Use of CYM50308 in preparation of anti-tumor drugs

Abstract

The application provides application of CYM50308 in preparation of an antitumor drug. Through in-vivo and in-vitro experimental researches, it is proved that CYM50308 has a down-regulation effect on the expression level of immune checkpoint protein PD-L1 in various tumor cells, has significant in-vivo antitumor activity, and can be used for tumor immunotherapy through a small-molecule compound which can play an antitumor role by interfering with the expression of PD-L1 protein, and further related researches can be carried out through regulation of the expression of PD-L1 protein by CYM50308. The application provides a small-molecule compound for clinical tumor immunotherapy, and provides a new disease treatment purpose for CYM50308.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and relates to application of CYM50308 in preparation of an antitumor drug. BACKGROUND

[0002] The T cell function inhibition caused by the combination of programmed death-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 immune suppression 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 durable anti-tumor activity in many solid tumor patients, significantly improving the survival and prognosis of tumor patients. However, due to the problems such as the immunorelated adverse reactions easily caused by monoclonal antibody drugs in clinical application, poor tissue penetration and the need for intravenous injection, the clinical application of monoclonal antibody drugs has been greatly limited, and new strategies for intervening PD-1 / PD-L1 axis are still needed 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 as follows: 1) strong tissue penetration, which can overcome the drug resistance problem caused by the difficulty of monoclonal antibody drugs to effectively penetrate into tumor tissues; 2) low inherent immunogenicity and short half-life, which makes the adverse reactions more controllable during drug use and is conducive to timely adjustment of drug strategies when adverse reactions occur; 3) can be administered orally, which is better for patient compliance and is conducive to the development of combination therapy. Therefore, finding small molecule inhibitors targeting PD-1 / PD-L1 axis can provide new intervention strategies for clinical tumor immunotherapy.

[0003] The current development of PD-L1 small molecule inhibitors mainly focuses on two aspects, i.e., intervening the interaction between PD-1 and PD-L1 or regulating the expression of PD-L1 protein. With the gradual resolution of the crystal structure of PD-1 / PD-L1 complex and the gradual revelation of the regulation mechanism of PD-L1 expression, the development of PD-L1 small molecule inhibitors has been rapidly developed, and multiple candidate small molecule compounds with potential anti-tumor activity have been screened. However, the 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 development of PD-L1 small molecule inhibitors has not made a breakthrough, and further research is needed.

[0004] S1PR4 is a member of the G protein-coupled receptor family S1PRs, 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 of patients, but its role in tumor development is not clear. CYM50308 is a modulator of S1PR4, which has high affinity and low intrinsic activity, and can specifically cause S1PR4 protein endocytosis degradation to inhibit the downstream signal of S1PR4. At present, CYM50308 is only used as a tool drug for research related to the function of S1PR4 protein, and has not been approved for clinical disease treatment. SUMMARY

[0005] The purpose of the present application is to provide the application 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-ketone, molecular formula C 20 H 21 F2N3O2S, molecular weight is 405.46.

[0006] Experiments have proved 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 experiments by Western blotting and flow cytometry. And in the humanized model of the immune system, 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 effect of the positive control drug nivolumab (PD-1 monoclonal antibody).

[0007] The drug is made of CYM50308 and pharmaceutically acceptable excipients, and the preparation form is injection liquid, once a day.

[0008] CYM50308 is a modulator of S1PR4, which has not yet entered the disease application stage. The present application proves by in vitro and in vivo experiments that CYM50308 has a down-regulating effect on the expression level of immune checkpoint protein PD-L1 in various tumor cells, and can exert significant anti-tumor activity, thereby expanding the clinical application of small molecule compound CYM50308 in tumor immunotherapy. The significance of the present application not only lies in the possibility of using CYM50308 for clinical treatment of solid tumors, but also provides a new idea and potential target for the development of small molecule inhibitors for intervention of PD-L1 expression.

[0009] The inventors found that a modulator CYM50308 of S1PR4 can significantly reduce the protein expression level of PD-L1 of various solid tumor cells and has significant anti-tumor activity in vivo in the previous research. There is no small molecule compound for tumor immunotherapy by intervening the expression of PD-L1 protein in the clinic at present, and there is no related research on the regulation of PD-L1 protein expression by CYM50308. The present application provides a small molecule compound for clinical tumor immunotherapy, and provides a new disease use for CYM50308. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 CYM50308 can inhibit the total protein expression of PD-L1 of tumor cells in a concentration-dependent manner, and the Western blotting diagram of non-small cell lung cancer cells H292 treated with CYM50308 (2.5 μM, 5 μM and 10 μM) is shown in Figure 1.

[0011] Figure 2 CYM50308 can significantly inhibit the membrane surface protein expression of PD-L1 of tumor cells, and the flow cytometry column chart of non-small cell lung cancer cells H292 treated with CYM50308 (2.5 μM, 5 μM and 10 μM) is shown in Figure 2.

[0012] Figure 3 CYM50308 can significantly inhibit the total protein expression of PD-L1 of hepatocarcinoma cells and breast cancer cells, and the Western blotting diagram of hepatocarcinoma cells Huh7 and breast cancer cells T47D treated with CYM50308 (10 μM) is shown in Figure 3.

[0013] Figure 4 CYM50308 can significantly promote the degradation of PD-L1 protein and shorten its half-life, and the immunoblotting diagram of the influence of CYM50308 (10 μM) on the stability of PD-L1 protein of non-small cell lung cancer cells H292 is shown in Figure 4.

[0014] Figure 5 CYM50308 can increase the infiltration of cytotoxic T lymphocytes in tumor tissues and inhibit tumor growth, and the statistical chart of CYM50308 (10 mg / kg) exerting significant anti-tumor activity by increasing the infiltration of cytotoxic T lymphocytes in tumor tissues in an immune system humanized mouse model is shown in Figure 5. DETAILED DESCRIPTION

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

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

[0017] Non-small cell lung cancer H292 cells were selected and seeded at a density of 1.0 x 10 6 The cells were seeded in 6-well plates at a density of 1.0 x 10 Figure 1 The 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-regulates PD-L1 protein level on the cell membrane of non-small cell lung cancer cell H292. The specific steps are as follows:

[0019] Non-small cell lung cancer H292 cells were selected and seeded at a density of 1.0 x 10 6 The cells were seeded in 6-well plates at a density of 1.0 x 10 Figure 2 The results showed that CYM50308 could inhibit the expression of PD-L1 protein on the cell membrane of tumor cells in a concentration-dependent manner.

[0020] Example 3: CYM50308 significantly down-regulates PD-L1 protein level in hepatocarcinoma cell Huh7 and breast cancer cell T47D. The specific steps are as follows:

[0021] