Methods for treating cancer

Abstract

To provide a method for treating cancer. [Solution] A method for inhibiting tumor growth or causing tumor regression in a subject with drug-resistant estrogen receptor alpha-positive cancer, comprising administering to the subject a therapeutically effective amount of RAD1901 ((6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-2-ol) or a salt or solvate thereof.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 154,699, filed Apr. 29, 2015; U.S. Provisional Patent Application No. 62 / 155,451, filed Apr. 30, 2015; U.S. Provisional Patent Application No. 62 / 252,085, filed Nov. 6, 2015; U.S. Provisional Patent Application No. 62 / 265,696, filed Dec. 10, 2015; U.S. Provisional Patent Application No. 62 / 158,469, filed May 7, 2015; U.S. Provisional Patent Application No. 62 / 252,916, filed Nov. 9, 2015; U.S. Provisional Patent Application No. 62 / 265,774, filed Dec. 10, 2015; U.S. Provisional Patent Application No. 62 / 192,940, filed Jul. 15, 2015; U.S. Provisional Patent Application No. 62 / 265,658, filed Dec. 10, 2015; U.S. Provisional Patent Application No. 62 / 323,572, filed Apr. 15, 2016; U.S. Provisional Patent Application No. 62 / 192,944, filed Jul. 15, 2015; U.S. Provisional Patent Application No. 62 / 265,663, filed Dec. 10, 2015; U.S. Provisional Patent Application No. 62 / 323,576, filed Apr. 15, 2016, all of which are hereby incorporated by reference in their entirety.

[0002] The present invention relates to a method for treating cancer.

Background Art

[0003] Breast cancer is classified into three subtypes based on the expression of three receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor - 2 (Her2). Over - expression of ER is found in many breast cancer patients. ER - positive (ER+) breast cancer accounts for two - thirds of all breast cancers. Estrogen and ER are associated with, for example, ovarian cancer, colon cancer, prostate cancer, and endometrial cancer other than breast cancer.

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

[0005] Agents that inhibit estrogen production, such as aromatase inhibitors (AIs, e.g., letrozole, anastrozole, and arimidex), or those that directly block ER activity, such as selective estrogen receptor modulators (SERMs, e.g., tamoxifen, toremifene, droloxifene, idoxifene, raloxifene, lasofoxifene, arzoxifene, mioproxifene, levormeloxifene, and EM-652 (SCH 57068)) and selective estrogen receptor degraders (SERDs, e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 / DPC974, SRN-927, ICI182782, and AZD9496) have already been used or are being developed for the treatment of ER-positive breast cancer.

[0006] SERMs and AIs are also commonly used as first-line adjuvant systemic therapy for ER-positive breast cancer. Tamoxifen is currently used for both early and advanced ER-positive breast cancer in premenopausal and postmenopausal women. However, tamoxifen can have serious side effects such as blood clots and stroke. Tamoxifen can prevent bone loss in postmenopausal women but can cause osteoporosis in premenopausal women. Tamoxifen also increases the risk of endometrial cancer because it acts as a partial agonist in the endometrium.

[0007] AI suppresses estrogen production in peripheral tissues by blocking the activity of aromatase, which converts androgens to estrogens in the body. However, AI cannot stop the ovaries from producing estrogen. Therefore, AI is mainly used to treat postmenopausal women. Furthermore, since AI has fewer severe side effects and is more effective than tamoxifen, AI can be used to treat premenopausal women and suppress ovarian function. See, for example, Non-Patent Document 3.

[0008] Initial treatment with these agents can be successful, but many patients will ultimately relapse with drug-resistant breast cancer. Mutations affecting ER appear as one potential mechanism for this resistance. See, for example, Non-Patent Document 4. Mutations in the ligand-binding domain (LBD) of ER are found in 21% of metastatic ER-positive breast cancer samples from patients who have received at least one endocrine therapy. Non-Patent Document 5.

[0009] 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 effectiveness, the utility of fulvestrant is limited by a decrease in bioavailability due to the amount of drug that can be administered in a single dose. Imaging studies using 18F-fluoroestradiol positron emission tomography (FES-PET) have shown that even at a dose level of 500 mg, some patients may not have complete ER inhibition, suggesting that inadequate dosing may be a reason for treatment failure.

[0010] Another issue related to estrogen - targeted therapies is that they can have undesirable effects on the uterus, bone, and other tissues. The ER directs the transcription of estrogen - responsive genes in a wide variety of tissues and cell types. These effects can be particularly prominent when the endogenous levels of estrogen and other ovarian hormones decline during menopause. For example, tamoxifen acts as a partial agonist in the endometrium, which can cause osteoporosis and increase the risk of endometrial cancer in pre - menopausal women. In post - menopausal women, AIs can cause more bone loss and fractures than tamoxifen. Patients treated with fulvestrant can also be exposed to an increased risk of osteoporosis due to its mechanism of action.

Prior Art Documents

Non - Patent Documents

[0011]

Non - Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Non - Patent Document 4

[0012] Therefore, there remains a need for a more sustained and effective ER - targeted therapy that overcomes some of the problems associated with current endocrine therapies and is effective in the development of resistance. [Means for Solving the Problems]

[0013] In one aspect, the present disclosure relates to a method of inhibiting tumor growth or causing tumor regression in a subject having drug - resistant estrogen receptor alpha - positive cancer. The method comprises administering to the subject a therapeutically effective amount of RAD1901 having the structure: [Chemical Formula] or a salt or solvate thereof.

[0014] In another aspect, the present disclosure relates to a method of inhibiting tumor growth or causing tumor regression in a subject having mutant estrogen receptor alpha - positive cancer. The method comprises administering to the subject a therapeutically effective amount of RAD1901 having the structure: [Chemical Formula] or a salt or solvate thereof.

[0015] In some embodiments, the cancer is selected from the group consisting of breast cancer, uterine cancer, ovarian cancer, and pituitary cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer 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, and combinations thereof, where X1 is S, N, or C, D538G, and X2 is R or Q. For example, the mutation is Y537S.

[0016] In some embodiments, the tumor is resistant to drugs selected from the group consisting of anti-estrogens, aromatase inhibitors, and combinations thereof. For example, the anti-estrogen is tamoxifen or fulvestrant, and the aromatase inhibitor is aromasin.

[0017] That is, the gist of the present invention relates to the following. Item 1 The invention described in the specification.

Effect of the Invention

[0018] The present invention can provide a method for treating cancer.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] As shown in the Examples section below, RAD1901 (the following structure) has been found to inhibit tumor growth and / or cause tumor regression in breast cancer xenograft models despite the ESR1 status and prior endocrine therapies (Example I(A)). The treated xenograft models had high or low Her2 expression and tumors expressing WT or mutant (e.g., Y537S) ERα with or without prior endocrine therapies (e.g., tamoxifen (tam), AI, chemotherapy (chemo), Her2 inhibitor (Her2i, e.g., trastuzumab, lapatinib), bevacizumab, and / or rituximab) (Figure 1). And in all cases, RAD1901 inhibited tumor growth. The WT ER PDx model and the mutant ER PDx model may have different levels of responsiveness to fulvestrant treatment. However, RAD1901 has been found to inhibit tumor growth despite the PDx model being responsive to fulvestrant treatment. Therefore, RAD1901 can be used as a fulvestrant replacement to treat the responsiveness of breast cancer that has improved inhibition of tumor growth and to treat breast cancer that is not treated effectively by fulvestrant. For example, RAD1901 caused tumor regression in WT ER+PDx models with diverse responsiveness to fulvestrant treatment (e.g., MCF-7 cell line xenograft models, PDx-4, PDx-2, and PDx-11 models responsive to fulvestrant treatment, and the PDx-12 model with little response to fulvestrant treatment), as well as mutant (e.g., Y537S) ER+PDx models with varying levels of responsiveness to fulvestrant treatment (e.g., the PDx-6 model responsive to fulvestrant treatment and the PDx-5 model with little response to fulvestrant treatment). RAD1901 showed sustained efficacy in inhibiting tumor growth after treatment was terminated while estradiol treatment continued (e.g., PDx-4 model).The results provided herein also show that RAD1901 can be delivered to the brain (Example II), and that such delivery improved mouse survival in an intracranial tumor model expressing wild-type ERα (MCF-7 xenograft model, Example I(B)). RAD1901 is a potent anti-ER+ breast cancer therapy.

Chemical formula

[0021] RAD1901 is also likely to produce fewer side effects compared to other endocrine therapies (e.g., other SERMs such as tamoxifen and SERDs such as fulvestrant). For example, tamoxifen can also increase the risk of endometrial cancer. Tamoxifen can also cause osteoporosis in premenopausal women. Fulvestrant can also increase the risk of bone loss in treated patients. RAD1901 is unlikely to have similar side effects. RAD1901 has been found to preferentially accumulate in tumors, with a RAD1901 level in up to about 35 tumors versus the RAD1901 level in plasma (T / P ratio) (Example II). Standard uptake values (SUVs) for the uterus, muscle, and bone were calculated for human subjects treated once daily with RAD1901 at doses of about 200 mg to up to about 500 mg (Example III(A)). The uterine signal after administration was close to the level from "non-target tissues" (tissues that do not express estrogen receptors), suggesting complete attenuation of FES-PET uptake after RAD1901 treatment. Little change was observed for pre- versus post-treatment PET scans in tissues that did not significantly express estrogen receptors (e.g., muscle, bone) (Example III(A)). RAD1901 treatment antagonized estradiol stimulation of uterine tissue in ovariectomized (OVX) rats (Example IV(A)) and greatly preserved the bone mass of treated subjects. Therefore, RAD1901 treatment is unlikely to harm the patient's bone structure as other endocrine therapies can. For example, OVX rats treated with RAD1901 showed maintained BMD and femoral microarchitecture (Example IV(A)). Therefore, RAD1901 treatment may be particularly useful in patients with osteoporosis or a higher risk of osteoporosis.

[0022] Furthermore, RAD1901 was found to degrade wild-type ERα in vivo and abrogate ER signaling in the MCF7 cell line xenograft model, and showed a dose-dependent decrease in PR in these MCF7 cell line xenograft models (Example III(B)). RAD1901 decreased proliferation in the MCF-7 cell line xenograft model and the PDx-4 model, as evidenced by a decrease in the proliferation marker Ki67 in tumors harvested from treated subjects. RAD1901 also decreased ER signaling in vivo in a mutant ER PDx model that was largely unresponsive to fulvestrant treatment (Example III(B)).

[0023] The unexpected efficacy of RAD1901 against tumors that are largely unresponsive to fulvestrant treatment and tumors expressing mutant ERα may be due to a unique interaction between RAD1901 and ERα. Structural models of ERα that binds RAD1901 and compounds that bind other ERαs were analyzed to obtain information about specific binding interactions (Example V). Computer modeling indicated that the RAD1901-ERα interaction is unlikely to be affected by mutants of the LBD of ERα, such as the Y537X mutant (where X is S, N, or C), D538G, and S463P, which account for approximately 81.7% of the LBD mutations found in recent studies of metastatic ER-positive breast tumor samples from patients treated with at least one of the endocrine therapies (Table 11, Example V). This led to the identification of specific residues in the C-terminal ligand-binding domain of ERα that are essential for binding and can be used to generate compounds that bind and antagonize not only wild-type ERα but also certain mutations and their variants, which is information crucial for binding.

[0024] Based on these results, provided herein is a method for inhibiting the growth of or causing regression of ERα-positive cancer or tumors in subjects in need thereof by administering to the subject a therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) or salt thereof. In certain embodiments, administration of RAD1901 or a salt or solvate (e.g., hydrate) thereof has additional therapeutic efficacy including, for example, inhibiting the proliferation of cancer cells or inhibiting ERα activity (e.g., by inhibiting estradiol binding or degrading ERα). In certain embodiments, the method does not produce negative effects on muscle, bone, breast, and uterus.

[0025] Also provided herein are methods of modulating and degrading ERα and mutant ERα, methods of treating conditions associated with the activity or expression of ERα and mutant ERα, compounds for use in these methods, and complexes and crystals of such compounds that bind to ERα and mutant ERα.

