A pharmaceutical composition and its use in the preparation of an antitumor drug

Abstract

The application provides a kind of pharmaceutical composition and its application in preparation anti-tumor drug.The composition is combined by everolimus and a small molecule conjugate, and the small molecule conjugate is BSJ-4-116 in English name.The small molecule conjugate BSJ-4-116 in the composition can significantly reduce CDK12 protein content, inhibit the proliferation of tumor cells.The in-vivo and in-vitro experimental researches prove that the pharmaceutical composition prepared by everolimus and small molecule conjugate BSJ-4-116 in proportion can significantly inhibit the proliferation of colorectal cancer in-vivo and in-vitro, and the anti-tumor effect is better than the effect of everolimus or small molecule conjugate BSJ-4-116 alone.The application provides a new strategy for anti-tumor of colorectal cancer.Lay a foundation for expanding the clinical indications of anti-tumor drug everolimus.Also provides a new medical use for everolimus and small molecule conjugate BSJ-4-116.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a pharmaceutical composition and application thereof in preparation of an antitumor drug, in particular to a pharmaceutical composition of everolimus and a small molecule conjugate BSJ-4-116 for treating tumors. BACKGROUND

[0002] Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for about 10% of all cancer cases. About 900,000 people die from CRC each year, and its mortality rate ranks second in the world in cancer-related deaths, seriously threatening human life and health. At present, the treatment method for colorectal cancer is mainly surgical resection, supplemented by comprehensive treatment of chemotherapy and radiotherapy, but its effect is limited in patients with advanced stage and distant metastasis. In recent years, although the rapid development of immunotherapy and molecular targeted therapy has brought a glimmer of hope to break through the treatment dilemma of colorectal cancer, but its efficacy is usually limited to a specific subgroup. For example, anti-PD-1 therapy is only effective for about 15% of colorectal cancer patients with microsatellite instability high (MSI-H) genotype. Therefore, the treatment means for colorectal cancer is still limited, and the treatment effect is still not satisfactory. The current situation, clinical needs to find new treatment methods and more effective treatment drugs to break through the existing treatment dilemma.

[0003] Everolimus is a highly effective and selective mTOR inhibitor. It forms a complex by combining with intracellular protein FK506 binding protein 12 (FKBP12) and combining with mTOR to inhibit its activity, thereby inhibiting the growth, differentiation and metabolism of tumor cells, and ultimately exerting an antitumor effect. At present, everolimus, as the only marketed oral mTOR inhibitor, has been approved for the treatment of advanced renal cell carcinoma (RCC), subependymal giant cell astrocytoma (SEGA) associated with tuberous sclerosis, renal angiomyolipoma (TSC-AML), advanced pancreatic neuroendocrine tumor (pNET), and postmenopausal estrogen receptor-positive / Her2-negative advanced breast cancer, and can also be used as an immunosuppressant to inhibit organ transplant rejection, and has a wide range of application scenarios. However, in clinical trials of colorectal cancer, everolimus alone or in combination with bevacizumab and linxiditing for the treatment of refractory metastatic colorectal cancer has not achieved the expected effect, which indicates that single treatment with everolimus or reliance on existing targeted drugs is difficult to effectively cope with the complexity of colorectal cancer. Therefore, a new combination therapy can be developed or an effective biomarker can be found to improve the efficacy of everolimus.

[0004] The small molecule conjugate BSJ-4-116 is the first reported proteolysis-targeting chimera (PROTAC) that can selectively degrade cyclin-dependent kinase 12 (CDK12). Its structure consists of three parts: (1) a warhead that specifically binds to the CDK12 protein; (2) an E3 ligand that binds and recruits CRBN; and (3) a linker that connects the two. Mechanistically, BSJ-4-116 specifically induces CDK12 degradation, causing DNA damage repair (DDR) genes to prematurely terminate transcription at intronic polyadenylic acid (PCPA) sites, reducing the expression of multiple DDR genes (such as FANCD2, FANCI, ATR, and BRCA1), thereby interfering with key processes such as DDR and transcriptional elongation in tumor cells, increasing genomic instability in tumor cells, and ultimately exerting anti-tumor activity. In addition, studies have shown that BSJ-4-116 also exhibits effective anti-tumor effects when used in combination with the poly (ADP-ribose) polymerase inhibitor Olaparib. Currently, research on the small molecule conjugate BSJ-4-116 is still in its early stages, but it has shown great therapeutic potential by targeting and degrading CDK12, especially when used in combination with other treatments, which may produce synergistic anti-tumor effects. Summary of the Invention

[0005] The object of the present invention is to address the deficiencies in the prior art and to provide a pharmaceutical composition comprising everolimus and a small molecule conjugate, wherein the molar ratio of everolimus to the small molecule conjugate is 1:1 to 10:1.

