A pharmaceutical composition for treating cancer

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a pharmaceutical composition for the prevention and / or treatment of cancer. In particular, it relates to a pharmaceutical composition comprising a CDK4 / 6 inhibitor compound of formula (I) or a pharmaceutically acceptable salt thereof, and a second therapeutic agent or a pharmaceutically acceptable salt thereof, in a fixed ratio or fixed dose combination; the use of the above pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of cancer; and a cassette containing the pharmaceutical composition.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pharmaceutical composition for the prevention and / or treatment of cancer, particularly a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in a fixed proportion or fixed dose combination with a second therapeutic agent or a pharmaceutically acceptable salt thereof, and the use of said pharmaceutical composition for the treatment of cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor in women, with approximately 2 million new cases worldwide each year. Breast cancer is a highly heterogeneous tumor at the molecular level, exhibiting significant differences in histological morphology, immunophenotype, biological behavior, and treatment response. Based on the expression of three receptors—estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2)—breast cancer is classified into three subtypes. Estrogen receptor-positive (ER+) breast cancer is the most common molecular subtype, with an incidence rate of approximately 65%-70% in the general population. It has the best prognosis among breast cancers, with a high proportion of early-stage patients. Compared to other molecular subtypes of breast cancer, ER+ breast cancer has a better prognosis, but still has a 5-year recurrence and metastasis rate of approximately 15-30%. The human epidermal growth factor receptor-2 (HER2) molecule is an independent factor indicating a poor prognosis in breast cancer; approximately 20%-30% of breast cancer patients in China have HER2 gene amplification / overexpression. HER2 overexpression is typically associated with aggressive, metastatic forms of breast cancer, which are associated with high recurrence rates and / or poor patient outcomes.

[0003] The general treatment goal for all patients with ER / PR-positive metastatic breast cancer is to prolong survival and improve quality of life. This can be achieved through surgical intervention and drug therapy (if possible). Endocrine (anti-estrogenic) drugs are usually used initially and maintained until resistance develops. Their use avoids the toxicity of chemotherapy-based regimens, but the toxicity of the drugs themselves and drug resistance still need to be well addressed.

[0004] Estrogen receptor (ER) drugs are mainly classified into selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs), and aromatase inhibitors. Marketed SERDs include fulvestrant (Astrrazeneca), while investigational SERDs include AZD-9496 (Astrrazeneca), RAD1901 (Eisai), and ZB-716 (Louisiana University).

[0005] Almost all oncogenes and tumor suppressor genes ultimately converge on the cell cycle, and regulating or blocking the cell cycle is one of the ways to treat tumors. Currently, many molecules related to cell cycle regulation have been discovered, among which cyclin-dependent kinases (CDKs) are the core molecules of the cell cycle regulatory network. Among the CDK subtypes involved in the cell cycle, CDK4 / 6 plays an irreplaceable role. CDK4 / 6-specific activation is closely related to the proliferation of some tumors; approximately 80% of human tumors show abnormalities in the cyclin D-CDK4 / 6-INK4-Rb pathway. Currently marketed CDK4 / 6 inhibitors include Pfizer's PD0332991 (Palbociclib, Ibrance), Eli Lilly's LY2835219 (Abemaciclib, Verzenio), and Novartis' LEE011 (Ribociclib, Kisqali).

[0006] Like other kinase inhibitors used to treat cancer, the effective use of CDK4 / 6 inhibitors is also limited by drug resistance. This invention provides a tumor treatment composition that effectively addresses the problem of drug resistance and has low toxicity, with resistance primarily targeting endocrine and CDK inhibitor therapies. Summary of the Invention

[0007] In one aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of cancer, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one second therapeutic agent or a pharmaceutically acceptable salt thereof.

[0008]

[0009] The second therapeutic agent is selected from selective estrogen receptor downregulators, wherein the weight ratio of the active ingredient of the compound of formula (I) or a pharmaceutically acceptable salt thereof to the second therapeutic agent or a pharmaceutically acceptable salt thereof is 1:1 to 3000:1.

