibitors.

[0251] Example II. RAD1901 preferentially accumulates in tumors and can be delivered to the brain.

[0252] LC-MS / MS was used to further evaluate the RAD1901 concentration in the plasma and tumor of the MCF-7 xenograft described in Example 1 (A) (i). At the end of the study, at 60 mg / kg dosage level, the concentration of RAD1901 in plasma was 344 ± 117 ng / mL, and the concentration in the tumor was 11,118 ± 3801 ng / mL. Similar tumors and plasma ratios were also observed at lower dosage levels where the tumor concentration was about 20-30 times the plasma concentration. The RAD1901 levels in the plasma, tumors, and brain of mice treated for 40 days are summarized in Table 1. A significant amount of RAD1901 is delivered to the brain of mice treated (e.g., see B / P ratio (RAD1901 concentration in the brain / RAD1901 concentration in plasma)), which shows that RAD1901 can pass through the blood-brain barrier (BBB). Unexpectedly, RAD1901 preferentially accumulates in tumors. See, e.g., the T / P (RAD1901 concentration in tumor / RAD1901 concentration in plasma) ratio shown in Table 1.

[0253] Table 1

[0254] RAD1901 levels in plasma, tumors, and brains of mice implanted with MCF7 cells after 40 days of treatment.

[0255]

[0256] *BLQ: Below the limit of quantification

[0257] Example III. RAD1901 inhibits the ER pathway and degrades ER.

[0258] III(A). RAD1901 reduces ER binding in the uterus and pituitary in healthy postmenopausal female subjects.

[0259] Subjects had a duration of amenorrhea of ​​at least 12 months and serum FSH consistent with menopause. Subjects were 40-75 years old with a BMI of 18.0-30 kg / m 2 Subjects had an intact uterus. Subjects were excluded if they had evidence of clinically relevant pathology, were at increased risk of stroke, or had a history of venous thrombotic events, or if they were using concomitant medications less than 14 days before entering the clinical study center (up to 3 days of prior use of paracetamol was allowed).

[0260] FES-PET was performed at baseline and after 6 days of exposure to RAD1901 to assess ER binding in the uterus. At the 200 mg (7 subjects) and 500 mg (6 subjects) dose levels, RAD1901 occupied 83% and 92% of the ER in the uterus, respectively.

[0261] After treatment with 200 mg or 500 mg (oral, once daily, for 6 days) of RAD1901, FES-PET imaging showed a significant decrease in the binding of labeled estradiol to the uterus and pituitary.

[0262] Due to high ER expression, the uterus showed strong FES-PET signal at baseline before RAD1901 treatment ( Fig. 9A , Baseline transverse view of the uterine FES-PET scan of Subject 3 treated with the 200 mg dose level; Fig. 9B , baseline sagittal and transverse views, respectively, of the uterine FES-PET scan of Subject 7 treated with the 500 mg dose level). However, when the scan was performed 4 hours after dosing on Day 6 of the study, the uterus was barely visible (at or near the background FES-PET signal level ( Fig. 9A , transverse view of subject 3 uterine scan on day 6; and FIG. 9B , sagittal and transverse views, respectively, of subject 7 uterine scan on day 6). These data are consistent with ER degradation and / or competition for receptor binding. Fig. 9A and 9B Also included was a CT scan of the uterus from a FES-PET scan, which showed the presence of the uterus before and after RAD1901 treatment.

[0263] The FES-PET uterine scans were further quantified to show changes in ER-binding from baseline after dosing in 7 subjects ( Fig. 9C ), which show subjects 1-3 and subjects 4-7 as examples of the 200 mg and 500 mg dose groups, respectively. RAD1901 showed stable ER binding at a lower dose level (200 mg).

[0264] Fig. 10A-B shows representative images of FES-PET scans of the uterus (A) and pituitary (B) before (baseline) and after (post-treatment) treatment with RAD1901 at 500 mg (oral, once daily) after 6 days. Fig. 10A FES-PET scans of the uterus are shown as follows: (a) lateral section; (b) longitudinal section; and (c) longitudinal section.

[0265] The FES-PET dose scans of the subjects' uterus and pituitary gland after administration showed that in the uterus ( Fig. 10A , after treatment) and pituitary ( Fig. 10B , after treatment), there was no obvious ER binding signal.

[0266] Thus, the results show that RAD1901 effectively binds to the ER in humans at doses of 200 mg and 500 mg (oral, once daily) after 6 days.

[0267] Standardized uptake values ​​(SUV) were calculated for uterus, muscle, and bone, and are summarized in Tables 2 and 3 for 200 mg and 500 mg (oral, once daily) of RAD1901 treatment, respectively. The uterine signal after administration was very close to the level from "non-target tissues", which showed that FES-PET uptake after RAD1901 treatment was completely attenuated. In tissues that do not significantly express estrogen receptors, almost no changes were observed in PET scans before and after treatment.

[0268] Table 2

[0269] Uterine, muscle, and bone SUV in human subjects treated with a 200 mg dose (oral, once a day) for 6 days

[0270]

[0271] Table 3

[0272] SUV of uterus, muscle and bone in human subjects (n=4) treated with a 500 mg dose (oral, 1 time / day) for 6 days.