[0006] The everolimus has an English name of everolimus, a chemical name of (1R,9S,12R,15R,16E,18R,19R,21R,23S,24E,26,E,28E,30S,32R,35R)-1,18-dihydroxy-12-{(2R)-1-[(1R,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-2-propyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,3, a molecular formula of C53H83NO14, and a CAS number of 159351-69-6.

[0007] The small molecule conjugate is named BSJ-4-116 in English, and its chemical name is acetamide, N-[7-[(3R)-3-[[5-chloro-4-[[2-[(1-methylethyl)sulfonyl]phenyl]amino]-2-pyrimidinyl]amino]-1-piperidinyl]heptyl]-2-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-4-yl]oxy]-, with a molecular formula of C40H49ClN8O8S and a CAS number of 2519823-34-6.

[0008] Another object of the present invention is to provide the use of the drug combination in the preparation of anti-tumor drugs. During the preparation process, the pharmaceutical composition contains everolimus with an effective in vitro therapeutic concentration of 0.1-1 μmol / L, preferably 1 μmol / L, and an effective in vivo therapeutic concentration of 1-5 mg / kg body weight / day (NSG immunodeficient mice), preferably 2.5 mg / kg body weight / day (NSG immunodeficient mice); the small molecule conjugate contained has an effective in vitro therapeutic concentration of 0.05-0.1 μmol / L, preferably 0.1 μmol / L, and an effective in vivo therapeutic concentration of 1-5 mg / kg body weight / day (NSG immunodeficient mice), preferably 2.5 mg / kg body weight / day (NSG immunodeficient mice). The tumor is colorectal cancer.

[0009] The pharmaceutical composition is a compound preparation of everolimus and a small molecule conjugate. When administering the pharmaceutical composition to tumor cells, the raw materials of everolimus and BSJ-4-116 are dissolved in dimethyl sulfoxide (DMSO) at the specified molar ratio, mixed with cell culture medium to the specified final concentration, and incubated with the tumor cells. When administering the pharmaceutical composition to tumor-bearing mice, the raw materials of everolimus and BSJ-4-116 are dissolved in a mixed solvent (the mixed solvent formula is a volume ratio of dimethyl sulfoxide: celiac sulfone: normal saline = 1:1:8) at the specified molar ratio, and the drug mixture is then injected intraperitoneally into the tumor-bearing mice at the specified dosage based on the mouse weight for treatment.

[0010] Everolimus is an active site inhibitor of mTOR protein, which is approved by China's drug regulatory agency and the US Food and Drug Administration. It is currently mainly used in the treatment of advanced renal cell carcinoma, advanced neuroendocrine tumor, tuberous sclerosis complex associated renal angiomyolipoma and prevention of organ transplant rejection. Small molecule conjugates can be used to degrade cancer proteins in tumor cells. The small molecule conjugate BSJ-4-116 described in the present application can significantly reduce the content of CDK12 protein and inhibit the proliferation of tumor cells. The present application proves through in vivo and in vitro experimental research that the drug composition prepared by mixing everolimus and the small molecule conjugate BSJ-4-116 in proportion can significantly inhibit the proliferation of colorectal cancer in vivo and in vitro, and the anti-tumor effect is better than that of everolimus or the small molecule conjugate BSJ-4-116 alone. The significance of the present application lies not only in the invention of a new anti-tumor strategy for colorectal cancer, but also in laying a foundation for expanding the clinical indications of the anti-tumor drug everolimus. At present, there is no better drug combination method for treating colorectal cancer based on everolimus in clinical practice. The present application provides a new medical use of everolimus and the small molecule conjugate BSJ-4-116. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a heat map of SRB method for detecting cell proliferation. After 96h of co-treatment of colorectal cancer cell lines HCT-15 and HCT116 with everolimus and small molecule conjugate BSJ-4-116, the cell proliferation inhibition changes with the concentration. The degree of cell proliferation inhibition is directly represented by color intensity. The darker the color, the stronger the cell proliferation inhibition.

[0012] Figure 2 is a representative picture and statistical chart of cell clone formation experiment, which reflects the cell clone formation after 10 days of co-treatment of colorectal cancer cell lines HCT-15 and HCT116 with everolimus (1 μmol / L) and small molecule conjugate BSJ-4-116 (0.1 μmol / L) alone or in combination. The column chart shows the average value ± standard error from 3 independent experiments.

