Use of sglt2 inhibitors for the preparation of a medicament for the treatment of a hormone receptor low responsive reproductive system disease

Abstract

The present application relates to a kind of SGLT2 inhibitor in the preparation for treating hormone receptor low reaction type reproductive system disease in the application of drug, in particular, SGLT2 inhibitor and hormone can improve or enhance the sensitivity and / or for reversing the drug resistance of endocrine therapy drug, such as hormone, can enhance the therapeutic effect of such drug, especially in drug-resistant reproductive system disease and tumor treatment has good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of new uses of drugs, and relates to application of an SGLT2 inhibitor in preparation of a drug for treating a hormone receptor low-response type of reproductive system disease, in particular, application of the SGLT2 inhibitor in preparation of a drug for treating a reproductive system disease with negative or low expression of a hormone receptor or with mutation or low expression of a thyroid hormone receptor. BACKGROUND

[0002] Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new type of hypoglycemic drugs, which can inhibit the reabsorption of glucose in the proximal tubule of the kidney and thus play a role in reducing blood glucose. At present, there are six SGLT2 inhibitors on the market worldwide, namely: Canagliflozin (Canagliflozin), Dapagliflozin (Dapagliflozin), Empagliflozin (Empagliflozin), Ipragliflozin (Ipragliflozin), Luseogliflozin (Luseogliflozin) and Tofogliflozin (Tofogliflozin). In recent years, large-scale clinical research results have shown that SGLT2 inhibitors can significantly improve the cardiovascular and renal adverse outcomes of patients with type II diabetes (N Engl J Med, 2017, 377 (7): 644-657; N Engl J Med, 2019, 380 (24): 2295-2306; N Engl J Med 2019, 380 (19): 1881-1882). Therefore, this type of drug has become the first choice for patients with type II diabetes combined with cardiovascular disease and chronic kidney disease.

[0003] The chemical name of Canagliflozin is (1S)-1,5-dehydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-D-glucitol. According to the current literature reports, Canagliflozin can be combined with metformin (Chen Yuying et al., Chinese General Medicine, 2022, 25 (15): 1888-1896+1905), pioglitazone (Huang Yuan, New World of Diabetes, 2023, 26 (11): 107-110) and the like for the treatment of type II diabetes. However, there is no report on whether Canagliflozin can be combined with endocrine drugs for the treatment of reproductive system diseases and related tumors, including endometrial cancer, breast cancer, prostate cancer, ovarian cancer and the like; in particular, there is no report on whether such drugs can be combined with hormone drugs to improve or enhance the efficacy of endocrine drugs, reverse the drug resistance of hormone drugs. In addition, there is no report on the influence of SGLT2 inhibitors represented by Canagliflozin on endocrine diseases / tumors with mutation or abnormal expression of thyroid hormone receptors.

[0004] Unlike other organ diseases and therapeutic drugs, the reproductive system diseases and tumors are treated with hormone drugs. Hormone drugs are effective means for treating dysmenorrhea, endometriosis, polycystic ovary, endometrial hyperplasia and prostatic hyperplasia, and endometrial cancer conservation treatment. Such drugs include hormone drugs such as estrogen, progestogen, anti-estrogen, anti-progestogen, gonadotropin releasing hormone agonist (GnRH-a) or antagonist, and aromatase inhibitors. Among them, progestogen is an important endocrine therapy drug, but due to the existence of drug insensitivity or drug resistance in some patients, the curative effect of the treatment by using such drugs alone is often poor.

[0005] Taking endometrial cancer (EC) and endometrial hyperplasia (EH) as examples, progestogen is the only drug treatment method for EH and EC patients who want to preserve fertility in clinical practice. However, some EH and EC patients have primary or secondary progestogen treatment insensitivity and drug resistance during treatment, mainly manifested as ineffective progestogen treatment of hyperplasia or tumor tissue or abnormal proliferation of endometrium during progestogen treatment, which makes about 30% of EC patients fail in treatment and ultimately can only choose surgery. This not only increases the social and economic burden, but also brings pain to some patients who cannot tolerate surgery. Therefore, how to solve the progestogen resistance has become a special concern in clinical endocrine therapy.

[0006] At present, the exact mechanism of progestogen insensitivity has not been elucidated, and therefore, there are few drugs for progestogen resistance. At present, there is no approved effective drug or treatment method for enhancing progestogen sensitivity. It is generally believed that down-regulation of progestogen receptor (PR) is the main cause of progestogen resistance, but the root cause of PR down-regulation is not clear. Therefore, the urgent task is still to find the cause of progestogen resistance in patients, to develop new drug therapy to enhance the therapeutic effect of progestogen, shorten the medication time of patients, improve the cure rate, and reduce the pain of patients. The present application especially provides a treatment scheme for those patients who are not sensitive to metformin and atorvastatin and insulin, cannot improve the curative effect by using metformin and / or atorvastatin in combination, or are not tolerant to the side effects of metformin and statin drugs.

[0007] It is reported that high frequency of somatic deletion and mutation in thyroid hormone receptor (TRa / b) loci exist in various tumor patients such as uterus, ovary and testis (Rohit Sinha & Paul M Yen, Cellular Action of Thyroid Hormone. Endotext. Last Update: June 20, 2018. https: / / www.ncbi.nlm.nih.gov / books / NBK285568). The inventors found in previous studies that the level of TRb in endometrial tissue of EC and EH patients with failed progesterone therapy is reduced, while the expression of TRa is not significantly associated with progesterone resistance. In in vitro experiments, after THRB gene silencing, the proliferation activity and adhesion ability of RL95-2 cells are enhanced, the inhibitory effect of progesterone is weakened, progesterone resistance is presented, and the cell proliferation signaling pathway mTOR-4EBP1 / eIF4G is activated, which indicates that progesterone resistance and progesterone insensitivity may be related to low expression of TRb (Int. J. Mol. Sci. 2022, 23(20), 12517. https: / / doi.org / 10.3390 / ijms232012517).

[0008] The inventors also found in previous studies that the alpha-glucosidase inhibitor acarbose has no obvious inhibitory effect on progesterone-resistant cells (RL95-2 / THRB (- / -) ) with thyroid hormone receptor b gene knockout when combined with progesterone, indicating that acarbose is ineffective when combined with progesterone and cannot reverse and reduce the drug resistance of progesterone, indicating that not all hypoglycemic and hypolipidemic drugs can enhance the efficacy of progesterone. SUMMARY

[0009] To solve the above problems, the inventors of the present application have provided, for the first time, a new use of SGLT2 inhibitors in enhancing the treatment of endocrine drugs. The present application uses SGLT2 inhibitors in the treatment of endocrine disease or tumor patients with negative or low expression of hormone receptors or low response to hormones or mutation or low expression of thyroid hormone receptor b, in order to improve the sensitivity of endocrine treatment drugs such as progesterone and enhance the therapeutic effect of such drugs, which is beneficial to the treatment of patients.

[0010] According to one aspect of the present application, there is provided a use of an SGLT2 inhibitor in the preparation of a drug for treating a reproductive system disease with negative expression of hormone receptors or low response to hormones.

[0011] According to another aspect of the present application, there is provided a use of an SGLT2 inhibitor and a hormone in the preparation of a drug for treating a reproductive system disease with negative expression of hormone receptors or low response to hormones.

[0012] According to still another aspect of the present application, there is provided a combination of an SGLT2 inhibitor and a hormone for treating a reproductive system disease which is negative for hormone receptor expression or is hormone hypo-responsive.

[0013] According to still another aspect of the present application, there is provided a composition for treating a reproductive system disease which is negative for hormone receptor expression or is hormone hypo-responsive, comprising an SGLT2 inhibitor and a hormone.

[0014] According to still another aspect of the present application, there is provided use of an SGLT2 inhibitor in the manufacture of a medicament for increasing or enhancing sensitivity of an endocrine therapy drug such as a hormone.

[0015] According to still another aspect of the present application, there is provided use of an SGLT2 inhibitor in the manufacture of a medicament for reversing resistance of an endocrine therapy drug such as a hormone.

[0016] According to the present application, the SGLT2 inhibitor is used for increasing or enhancing sensitivity of a hormone and / or for reversing resistance of a hormone.

[0017] According to the present application, the reproductive system disease is an endocrine disease or tumor which is negative or hypo-expresses for hormone receptor expression or is hormone hypo-responsive, particularly an endocrine disease or tumor which is mutant or hypo-expresses for thyroid hormone receptor β.

[0018] According to the above-mentioned application, the SGLT2 inhibitor is a compound targeting SGLT2, including Canagliflozin, Dapagliflozin, Phlorizin, Canagliflozin, Empagliflozin, Ipragliflozin, Luseogliflozin, Tofogliflozin, Ertugliflozin, Sotagliflozin, and Remogliflozin.

[0019] According to the above-mentioned application, the hormone includes various types of progestin (such as medroxyprogesterone acetate, norethisterone acetate, etc.), anti-progestin, estrogen and anti-estrogen, androgen and anti-androgen, aromatase inhibitor, etc.

[0020] According to the above-mentioned application, the reproductive system disease which is negative or hypo-responsive for hormone receptor expression, particularly an endocrine disease and / or tumor which is mutant or abnormally expresses for thyroid hormone receptor β, particularly includes endometriosis, endometrial hyperplasia, endometrial cancer, breast hyperplasia, breast cancer, prostate hyperplasia, prostate cancer, ovarian cancer, polycystic ovary syndrome, etc.

[0021] According to the present application, the two types of drugs can be used in succession or in combination with various conventional adjuvants to prepare capsules, tablets, injection solutions, pills, granules, powders, subcutaneous implants, intrauterine administration, and various other dosage forms, and the drug combinations or compositions include oral preparations, suppositories, injections, transdermal preparations, subcutaneous implants, and preparations for vaginal and intrauterine administration.