[0273]

[0274] Therefore, RAD1901 or its salt or solvate (e.g., hydrate thereof) can be used to treat cancers and / or tumor cells (e.g., breast cancer, uterine cancer, and ovarian cancer) with ER overexpression without negative effects on other organs (e.g., bone, muscle). RAD1901 or its salt or solvate (e.g., hydrate) may be particularly suitable for treating metastatic cancers and / or tumors with ER overexpression in other organs, such as primary breast cancer, uterine cancer, and / or ovarian cancer that metastasize to other organs (e.g., bone, muscle), to treat breast cancer, uterine cancer, and / or ovarian cancer lesions in other organs (e.g., bone, muscle) without negative effects on the organs.

[0275] III(B). RAD1901 reduces ER expression and inhibits the ER pathway.

[0276] IIIIII(B)(i)(1) The RAD1901-everolimus combination was more effective in reducing ER and PR expression in the MCF7 xenograft model than treatment with RAD1901, everolimus or fulvestrant alone or the fulvestrant-everolimus combination.

[0277] Treatment with the combination of RAD1901 and everolimus was also more effective in reducing ER and PR expression in vivo in the MCF7 xenograft model (as described in Example 1(A)(ii)) compared to treatment with RAD1901, everolimus, or fulvestrant alone, or treatment with the combination of fulvestrant and everolimus ( Fig.11 ); tumors were collected two hours after the last dose).

[0278] III(B)(i)(2) Comparison of RAD1901 and fulvestrant in MCF7 and T47D cell lines.

[0279] MCF-7 and T47D cell lines were used to compare the effects of different concentrations of RAD1901 and fulvestrant, both of which were human breast cancer cell lines. The different concentrations were 0.01 μM, 0.1 μM and 1 μM ( Fig. 12A For MCF-7 cell line analysis; Fig. 12B The following three ER target genes were used as markers: progesterone receptor (PgR), growth regulation by estrogen in breast cancer 1 (GREB1), and trefoil factor 1 (TFF1). RAD1901 caused almost complete ER degradation and inhibited ER signaling ( Fig. 12A-B). In particular, for the MCF-7 cell line, when administered at the same concentration, fulvestrant showed comparable or even slightly higher efficacy in inhibiting ER signaling. Unexpectedly, RAD1901 was comparable or more effective than fulvestrant in inhibiting tumor growth and causing tumor regression, as disclosed in Examples 1(A) and 1(B).

[0280] III(B)(i)(3) RAD1901 treatment resulted in ER degradation and abolished ER signaling in the MCF-7 xenograft model II described in Example I(A)(ii)(1) above.

[0281] RAD1901 treatment leads to ER degradation in vivo ( Fig.13A and 13B , Student's t-test: *p value < 0.05, **p value < 0.01) and inhibited ER signaling in vivo ( Fig.13A and 13C , Student's t test: *p value < 0.05, **p value < 0.01).

[0282] Tumors harvested from MCF-7 xenografts 2 hours after the final dose of RAD1901 (30 mg / kg, 60 mg / kg, oral, once daily) or fulvestrant (3 mg / dose, subcutaneous injection, once weekly) showed significant reductions in ER and PR expression ( Fig.13A -B). Tumors harvested from MCF-7 xenografts 8 hours after the final dose of fulvestrant treatment showed different PR and ER expression. However, tumors harvested from MCF-7 xenografts 8 hours after the final dose of RAD1901 treatment showed decreased PR and ER expression ( Fig.13A and 13C ).

[0283] 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, po, once daily) showed a rapid decrease in PR expression ( Fig.14A -C). Tumors harvested from MCF-7 xenografts 4 or 24 hours after the seventh dose of RAD1901 (30 mg / kg, 60 mg / kg, or 90 mg / kg, po, once daily) showed stable and consistent inhibition of ER signaling ( Fig. 14B Quantitative analysis of Western blot analysis of tumors harvested from MCF-7 xenografts at several time points during RAD1901 (30 mg / kg, 60 mg / kg, or 90 mg / kg, orally, once daily) treatment showed a dose-dependent decrease in PR ( Fig. 14C ).

[0284] RAD1901 treatment resulted in a rapid decrease in proliferation in the MCF-7 xenograft model. For example, tumor sections collected from the MCF-7 xenograft model 8 hours after a single dose of RAD1901 (90 mg / kg, oral, once daily) and 24 hours after the fourth dose of RAD1901 (90 mg / kg, oral, once daily) were stained, which showed a rapid decrease in the proliferation marker Ki67 ( Fig.15A and 15B ).

[0285] These results indicate that RAD1901 treatment leads to ER degradation and inhibits ER signaling in vivo in WT ER xenografts.

[0286] III(B)(i)(4) RAD1901 treatment results in ER degradation and abrogation of ER signaling in the PDx-4 model described in Example I(A)(ii).

[0287] RAD1901 treatment resulted in a rapid decrease in proliferation in the PDx-4 model. For example, tumor sections collected four hours after the last dose on the last day of the 56-day efficacy study from PDx-4 models treated with RAD1901 (30, 60, or 120 mg / kg, orally, once daily) or fulvestrant (1 mg / animal, once weekly) showed a rapid decrease in the proliferation marker Ki67 compared to the PDx-4 model treated with fulvestrant ( Fig.16 ).

[0288] These results indicate that RAD1901 treatment leads to ER degradation and inhibits ER signaling in vivo in WT ER xenografts.

[0289] III(B)(ii) RAD1901 treatment results in reduced ER signaling in the mutant ER xenograft model (PDx-5) described in Example I(A)(iii)(1) above.

[0290] Tumors were harvested at designated time points after the last day of dosing (unless otherwise stated) and homogenized in RIPA buffer with protease and phosphatase inhibitors using Tissuelyser (Qiagen). Equal amounts of protein were separated by MW, transferred to nitrocellulose membranes, and blotted with the following antibodies as described in the Materials and Methods section: progesterone receptor (PR, Cell Signaling Technologies; 3153).

[0291] The bands were quantified using 1D Quant software (GE), and the PR (Allred score) obtained from the PDx-5 model as described in Example 1(A)(iii)(1) is shown in Fig.17 Fulvestrant had little effect on PR expression, while RAD1901 had no significant effect at 60 mg / kg and 120 mg / kg (oral, once daily, Fig.17 ) doses showed efficacy.

[0292] These results suggest that RAD1901 is more effective than fulvestrant in inhibiting tumor growth in tumors expressing certain ERα mutations (e.g., Y537S) and is particularly effective in inhibiting the growth of tumors that have little response to fulvestrant treatment (e.g., at a dose of 3 mg / dose, subcutaneously, once weekly, Fig. 6A In addition, for tumors that did not respond well to fulvestrant therapy (e.g., PDx-5), RAD1901 was effective in reducing PR expression in vivo, whereas fulvestrant was ineffective ( Fig.17 ).

[0293] Example IV Effects of RAD1901 Treatment on Uterine Tissue and / or BMD

[0294] IV(A(1)): RAD1901 antagonizes the stimulation of uterine tissue by estradiol.

[0295] The effects of RAD1901 on the uterus were investigated by evaluating changes in uterine weight, histology, and C3 gene expression in immature rats. Results from a representative study are presented in Fig.18A -D is shown.

[0296] Assessing uterine activity

[0297] Sprague-Dawley rat pups were weaned at 19 days of age, randomly divided into groups (n=4), and administered for 3 consecutive days with vehicle (aqueous methylcellulose), E2 (0.01 mg / kg), raloxifene (3 mg / kg), tamoxifen (1 mg / kg), RAD1901 (0.3 to 100 mg / kg) alone, or a combination of RAD1901 (0.01 to 10 mg / kg) and E2 (0.01 mg / kg), subcutaneously or orally (see reagents above), once daily. 24 hours after the final dose, all animals were euthanized by inhalation of carbon dioxide. The body weight and uterine wet weight of each animal were recorded. Similar determinations were also performed using RAD1901 (0.03 to 100 mg / kg) in rats and mice (Charles River Laboratories, Montreal, Quebec).

[0298] Fresh uterine tissue from each rat was fixed in 4% paraformaldehyde, dehydrated with ethanol and embedded in JB4 plastic resin. Sections were cut into 8 μm and stained with 0.1% toluidine blue O. The thickness of the endometrial epithelium was measured using a Zeiss Axioskop 40 microscope using the Spot Advanced program; the average of 9 measurements per sample was calculated.

[0299] Uterine complement component 3 (C3) gene expression

[0300] To determine the relative expression level of C3 in treated uterine tissue, RNA was extracted from the remaining tissue using the Micro to Midi Total RNA Purification Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA was quantified, and equal amounts of RNA were reverse transcribed using the High Capacity cDNA Archiving Kit (Applied Biosystems, Foster City, CA).

[0301] Quantitative PCR was performed using an ABI Prism 7300 system (Applied Biosystems). PCR was performed using a Taqman universal master mixer with probe sets for C3 and for 18S ribosomal RNA as a reference gene. Thermal cycling conditions included an initial denaturation step of 10 minutes at 95°C and then 40 cycles of 15 seconds at 95°C and 1 minute at 60°C.

[0302] Relative gene expression was determined by normalizing each sample to an endogenous control (18S) and comparing with a calibrator (vehicle). Relative gene expression was determined using the following formula: 2-ΔΔCt (where Ct = cycle threshold or the number of cycles at which the PCR product was first detected, ΔCt = normalized sample value, ΔΔCt = normalized difference between the dosing subject and the vehicle). Gene expression measurements were repeated 5 times for each dose in each study.

[0303] 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 to vehicle alone, whereas treatment with RAD1901 at a dose range of 0.3-100 mg / kg did not significantly affect uterine wet weight ( Fig.18A The data shown ( Fig.18A ) are mean values ​​(±SEM); n=4 rats per group; P-vehicle: *<0.05; P-E2: Furthermore, when co-administered with E2 (0.01 mg / kg), RAD1901 antagonized E2-mediated uterine stimulation in a dose-dependent manner, exhibiting significant inhibition of uterine activity at doses of 0.1 mg / kg and higher, and completely inhibiting uterine activity at a dose of 3 mg / kg. 50 Similar results were obtained in mice in which RAD1901 doses from 0.03 to 100 mg / kg had no effect on uterine wet weight or epithelial thickness (data not shown).

[0304] Treatment-dependent changes in uterine tissue were further investigated by quantitative microhistology. After treatment with E2 at 0.01 and 0.3 mg / kg, there was a statistically significant increase in endometrial epithelial thickness ( Fig.18B Significant increases in epithelial thickness were also observed following treatment with tamoxifen (1 mg / kg) or raloxifene (3 mg / kg). In contrast, RAD1901 treatment did not increase endometrial epithelial thickness at the highest evaluated dose of 100 mg / kg. Fig.18C Representative images of the endometrial epithelium are shown in .

[0305] Consistent with the changes in uterine weight and endometrial epithelial thickness, E2, tamoxifen, and raloxifene all significantly increased the expression of the estrogen-regulated complement gene C3 ( Fig.18D ). In contrast, RAD1901 did not increase C3 gene expression at any dose tested (0.3 to 100 mg / kg). Moreover, RAD1901 at 1, 3, and 10 mg / kg significantly inhibited E2-stimulated C3 gene expression.

[0306] RAD1901 does not stimulate the uterus of immature female rats

[0307] Immature female rats were administered vehicle (VEH), estradiol (E2), raloxifene (RAL), tamoxifen (TAM), RAD1901, or RAD1901+E2 (oral, once daily) for 3 consecutive days. Uterine wet weights were measured. Data shown (Figure 18) are mean values ​​(± SEM); n = 4 rats per group; P-vehicle: * < 0.05; P-E2:

[0308] Example II(A)(2). RAD1901 treatment prevents bone loss in ovariectomized rats

[0309] The bone-specific effects of RAD1901 were examined in ovariectomized rats.

[0310] As a model of postmenopausal bone loss, ovariectomy was performed on anesthetized adult female Sprague-Dawley rats, with sham surgery as control. After surgery, vehicle, E2 (0.01 mg / kg) or RAD1901 (0.1, 0.3, 1, 3 mg / kg) were used to treat ovariectomized rats for 4 weeks (once a day, as described above), 20 animals per group. Animals in the sham operation group were treated with vehicle. 24 hours after the final dose, all animals were euthanized by inhalation of carbon dioxide. Bone mineral density was assessed at baseline using PIXImus dual emission x-ray absorptiometry, and reassessed after treatment for 4 weeks.

[0311] At necropsy, the left femur of each animal was removed, the soft tissue was dissected away, and stored in 70% ethanol before analysis. A detailed qualitative and quantitative 3-D evaluation was performed using a micro-CT40 system (Scanco Systems, Wayne, Pennsylvania). For each sample, 250 image slices of the distal femoral metaphysis were obtained. Morphometric parameters were determined using a direct 3D method in a pre-selected analysis area. Parameters measured in trabecular bone included bone volume density, bone surface density, trabecular number, trabecular thickness, trabecular spacing, connection density, and apparent bone density.

[0312] Following ovariectomy, untreated (vehicle control) rats experienced a decrease in bone mineral density throughout the femur and lumbar spine compared to baseline (Table 4). E2 treatment was associated with prevention of bone loss in both the femur and spine. RAD1901 treatment produced a dose-dependent and statistically significant inhibition of ovariectomy-induced bone loss (data shown for the 3 mg / kg treatment group). Bone mineral density in rats treated with RAD1901 was intact at doses from 0.1 mg / kg to 3 mg / kg, with no statistically significant differences from the E2 treatment group.

[0313] Table 4

[0314] Effect of RAD1901 on BMD in ovariectomized rats. a

[0315]

[0316] aAdult female rats were sham-operated or ovariectomized before initiation of treatment with vehicle, E2 (0.01 mg / kg) or RAD1901 (3 mg / kg) once daily (n=20 / treatment group). BMD was measured by dual emission x-ray absorptiometry at baseline and after 4 weeks of treatment. Data are expressed as mean ± SD. *P < 0.05 relative to the corresponding OVX + vehicle control. BMD is bone mineral density; E2 is beta estradiol: OVX is ovariectomy: Veh is vehicle.

[0317] Micro-CT analysis of the distal femur (Table 5) confirmed that ovariectomy induced significant changes in multiple key microstructural parameters compared to sham-operated animals. These changes were consistent with osteopenia and included decreased bone volume, decreased trabecular number, thickness, and density, and increased trabecular separation. Consistent with the preservation of bone mineral density observed after treatment with RAD1901, significant preservation of trabecular architecture was observed in key microstructural parameters (Table 5)

[0318] Table 5

[0319] Effects of RAD1901 on femoral microstructure in ovariectomized rats a

[0320]

[0321] a Adult female rats were sham-operated or ovariectomized before initiation of treatment with vehicle, E2 (0.01 mg / kg) or RAD1901 (3 mg / kg) once daily (n=20 treatment groups). Bone microarchitecture was evaluated using microcomputed tomography after 4 weeks. Data are expressed as mean ± SD. *P < 0.05 relative to the corresponding OVX + vehicle control. ABD is apparent bone density; BVTV is bone volume density; ConnD is connection density; E2 is beta estradiol; OVX is ovariectomy; TbN is trabecular number; TbTh is trabecular thickness; TbSp is trabecular spacing; Veh is vehicle.

[0322] Example IV(B): Phase 1 Dose Escalation Study of RAD10 in Healthy Postmenopausal Women

[0323] In the Phase 1 study, safety, tolerability, and pharmacokinetics were evaluated in 44 healthy postmenopausal women. No dose-limiting toxicity (DLT) was observed and the tolerable dose (MTD) was not determined. Within the dose range tested, plasma exposure increased proportionally with increasing dose.

