Rasofoxifen treatment for aromatase-resistant ER+ cancer

Abstract

The present disclosure provides a method for the treatment of estrogen receptor-positive (ER) breast cancer in patients who have progressed on aromatase inhibitors. + The present invention provides an effective amount of lasofoxifene, or a pharma- ceutically acceptable salt or functional derivative thereof, for use in a method for treating cancer. + Cancer is of particular interest: new in vitro and animal model experiments clearly show that lasofoxifene is more effective than fulvestrant (ICI) in a letrozole-inducible, AI-resistant breast tumor model (MCF-7 LTLT cells) that does not express ERα-activating mutations.

Description

[Technical Field]

[0001] 1. Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 280,769, filed November 18, 2021 (the disclosure thereof is incorporated herein by reference in its entirety). [Background technology]

[0002] 2. Background of the Invention Estrogen receptor positive (ER) + Breast cancer is a type of cancer that expresses estrogen receptor α (ERα), which is encoded by the ESR1 gene. Approximately 70% of breast cancers express ERα. + Therefore, it is treated with drugs that deplete circulating estrogen levels or block estrogen signaling in cancer cells (collectively, endocrine therapy). Selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), and aromatase inhibitors (AIs) are the main classes of endocrine therapy drugs. Endocrine therapy is used in ER + It resulted in a significant improvement in outcomes for women with breast cancer. However, the effectiveness of endocrine therapy is limited by intrinsic and, importantly, acquired endocrine resistance. In response to the selective pressures imposed by endocrine therapy (particularly aromatase inhibitors), ER + Tumors develop various evasion mechanisms. These include the acquisition of gain-of-function mutations in the ESR1 gene, which alters the ligand-binding domain of the ERα receptor, constitutively activating the receptor in the presence of low levels of estrogen or estrogen absence. Despite the benefits of endocrine therapy, ER + The majority of patients with this condition eventually develop resistance and their condition progresses. Rasofoxifene, a selective estrogen receptor modulator (SERM), is used in postmenopausal women with wild-type estrogen receptors, no history of breast cancer, and treatment for osteoporosis, to improve ER. +It has been shown to reduce the risk of breast cancer. LaCroix et al., J. Natl. Cancer Inst. 102:1706-1715 (2010). Raloxifene is an ER that has acquired a gain-of-function mutation in the ligand-binding domain of the ERα receptor + It was later shown to retain the ability to inhibit cancer progression. U.S. Patent Nos. 10,258,605; 10,905,659; WO2019 / 199891. Raloxifene as a single agent in the treatment of premenopausal and postmenopausal women with locally advanced or metastatic ER + The effectiveness of raloxifene as a single agent in the treatment of breast cancer is currently being confirmed in a Phase 2 clinical trial, NCT03781063 (ELAINE trial). A clinical trial to confirm the effectiveness of raloxifene in combination with the CDK4 / 6 inhibitor, abemaciclib, is also underway in a similar population of breast cancer patients (all of whom have gain-of-function mutations in the ERα receptor) (NCT04432454 (ELAINE II trial)). Raloxifene has been shown to be effective in inhibiting the progression of ER + breast cancer through the acquisition of ESR1 gain-of-function mutations, but ER + cancer cells develop other mechanisms to evade endocrine therapy. Therefore, there is still a need for therapeutic agents that are effective in inhibiting the progression and metastasis of ER + cancer that progresses under endocrine therapy and lacks mutations in the ESR1 gene.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0004] [Non-Patent Document 1] LaCroix et al., J. Natl. Cancer Inst. 102:1706-1715 (2010) [Overview of the project] [Means for solving the problem]

[0005] 3. Summary of the Invention In vitro and animal model experiments newly demonstrate that rasofoxifene is more effective than fulvestrant (ICI) in a letrozole-induced, AI-resistant breast tumor model (MCF-7 LTLT cells) that does not express ERα-activating mutations. These data show that rasofoxifene is effective in AI-resistant ERα-resistant cells that do not express ERα-activating mutations. + It clearly demonstrates that it is an effective treatment for cancer.

[0006] Therefore, in the first phase, in patients whose condition progressed under aromatase inhibitors, estrogen receptor positive (ER) + A method for reducing the progression of cancer is provided, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene. The method comprises administering an effective amount of rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug, or functional derivative to the patient.

[0007] In some embodiments, the above ER + The cancer is locally advanced or metastatic breast cancer, and here, if necessary, the cancer is HER2-negative.

[0008] In some embodiments, the aromatase inhibitor is exemestane (Aromasin®), letrozole (Femara®), or anastrozole (Arimidex®).

[0009] In some embodiments, the method further comprises an earlier step of determining that the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0010] In some embodiments, rasofoxifene is administered as rasofoxifene tartrate.

[0011] In some embodiments, rasofoxifen is administered orally, intravenously, transdermally, topically, or via a vaginal ring.

[0012] In some embodiments, rasofoxifene is administered orally. In certain of these embodiments, rasofoxifene is administered orally at a dose of 5 mg / day to about 10 mg / day.

[0013] In some embodiments, the method further comprises the step of treating the patient with at least one further endocrine therapy.

[0014] In some embodiments, the method further comprises the step of administering an effective amount of a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor. In certain embodiments, the CDK4 / 6 inhibitor is palbociclib, abemaciclib, or ribociclib. In some embodiments, the CDK4 / 6 inhibitor is abemaciclib.

[0015] In some embodiments, the method includes the step of administering an effective amount of AKT inhibitor. In certain embodiments, the AKT inhibitor is an afrecertib.

[0016] In some embodiments, the above method further includes the step of administering an effective amount of mTor inhibitor.

[0017] 4. Brief explanation of the drawing These and other features, aspects, and advantages of the present invention will be better understood with reference to the following description and accompanying drawings. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 shows a comparison of variant counts in AI-resistant breast tumor model MCF-7 LTLT cells against two publicly available reference genomes of MCF7 "WT" from the literature.

[0019] [Figure 2] Figure 2 shows data demonstrating that rasofoxifene inhibits primary tumor growth in MCF7 LTLT (AI-resistant ER+ cells lacking ESR1 gain-of-function mutations) tumors. The data are from in vivo imaging showing total photon flux quantified over time using live imaging software for each group. Mice were treated with vehicle, palbociclib, rasofoxifene, fulvestrant (ICI), rasofoxifene + palbociclib, or fulvestrant (ICI) + palbociclib.

[0020] [Figure 3] Figure 3 is a histogram summarizing data showing that rasofoxifen inhibits primary tumor growth in AI-resistant ER+ cells lacking ESR1 gain-of-function mutations. The histogram above shows the total photon flux of mammary glands at day 104. N=6-12 glands ± SEM. P values ​​are *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001.

[0021] [Figure 4] Figure 4 shows data demonstrating that rasofoxifen inhibits the increase in primary tumor weight in AI-resistant ER+ cells lacking ESR1 gain-of-function mutations. The histogram shows the average mammary gland weight on the day of slaughter. N=6-12 glands ± SEM. P values ​​are *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

[0022] [Figure 5] Figure 5 shows data demonstrating that tumor area is reduced by rasofoxifene and rasofoxifene + palbociclib. Percentage tumor area vs. total glandular area as revealed via H&E analysis of breast tissue sections. Histogram showing the percentage of tumor area relative to total glandular area of ​​H&E sections. N=3-6 glands ± SEM. P values ​​are *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001.

[0023] [Figure 6-1] Figure 6 shows data demonstrating that rasofoxifen, both as a monotherapy and in combination with palbociclib, reduces the Ki67 proliferation index in the Let+ cohort. Figure 6A shows % Ki67 in comparison of vehicle vs. Let- vs. Let+ cohorts. Figures 6B and 6C show % Ki67 for the Let- and Let+ cohorts, respectively. N=3-6 glands ± SEM. P values ​​are *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001. [Figure 6-2] Same as above.

[0024] [Figure 7A] Figure 7 shows exovivographic radius measurements in the liver, lungs, and brain. Exovivographic imaging of organs removed at slaughter. Radiance was measured in the liver (Figure 7A), lungs (Figure 7B), and brain (Figure 7C). N=3–6 for the liver, lungs, and brain, and N=6–12 for the bone. [Figure 7B] Same as above. [Figure 7C] Same as above.

[0025] [Figure 8] Figure 8 shows the exovivorage measurements in bone. Figure 8A: Radius was measured in bone at the time of slaughter. N=6-12 for bone. Figure 8B shows a representative image.

[0026] [Figure 9-1] Figure 9 shows data indicating that ERα and glucocorticoid receptor (GR) protein expression is lower in MCF7aro and LTLT compared to MCF7 and T47D. Figure 9A, Western blot showing ERα and actin. Figure 9B, Normalization of ERα levels relative to actin. Figure 9C, Western blot showing GR and actin. Figure 9D, Normalization of the GR protein band relative to actin. [Figure 9-2] Same as above.

[0027] [Figure 10-1] Figure 10 shows data comparing AR, HER2, and PR protein levels in MCF7aro and MCF7 LTLT compared to MCF7 and T47D. Figure 10A, Western blot showing AR and actin. Figure 10B, Normalized AR levels relative to actin. Figure 10C, Western blot showing Her2 expression in MCF7 LTLT. Figure 10D, Normalized Her2 relative to actin in T47D and MCF7 LTLT. Figure 10E, Western blot showing PR. For AR and Her2, one gel was run. For PR, one of two representative experiments is shown. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Modes for carrying out the invention]

[0028] 5. Detailed Description of the Invention Estrogen receptor positive (ERα +Postmenopausal patients with primary invasive breast cancer are typically treated with aromatase inhibitors (AIs) as first-line adjuvant therapy. Patients who become resistant to AIs are currently treated with SERD, fulvestrant, and / or CDK4 / 6 inhibitors (e.g., palbociclib) as second-line treatment. Rasofoxifen has previously been used to treat AI-resistant ERs through the acquisition of gain-of-function (activating) mutations in the ligand-binding domain of the ERα receptor. + It was clearly demonstrated that it retains its ability to inhibit tumor progression. AI-resistant ER avoids endocrine therapy through other mechanisms. + The ability of rasofoxifen to prevent cancer progression is unknown.

