Therapeutic and prophylactic method for tumor treatable with endocrine therapy by combined use of fibroblast growth factor receptor inhibitor with endocrine therapy

Abstract

To provide a novel therapeutic method exhibiting significantly excellent antitumor effect to a tumor treatable with endocrine therapy by using a FGFR inhibitor.SOLUTION: Provided is a novel treatment method for cancer tumors to which endocrine therapy is to be applied, using an FGFR inhibitor (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof. Further provided are a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt thereof for use in treatment and / or prevention of tumors to which endocrine therapy is applicable, as well as related compounds, uses, methods, and combinations.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority based on Japanese Patent Application No. 2018-220046, filed on November 26, 2018, the entire disclosure of which is incorporated herein by reference. The present disclosure relates to the treatment and prevention of tumors to which endocrine therapy is applied. [Background technology]

[0002] Fibroblast growth factors (FGFs) are expressed in a wide range of tissues and are one of the growth factors that control cell proliferation and differentiation. The physiological activity of FGFs is mediated by specific cell surface receptors, called fibroblast growth factor receptors (FGFRs). FGFRs belong to the receptor-type protein tyrosine kinase family and consist of an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular tyrosine kinase domain. Four types of FGFRs have been identified so far (FGFR1, FGFR2, FGFR3, and FGFR4). FGFRs form dimers upon binding with FGF and are activated by phosphorylation. Receptor activation induces the recruitment and activation of specific downstream signaling molecules, resulting in physiological functions. Abnormalities in FGF / FGFR signaling have been reported to be associated with various human tumors. Abnormal activation of FGF / FGFR signaling in human tumors is believed to result from overexpression of FGFR and / or gene amplification, gene mutation, chromosomal translocation / insertion / inversion, gene fusion, or autocrine or paracrine mechanisms due to overproduction of the ligand, FGF (Non-Patent Documents 1, 2, 3, and 4).

[0003] In tumors in hormone-responsive tissues such as the breast and prostate, there are types in which hormone stimulation targeting the tissue promotes tumor growth. Focusing on this, a treatment method that controls hormone stimulation to tumors is known as endocrine therapy (also called hormone therapy). For example, in estrogen receptor-positive breast tumors, endocrine therapy such as suppression of ovarian function, which is an estrogen-producing organ (by surgical methods, radiological methods, or drug administration), estrogen-producing enzyme (aromatase) inhibitors, and estrogen receptor inhibitors (anti-estrogens) can be considered. In addition, in prostate tumors, endocrine therapy such as suppression of testosterone secretion (testicular removal, which is the producing organ, or administration of drugs such as LH-RH agonists, LH-RH antagonists, or estrogen agents) and administration of anti-androgens is known for prostate tumors that are androgen-dependent. Anti-androgens are also used for benign prostate hyperplasia (BPH). LH-RH agonists are also sometimes used to treat ovarian cancer.

[0004] Abnormalities in FGF / FGFR signals have also been found in tumors in these hormone-responsive tissues, and their association with tumors has been reported. In ER-positive breast cancer patients, a correlation between FGFR1 amplification and poor prognosis has been reported (Non-Patent Document 5). In prostate cancer, abnormal expression of the FGF / FGFR system constitutively activates multiple downstream signals, which are involved in the progression of cancer (Non-Patent Document 6). Overexpression of FGFR1 localized in stromal cells has also been reported in benign prostatic hyperplasia (Non-Patent Document 7). Amplification of the FGFR1 gene, for example, has also been reported in ovarian cancer (Non-Patent Document 1).

[0005] Furthermore, with regard to endocrine therapy for hormone-responsive tissue cancer, although the initial treatment shows a suppressive response to progression, the response later disappears, a phenomenon known as resistance to endocrine therapy. It is known that FGF / FGFR system is involved in such endocrine therapy resistance, and it has been reported that FGF / FGFR system is involved in such endocrine therapy resistance. For example, FGFR1 gene amplification in breast cancer causes FGFR1 overexpression, activates FGFR1 downstream signaling in a ligand-dependent and independent manner (Non-Patent Document 4), and enhances gene expression downstream of estrogen receptor even in the absence of estrogen stimulation, causing resistance to endocrine therapy (Non-Patent Document 8). In addition, in prostate cancer, resistance to androgen deprivation therapy may be observed accompanied by neuroendocrine tumor-like differentiation without expressing androgen receptor, but further, a phenotype (double negative) that does not express androgen receptor and does not exhibit the characteristics of neuroendocrine tumor is also observed, and it has been reported that androgen receptor dependency is avoided by constitutive activation of FGF and downstream signaling pathways (Non-Patent Document 9). When resistance to these endocrine therapies appears, activation of MAPK and the like downstream of the FGFR signaling pathway has been commonly found (Non-Patent Document 4; Non-Patent Document 9).

[0006] In cancers for which endocrine therapy is applicable, several FGFR inhibitors are being clinically developed for use in combination with hormonal drugs. In a phase II study of breast cancer in which the multikinase inhibitor dovitinib, which includes FGFR kinases, was combined with the anti-estrogen drug fulvestrant, the median mean progression-free survival (PFS) was significantly improved in the combination group compared to the fulvestrant monotherapy group in a small group of patients with enhanced FGF pathways (10.9 vs. 5.5 months) (Non-Patent Document 10). In addition, in breast cancer, a phase II study of the pan-FGFR inhibitor AZD4547 in combination with fulvestrant or the aromatase inhibitor exemestane and in fulvestrant and placebo is being conducted (NCT01202591) (Non-Patent Document 11). In prostate cancer, a phase II trial is ongoing evaluating dovitinib in combination with androgen deprivation therapy (NCT02065323) and a phase II trial is ongoing evaluating dovitinib in combination with the androgen synthase inhibitor abiraterone (NCT01994590).

[0007] Disubstituted benzenealkynyl compounds having FGFR inhibitory effects have been reported (Patent Document 1). It has also been reported that these compounds are effective against cancers with specific FGFR2 mutations (Patent Document 2), that intermittent administration may be useful as an administration schedule (Patent Document 3), and that cancer can be treated by combining them with antitumor agents (Patent Document 4). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication WO2013 / 108809 Brochure [Patent Document 2] International Publication WO2015 / 008844 Brochure [Patent Document 3] International Publication WO2015 / 008839 Brochure [Patent Document 4] International Publication WO2017 / 150725 Brochure [Non-patent literature]

[0009] [Non-Patent Document 1] Nat.Rev.Cancer 10:116-129(2010) [Non-Patent Document 2] J. Clin. Oncol. 24, 3664-3671 (2006) [Non-Patent Document 3] Mol. Cancer Res. 3, 655-667 (2005) [Non-Patent Document 4] Cancer Res.70, 2085-2094 (2010) [Non-Patent Document 5] Breast Cancer Res 2007;9:R23 [Non-Patent Document 6] Clin Cancer Res 19:5856-5866(2013) [Non-Patent Document 7] Eur J Endocrinol 145:303-310(2001) [Non-Patent Document 8] Clin Cancer Res 23:6138-6150(2017) [Non-Patent Document 9] Cancer Cell 32:474-489(2017) [Non-Patent Document 10] Breast Cancer Research.19,18(2017) [Non-Patent Document 11] The Breast.37,126-133(2018) [Non-Patent Document 12] Oncotarget. 8:16052-16074(2017) Summary of the Invention

[0010] Disclosure Summary

[0011] Problem to be solved An objective of the present disclosure is to provide a novel method for treating tumors to which endocrine therapy is applicable, using an FGFR inhibitor that exhibits significantly excellent antitumor effects. [Means for solving the problem]

[0012] In the present disclosure, the combined administration of the FGFR inhibitor (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof in tumors to which endocrine therapy is applicable was investigated, and it was found that the antitumor effect was significantly enhanced compared to the administration of the same or endocrine therapy alone.

[0013] That is, in one aspect, the present disclosure provides the following [A1] to [A16], [B1] to [B14], [C1] to [C14], [D1] to [D14], and [E1] to [E14]. [A1] A pharmaceutical composition used for the treatment and / or prevention of a tumor to which endocrine therapy is applicable, comprising (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof, characterized in that it is administered in combination with endocrine therapy. [A2] The pharmaceutical composition according to [A1], wherein the tumor is a tumor expressing FGFR. [A3] The pharmaceutical composition according to [A1] or [A2], wherein the tumor is a tumor in which FGFR signaling is activated. [A4] The pharmaceutical composition according to any one of [A1] to [A3], wherein the tumor is a tumor having an abnormality in an FGFR gene. [A5] The pharmaceutical composition according to any one of [A1] to [A4], wherein the tumor to which endocrine therapy is applied is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer and benign prostatic hyperplasia. [A6] The pharmaceutical composition according to any one of [A1] to [A5], wherein the tumor is a tumor resistant to endocrine therapy. [A7] The pharmaceutical composition according to any one of [A1] to [A6], wherein the tumor is breast cancer expressing FGFR1. [A8] The pharmaceutical composition according to any one of [A1] to [A7], wherein the tumor is a breast cancer having amplification of the FGFR1 gene. [A9] The pharmaceutical composition according to any one of [A1] to [A8], wherein the tumor to which endocrine therapy is applied is breast cancer, and the endocrine therapy includes at least one selected from the group consisting of ovarian function suppression, administration of an aromatase inhibitor, administration of an anti-estrogen drug, and administration of a progesterone hormone. [A10] The pharmaceutical composition according to [A9], wherein the endocrine therapy is administration of an anti-estrogen drug. [A11] The pharmaceutical composition according to [A10], wherein the anti-estrogen drug is tamoxifen or fulvestrant, or a salt or derivative thereof. [A12] The pharmaceutical composition according to any one of [A1] to [A11], characterized in that the endocrine therapy and the administration of a composition comprising (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof are carried out simultaneously, separately, or successively. [A13] The pharmaceutical composition according to any one of [A1] to [A12], which is administered to a tumor patient who has undergone endocrine therapy. [A14] The pharmaceutical composition according to any one of [A1] to [A13], which is administered to a tumor patient who undergoes endocrine therapy after administration of the pharmaceutical composition. [A15] A method for treating and / or preventing a tumor to which endocrine therapy is applicable, comprising administering to a patient an effective amount of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof in combination with endocrine therapy. [A16] A combination of endocrine therapy and the administration of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof for use in the treatment and / or prophylaxis of tumors for which endocrine therapy is indicated. [B1] (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof for use in the treatment and / or prevention of a tumor to which endocrine therapy is applicable, characterized in that it is administered in combination with endocrine therapy. [B2] The compound according to [B1] or a pharma- ceutically acceptable salt thereof, wherein the tumor is a tumor expressing FGFR. [B3] The compound or a pharma- ceutically acceptable salt thereof according to [B1] or [B2], wherein the tumor is a tumor in which FGFR signaling is activated. [B4] The compound or a pharma- ceutically acceptable salt thereof according to any one of [B1] to [B3], wherein the tumor is a tumor having an abnormality in an FGFR gene. [B5] The compound or a pharma- ceutically acceptable salt thereof according to any one of [B1] to [B4], wherein the tumor to which endocrine therapy is applied is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer and benign prostatic hyperplasia. [B6] The compound or a pharma- ceutically acceptable salt thereof according to any one of [B1] to [B5], wherein the tumor is a tumor resistant to endocrine therapy. [B7] The compound or a pharma- ceutically acceptable salt thereof according to any one of [B1] to [B6], wherein the tumor is breast cancer expressing FGFR1. [B8] The compound or a pharma- ceutically acceptable salt thereof according to any one of [B1] to [B7], wherein the tumor is breast cancer having amplification of the FGFR1 gene. [B9] The compound or a pharma- ceutical acceptable salt thereof according to any one of [B1] to [B8], wherein the tumor to which endocrine therapy is applied is breast cancer, and the endocrine therapy includes at least one selected from the group consisting of ovarian function suppression, administration of an aromatase inhibitor, administration of an anti-estrogen drug, and administration of a progesterone hormone. [B10] The compound or a pharma- ceutically acceptable salt thereof according to [B9], wherein the endocrine therapy is administration of an anti-estrogen drug. [B11] The compound according to [B10] or a pharma- ceutically acceptable salt thereof, wherein the anti-estrogen drug is tamoxifen or fulvestrant, or a salt or derivative thereof. [B12] The compound or pharma- ceutically acceptable salt thereof according to any one of [B1] to [B11], characterized in that endocrine therapy and administration of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof are carried out simultaneously, separately, or successively. [B13] The compound or a pharma- ceutically acceptable salt thereof according to any one of [B1] to [B12], which is administered to a tumor patient who has been treated with endocrine therapy. [B14] The compound or a pharma- ceutically acceptable salt thereof according to any one of [B1] to [B13], wherein the compound or a pharma- ceutically acceptable salt thereof is administered to a tumor patient who is administered endocrine therapy after administration of the compound or a pharma- ceutically acceptable salt thereof. [C1] Use of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament used for the treatment and / or prevention of a tumor to which endocrine therapy is applicable, characterized in that the medicament is administered in combination with endocrine therapy. [C2] The use according to [C1], wherein the tumor is a tumor expressing FGFR. [C3] The use according to [C1] or [C2], wherein the tumor is a tumor in which FGFR signaling is activated. [C4] The tumor is a tumor having an abnormality in the FGFR gene, [C1 or the tumor according to claim 2] use. [C5] The tumor to which endocrine therapy is applied is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer and benign prostatic hyperplasia, [C1 or the use described in claim 2. [C6] The tumor is a tumor resistant to endocrine therapy, [C1 or the use according to claim 2. [C7] The tumor is a breast cancer expressing FGFR1, [C1 or the use described in claim 2. [C8] The tumor is a breast cancer having an amplification of the FGFR1 gene, [C1 or the use described in claim 2. [C9] The use described in C1 or claim 2, wherein the tumor to which endocrine therapy is applied is breast cancer, and the endocrine therapy includes at least one selected from the group consisting of ovarian function suppression, administration of an aromatase inhibitor, administration of an anti-estrogen drug, and administration of a progesterone hormone. [C10] The use according to [C9], wherein the endocrine therapy is administration of an anti-estrogen drug. [C11] The use according to [C10], wherein the anti-estrogen drug is tamoxifen or fulvestrant or a salt or derivative thereof. [C12] The use according to any one of [C1] to [C11], characterized in that the endocrine therapy and the administration of a medicine containing (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof are carried out simultaneously, separately, or successively. [C13] The use according to any one of [C1] to [C12], characterized in that the use is administered to a tumor patient who has undergone endocrine therapy. [C14] The use according to any one of [C1] to [C13], characterized in that the medicament is administered to a tumor patient who will undergo endocrine therapy after administration of the medicament. [D1] A method for treating and / or preventing a tumor to which endocrine therapy is applicable, comprising administering to a patient an effective amount of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof in combination with endocrine therapy. [D2] The method according to [D1], wherein the tumor is a tumor expressing FGFR. [D3] The method according to [D1] or [D2], wherein the tumor is a tumor in which FGFR signaling is activated. [D4] The method according to any one of [D1] to [D3], wherein the tumor is a tumor having an abnormality in an FGFR gene. [D5] The method according to any one of [D1] to [D4], wherein the tumor to which endocrine therapy is applied is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer and benign prostatic hyperplasia. [D6] The method according to any one of [D1] to [D5], wherein the tumor is a tumor resistant to endocrine therapy. [D7] The method according to any one of [D1] to [D6], wherein the tumor is breast cancer expressing FGFR1. [D8] The method according to any one of [D1] to [D7], wherein the tumor is a breast cancer having amplification of the FGFR1 gene. [D9] The method according to any one of [D1] to [D8], wherein the tumor to which endocrine therapy is applied is breast cancer, and the endocrine therapy includes at least one selected from the group consisting of ovarian function suppression, administration of an aromatase inhibitor, administration of an anti-estrogen drug, and administration of a progesterone hormone. [D10] The method according to [D9], wherein the endocrine therapy is administration of an anti-estrogen drug. [D11] The method according to [D10], wherein the anti-estrogen drug is tamoxifen or fulvestrant, or a salt or derivative thereof. [D12] The method according to any one of [D1] to [D11], characterized in that the endocrine therapy and the administration of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof are carried out simultaneously, separately, or successively. [D13] The method according to any one of [D1] to [D12], characterized in that the method is administered to a tumor patient who has undergone endocrine therapy. [D14] The method according to any one of [D1] to [D13], characterized in that (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof is administered to a tumor patient who is receiving endocrine therapy after administration of the compound or a pharma- ceutically acceptable salt thereof. [E1] A combination of endocrine therapy and the administration of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof for use in the treatment and / or prophylaxis of tumors for which endocrine therapy is indicated. [E2] The combination described in [E1], wherein the tumor is a tumor expressing FGFR. [E3] The combination according to [E1] or [E2], wherein the tumor is a tumor in which FGFR signaling is activated. [E4] The combination according to any one of [E1] to [E3], wherein the tumor is a tumor having an abnormality in an FGFR gene. [E5] The combination according to any one of [E1] to [E4], wherein the tumor to which endocrine therapy is applied is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer and benign prostatic hyperplasia. [E6] The tumor is resistant to endocrine therapy, either [E1] to [E5] The combination described in item . [E7] The combination according to any one of [E1] to [E6], wherein the tumor is breast cancer expressing FGFR1. [E8] The combination according to any one of [E1] to [E7], wherein the tumor is a breast cancer having amplification of the FGFR1 gene. [E9] The combination according to any one of [E1] to [E8], wherein the tumor to which endocrine therapy is applied is breast cancer, and the endocrine therapy includes at least one selected from the group consisting of ovarian function suppression, administration of an aromatase inhibitor, administration of an anti-estrogen drug, and administration of a progesterone hormone. [E10] The combination described in [E9], wherein the endocrine therapy is administration of an anti-estrogen drug. [E11] The combination according to [E10], wherein the anti-estrogen drug is tamoxifen or fulvestrant or a salt or derivative thereof. [E12] The combination according to any one of [E1] to [E11], characterized in that the administration of endocrine therapy and (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof is carried out simultaneously, separately or sequentially. [E13] The combination according to any one of [E1] to [E12], which is administered to a tumor patient who has undergone endocrine therapy. [E14] The combination according to any one of [E1] to [E13], characterized in that the compound or a pharma- ceutical acceptable salt thereof is administered to a tumor patient who is administered endocrine therapy after administration of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutical acceptable salt thereof.

[0014] Effect of the present disclosure According to the present disclosure, it is possible to realize a tumor treatment that exhibits a high antitumor effect, including a tumor shrinking effect and a tumor growth retarding effect, which can lead to inhibition of the progression of the disease, improvement of the pathology, and long-term survival of tumor patients. [Brief description of the drawings]

[0015] [Figure 1A] 1 shows the effect of the combination of Compound 1 and 4-hydroxytamoxifen on cell proliferation in MDA-MB-134VI cells. [Figure 1B] 1 shows the effect of combined use of Compound 1 and fulvestrant on cell proliferation in MDA-MB-134VI cells.

[0016] As an embodiment, the present disclosure provides a pharmaceutical composition for the treatment and / or prevention of a tumor to which endocrine therapy is applicable, comprising (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof, in combination with endocrine therapy, as well as related compounds, uses, methods, and combinations.

[0017] (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one is a disubstituted benzenealkynyl compound having the following structure. In this specification, the above compound is referred to as "compound 1" for convenience. Compound 1 is described as Example compound 2 in the above Patent Document 1 International Publication WO2013 / 108809. Compound 1 has an excellent FGFR inhibitory effect, and it has been reported that it inhibits the phosphorylation ability of the receptor protein tyrosine kinases of FGFR1, FGFR3, and FGFR4 to tyrosine in the substrate peptide sequence (Patent Document 1).

[0018] [ka]

[0019] Compound 1 or a pharma- ceutical acceptable salt thereof in the pharmaceutical composition of the present disclosure can be synthesized, for example, based on the production method described in International Publication WO2013 / 108809 (Patent Document 1), without particular limitation.

[0020] In the present disclosure, Compound 1 can be used as it is or in the form of a pharma- ceutically acceptable salt. The pharma- ceutically acceptable salt of Compound 1 includes, but is not limited to, addition salts with inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as acetic acid, citric acid, tartaric acid, and maleic acid, salts with alkali metals such as potassium and sodium, salts with alkaline earth metals such as calcium and magnesium, and salts with organic bases such as ammonium salts, ethylamine salts, and arginine salts.

[0021] The present disclosure can be used for the treatment and / or prevention of tumors to which endocrine therapy is applicable. Tumors to which endocrine therapy is applicable include tumors for which some biological response is expected to be observed when endocrine therapy is applied. Here, some biological response includes, for example, changes in indicators at the cell or tissue level (e.g., proliferation rate, staining image) in a specimen of the tumor, and changes in indicators in clinical trials (e.g., tumor volume reduction on an image, overall survival (OS), progression free survival (PFS), response rate (RR)). Tumors to which endocrine therapy is applicable include tumors for which the following occurs as a result of endocrine therapy: Tumors to which endocrine therapy is applied include those that initially showed a biological response but subsequently showed a weakened or lost biological response to endocrine therapy, i.e., those that have become resistant to endocrine therapy.

[0022] Tumors to which endocrine therapy is applicable include benign tumors, malignant tumors (cancer), and borderline malignant tumors.

[0023] Tumors to which endocrine therapy is applied include tumors in hormone target tissues. Hormone target tissues include the breast (mammary gland), prostate, and ovary. Hormones include sex hormones. Tumors to which endocrine therapy is applied include hormone-dependent tumors. Sex hormones include sex steroid hormones.

[0024] Cancers for which endocrine therapy is used include breast cancer and prostate cancer, and ovarian cancer.

[0025] In certain embodiments, the present disclosure is used for primary cancer, and metastatic and recurrent cancer.

[0026] Benign tumors for which endocrine therapy is applicable include benign prostatic hyperplasia (BPH).

[0027] In the present disclosure, treatment includes procedures performed with the aim of curing or remitigating a disease, or inhibiting the progression or recurrence of the disease, or alleviating symptoms. Treatment includes administration of drugs before or after a surgical procedure. Treatment includes post-operative and pre-operative therapy, as well as adjuvant and neo-adjuvant therapy.

[0028] Prevention in the present disclosure includes suppressing the onset of a cancer for which endocrine therapy is applicable when the patient has not yet reached the stage of the cancer for which endocrine therapy is applicable, and includes reducing the risk of onset of a cancer for which endocrine therapy is applicable in a patient at risk of developing a cancer for which endocrine therapy is applicable.

[0029] The present disclosure is characterized in that Compound 1 or a pharma- ceutically acceptable salt thereof is used in combination with endocrine therapy, also known as hormonal therapy, which is a preventive and therapeutic method that inhibits tumor progression by directly or indirectly controlling hormonal stimulation of tumors.

[0030] It is known that some breast cancers are aggravated by the presence of estrogen, and these breast cancers show expression of estrogen receptors and / or progesterone receptors (ER and / or PgR positive). Endocrine therapy for breast cancer is a treatment or prevention method that suppresses the aggravation of malignant tumors caused by the presence of estrogen.

[0031] The main endocrine therapies for breast cancer can be broadly classified into ovarian function suppression (OFS), aromatase inhibitor administration, anti-estrogen administration, and progesterone. Medication is administered.

[0032] Ovarian function suppression includes, for example, surgical treatment including oophorectomy, radiation treatment including elimination of ovarian function by irradiation, and administration of LH-RH agonists. LH-RH agonists suppress the action of pituitary hormones that promote the biosynthesis of estrogen in the ovaries, thereby reducing the estrogen production ability of the ovaries. Examples of LH-RH agonists include goserelin, leuprorelin, triptorelin, and salts and derivatives thereof.