[0026] In certain embodiments of the methods of inhibiting tumor growth or causing tumor regression provided herein, provided is a method for inhibiting the growth of or causing regression of ERα-positive tumors in subjects in need thereof by administering to the subject a therapeutically effective amount of RAD1901 or a salt or solvate (e.g., hydrate) thereof. In certain of these embodiments, the salt has the structure:

Chemical formula

[0027] As used herein, "inhibiting the growth of" an ERα-positive tumor can refer to slowing the rate of tumor growth or completely stopping tumor growth.

[0028] As used herein, "tumor regression" or "regression" of an ERα-positive tumor can refer to reducing the size of the largest tumor. In certain embodiments, administration of RAD1901 or a solvate (e.g., hydrate) or salt thereof can result in a decrease in tumor size relative to baseline (i.e., the size prior to the start of treatment), or even eradication or partial eradication of the tumor. Thus, in certain embodiments, the methods of tumor regression provided herein can alternatively be characterized as methods of decreasing tumor size relative to baseline.

[0029] As used herein, "tumor" is a malignant tumor and is used synonymously with "cancer".

[0030] Inhibition or regression of tumor growth can be limited to a solitary tumor or a series of tumors within a particular tissue or organ, or can be systemic (i.e., affecting tumors in all tissues or organs).

[0031] Since RAD1901 is known to preferentially bind to estrogen receptor alpha (ERα) over estrogen receptor beta (ERβ), unless otherwise stated, estrogen receptor, estrogen receptor alpha, ERα, ER, wild-type ERα, and ESR1 are used interchangeably herein. As used herein, "estrogen receptor alpha" or "ERα" refers to a polypeptide comprising, consisting of, or essentially consisting of the wild-type ERα amino acid sequence encoded by the gene ESR1. As used herein, a tumor that is "positive for estrogen receptor alpha", "ERα positive", "ER+", or "ERα+" refers to a tumor in which one or more cells express at least one isoform of ERα. In certain embodiments, these cells overexpress ERα. In certain embodiments, a patient has one or more cells within a tumor that expresses one or more types of estrogen receptor beta. In certain embodiments, an ERα-positive tumor and / or cancer is associated with breast, uterine, ovarian, or pituitary cancer. In certain of these embodiments, the patient has a tumor located in breast, uterine, ovarian, or pituitary tissue. In embodiments where the patient has a tumor located in the breast, the tumor may or may not be positive for HER2 and HER2+ tumors, may be associated with luminal breast cancer, and the tumor may express high or low HER2 (e.g., FIG. 1). In other embodiments, the patient has a tumor located in another tissue or organ (e.g., bone, muscle, brain), but nevertheless is not associated with breast, uterine, ovarian, or pituitary cancer (e.g., a tumor resulting from the metastasis or spread of breast, uterine, ovarian, or pituitary cancer). Thus, in certain embodiments of the methods of inhibiting or regressing tumor growth provided herein, the targeted tumor is a metastatic tumor and / or the tumor has overexpression of ER in other organs (e.g., bone and / or muscle). In certain embodiments, the targeted tumor is a brain tumor and / or cancer.In certain embodiments, the targeted tumor is more sensitive to treatment with RAD1901 than treatment with another SERD (e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810), GW5638 / DPC974, SRN-927, ICI182782, and AZD9496), a Her2 inhibitor (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine, and / or pertuzumab), chemotherapy (e.g., abraxane, doxorubicin, carboplatin, cyclophosphamide, daunorubicin, doxil, elence, fluorouracil, gemzar, herceptin, lxempra, methotrexate, mitomycin, micoxantrone, navelbine, paclitaxel, taxotere, thiotepa, vincristine, and xeloda), an aromatase inhibitor (e.g., anastrozole, exemestane, and letrozole), a selective estrogen receptor modulator (e.g., tamoxifen, raloxifene, lasofoxifene, and / or toremifene), an angiogenesis inhibitor (e.g., bevacizumab), and / or rituximab.

[0032] In certain embodiments of the methods of inhibiting or regressing tumor growth provided herein, the method further comprises determining whether the patient has a tumor that expresses ERα prior to administering RAD1901 or a solvate (e.g., hydrate) or salt thereof. In certain embodiments of the methods of inhibiting or regressing tumor growth provided herein, the method further comprises determining whether the patient has a tumor that expresses mutant ERα prior to administering RAD1901 or a solvate (e.g., hydrate) or salt thereof. In certain embodiments of the methods of inhibiting or regressing tumor growth provided herein, the method further comprises determining whether the patient has a tumor that expresses ERα that responds or does not respond to fulvestrant treatment prior to administering RAD1901 or a solvate (e.g., hydrate) or salt thereof. These determinations can be made using any of the methods of detecting expression well known in the art and can be performed in vitro using a tumor or tissue sample excised from the subject.

[0033] In addition to demonstrating the ability of RAD1901 to inhibit tumor growth in tumors expressing wild-type ERα, the results provided herein indicate that RAD1901 has an unexpected ability to inhibit the growth of tumors expressing a mutant form of ERα, namely Y537S ERα (Example I(A)). Computer modeling evaluations of examples of ERα mutations indicate that any of these mutations, such as ERα having one or more mutants selected from the group consisting of ERα having a Y537X mutant (wherein X is S, N, or C), ERα having a D538G mutant, and ERα having an S463P mutant, were not expected to affect the ligand-binding domain or specifically interfere with RAD1901 binding (Example V(A)). Based on these results, a method is provided herein for inhibiting the growth or causing regression of tumors that are positive for one or more mutants within the ligand-binding domain (LBD) selected from the group consisting of Y537X1 (wherein X1 is S, N, or C), D538G, L536X2 (wherein X2 is R or Q), P535H, V534E, S463P, V392I, E380Q, particularly Y537S ERα, in a subject suffering from cancer by administering to the subject a therapeutically effective amount of RAD1901 or a solvate (e.g., hydrate) or salt thereof. As used herein, "mutant ERα" refers to ERα and its variants that contain, consist of, or consist essentially of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the amino acid sequence of ERα and include one or more substitutions or deletions.

[0034] In addition to inhibiting breast cancer tumor growth in the animal xenograft model, the results disclosed herein show that RAD1901 exhibits significant accumulation within tumor cells and is able to cross the blood-brain barrier (Example II). The ability to cross the blood-brain barrier was confirmed by the fact that administration of RAD1901 significantly prolonged survival in the brain metastasis xenograft model (Example I(B)). Thus, in certain embodiments of the methods for inhibiting or regressing tumor growth provided herein, the targeted ERα-positive tumors are located in the brain or other locations in the central nervous system. In certain of these embodiments, the ERα-positive tumors are primarily associated with brain cancer. In other embodiments, the ERα-positive tumors are primarily metastatic tumors associated with another type of cancer, such as breast, uterine, ovarian, or pituitary cancer that has migrated from another tissue or organ. In certain of these embodiments, the tumor is a brain metastasis, such as breast cancer brain metastasis (BCBM). In certain embodiments of the methods disclosed herein, RAD1901 or a salt or solvate thereof accumulates in one or more cells within the target tumor.

[0035] In certain embodiments of the methods disclosed herein, RAD1901 or a solvate thereof (e.g., hydrate) or a salt preferably accumulates in the tumor at a T / P (concentration of RAD1901 in tumor / concentration of RAD1901 in plasma) ratio of about 15 or greater, about 18 or greater, about 19 or greater, about 20 or greater, about 25 or greater, about 28 or greater, about 30 or greater, about 33 or greater, about 35 or greater, about 40 or greater.

[0036] The results provided herein show that RAD1901 administration prevents bone mass loss in ovariectomized rats (Example IV(A)). Thus, in certain embodiments of the methods of inhibiting or regressing tumor growth provided herein, RAD1901 or a solvate (e.g., hydrate) or salt thereof does not have an undesirable effect on bone, including, for example, an undesirable effect on the bone volume density, bone surface density, bone mineral concentration, trabecular number, trabecular width, trabecular separation, connectivity density, and / or apparent bone density of a treated subject. RAD1901 may be useful in patients with osteoporosis or a higher risk of osteoporosis. Tamoxifen may be associated with bone loss in premenopausal women and may damage bone structure by its mechanism of action. RAD1901 may be particularly useful in premenopausal women and / or tumors that are resistant to tamoxifen or anti-estrogen therapy.

[0037] The results provided herein show that RAD1901 antagonizes estradiol stimulation of uterine tissue in ovariectomized rats (Example IV(A)). Further, in human subjects treated once daily with a dose of RAD1901 of 200 mg or up to 500 mg, the standard uptake values (SUVs) for uterine, muscle, and bone tissue that did not significantly express ER showed little change in the signals before and after treatment (Example III(A)). Thus, in certain embodiments, such administration also does not result in an undesirable effect on other tissues, including, for example, uterine, muscle, or breast tissue.

[0038] A therapeutically effective amount of RAD1901 for use in the methods disclosed herein is an amount that, when administered over a particular time interval, results in the achievement of one or more therapeutic criteria (e.g., slowing or halting tumor growth, causing tumor regression, etc.). Ideally, the therapeutically effective amount does not exceed the maximum tolerated dose at which more than 50% of the treated subjects experience nausea or other toxic reactions that preclude further drug administration. The therapeutically effective amount can vary in a subject depending on various factors, including the various symptoms, gender, age, weight range, or general health of the subject, the mode of administration and salt or solvate type, variations in sensitivity to the drug, and the particular type of disease, among others.

[0039] Examples of therapeutically effective amounts of RAD1901 for use in the methods disclosed herein include, in subjects with resistant ER-driven tumors or cancers, a dose of about 150 to about 1,500 mg, about 200 to about 1,500 mg, about 250 to about 1,500 mg, or about 300 to about 1,500 mg once daily; in subjects with both wild-type ER-driven tumors and / or cancers and resistant tumors and / or cancers, a dose of about 150 to about 1,500 mg, about 200 to about 1,000 mg, about 250 to about 1,000 mg, or about 300 to about 1,000 mg once daily; and in subjects mainly having wild-type ER-driven tumors and / or cancers, a dose of about 300 to about 500 mg, about 300 to about 550 mg, about 300 to about 600 mg, about 250 to about 500 mg, about 250 to about 550 mg, about 250 to about 600 mg, about 200 to about 500 mg, about 200 to about 550 mg, about 200 to about 600 mg, about 150 to about 500 mg, about 150 to about 550 mg, or about 150 to about 600 mg once daily, but are not limited thereto. In certain embodiments, in adult subjects, the dose of RAD1901 or its solvates (e.g., hydrates) or salts for use in the currently disclosed methods can be an oral dose of about 200 mg, 400 mg, 500 mg, 30 mg to 2,000 mg, 100 mg to 1,500 mg, or 150 mg to 1,500 mg once daily. This daily dose can be achieved via a single administration or multiple administrations.

[0040] The dosage of RAD1901 in the treatment of breast cancer, including examples of expressing resistant strains and mutant receptor(s), is in the range of 100 mg to 1,000 mg per day. For example, RAD1901 can be administered at 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mg per day. In particular, 200 mg, 400 mg, 500 mg, 600 mg, 800 mg, and 1,000 mg per day are known. In humans after oral administration, RAD1901, which has a surprisingly long half-life, creates this particularly feasible option. Thus, the drug can be administered twice a day at 200 mg (total 400 mg per day), twice a day at 250 mg (total 500 mg per day), twice a day at 300 mg (total 600 mg per day), twice a day at 400 mg (800 mg per day), or twice a day at 500 mg (total 1,000 mg per day). Preferably, the administration is oral.