[0029] As described in the following example, the inventors compared the efficacy of rasofoxifene (both alone and in combination with palbociclib) to fulvestrant (both alone and in combination with palbociclib) in a breast tumor model using letrozole-inducible AI-resistant cells (LTLT cells) that do not have activating mutations in the ligand-binding domain of ERα. Rasofoxifene (both alone and in combination with palbociclib) was significantly more effective than fulvestrant (both alone and in combination with palbociclib) in inhibiting primary tumor growth. Furthermore, all treatments except fulvestrant alone inhibited bone metastases to the vehicle. These data demonstrate that rasofoxifene is more effective than fulvestrant in this tumor model and clearly indicate that rasofoxifene is an effective treatment for AI-resistant breast cancer that does not express ERα activating mutations. The data show that the combination of rasofoxifene and a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor is a more effective treatment for AI-resistant breast cancer that does not express ERα-activating mutations. Furthermore, rasofoxifene (alone or in combination with a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor) inhibits bone and brain metastases.

[0030] 5.1 Treatment Method Therefore, in the first phase, in patients whose condition progressed under aromatase inhibitors, estrogen receptor positive (ER) + Disclosed herein is a method for reducing the progression of cancer, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene. The method comprises administering an effective amount of rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug, or functional derivative to the patient.

[0031] In some embodiments, the method further comprises an earlier step of determining that the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0032] The above method involves combining an effective amount of rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug, or functional derivative with a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor (e.g., palbociclib, abemaciclib, or ribociclib) and / or an aromatase inhibitor (e.g., exemestane (aromasin)). (登録商標) ), letrozole (Femara (登録商標) ), or anastrozole (Arimidex) (登録商標) In combination with the above, this may include the step of administering the drug to the patient.

[0033] 5.1.1. ER + Patients with cancer In various embodiments, the patient is in the ER +The patient has been diagnosed with cancer. In some embodiments, the ER status is determined by immunohistochemistry (IHC), RT-PCR, large-scale parallel next-generation sequencing (NGS), or other conventional techniques performed on a sample of the patient's cancer. In some embodiments, the sample is tumor tissue derived from a biopsy. In some embodiments, the sample is liquid biopsy material from blood, saliva, or other bodily fluids (e.g., serum, circulating DNA of tumor biomarkers).

[0034] In some embodiments, the patient is in the ER + She has been diagnosed with breast cancer. In some embodiments, the above cancer is ER + / HER2 - The patient has breast cancer. In some embodiments, the patient is in the ER + The patient has been diagnosed with primary breast cancer. In some embodiments, the cancer is locally advanced or metastatic ER + The patient has breast cancer. In some embodiments, the above patient has previously received exemestane (aromatherapy). (登録商標) ), letrozole (Femara (登録商標) ), or anastrozole (Arimidex) (登録商標) It is treated with an aromatase inhibitor selected from the following.

[0035] In some embodiments, the patient is premenopausal, around menopause, or postmenopausal. In some embodiments, the patient is premenopausal and has primary ER + The patient has breast cancer. In some embodiments, the patient is premenopausal and has locally advanced or metastatic ER. + The patient has breast cancer. In some embodiments, the patient is perimenopause or has primary ER. + The patient has breast cancer. In some embodiments, the patient is pre- or post-menopausal and has locally advanced or metastatic ER. + The patient has breast cancer. In some embodiments, the patient is postmenopausal and has primary ER +The patient has breast cancer. In some embodiments, the patient is postmenopausal and has locally advanced or metastatic ER. + She has breast cancer.

[0036] In some embodiments, the patient is premenopausal and has primary breast cancer or locally advanced or metastatic ER + The patient has breast cancer and has previously been treated with an aromatase inhibitor in combination with a second treatment. For example, the patient in question received a second treatment (e.g., goserelin (zoladex)). (登録商標) ) or leuprolide (leupron) (登録商標) In combination with ), treatment with letrozole may have been performed.

[0037] In some embodiments, the patient described above has ER other than breast cancer. + He has been diagnosed with cancer. In some of these embodiments, the patient is in the ER + She has been diagnosed with ovarian cancer. In some of these embodiments, the patient is in the ER + She has been diagnosed with lung cancer. In a particular embodiment, the cancers mentioned above are gynecological cancers selected from uterine cancer, cervical cancer, peritoneal cancer, vulvar cancer, and vaginal cancer.

[0038] In some embodiments, the patient has primary ER + The patient has been diagnosed with uterine cancer. In certain embodiments, the uterine cancer is selected from endometrioid, clear cell carcinoma, papillary serous, carcinosarcoma, leiomyosarcoma, and endometrial stromal sarcoma (ESS). In certain embodiments, the uterine cancer is endometrial stromal sarcoma, endometrial adenosarcoma, adenosquamous cell carcinoma, uterine leiomyosarcoma, or endometrial cancer.

[0039] In some of these embodiments, the patient is in the ER + She has been diagnosed with cervical cancer. In a particular embodiment, the cervical cancer is clear cell carcinoma of the cervix.

[0040] In some of these embodiments, the patient is in the ER + The patient has been diagnosed with vulvar / vaginal cancer. In certain embodiments, the vulvar or vaginal cancer is squamous cell carcinoma (SCC) or adenocarcinoma.

[0041] In some of these embodiments, the patient is in the ER + The patient has been diagnosed with lung cancer. In certain embodiments, the lung cancer is adenosarcoma, squamous cell carcinoma of the lung, or small cell lung cancer.

[0042] In a particular embodiment, the cancer is a cancer of the digestive system selected from esophageal cancer, gastric cancer, small intestine cancer, colon cancer, rectal cancer, and colorectal cancer.

[0043] In some embodiments, the patient has primary ER + He has been diagnosed with esophageal cancer. In certain embodiments, the esophageal cancer is adenocarcinoma or squamous cell carcinoma.

[0044] In some embodiments, the patient has primary ER + He has been diagnosed with stomach cancer. In a particular embodiment, the stomach cancer is gastric adenocarcinoma.

[0045] In some embodiments, the patient has primary ER + The patient has been diagnosed with small intestine cancer. In certain embodiments, the small intestine cancer is an adenocarcinoma, carcinoid tumor, lymphoma, or sarcoma (e.g., leiomyosarcoma). In certain embodiments, the small intestine cancer is a malignant small intestine neoplasm.

[0046] In some embodiments, the patient has primary ER + He has been diagnosed with colon cancer. In certain embodiments, the colon cancer is colon adenocarcinoma.

[0047] In some embodiments, the patient has primary ER +He has been diagnosed with rectal cancer. In certain embodiments, the rectal cancer is rectal adenocarcinoma.

[0048] In some embodiments, the patient has primary ER + The patient has been diagnosed with colorectal cancer. In certain embodiments, the colorectal cancer is colorectal adenocarcinoma and colorectal mucinous adenocarcinoma.

[0049] In a particular embodiment, the cancer is selected from bladder cancer, e.g., urothelial carcinoma of the bladder; glioblastoma, e.g., conventional glioblastoma multiforme; skin cancer, e.g., squamous cell carcinoma of the skin; melanoma, e.g., cutaneous melanoma; invasive renal pelvis cancer; pancreatic cancer, e.g., pancreatic adenocarcinoma; and cancer of unknown primary origin.

[0050] In some embodiments, the above ER + The cancer is a primary cancer. In some embodiments, the above ER + The cancer is localized. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is metastatic ER + It's cancer.

[0051] In some embodiments, the patient's cancer relapses or progresses after tamoxifen treatment. In some embodiments, the patient's cancer relapses or progresses after fulvestrant treatment. In some embodiments, the patient's cancer relapses or progresses after aromatase inhibitor treatment. In some of these embodiments, the patient's cancer relapses or progresses after multiple lines of endocrine therapy treatment.

[0052] 5.1.1.1. Detection of ESR1 gene mutations In various embodiments, the patient is predetermined to be free of mutations in the LBD of the ESR1 gene. Some embodiments of the methods described herein further include a step of detecting mutations in the ESR1 gene.

[0053] In some embodiments, large-scale parallel next-generation sequencing (NGS) is used to detect estrogen receptor mutations in the cancer of the patient described above. In certain embodiments, the entire genome is sequenced. In certain embodiments, a selected gene panel of cancer-related genes is sequenced. In certain embodiments, all coding exons within a given set of genes are sequenced. In certain embodiments, known "hotspot" regions within a given set of genes are sequenced. However, the inherent error rate of current next-generation sequencing techniques is up to 1%, limiting detection sensitivity and specificity. In some embodiments, targeted sequencing is used to detect the presence of ESR1 mutations. Targeted sequencing allows for deeper sequencing, but it is also currently limited by an error rate of 1%. In some embodiments, methods with reduced sequencing error rates are used. In certain embodiments, a Safe-Sequencing System (Safe-SeqS) is used. This involves tagging each template molecule to enable reliable identification of rare variants. See Kinde et al., Proceedings of the National Academy of Sciences 108(23):9530-9535 (2011). In certain embodiments, ultrasensitive duplex sequencing is used, which independently tags and sequences each of the two strands of a DNA double helix. See Schmitt et al., Proceedings of the National Academy of Sciences 109(36):14508-14513 (2012). In some embodiments, digital droplet PCR is used, which emulsifies DNA in thousands to millions of droplets to encapsulate a single DNA molecule, designed with mutant-specific primers.See Vogelstein and Kinzler, Proceedings of the National Academy of Sciences 96(16):2322-2326 (1999) and Huggett et al., Clinical Chemistry 61(1):79-88 (2014).