[0010] In some embodiments, the active ingredient of formula (I) compound or a pharmaceutically acceptable salt thereof is in a weight ratio of 1:1 to 2672:1 to a second therapeutic agent or a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the weight ratio of the active ingredient of formula (I) compound or a pharmaceutically acceptable salt thereof to that of the second therapeutic agent or a pharmaceutically acceptable salt thereof is 5.2:1 to 2672:1.

[0012] In some embodiments, the active ingredient of formula (I) compound or a pharmaceutically acceptable salt thereof is in a weight ratio of 1:1 to 800:1 to a second therapeutic agent or a pharmaceutically acceptable salt thereof.

[0013] In some embodiments, the weight ratio of the active ingredient of formula (I) compound or a pharmaceutically acceptable salt thereof to that of the second therapeutic agent or a pharmaceutically acceptable salt thereof is 1:1 to 500:1.

[0014] In some of the above embodiments, the second therapeutic agent is fulvestrant.

[0015] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 10:1 to 1200:1.

[0016] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 10:1 to 800:1.

[0017] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 10:1 to 600:1.

[0018] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 10:1 to 400:1.

[0019] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 10:1 to 200:1.

[0020] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 20:1 to 150:1.

[0021] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 30:1 to 150:1.

[0022] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 40:1 to 150:1.

[0023] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 20:1 to 120:1.

[0024] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 30:1 to 120:1.

[0025] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 40:1 to 120:1.

[0026] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 20:1 to 100:1.

[0027] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 30:1 to 100:1.

[0028] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 40:1 to 100:1.

[0029] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 1:1-150:1, preferably 10:1-20:1, 20:1-30:1, 30:1-40:1, 40:1-50:1, 50:1-60:1, 60:1-70:1, 70:1-80:1, 80:1-90:1, 90:1-100:1, 100:1-110:1, 110:1-120:1, 120:1-130:1, 130:1-140:1, or 140:1-150:1.

[0030] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 107.52:1, 94.08:1, 80.64:1, 67.2:1, 62.72:1, 53.76:1, 47.04:1, 40.32:1, 33.6:1, 31.36:1, 26.88:1, 23.52:1, 20.16:1, 16.8:1, 15.68:1, 13.44:1, or 11.76:1.

[0031] In some embodiments, the second therapeutic agent is fulvestrant, and the weight ratio of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof to fulvestrant or a pharmaceutically acceptable salt thereof is 115.2:1, 100.8:1, 86.4:1, 72:1, 67.2:1, 57.6:1, 50.4:1, 43.2:1, 36:1, 33.6:1, 28.8:1, 25.2:1, 21.6:1, 18:1, 16.8:1, 14.4:1, or 12.6:1.

[0032] In some embodiments, the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof is used in a daily dose of 200-2000 mg.

[0033] In some embodiments, the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof is used in a daily dose of 200-1000 mg.

[0034] In some embodiments, the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof is used in a daily dose of 400-1000 mg.

[0035] In some embodiments, the daily dose of the active ingredient of formula (I) or a pharmaceutically acceptable salt thereof is 960 mg, 840 mg, 720 mg, 600 mg, 560 mg, 480 mg or 420 mg.

[0036] In some implementations, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once, twice, or three times a day.

[0037] In some embodiments, the second therapeutic agent or its pharmaceutically acceptable salt is administered once a month, once every 28 days, once every two weeks, or once every 14 days.

[0038] In some embodiments, the effective ingredient of the second therapeutic agent or its pharmaceutically acceptable salt is used in doses of 1-1000 mg, such as 10-1000 mg, 100-1000 mg, or 250-1000 mg.

[0039] In some of the above embodiments, the second therapeutic agent is fulvestrant.

[0040] In some embodiments, the second therapeutic agent is fulvestrant, or an active ingredient of fulvestrant or a pharmaceutically acceptable salt thereof, in a dose of 250 mg or 500 mg.

[0041] In some embodiments, the second therapeutic agent is fulvestrant, the active ingredient of which is fulvestrant or a pharmaceutically acceptable salt thereof, in a dose of 250 mg or 500 mg, administered once a month, once every 28 days, once every two weeks, or once every 14 days.

[0042] In some embodiments, the compound of formula (I) is administered simultaneously or sequentially with the second therapeutic agent.

[0043] In some embodiments, the compound of formula (I) or its pharmaceutically acceptable salt is administered orally.

[0044] In some embodiments, the second therapeutic agent or its pharmaceutically acceptable salt is administered orally or by injection.

[0045] In another aspect, the present invention also provides a pharmaceutical formulation comprising the aforementioned pharmaceutical composition and one or more pharmaceutically acceptable excipients, wherein the pharmaceutical formulation may be any pharmaceutically acceptable dosage form. Pharmaceutically acceptable excipients are substances that are non-toxic, compatible with the active ingredient, and otherwise biologically suitable for use in organisms. The selection of a particular excipient will depend on the route of administration or the type and state of disease for treating a particular patient.

[0046] In some embodiments, the above-described pharmaceutical preparation can be administered to patients or subjects requiring this treatment via oral, parenteral, rectal, or pulmonary administration. For oral administration, the pharmaceutical composition can be formulated as an oral preparation, such as conventional oral solid dosage forms like tablets, capsules, pills, granules, etc.; or as an oral liquid preparation, such as an oral solution, oral suspension, syrup, etc. For parenteral administration, the above-described pharmaceutical preparation can also be formulated as an injectable preparation, including injection solutions, sterile powders for injection, and concentrated solutions for injection. For rectal administration, the pharmaceutical composition can be formulated as suppositories, etc. For pulmonary administration, the pharmaceutical composition can be formulated as an inhalation preparation, aerosol, powder inhaler, or spray, etc.

[0047] In another aspect, the present invention also relates to the use of the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of cancer, wherein the cancer is selected from brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma, preferably breast cancer.

[0048] In some implementations, the cancer is selected from HR. + HER2 - Breast cancer.

[0049] In some implementations, the cancer is selected from postmenopausal HR. + HER2 - Breast cancer.

[0050] In some implementations, the cancer is selected from locally advanced or metastatic breast cancer.

[0051] In some implementations, the cancer is selected from HR. + HER2 - Advanced or metastatic breast cancer.

[0052] In some embodiments, the cancer is selected from HR patients who have progressed after endocrine therapy alone. + HER2 - Advanced or metastatic breast cancer.

[0053] In another aspect, the present invention also relates to the use of a combination of a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and fulvestrant or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of cancer, wherein the cancer is selected from brain tumors, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female reproductive tract cancer, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma or sarcoma, preferably breast cancer.

[0054] In some implementations, the cancer is selected from HR. + HER2 - Breast cancer.

[0055] In some implementations, the cancer is selected from postmenopausal HR. + HER2 - Breast cancer.

[0056] In some implementations, the cancer is selected from locally advanced or metastatic breast cancer.

[0057] In some implementations, the cancer is selected from HR. + HER2 - Advanced or metastatic breast cancer.

[0058] In some embodiments, the cancer is selected from HR patients who have progressed after endocrine therapy alone. + HER2 - Advanced or metastatic breast cancer.

[0059] Furthermore, the present invention also relates to the use of pharmaceutical preparations containing the aforementioned pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of cancer.

[0060] In another aspect, the present invention also provides a medicine box containing the aforementioned pharmaceutical composition and instructions for use of the pharmaceutical composition or the compound of formula (I) contained therein and the second therapeutic agent.

[0061] In another aspect, the present invention also provides a method for treating cancer, the method comprising administering to a patient in need an effective amount of the aforementioned pharmaceutical composition and a pharmaceutical preparation containing the pharmaceutical composition; wherein the cancer is as described above.

[0062] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a second therapeutic agent or a pharmaceutically acceptable salt thereof according to a specific dosage regimen.

[0063] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally at a dose of 400-1000 mg per day.