[0041] In certain embodiments, the cancer or tumor is a resistant ER-driven cancer or tumor (e.g., having a mutant ER binding domain (e.g., Y537X1 (wherein X1 is S, N, or C), D538G, L536X2 (wherein X2 is R or Q), P535H, V534E, S463P, V392I, E380Q, and combinations thereof, including but not limited to, one or more mutations of ERα)), and the overexpressors of ER or tumor and / or cancer proliferation become ligand-independent, or SERDs (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, ado-trastuzumab emtansine, and / or pertuzumab), chemotherapy (e.g., abraxane, adriamycin, carboplatin, cytoxan, daunorubicin, doxil, eleence, fluorouracil, gemzar, heraben, lxempra, methotrexate, mitomycin, micoxantrone, navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda), aromatase inhibitors (e.g., anastrozole, exemestane, and letrozole), selective estrogen receptor modulators (e.g., tamoxifen, raloxifene, lasofoxifene, and / or toremifene), angiogenesis inhibitors (e.g., bevacizumab), and / or tumors and / or cancers that progress with treatment of rituximab.

[0042] RAD1901 or a solvate (e.g., hydrate) or salt thereof for use in the presently disclosed methods can be administered to a subject one or more times. In these embodiments where the compound is administered multiple times, they can be administered at a series of intervals, e.g., daily, every other day, weekly, or monthly. Alternatively, they can be administered at irregular intervals as needed, e.g., based on symptoms, the health status of the patient, etc.

[0043] RAD1901 or a solvate (e.g., hydrate) or salt thereof for use in the presently disclosed methods can be formulated into a single dosage form, meaning physically discrete units suitable as a single dosage for a subject being treated, each unit optionally containing a predetermined amount of the active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier. The unit dosage form can be one of a single daily dose or multiple daily doses (e.g., once, about 1 to 4 times or more per day). When multiple daily doses are used, the unit dosage form can be the same for each dose or different. In certain embodiments, the compound can be formulated for controlled release.

[0044] RAD1901 or a solvate (e.g., hydrate) or salt thereof for use in the presently disclosed method can be formulated according to any available conventional method. Examples of preferred dosage forms include tablets, powders, fine granules, granules, coated tablets, capsules, syrups, troches, inhalants, suppositories, injections, ointments, ophthalmic ointments, eye drops, nasal drops, ear drops, cataplasms, lotions, etc. For formulation, diluents, binders, disintegrants, lubricants, coloring agents, flavoring agents, and, if necessary, commonly used additives such as stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants can be used. In addition, formulation is also carried out by mixing components generally used as raw materials for pharmaceutical formulations according to conventional methods. Examples of these compositions include, for example, (1) oils such as soybean oil, beef tallow, and synthetic glycerides, (2) hydrocarbons such as liquid paraffin, squalane, and solid paraffin, (3) ester oils such as octyldodecyl myristate and isopropyl myristate, (4) higher alcohols such as cetostearyl alcohol and behenyl alcohol, (5) silicone resins, (6) silicone oils, (7) 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) polyhydric alcohols such as glycerol, propylene glycol, dipropylene glycol, and sorbitol, (11) saccharides 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 use in the above formulations include, for example, 1) as diluents, lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide, 2) as binders, polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, polypropylene glycol-polyoxyethylene-block copolymer, meglumine, calcium citrate, dextrin, pectin, etc., 3) as disintegrants, starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, carboxymethyl cellulose / calcium, etc., 4) as lubricants, magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, etc., 5) any coloring agent sufficient as a pharmaceutically acceptable coloring agent for addition, 6) as flavoring agents, cocoa powder, menthol, aromatizer, peppermint oil, and cinnamon powder, 7) may contain antioxidants such as ascorbic acid or alpha-tocopherol whose addition is pharmaceutically acceptable.

[0045] RAD1901 or its solvate (e.g., hydrate) or salt for use in the presently disclosed method can be formulated into a pharmaceutical composition with any one or more of the 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 RAD1901 used in the method of the present invention, as well as mixtures containing two or more of such compounds, pharmaceutically acceptable salts of such compounds, and pharmaceutically acceptable solvates of such compounds. Such compositions are prepared according to acceptable pharmaceutical procedures as described in Remington’s Pharmaceutical Sciences, 17th edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Eaton, Pa. (1985) (incorporated herein by reference).

[0046] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause allergic reactions or other adverse reactions in patients, is administered to the patient, and is capable of being mixed with other materials in the formulation. Pharmaceutically acceptable carriers include, for example, pharmaceutically diluents, excipients, or carriers that are appropriately selected for the intended dosage form and are consistent with conventional pharmaceutical practice. For example, solid carriers / diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethyl acrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. Pharmaceutically acceptable carriers may further contain small amounts of auxiliary substances such as wetting agents or emulsifiers, preservatives or buffers that enhance the storage period or effectiveness of the therapeutic agent.

[0047] RAD1901 can be converted to a salt by conventional methods in its free form. As used herein, the term "salt" is not limited as long as the salt is formed with RAD1901 and is pharmaceutically acceptable. Preferred examples of salts include hydrohalides (e.g., hydrochloride, hydrobromide, hydroiodide, etc.), inorganic acid salts (e.g., sulfate, nitrate, perchlorate, phosphate, carbonate, bicarbonate, etc.), organic carboxylates (e.g., acetate, maleate, tartrate, fumarate, citrate, etc.), organic sulfonates (e.g., methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, camphorsulfonate, etc.), amino acid salts (e.g., aspartate, glutamate, etc.), quaternary ammonium salts, alkali metal salts (e.g., sodium salt, potassium salt, etc.), alkaline earth metal salts (magnesium salt, calcium salt, etc.), and the like. In addition, hydrochloride, sulfate, methanesulfonate, acetate, etc. are preferred as "pharmaceutically acceptable salts" of the compounds according to the present invention.

[0048] RAD1901 or its solvate (e.g., hydrate) or salt and / or isomers (e.g., geometric isomers, optical isomers, rotational isomers, tautomers, etc.) of a second therapeutic agent(s) (e.g., everolimus) can be purified to a single isomer using conventional separation methods including, for example, optical resolution such as recrystallization, diastereomeric salt method, enzymatic resolution method, various chromatographic methods (e.g., thin layer chromatography, column chromatography, glass chromatography, etc.). As used herein, the term "single isomer" includes not only isomers having 100% purity but also isomers containing isomers other than the target that exist even through conventional purification operations. Crystal polymorphs sometimes exist for RAD1901 or its salts, and all of its crystal polymorphs are included in the present invention. Crystal polymorphs can sometimes be single and sometimes be mixtures, both of which are included herein.

[0049] In certain embodiments, RAD1901 can be in prodrug form, meaning that it must undergo some changes (e.g., oxidation or hydrolysis) to achieve its active form. Alternatively, RAD1901 can be a compound produced by the change of a parent prodrug to its active form.

[0050] In certain embodiments, the method of inhibiting tumor growth provided herein further comprises profiling a subject, and the profiled genes are one or more genes selected from ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1, IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2, and VHL.

[0051] In some embodiments, the present invention provides a method of treating a subpopulation of breast cancer patients, the method of treating comprising increasing the expression of one or more of the following genes, and treating the subpopulation with an effective dose of RAD1901 (or combination) according to the administration embodiments described in the present disclosure.

[0052] In addition to establishing the ability of RAD1901 to inhibit tumor growth, the results provided herein indicate that RAD1901 inhibits estradiol binding to ER in the uterus and pituitary gland (Example III(A)). In these experiments, estradiol binding to ER in uterine and pituitary tissues was evaluated by FES-PET imaging. After treatment with RAD1901, the observed levels of ER binding were at or below background levels. These results indicate that the antagonistic effect of RAD1901 on ER activity can be evaluated using real-time scanning. Based on these results, a method is provided herein for monitoring the efficacy of treatment with RAD1901 or a salt or solvate thereof by measuring estradiol-ER binding in one or more target tissues, where a decrease or loss of binding indicates efficacy.

[0053] Methods are further provided for adjusting the dosage of RAD1901 or a salt or solvate thereof based on estradiol-ER binding. In certain embodiments of these methods, binding is measured at several time points after administration of one or more of a first dosage of the compound. If the estradiol-ER binding is not affected or shows a decrease below a predetermined threshold (e.g., a decrease in binding relative to baseline of less than 5%, less than 10%, less than 20%, less than 30%, or less than 50%), the first dosage is considered too low. In certain embodiments, these methods include a further step of administering an increased second dosage of the compound. These steps can be repeated to repeatedly increase the dosage until a desired decrease in estradiol-ER binding is achieved. In certain embodiments, these steps can be incorporated into the methods for inhibiting tumor growth provided herein. In these methods, estradiol-ER binding can serve as a surrogate for inhibition of tumor growth or as an adjunct means for assessing inhibition of growth. In other embodiments, these methods can be used in combination with administration of RAD1901 for purposes other than inhibition of tumor growth, including, for example, inhibition of cancer cell proliferation.

[0054] In certain embodiments, the methods provided herein for modulating the dosage of RAD1901 or a salt or solvate thereof (e.g., hydrate) are: (1) administering a first dosage of RAD1901 or a solvate thereof (e.g., hydrate) or a salt (e.g., from about 350 to about 500 mg, or from about 200 to about 600 mg / day) for 3, 4, 5, 6, or 7 days, (2) detecting estradiol-ER binding activity, for example, by using FES-PET imaging disclosed herein, (i) if the ER binding activity is undetectable or below a predetermined threshold level, continuing to administer the first dosage (i.e., maintaining the dosage level), or (ii) if the ER binding activity is detectable or above a predetermined threshold level, administering a second dosage greater than the first dosage (e.g., the first dosage + from about 50 to about 200 mg) for 3, 4, 5, 6, or 7 days, and then proceeding to step (3), (3) detecting estradiol-ER binding activity, for example, by using FES-PET imaging disclosed herein, (i) if the ER binding activity is undetectable or below a predetermined threshold level, continuing to administer the second dosage (i.e., maintaining the dosage level), or (ii) if the ER binding activity is detectable or above a predetermined threshold level, administering a third dosage greater than the second dosage (e.g., the second dosage + from about 50 to about 200 mg) for 3, 4, 5, 6, or 7 days, and then proceeding to step (4), (4) repeating the above steps through a fourth dosage, a fifth dosage, etc. until ER binding activity is no longer detected.

[0055] In certain embodiments, the invention includes the use of PET imaging for detecting and / or administering ER-sensitive or ER-resistant cancer.

[0056] Routes of administration of RAD1901 or its solvate (e.g., hydrate) or salt disclosed in this specification 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.

[0057] RAD1901-ERα Interaction (1) Mutant ERα in ER-positive breast cancer tumor samples from patients receiving at least one regimen of endocrine therapy In five studies reported in the past two years, a total of 187 metastatic ER-positive breast cancer tumor samples from patients receiving at least one regimen of endocrine therapy were sequenced, and ER LBD mutations were identified in 39 patients (21%) (Jeselsohn). Among the 39 patients, the six most frequent LBD mutations are shown in Scheme 1 adapted from Jeselsohn, as shown in Figure 42.

[0058] The frequencies of all LBD mutations are summarized in Table 11.

[0059] Computer modeling indicates that the RAD1901-ERα interaction is unlikely to be affected by mutants of the LBD of ERα, such as the Y537X mutant (where X is S, N, or C), D538G, and S463P, which account for approximately 81.7% of the LBD mutations found in recent studies of metastatic ER-positive breast tumor samples from patients who received at least one endocrine therapy (Table 11, Example V).

[0060] Complexes and crystals of RAD1901 that bind to ERα and / or mutant ERα are provided herein, and the mutant ERα includes, but is not limited to, one or more mutations such as Y537X1 (where X1 is S, N, or C), D538G, L536X2 (where X2 is R or Q), P535H, V534E, S463P, V392I, E380Q, and combinations thereof.

[0061] In certain embodiments of the methods provided herein, the LBDs of ERα and mutant ERα include AF-2. In other embodiments, the LBD consists of, consists essentially of, or comprises amino acids 299-554 of ERα. In certain embodiments, the LBD of mutant ERα includes one or more mutations such as Y537X1 (wherein X1 is S, N, or C), D538G, L536X2 (wherein X2 is R or Q), P535H, V534E, S463P, V392I, E380Q, and combinations thereof, but is not limited thereto. As used herein, the term "and / or" includes both the "and" case and the "or" case.