[0022] The present application is confirmed by experiments that SGLT2 inhibitor canagliflozin can inhibit the proliferation of various types of endometrial cancer cells, especially the proliferation of hormone receptor low expression type II cells, lymphatic metastasis EC cells, and thyroid hormone receptor beta gene knockout progesterone resistant cells (RL95-2 / THRB (- / -) ), and significantly enhance the inhibitory activity of low concentration progesterone on endometrial cancer type I, type II, and RL95-2 / THRB (- / -) resistant cells; in the subcutaneous transplanted tumor of RL95-2 / THRB (- / -) cell inoculation, the subcutaneous transplanted tumor volume of RL95-2 / THRB (- / -) resistant cell-bearing nude mice is significantly reduced, indicating that canagliflozin can significantly enhance the inhibitory activity of progesterone, and reverse the progesterone-promoting effect on RL95-2 / THRB (- / -) resistant cell transplanted tumor. This shows that SGLT2 inhibitors can be combined with progesterone for the treatment of drug-resistant endometrial hyperplasia, endometrial cancer, and endometriosis and other various reproductive system-related diseases, especially for patients with low expression of thyroid receptor TRβ and hormone receptor expression negative or hormone low response type who are resistant and insensitive to hormone therapy. Therefore, the combined use of canagliflozin and progesterone has a good application prospect in the treatment of drug-resistant reproductive system diseases and tumors, and provides a new treatment method for improving the efficacy of progesterone and reducing the resistance of endocrine therapy drugs.

[0023] The content described in the present application is the combined use of SGLT2 inhibitors and endocrine therapy drugs such as hormones, or the formation of a single compound by structural modification of the two compounds.

[0024] For example, in one embodiment, a combination of progesterone (MPA (medroxyprogesterone acetate) or NOMAc (norgestimate acetate)) and canagliflozin is used, wherein the concentration of progesterone can be 10-100 μM, and the molar ratio of progesterone:canagliflozin can be in the range of 1:1-10:1.

[0025] The SGLT2 inhibitor canagliflozin can enhance the inhibitory effect of progestogen on the growth of endometrial cancer cells and transplanted tumors in nude mice. In the progestogen-resistant cell line RL95-2 / THRB (- / -) ) with the thyroid hormone receptor β gene knocked out, the SGLT2 inhibitor canagliflozin can also enhance the inhibitory activity of progestogen, reverse the pro-proliferation effect of progestogen on the in-vitro cultured resistant cells and subcutaneously transplanted tumors in nude mice, and inhibit the growth. Therefore, the SGLT2 inhibitor can be applied to the preparation of an endocrine adjuvant therapy drug. The use is disclosed for the first time, and has obvious significance for improving the efficacy of endocrine drugs, and improving the pain of patients, especially for those patients resistant to progestogen to improve their quality of life.

[0026] Therefore, the combination of canagliflozin and progestogen has a good application prospect in the treatment of drug-resistant reproductive system diseases and tumors. The present application provides a new treatment method for improving the efficacy of progestogen and reducing the resistance of endocrine therapy drugs. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The inhibitory effect of canagliflozin, metformin and atorvastatin on the growth of KLE cells, which are endometrial cancer cells negative for hormone receptor expression, in combination with medroxyprogesterone acetate (MPA) is shown (n=9);

[0028] Figure 2 The inhibitory effect of canagliflozin, metformin and atorvastatin on the growth of KLE cells, which are endometrial cancer cells negative for hormone receptor expression, in combination with norethisterone acetate (NOMAc) is shown (n=9);

[0029] Figure 3 The inhibitory effect of canagliflozin and atorvastatin on the growth of AN3CA cells, which are lymph node metastatic endometrial cancer cells negative for hormone receptor expression, in combination with medroxyprogesterone acetate (MPA) is shown (n=9);

[0030] Figure 4 The inhibitory effect of canagliflozin and atorvastatin on the growth of AN3CA cells, which are lymph node metastatic endometrial cancer cells negative for hormone receptor expression, in combination with norethisterone acetate (NOMAc) is shown (n=9);

[0031] Figure 5 The inhibitory effect of canagliflozin and metformin on the growth of RL95-2 / THRB (- / -) cells, which are endometrial cancer cells with the THRB gene knocked out, in combination with medroxyprogesterone acetate (MPA) is shown (n=9);

[0032] Figure 6 The inhibitory effect of canagliflozin and metformin on the growth of RL95-2 / THRB(- / -) Inhibition of cell growth (n = 9);

[0033] Figure 7 The combination of canagliflozin with norethindrone acetate (NOMAc) and medroxyprogesterone acetate (MPA) on RL95-2 / THRB (- / -) Effects of cell inoculation on the growth of subcutaneous transplanted tumors in nude mice, wherein (A) solvent control group; (B) MPA 100 mg / kg group; (C) MPA 100 mg / kg + metformin 100 mg / kg combination group; (D) MPA 100 mg / kg + canagliflozin 15 mg / kg combination group; (E) NOMAc 100 mg / kg group; (F) NOMAc 100 mg / kg + metformin 100 mg / kg combination group; (G) NOMAc 100 mg / kg + canagliflozin 15 mg / kg combination group,

[0034] wherein: a,b,e,f,g,h,I,j,k P < 0.05, indicating compared with the same dose of progestin (MPA or NOMAc), canagliflozin, metformin, metformin + progestin and / or atorvastatin, atorvastatin + progestin. DETAILED DESCRIPTION