[0033] Aromatase inhibitors inhibit aromatase, which converts male hormones into estrogen, thereby reducing the ability to produce estrogen. Examples of aromatase inhibitors include anastrozole, exemestane, letrozole, and salts thereof, as well as derivatives thereof.

[0034] Anti-estrogen drugs include, for example, selective estrogen receptor modulators that inhibit the binding of estrogen to estrogen receptor, and selective estrogen receptor down-regulators that inhibit binding and promote the degradation of estrogen receptor.For example, the former include tamoxifen, toremifene, raloxifene, and their salts, and their derivatives.For example, the latter include fulvestrant, and their salts, and their derivatives.

[0035] Progestins exert antitumor effects on breast cancer through various mechanisms, including antiestrogenic activity, and include, for example, the synthetic progestin medroxyprogesterone acetate and its salts, as well as derivatives thereof.

[0036] The present disclosure is used in some embodiments for premenopausal breast cancer, and the present disclosure is used in some embodiments for postmenopausal breast cancer.

[0037] In one embodiment, the present disclosure is used for estrogen receptor and / or progesterone receptor positive (ER and / or PgR positive) breast cancer. Positivity for ER and / or PgR can be determined, for example, by immunohistochemical (IHC) methods, by determining the occupancy rate of positive cells, or by combining the occupancy rate and staining intensity. When determining by the occupancy rate, the presence of even one positive cell (any positive cell) can be used as an indicator. In addition, A cut-off index can be set to a positive cell occupancy rate of 1% or 10%. In addition, staining intensity can also be taken into consideration (Arch Pathol Lab Med. 2010;134(7):e48-72). The positivity of ER and / or PgR can also be determined by a method that can show scientifically similar results to the above-mentioned methods. ER and / or PgR positive breast cancer includes not only breast cancer that is determined to be positive at the time of primary diagnosis, but also breast cancer that is determined to be positive for ER and / or PgR at least once at any point in the progression of breast cancer, including metastasis and recurrence. There are two highly homologous isoforms of estrogen receptors, ERα and ERβ, which are encoded by different genes ESR1 and ESR2, respectively (Arch Pathol Lab Med., ibid), but when simply referring to estrogen receptor or ER in this specification, it means ERα (ESR1 gene product).

[0038] The present disclosure, in one embodiment, is used against prostate cancer. It is known that some prostate cancers are aggravated by the presence of androgens, and these prostate cancers show expression of the androgen receptor (AR positive). Endocrine therapy for prostate cancer is a treatment or preventive method to suppress the aggravation of malignant tumors caused by the presence of androgens.

[0039] The main endocrine therapies for prostate cancer can be broadly categorized into those for androgen-dependent prostate cancer, such as suppression of secretion of testosterone, a type of androgen (removal of the testicle, which is the producing organ, or administration of drugs such as LH-RH agonists, LH-RH antagonists or estrogen agents), and administration of antiandrogens and androgen synthesis inhibitors.

[0040] LH-RH agonists act on the pituitary gland to reduce the production of testosterone, and include, for example, goserelin, leuprorelin, buserelin, and salts thereof, as well as derivatives thereof.

[0041] There are several known mechanisms of antiandrogen drugs, including inhibition of ligand binding to androgen receptor, inhibition of nuclear translocation of androgen receptor, and suppression of androgen receptor signaling. Examples of antiandrogen drugs include chlormadinone acetate, biphenylsulfonyl ester, and phenylmethylsulfonyl ester. These include carutamide, flutamide, enzalutamide, nilutamide, compounds disclosed in International Publication No. WO2015 / 182712, and salts thereof, and derivatives thereof.

[0042] Endocrine therapy also includes, for example, androgen synthesis inhibitors that inhibit enzymes in the androgen synthesis pathway, such as ketoconazole, aminoglutetide, abiraterone acetate and salts thereof, and derivatives thereof.

[0043] In the present disclosure, in one embodiment, Compound 1 or a pharma- ceutically acceptable salt thereof is administered in combination with the administration of a compound disclosed in International Publication No. WO2015 / 182712, which is an antiandrogen drug, as endocrine therapy, for the treatment and / or prevention of a tumor to which endocrine therapy is applied. In the present disclosure, in one embodiment, Compound 1 or a pharma- ceutically acceptable salt thereof is administered in combination with the administration of an antiandrogen drug other than a compound disclosed in International Publication No. WO2015 / 182712, as endocrine therapy, for the treatment and / or prevention of a tumor to which endocrine therapy is applied.

[0044] The surgical procedures, radiation procedures, and drug administration in the above endocrine therapy are known, and can be performed by a person skilled in the art. The action mechanism, dosage, and therapeutic application of the above drugs are known, and are available along with necessary information about the drugs. When the endocrine therapy is by administration of drugs, a person skilled in the art can administer an effective amount of the drug.

[0045] In some embodiments, the present disclosure is used to treat ovarian cancer. In some embodiments, Compound 1 or a pharma- ceutically acceptable salt thereof is used in combination with an LH-RH agonist to treat ovarian cancer.

[0046] In some embodiments, the present disclosure is used for benign prostatic hyperplasia (BPH). In some embodiments, compound 1 or a pharma- ceutically acceptable salt thereof is used in combination with an antiandrogen drug for benign prostatic hyperplasia (BPH).

[0047] In some embodiments, the present disclosure is used for tumors expressing FGFR. In some embodiments, the FGFR is selected from the group consisting of FGFR1, FGFR2, FGFR3 and FGFR4. In some preferred embodiments, the FGFR is FGFR1.

[0048] The presence or absence of FGFR expression can be detected by methods known to those skilled in the art. Detection of gene expression and gene product proteins can be performed by known methods, such as antibody-based methods (immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoblotting, etc.), nucleic acid-based methods (in situ hybridization, PCR, Northern blotting, etc.), and methods based on principles common to these. Detection devices can be known devices (Genechip, microarray, etc.).