[0054] In some embodiments, detection of the ESR1 mutation is performed at the time of initial diagnosis. In some embodiments, detection of the mutation is performed during disease progression, relapse, or recurrence. In some embodiments, detection of the mutation is performed during disease progression. In some embodiments, detection of the mutation is performed when the disease is stable.

[0055] In some embodiments, one or more tissue samples are obtained for the detection of the mutation. In a particular embodiment, the tissue sample is a tumor biopsy sample. In a particular embodiment, the tissue sample is a metastatic biopsy sample. In some other embodiments, a liquid biopsy sample is obtained for the detection of the mutation. In a particular embodiment, the liquid biopsy sample is circulating tumor cells (CTCs). In a particular other embodiment, the liquid biopsy sample is cell-free DNA derived from a blood sample.

[0056] In specific embodiments, the ESR1 mutation is monitored by circulating tumor DNA (ctDNA) analysis. In some embodiments, the ctDNA analysis is performed throughout the treatment cycle. In some of these embodiments, the ctDNA is extracted from a patient blood sample. In certain embodiments, the ctDNA is evaluated by digital PCR analysis of the ESR1 mutation.

[0057] 5.1.2. Supportive measures In various embodiments, rasofoxifene is administered to the patient as an adjunct treatment. In certain embodiments, rasofoxifene is administered to the patient as an adjunct treatment alone. In certain other embodiments, rasofoxifene is administered to the patient as an adjunct treatment in combination with other endocrine therapies. In some embodiments, rasofoxifene is administered to the patient after primary treatment. In some of these embodiments, rasofoxifene is administered to the patient after surgical removal or reduction of cancer.

[0058] In some embodiments, rasofoxifen is administered to the patient as adjunctive therapy in combination with an aromatase inhibitor (AI). In various embodiments, the aromatase inhibitor is exemestane (aromasin). (登録商標) ), letrozole (Femara (登録商標) ), or anastrozole (Arimidex) (登録商標) )

[0059] In various embodiments, the aromatase inhibitor makes the patient more susceptible to bone-related toxic effects. In some embodiments, the aromatase inhibitor makes the patient more susceptible to osteoporosis. In some embodiments, the aromatase inhibitor makes the patient more susceptible to bone loss. In some embodiments, the aromatase inhibitor makes the patient more susceptible to fractures. In some embodiments, the aromatase inhibitor makes the patient more susceptible to bone pain.

[0060] In various embodiments, the aromatase inhibitor makes the patient more susceptible to vulvovaginal atrophy (VVA).

[0061] In some embodiments, rasofoxifene is administered sequentially during the administration of the aromatase inhibitor. In some other embodiments, rasofoxifene is administered periodically between the administrations of the aromatase inhibitor. In some embodiments, rasofoxifene and the aromatase inhibitor are administered together (simultaneously). In some other embodiments, rasofoxifene and the aromatase inhibitor are administered separately (sequentially).

[0062] In certain embodiments, the administration regimen for rasofoxifen differs from that of the aromatase inhibitor. In some of these embodiments, the dose of rasofoxifen differs from that of the aromatase inhibitor. In some embodiments, the administration schedule for rasofoxifen differs from that of the aromatase inhibitor. In some embodiments, the route of administration for rasofoxifen differs from that of the aromatase inhibitor.

[0063] In certain embodiments, the administration regimen for rasofoxifen is the same as the administration regimen for the aromatase inhibitor. In some embodiments, the dose of rasofoxifen is the same as the dose of the aromatase inhibitor. In some embodiments, the administration schedule for rasofoxifen is the same as the administration schedule for the aromatase inhibitor. In some embodiments, the route of administration for rasofoxifen is the same as the route of administration for the aromatase inhibitor.

[0064] In some embodiments, rasofoxifen is administered to the patient for one year as adjunctive therapy in combination with an aromatase inhibitor. In some embodiments, rasofoxifen is administered to the patient for two years as adjunctive therapy in combination with an aromatase inhibitor. In some embodiments, rasofoxifen is administered to the patient for three years as adjunctive therapy in combination with an aromatase inhibitor. In some embodiments, rasofoxifen is administered to the patient for four years as adjunctive therapy in combination with an aromatase inhibitor. In some embodiments, rasofoxifen is administered to the patient for five years as adjunctive therapy in combination with an aromatase inhibitor. In some embodiments, rasofoxifen is administered to the patient for six years as adjunctive therapy in combination with an aromatase inhibitor. In some embodiments, rasofoxifen is administered to the patient for seven years as adjunctive therapy in combination with an aromatase inhibitor. In some embodiments, rasofoxifene is administered to the patient as adjuvant therapy in combination with an aromatase inhibitor for 8 years. In some embodiments, rasofoxifene is administered to the patient as adjuvant therapy in combination with an aromatase inhibitor for 9 years. In some embodiments, rasofoxifene is administered to the patient as adjuvant therapy in combination with an aromatase inhibitor for 10 years. In some other embodiments, rasofoxifene is administered to the patient as adjuvant therapy in combination with an aromatase inhibitor for longer than 10 years. In a particular embodiment, rasofoxifene is administered as adjuvant therapy in combination with an aromatase inhibitor until the patient's cancer progresses under treatment.

[0065] In some embodiments, rasofoxifene is administered as adjuvant therapy in combination with an aromatase inhibitor to increase disease-free survival in cancer patients. In some embodiments, rasofoxifene is administered as adjuvant therapy in combination with an aromatase inhibitor to reduce the incidence of contralateral breast cancer. In some embodiments, rasofoxifene is administered as adjuvant therapy in combination with an aromatase inhibitor to prevent cancer recurrence or progression.

[0066] 5.2. Rasofoxifen In various embodiments, selected patients are treated with an effective dose of rasofoxifene, a pharmaceutically acceptable salt thereof, a prodrug thereof, or a functional derivative. Rasofoxifene has the following structure: [ka]

[0067] In some embodiments, rasofoxifene is administered to the selected patients as rasofoxifene tartrate.

[0068] The term "pharmaceutically acceptable salt" refers to a non-toxic, pharmaceutically acceptable salt. See Gould, International Journal of Pharmaceutics 33: 201-217 (1986) and Berge et al., Journal of Pharmaceutical Sciences 66(1):1-19 (1977). However, other salts well known to those skilled in the art may be used. Representative organic or inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, hydroxyethanesulfonic acid, benzenesulfonic acid, oxalic acid, pamoic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, salicylic acid, saccharic acid, or trifluoroacetic acid. Representative organic or inorganic bases include, but are not limited to, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, and basic or cationic salts of aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.

[0069] Embodiments also include prodrugs of the compounds disclosed herein. Generally, such prodrugs include functional derivatives of the compounds described herein that are readily convertible in vivo to the compound of interest. Accordingly, in the treatment methods of the present invention, the term “administering” encompasses the treatment of the various disorders described with the compounds specifically disclosed or compounds that may not be specifically disclosed but are converted in vivo to a particular compound after administration to a subject. Conventional procedures for the selection and preparation of appropriate prodrug derivatives are described, for example, in “Design of Prodrugs,” H. Bundgaard, Elsevier, 1985.

[0070] In certain embodiments, functional derivatives of rasofoxifene include proteolysis-targeting chimeras (PROTACs) containing rasofoxifene. In certain embodiments, PROTACs are heterobifunctional small molecules having three chemical components: rasofoxifene, a ubiquitin ligand-binding moiety or ULM group, and a ligand for conjugating these two components. In some embodiments, rasofoxifene is covalently conjugated to the ubiquitin ligand-binding moiety or ULM group via a linker. Non-limiting examples of such linkers include ester linkers, amide linkers, maleimide or maleimide-based linkers; valine-citrulline linkers; hydrazone linkers; N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linkers; succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers; vinyl sulfone-based linkers; linkers containing polyethylene glycol (PEG) (e.g., tetraethylene glycol); linkers containing propanols acid; linkers containing caproleic acid; and linkers containing any combination thereof. In embodiments, the linker is a chemically unstable linker (for example, an acid-cleavable linker that is stable at a neutral pH (blood flow pH 7.3-7.5) but undergoes hydrolysis upon internalization into the slightly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of target cells (e.g., cancer cells)). Examples of chemically unstable linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, and ester-based linkers.In some embodiments, the linker is an enzyme-unstable linker (for example, an enzyme-unstable linker that is stable in the bloodstream but is enzymatically cleaved by a lysosomal protease (e.g., cathepsin or plasmin) in the lysosome of a target cell (e.g., a cancer cell) during internalization to the target cell). Examples of enzyme-unstable linkers include, but are not limited to, linkers containing peptide bonds, such as dipeptide-based linkers (e.g., valine-citrulline linkers (e.g., maleimidocaproyl-valine-citrulline-p-aminobenzyl (arninobenzyl) (MC-vc-PAB) linker, valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, etc.)). Chemically unstable, enzyme-unstable, and uncleavable linkers are described in detail, for example, Ducry & Stump (2010) Bioconjugate Chem. 21:5-13. In a particular embodiment, the ULM is selected from the group consisting of the following: [ka] [ka] Here, the ULM group is covalently bonded to a linker to which rasofoxyfen, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, or prodrug thereof, is attached.