[0035] This study takes the inhibitory effect of canagliflozin on endometrial cancer cells as an example, and provides the inhibitory effect of SGLT2 inhibitors on hormone receptor low response type (estrogen and progesterone receptor expression negative) endometrial cancer (EC) type II (AN3CA and KLE) cells and thyroid hormone receptor beta gene knockout progestin-resistant cells (RL95-2 / THRB (- / -) ). On the one hand, canagliflozin alone can inhibit the proliferation of different types of EC cells and RL95-2 / THRB (- / -) progestin-resistant cells; on the other hand, canagliflozin can be combined with progestin (such as medroxyprogesterone acetate or norethindrone acetate, etc.) to enhance the inhibitory effect of progestin on RL95-2 / THRB (- / -) progestin-resistant cells and their transplanted tumors in vitro and in vivo. Compared with progestin alone, the half maximal inhibitory concentration (IC50) of AN3CA and KLE endometrial cancer cells and RL95-2 / THRB (- / -) progestin-resistant cells treated with canagliflozin and progestin combination was significantly reduced, and the cell proliferation activity was significantly weakened; in RL95-2 / THRB (- / -) progestin-resistant cell-bearing mice, the combination of canagliflozin and progestin can significantly reduce the volume of subcutaneous transplanted tumors and reverse the pro-proliferation effect of progestin on subcutaneous transplanted tumors. The present application will be further described in detail by the following examples, but the protection scope of the present application is not limited by any of the specific examples, but is defined by the claims.

[0036] Experimental Example 1, Inhibitory Effect of Canagliflozin on the Growth of Two Types of In Vitro Cultured Low Hormone Receptor Type II Endometrial Cancer Cells

[0037] 1.1 Experimental Materials

[0038] DMEM-F12 and MEM cell culture medium, fetal bovine serum FBS, HBSS were purchased from Gibco, endometrial cancer AN3CA cells were purchased from American Type Culture Collection (ATCC), endometrial cancer KLE cells were purchased from China Typical Culture Collection, CCK-8 was purchased from Dojindo Laboratories, DMSO was purchased from Sigma, Canagliflozin (Canagliflozin, Canag) was purchased from MedChemExpress, Catalog No: HY-10451, purity 99.80%; Metformin (Metformin, Met) was purchased from Sigma-Aldrich, Catalog No: D150959, purity 97%. Atorvastatin (Atorvastatin, Atorva) was purchased from MedChemExpress, Catalog No: HY17379, purity 99.67%. Norethisterone acetate (NOMAC) and medroxyprogesterone acetate (MPA) were donated by Zhejiang Xianyu Pharmaceutical Co., Ltd., purity >98%, both of which were dissolved in DMSO for standby.

[0039] 1.2 Experimental Methods

[0040] 1.2.1. Tumor Proliferation Detection

[0041] KLE or AN3CA cells cultured to the logarithmic growth phase were trypsinized, centrifuged and the culture medium was discarded, washed with HBSS, and resuspended with DMEM-F12 medium containing 10% FBS. Cell counting plate was used to count and adjust the cell density to 1×10^5 / mL. 100 μL of cell suspension was plated into a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 48 h, and the medium was changed. The single drug group added 100 fresh medium, the combined drug group added 200 μL fresh medium. Three replicates were set for each concentration, 1 μL of drug was added to each well to meet the specified requirements. The cells were incubated in a 37°C, 5% CO2 incubator for 48 h, 10 μL of CCK-8 solution was added to each well, and the OD value was measured at 450 nm on a microplate reader after incubation at 37°C for 2 h.

[0042] 1.2.2 Statistical Methods

[0043] The cell growth inhibition rate (%) of the drug = (1-OD drug treatment / OD solvent) x 100%, all statistical analysis was performed using Graphpad Prism 9.5.1. The mean ± standard error was used to represent the measurement data. The single factor analysis of variance method was used to compare the changes of cell number in different groups to determine the statistical significance, and Turkey's multiple comparison test was used as a post-hoc test when comparing the differences between multiple groups. P<0.05 was statistically significant. IC50 refers to the concentration at which the drug has a 50% inhibitory effect on cell growth. By inputting a series of drug concentrations and their corresponding cell growth inhibition rates into the Graphpad Prism calculation software, the software calculates the IC50 value.

[0044] 1.3 Experimental results

[0045] In KLE cells, when administered alone, norethisterone acetate, canagliflozin, atorvastatin and metformin had no significant inhibitory effect on endometrial cancer KLE cells at a concentration range of 3-30 μM after 48 hours of treatment, and the half inhibitory concentration (IC50 value) could not be calculated. The IC50 value and 95% confidence interval of medroxyprogesterone acetate (MPA) for endometrial cancer KLE cells were calculated as 155.1 μM (77.06-693.8 μM) at a concentration range of 3-30 μM after 48 hours of treatment, indicating that MPA alone had weak inhibitory effect on KLE cell growth. As shown in Table 1 and Figures 1-2 .

[0046] Compared with MPA and NOMAc alone, the inhibitory effect of canagliflozin on KLE cell viability was enhanced when combined with MPA and NOMAc; among them, NOMAc combined with canagliflozin (3, 10, 30 μM) at 10 and 30 μM, the IC50 value decreased to 39.73 μM and 37.14 μM, respectively, which was significantly lower than the IC50 of NOMAc and canagliflozin alone by more than 10 times (P<0.05); MPA combined with canagliflozin (3, 10, 30 μM) at 10 μM, the IC50 value decreased to 52.55 μM, which was 3 times lower than the IC50 value of MPA alone (P<0.05). The above results showed that the combination of canagliflozin and progestin could enhance the inhibitory effect of progestin on KLE cells. As shown in Table 1 and Figures 1-2 .