[0049] In addition, in one embodiment, the present disclosure is used for tumors in which FGFR signaling is activated. FGFR forms a dimer upon binding to FGF and is activated by phosphorylation, thereby inducing the recruitment and activation of specific downstream signaling molecules, thereby exerting physiological functions.

[0050] Activation of FGFR signaling can be detected by a method known to those skilled in the art. Detection of signaling activation can be, for example, detection of FGF, which is a substrate of FGFR, direct phosphorylation state or biological activity including enzymatic activity of FGFR, which is a phosphorylation enzyme, or phosphorylation state or enzymatic activity of intracellular substrates and intracellular proteins present downstream of the FGFR signaling cascade. This can be achieved by detection of biological activity, including affinity.

[0051] In addition, in one embodiment, the present disclosure is used for tumors having abnormalities in FGFR genes. Abnormalities in FGFR genes include gene amplification, gene mutation, chromosomal translocation / insertion / inversion, and gene fusion. Some abnormalities in FGFR genes in tumors have already been reported in literature available to those skilled in the art (Non-Patent Document 11, Non-Patent Document 12). Abnormalities in FGFR1 genes can be detected by methods known to those skilled in the art, such as DNA sequencing including pyrosequencing and NGS (next generation sequencing), PCR-based methods including allele-specific PCR chain reaction, microarray-based comparative genomic hybridization (aCGH), fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CGH), and other methods. It can be detected by methods such as CISH.

[0052] In one embodiment, the present disclosure is used for breast cancer with FGFR1 gene amplification.Breast cancer with FGFR1 gene amplification refers to breast cancer with more copies of FGFR1 gene on genome than normal cells.FGFR1 gene amplification can be detected by methods known to those skilled in the art, such as DNA sequencing including NGS (next generation sequencing), microarray-based comparative genomic hybridization (aCGH), fluorescent in situ hybridization (FISH), colorimetric in situ hybridization (CISH), etc.

[0053] In the present disclosure, two treatments, administration of compound 1 or a pharma- ceutically acceptable salt thereof and endocrine therapy, are used in combination. The term "combined use" refers to the two treatments being performed simultaneously, separately, or consecutively in a series of treatments. Thus, the present disclosure includes an embodiment in which compound 1 or a pharma- ceutically acceptable salt thereof is administered to a tumor patient who has previously undergone endocrine therapy. The present disclosure also includes an embodiment in which compound 1 or a pharma- ceutically acceptable salt thereof is administered simultaneously with endocrine therapy. In this case, when endocrine therapy is performed by administration of a drug, the drug for endocrine therapy and compound 1 or a pharma- ceutically acceptable salt thereof can be a single pharmaceutical preparation or a single administration kit. The present disclosure also includes an embodiment in which endocrine therapy is performed on a tumor patient who has previously been administered compound 1 or a pharma- ceutically acceptable salt thereof. When endocrine therapy is performed by administration of a drug, the administration interval when the drug for endocrine therapy and compound 1 or a pharma- ceutically acceptable salt thereof are administered separately is not particularly limited, and can be selected so that the individual effects of compound 1 or a pharma- ceutically acceptable salt thereof, and the other compound having an antitumor effect or a pharma- ceutically acceptable salt thereof, as well as the combined effect, are optimally exhibited.

[0054] In the present disclosure, in addition to the two treatments of administration of compound 1 or a pharma- ceutically acceptable salt thereof and endocrine therapy being performed in combination, other antitumor agents and other treatments are also included. In the present disclosure, the endocrine therapy combined with administration of compound 1 or a pharma- ceutically acceptable salt thereof may be performed once, or the same or different endocrine therapies may be performed multiple times.

[0055] In one embodiment of the present disclosure, compound 1 or a pharma- ceutically acceptable salt thereof enhances the antitumor effect of endocrine therapy in combination. In one embodiment of the present disclosure, a pharmaceutical composition is provided for enhancing the antitumor effect of endocrine therapy, comprising compound 1 or a pharma- ceutically acceptable salt thereof. In one embodiment of the present disclosure, compound 1 or a pharma- ceutically acceptable salt thereof is provided for use in enhancing the antitumor effect of endocrine therapy. In one embodiment of the present disclosure, a use of compound 1 or a pharma- ceutically acceptable salt thereof is provided for the manufacture of a medicament for enhancing the antitumor effect of endocrine therapy. In one embodiment of the present disclosure, a method for enhancing the antitumor effect of endocrine therapy is provided, comprising administering an effective amount of compound 1 or a pharma- ceutically acceptable salt thereof to a patient. In one embodiment of the present disclosure, a combination of endocrine therapy and administration of compound 1 or a pharma- ceutically acceptable salt thereof for enhancing the antitumor effect of endocrine therapy is provided.

[0056] In the present disclosure, compound 1 or a pharma- ceutically acceptable salt thereof and a drug used in endocrine therapy may be formulated separately into a plurality of preparations or may be formulated together into a single preparation. In addition, in the present disclosure, compound 1 or a pharma- ceutically acceptable salt thereof and other compounds having antitumor effects or pharma- ceutically acceptable salts thereof may further contain other active ingredients.

[0057] When compound 1 or a pharma- ceutically acceptable salt thereof and other drugs used in endocrine therapy are contained in a formulation as active ingredients, each active ingredient may be mixed with a pharmaceutical carrier as necessary, and various administration forms may be adopted according to the purpose of prevention or treatment. Examples of administration forms include oral agents, injections, suppositories, ointments, patches, etc., and oral agents are preferred. Oral agents may be in the form of tablets, capsules, granules, powders, syrups, etc., and are not particularly limited. Each of these administration forms can be prepared by a formulation method known and commonly used by those skilled in the art. Depending on the administration form, and as necessary, carriers such as appropriate excipients, diluents, fillers, and disintegrants may be added to the formulation or pharmaceutical composition.