[0062] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are recited, they are for illustrative purposes only and are not intended to limit the invention. One of ordinary skill in the art will develop equivalent means or reactions without the exercise of inventive faculty and without departing from the scope of the invention. It will be understood that many changes may be made to the procedures described herein while still remaining within the scope of the invention. It is the intention of the inventors that such changes be included within the scope of the invention.

Examples

[0063] Materials and Methods Test Compounds The RAD1901 used in the following examples was (6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-2-ol dihydrochloride manufactured by IRIX Pharmaceuticals, Inc. (Florence, SC). RAD1901 was stored as a dry powder and formulated for use as a uniform suspension in 0.5% (w / v) methylcellulose in deionized water and was administered by oral gavage in animal models. Tamoxifen, raloxifene, and estradiol (E2) were obtained from Sigma-Aldrich (St. Louis, MO) and administered by subcutaneous injection. Fulvestrant was obtained from Tocris Biosciences (Minneapolis, MN) and administered by subcutaneous injection. All other experimental reagents were purchased from Sigma-Aldrich unless otherwise described.

[0064] Cell line MCF-7 cells (human metastatic adenocarcinoma of the breast) were purchased from the American Type Culture Collection (Rockville, MD) and maintained in phenol red-free minimum essential medium (MEM) containing 2 mM L-glutamine and Earle's BSS, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate, supplemented with 0.01 mg / ml bovine insulin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA), usually at 5% CO2.

[0065] T47D cells were cultured in a 10 cm dish in a 5% CO2 incubator in RPMI growth medium supplemented with 10% FBS and 5 μg / mL human insulin until approximately 75% confluent.

[0066] Xenograft model in vivo All mice were housed in a sterile environment in individually ventilated cages with sterile dust-free bedding cob, under a light-dark cycle (a circadian cycle of 12 - 14 hours of artificial light) and controlled room temperature and humidity, with continuous access to sterilized food and water. Tumors were measured twice a week with calipers, and volume was calculated using the formula: (L * W 2 ) * 0.52.

[0067] PDx model Some examples of patient-derived xenograft models (PDx models) are shown in Figure 1. PDx models with patient-derived breast tumors were constructed from viable human tumor tissue or fluid that was serially passaged a limited number of times in animals (athymic nude mice (Nu(NCF)-Foxn1nu)) to maintain tumor heterogeneity. Pretreatment tumor volumes were recorded in each experiment starting approximately one week before the estimated start date. When tumors reached approximately the tumor volume initiation (TVI) range (150 - 250 mm 3 ), animals were randomized into treatment and control groups and dosing was initiated (day 0, 8 - 10 animals per group), and animals in all studies were individually followed in each experiment. Dosing initiation started on day 0, and animals in all groups were dosed by body weight (0.01 mL per gram, 10 ml / kg). Each group was treated with vehicle (control, oral administration / once daily until endpoint), tamoxifen (1 mg / animal, subcutaneous injection / once daily until endpoint), fulvestrant (Faslodex®; 1 mg / animal or 3 mg / animal as needed, subcutaneous injection / 5 times weekly, extended as needed), or RAD1901 (30 or 60 or 120 mg / kg / animal, oral administration / once daily until endpoint). The treatment period continued for 56 - 60 days depending on the model. The drinking water for these PDx models was supplemented with 17β-estradiol.

[0068] Efficacy of the drug In all tests, starting on day 0, tumor dimensions were measured with a digital caliper, including the individual and mean estimated tumor volumes (mean TV ± standard error) recorded for each group, and tumor volume was calculated using the formula (Yasui et al. Invasion Metastasis 17:259-269 (1997), which is incorporated herein by reference): TV = width 2 × length × 0.52. Immediately upon reaching the tumor volume (TV) endpoint, each group or test was terminated (the time endpoint was 60 days and the volume endpoint was a group mean of 2 cm 3 ), and individual mice reaching a tumor volume of 2 cm 3 or greater were excluded from the test, and the final measurement was included in the group mean until the mean reached the volume endpoint or the test reached the time endpoint.

[0069] Calculation of Efficacy and Statistical Analysis Tumor growth inhibition (%) (TGI (%)) values were calculated at a single time point (when the control group reached the tumor volume or time endpoint) and reported for each treatment group (T) versus control (C) using the initial (i) and final (f) tumor measurements according to the formula (Corbett TH et al. In vivo methods for screening and preclinical testing. In: Teicher B, ed., Anticancer Drug Development Guide. Totowa, NJ: Humana. 2004:99-123.): TGI (%) = 1 - Tf - Ti / Cf - Ci.

[0070] Statistics TGI test - one-way analysis of variance + Dunnett's multiple comparison test (Corbett TH et al.).

[0071] Sample Collection At the endpoint, the tumors were removed. One fragment was snap-frozen, while another fragment was placed in 10% NBF for at least 24 hours and formalin-fixed paraffin-embedded (FFPE). The snap-frozen samples were stored at -80°C and the FFPE blocks were stored at room temperature.

[0072] Western blot Cells were harvested and protein expression was analyzed using standard techniques. Tumors were harvested at the time point indicated after the last day of administration and homogenized in RIPA buffer with protease and phosphatase inhibitors using a Tissuelyser (Qiagen). Equal amounts of protein were separated by MW, transferred to nitrocellulose membranes, and blotted with the following antibodies using standard techniques: ● Estrogen receptor (Santa Cruz (HC-20); sc-543) ● Progesterone receptor (Cell Signaling Technologies; 3153) ● Vinculin (Sigma-Aldrich, v9131)

[0073] qPCR analysis was performed as follows: Cells were harvested, mRNA was extracted, and equal amounts were used for cDNA synthesis and qPCR with primers specific for progesterone receptor, GREB1, and TFF1 (LifeTech). Bands were quantified using 1D Quant software (GE).

[0074] Immunohistochemistry Tumors were harvested, fixed in formalin, and embedded in paraffin. The embedded tumors were sectioned (6 μM) and stained with antibodies specific for ER, PR, and Her2. Quantification was performed as follows: Five fields were counted for positive cells (0 - 100%) and staining intensity (0 - 3+). The H score (0 - 300) was calculated using the following formula: positive (%) * intensity.

[0075] Example I. RAD1901 inhibited tumor growth in tumors and / or cancers expressing WT ER or mutant ER (e.g., Y537S) using different pre-endocrine therapies.

[0076] I(A). Efficacy of RAD1901 in an animal xenograft model I(A)(i) RAD1901 inhibited tumor growth in PDx models (PDx-1 to PDx-12) despite the ER status and prior endocrine therapy.

[0077] Figure 1 demonstrates the inhibition of tumor growth in various PDx models in mice treated with RAD1901 alone. Twelve patient-derived xenograft models were screened to test the RAD1901 response in various genetic backgrounds with different levels of ER, PR, and Her2. A complete efficacy study was conducted on PDx models (PDx-1 to PDx-4, and PDx-12) marked with "*" having n = 8 - 10. These PDx models were treated with a vehicle (negative control) or RAD1901 at a dose of 60 mg / kg once daily orally for 60 days. The screening study was conducted on other PDx models (PDx-5 to PDx-11) having n = 3, which were treated with a vehicle (negative control) or RAD1901 at a dose of 90 mg / kg once daily orally for 60 days. As shown in Figure 1, RAD1901 treatment was effective against PDx models that were driven by ER and additional drivers (e.g., PR+ and / or Her2+). RAD1901 was effective in inhibiting tumor growth in models with ER mutations and / or high levels of Her2 (PDx) expression, regardless of prior treatment, naive (Rx-negative), or treatment with any of aromatase inhibitors, tamoxifen (tam), chemotherapy (chemo), Her2 inhibitors (Her2i, e.g., trastuzumab, lapatinib), bevacizumab, fulvestrant, and / or rituximab.

[0078] I(A)(ii) RAD1901 caused regression in xenograft models expressing WT ER.

[0079] I(A)(ii)(1) RAD1901 caused regression in MCF-7 xenografts that responded to fulvestrant treatment.

[0080] MCF-7 Xenograft Model - I The anti-cancer effect of RAD1901 was tested using a first MCF-7 xenograft model (MCF-7 xenograft model-I) in female thymus-deficient nude mice (control: NU(NCr)-Foxn1nu) with estradiol administration to stimulate tumor growth. Three days prior to tumor cell transplantation, an estrogen pellet (0.36 mg of E2, 60-day release; Innovative Research of America, Sarasota, FL) was subcutaneously implanted between the scapulae of all test animals using a sterile trocar. MCF-7 human breast cancer cells were cultured to mid-log phase in RPMI-1640 medium containing 10% fetal bovine serum, 100 units / mL of penicillin G, 100 μg / mL of streptomycin sulfate, 2 mM glutamine, 10 mM HEPES, 0.075% sodium bicarbonate, and 25 μg / mL of gentamicin. On the day of tumor cell transplantation, the MCF-7 cells were trypsinized, pelleted, and resuspended in phosphate-buffered saline at a concentration of 5×10 7 cells / mL. Each test mouse was subcutaneously implanted with 1×10 7 MCF-7 cells in the right flank, and tumor growth was monitored. As needed, tumor weight was estimated based on the assumption that a tumor volume of 1 mm 3 corresponds to a tumor wet weight of 1 mg. Body weight was measured once daily for 5 days after MCF-7 cell transplantation and then twice weekly throughout the remainder of the study.

[0081] Fourteen days after tumor cell transplantation (designated as day 1 of the study), the mice were 9 weeks old with body weights ranging from 21.4 to 32.5 grams, and individual tumor volumes ranged from 75 to 144 mm 3 and the group mean tumor volume (MTV) was 108 mm 3 . The mice were randomized into 9 groups of 15 animals each and treated with vehicle oral administration, tamoxifen (1 mg / animal, subcutaneous injection daily), fulvestrant (0.5 mg / animal, subcutaneous injection once daily), or RAD1901 (0.3, 1, 3, 10, 30, 60, 90, and 120 mg / kg oral administration once daily).

[0082] Tumor volume was evaluated twice a week. The tumor endpoint was defined as the MTV of 1,500 mm 3 in the control group. Treatment tolerance was evaluated by frequent observation of body weight measurement and clinical symptoms of side effects related to treatment. Animals with a weight loss of more than 30% in one measurement or more than 25% in three measurements were humanely euthanized and classified as treatment-related deaths. The acceptable toxicity was defined as less than 20% mean body weight loss in the group during the test period, and at most one treatment-related death per 10 treated animals, or 10%. At the end of the test, the animals were euthanized by terminal cardiac puncture under isoflurane anesthesia.

[0083] Treatment outcomes were evaluated based on the percentage of tumor growth inhibition (TGI) defined as the difference in the ratio between the baseline (i.e., day 1) tumor volume and the tumor volume at the end of the test (i.e., day 42). The dataset for TGI analysis included all animals in each group and did not include any that died due to treatment-related or non-treatment-related causes. The threshold for potential therapeutic activity was defined as a treatment effect of 60% or more TGI. The results were analyzed using the Kruskal-Wallis or Mann-Whitney test with a pre-specified alpha of 0.05.

[0084] Treatment with RAD1901 at doses of 30 and 60 mg / kg resulted in significant TGI (66% TGI (P < 0.05) and 88% TGI (P < 0.001) at day 40, respectively). These results were similar to those obtained with tamoxifen (86% TGI at day 40) and fulvestrant (88% TGI at day 40) (Figure 2A). In Figure 2A, the boxes represent the 25-75 percentile observations, the lines represent the median of the observations, and the whiskers represent the extreme observations.

[0085] To investigate whether higher doses of RAD1901 induced a stronger effect in this model, doses up to a maximum of 120 mg / kg of RAD1901 were evaluated (Figure 2B - D). Consistent with earlier results, RAD1901 induced significant tumor inhibition at a dose of 60 mg / kg (94% TGI on day 42, with 2 / 10 partial regressions). Higher doses resulted in even higher TGI (97% TGI with 8 / 10 PR at 90 mg / kg, 96% TGI with 7 / 10 PR at 120 mg / kg). At all doses tested, RAD1901 inhibited tumor growth to a greater extent than either tamoxifen or fulvestrant. Tamoxifen treatment resulted in 90% TGI (with 2 / 10 PR), while fulvestrant treatment resulted in 87% TGI (with 1 / 10 PR). The inhibition by RAD1901 in the 90 and 120 mg / kg groups was significantly higher compared to both tamoxifen (P < 0.05) and fulvestrant (P < 0.05).