[0064] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally at doses of 960 mg, 840 mg, 720 mg, 600 mg, 560 mg, 480 mg or 420 mg per day.

[0065] In some implementations, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once or twice a day.

[0066] In some embodiments, the second therapeutic agent is fulvestrant, administered at a dose of 250 mg or 500 mg or a pharmaceutically acceptable salt thereof on day 1, day 15, day 29, and thereafter once a month or every 28 days.

[0067] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. However, for a better understanding of this invention, definitions of some terms are provided below. When the definitions and interpretations of the terms provided in this invention differ from the meanings commonly understood by those skilled in the art, the definitions and interpretations provided in this invention shall prevail.

[0068] The HR positive (HR) of this invention + ) refers to positive expression of ER (estrogen receptor) and / or PR (progesterone receptor), i.e., HR + Including ER + PR + or ER + / PR + .

[0069] The HER2 described in this invention - This refers to a negative expression of human epidermal growth factor receptor 2 (HER2).

[0070] The "active ingredient weight ratio" mentioned in this invention can be a daily dosage ratio, a weekly dosage ratio, a monthly dosage ratio, or a dosage ratio for each dosing cycle. If the dosing frequencies of two drugs are different, the same standard is selected for conversion to obtain the active ingredient weight ratio.

[0071] The "daily dosage" mentioned in this invention can be the actual daily dosage or a daily dosage obtained through conversion. For example, if the dosing frequency is twice a day at 360mg each time, the daily dosage is 720mg; if the dosing frequency is once every 15 days at 300mg each time, the daily dosage is 20mg.

[0072] The "second therapeutic agent" described in this invention refers to a drug that has certain preventive and / or therapeutic effects on tumors, including but not limited to mitotic inhibitors, alkylating agents, selective estrogen receptor modulators, selective estrogen receptor downregulators, DNA chimeras, antitumor antibiotics, growth factor inhibitors, vitamin A receptor modulators, topoisomerase inhibitors, hormonal drugs, and angiogenesis inhibitors. The "effective dose" refers to the drug dosage that can prevent, alleviate, delay, inhibit, or cure the subject's symptoms. The dosage is related to the route of drug administration, the pharmacokinetics of the drug, the severity of the disease, and the subject's individual characteristics (gender, weight, height, age).

[0073] The "pharmaceutical composition" of the present invention includes a composition formed by preparing the compound of formula (I) and the second therapeutic agent and other active ingredients into the same compound preparation, and also includes a composition formed by preparing the compound of formula (I) and the second therapeutic agent and other active ingredients into separate single preparations.

[0074] The simultaneous administration of the compound of formula (I) and the second therapeutic agent described in this invention includes administering the compound of formula (I) and the second therapeutic agent after they are prepared into the same compound preparation, or administering the compound of formula (I) and the second therapeutic agent simultaneously after they are prepared into separate preparations.

[0075] The "separate administration" of the compound of formula (I) and the second therapeutic agent described in this invention means that after the compound of formula (I) and the second therapeutic agent are prepared into formulations, they are administered sequentially according to their respective formulation administration methods.

[0076] The "pharmaceutically acceptable salt" as described in this invention refers to a salt formed by an acidic functional group (e.g., -COOH, -OH, -SO3H, etc.) present in a compound and a suitable inorganic or organic cation (base), including salts formed with alkali metals or alkaline earth metals, ammonium salts, and salts formed with nitrogen-containing organic bases; and a salt formed by a basic functional group (e.g., -NH2, etc.) present in a compound and a suitable inorganic or organic anion (acid), including salts formed with inorganic acids or organic acids (e.g., carboxylic acids, etc.).

[0077] Beneficial effects of the invention

[0078] 1. The pharmaceutical composition of the present invention has excellent anti-tumor effects, especially significant efficacy in treating breast cancer.

[0079] 2. The pharmaceutical composition of the present invention can effectively solve the problem of drug resistance to endocrine therapy and the drug resistance problem of long-term use of CDK inhibitors.