[0058] Those skilled in the art can administer an effective amount of Compound 1 or a pharma- ceutically acceptable salt thereof to a patient. The amount of Compound 1 or a pharma- ceutically acceptable salt thereof to be incorporated in each of the above-mentioned dosage unit forms varies depending on the symptoms of the patient to which it is to be administered or on the dosage form, but it is generally desirable that the dosage unit form be 0.05 to 1000 mg for oral agents, 0.01 to 500 mg for injections, and 1 to 1000 mg for suppositories.

[0059] The daily dose of Compound 1 or a pharma- ceutically acceptable salt thereof varies depending on the symptoms, body weight, age, sex, etc. of the patient and cannot be determined in general, but is usually about 1 to 1000 mg of Compound 1 or a pharma- ceutically acceptable salt thereof per day for an adult (body weight 60 kg), preferably about 10 to 500 mg per day, and more preferably about 20 to 300 mg per day.

[0060] When the daily dose of compound 1 or a pharma- ceutically acceptable salt thereof is administered every day, the dose is, for example, about 1 to 200 mg of compound 1 or a pharma- ceutically acceptable salt thereof per day, preferably 2 to 100 mg per day, more preferably 4 to 50 mg per day, and even more preferably 10 to 40 mg per day.

[0061] When the daily dose of compound 1 or a pharma- ceutically acceptable salt thereof is administered every other day, the dose is, for example, about 2 to 1000 mg of compound 1 or a pharma- ceutically acceptable salt thereof per day, preferably 10 to 500 mg, more preferably 20 to 200 mg, and even more preferably 50 to 160 mg.

[0062] In the present disclosure, the administration routes of the formulation containing compound 1 or a pharma- ceutically acceptable salt thereof and the drug used in endocrine therapy may be the same or different. For example, the formulation containing compound 1 or a pharma- ceutically acceptable salt thereof and the drug used in endocrine therapy may both be orally administered. Alternatively, for example, the formulation containing compound 1 or a pharma- ceutical acceptable salt thereof may be orally administered, and the drug used in endocrine therapy may be intravenously injected. The administration route may be appropriately determined depending on the active ingredient to be administered, and taking into consideration the progression of the malignant tumor in the patient, the overall condition of the patient, and the like.

[0063] In the present disclosure, compound 1 or a pharma- ceutically acceptable salt thereof can be administered to a patient either before or after surgery, and can also be administered to a patient who cannot undergo surgery. In addition, the present disclosure can further include administration of a drug to enhance the antitumor effect, and can also include administration of a drug to reduce side effects.

[0064] In one embodiment of the present disclosure, for example for the treatment of breast cancer, compound 1 or a pharma- ceutically acceptable salt thereof can be used in combination with an LH-RH agonist, an aromatase inhibitor, an anti-estrogen, and / or a progestin, either of which can be orally administered as the active ingredient or as the active ingredient of a pharmaceutical composition described in the present disclosure.

[0065] In one embodiment of the present disclosure, for example for the treatment of prostate cancer, compound 1 or a pharma- ceutically acceptable salt thereof can be used in combination with an LH-RH agonist, an LH-RH antagonist, an estrogen, an antiandrogen, and / or an androgen synthesis inhibitor, either of which can be orally administered as the active ingredient or as the active ingredient of a pharmaceutical composition described in the present disclosure.

[0066] The administration or mixing ratio of compound 1 or a pharma- ceutically acceptable salt thereof to a drug used in endocrine therapy is not particularly limited as long as it is within a range that exhibits an antitumor effect. For example, the amount of compound 1 or a pharma- ceutically acceptable salt thereof, in terms of free form, may be about 0.001 to 1000 moles, preferably about 0.01 to 100 moles, per mole of another compound having an antitumor effect.

[0067] For example, when the other compound having an antitumor effect is an anti-estrogen drug, the suitable amount of compound 1 or a pharma- ceutically acceptable salt thereof to be used per mole of the anti-estrogen drug is 0.001 to 10 moles, preferably 0.001 to 1 mole, and more preferably 0.001 to 0.1 mole.

[0068] The present disclosure can also be effectively used against tumors that are resistant to FGFR inhibitors other than Compound 1 or a pharma- ceutically acceptable salt thereof. EXAMPLES

[0069] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited thereto. The present disclosure has been fully explained with reference to examples, but it is understood that various changes and modifications may be made by those skilled in the art. Therefore, as long as such changes and modifications do not deviate from the scope of the present disclosure, they are included in the present disclosure. The various reagents used in the examples were commercially available unless otherwise specified.

[0070] [Example 1: Evaluation of the cell proliferation inhibitory effect of the combination of Compound 1 with 4-hydroxytamoxifen or fulvestrant in vitro] (combination index) A. Materials and Methods Human breast cancer cell line T47D (Dainippon Pharmaceutical Co., Ltd.) was cultured in RPMI-1640 medium containing 10% fetal bovine serum, with reference to British Jounal of Cancer (2006) 94, 661-671. The cells were maintained under conditions of 37°C and 5% CO2, and subcultured once or twice a week at a ratio of 1:5 to 1:20. Human breast cancer cell line MDA-MB-134VI (American Type Culture Collection) was cultured in RPMI-1640 medium containing 10% fetal bovine serum, with reference to British Jounal of Cancer (2006) 94, 661-671. The cells were maintained under conditions of 37°C and 5% CO2, and subcultured once or twice a week at a ratio of 1:2 to 1:3.

