Application of CYM50308 in preparation of antitumor drugs

Abstract

The invention provides an application of CYM50308 in preparation of an antitumor drug. In-vivo and in-vitro experimental studies prove that CYM50308 has a down-regulation effect on the expression level of immune checkpoint protein PD-L1 in various tumor cells, has remarkable in-vivo anti-tumor activity, and can be used for tumor immunotherapy by a small molecular compound which plays an anti-tumor role by intervening PD-L1 protein expression. Meanwhile, the expression of the PD-L1 protein can be regulated and controlled through CYM50308, and further related research can be carried out. The invention provides a small molecule compound for clinical tumor immunotherapy, and provides a new disease treatment application for CYM50308.

Description

Technical Field

[0001] The invention belongs to the pharmaceutical field and relates to the application of CYM50308 in the preparation of anti-tumor drugs. Background Art

[0002] The inhibition of T cell function caused by the binding of programmed death receptor-1 (PD-1) and its ligand (PD-L1) plays a key role in tumor immune escape. Blocking the interaction between PD-1 and PD-L1 can relieve immunosuppression and restore the body's anti-tumor immune response. Multiple PD-1 / PD-L1 monoclonal antibody drugs have been widely used in clinical tumor immunotherapy and have shown extensive and lasting anti-tumor activity in many solid tumor patients, significantly improving the survival and prognosis of tumor patients. However, due to the fact that monoclonal antibody drugs are prone to induce immune-related adverse reactions in clinical applications, have poor tissue permeability, and require intravenous administration, their clinical application has been greatly limited. It is still necessary to find new strategies to intervene in the PD-1 / PD-L1 axis to improve the current difficulties faced by monoclonal antibody drugs. In recent years, the development of PD-L1 small molecule inhibitors has become a research hotspot in this field. Compared with monoclonal antibody drugs, the advantages of small molecule inhibitors are reflected in the following aspects: 1) Strong tissue permeability, which can overcome the drug resistance problem caused by the difficulty of monoclonal antibody drugs to effectively penetrate into tumor tissues to a certain extent; 2) Low inherent immunogenicity and short half-life make adverse reactions during medication more controllable and are conducive to timely adjustment of medication strategies when adverse reactions occur; 3) Oral administration is often used, which improves patient compliance and is conducive to combined medication. Therefore, finding small molecule inhibitors targeting the PD-1 / PD-L1 axis may provide new intervention strategies for clinical tumor immunotherapy.

[0003] At present, the research and development of PD-L1 small molecule inhibitors mainly revolves around two aspects, namely: intervening the interaction between PD-1 and PD-L1 or regulating the expression of PD-L1 protein. As the crystal structure of the PD-1 / PD-L1 complex is gradually resolved and the PD-L1 expression regulation mechanism is gradually revealed, the research and development of PD-L1 small molecule inhibitors has developed rapidly, and a number of candidate small molecule compounds with potential anti-tumor activity have been screened. However, related compounds are still in the clinical and preclinical research stage, and no PD-L1 small molecule inhibitor has been approved for clinical tumor treatment. Therefore, the research and development of PD-L1 small molecule inhibitors has not made a breakthrough, and continued in-depth research is needed.

[0004] S1PR4 is a member of the G protein-coupled receptor family S1PRs and is mainly distributed in lymphocytes. Recent studies have shown that S1PR4 is expressed in tumor tissues such as lung cancer and is usually associated with poor prognosis in patients, but its role in tumor development is not clear. CYM50308 is a regulator of S1PR4 with high affinity and low intrinsic activity. It can inhibit S1PR4 downstream signals by specifically causing endocytic degradation of S1PR4 protein. At present, CYM50308 is only used as a tool drug for research related to the function of S1PR4 protein and has not yet been approved for clinical disease treatment. Summary of the invention

[0005] The purpose of the present invention is to provide the use of CYM50308 in the preparation of anti-tumor drugs, especially in the preparation of drugs for treating solid tumors. The chemical name of CYM50308 is (2Z, 5Z)-5-((1-(2,4-difluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)-2-((2-methoxyethyl)imino)-3-methylthiazolidine-4-one, and the molecular formula is C 20 H 21 F 2 N 3 O 2 S, molecular weight is 405.46.

[0006] The experiment confirmed that CYM50308 (10 μM) can significantly reduce the expression of PD-L1 in non-small cell lung cancer cells, liver cancer cells and breast cancer cells in vitro through Western blotting and flow cytometry. In addition, in the humanized immune system model, CYM50308 can effectively inhibit the growth of transplanted tumors and increase the infiltration of cytotoxic T lymphocytes in tumor tissues, which is similar to the positive control drug nivolumab (PD-1 monoclonal antibody).