[0080] 3. The pharmaceutical composition of the present invention has low or no toxicity and few side effects. Attached Figure Description

[0081] Figure 1 The curves show the cell proliferation inhibition of compound (I) and fulvestrant in a fixed ratio. The horizontal axis represents the logarithm of the test concentration (c) of compound (I), i.e., lg c; the vertical axis represents the inhibition rate (%). Detailed Implementation Plan

[0082] Experimental protocol

[0083] The following provides exemplary experimental schemes for some compounds of the present invention to demonstrate the beneficial activity and technical effects of the compounds. However, it should be understood that the following experimental schemes are merely examples of the content of the present invention and not limitations on the scope of the present invention.

[0084] Experimental Example 1: In vitro cellular inhibition of the compound of formula (I) of the present invention, and fulvestrant, alone and in combination. active Test sample:

[0085] The compound of formula (I) of this invention is prepared according to prior art methods;

[0086] Fluvestrant: Purchase or prepare according to existing technical methods.

[0087] The abbreviations used in the following experiments have the following meanings:

[0088]

[0089] Experimental methods: Cell proliferation was detected using the BrdU method (BrdU cell proliferation assay kit, PerkinElmer).

[0090] 1. Preparation of reagents and compounds

[0091] 1.1 Preparation of 1x washing solution:

[0092] Dilute the washing solution with a mother liquor concentration of 25 times with ultrapure water to a washing solution concentration of 1.

[0093] 1.2 Preparation of 1x detection antibody solution:

[0094] Dilute the BrdU-Eu detection antibody, which has a stock solution concentration of 200 times, with the detection antibody diluent to a 1-fold detection antibody solution.

[0095] 1.3 100x BrdU-labeled solution:

[0096] The BrdU stock solution with a stock concentration of 1000 times was diluted with the corresponding cell culture medium to make a BrdU-labeled solution with a concentration of 100 times.

[0097] 1.4 Preparation of test compounds:

[0098] Compound (I) was diluted with DMSO to 0.256 mM, and then serially diluted 2-fold to 9 concentrations starting from 0.256 mM; fulvestrant was diluted with DMSO to 1.28 μM, and then serially diluted 2-fold to 9 concentrations starting from 1.28 μM; fulvestrant was diluted with DMSO to 80 nM and 160 nM.

[0099] Compounds used alone: ​​Mix the compound of formula (I) or fulvestrant gradient dilution with DMSO at a 1:1 ratio;

[0100] A fixed ratio of compound used in combination: Compound of formula (I) and fulvestrant gradient dilution solution are mixed at a 1:1 ratio;

[0101] Fixed concentration of compound: The compound of formula (I) and 80 nM and 160 nM of fulvestrant were mixed at a ratio of 1:1.

[0102] 1.5 Culture medium preparation:

[0103] MCF7 medium: MEM + 10% FBS + 1% penicillin antibody + 10 μg / mL recombinant human insulin

[0104] 2. Test Procedure

[0105] 2.1 Cells were cultured routinely until the cell confluence reached 80%-90%. Cells were then collected by trypsin digestion and centrifuged. The cells were resuspended in the appropriate culture medium, counted, and seeded into 96-well plates. MCF7 cells were seeded at 8000 cells / well / 100μL and cultured in a 37℃ cell culture incubator. After cell adhesion, the medium was replaced with serum-free medium.

[0106] 2.2 Add 99 μL of growth medium containing 20% ​​FBS to each well, then add 1 μL of the diluted 200× analyte to each well. The final concentration of the analyte is shown in the table below. Incubate at 37℃ for 48 hours.

[0107] Solvent control: 0.5% DMSO; Blank control: culture medium only, no cells.

[0108]

[0109] 2.3 Add 2 μL of 100-fold BrdU-labeled solution to each well, incubate overnight in a cell culture incubator, then equilibrate at room temperature for 0.5 hours, and remove the culture medium using a vacuum pump;

[0110] 2.4 Add 100 μL of fixative to each well, incubate at room temperature for 30 minutes, and then discard the solution;

[0111] 2.5 Add 100 μL of 1x BrdU-Eu detection antibody solution to each well, incubate at room temperature for 1 hour, discard the solution, and wash 5 times with 250 μL of 1x washing buffer per well;

[0112] 2.6 Add 200 μL of time-resolved immunofluorescence induction solution to each well, incubate at room temperature for 15 minutes, and detect the fluorescence value (Flu value) using a microplate reader.