[0047] However, atorvastatin and metformin still could not calculate the IC50 value when combined with MPA and NOMAc (10 and 30 μM) at a concentration range of 3, 10, 30 μM to treat KLE cells for 48 hours, indicating that atorvastatin and metformin had no significant inhibitory effect when combined with progestin. As shown in Table 1 and Figures 1-2Figure 2. The effect of MPA, NOMAc, Canagliflozin and Atorvastatin on the viability of KLE cells. The x-axis represents the concentration of the drugs (alone) or the other drugs when combined with MPA. The y-axis represents the viability of KLE cells.

[0048] Table 1. The effect of MPA, NOMAc, Canagliflozin, Atorvastatin and Metformin alone and in combination on the median inhibitory concentration (IC50) and 95% confidence interval of KLE cells (n = 9).

[0049]

[0050] MPA (medroxyprogesterone acetate), NOMAc (norethisterone acetate), Canag (Canagliflozin), Met (Metformin), Atorva (Atorvastatin)

[0051] *P < 0.05 compared with MPA and NOMAc alone; "very wide" indicates that the corresponding 95% confidence interval is very wide and cannot be calculated; " / " indicates that the inhibition rate did not exceed 50% and the IC50 cannot be calculated.

[0052] In type II AN3CA cells, when administered alone, norethisterone acetate (NOMAc), medroxyprogesterone acetate (MPA), Canagliflozin and Atorvastatin had median inhibitory concentrations (IC50 values) of 89.67, 50.30, 51.46 and ~ 55.81 μΜ, respectively, after treatment for 48 hours at a concentration range of 3-30 μΜ, indicating that MPA and NOMAc and Canagliflozin and Atorvastatin had weak inhibitory effects on the growth of type II AN3CA cells when used alone. As shown in Table 2 and Figures 3-4 Figure 2. The effect of MPA, NOMAc, Canagliflozin and Atorvastatin on the viability of KLE cells. The x-axis represents the concentration of the drugs (alone) or the other drugs when combined with MPA. The y-axis represents the viability of KLE cells.

[0053] Compared with MPA and NOMAc alone, the inhibitory effect of Canagliflozin on the viability of AN3CA cells was enhanced when combined with MPA and NOMAc. When 10 μΜ MPA was combined with Canagliflozin (3, 10, 30 μΜ), the IC50 value decreased to 41.24 μΜ; when 30 μΜ MPA was combined with Canagliflozin, the IC50 value decreased to 27.51 μΜ, which was significantly lower than the IC50 values of MPA and Canagliflozin alone (P < 0.05). As shown in Table 2 and Figures 3-4 Figure 2. The effect of MPA, NOMAc, Canagliflozin and Atorvastatin on the viability of KLE cells. The x-axis represents the concentration of the drugs (alone) or the other drugs when combined with MPA. The y-axis represents the viability of KLE cells.

[0054] The inhibitory effect of canagliflozin combined with NOMAc on the viability of AN3CA cells was significantly enhanced. When 10 μM NOMAc was combined with canagliflozin (3, 10, 30 μM), the IC50 value was 36.06 μM, which was about 60% lower than that of NOMAc alone. When 30 μM NOMAc was combined with canagliflozin, the IC50 value was 27.97 μM, which was about 70% and 30% lower than that of NOMAc and canagliflozin alone, respectively (P<0.05). The above results showed that the combination of canagliflozin and progestin could enhance the inhibitory effect of progestin on AN3CA cells. As shown in Table 2 and Figures 3-4

[0055] When atorvastatin was combined with MPA (10 and 30 μM) at a concentration range (3, 10, 30 μM) to treat AN3CA cells for 48 hours, the IC50 values were 288.9 μM or could not be calculated, respectively. When atorvastatin was combined with NOMAc (10 and 30 μM), the IC50 values were both >500 μM. These results showed that the combination of atorvastatin and progestin had no inhibitory effect on AN3CA cells. As shown in Table 2 and Figures 3-4

[0056] Table 2. Effects of norethisterone acetate, medroxyprogesterone acetate, canagliflozin, and atorvastatin alone and in combination on the half inhibitory concentration (IC50) and 95% confidence interval of KLE cells (n=9).

[0057]

[0058] MPA (medroxyprogesterone acetate), NOMAc (norethisterone acetate), Canag (canagliflozin), Met (metformin), Atorva (atorvastatin)

[0059] *P<0.05 compared with MPA and NOMAc alone; "very wide" indicates that the corresponding 95% confidence interval range is very wide and cannot be calculated; " / " indicates that the inhibition rate did not exceed 50% and the IC50 cannot be calculated.

[0060] Example 2, canagliflozin enhances the growth inhibitory effect of progestin on RL95-2 / THRB (- / -) drug-resistant cells

[0061] 2.1 Experimental materials

[0062] ​​DMEM-F12 medium, fetal bovine serum FBS, HBSS were purchased from Gibco company, endometrial cancer RL95-2 cells: from American Type Culture Collection (ATCC); RL95-2 cell line with thyroid hormone receptor β gene (THRB) knocked out (RL95-2 / THRB (- / -) ) drug-resistant cell line was prepared by Sanye (Suzhou) Biotechnology Co., Ltd.; CCK-8 was purchased from Dojindo Laboratories; DMSO was purchased from Sigma Company, USA; Canagliflozin (Canag) was purchased from MedChemExpress, item number: HY-10451, purity 99.80%; Metformin (Met) was purchased from Sigma-Aldrich, item number: D150959, purity 97%. Nomegestrol acetate (NOMAc) and medroxyprogesterone acetate (MPA) were donated by Zhejiang Xianju Pharmaceutical Co., Ltd., purity >98%. The above two test samples were dissolved with DMSO and used as stock solutions.