[0324] Subjects

[0325] 44 healthy postmenopausal women were recruited as subjects for the Phase 1 study. The subjects had been amenorrhea for at least 12 months and their serum FSH was consistent with menopause. The subjects were 40-75 years old and had a BMI of 18.0-30 kg / m 2 Subjects with evidence of clinically relevant pathology, increased risk of stroke or a history of venous thrombotic events, or use of concomitant medications less than 14 days before admission to the clinical study center (paracetamol use was allowed up to 3 days prior) were excluded.

[0326] Drug administration

[0327] Subjects were treated for 7 days with placebo or at least one dose (oral, once daily) after a small breakfast at doses of 200 mg, 500 mg, 750 mg, and 1000 mg. The key baseline demographics of the 44 healthy postmenopausal women enrolled in the Phase 1 study are summarized in Table 6.

[0328] Table 6

[0329] Key Baseline Demographics of the Phase 1 Dose-Escalation Study of RAD1901

[0330]

[0331] Treatment-emergent adverse events (TEAEs)

[0332] TEAEs were recorded, and the most frequent (>10% of patients in the total active group with any relevant TEAE) adverse events (AEs) are summarized in Table 7, "n" is the number of subjects with at least one treatment-related AE in a given category (AEs were graded according to Common Criteria for Adverse Events (CTCAE) v4.0), and any patient experiencing multiple instances of the same priority item was counted only once with the most severe grade. No dose-limiting toxicity was observed, and the maximum tolerated dose (MTD) was not determined.

[0333] Table 7

[0334] Most frequent (>10%) treatment-related AEs in the RAD1901 Phase 1 dose-escalation study

[0335]

[0336] Pharmacokinetic evaluation

[0337] A series of blood samples were collected during the study for analysis of RAD1901 in plasma. Each 5 mL blood sample was collected by indwelling IV catheter or direct venipuncture into a tube containing K3-EDTA as an anticoagulant. Steady state was reached on the 5th day of treatment. The geometric mean (Geo-Mean) plasma concentration-time curve of RAD1901 was evaluated. As an example, the plasma pharmacokinetic results of the group treated with RAD1901 (200, 500, 750 or 1000 mg) on ​​the 7th day of the study (N=35) are provided in Tables 8 and Fig.19 Middle. Median t 1 / 2 Between 37.5 and 42.3 hours (Table 8). After multiple doses of RAD1901, the median t max Between 3-4 hours after administration.

[0338] Table 8

[0339] Pharmacokinetic Parameters of RAD1901 in Phase 1 Dose Escalation Study (Day 7)

[0340]

[0341] Example V(A)-1. Modeling of RAD1901-ERα binding using selected published ER structures.

[0342] Unless otherwise specified, when showing a structure by its stick model, color each end of a bond the same color as the atom it is attached to, with gray being carbon, red being oxygen, blue being nitrogen, and white being hydrogen.

[0343] Through careful evaluation, 14 published structures (i.e., models) of the ERα ligand binding domain (LBD) in complex with several ER ligands were selected from 96 published models. One of the 14 models is 3ERT (human ERα LBD bound to 4-hydroxytamoxifen (OHT)). OHT is an active metabolite of tamoxifen and a first-generation SERM that acts as an antagonist in breast tissue.

[0344] In 3ERT( Fig. 20 and 21 ), the ERα binding site adopts a three-layer "helical sandwich" (H3), a hydrophobic pocket consisting of helix 3 (H3), helix 5 (H5), and helix 11 (H11). Fig. 20 ). Fig.21The dashed boxes in the figure indicate the binding site and residues within the binding site that are important or affected by OHT binding. OHT acts as an antagonist by displacing H12 into the site where the LXXLL coactivator binds. OHT occupies the space normally filled by L540 and modifies the conformation of four residues (G521, H524, L525, and M528) on the C-terminal end of helix 11. OHT also forms a salt bridge with D351, resulting in charge neutralization.

[0345] Thirteen additional ERα LBD-ER ligand models were compared with 3ERT. The differences in their residue configurations are summarized in Table 10. Fourteen models ( Fig. 22 ) showed that these structures differed significantly at residues E380, M421, G521, M522, H524, Y526, S527, M528, P535, Y537, L540 and various combinations thereof.

[0346] Table 10

[0347] Differences in residue configurations between the ER-α LBD-antagonist complex and 3ERT

[0348]

[0349] The root mean square deviation (RMSD) calculations for any pair of models for each of the fourteen models are summarized in Table 11. Table 11 shows that the RMSD of all fourteen models is Analysis using conditional formats showed that 1R5K and 3UUC were the least similar to the other models (analysis not shown). Therefore, 1R5K and 3UUC were considered as unique and independent structural groups.

[0350] Table 11

[0351] Structural overlap of the ER-α LBD-antagonist complex was evaluated by RMSD calculations:

[0352]

[0353] The ERα residues bound by ligand in the fourteen models are summarized in Table 12. Table 12 also shows the ECs in the ERα LBD-antagonist complex 50Among the fourteen models, thirteen models showed hydrogen-bonding interactions between the ligand and E353; twelve models showed π interactions between the ligand and F404; five models showed hydrogen-bonding interactions between the ligand and D351; six models showed hydrogen-bonding interactions between the ligand and H524; four models showed hydrogen-bonding interactions between the ligand and R394; and one model (3UUC) showed interactions between the ligand and T347.