[0013] Figure 3 is an immunoblotting chart of apoptosis marker protein. After 72h of treatment of colorectal cancer cell lines HCT-15 and HCT116 with everolimus (1 μmol / L) and small molecule conjugate BSJ-4-116 (0.1 μmol / L), the changes of apoptosis marker proteins Cleaved-PARP and Cleaved-caspase3.

[0014] Figure 4Figure 1 is a growth curve plot of a xenograft tumor model of HCT-15 cell line on NSG mice. The change of tumor volume after mice transplanted tumor were treated with everolimus (5 mg / kg body weight) and small molecule conjugate BSJ-4-116 (5 mg / kg body weight). The drug was administered and the tumor volume was measured every other day. Five mice in each group, the broken line graph shows the average value ± standard error of tumor volume of 5 mice in each group. DETAILED DESCRIPTION

[0015] The present application is further illustrated in conjunction with the accompanying drawings and examples.

[0016] Example 1, the cell proliferation inhibition rate of colorectal cancer HCT-15, HCT116 cell lines after 24h treated with different concentrations and different molar ratios of everolimus and small molecule conjugate BSJ-4-116.

[0017] The specific steps are as follows: select colorectal cancer HCT-15, HCT116 cells and inoculate them in 96-well plates, with a cell density of 2000 / well. Incubate in a 37℃, 5% CO2 incubator overnight. Then prepare different concentrations and different molar ratios of everolimus and small molecule conjugate BSJ-4-116, and incubate with the above tumor cells for 96h. Discard the cell culture solution in the 96-well plate, add 70μL of 10% trichloroacetic acid solution to the 96-well plate and fix for 1h, then discard the supernatant and wash the 96-well plate with pure water for 5 times to wash away the residual trichloroacetic acid in the plate. Dry the 96-well plate at 60℃ for 1h, add 70μL of 0.4% sulforhodamine B (SRB) dye to the 96-well plate and stain for 0.5h, then discard the supernatant and wash the 96-well plate with 1% glacial acetic acid solution for 5 times to wash away the residual SRB dye in the plate. Dry the 96-well plate at 60℃ for 1h, add 100μL of 10mM Tris-base solution to the 96-well plate to dissolve, and measure the absorbance at 540nm wavelength with a microplate reader. The cell proliferation inhibition rate of different concentrations and different molar ratios of everolimus and small molecule conjugate BSJ-4-116 combination is shown in Figure 1 As shown in the table, the combination of everolimus and small molecule conjugate BSJ-4-116 can significantly inhibit the proliferation of colorectal cancer cell lines HCT-15, HCT116, and the inhibition effect is significantly better than that of each single drug. According to the results of the two tumor cells, when the concentration of everolimus is 1μmol / L and the concentration of small molecule conjugate is 0.1μmol / L, the anti-tumor effect of the drug combination is the best, and the molar ratio is 10:1.

[0018] Example 2, the cell clone formation of colorectal cancer HCT-15, HCT116 cell lines after 10 days of treatment with everolimus at a concentration of 1 μmol / L and small molecule conjugate BSJ-4-116 at a concentration of 0.1 μmol / L alone or in the form of a pharmaceutical composition.

[0019] The specific steps are as follows: colorectal cancer HCT-15, HCT116 cells are selected and inoculated in a 6-well plate at a cell density of 1000 / well, and cultured in a 37°C, 5% CO2 incubator overnight. Then, everolimus at a concentration of 1 μmol / L and small molecule conjugate BSJ-4-116 at a concentration of 0.1 μmol / L are prepared and incubated with the above tumor cells for 10 days. The cell culture solution in the 6-well plate is discarded, the 6-well plate is gently washed with PBS for 2 times, 1 mL of 10% trichloroacetic acid solution is added to the 6-well plate for 1 h of fixation, the supernatant is discarded, and the 6-well plate is washed with pure water for 5 times to wash away the residual trichloroacetic acid in the plate. The 96-well plate is dried at 60°C for 1 h, 1 mL of 0.4% sulforhodamine B (SRB) dye is added to the 6-well plate for 0.5 h of staining, the supernatant is discarded, and the 6-well plate is washed with 1% glacial acetic acid aqueous solution for 5 times to wash away the residual SRB dye in the plate. The 96-well plate is dried at 60°C for 1 h, the cell clones on the 6-well plate are photographed, and the clone number is counted using ImageJ software. The inhibition rate of cell clone formation is calculated according to the cell clone number, and the clone formation inhibition rate of everolimus at a concentration of 1 μmol / L and small molecule conjugate BSJ-4-116 at a concentration of 0.1 μmol / L is shown in Figure 2 The combination of everolimus and small molecule conjugate BSJ-4-116 can significantly inhibit the cell clone formation of colorectal cancer cell lines HCT-15, HCT116, and the inhibition effect is significantly better than that of each alone.