[0113] 3. Data Processing

[0114] 1) Cell inhibition rate (%) = 1 - (Flu) 测试物 -Flu 空白对照 ) / (Flu 溶剂对照 -Flu 空白对照 )×100%,

[0115] Flu 空白对照 Blank control value, Flu 溶剂对照 Solvent reference value;

[0116] 2) The data was plotted using GraphPad Prism 6 software to obtain curves and IC values. 50 value.

[0117] 3) The combination index CI was fitted using CompuSyn software. CI < 1 indicates that the two drugs have a synergistic effect when used together.

[0118] Experimental results:

[0119] Table 1 shows the in vitro cellular activities of compound (I) and fulvestrant, both alone and in combination.

[0120]

[0121] Table 2 shows the combined use of compound (I) and fulvestrant in MCF7 in vitro cell culture.

[0122]

[0123] Experimental conclusion:

[0124] As can be seen from the data in Tables 1 and 2, the compound of formula (I) of this invention has high inhibitory activity against cells in vitro and has a synergistic effect when used in combination with fulvestrant.

[0125] Experimental Example 2: Effects of compound (I) of the present invention, alone and in combination with fulvestrant, on xenografts in different cancer types. In vivo efficacy evaluation test of plant model

[0126] Test sample:

[0127] The compound of formula (I) of this invention is prepared according to prior art methods;

[0128] Fluvestrant: Purchase or prepare according to existing technical methods.

[0129] The meanings of the abbreviations for experiments below are as follows:

[0130]

[0131] Experimental methods:

[0132] 1. Tumor inoculation and grouping

[0133] Animals were given subcutaneous estrogen injections starting one day prior to inoculation, twice a week, 40 μg / 20 μL each time (estrogen strength: 4 mg / 2 mL), until the end of the experiment. MCF7 cells resuspended in serum-free medium were then inoculated at 1.5 × 10⁻⁶ cells / mL. 7 +0.2 mL of matrix gel (cells:matrix gel = 1:1) was inoculated subcutaneously in the right rib area of ​​female Balb / c nude mice. The tumors grew to a mean volume of 163 mm². 3 The animals were administered the compound (I) orally once a day at a dose of 30 mg / kg for 21 consecutive days. The fulvestrant group was administered subcutaneously once a week at a dose of 5 mg per animal for 3 consecutive weeks. When compound (I) and fulvestrant were used in combination, the administration method was the same as when they were used alone.

[0134] 2. Measurement and experimental indicators of tumors

[0135] The tumor volume was measured twice a week using calipers, measuring both the long and short diameters. The volume was calculated using the formula: Volume = 0.5 × Long diameter × Short diameter 2 Calculate the relative tumor volume (RTV) and relative tumor volume increase ratio (T / C) based on the measurement results. RTV = V t / V0, where V t Vt represents the mean tumor volume on day t after group administration, and V0 represents the mean tumor volume on the day of grouping. T / C (%) = RTV of treatment group / RTV of control group × 100%. Tumor growth inhibition rate TGI (%) = (1 - T / C) × 100%.

[0136] 3. Statistical Analysis

[0137] The tumor volume was analyzed between groups using SPSS statistical analysis software, and p < 0.05 was considered statistically significant.

[0138] Experimental results:

[0139] Table 3 shows the tumor growth inhibition results of compound (I) and fulvestrant alone and in combination in the MCF7 human xenograft model.

[0140]

[0141] Note: a. Mean ± standard error; b. Compared with the control group.

[0142] Experimental conclusion:

[0143] As shown in Table 3, the combination of compound (I) and fulvestrant significantly inhibited the growth of MCF7 human breast cancer cells (tumor inhibition rate TGI was 83.3%), which was significantly better than fulvestrant alone.