[0086] Overall, these results indicate that RAD1901 inhibits estrogen - induced tumor growth in a dose - dependent manner. The tumor volume at the end of treatment in mice treated with RAD1901 was equal to or lower than baseline, indicating that RAD1901 not only inhibits tumor growth but can also cause regression of tumor size in the mouse xenograft model.

[0087] RAD1901 showed good tolerance at all dose levels without side effects on body weight (Figure 2E).

[0088] The anti - cancer effect of RAD1901 was further tested using a second MCF - 7 xenograft (MCF - 7 xenograft model - II) prepared as described below.

[0089] MCF - 7 xenograft model - II Two days prior to cell transplantation, Balb / C-nude mice were incubated with 17β-estradiol pellets at a release rate of 0.18 / 90 days. MCF-7 cells (PR+, Her2-) were harvested and 1×10 7 cells were subcutaneously transplanted into the right flank of Balb / C-nude mice. Tumors were treated with the test compound when they reached an average of 200 mm 3 . Mice were randomized into treatment groups based on tumor volume and treated with the test compound. Each group was treated with vehicle (control, oral administration once a day until the endpoint), fulvestrant (Faslodex®; 3 mg / subject, subcutaneous injection once a week × 5, extended as needed), RAD1901 (30 mg / kg or 60 mg / kg per subject, oral administration once a day until the endpoint) as specified from day 0. The treatment period continued for 28 days.

[0090] Figures 3A - B show that in the MCF-7 xenograft model-II, RAD1901 (30 mg / kg and 60 mg / kg, oral administration once a day) produced a more significant inhibition of tumor growth than fulvestrant (3 mg / subject, subcutaneous injection once a week). Surprisingly, only 1 out of 10 subjects in the fulvestrant treatment group showed slight tumor regression at the end of the study, while 5 out of 10 subjects in the 30 mg / kg RAD1901 treatment group and 8 out of 10 subjects in the 60 mg / kg RAD1901 treatment group showed various levels of regression (Figure 3B).

[0091] I(A)(ii)(2) RAD1901 caused tumor regression in WT ER PDx models (e.g., PDx-4, PDx-2, and PDx-11) that responded to fulvestrant treatment.

[0092] Different WT ER PDx models (PDx-4, PR+, Her2−, and PDx-2 models, PR+, Her2+, both treatment-naïve; and PDx-11, PR+, Her2+, AI, fulvestrant, and chemotherapy-treated) showed different responses to fulvestrant (1 mg / dose or 3 mg / dose, subcutaneous injection once a week), but RAD1901 treatment at various doses (30 mg / kg, 60 mg / kg, and / or 120 mg / kg, oral administration once a day) resulted in more significant inhibition of tumor growth in all PDx models than fulvestrant (Figure 4A for the PDx-4 model, Figure 6 for the PDx-2 model, and Figure 7 for the PDx-11 model). For example, in the PDx-4 model that responded to fulvestrant treatment (1 mg / dose), RAD1901 unexpectedly stopped tumor growth or caused further tumor regression in the subjects compared to fulvestrant (1 mg / subject) (Figure 4B). Furthermore, in the PDx-11 model that responded to fulvestrant treatment (3 mg / dose), RAD1901 unexpectedly stopped tumor growth or caused tumor regression in all treated subjects, while only fulvestrant (3 mg / subject) caused regression in 2 out of 10 treated subjects (Figure 7B).

[0093] Oral administration of 60 mg / kg of RAD1901 alone achieved inhibition of tumor growth in the PDx-2 model similar to subcutaneous injection of 3 mg / dose of fulvestrant (Figure 6). Furthermore, the combination of RAD1901 and fulvestrant did not result in additional benefit.

[0094] Finally, in the PDx-4 model that responded to fulvestrant treatment (1 mg / dose, subcutaneous injection once a week), RAD1901-mediated inhibition of tumor growth was maintained for at least 2 months in the absence of treatment after the RAD1901 treatment (30 mg / kg, oral administration once a day) period ended but estradiol treatment continued (Figure 5).

[0095] I(A)(ii)(3) RAD1901 caused regression in WT ER PDx models (e.g., PDx-12) that were mostly unresponsive to fulvestrant treatment.

[0096] In WT ER PDx-12 (PR+, Her2-, treatment-naïve) that was mostly unresponsive to fulvestrant (1 mg / dose, subcutaneous injection once a week), RAD1901 treatment at various doses (30 mg / kg or 60 mg / kg, oral administration once a day) unexpectedly caused tumor regression in the PDx-12 model (Figure 8).

[0097] I(A)(iii) RAD1901 inhibited tumor growth and / or caused regression in xenograft models expressing mutant ER (ERα Y537S).

[0098] I(A)(iii)(1) RAD1901 inhibited tumor growth in the PDx-5 model that was mostly unresponsive to fulvestrant treatment.

[0099] The PDx-5 model prepared the following similar protocol as described above for the PDx model. The tumor size of each treatment group was measured twice a week with calipers, and the volume was calculated using the formula (L*W2)*0.52.

[0100] RAD1901 was more effective than fulvestrant in inhibiting tumor growth (60 mg / kg or 120 mg / kg, oral administration once a day) in the PDx-5 model that was mostly unresponsive to fulvestrant treatment (3 mg / dose, subcutaneous injection once a week) (Figures 9A - C). RAD1901 treatment at a high dose (120 mg / kg) was more effective than RAD1901 treatment at a low dose (60 mg / kg) (Figures 9A - C). The tumor size of individual animals was measured on day 17 (Figure 9B) and day 56 (Figure 9C), respectively.

[0101] I(A)(iii)(2) RAD1901 caused regression in the PDx-6 model that responded to fulvestrant treatment.

[0102] PDx models expressing mutant ER (e.g., Y537S) can respond to fulvestrant treatment (1 mg / dose, subcutaneous injection once a week) and tamoxifen (1 mg / dose, subcutaneous injection three times a week) treatment, e.g., PDx-6 (PR+, Her2:1+, previously treated with tamoxifen, AI, and fulvestrant) (Figures 10A - B). RAD1901 (30 mg / kg, 60 mg / kg, and 120 mg / kg, oral administration once a day) was more effective in inhibiting tumor growth than fulvestrant and tamoxifen (Figures 10A - B). For example, RAD1901 treatment of the PDx-6 model showed tumor regression, while fulvestrant treatment (1 mg / dose) did not (Figure 10B, showing the change in individual tumor size at the end of the test from baseline).

[0103] I(A)(iv) Pharmacokinetic evaluation of fulvestrant treatment in non-tumor-bearing mice.

[0104] Various doses of fulvestrant were administered to mice, and these showed significant dose exposure to the subjects (Figure 11).

[0105] 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 the indicated time points from 168 hours after the second administration, centrifuged, and plasma was analyzed by liquid chromatography-mass spectrometry.

[0106] I(B) RAD1901 promoted survival in a mouse xenograft model of brain metastasis (MCF-7 intracranial model).

[0107] 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.

[0108] Female athymic nude mice (Crl:NU(NCr)-Foxn1nu) were used for the tumor xenograft experiments. Three days prior to tumor cell transplantation, an estrogen pellet (0.36 mg of E2, 60-day release; Innovative Research of America, Sarasota, FL) was subcutaneously implanted between the scapulae of all test animals using a sterile trocar. MCF-7 human breast cancer cells were cultured to mid-log phase in RPMI-1640 medium containing 10% fetal bovine serum, 100 units / mL of penicillin G, 100 μg / mL of streptomycin sulfate, 2 mM glutamine, 10 mM HEPES, 0.075% sodium bicarbonate, and 25 μg / mL of gentamicin. On the day of tumor cell transplantation, the cells were trypsinized, pelleted, and resuspended in phosphate-buffered saline at a concentration of 5×10 7 cells / mL. 1×10 6 MCF-7 cells were implanted intracranially into each test mouse.

[0109] Five days after tumor cell transplantation (designated as day 1 of the study), the 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.

[0110] The endpoint was defined as either mortality or the earlier of three-fold survival of the control group. Treatment tolerance was evaluated by frequent observations of body weight measurements and clinical signs of side effects related to treatment. Animals with a weight loss of more than 30% in a single measurement or more than 25% in three measurements were humanely euthanized and classified as treatment-related deaths. Acceptable toxicity was defined as less than 20% mean body weight loss in the group during the study period and at most one treatment-related death out of 10 treated animals, or 10%. At the end of the study, the animals were euthanized by terminal cardiac puncture under isoflurane anesthesia. RAD1901 and fulvestrant concentrations in plasma and tumors were determined using LC-MS / MS.

[0111] Kaplan-Meier survival analysis showed that RAD1901 significantly prolonged survival compared to fulvestrant (P < 0.0001; Figure 12). None of the animals in the control or fulvestrant groups survived beyond day 20 and day 34, respectively, while 41% (5 / 12) of the animals treated with RAD1901 survived until the end of the 54-day study.

[0112] The concentration of RAD1901 in plasma was 738 ± 471 ng / mL, and in intracranial tumors was 462 ± 105 ng / g, which supported the hypothesis that RAD1901 can effectively cross the blood-brain barrier. In contrast, the concentration of fulvestrant was substantially lower in plasma (21 ± 10 ng / mL) and intracranial tumors (8.3 ± 0.8 ng / g).

[0113] I(C). Phase 1 trial of RAD1901 treatment for ER+ advanced breast cancer.

[0114] In the phase 1 trial, safety, tolerance, and pharmacokinetics were evaluated in 44 healthy postmenopausal women. Dose-limiting toxicity was not observed, and the maximum tolerated dose (MTD) was not established. Plasma exposure increased more than dose-proportionally across the tested dose range.

[0115] Subjects Eight postmenopausal women with progressive breast adenocarcinoma (ER+ tumors with ≥1% staining by IHC, HER2-negative tumors with an ECOG performance status of 0 or 1) were enrolled as subjects in this phase 1 trial. Subjects must have received no prior treatment as follows: ● Two or fewer prior chemotherapy regimens in a progressive / metastatic setting ● Six months of prior endocrine therapy and progression on prior endocrine therapy ● Patients with untreated or symptomatic CNS metastases, or prior anticancer treatment within the following windows were excluded: ● Tamoxifen < 14 days prior to the first dose of study treatment ● Fulvestrant < 90 days prior to the first dose of study treatment ● Chemotherapy <28 days before the first dose test treatment ● LHRH analog <12 months before the first dose test treatment DLT criteria ● Any grade 3 or higher non-hematological toxicity (excluding alopecia, nausea, vomiting, or diarrhea not treated with the optimal agent) ● Any grade 3 or higher hematological toxicity ● Any grade toxicity resulting in a test drug interruption exceeding 7 days ● The observation period for dose-limiting toxicity is days 1 - 28 in cycle 1

[0116] Administration and tumor evaluation Subjects were treated with once-daily oral doses of 200 mg or 400 mg and evaluated every 8 weeks until disease progression (Figure 13). The main baseline demographics of 8 postmenopausal women with progressive breast adenocarcinoma enrolled in the phase 1 trial are summarized in Table 1

[0117] The pre-cancer treatment of the subjects is shown in Figure 14A, the RAD1901 treatment received is shown in Figure 14B, and subjects numbered 1 - 3 were treated with once-daily oral doses of 200 mg of RAD1901, and subjects numbered 4 - 7 were treated with once-daily oral doses of 400 mg of RAD1901. Arrows indicate ongoing trials, and bars indicate interrupted treatments. In Figure 14A, "AC" is doxorubicin / cyclophosphamide, and "FAC" is 5-fluorouracil / doxorubicin / cyclophosphamide

[0118] Treatment-emergent adverse events (TEAE) TEAE was recorded throughout the trial. The pre - data are summarized in Table 2. "n" is the number of subjects having an AE related to at least one treatment within a given category. AEs were graded, such as by the Common Terminology Criteria for Adverse Events (CTCAE) v4.0, and any patient having multiple scenarios of the same preferred term was counted only once up to the most severe grade. No deaths or dose - limiting toxicities were observed, and the maximum tolerated dose (MTD) was not constructed. Most AEs were grade 1 or 2. The most common treatment - related AEs were dyspepsia (5 / 8 patients) and nausea (3 / 8 patients). Two serious adverse events (SAEs) were observed. One was grade 3 treatment - related constipation, and the other was shortness of breath (pleural effusion), which was not related to the treatment.