[0071] Cell viability assay The cell viability was measured using CellTiter-Glo2.0 (Promega). After harvesting the cells by a conventional method, they were suspended in RPMI-1640 (phenol red-free) medium containing 1% fetal bovine serum (Chacoal Stripped), 0.5 ng / mL estradiol, and 25 ng / mL human FGF2, and then plated on a 96-well Ultralow Attach. The cells were seeded on a ment plate. The seeding number was 500 / 80μL per well. After incubation at 37℃ and 5% CO2 for 24 hours, 10μL of medium containing the drug solution was added. The drug solution was prepared in a dilution series of vehicle (DMSO), compound 1, tamoxifen and fulvestrant, and the experiment was performed in a total of 7 groups, including vehicle, compound 1, tamoxifen and fulvestrant, and their combined groups (compound 1 + vehicle, compound 1 + tamoxifen, compound 1 + fulvestrant). The concentration of compound 1 was set to 1μM as the maximum final concentration, and 5 concentrations were set in a 10-fold dilution series. 4-hydroxytamoxifen and fulvestrant were set to 4 concentrations each in a 10-fold dilution series with a maximum concentration of 10μM. After the drug was added to the cells, the cells were further incubated at 37℃ and 5% CO2 for 120 hours. The experiment was performed in duplicate (2 wells per treatment group). Cell viability was calculated by adding 100 μL of CellTiter-Glo 2.0 solution per well, incubating at room temperature for 30 minutes, and then measuring the amount of chemiluminescence in each well using a plate reader (Enspire). The cell viability upon drug addition was calculated as a ratio to the control group, which was taken as 100%, according to the following formula, and the average data from each of the two wells was used for analysis.

[0072] Cell viability (%) = (chemiluminescence intensity upon addition of drug) / (chemiluminescence level of control group) x 100 The Fa (Fraction of Affect) value was calculated by subtracting 1 / 100 of the cell survival rate from 1.

[0073] Next, the combination index (CI) value was determined for each combination concentration of the drugs. The presence or absence of a combined effect of two drugs was judged to indicate antagonism, additive action, or synergy when the CI value was greater than 1, equal to 1, or less than 1, respectively (Table 1, Pharmacol Rev. 2006;58(3):621-81, BMC Complement Altern Med. 2013;13:212., Anticancer Res. 2005;25(3B):1909-17.).

[0074] [Table 1]

[0075] In addition, the Fa values ​​calculated by combining the concentrations of Compound 1 and 4-hydroxytamoxifen or fulvestrant in T47D cells and MDA-MB-134VI cells were Fa = 0.5 (corresponding to ED50), 0.75 (corresponding to ED75), 0.9 (corresponding to ED Combinations of concentrations of both drugs that yielded a CI of 90 were extracted and subjected to linear curve fitting using CalcuSyn (Hulinks Corporation).

[0076] B Results In the T47D breast cancer cell line expressing FGFR1 and the MDA-MB-134VI breast cancer cell line with FGFR1 gene amplification, the combination of compound 1 and 4-hydroxytamoxifen showed synergistic effects in all four combinations of concentration ratios (Table 2). Table 2 below shows the combination index values ​​for the ratios of 4-hydroxytamoxifen and compound 1 of 1:1, 10:1, 100:1, and 1000:1, at the time points (ED50, ED75, and ED90) at which the cell proliferation inhibitory effect of the combination was 50%, 75%, and 90%.

[0077] [Table 2]

[0078] [Example 2: Evaluation of the cell proliferation inhibitory effect of the combination of Compound 1 with 4-hydroxytamoxifen or fulvestrant in vitro] A. Materials and Methods Human breast cancer cell line MDA-MB-134VI (American Type Culture Collection) was cultured in RPMI-1640 medium containing 10% fetal bovine serum, with reference to British Journal of Cancer (2006) 94, 661-671. The cells were maintained under conditions of 37°C and 5% CO2, and subcultured once or twice a week at a ratio of 1:2 to 1:3.

[0079] Cell viability assay The cell viability was measured using CellTiter-Glo2.0 (Promega). After collecting the cells by a conventional method, they were suspended in RPMI-1640 (phenol red-free) medium containing 1% fetal bovine serum (Chacoal Stripped), 0.5 ng / mL estradiol, and 25 ng / mL human FGF2, and seeded on a 96-well ultra-low attachment plate. The seeding number was 500 cells / 80 μL per well. After incubation at 37°C and 5% CO2 for 24 hours, 10 μL of medium containing compound 1, 4-hydroxytamoxifen, or fulvestrant, or vehicle (DMSO) containing no drug as a control, was added. Compound 1 was 0.1 μM, 4-hydroxytamoxifen was 0.1 μM, and 10 μL of medium containing fulvestrant was 0.1 μM. The maximum concentrations of tamoxifen and fulvestrant were set at 1 μM and 0.1 μM, respectively. After adding the drugs to the cells, the cells were incubated at 37°C and 5% CO2 for an additional 120 hours. The experiment was performed in triplicate (3 wells per treatment group). Cell viability was calculated by adding 100 μL of CellTiter-Glo2.0 solution per well, incubating at room temperature for 30 minutes, and then measuring the amount of chemiluminescence in each well using a plate reader (Enspire). The cell viability upon drug addition was calculated as a ratio when the control group was set to 100% according to the following formula, and the average data for each of the three wells was used for analysis.

[0080] Cell viability (%) = (chemiluminescence intensity upon addition of drug) / (chemiluminescence level of control group) x 100

[0081] B Results Treatment with compound 1 (0.1 μM), 4-hydroxytamoxifen (1 μM) and fulvestrant (0.1 μM) inhibited the proliferation of MDA-MB-134VI cells, respectively, with T / C (treated / control) (%) on the day of evaluation being 35%, 65% and 75%. In contrast, combined treatment with 0.1 μM compound 1 and 1 μM 4-hydroxytamoxifen, and combined treatment with 0.1 μM compound 1 and 0.1 μM fulvestrant further inhibited cell proliferation compared to treatment with each drug alone, with T / C (%) being 7% and 16%, respectively.

[0082] The cell proliferation inhibitory effect of the combination of compound 1 with 4-hydroxytamoxifen or fulvestrant exceeded the combined effect of the two drugs (T / C = 23% and 27%) calculated using the effect of each drug alone (Bliss method, Ann Appl Biol. 1939;26:585-615), indicating a synergistic combination effect (P<0.05; Student's t-test) (Figures 1A and B).

Claims

[Claim 1] A pharmaceutical composition used for the treatment and / or prevention of a tumor to which endocrine therapy is applicable, comprising (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharma- ceutically acceptable salt thereof, characterized in that it is administered in combination with endocrine therapy.

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