Pharmaceutical composition and application thereof in preparation of antitumor drugs

Abstract

The invention provides a pharmaceutical composition and application thereof in preparation of antitumor drugs. The everolimus composition is formed by combining everolimus and a small molecule conjugate, and the English name of the small molecule conjugate is BSJ-4-116. The small molecule conjugate BSJ-4-116 in the composition can be used for remarkably reducing the content of CDK12 protein and inhibiting the proliferation of tumor cells. In-vivo and in-vitro experimental researches prove that the pharmaceutical composition prepared from the everolimus and the small molecule conjugate BSJ-4-116 according to the proportion can remarkably inhibit proliferation of colorectal cancer in vivo and in vitro, and the anti-tumor effect of the pharmaceutical composition is superior to that of single action of the everolimus or the small molecule conjugate BSJ-4-116. The invention provides a new strategy for resisting tumors of colorectal cancer. And a foundation is laid for expanding clinical indications of the antitumor drug everolimus. The invention also provides a new medical application for the everolimus and the small molecule conjugate BSJ-4-116.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to a pharmaceutical composition and its application in the preparation of anti-tumor drugs, and in particular to a pharmaceutical composition of everolimus and a small molecule conjugate BSJ-4-116 for treating tumors. Background Art

[0002] Colorectal cancer (CRC) is the third most common cancer in the world, accounting for about 10% of all cancer cases. About 900,000 people die from CRC each year, and its mortality rate ranks second among cancer-related deaths worldwide, seriously threatening human life and health. At present, the treatment method for colorectal cancer is mainly surgical resection, supplemented by chemotherapy and radiotherapy, but its efficacy is limited in patients with advanced and distant metastasis. In recent years, although the rapid development of immunotherapy and molecular targeted therapy has brought a ray of hope to break the treatment dilemma of colorectal cancer, its efficacy is usually limited to specific subgroups. For example, anti-PD-1 therapy is only effective for about 15% of colorectal cancer patients with microsatellite instability-high (MSI-H) genotype. Therefore, in view of the current situation that the treatment methods for colorectal cancer are still limited and the treatment effect is still unsatisfactory, the clinic urgently needs to find new treatment methods and more effective therapeutic drugs to break through the existing treatment dilemma.

[0003] Everolimus is a highly effective and selective mTOR inhibitor. It binds to the intracellular protein FK506 binding protein 12 (FKBP12) to form a complex and binds to mTOR to inhibit its activity, thereby inhibiting the growth, differentiation and metabolism of tumor cells, and ultimately exerting an anti-tumor effect. At present, as the only oral mTOR inhibitor on the market, everolimus has been approved for the treatment of advanced renal cell carcinoma (RCC), tuberous sclerosis complex-associated subependymal giant cell astrocytoma (SEGA), renal angiomyolipoma (TSC-AML) tumor, advanced pancreatic neuroendocrine tumor (pNET) and postmenopausal estrogen receptor-positive / Her2-negative advanced breast cancer. It 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, most of the cases of everolimus alone or in combination with bevacizumab or lincitinib for the treatment of refractory metastatic colorectal cancer did not achieve the expected therapeutic effect, which shows that everolimus alone or relying on existing targeted drugs is difficult to effectively deal with the complexity of colorectal cancer. Therefore, a new combination therapy or effective biomarkers can be developed 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 to 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 terminate transcription prematurely 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 the genomic instability of 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. At present, the research on the small molecule conjugate BSJ-4-116 is still in its early stages, but it has shown great therapeutic potential in the mechanism of targeting and degrading CDK12, especially when used in combination with other treatments, it may produce a synergistic anti-tumor effect. Summary of the invention

[0005] The purpose of the present invention is to provide a pharmaceutical composition in view of the deficiencies in the prior art. The pharmaceutical composition is composed of 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 has an English name of BSJ-4-116, a chemical name of 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]-, 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 form of the pharmaceutical composition is a compound preparation of everolimus and a small molecule conjugate. When the pharmaceutical composition is applied to tumor cells, the raw materials of everolimus and BSJ-4-116 are dissolved in dimethyl sulfoxide (DMSO) at the molar ratio, and then mixed with the cell culture fluid to the final concentration and co-incubated with the tumor cells. When the pharmaceutical composition is applied to tumor-bearing mice, the raw materials of everolimus and BSJ-4-116 are dissolved in a mixed solvent (the mixed solvent formula is dimethyl sulfoxide: celiac sulfone: normal saline = 1:1:8 according to the volume ratio) at the molar ratio, and then the drug mixture is injected into the tumor-bearing mice through the abdominal cavity for treatment according to the dosage according to the weight of the mice.