[0020] Example 3, the apoptosis of colorectal cancer HCT-15, HCT116 cell lines after 72 h of treatment with everolimus at a concentration of 1 μmol / L and small molecule conjugate BSJ-4-116 at a concentration of 0.1 μmol / L alone or in the form of a pharmaceutical composition.

[0021] The specific steps are as follows: select colorectal cancer HCT-15, HCT116 cells are inoculated in 6-hole plate, the cell density is 80000 / hole, and the cells are cultured in a 37°C, 5% CO2 incubator overnight. Then prepare 1 μmol / L of everolimus and 0.1 μmol / L of small molecule conjugate BSJ-4-116, and incubate with the tumor cells for 72 hours. Then collect the cells, lyse the cells with 4% SDS lysis buffer, use BCA quantitative kit to determine the total protein concentration of the cell lysate and prepare the sample buffer with a concentration of 50 mg / mL. Then the sample is subjected to SDS gel electrophoresis separation, and Western blot immunoblotting is performed with apoptosis marker protein antibodies Cleaved-PARP and Cleaved-Caspase3, and the internal reference protein antibody GAPDH is used as an internal reference. The results are shown in Figure 3 Fig. 4, the combination of everolimus and small molecule conjugate BSJ-4-116 can significantly induce apoptosis of colorectal cancer cell lines HCT-15 and HCT116, and the induction effect of apoptosis is significantly stronger than that of each single action.

[0022] Example 4, the dose of 2.5 mg / kg body weight / day of everolimus and the dose of 2.5 mg / kg body weight / day of small molecule conjugate BSJ-4-116 are used alone or prepared into a pharmaceutical composition to act on the tumor-bearing mice of colorectal cancer cell HCT-15 xenograft tumor, and the growth of the mouse xenograft tumor is observed.

[0023] The specific steps are as follows: select NSG immunodeficient mice from Shanghai Southern Model Company, 4 weeks old. The NSG mice are randomly divided into 5 groups, namely control group, everolimus group, small molecule conjugate BSJ-4-116 group, everolimus and small molecule conjugate BSJ-4-116 combination group, 5 mice in each group. Each mouse is subcutaneously inoculated with 10 6 cells of HCT-15 cell strain, and the tumor volume is measured every other day and recorded. When the tumor volume grows to 70 mm 3 , the control group is given normal saline, the everolimus group is given 5 mg / kg body weight of everolimus, the small molecule conjugate BSJ-4-116 group is given 5 mg / kg body weight of small molecule conjugate BSJ-4-116, and the everolimus and small molecule conjugate BSJ-4-116 combination group is given 5 mg / kg body weight of everolimus and 5 mg / kg body weight of BSJ-4-116, every other day. The experimental period is about 4 weeks. The results are shown in Figure 4 Fig. 6, the combination of everolimus and small molecule conjugate BSJ-4-116 can significantly inhibit the growth of HCT-15 mouse xenograft tumor, and the inhibition effect is significantly stronger than that of each single action.

Claims

1. A pharmaceutical composition, characterized in that The drug is composed of everolimus and a small molecule conjugate. The English name of everolimus is everolimus, and its CAS number is 159351-69-6. The English name of the small molecule conjugate is BSJ-4-116, and its CAS number is 2519823-34-6.

2. A pharmaceutical composition according to claim 1, characterized in that: When applied to tumor cells in vitro, the pharmaceutical composition consists of everolimus with a final concentration of 0.1-1 μmol / L and a small molecule conjugate with a final concentration of 0.05-0.1 μmol / L, wherein the molar ratio of everolimus to the small molecule conjugate is 1:1 to 10:1; when applied to solid tumors in vivo, the pharmaceutical composition consists of everolimus with a final concentration of 1-5 mg / kg body weight and a small molecule conjugate with a final concentration of 1-5 mg / kg body weight, wherein the molar ratio of everolimus to the small molecule conjugate is 1:

1.

3. Use of a pharmaceutical composition according to claim 1 or 2 in the preparation of an anti-tumor drug, characterized in that: The tumor is colorectal cancer.

4. The use according to claim 3, characterized in that The anti-tumor effect is that the composition significantly inhibits the proliferation of tumor cells in vitro and solid tumors in vivo.

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