Pharmaceutical composition for treating cancer and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating cancer, and belongs to the technical field of biological medicine, the pharmaceutical composition is composed of a pharmaceutical active component and a pharmaceutically acceptable carrier, and the pharmaceutical active component is composed of a PARP1 / 2 inhibitor and an IDO1 inhibitor. A PARP1 / 2 inhibitor and an IDO1 inhibitor have the limitations of narrow indications, easy generation of drug resistance and the like when being independently used for treating tumors, and the two inhibitors are combined together according to a specific ratio, so that a very strong anti-tumor effect can be obtained, and a relatively good synergistic interaction effect is shown. Particularly, the pharmaceutical composition has a better effect on inhibiting the growth of tumors such as triple negative breast cancer and pancreatic cancer than a single drug, and a new way is provided for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition for treating cancer and application thereof. Background Art

[0002] Cancer is one of the diseases that poses the greatest threat to human life and health, and a large number of people die from cancer every year. Among them, the incidence of breast cancer is very high, and it is one of the most common cancers threatening women worldwide. Triple-negative breast cancer is a highly heterogeneous tumor, accounting for 15%-20% of breast cancer pathological subtypes. Because it does not express estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2, triple-negative breast cancer cannot be targeted by endocrine therapy and is a difficult-to-treat breast cancer subtype. Although cytotoxic drugs such as paclitaxel and cisplatin are effective in treating early triple-negative breast cancer, the effect of treating late triple-negative breast cancer is poor. Pancreatic cancer is also one of the most malignant digestive tract tumors, with an overall 5-year survival rate of less than 8%. Although many treatment options have been proposed, the prognosis of pancreatic cancer patients has not been significantly improved. Therefore, there is an urgent need to find new strategies to treat a variety of cancers, including triple-negative breast cancer and pancreatic cancer.

[0003] PARPs are signaling molecules and transduction proteins for DNA damage response, and are abnormally expressed in a variety of tumors. + As a substrate, PARP1 and PARP2 regulate various cellular responses by transferring one or more ADP ribose units to receptor proteins or themselves. PARP1 and PARP2, which catalyze poly-ADP ribosylation, have been studied in depth and have been shown to be involved in regulating the base excision repair pathway of single-strand DNA damage. Genes such as BRCA1 / 2 and Rad51 regulate the precise repair of damaged DNA double strands through pathways such as homologous recombination (HR). BRCA1 / 2 is prone to mutation, and mutated BRCA1 / 2 blocks the homologous recombination repair pathway, leading to chromosomal instability in cells. BRCA1 / 2 mutations exist in a variety of cancer tissues, including triple-negative breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer. Inhibiting the expression and activity of PARP1 / 2 in such cancer cells can lead to the inability of damaged DNA in cells to be repaired by the HR pathway and cause death. Based on the theory of "synthetic lethality", multiple PARP1 / 2 inhibitors represented by Olaparib have been launched on the market and are widely used to treat various cancers with HR repair defects. However, several PARP1 / 2 inhibitors, including olaparib, have limitations such as narrow indications and easy development of drug resistance.

[0004] IDO1 belongs to the indoleamine-2,3-dioxygenase family and is a type of oxygenase responsible for catalyzing the metabolism of tryptophan into kynurenine and NAD+. IDO1 regulates tumor immune escape, DNA damage and repair by catalyzing the degradation of tryptophan into kynurenine and NAD+, and plays an important regulatory role in inflammation, tumors and autoimmune diseases. IDO1 has been shown to be overexpressed in a variety of tumors, including melanoma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, ovarian cancer and gastric cancer, and is closely involved in the malignant proliferation, invasion and metastasis of tumors, and resistance to chemotherapeutic drugs. Epacadostat (1-(2-amino-4-chloro-5-methylphenyl)-3-(2-fluorophenyl)urea, CAS No.: 1204669-58-8) is the IDO1 inhibitor with the most clinical trials currently. It can be used to treat a variety of cancers. However, when it is used alone to treat cancer, it has weak anti-tumor effects, narrow indications, and drug resistance. In April 2018, the Phase III clinical trial of Epacadostat combined with PD-1 monoclonal antibody Pembrolizumab for the treatment of metastatic melanoma failed, and the FDA still has no IDO1 inhibitor approved for marketing. Summary of the invention

[0005] The purpose of the present invention is to solve the above-mentioned deficiencies in the prior art and provide a pharmaceutical composition for treating cancer.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] A pharmaceutical composition for treating cancer, consisting of a pharmaceutically active ingredient and a pharmaceutically acceptable carrier, wherein the pharmaceutically active ingredient consists of a PARP1 / 2 inhibitor and an IDO1 inhibitor.