[0063] 2.2 Experimental method

[0064] 2.2.1 Detection of tumor proliferation ability

[0065] RL95-2 / THRB (- / -) cells cultured to the logarithmic growth phase were trypsinized, centrifuged and the culture medium was discarded, washed with HBSS, and resuspended with DMEM-F12 medium containing 10% FBS. Counted using a cell counting plate, and adjusted the cell density to 1×10^5 / mL. 100 μL of cell suspension was plated into a 96-well plate, and incubated in a 37℃, 5% CO2 incubator for 48 h, and the old culture medium was discarded. The single drug group added 100 μL of fresh culture medium, and the combined drug group added 200 μL of fresh culture medium. Three replicate wells were set for each concentration, and 1 μl of drug was added to each well to meet the specified requirements. The cells were incubated in a 37℃, 5% CO2 incubator for 48 h, 10 μL of CCK-8 solution was added to each well, and incubated in a 37℃ incubator for 2 h. The OD value was measured at 450 nm on a microplate reader.

[0066] 2.2.2 Statistical method

[0067] The growth inhibition rate of drug on cells (%) = (1-OD drug treatment / OD solvent) x 100%, all statistical analysis was performed using Graphpad Prism 9.5.1. The mean ± standard error was used to represent the measurement data. The single factor analysis of variance method was used to compare the changes of cell number in different groups to determine the statistical significance, and Turkey's multiple comparison test was used as post hoc test when comparing the differences between multiple groups. P<0.05 was statistically significant. The calculation method of IC50 was the same as 1.2.2

[0068] 2.3 Experimental results

[0069] In RL95-2 / THRB (- / -) cells, 1-30 μM medroxyprogesterone acetate (MPA), norethindrone acetate (NOMAc), canagliflozin and metformin treatment for 48 hours, the IC50 values of RL95-2 / THRB (- / -) cell proliferation inhibition rate were all greater than 100 μM, and the 95% confidence interval could not be calculated, indicating that in RL95-2 / THRB (- / -) cells, progestogens had no significant inhibitory effect on cell growth, and canagliflozin and metformin also had no significant inhibitory activity, as shown in Table 3 and Figures 5-6 .

[0070] When canagliflozin was combined with MPA and NOMAc, the inhibitory effect on RL95-2 / THRB (- / -) cells was significantly enhanced, among which, 10 μM MPA combined with canagliflozin (3, 10, 30 μM), the IC50 value was 54.63 μM; 30 μM MPA combined with canagliflozin, the IC50 value was 10.40 μM, which was reduced by 50% and 90% (P<0.05) compared with the IC50 of MPA alone, and was 1 / 10-1 / 60 (P<0.05) of canagliflozin alone. In addition, 10 μM NOMAc combined with canagliflozin (3, 10, 30 μM), the IC50 value was 49.11 μM; 30 μM NOMAc combined with canagliflozin, the IC50 value was 24.95 μM, which was reduced by about 89% and 95% (P<0.05) compared with the IC50 of NOMAc alone, and was 1 / 8.5-1 / 16 (P<0.05) of canagliflozin alone. As shown in Table 3 and Figures 5-6 , in which the abscissa is the concentration of the drug (single use) or progestogen + other drugs combined with other drugs as shown in the legend.

[0071] The results showed that the combination of canagliflozin and progestogen could enhance the inhibitory effect of progestogen on endometrial cancer cells RL95-2 / THRB (- / -) ; and the combination of metformin and MPA or NOMAc had no effect on RL95-2 / THRB(- / -) Cell viability was significantly inhibited, and thus IC50values could not be calculated. The above results show that the inhibitory activity of canagliflozin combined with progestins on RL95-2 / THRB(- / -) drug-resistant cells was significantly stronger than that of metformin combined with progestins.

[0072] In addition, atorvastatin alone had no significant inhibitory effect on the growth of RL95-2 / THRB(- / -) drug-resistant cells. (- / -) Cell viability was slightly inhibited, with an IC50value of about 50 μM, and when it was combined with 10 and 30 μM NOMAc, the IC50values increased to 277.6 and 163.78 μM, respectively, which was about 5.5 times and 3.2 times the IC50value of atorvastatin alone (P < 0.05). This result shows that atorvastatin combined with progestins (such as NOMAc) has no significant inhibitory effect on the growth of RL95-2 / THRB(- / -) drug-resistant cells, and may even promote cell proliferation, indicating that atorvastatin is not suitable for combination with some progestins (such as NOMAc). (- / -) Drug-resistant cells not only had no significant inhibitory effect; on the contrary, they may even promote cell proliferation, indicating that atorvastatin is not suitable for combination with some progestins (such as NOMAc). As shown in Table 3.

[0073] Table 3 Effects of norethisterone acetate, medroxyprogesterone acetate, canagliflozin, metformin, and atorvastatin alone and in combination on the half maximal inhibitory concentration (IC50) and 95% confidence interval of RL95-2 / THRB(- / -) drug-resistant cells (n = 9). (- / -) Effects of norethisterone acetate, medroxyprogesterone acetate, canagliflozin, metformin, and atorvastatin alone and in combination on the half maximal inhibitory concentration (IC50) and 95% confidence interval of RL95-2 / THRB(- / -) drug-resistant cells (n = 9).