[0071] Some of the crystalline forms of the above compounds may exist as polymorphs and are therefore intended to be included in the present invention. Furthermore, some of the above compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are intended to be included by some embodiments.

[0072] If the process for preparing the compounds as disclosed herein results in a mixture of stereoisomers, these isomers can be separated by prior art (e.g., preparative chromatography). The compounds can be prepared in racemic form or as individual enantiomers or diastereomers by either stereospecific synthesis or resolution. The compounds can be separated into enantiomers or diastereomers of their components by, for example, standard techniques (e.g., salt formation with an optically active base, followed by fractional crystallization and regeneration of the free acid to form stereoisomer pairs). The compounds can also be separated by the formation of esters or amides of the stereoisomers, followed by chromatographic separation and removal of a chiral auxiliary. Alternatively, the compounds can be separated using a chiral HPLC column. It should be understood that all stereoisomers, racemic mixtures, diastereomers, cis-trans isomers, and their enantiomers are encompassed by several embodiments.

[0073] 5.3. Pharmaceutical Compositions Estrogen receptor positive (ER) + A method for treating cancer comprises the step of administering a therapeutically effective amount of rasofoxifen, a pharmaceutically acceptable salt thereof, a prodrug, or a functional derivative. The rasofoxifen, pharmaceutically acceptable salt thereof, or prodrug of the present invention may be formulated in a pharmaceutical composition. In addition to rasofoxifen, a pharmaceutically acceptable salt thereof, or a prodrug thereof, the composition further comprises a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other substance well known to those skilled in the art. Such substance should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance may depend on the route of administration, e.g., oral, intravenous, transdermal, topical vaginal, or vaginal ring.

[0074] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier (e.g., gelatin or an adjuvant). Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal oil, vegetable oil, mineral oil, or synthetic oil. Physiological saline solutions, dextrose or other saccharide solutions, or glycols (e.g., ethylene glycol, propylene glycol, or polyethylene glycol) may also be included.

[0075] For parenteral administration, the rasofoxifen described above is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. Those skilled in the art can easily prepare a suitable solution using, for example, an isotonic vehicle (e.g., sodium chloride injection, Ringer's injection, lactose-added Ringer's injection). Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included if necessary.

[0076] Pharmaceutical compositions for topical vaginal administration may be in the form of ointments, creams, gels, or lotions. Pharmaceutical compositions for topical vaginal administration often contain water, alcohol, animal oils, vegetable oils, mineral oils, or synthetic oils. Hydrocarbons (paraffin), lanolin, beeswax, macrogol, emulsifying waxes, or cetrimides may also be included.

[0077] Depending on the condition being treated, the composition may be administered either alone or in combination with other treatments, simultaneously or sequentially.

[0078] 5.4 Treatment regimen ER +In relation to the treatment of cancer, in a method of administering an effective amount of rasofoxifen, a pharmaceutically acceptable salt thereof, a prodrug, or a functional derivative thereof in the form of a pharmaceutical composition as described above, the terms “treatment,” “to treat,” etc., are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease, condition, or its symptoms, and / or therapeutic in terms of partially or completely treating a disease or condition and / or adverse effect (e.g., symptoms that may be caused by the disease or condition). “Treatment,” as used herein, encompasses any treatment of a disease or condition in a mammal, in particular a human, including (a) preventing the disease or condition from occurring in a subject that may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., stopping its occurrence); or (c) alleviating the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms). Improvement in any given condition can be readily evaluated according to standard methods and techniques known in the art. The population of subjects treated for the above-mentioned diseases by the above-mentioned methods includes subjects suffering from undesirable conditions or diseases, and subjects at risk of developing the above-mentioned conditions or diseases.

[0079] The term "effective dose" refers to the amount administered to produce the desired effect. The exact dose depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques. See Lloyd, *The Art, Science and Technology of Pharmaceutical Compounding* (1999).

[0080] 5.4.1. Route of administration In various embodiments, rasofoxifen, a pharmaceutically acceptable salt thereof, a prodrug, or a functional derivative thereof is administered orally, intravenously, transdermally, topically, or via a vaginal ring.

[0081] In some embodiments, rasofoxifene is administered orally to the patient. In certain embodiments, rasofoxifene is administered orally at a dose of approximately 5 mg / day to approximately 10 mg / day. For example, in some embodiments, rasofoxifene is administered orally at a dose of approximately 5 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 6 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 7 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 8 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 9 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 10 mg / day. In some embodiments, rasofoxifen is administered orally at doses ranging from approximately 0.5 mg / day to approximately 10 mg / day (for example, approximately 0.5 mg / day to approximately 5 mg / day, approximately 1 mg / day to approximately 5 mg / day, approximately 2 mg / day to approximately 5 mg / day, approximately 3 mg / day to approximately 5 mg / day, approximately 4 mg / day to approximately 5 mg / day, approximately 0.5 mg / day to approximately 4 mg / day, approximately 1 mg / day to approximately 4 mg / day, approximately 2 mg / day to approximately 4 mg / day, approximately 3 mg / day to approximately 4 mg / day, approximately 0.5 mg / day to approximately 3 mg / day, approximately 1 mg / day to approximately 3 mg / day, approximately 2 mg / day to approximately 3 mg / day, approximately 0.5 mg / day to approximately 2 mg / day). The above-mentioned patients are administered orally (by mouth) at doses of approximately 1 mg / day, or approximately 2 mg / day, or approximately 0.5 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 0.5 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 1 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 1.5 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 2 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 2.5 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 3 mg / day.In some embodiments, rasofoxifene is administered orally at a dose of approximately 3.5 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 4 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 4.5 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 5 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 6 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 7 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 8 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 9 mg / day. In some embodiments, rasofoxifene is administered orally at a dose of approximately 10 mg / day. In some other embodiments, rasofoxifene is administered orally at a dose greater than 10 mg / day. In some embodiments, rasofoxifene is administered at a dose of approximately 0.5 mg / day to approximately 10 mg / day. In some embodiments, rasofoxifen is administered at doses of approximately 0.5 mg / day, 1 mg / day, 1.5 mg / day, 2 mg / day, 2.5 mg / day, 3 mg / day, 3.5 mg / day, 4 mg / day, 5 mg / day, 5.5 mg / day, 6 mg / day, 6.5 mg / day, 7 mg / day, 7.5 mg / day, 8 mg / day, 8.5 mg / day, 9 mg / day, 9.5 mg / day, or 10 mg / day. In some embodiments, rasofoxifen is administered orally at a dose of approximately 5 mg / day.

[0082] In certain embodiments, rasofoxifene is administered once daily. In certain embodiments, rasofoxifene is administered once every two days. In certain embodiments, rasofoxifene is administered once every three days. In certain embodiments, rasofoxifene is administered once every four days. In certain embodiments, rasofoxifene is administered once every five days. In certain embodiments, rasofoxifene is administered once every six days. In certain embodiments, rasofoxifene is administered once a week. In certain embodiments, rasofoxifene is administered once every two weeks. In certain embodiments, rasofoxifene is administered once every three weeks. In certain embodiments, rasofoxifene is administered once a month.

[0083] In some embodiments, rasofoxifen is administered to the patient via a vaginal ring. In some of these embodiments, rasofoxifen is administered once every two weeks. In some of these embodiments, rasofoxifen is administered once every three weeks. In some of these embodiments, rasofoxifen is administered once every month. In some of these embodiments, rasofoxifen is administered once every two months. In some of these embodiments, rasofoxifen is administered once every three months. In some of these embodiments, rasofoxifen is administered once every four months.

[0084] In some embodiments, rasofoxifen is used in the above ER + This drug is administered to breast cancer patients until their cancer progresses under treatment, they achieve complete remission, or they can no longer tolerate the side effects.

[0085] 5.4.2. Combination Therapy In various embodiments, rasofoxifene, a pharmaceutically acceptable salt thereof, a prodrug, or a functional derivative thereof is administered either alone or in combination with other treatments. In certain embodiments, rasofoxifene is administered in combination with at least one other treatment. In some embodiments, rasofoxifene and other treatments are administered together (simultaneously). In some other embodiments, rasofoxifene and other treatments are administered at different times (sequentially).

[0086] In certain embodiments, the further treatment to which the patient is treated is endocrine therapy. In various embodiments, the patient is treated with at least one line of further endocrine therapy. In some embodiments, the patient is treated with one line of further endocrine therapy. In some other embodiments, the patient is treated with multiple lines of further endocrine therapy. In certain embodiments, the patient's cancer has relapsed or progressed after previous treatment.

[0087] In some embodiments, the patient is treated with further endocrine therapy at the original dose. In some other embodiments, the patient is treated with further endocrine therapy at a dose higher than the original dose. In a particular embodiment, the patient is treated with further endocrine therapy at a dose lower than the original dose.

[0088] In certain embodiments, the further endocrine therapy described above is treatment with a selective ER regulator (SERM) other than rasofoxifene. In some of these embodiments, the selective ER regulator is selected from tamoxifen, raloxifene, bazedoxifene, toremifene, and ospermifene, broparestrol, ormeloxifene, OP-1074, and GDC-0945. In certain embodiments, the selective ER regulator is tamoxifen.

[0089] In certain embodiments, the further endocrine therapy described above is treatment with a selective ER degradation factor (SERD). In some of these embodiments, the selective ER degradation factor binds to the estrogen receptor, resulting in proteasomal degradation of the receptor. In some embodiments, the selective ER degradation factor is selected from fulvestrant, RAD1901 (elacestrant), ARN-810 (GDC-0810), giredestrant (GDC-9545), amcenestrant (SAR439859), rintodestrant (G1T48), LSZ102, LY3484356, zN-c5, D-0502, SHR9549, camizestrant (AZD9833), and AZD9496. In a particular embodiment, the endocrine therapy described above is fulvestrant.