[0008] Further, the PARP1 / 2 inhibitor is Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, Veliparib or a pharmaceutically acceptable salt thereof, more preferably Olaparib.

[0009] Furthermore, the IDO1 inhibitor is Epacadostat (chemical name: 1-(2-amino-4-chloro-5-methylphenyl)-3-(2-fluorophenyl)urea) or a pharmaceutically acceptable salt thereof.

[0010] Furthermore, in the pharmaceutical composition, the single dose of the PARP1 / 2 inhibitor is 0.1 to 50 mg / kg, and the single dose of the IDO1 inhibitor is 0.1 to 50 mg / kg.

[0011] Furthermore, the pharmaceutical composition is in the form of capsules, tablets, pills, granules or liquid preparations.

[0012] Application of the above pharmaceutical composition in the preparation of drugs for treating cancer.

[0013] Furthermore, the cancer is triple-negative breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, prostate cancer, pancreatic cancer or colon cancer.

[0014] Beneficial effects of the present invention:

[0015] The present invention provides a pharmaceutical composition for treating cancer, wherein the pharmaceutical active ingredients consist of a PARP1 / 2 inhibitor and an IDO1 inhibitor. Pharmacodynamic experiments have shown that the combined use of the PARP1 / 2 inhibitor and the IDO1 inhibitor has a synergistic effect in therapeutic effect, and the therapeutic effect is better than the use of the PARP1 / 2 inhibitor alone or the use of the IDO1 inhibitor alone. The pharmaceutical composition of the present invention is suitable for treating triple-negative breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, prostate cancer, pancreatic cancer or colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of the proliferation test of SW1990 cells after treatment with Olaparib, Epacadostat, and Olaparib and Epacadostat in combination;

[0017] Figure 2 The results of proliferation test of MDA-MB-231 cells after treatment with Olaparib, Epacadostat, and Olaparib and Epacadostat in combination;

[0018] Figure 3 The results of cell clone number detection of SW1990 cells and MDA-MB-231 cells after treatment with Olaparib, Epacadostat and Olaparib + Epacadostat;

[0019] Figure 4 The results of apoptosis test of SW1990 cells and MDA-MB-231 cells after treatment with Olaparib, Epacadostat and Olaparib + Epacadostat;

[0020] Figure 5 These are photos of tumors in mice after different drug treatments;

[0021] Figure 6 These are the test results of tumor volume and tumor weight in mice after different drug treatments. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below through the accompanying drawings and specific embodiments, but it should not be understood that the above subject matter of the present invention is limited to the following embodiments. All technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0023] It should be noted that in the following examples, the PARP1 / 2 inhibitor used is Olaparib, and the IDO1 inhibitor used is Epacadostat. Other PARP1 / 2 inhibitors or pharmaceutically acceptable salts thereof and IDO1 inhibitors or pharmaceutically acceptable salts thereof are used in combination to have the same therapeutic effect.

[0024] In the following examples, the triple-negative breast cancer cell line MDA-MB-231 and the pancreatic cancer cell line SW1990 used were purchased from the National Biomedical Experimental Cell Resource Bank; L15 culture medium was purchased from Jiangsu KeyGen Biotechnology Co., Ltd., which is a culture medium containing double antibodies (penicillin and streptomycin); Epacadostat was purchased from MedChemExpress); Olaparib was purchased from Selleck; apoptosis detection kit was purchased from KeyGEN; and crystal violet staining solution was purchased from KeyGEN.

[0025] Example 1 PARP1 / 2 inhibitor combined with IDO1 inhibitor inhibits the growth of triple-negative breast cancer and pancreatic cancer cells in vitro

[0026] 1. Experimental Methods

[0027] (1) MTT assay for cell proliferation

[0028] The experiment set up a DMSO control group, an olaparib experimental group, an epacadostat experimental group, and an olaparib + epacadostat combined treatment experimental group, respectively treating SW1990 and MDA-MB-231 two tumor cells, in the three experimental groups, the drug concentrations of olaparib and epacadostat all started at 50μM, and were diluted down by 2 times to 8 gradient concentrations for treatment, among which, in the epacadostat combined treatment experimental group, olaparib and epacadostat were co-administered at a molar ratio of 1:1 (such as 50μM:50μM), 2:1 (such as 50μM:25μM), and 4:1 (such as 50μM:12.5μM), and were also diluted down by 2 times to 8 gradient concentrations for treatment. After 7 days, MTT was used to detect cell proliferation.