[0144] Experimental Example 3: Clinical trial of the combination of compound (I) of the present invention and fulvestrant for the treatment of breast cancer patients. Test

[0145] Test sample:

[0146] The compound of formula (I) of this invention is prepared according to prior art methods;

[0147] Fluvestrant: Purchase or prepare according to existing technical methods.

[0148] Inclusion criteria for the trial:

[0149] 1. Age 18 to 70 (inclusive);

[0150] 2. Patients with locally advanced or metastatic breast cancer who are confirmed by laboratory results to be hormone receptor positive (HR+) and HER2 negative (HER2-), who are not suitable for surgical resection or radiotherapy for the purpose of cure, have no clinical indication for chemotherapy, have received ≤1 line of endocrine therapy and are allowed to receive 1 chemotherapy regimen.

[0151] 3. Must have at least one measurable lesion that meets the definition of RECIST v1.1;

[0152] 4. ECOG score is 0-1;

[0153] 5. All acute toxicities from previous cancer treatment or surgery have resolved to baseline or ≤ grade 1 (NCI-CTCAE v4.03, excluding alopecia or other toxicities that the investigator considers to pose no safety risk to the subject);

[0154] 6. Subjects had good organ function at enrollment, and their laboratory test data met the following criteria:

[0155] • Complete blood count: Absolute neutrophil count ≥2.0×10⁻⁶ 9 / L (or greater than the lower limit of normal in the research center laboratory), platelet count ≥100×10 9 / L, hemoglobin ≥100g / L;

[0156] • Liver function: Serum total bilirubin ≤ 1.5 times ULN, AST and ALT ≤ 1.5 times ULN (if liver metastasis is present, AST and ALT ≤ 3 times ULN);

[0157] • Kidney function: CrCl ≥ 60 ml / min / 1.73 m 2(Calculated according to the Cockcroft-Gault formula);

[0158] 7. The researchers determined that the expected lifespan of the subjects was ≥12 weeks;

[0159] 8. Male or female participants of fertility must agree to use effective contraception during the study period and for 3 months after the last study dose, such as double barrier contraception, condoms, oral or injectable contraceptives, intrauterine devices, etc.

[0160] 9. Before the study begins, participants must provide written informed consent.

[0161] Evaluation criteria for target lesions

[0162]

[0163]

[0164] Dosage regimen:

[0165] The subject was given compound of formula (I) and fulvestrant.

[0166] The compound of formula (I) is administered orally at a dose of 360 mg twice daily. Each treatment cycle lasts 28 days.

[0167] The administration method for fulvestrant is as follows: Day 1 of the first cycle (C1D1), Day 15 of the first cycle (C1D15), and Day 1 of each subsequent cycle (CnD1): 500 mg intramuscular injection. Each treatment cycle consists of 28 days.

[0168] Experimental results:

[0169] Table 4 shows the tumor-suppressive effects of compound (I) and fulvestrant in combination on subjects.

[0170]

[0171] Experimental conclusion:

[0172] As can be seen from the data in Table 4, after 2-3 cycles of combined treatment in the first phase, the lesions of the subjects had already shrunk significantly. In some sensitive patients, the lesions shrank by as much as 50%. Two of the five subjects achieved partial response (PR) and the other two achieved stable disease (SD), demonstrating the significant benefits of combined treatment.

[0173] Experimental Example 4: Combination therapy of compound (I) of the present invention and fulvestrant for disease progression following prior endocrine therapy. Clinical trials of breast cancer patients

[0174] Test sample:

[0175] The compound of formula (I) of this invention is prepared according to prior art methods;

[0176] Fluvestrant: Purchase or prepare according to existing technical methods.

[0177] Inclusion criteria for the trial:

[0178] 1. Age 18 to 70 (inclusive);

[0179] 2. Laboratory results confirm that patients with hormone receptor-positive (HR+) and HER2-negative (HER2-) locally advanced, recurrent, or metastatic disease have progressed after receiving first-line endocrine therapy, and patients are allowed to receive no more than one chemotherapy regimen.