[0354] Table 12

[0355] Analysis of ligand binding in the ER-α LBD-antagonist complex

[0356]

[0357] Each of the fourteen models was docked to a random library of 1000 compounds plus ligands, (the models were published with known antagonists) to determine whether the model could recognize and prioritize known antagonists. If the model could recognize known antagonists, it was determined that the model could predict the configuration of its own published ligand. EF was then calculated. 50 The model strength was quantified to see how much better it was than a random choice. RAD1901 was docked in the selected model (e.g., Figures 23A&B-27A&B). The docking scores of the disclosed ligands and RAD1901 in the model were determined. EC 50 Visual inspection of RAD1901 showed that it "complies" with the interactions shown with the disclosed ligands in 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. No steric clashes were found. In certain embodiments, such as in 1R5k and 2BJ4, RAD1901 had a higher docking score than the disclosed ligands.

[0358] Table 13 summarizes the evaluation results of nine models (1ERR, 3ERT, 3UCC, 2IOK, 1R5K, 1JJ0, 2JFA, 2BJ4, and 2OUZ).

[0359] Table 13

[0360] Model evaluation of RAD1901 docking

[0361]

[0362] 1ERR and 3ERT could not predict the correct configuration of their crystallization ligands. RAD1901 was not docked in 3UCC. The tetrahydronaphthylamine in 2IOK-RAD1901 was bound in an unconventional manner.

[0363] The major differences between models 1R5K, 1SJ0, 2JFA, 2BJ4 and 2OUZ are the residues in the C-terminus of helix 11 (G521-M528).

[0364] Fig.23A & B show the modeling of RAD1901-1R5K (A) and GW5-1R5K (B). RAD1901 binds to E353, R394, and L536 using hydrogen bond interactions; and binds to F404 using p-interactions.

[0365] Fig.24A & B show the modeling of RAD1901-1SJ0 (A) and E4D-1SJ0 (A). RAD1901 binds to E353 and D351 using hydrogen bonds; and binds to F404 using p-interactions.

[0366] Fig.25A & B show the modeling of RAD1901-2JFA (A) and RAL-2JFA (B). RAD1901 binds to F404 using p-interaction.

[0367] Fig.26A & B show the modeling of RAD1901-2BJ4 (A) and OHT-2BJ4 (B). RAD1901 binds to E353 and R394 using hydrogen bond interactions; and binds to F404 using p-interactions.

[0368] Fig.27A & B show the modeling of RAD1901-2IOK (A) and IOK-2IOK (B). RAD1901 binds to E353, R394, and D351 using hydrogen bond interactions; and binds to F404 using p-interactions.

[0369] The public ligand in the model has the following structure:

[0370]

[0371] GW5, (2E)-3-{4-[(1E)-1,2-diphenylbut-1-enyl]phenyl}acrylic acid

[0372]

[0373] E4D, (2S,3R)-2-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-2,3-dihydro-3-(4-hydroxyphenyl)benzo[B][1,4]oxathiin-6-ol

[0374]

[0375] OHT, 4-hydroxytamoxifen

[0376]

[0377] IOK, N-[(1R)-3-(4-hydroxyphenyl)-1-methylpropyl]-2-(2-phenyl-1H-indol-3-yl)acetamide

[0378] Example V(A)-2. Induced Fit Docking (IFD) of ERα with RAD1901 and Fulvestrant

[0379] The binding conformation of RAD1901 in ERα was further optimized by IFD analysis of five ERα crystal structures: 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. The IFD analysis takes into account the flexibility of the receptor to adapt to its correct binding conformation (after ligand binding).

[0380] A library of different conformations of each ligand (eg RAD1901 and fulvestrant) was generated by finding local minima as a function of rotation about a rotatable bond. The library for RAD1901 had 25 different conformations.

[0381] Five ERα crystal structures were prepared and minimized. The ERα binding pocket was defined using the corresponding ligands in the published X-ray structures.

[0382] The RAD1901 conformation is docked into the prepared ERα structure, wherein they are made to induce side chain or main chain movements to residues located in the binding pocket. These movements enable ERα to change its binding site so that it conforms more closely to the shape and binding mode of the RAD1901 conformation. In some embodiments, a small amount of main chain relaxation and significant side chain conformational changes in the receptor structure are allowed in the IFD analysis.

[0383] An empirical scoring function is used to approximate the ligand binding free energy to provide a docking score or G score. The G score is also called the GlideScore, which can be used interchangeably with the docking score in this embodiment. The docking score is an estimate of the binding affinity. Therefore, the lower the value of the docking score, the "better" the ligand binds to its receptor. A docking score of -13 to -14 corresponds to a very good binding interaction.

[0384] The RAD1901 conformations obtained by IFD analysis using 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ were superimposed to show their differences ( Figure 28-30A &B, shown as a rod model). All bonds in each RAD1901 conformation are Fig.28 , 29 The same colors are used in 30A.