[0029] MTT method for detecting cell proliferation:

[0030] The cells were seeded at a density of 3000-5000 cells / well in a 96-well plate, cultured at 5% CO2, 37°C for 24 hours, treated with the corresponding drugs for 7 days, added with thiazolyl blue dye (MTT) and incubated for 3-4 hours, the culture medium was discarded, DMSO was added to fully dissolve and mix, and the cell proliferation was detected. Cell growth inhibition rate = (1-absorbance value of the experimental group / absorbance value of the control group) × 100%.

[0031] (2) Clone formation assay to detect cell number

[0032] SW1990 cells and MDA-MB-231 cells were seeded in 24-well plates at 800-2000 cells / well, cultured overnight to allow the cells to adhere to the wall, and then treated with corresponding drugs according to the grouping:

[0033] The experiment set up a DMSO control group, an Olaparib experimental group, an Epacadostat experimental group, and an Olaparib + Epacadostat combined treatment group. When treating MDA-MB-231 cells, the dosage of Olaparib was 5.0 μM, the dosage of Epacadostat was 5.0 μM, and the combined treatment group was treated with Olaparib 5.0 μM + Epacadostat 5.0 μM; when treating SW1990 cells, the dosage of Olaparib was 4.0 μM, the dosage of Epacadostat was 4.0 μM, and the combined treatment group was treated with Olaparib 4.0 μM + Epacadostat 4.0 μM.

[0034] After 10 days of treatment, the cells were fixed with 4% paraformaldehyde for 15 min, stained with crystal violet for 10 min, and observed and photographed with an inverted fluorescence microscope.

[0035] (3) Observation of cell apoptosis

[0036] SW1990 cells and MDA-MB-231 cells were seeded in 35 mm culture dishes at a density of 30%-50% per dish, cultured overnight to allow the cells to adhere to the wall, and then treated with corresponding drugs according to the grouping:

[0037] The experiment set up a DMSO control group, an Olaparib experimental group, an Epacadostat experimental group, and an Olaparib + Epacadostat combined treatment group. When treating MDA-MB-231 cells, the dosage of Olaparib was 5.0 μM, the dosage of Epacadostat was 5.0 μM, and the combined treatment group was treated with Olaparib 5.0 μM + Epacadostat 5.0 μM; when treating SW1990 cells, the dosage of Olaparib was 4.0 μM, the dosage of Epacadostat was 4.0 μM, and the combined treatment group was treated with Olaparib 4.0 μM + Epacadostat 4.0 μM.

[0038] After 7 days of treatment, the cells were digested with trypsin and washed twice with PBS. The cells were then stained with propidium iodide and annexin V-FITC, and apoptosis was analyzed using a Miltenyi MACSQuant flow cytometer.

[0039] 2. Experimental results

[0040] (1) MTT was used to detect cell proliferation, and the results were used to calculate the synergistic index (CI) using the Chou-Talalay formula, where CI < 1.0 indicated a synergistic effect, CI = 1.0 indicated only an additive effect, and CI > 1.0 indicated an antagonistic effect.

[0041] Figure 1 These are the test results of the proliferation of SW1990 cells after treatment with Olaparib, Epacadostat, and the combination of Olaparib and Epacadostat. Figure 1 A is the test result of the proliferation of SW1990 cells after treatment. Figure 1 B is the calculation result of the synergy index; Figure 2 These are the test results of proliferation of MDA-MB-231 cells after treatment with Olaparib, Epacadostat, and Olaparib and Epacadostat in combination. Figure 2 A is the test result of the proliferation of MDA-MB-231 cells after treatment. Figure 2 B is the result of synergy index calculation. Figure 1 and Figure 2 It can be seen that when the combined molar ratio of Epacadostat and Olaparib is in the range of 1:(1-4), the CI values ​​in both tumor cells are less than 1, indicating that the combination of Olaparib and Epacadostat can synergistically inhibit the growth of tumor cells within the measured molar ratio range.

[0042] (2) Cell clone number test results

[0043] Figure 3 The results of cell clone number detection of SW1990 cells and MDA-MB-231 cells after treatment with olaparib, epacadostat and olaparib + epacadostat show that after olaparib and epacadostat were used in combination at a molar ratio of 1:1 to treat triple-negative breast cancer cells MDA-MB-231 and pancreatic cancer cells SW1990, the cell clone number was significantly less than that of the group using olaparib or epacadostat alone. This further verifies the long-term effect of olaparib combined with epacadostat on tumor cell survival.