[0074]

[0075]

[0076] MPA (medroxyprogesterone acetate), NOMAc (norethisterone acetate), Canag (canagliflozin), Met (metformin), Atorva (atorvastatin)

[0077] *P < 0.05 compared with MPA and NOMAc alone; # P < 0.05 compared with canagliflozin alone; “very wide” indicates that the corresponding 95% confidence interval range is very wide and cannot be calculated; “ / ” indicates that the inhibition rate did not exceed 50%, and the IC50could not be calculated, or the confidence limit range could not be calculated.

[0078] Example 3: Canagliflozin enhances the inhibitory effect of progestins on RL95-2 / THRB(- / -) drug-resistant cells in nude mice (- / -) Inhibitory effect on RL95-2 / THRB(- / -) drug-resistant cell xenografts

[0079] 3.1 Experimental materials

[0080] Clean level immunodeficient female nude mice (BABL / c-nu), 6 weeks old, body weight 16-18 g when purchased, purchased from Shanghai Slek Experimental Animal Co., Ltd., license number: SCXK (Shanghai) 2017-0005. Raising in SPF level animal room, temperature 25±2℃, relative humidity 50±10%, using 12 hours of light / dark cycle, the cages, bedding, feed and water used for nude mice were sterilized by high temperature, feed was purchased from Shanghai Silin Biological Technology Co., Ltd. This experiment was reviewed by Shanghai Institute of Biomedicine Technology Animal Ethics Committee, batch number 2017-07. Endometrial cancer RL95-2 cells: from American Type Culture Collection (ATCC); RL95-2 / THRB (- / -) Drug-resistant cell lines were prepared by Saiye (Suzhou) Biotechnology Co., Ltd.; Canagliflozin (Canagliflozin, Canag) was purchased from MedChemExpress, item number: HY-10451, purity 99.80%; medroxyprogesterone acetate (Medroxyprogesterone acetate, MPA) and nomegestrol acetate (Nomegestrol acetate, NOMAc) were donated by Zhejiang Xianju Pharmaceutical Co., Ltd., purity >98%. After grinding and dissolving, 0.05% Tween-80 and 0.5% CMC-Na were used for standby.

[0081] 3.2 Experimental method

[0082] After one week of adaptive feeding of nude mice, the body weight was 20±2g. The logarithmic growth phase RL95-2 / THRB (- / -) cells were collected and the cell density was adjusted, and the RL95-2 / THRB (- / -) cell suspension 0.2ml was injected subcutaneously in the left axillary under sterile conditions. Each nude mouse was inoculated with (8-10)×10 6 cells. The nude mice were observed for eating and excretion every other day, and the state of the nude mice and the growth changes of the tumor were observed every day. The body weight was measured once a week, and the long diameter (a) and short diameter (b) of the tumor were measured 1-2 times a week, and the tumor volume was calculated according to V=ab 2 / 2. The tumor volume was measured twice continuously, and reached about 50mm 3Right and left, as a successful modeling, according to the principle of random grouping and dosing tumor volume. The success of the nude mice were randomly divided into 7 groups, respectively, ① solvent control group, ② MPA 100mg / kg group, ③ MPA 100mg / kg + Met 100mg / kg combined application group, ④ MPA 100mg / kg + Canagliflozin 15mg / kg combined group, and ⑤ NOMAc 100mg / kg group, ⑥ NOMAc 100mg / kg + Met 100mg / kg combined application group, ⑦ NOMAc 100mg / kg + Canagliflozin 15mg / kg combined application group. Continuous intragastric administration for 28 days. 2 hours after the last administration, the animals were anesthetized, blood was taken and dissected, the tumor growth inhibition rate was calculated, and pathological examination was performed to evaluate the endometrial cancer transplanted tumor.

[0083] Tumor growth inhibition rate / % = (model control group tumor volume - drug group tumor volume) / model control group tumor volume x 100%

[0084] Volume / mm 3 = 1 / 2 x long diameter x short diameter x short diameter

[0085] Organ coefficient / % = organ wet weight / body weight x 100%

[0086] 3.3 Experimental results

[0087] Each group of Balb / c-nu nude mice in good condition during the experiment. RL95-2 / THRB (- / -) Cells were inoculated into the subcutaneous of nude mice for about 5 weeks to grow into a volume of > 50mm 3 Transplanted tumor. Random grouping according to tumor volume.

[0088] Compared with the solvent control group, there was no significant difference in tumor volume of each group of animals before drug treatment (P> 0.05). After 4 weeks of different drug treatment, the tumor volume of each group of nude mice showed different changes, as shown in Table 4 and Figure 7 A-G. Among them, the MPA 100mg / kg treatment group transplanted tumor grew rapidly, the transplanted tumor volume increased, and the average tumor inhibition rate was (-) 18.27%, indicating that MPA 100mg / kg had no inhibitory effect on drug-resistant RL95-2 / THRB (- / -) Cell transplanted tumor, that is, RL95-2 / THRB (- / -)The cell transplanted tumor was not sensitive to the treatment of the progestogen MPA. The combination of MPA and metformin slightly inhibited the growth of the transplanted tumor, but there was no significant difference compared with the solvent control group (P>0.05), and the average tumor growth inhibition rate was 24.47%. In contrast, the transplanted tumor volume in the MPA+canagliflozin group was significantly lower than that in the model control group (P<0.05), and the average tumor growth inhibition rate was 41.95%, indicating that canagliflozin can enhance the inhibitory effect of MPA on the RL95-2 / THRB(- / -) cell transplanted tumor.