[0010] Everolimus is an active site inhibitor of mTOR protein. It is a drug approved for marketing by the China Food and Drug Administration and the U.S. Food and Drug Administration. It is currently mainly used in the clinic to treat advanced renal cell carcinoma, advanced neuroendocrine tumors, renal angiomyolipomas associated with tuberous sclerosis, and to prevent organ transplant rejection. Small molecule conjugates can be used to degrade oncoproteins in tumor cells. The small molecule conjugate BSJ-4-116 described in the present invention can significantly reduce the content of CDK12 protein and inhibit the proliferation of tumor cells. The present invention has been proved by in vitro and in vivo experimental studies that the pharmaceutical composition prepared by 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 acting alone. The significance of the present invention is not only to invent a new anti-tumor strategy for colorectal cancer, but also to lay the foundation for expanding the clinical indications of the anti-tumor drug everolimus. Currently, there is no good everolimus-based drug combination method for treating colorectal cancer in clinic. The present invention provides a new medical use for everolimus and the small molecule conjugate BSJ-4-116. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0012] Figure 2 Representative images and statistical graphs of cell clone formation experiments, reflecting the cell clone formation of colorectal cancer cell lines HCT-15 and HCT116 after 10 days of treatment with everolimus (1μmol / L) and small molecule conjugate BSJ-4-116 (0.1μmol / L) alone or in combination. The bar graphs show the mean ± standard error from 3 independent experiments.

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

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

[0015] The present invention is further described in conjunction with the accompanying drawings and embodiments.

[0016] Example 1: The cell proliferation inhibition rate of colorectal cancer HCT-15 and HCT116 cell lines after 24 hours of co-action of everolimus at different concentrations and molar ratios with the small molecule conjugate BSJ-4-116.

[0017] The specific steps are as follows: select colorectal cancer HCT-15 and HCT116 cells and inoculate them in a 96-well plate with a cell density of 2000 / well, and culture them in a 37°C, 5% CO2 incubator overnight. Subsequently, different concentrations and molar ratios of everolimus and small molecule conjugate BSJ-4-116 were prepared and incubated with the above tumor cells for 96 hours. The cell culture medium in the 96-well plate was discarded, 70 μL of 10% trichloroacetic acid solution was added to the 96-well plate for 1 hour, and the supernatant was discarded, and the 96-well plate was washed 5 times with pure water to wash away the residual trichloroacetic acid in the well plate. The 96-well plate was dried at 60°C for 1 hour, 70 μL of 0.4% sulforhodamine B (SRB) dye was added to the 96-well plate for staining for 0.5 hours, and the supernatant was discarded, and the 96-well plate was washed 5 times with 1% glacial acetic acid aqueous solution to wash away the residual SRB dye in the well plate. The 96-well plate was dried at 60°C for 1 h, 100 μL of 10 mM Tris-base solution was added to the 96-well plate to dissolve, and the absorbance at a wavelength of 540 nm was measured using an ELISA reader. The cell proliferation inhibition rate of the combination of everolimus and the small molecule conjugate BSJ-4-116 at different concentrations and different molar ratios is shown in Figure 1 As shown, the combination of everolimus and the small molecule conjugate BSJ-4-116 can significantly inhibit the proliferation of colorectal cancer cell lines HCT-15 and HCT116, and the inhibitory effect is significantly better than their respective effects alone. Based on the results of the two tumor cells, when the concentration of everolimus is 1μmol / L and the concentration of the small molecule conjugate is 0.1μmol / L, the anti-tumor effect of the drug combination is the best, with a molar ratio of 10:1.

[0018] Example 2: Everolimus at a concentration of 1 μmol / L and the small molecule conjugate BSJ-4-116 at a concentration of 0.1 μmol / L act alone or as a pharmaceutical composition and act on colorectal cancer HCT-15 and HCT116 cell lines for 10 days to determine the cell clone formation.