[0385] The conformations of RAD1901 obtained from IFD analysis of 1R5K (blue) and 2OUZ (yellow) have the N-benzyl-N-ethylaniline group of RAD1901 on the front side ( Fig.28 ). The RAD1901 conformation obtained by IFD analysis using 2BJ4 (green) and 2JFA (pink) has the N-benzyl-N-ethylaniline group of RAD1901 on the back ( Fig.29 ). The RAD1901 conformations obtained by IFD analysis using 2BJ4 (green), 2JFA (pink), and 1SJ0 (brown) are compared with the conformations shown by their superposition ( Fig. 30A and 30B The RAD1901 IFD docking scores are summarized in Table 14.

[0386] Table 14

[0387] Induced fit docking scores of RAD1901 with 1R5K, 1SJ0, 2IFA, 2BJ4 and 2OUz

[0388] ER-α crystal structure RAD1901IFD docking score 1R5K -14.1 1SJO -13.1 2JFA -13.9 2BJ4 -13.8 2U -13.4

[0389] IFD of RAD1901 using 2BJ4 showed hydrogen bonding interactions with E353 and D351 and π-interaction with F404 ( Figures 31A-31C ). Fig.31A Regions within the binding site that are suitable for hydrogen bond acceptor groups (red), hydrogen bond donor groups (blue), and hydrophobic groups (yellow) are shown. Fig.31A and 31B In the middle, the light blue is the carbon of RAD1901. Figures 32A-32C Shown is the protein-surface interaction of the IFD of RAD1901 using 2BJ4. Fig.32A and 32B is a front view, Fig.32C This is a side view. The molecular surface of RAD1901 is Fig.32A In the middle is blue, and in Fig.32C The middle color is green. Fig.32B and 32C is an electrostatic representation of the solvent accessible surface of ERα, where red represents electronegativity and blue represents electronpositivity.

[0390] A similar IFD analysis of fulvestrant was performed using 2BJ4 as described above. The fulvestrant-2BJ4 IFD resulted in a G score of -14.945 and showed hydrogen bonding interactions with E353, Y526, and H524 and a π-interaction with F404 ( Figures 33A-33C ). Fig.33AThe regions within the binding site that are suitable for hydrogen bond acceptor groups (red), hydrogen bond donor groups (blue), and hydrophobic groups (yellow) are shown. Fig.33A In the middle, the light blue is the carbon of RAD1901.

[0391] Fig.34A and 34B RAD1901 and fulvestrant docked in 2BJ4 by IFD are shown, both of which have π-interactions with F404 and hydrogen-bonded interactions with E353. In addition, RAD1901 has hydrogen-bonded interactions with D351 (blue indicates the surface of the RAD1901 molecule, Fig.34B ), while fulvestrant has hydrogen bond interactions with Y526 and H524. Fig.35A and 35B A superposition of 2BJ4 docked with RAD1901 and fulvestrant is shown in FIG. Fig.35A In the figure, green represents the molecular surface of Fulvestrant, and blue represents the molecular surface of RAD1901. Fig.35B In the figure, the brown structure is fulvestrant and the blue structure is RAD1901.

[0392] Example Example V(A)-3. Modeling evaluation of selected ERα mutations.

[0393] Several ERa mutations were evaluated for their effects on the C-terminal ligand binding domain. The specific ERa mutations evaluated were the Y537X mutant, where X is S, N, or C; D538G; and S463P.

[0394] Y537 is located in helix 12. It may regulate ligand binding, homodimerization, and DNA binding upon phosphorylation and may allow ERα to escape phosphorylation-mediated control and provide cells with a potential selective tumorigenic advantage. In addition, it may induce conformational changes that render the receptor constitutively active.

[0395] The Y537S mutation favors a closed pocket conformation that is transcriptionally active, regardless of whether it is occupied by a ligand. A closed but unoccupied pocket could explain the constitutive activity of ERα (Carlson et al., Biochemistry 36: 14897-14905 (1997)). Ser537 forms a hydrogen bond interaction with Asp351, resulting in a conformational change in the helix 11-12 loop and causing Leu536 to be buried in a position that is inaccessible to the solvent. This may promote the constitutive activity of the Y537S mutant protein. The Y537S surface mutation has no effect on the structure of the LBD pocket.

[0396] Y537N is commonly found in ERα-negative metastatic breast cancer. Mutations at this site may free ERα from phosphorylation-mediated control and provide cells with a potential selective tumorigenic advantage. Specifically, the Y537N substitution induces a conformational change in ERα that mimics hormone binding, does not affect the receptor's ability to dimerize, but provides a constitutive transactivation function for the receptor (Zhang et al., Cancer Res 57: 1244-1249 (1997)).

[0397] Y537C has a similar effect to Y537N.

[0398] D538G can change the entire energy landscape by stabilizing both active and inactive conformations, but prefers the active conformation. This may cause this mutant to be constitutively active in the absence of hormones, 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)).

[0399] None of these mutations are expected to affect the ligand binding domain nor specifically block RAD1901 binding. Y537 and D538 may cause conformational changes that lead to constitutive receptor activation independent of ligand binding.

[0400] Example V(B). In vitro binding assay of wild-type and LBD mutant ERα constructs with RAD1901 and other compounds

[0401] In vitro binding assays of wild-type (WT) and LBD mutant ERα constructs with RAD1901 showed that RAD1901 bound to mutant ERα with similar affinity as that to WT ERα.

[0402] Constructs of WT and LBD mutant ERα were made by expressing and purifying the corresponding LBD residues 302–552 with an N-terminal thioredoxin and 6xHis tag cleaved by TEV protease.