[0089] The average tumor inhibition rate of 100 mg / kg NOMAc on the RL95-2 / THRB(- / -) cell transplanted tumor was 7.17%, indicating that NOMAc had no obvious inhibitory effect on the RL95-2 / THRB(- / -) cell transplanted tumor, that is, the RL95-2 / THRB(- / -) cell transplanted tumor was not sensitive to the treatment of the progestogen NOMAc. (- / -) The cell transplanted tumor was not sensitive to the treatment of the progestogen MPA. The combination of MPA and metformin slightly inhibited the growth of the transplanted tumor, but there was no significant difference compared with the solvent control group (P>0.05), and the average tumor growth inhibition rate was 24.47%. In contrast, the transplanted tumor volume in the MPA+canagliflozin group was significantly lower than that in the model control group (P<0.05), and the average tumor growth inhibition rate was 41.95%, indicating that canagliflozin can enhance the inhibitory effect of MPA on the RL95-2 / THRB(- / -) cell transplanted tumor. (- / -) The cell transplanted tumor was not sensitive to the treatment of the progestogen MPA. The combination of MPA and metformin slightly inhibited the growth of the transplanted tumor, but there was no significant difference compared with the solvent control group (P>0.05), and the average tumor growth inhibition rate was 24.47%. In contrast, the transplanted tumor volume in the MPA+canagliflozin group was significantly lower than that in the model control group (P<0.05), and the average tumor growth inhibition rate was 41.95%, indicating that canagliflozin can enhance the inhibitory effect of MPA on the RL95-2 / THRB(- / -) cell transplanted tumor.

[0090] Compared with before administration, the body weight of the animals in each group after drug treatment increased to different degrees, but there was no significant difference (P>0.05). After 4 weeks of administration, there was no significant difference in the liver and kidney organ coefficients of the nude mice in each group (P>0.05), indicating that the combination of canagliflozin, NOMAc and MPA in the present application is safe and has no obvious effect on body weight, kidney and liver organ weight. See Table 5.

[0091] In summary, the combination of canagliflozin and progestogen shows a strong inhibitory effect on the growth of the drug-resistant cell RL95-2 / THRB(- / -) transplanted tumor, indicating that the combination of canagliflozin and progestogen can become a new method for improving the treatment of drug-resistant tumors and enhancing the activity of endocrine drug treatment.

[0092] Table 4 Inhibitory effect of the combination of canagliflozin, NOMAc and MPA on the RL95-2 / THRB(- / -) progestogen-resistant cell-induced transplanted tumor in nude mice (mean ± standard deviation)

[0093]

[0094] *P<0.05, compared with the solvent control group.

[0095] Table 5 Effect of canagliflozin in combination with NOMAc and MPA on body weight and organ coefficients of RL95-2 / THRB(- / -) cell inoculated nude mice (mean ± standard deviation)

[0096]

[0097] The above merely describes preferred embodiments of the present application, but does not limit the scope of the technical content of the present application. The technical content of the present application is broadly defined in the scope of the claims of the application. Any technical entity or method completed by others, if it is exactly the same as or an equivalent modification of the scope defined by the claims of the application, is considered to be covered in the scope of the claims.

Claims

1. Use of an SGLT2 inhibitor and a progestogen for the manufacture of a medicament for the treatment of a disease of the reproductive system which is negative for hormone receptor expression or which is of low hormonal reactivity, wherein, The SGLT2 inhibitor is selected from canagliflozin and ipragliflozin, and the reproductive system disease is selected from endometrial cancer and ovarian cancer.

2. A combination of an SGLT2 inhibitor and a progestogen, wherein, The combination is used for treating a hormone receptor expression negative or hormone low responsive type of reproductive system disease, wherein the SGLT2 inhibitor is selected from canagliflozin and ipragliflozin, and the reproductive system disease is selected from endometrial cancer and ovarian cancer.

3. A composition for treating a reproductive system disease that is hormone receptor expression negative or hormone hypo-responsive, comprising an SGLT2 inhibitor and a progestogen, wherein, The SGLT2 inhibitor is selected from canagliflozin and ipragliflozin, and the reproductive system disease is selected from endometrial cancer and ovarian cancer.

4. Use according to claim 1, or a combination according to claim 2, or a composition according to claim 3, wherein, The SGLT2 inhibitor is used for improving or enhancing the sensitivity of hormones and / or for reversing the drug resistance of hormones.

5. Use according to claim 1, or a combination according to claim 2, or a composition according to claim 3, wherein, The progestogen is selected from medroxyprogesterone acetate and norethisterone acetate.

6. Use according to claim 1, or a combination according to claim 2, or a composition according to claim 3, wherein, The progestogen is selected from medroxyprogesterone acetate and norethisterone acetate, the SGLT2 inhibitor is canagliflozin, and the molar ratio of progestogen: canagliflozin is in the range of 1:1-10:1.

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