[0090] In certain embodiments, the further endocrine therapy described above is treatment with an aromatase inhibitor (AI). In some of these embodiments, the aromatase inhibitor is exemestane (aromasin). (登録商標) ), letrozole (Femara (登録商標) ), and anastrozole (Arimidex) (登録商標) ) will be selected from.

[0091] In some embodiments, the endocrine therapy described above is ovarian suppression. In various embodiments, ovarian suppression is achieved by oophorectomy or treatment with a GnRH antagonist. In some embodiments, ovarian suppression is achieved by treatment with goserelin (Zoladex®) or leuprolide (Lupron®).

[0092] In various embodiments, the further treatment is the administration of an effective dose of a cell cycle inhibitor to the patient. In certain embodiments, the further treatment is the administration of an effective dose of a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor. In some embodiments, the further treatment is a CDK4 / 6 inhibitor selected from the group of palbociclib, abemaciclib, and ribociclib.

[0093] In various embodiments, the further treatment is the administration of an effective amount of a cell cycle inhibitor to the patient. In certain embodiments, the further treatment is the administration of an effective amount of an AKT kinase inhibitor. In some embodiments, the further treatment is an AKT inhibitor selected from the group of afrecertib, capivacertib, and ipatasertib.

[0094] In some embodiments, the further treatment is the administration of an inhibitor to the patient that crosstalks with and activates ER transcriptional activity. In certain embodiments, the further treatment is a mammalian target (mTOR) inhibitor of rapamycin. In specific embodiments, the mTOR inhibitor is everolimus. In some of these embodiments, rasofoxifene in combination with everolimus is administered either as monotherapy or in combination with a CDK4 / 6 inhibitor to postmenopausal women with locally advanced or metastatic cancer that has progressed under nonsteroidal AI and / or fulvestrant. In various embodiments, the further treatment is a phosphoinositide 3-kinase (PI3K) inhibitor or a heat shock protein 90 (HSP90) inhibitor.

[0095] In various embodiments, the further treatment is the administration of an effective amount of growth factor inhibitor to the patient. In certain embodiments, the further treatment is a human epidermal growth factor receptor 2 (HER2) inhibitor. In some embodiments, the HER2 inhibitor is trastuzumab (Herceptin). (登録商標) ) In some other embodiments, the above HER2 inhibitor is ado-trastuzumab emtansine (kadcyla). (登録商標) )

[0096] In some embodiments, the further treatment involves administering an effective dose of a histone deacetylase (HDAC) inhibitor to the patient. In various embodiments, the HDAC inhibitor is vorinostat (Zolinza (登録商標) ), Romidepsin (Istodax (登録商標) ), chidamide (epidaza) (登録商標) ), Panovinostat (Faryduck (登録商標) ), Belinostat (Beleodaq) (登録商標) PXD101), valproic acid (Depacoat) (登録商標) Depakene (登録商標) Stavzor (登録商標)), mosetinostat (MGCD0103), avexinostat (PCI-24781), entinostat (MS-275), prasinostat (SB939), resminostat (4SC-201), givinostat (ITF2357), xinostat (JNJ-26481585), kevetrin, CUDC-101, AR-42, tefinostat (CHR-2835), CHR-3996, 4SC202, CG200745, rocilinostat (ACY-1215), or sulforaphane. In a particular embodiment, the HDAC inhibitor is entinostat (MS-275), provided that the patient is not treated with a HER2 inhibitor. In a particular other embodiment, the HDAC inhibitor is vorinostat (Zolinza (登録商標) ) Furthermore, in certain other embodiments, the HDAC inhibitor is romidepsin (istodax). (登録商標) )

[0097] In some embodiments, the further treatment involves administering an effective dose of a checkpoint inhibitor to the patient. In certain embodiments, the checkpoint inhibitor is an antibody. In some of these embodiments, the checkpoint inhibitor is an antibody specific to programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). In some embodiments, the PD-1 antibody is pembrolizumab (Keytruda). (登録商標) ) or nivolumab (Opdivo) (登録商標) ) In some embodiments, the above CTLA-4 antibody is ipilimumab (Yervoy). (登録商標) )

[0098] In a particular embodiment, the further treatment is the administration of an effective dose of the cancer vaccine to the patient.

[0099] In some embodiments, the further treatment involves administering an effective dose of denosumab to the patient.

[0100] In some embodiments, the further treatment described above is the administration of an effective dose of a serotonin-norepinephrine reuptake inhibitor (SNRI), a selective serotonin reuptake inhibitor (SSRI), or gabapentin to the patient. In certain embodiments, the SNRI described above is venlafaxine (Effexor). (登録商標) )

[0101] In some embodiments, the further treatments described in the preceding paragraphs may be used in combination. Rasofoxifen, pharmaceutically acceptable salts thereof, prodrugs, or functional derivatives thereof may be administered in combination with two treatments (e.g., endocrine therapy (e.g., aromatase inhibitor (e.g., letrozole)) and cell cycle inhibitor (e.g., CDK4 / 6 inhibitor (e.g., palbociclib, abemaciclib, and ribociclib))).

[0102] 5.4.3. Clinical Endpoints 5.4.3.1. Primary Clinical Endpoints In various embodiments, the above method is used in the above ER + The method comprises administering an effective amount of rasofoxifen, a pharmaceutically acceptable salt thereof, a prodrug, or a functional derivative thereof to increase the disease-free survival of cancer patients. In some embodiments, the above method is used in ER + The method includes the step of administering rasofoxifen in an amount effective to reduce cancer recurrence. In some embodiments, the above method is used in ER + The method includes the step of administering rasofoxifen in an amount effective to increase the time to cancer recurrence. In some embodiments, the above method is used in ER+ administering raloxifene in an amount effective to reduce cancer metastasis. In some embodiments, the method is the above ER + administering raloxifene in an amount effective to increase the duration of the progression-free survival period of a cancer patient.

[0103] In various embodiments, the method is the above ER + increases the disease-free survival period of a cancer patient. In certain embodiments, the method is ER + reduces cancer recurrence. In certain embodiments, the method is ER + increases the time to cancer recurrence. In certain embodiments, the method is ER to bone + reduces cancer metastasis. In certain embodiments, the method is ER to tissues other than bone + reduces cancer metastasis. In certain embodiments, the method is ER to brain + reduces cancer metastasis. In certain embodiments, the method is ER to lung + reduces cancer metastasis. In certain embodiments, the method is ER to liver + reduces cancer metastasis. In certain embodiments, the method is the above ER + increases the duration of the progression-free survival period of a cancer patient. The method may include administering raloxifene in combination with one or more additional therapeutic agents (as described herein).

[0104] In various embodiments, the method is ER having AI resistance + increases the disease-free survival period in cancer patients. In some embodiments, the method reduces cancer recurrence in patients having AI resistance. In some embodiments, the method increases the time to cancer recurrence in patients having AI resistance. In some embodiments, the method reduces cancer metastasis in patients having AI resistance. In some embodiments, the method is ER having AI resistance+ Increase the duration of progression-free survival in cancer patients.

[0105] In some embodiments, the method generates AI-resistant ER + In patients with locally advanced or metastatic cancer, increase disease-free survival, reduce recurrence, increase time to recurrence, reduce metastasis, and / or increase the duration of progression-free survival. In certain embodiments, the cancer has acquired one or more mutations other than gain-of-function mutations in the LBD of ESR1 discussed herein, thereby generating AI resistance. In some embodiments, the method reduces selection pressure and ER + Prevent the expansion of AI-resistant clones in locally advanced or metastatic cancer.

[0106] 5.4.3.2. Secondary Clinical Endpoints In some embodiments, the method is effective for preventing fractures and bone loss in women being treated concurrently with one or more drugs that cause or predispose to osteoporosis.

[0107] In some embodiments, the method is effective for reducing vaginal pH, increasing vaginal lubrication, and / or improving the vaginal cell maturation index in women being treated concurrently with one or more drugs that cause or predispose to vulvovaginal atrophy (VVA).

[0108] In some embodiments, the method reduces one or more symptoms of sexual dysfunction in women being treated concurrently with one or more drugs that cause or predispose to sexual dysfunction.

[0109] In some embodiments, the above method treats a hot flash in a woman who is simultaneously treated with one or more drugs that cause or make her susceptible to hot flashes.

[0110] In some embodiments, the above method increases one or more measures of quality of life selected from joint pain, genitourinary symptoms, osteoporosis, and fractures.

[0111] Embodiments of the present invention

[0112] A1a. Rasofoxifene or a pharmaceutically acceptable salt thereof, prodrug or functional derivative for use in a method for reducing the progression of estrogen receptor-positive (ER+) cancer in patients who have progressed under aromatase inhibitor, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, and the method comprises the step of administering an effective amount of rasofoxifene or a pharmaceutically acceptable salt thereof, prodrug or functional derivative to the patient.

[0113] A1b. In patients whose condition has progressed under aromatase inhibitors, ER + Rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug or functional derivative for use in a method for reducing cancer metastasis, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, wherein the method, if applicable, reduces ER to bone or brain. + To reduce cancer metastasis, preferably the method involves the ER to the brain. + Rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug, or functional derivative that reduces cancer metastasis.

[0114] A1c. Combinations comprising rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug or functional derivative, and a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor for separate, simultaneous or sequential use in a patient whose cancer has progressed under an aromatase inhibitor, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, wherein the combination further comprises an aromatase inhibitor (e.g., letrozole) as appropriate.