[0019] The specific steps are as follows: select colorectal cancer HCT-15 and HCT116 cells and inoculate them in 6-well plates with a cell density of 1000 / well, and culture them in a 37°C, 5% CO2 incubator overnight. Then, prepare everolimus at a concentration of 1 μmol / L and a small molecule conjugate BSJ-4-116 at a concentration of 0.1 μmol / L, and incubate them with the above tumor cells for 10 days. Discard the cell culture medium in the 6-well plate, gently wash the 6-well plate with PBS twice, add 1 mL of 10% trichloroacetic acid solution to the 6-well plate and fix it for 1 hour, then discard the supernatant, and wash the 6-well plate 5 times with pure water to wash away the residual trichloroacetic acid in the well plate. The 96-well plate was dried at 60°C for 1 hour, 1 mL of 0.4% sulforhodamine B (SRB) dye was added to the 6-well plate and stained for 0.5 hours, then the supernatant was discarded and the 6-well plate was washed 5 times with 1% glacial acetic acid aqueous solution to wash away the residual SRB dye in the plate. The 96-well plate was dried at 60°C for 1 hour, the cell clones on the 6-well plate were photographed, and the number of clones was counted using ImageJ software. The inhibition rate of cell clone formation was calculated based on the number of cell clones. The cloning inhibition rate of 1 μmol / L everolimus and 0.1 μmol / L small molecule conjugate BSJ-4-116 is shown in the figure. Figure 2 As shown, the combination of everolimus and the small molecule conjugate BSJ-4-116 can significantly inhibit the cell clone formation of colorectal cancer cell lines HCT-15 and HCT116, and the inhibitory effect is significantly better than that of each of them alone.

[0020] Example 3: Cell apoptosis after 1 μmol / L of everolimus and 0.1 μmol / L of the small molecule conjugate BSJ-4-116 were used alone or as a pharmaceutical composition on colorectal cancer HCT-15 and HCT116 cell lines for 72 hours.

[0021] The specific steps are as follows: Select colorectal cancer HCT-15 and HCT116 cells and inoculate them in a 6-well plate with a cell density of 80,000 / well, and culture them in a 37°C, 5% CO2 incubator overnight. Then, everolimus at a concentration of 1 μmol / L and the small molecule conjugate BSJ-4-116 at a concentration of 0.1 μmol / L were prepared respectively and incubated with the above tumor cells for 72 hours. The cells were then collected and lysed with 4% SDS lysis buffer. The total protein concentration of the cell lysate was determined using the BCA quantitative kit and prepared into a loading buffer with a concentration of 50 mg / mL. The samples were then separated by SDS gel electrophoresis, and Western blot was performed using the cell apoptosis marker protein antibodies Cleaved-PARP and Cleaved-Caspase3, and the internal reference protein antibody GAPDH was used as the internal reference. The results are as follows Figure 3 As shown, the combination of everolimus and the small molecule conjugate BSJ-4-116 can significantly induce apoptosis in colorectal cancer cell lines HCT-15 and HCT116, and the apoptosis induction effect is significantly stronger than that of each of them alone.

[0022] Example 4: Everolimus at a dose of 2.5 mg / kg body weight / day and the small molecule conjugate BSJ-4-116 at a dose of 2.5 mg / kg body weight / day were used alone or prepared as a pharmaceutical composition and acted on colorectal cancer cell HCT-15 xenograft tumor-bearing mice to study the growth of mouse xenograft tumors.

[0023] The specific steps are as follows: NSG immunodeficient mice from Shanghai Nanfang Model Company, 4 weeks old, were selected. The NSG mice were randomly divided into 5 groups, namely control group, everolimus group, small molecule conjugate BSJ-4-116 group, and everolimus and small molecule conjugate BSJ-4-116 combination group, with 5 mice in each group. Each mouse was subcutaneously inoculated with HCT-15 cell line 10 6 The tumor volume was measured and recorded every other day. 3 The control group was given normal saline, the everolimus group was given 5 mg / kg body weight of everolimus, the small molecule conjugate BSJ-4-116 group was 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 was given 5 mg / kg body weight of everolimus and 5 mg / kg body weight of BSJ-4-116, and the administration was done every other day. The experimental period was about 4 weeks. Figure 4 As shown, the combination of everolimus and the small molecule conjugate BSJ-4-116 can significantly inhibit the growth of HCT-15 mouse xenograft tumors, and the inhibitory effect is significantly stronger than the effect of each alone.

Claims

1. A pharmaceutical composition, characterized in that It 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 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 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.

Images