[0119] The subjects who had been treated previously in this phase 1 trial included those who had been treated previously with multiple endocrine and targeted agents, such as CDK4 / 6, PI3K, and mTOR inhibitors. After treatment with once - daily oral administration of RAD1901 at a dose of 200 mg for up to 6 months and then at a dose of 400 mg for up to 2 months, no dose - limiting toxicities were observed. Therefore, RAD1901 showed the potential to treat ER + advanced breast cancer, particularly in subjects who had been treated previously with endocrine and / or targeted agents such as CDK4 / 6, PI3K, and mTOR inhibitors.

[0120] Example II. RAD1901 preferably accumulates in tumors and can be delivered to the brain.

[0121] As described in Example I(A)(i), MCF-7 xenografts were further evaluated for RAD1901 concentration in plasma and tumors using LC-MS / MS. At the end of the study, the concentration of RAD1901 in plasma was 344 ± 117 ng / mL, and in tumors it was 11,118 ± 3,801 ng / mL for a dose level of 60 mg / kg. Similar tumor-to-plasma ratios were observed at lower dose levels when the tumor concentration was approximately 20- to 30-fold higher than in plasma. The RAD1901 levels in plasma, tumors, and brains of mice treated for 40 days are summarized in Table 3. A significant amount of RAD1901 was delivered to the brains of treated mice (see, for example, the B / P ratio (RAD1901 concentration in brain / RAD1901 concentration in plasma)), indicating that RAD1901 was able to cross the blood-brain barrier (BBB). Unexpectedly, RAD1901 preferably accumulated in tumors. See, for example, the T / P (RAD1901 concentration in tumor / RAD1901 concentration in plasma) ratio shown in Table 3.

[0122] Example III. RAD1901 inhibited the ER pathway and degraded the ER.

[0123] III(A). RAD1901 reduced ER involvement in the uterus and pituitary gland of healthy postmenopausal human female subjects.

[0124] Subjects were amenorrheic for at least 12 months and had serum FSH consistent with menopause. Subjects were 40-75 years old with a BMI of 18.0-30 kg / m 2 and had an intact uterus. Patients with evidence of clinically relevant pathology, an increased risk of stroke or past venous thromboembolic events, or use of concomitant medications (paracetamol was allowed up to 3 days prior) within 14 days of registration at the clinical research center were excluded.

[0125] FES-PET was performed 6 days after baseline and exposure to RAD1901 to evaluate ER involvement in the uterus. RAD1901 occupied 83% and 92% of ER in the uterus at dose levels of 200 mg (7 subjects) and 500 mg (6 subjects), respectively.

[0126] FES-PET images showed a significant decrease in the binding of labeled estradiol to both the uterus and pituitary after treatment with 200 mg or 500 mg of RAD1901 (oral administration once a day for 6 days).

[0127] Due to high ER expression, the uterus showed a strong FES-PET signal at baseline before RAD1901 treatment (Figure 15A), cross-sectional view at baseline in the uterine FES-PET scan of Subject 3 treated at a dose level of 200 mg; Figure 15B, sagittal and cross-sectional views, respectively, in the baseline uterine FES-PET scan of Subject 7 treated at a dose level of 500 mg). However, when scanned 4 hours after administration on Day 6 during the trial, the uterus was hardly visible (at or near the background FES-PET signal (Figure 15A, cross-sectional view on Day 6 in the uterine scan of Subject 3; and Figure 15B, sagittal and cross-sectional views on Day 6, respectively, in the uterine scan of Subject 7). Such data was consistent with the degradation of ER and / or competition for binding to the receptor. Figures 15A and 15B also include CT scans of the uterus scanned by FES-PET showing the presence of the uterus before and after RAD1901 treatment.

[0128] The results of the uterine scans of FES-PET were further quantified to show the change in ER binding from baseline to post-administration for 7 subjects, showing Subjects 1 - 3 and Subjects 4 - 7 as examples of the 200 mg dose group and 500 mg dose group, respectively (Figure 15C). RAD1901 showed strong ER involvement at a low dose (200 mg).

[0129] Figure 16 shows representative images of FES-PET scans of the uterus (A) and pituitary gland (B) before treatment (baseline) and after treatment with once-daily oral administration of 500 mg of RAD1901 for 6 days. Figure 16A shows FES-PET scans of the uterus by (a) lateral cross-sectional view, (b) longitudinal cross-sectional view, and (c) longitudinal cross-sectional view.

[0130] Post-treatment FES-PET scans of the uterus and pituitary gland showed no significant signal of ER binding in the uterus (Figure 16A, after treatment) and pituitary gland (Figure 16B, after treatment), respectively.

[0131] Therefore, the results showed that ER was effectively involved in humans orally administered RAD1901 at doses of 200 and 500 mg once daily for 6 days.

[0132] Standard uptake values (SUVs) for the uterus, muscle, and bone were calculated and summarized in Tables 4 and 5 for treatment with RAD1901 orally administered once daily at 200 mg and 500 mg, respectively. The post-treatment uterine signal was very close to the level from "non-target tissues", suggesting complete attenuation of FES-PET uptake after RAD1901 treatment. Little change was observed for pre-treatment versus post-treatment PET scans in tissues that did not significantly express estrogen receptor.

[0133] Therefore, RAD1901 or its salt or solvate (e.g., hydrate) can be used in the treatment of cancers and / or tumor cells (e.g., breast cancer, uterine cancer, and ovarian cancer) having overexpression of ER without having adverse effects on other organs (e.g., bone, muscle). RAD1901 or its salt or solvate (e.g., hydrate) is particularly useful in treating metastatic cancers and / or tumors having overexpression of ER in other organs, such as original breast cancer, uterine cancer, and / or ovarian cancer that metastasize to other organs (e.g., bone, muscle), without having adverse effects on such organs, for treating breast cancer, uterine cancer, and / or ovarian cancer lesions in such organs.

[0134] III(B). RAD1901 decreased ER expression and inhibited the ER pathway.

[0135] III(B)(i)(1) Comparison of RAD1901 and fulvestrant in MCF-7 and T47D cell lines

[0136] The effects of RAD1901 and fulvestrant were compared at various concentrations of 0.01 μM, 0.1 μM, and 1 μM using MCF7 and T47D cell lines (both are human breast cancer cell lines) (Figure 17A for the MCF7 cell line assay and Figure 17B for the T47D cell line). Three ER target genes, progesterone receptor (PgR), growth regulation by estrogen in breast cancer 1 (GREB1), and trefoil factor 1 (TFF1), were used as markers. RAD1901 caused almost complete degradation of ER and inhibited ER signaling (Figures 17A - B). Unexpectedly, in Examples I(A) and I(B), as disclosed above, RAD1901 was as effective as or more effective than fulvestrant in inhibiting tumor growth and causing tumor regression.

[0137] III(B)(i)(2) RAD1901 treatment resulted in degradation of ER and abrogation of ER signaling in the MCF-7 xenograft model-II as described above in Example I(A)(ii)(1).

[0138] RAD1901 treatment caused degradation of ER in vivo (Figures 18A - B, Student's t-test: *p-value < 0.05, **p-value < 0.01) and inhibited ER signaling in vivo (Figures 19A and 19C, Student's t-test: *p-value < 0.05, **p-value < 0.01).

[0139] Tumors collected from MCF-7 xenografts 2 hours after the final administration of RAD1901 (30 mg / kg, 60 mg / kg, once daily oral administration) or fulvestrant (3 mg / dose, once weekly subcutaneous injection) showed a significant decrease in the expression of ER and PR (Figures 18A - B). Tumors collected from MCF-7 xenografts 8 hours after the final administration of fulvestrant treatment showed a change in the expression of PR and ER. However, tumors collected from MCF-7 xenografts 8 hours after the final administration of RAD1901 treatment showed a decrease in the expression of PR and ER (Figures 18A and 18C).

[0140] Tumors collected from MCF-7 xenografts 8 or 12 hours after a single administration of RAD1901 (30 mg / kg, 60 mg / kg, or 90 mg / kg, once daily oral administration) showed a rapid decrease in the expression of PR (Figure 19A). Tumors collected from MCF-7 xenografts 4 or 24 hours after the 7th administration of RAD1901 (30 mg / kg, 60 mg / kg, or 90 mg / kg, once daily oral administration) showed a consistent and stable inhibition of ER signaling (Figure 19B). Quantification of Western blot analysis of tumors collected from MCF-7 xenografts at various time points during treatment with RAD1901 (30 mg / kg, 60 mg / kg, or 90 mg / kg, once daily oral administration) showed a dose-dependent decrease in PR (Figure 19C).

[0141] RAD1901 treatment caused a rapid decrease in proliferation in the MCF-7 xenograft model. For example, tumors collected from the MCF-7 xenograft model 8 hours after a single administration of RAD1901 (90 mg / kg, once daily oral administration) and 24 hours after the 4th administration of RAD1901 (90 mg / kg, once daily oral administration) were sectioned and stained, showing a rapid decrease in the proliferation marker Ki67 (Figures 20A and 20B).

[0142] These results suggest that RAD1901 treatment results in in vivo degradation of ER and inhibition of ER signaling in WT ER xenografts.

[0143] III(B)(i)(3) As described above in Example I(A)(ii), RAD1901 treatment resulted in the degradation of ER and the abolition of ER signaling in the PDx-4 model.

[0144] RAD1901 treatment caused a rapid decrease in growth in the PDx-4 model. For example, at 4 hours after the final dose on the last day of the 56-day efficacy study, tumors were excised from PDx-4 models treated with RAD1901 (30, 60, or 120 mg / kg, administered orally once a day) or fulvestrant (1 mg / animal, once a week), which showed a rapid decrease in the proliferation marker Ki67 compared to PDx-4 models treated with fulvestrant (Figure 21).

[0145] These results suggest that RAD1901 treatment results in the degradation of ER and the inhibition of ER signaling in vivo in WT ER xenografts.

[0146] III(B)(ii) RAD1901 treatment resulted in a decrease in ER signaling in the mutant ER xenograft model (PDx-5) as described above in Example I(A)(iii)(1).

[0147] Tumors were harvested at the time point indicated after the last day of dosing (unless otherwise specified) and homogenized in RIPA buffer with protease and phosphatase inhibitors using a Tissuelyser (Qiagen). Equal amounts of protein were separated by MW, transferred to nitrocellulose membranes, and blotted with the following antibodies as described in the Materials and Methods section: Progesterone Receptor (PR, Cell Signaling Technologies; 3153).

[0148] The band was quantified using 1D Quant software (GE), and the PR (Allred score) obtained from the PDx-5 model, as described in Example I(A)(iii)(1), is shown in Figure 22. Fulvestrant had little effect on the expression of PR, whereas RAD1901 showed efficacy at both doses of 60 mg / kg and 120 mg / kg (oral administration once a day, Figure 22).

[0149] These results indicate that for tumors expressing certain ERα mutations (e.g., Y537S), RAD1901 is more effective than fulvestrant in inhibiting tumor growth, regardless of whether the tumor responded to fulvestrant / tamoxifen treatment (Figure 9 for PDx-5 and Figure 10 for PDx-6), and is particularly effective in inhibiting the growth of tumors that responded little to fulvestrant treatment (e.g., subcutaneous injection once a week at a dose of 3 mg / dose, Figure 9 for PDx-5). Furthermore, for tumors that responded little to fulvestrant treatment (e.g., PDx-5), RAD1901 was effective in reducing the expression of PR in vivo, whereas fulvestrant was not (Figure 23).