[0115] A1d. Rasofoxifene or a pharmaceutically acceptable salt thereof, prodrug or functional derivative for use in the treatment of AI-resistant breast cancer that does not express ERα-activating mutations.

[0116] A1e. In patients whose condition has progressed under aromatase inhibitors, ER + A combination comprising rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug or functional derivative, and a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor for separate, simultaneous or sequential use in a method for reducing cancer metastasis, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, wherein the combination further comprises an aromatase inhibitor (e.g., letrozole) as appropriate.

[0117] A2. Said ER + The cancer is locally advanced or metastatic breast cancer, where the cancer is HER2-, and the use for which is rasofoxifen, or a pharmaceutically acceptable salt thereof, prodrug, or functional derivative, as described in any one of embodiments A1a to A1e.

[0118] A3. The aromatase inhibitor is exemestane, letrozole, or anastrozole, rasofoxifen for use as described in any of the embodiments described above, or a pharmaceutically acceptable salt, prodrug, or functional derivative thereof.

[0119] A4. The above method is, (i) A step of determining that the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, and (ii) The step of administering to the patient an effective amount of rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug or functional derivative thereof, Rasofoxifen, or a pharmaceutically acceptable salt, prodrug, or functional derivative thereof, for use as described in any of the embodiments described above.

[0120] A5. Rasofoxifene for use as described in any of the embodiments described above, administered as rasofoxifene tartrate.

[0121] A6. Rasofoxifen, or a pharmaceutically acceptable salt, prodrug, or functional derivative thereof, for use as described in any of the embodiments described above, administered orally, intravenously, transdermally, topically, or via a vaginal ring.

[0122] A7. Rasofoxifene, administered orally, for use as described in any of the embodiments described above, or a pharmaceutically acceptable salt, prodrug or functional derivative thereof.

[0123] A8. Rasofoxifene, or a pharmaceutically acceptable salt, prodrug, or functional derivative thereof, administered orally in doses of 5 mg / day to approximately 10 mg / day, as described in Embodiment A7.

[0124] A9. The cancer having mutations in the genes listed in Table 1, rasofoxifen for use as described in any of the embodiments described above, or a pharmaceutically acceptable salt, prodrug, or functional derivative thereof.

[0125] A10. The method further comprises the step of treating the patient with at least one further endocrine therapy, comprising rasofoxifen for use according to any of the embodiments described above, or a pharmaceutically acceptable salt, prodrug or functional derivative thereof.

[0126] A11. The method further comprises administering an effective amount of a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor to the patient, wherein the CDK4 / 6 inhibitor is, if necessary, rasofoxifen for use as described in any of the embodiments above, or a pharmaceutically acceptable salt thereof, prodrug or functional derivative, administered orally at a dose of, for example, 70 mg / kg.

[0127] A12. Rasofoxifene, or a pharmaceutically acceptable salt thereof, prodrug or functional derivative thereof, and a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor for simultaneous, separate, or sequential use in a method for reducing the progression of estrogen receptor-positive (ER+) cancer in patients progressing under aromatase inhibitor, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0128] A13. Embodiment A11 or A12, wherein the CDK4 / 6 inhibitor is palbociclib, abemaciclib, or ribociclib, where the CDK4 / 6 inhibitor is palbociclib if necessary, and where the CDK4 / 6 inhibitor is preferably abemaciclib.

[0129] A14. The method further comprises the step of administering an effective amount of AKT inhibitor to the patient, for use according to any one of Embodiments A1 to A10, rasofoxifen, or a pharmaceutically acceptable salt thereof, prodrug, or functional derivative thereof.

[0130] A15. Rasofoxifene, or a pharmaceutically acceptable salt thereof, prodrug or functional derivative, and an AKT inhibitor for simultaneous, separate or sequential use in a method for reducing the progression of estrogen receptor-positive (ER+) cancer in a patient whose cancer has progressed under an aromatase inhibitor, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

[0131] A16. Embodiment A14 or A15, in which the AKT inhibitor is an adsorbent.

[0132] A17. Rasofoxifen, or a pharmaceutically acceptable salt thereof, prodrug, or functional derivative, for use according to any one of Embodiments A1 to A10, further comprising the step of administering an effective amount of mTor inhibitor to the patient.

[0133] A18. Rasofoxifene, or a pharmaceutically acceptable salt thereof, prodrug or functional derivative, and an mTor inhibitor for simultaneous, separate, or sequential use in a method for reducing the progression of estrogen receptor-positive (ER+) cancer in a patient whose cancer has progressed under an aromatase inhibitor, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene. [Examples]

[0134] 5.5. Examples The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. While efforts have been made to ensure accuracy with respect to the figures used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should naturally be acceptable.

[0135] The invention will be implemented using conventional methods within the scope of the art of molecular biology, cell biology, biochemistry, genetics, cancer biology, and pharmacology, unless otherwise specified. Such techniques are well described in the literature.

[0136] 5.5.1. Example 1: Generation of a letrozole-induced AI-resistant breast tumor model (MCF-7 cells) that does not express ERα-activating mutations. Estrogen receptor positive (ERα + Postmenopausal patients with primary invasive breast cancer are typically treated with aromatase inhibitors (AIs) as first-line adjuvant therapy. Patients who become resistant to AIs are treated with fulvestrant and / or CDK4 / 6 inhibitors (e.g., palbociclib) as second-line treatment. Rasofoxifene, a selective estrogen receptor modulator (SERM), was developed for the treatment of vaginal atrophy and osteoporosis. Our previous studies (conducted in an MCF-7 xenograft metastatic breast cancer mouse model with activated ERα mutations) showed that rasofoxifene (both alone and in combination with palbociclib) was more effective than fulvestrant in inhibiting tumor growth and metastasis to the liver, lungs, bone, and brain in the context of gain-of-function activating mutations in the ERα receptor (encoded by the ESR1 gene).

[0137] In recent studies, the inventors compared the efficacy of rasofoxifene (both alone and in combination with palbociclib) to fulvestrant (both alone and in combination with palbociclib) in a breast tumor model using letrozole-inducible AI-resistant cells (LTLT cells) that do not express ERα with an activating mutation in the ligand-binding domain.

[0138] Luciferase-GFP-tagged LTLT cells were injected into the mammary ducts of NSG mice (MIND model), and tumor progression was monitored by hepatic luminescence imaging of the primary tumor, as well as exovivo imaging and histochemical analysis of metastatic sites at the study endpoint. The area of ​​the primary tumor was also measured at the study endpoint. Rasofoxifene (both alone and in combination with palbociclib) was significantly more effective than fulvestrant (both alone and in combination with palbociclib) in inhibiting primary tumor growth. Furthermore, all treatments except fulvestrant alone inhibited bone metastases to the vehicle. These data demonstrate that rasofoxifene is more effective than fulvestrant in this tumor model and clearly indicate that rasofoxifene is an effective treatment for AI-resistant breast cancer that does not express ERα-activating mutations.

[0139] Cell culture, lentivirus generation and infection, and creation of stable cell lines. MCF7 LTLT cells (also known as LTLT-Ca cells (Sabnis, G. et al., Cancer Res. 69, 1416-1428 (2009))) are hormone receptor-positive (ER) cells with WT and mutant ERα. + PR + , GR +MCF7 LTLT cells are derivatives of the human breast cancer cell line MCF7. MCF7 LTLT cells have acquired resistance to aromatase inhibitors. They were originally obtained in the Brodie lab by long-term treatment of MCF7aro cells with letrozole, the aforementioned aromatase inhibitor (Sabnis et al., 2009). MCF-7aro cells are stably transfected with aromatase genes (Sun, XZ et al., J. Steroid Biochem. Mol. Biol. 63, 29-36 (1997)). MCF7 LTLT cells were kindly donated by Ganesh Raj of UT Southwestern. MCF7aro cells were kindly provided by Shiuan Chen of City of Hope.

[0140] To enable in vivo measurement of tumor cell proliferation, the inventors transfected MCF7 LTLT cells with an L2G lentiviral vector (pFU-Luc2-eGFP) containing luciferase and GFP in a suspension at MOI=5 under the control of a ubiquitin promoter (Liu, H. et al., Proc. Natl Acad. Sci. USA 107, 18115-18120 (2010)), plated the cells, and grew them in RPMI containing 10% FBS and 1 μM letrozole. For cell testing, MCF7 LTLT and MCF7aro cells were transfected with lentiviral nucleus-GFP staining (Nuclight GFP / puro, catalog no. 4475 Essen Bioscience) according to the manufacturer's recommendations.

[0141] Genotype evaluation To confirm that aromatase-resistant MCF7 LTLT cells lack gain-of-function mutations in the ESR1 ligand-binding domain and to investigate non-ESR1 mutations that could confer an endocrine therapy-resistant phenotype to MCF7 LTLT cells, we performed whole-genome exon sequencing and compared the sequence data with two previously reported MCF7 reference sequences (Reference 1 and Reference 2) (Figure 1).

[0142] The MCF7 LTLT cells (LT-LT cells in Figure 1) lacked the ESR1 mutation and possessed 18,687 novel variants, including 1,508 exonic nonsynonymous variants. Notable genes containing variants (63 genes, 85 variants) are listed in Table 1. [Table 1-1] [Table 1-2]

[0143] Phenotypic characterization of MCF7LTLT and MCF7aro via Western blotting To characterize the MCF7aro and MCF7 LTLT cell lines, the inventors performed Western blotting and probed ERα, progesterone receptor (PR), androgen receptor (AR), and glucocorticoid receptor (GR).