[0007] The drug is prepared from CYM50308 and pharmaceutically acceptable excipients, and is in the form of an injection, which is taken once a day.

[0008] CYM50308 is a regulator of S1PR4 and has not yet entered the disease application stage. The present invention has proved through in vitro and in vivo experimental studies that CYM50308 has a down-regulating effect on the expression level of the immune checkpoint protein PD-L1 in various tumor cells, and can exert significant anti-tumor activity, thereby expanding the clinical application of the small molecule compound CYM50308 in tumor immunotherapy. The significance of the present invention is not only to clarify the possibility of CYM50308 for clinical solid tumor treatment, but also to provide new ideas and potential targets for the research and development of small molecule inhibitors that intervene in PD-L1 expression.

[0009] In the previous study, the inventors systematically screened the small molecule drug library and found that the S1PR4 regulator CYM50308 can significantly reduce the protein expression level of PD-L1 in various solid tumor cells and has significant in vivo anti-tumor activity. At present, there are no small molecule compounds that exert anti-tumor effects by intervening in PD-L1 protein expression for tumor immunotherapy in the clinic, and there is no related research on CYM50308 regulating PD-L1 protein expression. The present invention provides a small molecule compound for clinical tumor immunotherapy and provides a new disease use for CYM50308. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 CYM50308 can inhibit the total protein expression of PD-L1 in tumor cells in a concentration-dependent manner. It is a protein immunoblot image of CYM50308 (2.5μM, 5μM and 10μM) acting on non-small cell lung cancer cells H292.

[0011] Figure 2 CYM50308 can significantly inhibit the expression of PD-L1 protein on the surface of tumor cell membrane. CYM50308 (2.5μM, 5μM and 10μM) is a flow cytometry bar graph of non-small cell lung cancer cells H292.

[0012] Figure 3 CYM50308 can significantly inhibit the total protein expression of PD-L1 in liver cancer cells and breast cancer cells. It is the protein immunoblot image of CYM50308 (10 μM) acting on liver cancer cells Huh7 and breast cancer cells T47D.

[0013] Figure 4 CYM50308 can significantly promote the degradation of PD-L1 protein and shorten its half-life. It is an immunoblot image showing the effect of CYM50308 (10 μM) on the stability of PD-L1 protein in non-small cell lung cancer cells H292.

[0014] Figure 5 CYM50308 can increase the infiltration of cytotoxic T lymphocytes in tumor tissues and inhibit tumor growth. It is a statistical graph showing that CYM50308 (10 mg / kg) exerts significant anti-tumor activity by increasing the infiltration of cytotoxic T lymphocytes in tumor tissues in a humanized immune system mouse model. DETAILED DESCRIPTION

[0015] The present invention is further described in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0016] Example 1: CYM50308 significantly down-regulated the PD-L1 protein level in non-small cell lung cancer cells H292. The specific steps are as follows:

[0017] Non-small cell lung cancer H292 cells were selected and cultured at 1.0×10 6 The cells were seeded in 6-well plates at a density of 1.5 × 10 / well and incubated at 37°C and 5% CO 2 The cells were cultured overnight in an incubator; CYM50308 (2.5 μM, 5 μM and 10 μM) was administered for 24 h the next day, and the cells were collected and lysed, proteins were extracted, and Western blotting was used to detect the expression of PD-L1 in the cells. The inhibitory effect of CYM50308 on PD-L1 protein expression in non-small cell lung cancer H292 cells is shown in Figure 1 The experimental results showed that CYM50308 could inhibit the expression of PD-L1 protein in tumor cells in a concentration-dependent manner.

[0018] Example 2: CYM50308 significantly down-regulated the level of PD-L1 protein on the cell membrane surface after acting on non-small cell lung cancer cells H292. The specific steps are as follows:

[0019] Non-small cell lung cancer H292 cells were selected and cultured at 1.0×10 6 The cells were seeded in 6-well plates at a density of 1.5 × 10 / well and incubated at 37°C and 5% CO 2 The cells were cultured overnight in an incubator; CYM50308 (2.5 μM, 5 μM and 10 μM) was administered for 24 h the next day, and the cells were collected and flow cytometry was used to detect the expression of PD-L1 on the cell membrane surface. The inhibitory effect of CYM50308 on the expression of PD-L1 protein on the cell membrane of non-small cell lung cancer cells H292 is shown in Figure 2 The experimental results showed that CYM50308 could inhibit the expression level of PD-L1 on the surface of tumor cell membrane in a concentration-dependent manner.