[0180] 3. Must have at least one measurable lesion that meets the definition of RECIST v1.1;

[0181] 4. ECOG score is 0-1;

[0182] 5. All acute toxicities from previous cancer treatment or surgery have resolved to baseline or ≤ grade 1 (NCI-CTCAE v4.03, excluding alopecia or other toxicities that the investigator considers to pose no safety risk to the subject);

[0183] 6. Subjects had good organ function and met the standards for laboratory test data at enrollment:

[0184] 7. The researchers determined that the expected lifespan of the subjects was ≥12 weeks;

[0185] 8. Male or female participants of fertility must agree to use effective contraception during the study period and for 3 months after the last study dose, such as double barrier contraception, condoms, oral or injectable contraceptives, intrauterine devices, etc.

[0186] 9. Before the study begins, participants must provide written informed consent.

[0187] Dosage regimen:

[0188] The subject was given compound of formula (I) and fulvestrant.

[0189] The compound of formula (I) is administered orally at a dose of 360 mg twice daily. Each treatment cycle lasts 28 days.

[0190] The administration method for fulvestrant is as follows: Day 1 of the first cycle (C1D1), Day 15 of the first cycle (C1D15), and Day 1 of each subsequent cycle (CnD1): 500 mg intramuscular injection. Each treatment cycle consists of 28 days.

[0191] Experimental results:

[0192] Table 5 shows the treatment response of compound (I) in combination with fulvestrant.

[0193]

[0194] Note: DCR = Disease Control Rate.

[0195] The compound of formula (I) of this invention, in combination with fulvestrant, has excellent therapeutic effects on breast cancer patients whose disease has progressed after prior endocrine therapy, with a disease control rate (DCR) of 92.3%, and the benefits of combination therapy are significant.

Claims

1. A pharmaceutical composition for treating cancer, said pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a second therapeutic agent or a pharmaceutically acceptable salt thereof. Equation (I), The second therapeutic agent is fulvestrant, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 5.2:1-2672:

1.

2. The pharmaceutical composition of claim 1, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 10:1 to 1200:

1.

3. The pharmaceutical composition of claim 1, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 10:1 to 600:

1.

4. The pharmaceutical composition of claim 1, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 10:1 to 200:

1.

5. The pharmaceutical composition of claim 1, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 20:1 to 150:

1.

6. The pharmaceutical composition of claim 1, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 20:1 to 120:

1.

7. The pharmaceutical composition of claim 1, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 20:1 to 100:

1.

8. The pharmaceutical composition of claim 1, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 40:1-50:1 or 80:1-90:

1.

9. The pharmaceutical composition of claim 1, wherein, The weight ratio of the active ingredient of formula (I) or its pharmaceutically acceptable salt to fulvestrant or its pharmaceutically acceptable salt is 107.52:1, 94.08:1, 80.64:1, 67.2:1, 62.72:1, 53.76:1, 47.04:1, 40.32:1, 33.6:1, 31.36:1, 26.88:1, 23.52: 1, 21.6:1, 20.16:1, 18:1, 16.8:1, 15.68:1, 14.4:1, 13.44:1, 12.6:1, 11.76:1, 115.2:1, 100.8:1, 86.4:1, 72:1, 57.6:1, 50.4:1, 43.2:1, 36:1, 28.8:1, 25.2:

1.

10. The use of the pharmaceutical composition according to any one of claims 1-9 in the preparation of a medicament for treating cancer, wherein, The cancer mentioned is breast cancer.

11. The application as described in claim 10, wherein, The cancer mentioned was selected from HR + HER2 - Breast cancer.

12. The application as described in claim 10, wherein, The cancer in question is locally advanced or metastatic breast cancer.

13. A medicine box comprising a pharmaceutical composition according to any one of claims 1-9, and instructions for use of the pharmaceutical composition, or a compound of formula (I) contained therein, and a second therapeutic agent.

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