[0144] Figures 9A–9D show the relative protein levels of ERα and GR proteins in MCF7aro and MCF7 LTLT cells compared to ERα in normal MCF7 and T47D cells. Actin expression was used as an internal control for each cell line. As described, MCF7 LTLT cells expressed less ERα protein than normal MCF7 cells and similarly less than T47D cells (Figure 9A), which is also indicated by the ER to actin expression ratio (Figure 9B). GR protein levels were low in MCF7 LTLT and MCF7aro cells but similar to T47D cells (Figures 9C–9D). In both assays, actin expression was consistent across cell lines (Figures 9A and 9C).

[0145] Figures 10A–10B show that AR levels are lower in MCF7aro and MCF7 LTLT compared to MCF7 and T47D. Figures 10C and 10D show the presence of HER2, which has been previously reported to be upregulated in the MCF7 LTLT cell line. PR was not detected in any of the MCF7, MCF7aro, or MCF7 LTLT cells (Figure 10E).

[0146] 5.5.2. Example 2: Efficacy of rasofoxifen in AI-resistant breast cancer lacking gain-of-function mutations in the ESR1 gene method For the letrozole resistance test, cells were plated in either RPMI or CS RPMI (SRPMI) in 96-well plates at 2500–3000 cells / well and treated with letrozole at various concentrations from 0.1 nM to 10 μM. For experiments performed in SRPMI, cells were cultured in CS serum for 48 hours prior to treatment. Treatment with estradiol and SERM (rasofoxifene) was performed in SRPMI. The 96-well plates were scanned every 6 hours for up to one week using an IncuCyte S3 (Essen Bioscience). Analysis was performed by counting GFP-tagged nuclei via IncuCyte software.

[0147] Animal testing and injection Mouse studies were conducted in compliance with approved animal experimentation committee protocols. NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) mice were purchased from The Jackson Laboratories. Prior to injection, mice were anesthetized by inhalation with 2-3% isoflurane in oxygen. Single-cell suspensions of 250,000 MCF7 cells were injected into the mammary ducts of inguinal glands 4 and 9 as described (Behbod, F. et al., Breast Cancer Res. 11, R66 (2009); Sflomos, G. et al., Cancer Cell 29, 407-422 (2016); Laine, M. et al., Breast Cancer Res. 23, 54 (2021)). In-situ tumor growth was tracked via imaging using the Xenogen IVIS 200 instrument at the Integrated Small Animal Imaging Research Resource of the University of Chicago. For IVIS imaging, mice were injected with 100 μl of 0.1 M luciferin solution in PBS (Perkin Elmer XenoLight #122799).

[0148] After cell injection, mice were divided into two equal groups; one group received 10 μg / day of letrozole in 15% PEG400 in PBS via subcutaneous injection ("LTLT-Let"), while the other group did not receive letrozole ("LTLT"). Two to three weeks after cell injection, mice in each group were randomized and treated with rasofoxifene (10 mg / kg in 100 μl of PBS containing 15% PEG400), palbociclib (70 mg / kg in 50 mM sodium lactate buffer (pH 4), Med Chem Express #HY-50567), or a vehicle every five days / week. Rasofoxifene and the vehicle were administered subcutaneously. Palbociclib was administered five times / week via oral gastric tube feeding. Fulvestrant (Med Chem Express #HY-13636) ("ICI") (5 mg / mouse) was administered once a week via subcutaneous injection in 100 μl of mineral oil. Mice were also treated with rasofoxifen + palbociclib, fulvestrant + palbociclib, rasofoxifen + letrozole, and combinations of palbociclib or fulvestrant + palbociclib, or letrozole. [Table 2-1] [Table 2-2]

[0149] After 90–93 days of treatment, mice were sacrificed, mammary tumors were excised, and weighed. Following in vivo injection of luciferin 8 minutes prior to sacrifice, the liver, bones, brain, and lungs were removed and imaged ex vivo with IVIS 200 to measure luciferase activity.

[0150] IHC and H&E analysis The collected tissues were fixed in formalin for IHC and H&E staining. Histological examination was performed at the Human Tissue Resource Center (HRTC) at the University of Chicago. Primary glands excised from mice were sectioned and stained with antibodies against estrogen receptor α (ThermoScientific, RM-9101-S0, clone SP1), progesterone receptor (ThermoScientific, RM-9102-S0, clone SP2), glucocorticoid receptor (Cell Signaling, catalog number 3660), androgen receptor (Abcam, ab133273, clone EPR1535), Ki67 (ThermoScientific, catalog number RM-9106-s, clone: ​​SP6), and a human-specific mouse monoclonal antibody against mitochondria (Abcam, ab92824, clone 112-1).

[0151] H&E and IHC slides were scanned using a Nikon eclipse Ti2 microscope equipped with a 10× objective lens for high-resolution imaging. Ki67 scores were determined using standardized manual counting.

[0152] Western blot analysis Cells were lysed in M-PER lysis buffer (Thermo Fisher, catalog no. 78501) in the presence of protease inhibitor cocktail 3 (Calbiochem, catalog no. 535140). Samples were loaded onto the WES Protein Simple platform. The antibodies used were: estrogen receptor (Santa Cruz, F10, catalog no. sc8002), glucocorticoid receptor (Cell Signaling, catalog no. 12041s), Her2 (Cell Signaling, catalog no. 2242s), androgen receptor (sigma, catalog no. sp242), and progesterone receptor (KD68), and an in-house produced rat monoclonal antibody (Greene, GL et al., Mol. Endocrinol 2, 714-726 (1988)). Graphs and statistical analysis Graphs and box plots were created using Graphpad Prism 7 software. An independent two-tailed t-test was used to determine the p-values.

[0153] result : Rasofoxifen alone and in combination with palbociclib inhibit the growth of primary MCF7 LTLT tumors. MCF7 LTLT cells labeled with GFP luciferase were injected into the mammary ducts of NSG mice to establish tumors. This represents an AI-resistant, non-ESR1 mutant breast cancer model. Treatment with rasofoxifene, palbociclib, and a combination of rasofoxifene and palbociclib was performed. - To account for the potential loss ("recovery") of letrozole resistance in the cohort, in the presence of letrozole (Let + ) or non-existent (Let -The study was conducted using a Xenogen IVIS scanner. Mice were imaged every two weeks to estimate tumor growth. At the end of the study, the mammary glands were removed and weighed. In this model, tumors were not palpable; however, whole-photon flux (darkly filled circles) and fulvestrant (inverted triangles (also called "ICI")) treatments of the vehicle were more effective than all other treatments (Figures 2 and 3; Let - (Only cohorts are shown).

[0154] Photon flux readings for different treatment groups showed similar patterns in the presence or absence of letrozole; readings for single treatment with palbociclib or rasofoxifen were 1 / 2 to 1 / 3 of those for the vehicle, and readings for the combination of palbociclib and rasofoxifen were 1 / 3 to 1 / 4 of those for the vehicle (Figures 2 and 3; Let - (Only cohort data are shown). Photon flux for each treatment at day 104 shows that rasofoxifene, palbociclib, and rasofoxifene + palbociclib were significantly lower than vehicle and fulvestrant (ICI) treatments, regardless of the presence of letrozole. Surprisingly, fulvestrant appeared to increase tumor photon flux in the presence of letrozole rather than inhibiting it as predicted (data not shown). This result is an unexplained exception.

[0155] Similar results were observed with respect to breast tissue weight at the end of the study (Figure 4; Let - (Only cohorts are shown). However, the differences between glandular weights are not as dramatic as those with respect to radiance (data not shown). This is probably because these weights include some normal breast tissue that dilutes the tumor weight estimate, whereas radiance measurements reflect only luciferase-expressing tumor cells. In any case, fulvestrant did not increase breast weight in either group, and in fact, Let - In the cohort, it inhibits breast tissue weight, but Let +The trends between weight and radiance are similar, except that there is no inhibition in the cohort (data not shown).

[0156] Figure 5 shows Let - In the cohort, tumor area is expressed as the percentage (%) of total tissue in the breast based on H&E staining of representative sections. H&E slides were scanned with a Nikon microscope and analyzed with NSI element software. H&E staining was performed using Let - The results show that the % tumor area in the cohort was significantly lower with rasofoxifen alone compared to the vehicle, and that rasofoxifen + palbociclib was significantly lower than with the vehicle. Palbociclib + fulvestrant (ICI) also showed a significant decrease in tumor area. - In the cohort, the risk was significantly lower than that of the vehicle. Rasofoxifen + palbociclib appeared to be lower than fulvestrant (ICI) + palbociclib, but this difference was not statistically significant. Let + In the cohort (data not shown), rasofoxifen + palbociclib was significantly lower than palbociclib + fulvestrant. Other treatments (e.g., palbociclib or fulvestrant alone) did not reach statistical significance against or against the vehicle.

[0157] Considering these results, we believe that rasofoxifen (alone and in combination with palbociclib, a CDK4 / 6 inhibitor) inhibits primary tumor growth, in particular, Let -It can be concluded that significant inhibition occurs in the cohort. Overall, the rasofoxifene + palbociclib combination is clearly superior to all other treatments, and especially to the conventional treatment regimen of fulvestrant (alone or in combination with palbociclib). The data indicate that rasofoxifene or the rasofoxifene + palbociclib combination is effective in inhibiting primary tumor growth in cancer cells that have developed resistance to letrozole (an aromatase inhibitor (AI) commonly prescribed to treat ER+ breast cancer) through mechanisms other than the acquisition of gain-of-function mutations in the ligand-binding domain of the ERα receptor (encoded by the ESR1 gene).