[0020] Example 3: CYM50308 significantly down-regulated the PD-L1 protein level in liver cancer cells Huh7 and breast cancer cells T47D. The specific steps are as follows:

[0021] Hepatoma cell Huh7 and breast cancer cell T47D were selected and the cells were cultured at 1.0×10 6 The cells were seeded in 6-well plates at a density of 1.5 × 10 / well and incubated at 37°C with 5% CO. 2The cells were cultured overnight in an incubator; the next day, CYM50308 (10 μM) was given for 24 h under the conditions of IFN-γ (10 ng / mL) induction or non-induction, and the cells were collected and lysed, proteins were extracted, and Western blotting was used to detect the expression of PD-L1 in the cells. The inhibitory effect of CYM50308 on PD-L1 protein expression in liver cancer cells Huh7 and breast cancer cells T47D is shown in Figure 3 The experimental results showed that CYM50308 can significantly inhibit the expression of PD-L1 protein in tumor cells.

[0022] Example 4: Effect of CYM50308 on the stability of PD-L1 protein in non-small cell lung cancer cells H292. The specific steps are as follows:

[0023] Non-small cell lung cancer H292 cells were selected and cultured at 1.0×10 6 The cells were seeded in 6-well plates at a density of 1.5 × 10 / well and incubated at 37°C and 5% CO 2 The cells were cultured overnight in an incubator; the next day, the protein synthesis inhibitor cycloheximide (CHX, 10 μg / mL) or CHX (10 μg / mL) + CYM50308 (10 μM) was given for 0 h, 2 h, 4 h, 6 h, 8 h, and 12 h, and the cells were collected and lysed to extract proteins. Western blotting was used to detect the expression of PD-L1 protein in the cells. The experimental results were then statistically analyzed with the help of Image J software and statistical graphs were drawn. The effect of CYM50308 on the stability of PD-L1 protein in non-small cell lung cancer cells H292 is shown in Figure 4 The experimental results showed that CYM50308 can significantly promote the degradation of PD-L1 protein and shorten the half-life of PD-L1 protein.

[0024] Example 5: CYM50308 (10 mg / kg) exerts significant anti-tumor activity in a humanized immune system mouse model by increasing the infiltration of cytotoxic T lymphocytes in tumor tissues. The specific steps are as follows:

[0025] Peripheral blood mononuclear cells (PBMCs) were isolated from human blood samples using Ficoll separation medium. Freshly extracted PBMC cells (1.5×10 7 cells / mouse) were transfused back into the tail vein of NSG mice to establish a humanized immune system mouse model. Five days after the PBMC transfusion, H292 cells were transfused at a rate of 5×10 6 The amount of cells / mouse was subcutaneously inoculated into the armpit of NSG mice, and the tumor was grown to about 100 mm. 3The mice were then randomly divided into a control group, a CYM50308 (10 mg / kg) group, and a PD-1 monoclonal antibody group (5 mg / kg). The CYM50308 group was given 10 mg / kg CYM50308 via tail vein injection every day, and the PD-1 monoclonal antibody group was given 5 mg / kg nivolumab twice a week. A vernier caliper was used to measure the changes in tumor growth in mice every 2 days, and the tumor volume (TV) was calculated as 0.5× tumor long diameter × short diameter. 2 Calculation. At the end of the experiment, tumor tissues were removed and flow cytometry was used to detect CD8 + T cell infiltration and PD-L1 expression level. Figure 5 CYM50308 can significantly inhibit tumor growth, downregulate PD-L1 protein expression and increase CD8 + Infiltration of T cells.

Claims

1. A use of CYM50308 in the preparation of an anti-tumor drug, wherein the chemical name of CYM50308 is (2Z, 5Z)-5-((1-(2,4-difluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)-2-((2-methoxyethyl)imino)-3-methylthiazolidin-4-one, and the molecular formula is C 20 H 21 F2N3O2S, molecular weight 405.46, characterized in that Application in the preparation of drugs for treating solid tumors.

2. The use according to claim 1, characterized in that: The solid tumor is non-small cell lung cancer, liver cancer or breast cancer.

3. The use according to claim 1, characterized in that: The application is that CYM50308 significantly reduces the expression of PD-L1 in tumor cells, can effectively inhibit the growth of transplanted tumors and increase the infiltration of cytotoxic T lymphocytes in tumor tissues.

4. The use according to claim 1, characterized in that: The drug is prepared from CYM50308 and pharmaceutically acceptable excipients, and the preparation form is injection.

Images