[0150] Example IV Effects of RAD1901 treatment on uterine tissue and / or BMD IV(A(1)): RAD1901 antagonized estradiol stimulation of uterine tissue.

[0151] The uterine tropic effect of RAD1901 was investigated in immature rats by evaluating changes in uterine weight, tissue, and C3 gene expression. The results from a representative study are shown in Figure 23.

[0152] Evaluation of uterine tropic activity Sprague-Dawley neonatal rats were weaned at 19 days of age, randomized into groups (n = 4), and administered vehicle (aqueous methylcellulose), E2 (0.01 mg / kg), raloxifene (3 mg / kg), tamoxifen (1 mg / kg), RAD1901 alone (0.3 - 100 mg / kg), or RAD1901 (0.01 - 10 mg / kg) combined with E2 (0.01 mg / kg) either subcutaneously or orally once daily for 3 consecutive days (see the above reagents). Twenty-four hours after the final administration, all animals were sacrificed by carbon dioxide inhalation. Body weight and wet uterine weight were recorded for each animal. Similar assays were also performed in rats and mice (Charles River Laboratories, Montreal, QC) with RAD1901 (0.03 - 100 mg / kg).

[0153] Fresh uterine tissue from each rat was fixed in 4% paraformaldehyde, dehydrated in ethanol, and embedded in JB4 plus plastic resin. Sections were cut at 8 μm and stained with 0.1% toluidine blue O. The thickness of the endometrial epithelial tissue was measured using a Zeiss Axioskop 40 microscope and the Spot Advanced program, and the mean of 9 measurements per specimen was calculated.

[0154] Component 3 (C3) gene expression of uterine components To determine the relative expression levels of C3 in the treated uterine tissue, RNA was extracted from the remaining tissue using a Micro to Midi Total RNA purification kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The RNA was quantified and equal amounts were reverse transcribed using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA).

[0155] Quantitative PCR was performed using the ABI Prism 7300 System (Applied Biosystems). PCR was carried out using Taqman Universal Master Mix with probe sets for C3 and for 18S ribosomal RNA as the reference gene. The thermal cycle conditions included an initial denaturation step at 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute.

[0156] Relative gene expression was determined by normalizing each sample to the endogenous control (18S) and comparing to a calibrator (vehicle). Relative gene expression was determined using the equation: 2-ΔΔCt where Ct = cycle threshold or the cycle number at which the PCR product was first detected, ΔCt = normalized sample value, and ΔΔCt = normalized difference between the dosed subject and the vehicle. Determination of five replicate gene expressions was performed for each dose within each study.

[0157] 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, while RAD1901 treatment at doses in the range of 0.3 - 100 mg / kg had no significant effect on uterine wet weight (Figure 23A). The data shown (Figure 23A) are mean (± standard error), n = 4 rats per group, P vs vehicle: * < 0.05; vs E2: ‡ < 0.05. Furthermore, when co-administered with E2 (0.01 mg / kg), RAD1901 antagonized E2-mediated uterine stimulation in a dose-dependent manner, showing significant inhibition of uterotropic activity at doses of 0.1 mg / kg and above and complete inhibition at 3 mg / kg. The EC 50 of RAD1901 was approximately 0.3 mg / kg. Similar results were obtained in mice in which administration of 0.03 - 100 mg / kg of RAD1901 also had no effect on uterine wet weight or epithelial thickness (data not shown).

[0158] Treatment-dependent changes in uterine tissue were further investigated by quantitative microscopic histology. There were statistically significant increases in the thickness of the endometrial epithelium after treatment with E2 at both 0.01 and 0.3 mg / kg (Figure 23B). A significant increase in epithelial thickness was also observed after treatment with tamoxifen (1 mg / kg) or raloxifene (3 mg / kg). In contrast, RAD1901 treatment did not increase the thickness of the endometrial epithelium up to the highest evaluated dose of 100 mg / kg. Representative images of the endometrial epithelium are shown in Figure 23C.

[0159] Consistent with the changes in both uterine weight and endometrial epithelial thickness, E2, tamoxifen, and raloxifene all significantly increased the expression of the estrogen-regulated component gene C3 (Figure 23D). In contrast, RAD1901 did not increase C3 gene expression at any of the tested doses (0.3 - 100 mg / kg). Furthermore, RAD1901 at 1, 3, and 10 mg / kg significantly suppressed C3 gene expression stimulated by E2.

[0160] RAD1901 did not stimulate the uterus of immature female rats Immature female rats were orally administered once daily for 3 consecutive days with vehicle (VEH), estradiol (E2), raloxifene (RAL), tamoxifen (TAM), RAD1901, or RAD1901 + E2. Uterine wet weight was measured. The data shown (Figure 23A) are mean (± standard error of the mean), n = 4 rats per group, P vs vehicle: * < 0.05; vs E2: ‡ < 0.05.

[0161] Example II(A)(2). Treatment with RAD1901 prevented bone mass loss in ovariectomized rats. The bone-specific effects of RAD1901 were tested in ovariectomized rats.

[0162] As a model of postmenopausal bone loss, ovariectomy was performed on anesthetized female Sprague-Dawley rats, and sham surgery was performed as a control. After surgery, ovariectomized rats with 20 animals per group were treated once a day for 4 weeks with vehicle, E2 (0.01 mg / kg), or RAD1901 (0.1, 0.3, 1, 3 mg / kg) and administered as described above. Animals in the sham surgery group were treated with vehicle. All animals were sacrificed by carbon dioxide inhalation 24 hours after the final dose. Bone mineral density was evaluated at baseline and 4 weeks after treatment using PIXImus dual-energy X-ray absorptiometry.

[0163] At necropsy, the left femur of each animal was removed, dissected free of soft tissue, and stored in 70% ethanol prior to analysis. Detailed qualitative and quantitative 3D evaluations were performed using a micro-CT40 system (Scanco Systems, Wayne, PA). For each specimen, 250 image slices of the distal femoral diaphysis were required. Morphological parameters were determined using a direct 3D approach in a preselected analysis region. Parameters determined in the trabecular bone included bone volume density, bone surface density, trabecular number, trabecular width, trabecular separation, connectivity density, and apparent bone density.

[0164] After ovariectomy, untreated (vehicle control) rats had decreased bone mineral density in both the entire femur and lumbar spine compared to baseline (Table 6). Treatment with E2 was associated with prevention of bone loss in both the femur and spine. Treatment with RAD1901 resulted in a dose-dependent and statistically significant suppression of bone loss induced by ovariectomy (data shown for the 3 mg / kg treatment group). At doses of 0.1 mg / kg to 3 mg / kg, bone mineral density in rats treated with RAD1901 was complete and not statistically different from the group treated with E2.

[0165] Micro-CT analysis of the distal femur (Table 7) showed that ovariectomy induced significant changes in a number of major microarchitecture parameters when compared to sham-operated animals. These changes were consistent with a decrease in bone mass and included a decrease in bone volume, trabecular number, thickness, and density, as well as an increase in trabecular separation. Consistent with the preservation of bone mineral concentration observed after treatment with RAD1901, significant preservation of trabecular architecture was observed in the major microstructural parameters (Table 7).

[0166] Example IV (B): Phase 1 Dose Escalation Study of RAD101 in Healthy Postmenopausal Women In the Phase 1 study, safety, tolerability, and pharmacokinetics were evaluated in 44 healthy postmenopausal women. Dose-limiting toxicity (DLT) was not observed and a maximum tolerated dose (MTD) was not established. Plasma exposure increased more than dose-proportionally across the dose range tested.

[0167] Subjects Forty-four healthy postmenopausal women were enrolled as subjects in this Phase 1 study. Subjects had been amenorrheic for at least 12 months and serum FSH was consistent with menopause. Subjects were 40 - 75 years old with a BMI of 18.0 - 30 kg / m 2 Patients with evidence of clinically relevant pathology, a history of stroke or increased risk of past venous thromboembolic events, or use of concomitant medications (paracetamol was allowed up to 3 days prior) within 14 days of enrollment in the clinical research center were excluded.

[0168] Administration Subjects were treated once daily for 7 days with placebo or at least one oral dose at dose levels of 200 mg, 500 mg, 750 mg, and 1000 mg, respectively, after a light breakfast. The major baseline demographics of the 44 postmenopausal women enrolled in the Phase 1 study are summarized in Table 8.

[0169] Treatment Emergent Adverse Events (TEAE) TEAE was recorded and the most frequently occurring adverse events (AEs) (occurring in more than 10% of patients in the active group with any related TEAE) were summarized in Table 9. "n" is the number of subjects having an AE related to at least one treatment in a given category. AEs were graded as in the Common Terminology Criteria for Adverse Events (CTCAE) v4.0, and any patient with multiple scenarios of the same preferred term was counted only once up to the most severe grade. Dose-limiting toxicity was not observed and a maximum tolerated dose (MTD) was not constructed.

[0170] Pharmacokinetic evaluation During the study period for the analysis of RAD1901 in plasma, a series of blood samples were collected. 5 mL of blood samples were collected via an indwelling IV catheter or by direct venipuncture into tubes containing K3-EDTA as an anticoagulant, respectively. Steady state was achieved on day 5 after treatment. The geometric mean (Geo-Mean) plasma concentration-time profile of RAD1901 was evaluated. In the study, the plasma pharmacokinetic results of the group (N = 35) treated with RAD1901 (200, 500, 750, or 1,000 mg) on day 7 are provided as an example in Table 10 and Figure 24. The median t 1 / 2 was 37.5 - 42.3 hours (Table 10). After multiple administrations of RAD1901, the median t max was 3 - 4 hours after dosing.

[0171] Example V(A)-1. Modeling of RAD1901-ERα binding using a selected published ER structure. Unless otherwise described, when structures are shown in their stick models, each end of the bond is colored the same color as the atom to which it is bonded, with gray for carbon, red for oxygen, blue for nitrogen, and white for hydrogen.

[0172] Fourteen published structures (i.e., models) of the ERα ligand-binding domain (LBD) complexed with various ER ligands were selected from 96 published models by careful evaluation. One of these 14 models was 3ERT (human ERα LBD (OHT) bound to 4-hydroxytamoxifen). OHT is an active metabolite of tamoxifen and is a first-generation SERM that acts as an antagonist in breast tissue.

[0173] In 3ERT (Figs. 25 and 26), the ERα binding site introduces a three-layer "helix sandwich" that forms a hydrophobic pocket containing helix 3 (H3), helix 5 (H5), and helix 11 (H11) (Fig. 25). The dotted boxes in Fig. 26 represent the binding site and residues within the binding site that are important or achieved by OHT binding. OHT acts as an antagonist by replacing H12 at the site that binds the LXXLL coactivator(s). OHT occupies the space normally filled by L540 and modifies the conformation of four residues (G521, H524, L525, and M528) on the C-terminus of helix 11. OHT also forms a salt bridge with D351, resulting in charge neutralization.

[0174] The other 13 ERα LBD-ER ligand models were compared with 3ERT. The differences in their residue structures are summarized in Table 12. Superposition of the ERα structures of the 14 models (Fig. 27) shows that these structures differ significantly at residues E380, M421, G521, M522, H524, Y526, S527, M528, P535, Y537, L540, and various combinations of these.

[0175] The calculation of the root mean square deviation (RMSD) for any pair of the 14 models is summarized in Table 13. Structures were considered to overlap if the RMSD was less than 2 Å. Table 13 shows that all 14 models had an RMSD of less than 1.5 Å. The use of the format analysis suggested that 1R5K and 3UUC were not very similar to the other models (the analysis is not shown). Thus, 1R5K and 3UUC were considered to be distinct separate structural clusters tested.