[0403] Binding of test compounds (RAD1901, fulvestrant, bazedoxifene, raloxifene, tamoxifen, and AZD9496) to ERα was determined using fluorescence polarization (FP) using 2 nM fluorone, 100 nM WT or LBD mutant ERα constructs according to the manufacturer's instructions (Polar Screen, Invitrogen). Each group was tested twice, and one test compound was tested to determine the IC of different ERα constructs. 50 ( Fig.36 RAD1901 binding assay is shown).

[0404] As mentioned above, the foregoing is intended to illustrate several embodiments of the present invention only. The specific modifications discussed above should not be construed as limiting the scope of the present invention. It is apparent to those skilled in the art that various equivalents, variations and modifications can be made without departing from the scope of the present invention, and it should be understood that such equivalent embodiments are also included herein. All references cited herein are incorporated herein by reference, as if fully recorded herein.

Claims

1. Use of a combination of everolimus and RAD1901 or a salt thereof in the preparation of a medicament for treating a tumor in a subject with mutant estrogen receptor α-positive cancer, wherein: The RAD1901 has the following structure: wherein the cancer is breast cancer, is a metastatic cancer, and is positive for a mutant estrogen receptor alpha comprising one or more mutations selected from the group consisting of Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q, D538G, and combinations thereof, wherein: X1 is S, N, or C; X2 is R or Q; Wherein, the drug comprises a therapeutically effective amount of RAD1901 or a salt thereof selected in a method comprising: (1) administering a first dose of RAD1901 or a salt thereof for 3, 4, 5, 6 or 7 days; (2) detecting estradiol-ER binding activity, for example using FES-PET imaging; wherein: (i) if the ER binding activity is undetectable or below a predetermined threshold level, continuing to administer the first dose; or (ii) if the ER binding activity is detectable or above a predetermined threshold level, administering a second dose greater than the first dose for 3, 4, 5, 6 or 7 days, and then performing step (3); (3) using FES-PET imaging to detect estradiol-ER binding activity; wherein, (i) if the ER binding activity is undetectable or below a predetermined threshold level, continuing with a second dose; or (ii) if the ER binding activity is detectable or above a predetermined threshold level, administering a third dose that is greater than the second dose for 3, 4, 5, 6, or 7 days, and then performing step (4); (4) Repeat the above steps with the fourth dose, the fifth dose, and so on, until no ER binding activity can be detected.

2. The use according to claim 1, wherein: The mutation is Y537X1, and X1 is S.

3. The use according to claim 1, wherein: After administration, the ratio of the concentration of RAD1901 or its salt in the tumor to the concentration of RAD1901 or its salt in plasma is at least 15.

4. The use according to claim 1, wherein The subject suffers from osteoporosis or has a high risk of osteoporosis.

5. The use according to claim 1, wherein: The subjects are premenopausal women.

6. The use according to claim 1, wherein: The subjects are postmenopausal women with relapse or malignant progression following previous treatment with SERMs and / or AIs.

7. The use according to claim 1, comprising administering to the subject a therapeutically effective amount of a combination of everolimus and RAD1901 or a salt thereof, wherein: The therapeutically effective amount is 150 to 1,500 mg / day.

8. The use according to claim 1, wherein: The salt is RAD1901 dihydrochloride.

9. The use according to claim 1, wherein: The tumor is resistant to a drug selected from the group consisting of antiestrogen, aromatase inhibitors, and combinations thereof.

10. The use according to claim 9, wherein: The anti-estrogen drug is tamoxifen or fulvestrant.

11. The use according to claim 9, wherein: The aromatase inhibitor is aromasin.

12. The use according to claim 1, comprising administering to the subject a therapeutically effective amount of a combination of everolimus and RAD1901 or a salt thereof, wherein: The therapeutically effective amount is 150 mg to 2,000 mg.

13. The use according to claim 1, wherein: The first dose of the drug administered in step (1) is 200 mg / day to 600 mg / day.

14. The use according to claim 1, wherein: The first dose of the drug administered in step (1) is 350 mg / day to 500 mg / day.

15. The use according to claim 1, wherein: The second dose that is greater than the first dose is the first dose plus 50 mg / day to 200 mg / day.

16. The use according to claim 1, wherein: A third dose greater than the second dose is the second dose plus 50 mg / day to 200 mg / day.

17. Use of a combination of everolimus and RAD1901 or a salt thereof in the preparation of a medicament for treating a tumor in a subject with mutant estrogen receptor alpha-positive cancer, wherein: The RAD1901 has the following structure: wherein the cancer is breast cancer and is positive for a mutant estrogen receptor alpha comprising one or more mutations selected from the group consisting of Y537X1, L536X2, P535H, V534E, S463P, V392I, E380Q, D538G, and combinations thereof, wherein: X1 is S, N, or C; X2 is R or Q; Wherein, the drug comprises a pharmaceutical composition, which comprises everolimus, RAD1901 or a salt thereof and a pharmaceutically acceptable carrier, or wherein the drug comprises two independent pharmaceutical compositions, wherein one pharmaceutical composition comprises RAD1901 or a salt thereof and a pharmaceutically acceptable carrier, and the other pharmaceutical composition comprises everolimus and a pharmaceutically acceptable carrier.

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