[0044] (3) Cell apoptosis test results

[0045] Figure 4 The results of the apoptosis test of SW1990 cells and MDA-MB-231 cells after treatment with olaparib, Epacadostat and olaparib + Epacadostat show that compared with the single-drug group, the olaparib + Epacadostat (molar ratio 1:1) combined treatment group can significantly promote the apoptosis of MDA-MB-231 cells and SW1990 cells. The experimental results show that the combination of olaparib and Epacadostat has an excellent synergistic anti-tumor effect.

[0046] Example 2 Olaparib combined with Epacadostat inhibits the growth of triple-negative breast cancer xenografts in vivo

[0047] (1) Test drug

[0048] PARP1 / 2 inhibitor: Olaparib; IDO1 inhibitor: Epacadostat

[0049] Preparation method: Olaparib is prepared with 10% DMSO and 10% β-cyclodextrin; Epacadostat is prepared with 10% DMSO, 10% Tween and 10% β-cyclodextrin.

[0050] (2) Experimental animals

[0051] BALB / cA-nude mice, 6-8 weeks old, female. Housing environment: SPF grade.

[0052] (3) Experimental procedures

[0053] Nude mice (from Hangzhou Medical College, 20 mice) were subcutaneously inoculated with human triple-negative breast cancer MDA-MB-231 cells and the tumors were grown to 100-300 mm 3 Afterwards, the animals were randomly divided into the model group (control), olaparib group, epacadostat group, and olaparib + epacadostat combined administration group. The olaparib group was intraperitoneally injected with olaparib (50 mg / kg) once a day; the epacadostat group was intraperitoneally injected with epacadostat (50 mg / kg) once a day; the combined administration group was intraperitoneally injected with olaparib (25 mg / kg) and epacadostat (25 mg / kg) (the molar ratio of olaparib to epacadostat was 1:1) once a day. The transplanted tumor volume was measured every two days, the mouse weight was weighed, the data was recorded, and the animals were killed on the 28th day.

[0054] (4) Experimental results

[0055] Figure 5These are photos of tumors in mice after different drug treatments. Figure 6 The results of the tumor volume and tumor weight test in mice after different drug treatments, among which, Figure 6 A is the tumor volume test result, Figure 6 B is the tumor weight test result, and the results show that compared with the model group, the tumor growth of the single-drug group and the Epacadostat group was significantly inhibited. Compared with the single-drug group and the model group, the tumor in the combination group (molar ratio 1:1) was significantly reduced in weight and volume. The inhibitory effect of the combination group on triple-negative breast cancer cell MDA-MB-231 xenograft tumors was stronger than that of the single-drug group or the Epacadostat group, and the difference was significant, indicating that the combination of Olaparib and Epacadostat can exert a better anti-tumor effect in vivo.

[0056] In summary, the combined drug disclosed in the present invention can significantly inhibit the growth of tumor cells in vivo and in vitro, and has good prospects for clinical application.

Claims

1. A pharmaceutical composition for treating cancer, characterized in that: The pharmaceutical composition consists of a pharmaceutically active ingredient and a pharmaceutically acceptable carrier, and the pharmaceutically active ingredient consists of a PARP1 / 2 inhibitor and an IDO1 inhibitor.

2. The pharmaceutical composition for treating cancer according to claim 1, characterized in that The PARP1 / 2 inhibitor is olaparib, rucaparib, niraparib, talazoparib, fluzoparib, pamiparib, velaparib or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition for treating cancer according to claim 2, characterized in that The PARP1 / 2 inhibitor is olaparib.

4. The pharmaceutical composition for treating cancer according to claim 1, characterized in that The IDO1 inhibitor is Epacadostat or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition for treating cancer according to claim 1, characterized in that In the pharmaceutical composition, the single dosage of the PARP1 / 2 inhibitor is 0.1 to 50 mg / kg, and the single dosage of the IDO1 inhibitor is 0.1 to 50 mg / kg.

6. The pharmaceutical composition for treating cancer according to any one of claims 1 to 5, characterized in that: The dosage form of the pharmaceutical composition is capsule, tablet, pill, granule or liquid preparation.

7. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of drugs for treating cancer.

8. The use according to claim 7, characterized in that The cancer is triple-negative breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, prostate cancer, pancreatic cancer or colon cancer.

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