[0176] The ERα residues bound by the ligand in the 14 models are summarized in Table 14. Table 14 also shows the EC in the ERα LBD-antagonist complex 50 is shown. Of the 14 models, 13 models showed H-bonding interactions between the ligand and E353, 12 models showed π-interactions between the ligand and F404, 5 models showed H-bonding interactions between the ligand and D351, 6 models showed H-bonding interactions between the ligand and H524, 4 models showed H-bonding interactions between the ligand and R394, and 1 model (3UUC) showed an interaction between the ligand and T347.

[0177] Each of the 14 models was used to dock a random library of 1,000 compounds in addition to the ligand, and this model was published with (well-known antagonists) to determine whether this model could identify and rank well-known antagonists. If this model could identify well-known antagonists, it was determined that this model could predict the structure of the published ligand itself. Then, EF 50 was calculated to quantify the strength of the model to know how much better it was than random selection. RAD1901 was docked to the selected model (e.g., Figures 28 - 32). The docking scores of the published ligand and RAD1901 in the model were determined. Also, EC 50It was also determined. Visual inspection of RAD1901 showed that it "followed" the interactions shown with the ligands published at 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. No steric clashes were observed. In certain embodiments, for example, at 1R5k and 2BJ4, RAD1901 had a higher docking score than the published ligands.

[0178] The evaluation results of nine models (1ERR, 3ERT, 3UCC, 2IOK, 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ) are summarized in Table 15.

[0179] 1ERR and 3ERT were unable to predict the exact structure of their crystal ligands. RAD1901 did not dock to 3UCC. In 2IOK - RAD1901, tetrahydronaphthalene bound in an unconventional manner.

[0180] The major difference among the models 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ was the C-terminal residues (G521 - M528) of helix 11.

[0181] Figures 28A - B show the modeling of RAD1901 - 1R5K (A) and GW5 - 1R5K (B). RAD1901 bound through H-bonding interactions with E353, R394, and L536 and a π - interaction with F404.

[0182] Figures 29A - B show the modeling of RAD1901 - 1SJ0 (A) and E4D - 1SJ0 (B). RAD1901 bound through H-bonding interactions with E353 and D351 and a π - interaction with F404.

[0183] Figures 30A - B show the modeling of RAD1901 - 2JFA (A) and RAL - 2JFA (B). RAD1901 bound through a π - interaction with F404.

[0184] Figures 31A - B show the modeling of RAD1901 - 2BJ4 (A) and OHT - 2BJ4 (B). RAD1901 bound through H - bond interactions with E353 and R394 and p - interaction with F404.

[0185] Figures 32A - B show the modeling of RAD1901 - 2IOK (A) and IOK - 2IOK (B). RAD1901 bound through H - bond interactions with E353, R394, and D351 and p - interaction with F404.

[0186] The ligands disclosed in the model have the following structures: [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula]

[0187] Example V(A) - 2. Induced - fit docking (IFD) of ERα by RAD1901 and fulvestrant In ERα, the binding conformation of RAD1901 was further optimized by IFD analysis of five ERα crystal structures 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. The IFD analysis revealed the receptor flexibility (upon ligand binding) to fit its exact binding conformation.

[0188] For each ligand (e.g., RAD1901 and fulvestrant), a library of different conformations was generated by searching for local minima as a function of rotation for rotatable bonds. The library of RAD1901 had 25 different conformations.

[0189] Five ERα crystal structures were prepared and minimized. The corresponding ligands in the published X-ray structures were used to define the ERα binding pocket.

[0190] The conformation of RAD1901 was docked to the prepared ERα structures, enabling the induction of side-chain or backbone motions to residues located in the binding pocket. These motions allowed the ERα to change its binding site, thus adapting more closely to the shape and binding mode of the RAD1901 conformation. In some cases, small backbone relaxations and significant side-chain conformational changes in the receptor structure were enabled in the IFD analysis.

[0191] Empirical scoring functions were used to estimate the ligand binding free energy to provide a docking score or G score. The G score is also known as GlideScore and may be used synonymously with the docking score in this example. The docking score was an estimate of the binding affinity. Thus, the lower the value of the docking score, the "better" the ligand bound to its receptor. Docking scores of -13 to -14 corresponded to very good binding interactions.

[0192] The conformations of RAD1901 obtained from the IFD analysis with 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ were superimposed to show their differences (shown in the stick models in FIGS. 33-35). All the bonds in each RAD1901 conformation were shown in the same color in FIGS. 33, 34, and 35A.

[0193] The three-dimensional structure of RAD1901 obtained from the IFD analysis with 1R5K (cyan) and 2OUZ (yellow) had the N-benzyl-N-ethylaniline group of RAD1901 on the front (Figure 33). The three-dimensional structure of RAD1901 obtained from the IFD analysis with 2BJ4 (green) and 2JFA (pink) had the N-benzyl-N-ethylaniline group of RAD1901 on the back (Figure 34). The three-dimensional structures of RAD1901 obtained from the IFD analysis with 2BJ4 (green), 2JFA (pink), and 1SJ0 (brown) were almost the same as shown by their superposition (Figures 34A and 34B). The IFD docking scores of RAD1901 are summarized in Table 16.

[0194] The IFD of RAD1901 with 2BJ4 showed hydrogen bond interactions with E353 and D351 and π interaction with F404 (Figures 36A - 36C). Figure 36A shows the region within the binding site suitable for the H-bond acceptor group (red), H-bond donor group (blue), and hydrophobic group (yellow). In Figures 36A - 36B, light blue was for the carbon of RAD1901. Figures 37A - 37C show the interaction of the protein surface of the IFD of RAD1901 and 2BJ4. Figures 37A and 37B are front views, and Figure 37C is a side view. The molecular surface of RAD1901 was blue in Figure 37A and green in Figure 37C. Figures 37B and 37C show the electrostatics of the solvent-accessible surface of ERα, with red indicating negative and blue indicating positive.

[0195] As described above, a similar IFD analysis was performed on fulvestrant with 2BJ4. The IFD of fulvestrant - 2BJ4 resulted in a G score of -14.945 and showed hydrogen bond interactions with E353, Y526, and H524, as well as π interaction with F404 (Figures 38A - 38C). Figure 38A shows the region within the binding site suitable for the H-bond acceptor group (red), H-bond donor group (blue), and hydrophobic group (yellow). In Figure 38A, light blue was for the carbon in RAD1901.

[0196] Figures 39A and 39B show that RAD1901 docked to 2BJ4 by IFD and fulvestrant had both π interactions with F404 and hydrogen bond interactions with E353. Further, RAD1901 had hydrogen bond interactions with D351 (blue represents the molecular surface of RAD1901, Figure 39B), while fulvestrant had hydrogen bond interactions with Y526 and H524 (green represents the molecular surface of fulvestrant, Figure 39C). Superimpositions of 2BJ4 docked with RAD1901 and fulvestrant are shown in Figures 40A and 40B. In Figure 40A, green represents the molecular surface of fulvestrant and blue represents the molecular surface of RAD1901. In Figure 40B, the brown structure is fulvestrant and the blue structure is RAD1901.

[0197] Example V(A)-3. Modeling evaluation of selected ERα mutations. The effects of various ERα mutations on the C-terminal ligand-binding domain were evaluated. The specific ERα mutations evaluated were the Y537X mutants (where X is S, N, or C), D538G, and S463P.

[0198] The Y537 residue in helix 12. Once phosphorylated, it can regulate ligand binding, homodimerization, and DNA binding, enabling ERα to escape phosphorylation-mediated control and potentially provide cells with selective tumorigenic advantages. Additionally, it can change the conformation that makes the receptor structurally active.

[0199] The Y537S mutation prefers a transcriptionally active closed pocket structure regardless of whether it is occupied by a ligand. A closed but unoccupied pocket can account for the constitutive activity of ERα (Carlson et al. Biochemistry 36:14897-14905 (1997)). Ser537 constructs a hydrogen bond interaction with Asp351, resulting in a changed structure of the loop between helices 11 and 12 and the burial of Leu536 at a position where the solvent is difficult to reach. This can contribute to the constitutive activity of the Y537S mutant protein. The Y537S surface mutation does not affect the surface of the LBD pocket.

[0200] Y537N is frequently seen in ERα-negative metastatic breast cancer. Mutations at this site may enable ERα to escape phosphorylation-mediated control and potentially provide cells with selective advantages in tumor formation. Specifically, the Y537N substitution induces a structural change in ERα that can mimic hormone binding, does not affect the receptor's ability to dimerize, but confers a constitutive transactivation function to the receptor (Zhang et al. Cancer Res 57:1244-1249 (1997)).

[0201] Y537C has an effect similar to that of Y537N.

[0202] D538G is more preferably an active structure, but can change the overall energy landscape by stabilizing both active and inactive structures. This can result in the constitutive activity of this mutant in the absence of hormone, as observed in hormone-resistant breast cancer (Huang et al., “A newfound cancer activating mutation reshapes the energy landscape of estrogen-binding domain,” J. Chem. Theory Comput. 10:2897-2900 (2014)).

[0203] None of these mutations are expected to affect the ligand-binding domain or, specifically, to interfere with RAD1901 binding. Y537 and D538 can change the conformation that leads to activation of the constitutive receptor independent of ligand binding.

[0204] Example V(B). In Vitro Binding Assays of Wild-Type and LBD Mutant ERα Constructs with RAD1901 and Other Compounds. In vitro binding assays of wild-type (WT) and LBD mutant ERα constructs with RAD1901 showed that RAD1901 bound to mutant ERα with an affinity similar to that of WT ERα.

[0205] WT and LBD mutant ERα constructs were prepared by expressing and purifying the corresponding LBD residues 302 - 552 with an N-terminal thioredoxin and a 6xHis tag cleaved by TEV protease.

[0206] Fluorescence polarization (FP) was used to determine the binding of test compounds (RAD1901, fulvestrant, bazedoxifene, raloxifene, tamoxifen, and AZD9496) to ERα using 2 nM fluoromone, 100 nM WT or LBD mutant ERα constructs as per the manufacturer's instructions (Polar Screen, Invitrogen). Each set was performed in duplicate, and the IC 50 for a certain test compound was determined to determine the IC for different ERα constructs (Figure 41 for the RAD1901 binding assay).

[0207] As described above, the foregoing is merely intended to illustrate various embodiments of the present invention. The specific modifications discussed above should not be construed as limitations on the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present invention, and thus it should also be understood that such equivalent embodiments should be included herein. All references cited herein are incorporated by reference as if fully set forth herein.

[0208] The following are examples of aspects of the present invention. [1] A method of inhibiting tumor growth or causing tumor regression in a subject having drug-resistant estrogen receptor alpha-positive cancer, comprising administering to the subject a therapeutically effective amount of RAD1901 having the structure:

Chemical formula

Chemical formula

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

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

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

[13] The method according to [1] or [2], wherein the tumor shows resistance to a drug selected from the group consisting of anti-estrogens, aromatase inhibitors, and combinations thereof.

[14] The method according to

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

[15] The method according to

[13] , wherein the aromatase inhibitor is aromasin.

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

[17] The method according to

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

Claims

1. A composition for inhibiting tumor growth or causing tumor regression in premenopausal or postmenopausal women having estrogen receptor alpha positive breast cancer, said composition comprising a therapeutically effective amount of a structure: 【Chemical 1】 comprising RAD1901 having the formula or a salt or solvate thereof, wherein the estrogen receptor alpha has a mutation selected from Y537S, Y537N, Y537C, D538G and S463P, and said therapeutically effective amount of RAD1901 is from 150 mg to 2,000 mg.

2. The composition according to claim 1, wherein said cancer is metastatic cancer.

3. The composition according to claim 1, wherein said mutation is Y537S.

4. The composition according to claim 1, wherein said woman has osteoporosis or a high risk of osteoporosis.

5. The composition according to claim 1, wherein said woman is a postmenopausal woman who has relapsed or progressed after previous treatment with SERM and / or AI.

6. The composition according to claim 1, wherein said therapeutically effective amount is from about 150 to about 1,500 mg per day.

7. The composition according to claim 1, wherein said salt is RAD1901 dihydrochloride.

8. The composition according to claim 6, wherein said therapeutically effective amount is 200 mg, 400 mg, or 500 mg.

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