[0158] The tumor growth index in the MCF7 LTLT model is letrozole. + Low only in the group Immunohistochemistry with anti-Ki67 antibody was performed on fixed mammary gland tissue to determine the growth index of MCF7 LTLT primary tumors.

[0159] Letrozole itself significantly reduces the Ki67 percentage from 56.7±9 to 39.5±7.7 compared to the vehicle (Figure 6A). +Observations were made in the cohort (Figure 6C). Here, rasofoxifen alone and rasofoxifen plus palbociclib reduced Ki67 percentage by 6.5% and 12.4%, respectively (Figure 6C and Table 3). Table 3 shows the manually measured mean % Ki67 and ± standard deviation for each treatment group. Examination of the data shows that the effect of rasofoxifen alone was not significant compared to letrozole control, while rasofoxifen plus palbociclib significantly reduced Ki67 compared to palbociclib alone. Interestingly, palbociclib alone or fulvestrant (ICI) alone did not reduce Ki67 % levels in this model. However, fulvestrant + palbociclib significantly reduced the Ki67 percentage to 18.8±12.3 compared to letrozole control (39.5±7.7) and palbociclib (45.3±19.0) (Table 3). These results suggest that the growth index alone may not be the best measure of rasofoxifen activity in the MCF7 LTLT model. [Table 3]

[0160] Rasofoxifene and rasofoxifene + palbociclib appear to reduce bone metastases. During slaughter, the liver, lungs, bones, and brain were removed and imaged with IVIS and exovivo. Figures 7A-7C and 8A are shown. - Box plots of the mean radiance for the liver (Figure 7A), lungs (Figure 7B), brain (Figure 7C), and bones (Figure 8A) in the treatment groups are shown. + Data for the group were evaluated but not presented. The mean radiance for all organs was low, indicating that only minimal metastases were observed. No statistically significant patterns were observed for the liver, lungs, and brain, but the rasofoxifen + palbociclib signaling pathway was observed. - and Let + Visually lower brain activity compared to any other treatment for both cohorts compared to the vehicle.

[0161] Possible bone metastases were detected (Figure 8B). However, only three readings in the vehicle group, two in the fulvestrant (ICI) group, and one in the fulvestrant (ICI) + palbociclib group showed signals exceeding the threshold. In both cohorts, in particular, Let - In the cohort, rasofoxifen + palbociclib appeared to inhibit the vehicle's radius of action, but this was not statistically significant.

[0162] In conclusion, these data demonstrate that rasofoxifene (alone or in combination with palbociclib) inhibits primary tumor growth in this model of AI-resistant breast cancer where resistance is due to mechanisms other than acquisition of ESR ligand-binding domain gain-of-function mutations. Furthermore, rasofoxifene (alone or in combination with palbociclib) appears to inhibit bone metastases. Generally, rasofoxifene in combination with palbociclib is more effective than the fulvestrant and palbociclib combination in inhibiting primary tumor and metastatic growth in these cells. The data have significant clinical implications and clearly indicate the potential for using rasofoxifene as an effective treatment for women with advanced or metastatic ER+ breast cancer that is resistant to aromatase inhibitors and lacks ESR1 gain-of-function mutations.

[0163] 6. Equivalents and references While the present invention has been described and illustrated in detail with reference to major embodiments and various alternative embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made therein without departing from the spirit and scope of the invention.

[0164] All references, published patents, and patent applications cited herein are incorporated herein by reference in their entirety for the sole purpose of this specification. In certain embodiments, for example, the following items are provided: (Item 1) In patients whose disease has progressed under aromatase inhibitors, estrogen receptor-positive (ER) + ) A method for reducing the progression of cancer, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, and the method is The step of administering an effective amount of rasofoxifen or a pharmaceutically acceptable salt thereof, prodrug, or functional derivative to the patient. A method that includes (Item 2) The aforementioned ER + The cancer is locally advanced or metastatic breast cancer, where the cancer is HER2-positive as appropriate. - The method described in item 1. (Item 3) The aforementioned aromatase inhibitors include exemestane (Aromasin®), letrozole (Femara®), or anastrozole (Arimidex). (登録商標) ) The method described in any of the above items. (Item 4) The method described in any of the preceding items further includes determining that the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene. (Item 5) The method described in any of the above items, wherein rasofoxifene is administered as rasofoxifene tartrate. (Item 6) Rasofoxifen is administered orally, intravenously, transdermally, topically, or via a vaginal ring, as described in any of the preceding items. (Item 7) Rasofoxifen is administered orally, as described in any of the above-mentioned methods. (Item 8) Rasofoxifen is administered orally at a dose of 5 mg / day to approximately 10 mg / day, as described in item 7. (Item 9) The method of any of the preceding items, further comprising the step of treating the patient with at least one further endocrine therapy. (Item 10) The method of any of the above items, further comprising the step of administering an effective amount of cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor to the patient. (Item 11) The method according to item 10, wherein the CDK4 / 6 inhibitor is palbociclib, abemaciclib, or ribociclib, where preferably the inhibitor is abemaciclib. (Item 12) The method according to any one of items 1 to 9, further comprising the step of administering an effective amount of AKT inhibitor to the patient. (Item 13) The method according to item 12, wherein the AKT inhibitor is an adsorbent. (Item 14) The method according to any one of items 1 to 9, further comprising the step of administering an effective amount of mTor inhibitor to the patient.

Claims

1. In patients with progressive breast cancer under aromatase inhibitors, estrogen receptor-positive and human epidermal growth factor receptor 2-negative (ER) ＋ / HER2 － A composition for use in a method for reducing the progression of breast cancer, wherein the composition comprises rasofoxifen or a pharmaceutically acceptable salt thereof, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

2. The aforementioned ER ＋ / HER2 － The composition according to claim 1, wherein the cancer is locally advanced or metastatic breast cancer.

3. The composition according to claim 1, wherein the aromatase inhibitor is exemestane, letrozole, or anastrozole.

4. The composition according to claim 1, further comprising determining that the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

5. The composition according to claim 1, characterized in that rasofoxifen is administered as rasofoxifen tartrate.

6. The composition according to claim 5, characterized in that the lasofoxifen tartrate is administered orally.

7. The composition according to claim 6, characterized in that rasofoxifen tartrate is administered orally at a dose of 5 mg rasofoxifen / day to approximately 10 mg rasofoxifen / day.

8. The composition according to claim 7, characterized in that rasofoxifen tartrate is administered orally at a dose of 5 mg rasofoxifen / day.

9. The composition according to claim 1, characterized in that it is administered to the patient in combination with an effective amount of cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor.

10. The composition according to claim 9, wherein the CDK4 / 6 inhibitor is palbociclib, abemaciclib, or ribociclib.

11. The composition according to claim 10, wherein the CDK4 / 6 inhibitor is palbociclib.

12. The composition according to claim 10, wherein the CDK4 / 6 inhibitor is abemaciclib.

13. The composition according to claim 10, wherein the CDK4 / 6 inhibitor is ribociclib.

14. The composition according to claim 1, characterized in that it is administered to the patient in combination with an effective amount of AKT inhibitor or mTo inhibitor.

15. In patients with progressive breast cancer under aromatase inhibitors, estrogen receptor-positive and human epidermal growth factor receptor 2-negative (ER) ＋ / HER2 － A composition for use in a method for reducing the progression of breast cancer, wherein the composition comprises rasofoxifen or a pharmaceutically acceptable salt thereof, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, and the composition is administered to the patient in combination with a cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor.

16. The composition according to claim 15, wherein the CDK4 / 6 inhibitor is palbociclib, abemaciclib, or ribociclib.

17. The composition according to claim 16, wherein the CDK4 / 6 inhibitor is palbociclib.

18. The composition according to claim 16, wherein the CDK4 / 6 inhibitor is abemaciclib.

19. The composition according to claim 16, wherein the CDK4 / 6 inhibitor is ribociclib.

20. The composition according to claim 15, further comprising determining that the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene.

21. The composition according to claim 15, characterized in that rasofoxifen is administered as rasofoxifen tartrate.

22. The composition according to claim 21, characterized in that rasofoxifen tartrate is administered orally at a dose of 5 mg rasofoxifen / day.

23. The composition according to claim 15, characterized in that it is administered to the patient in combination with an effective amount of AKT inhibitor or mTOR inhibitor.

24. The composition according to claim 15, wherein the combination of rasofoxifen and CDK4 / 6 inhibitor inhibits the metastasis of breast cancer to bone.

25. In patients with progressive breast cancer under aromatase inhibitors, estrogen receptor-positive and human epidermal growth factor receptor 2-negative (ER) ＋ / HER2 － A composition for use in a method for reducing the progression of breast cancer, wherein the composition comprises rasofoxifen or a pharmaceutically acceptable salt thereof, wherein the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, and the cancer is as shown in Table 1. Table 1-1 A composition having at least one variant in at least one gene shown in [the specified formula].

26. The composition according to claim 25, characterized in that rasofoxifen is administered orally as rasofoxifen tartrate.

27. The composition according to claim 26, characterized in that rasofoxifen tartrate is administered orally at a dose of 5 mg rasofoxifen / day.

28. The above method can be performed in any order. To determine that the cancer does not have a gain-of-function missense mutation in the ligand-binding domain (LBD) of the estrogen receptor 1 (ESR1) gene, and The aforementioned cancers are shown in Table 1. Table 1-2 To determine that the individual has at least one variant in at least one gene shown in [the specified field]. The composition according to claim 25, further encompassing the following:

29. The composition according to claim 25, characterized in that it is administered to the patient in combination with an effective amount of cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitor.

30. The composition according to claim 29, wherein the CDK4 / 6 inhibitor is palbociclib, abemaciclib, or ribociclib.

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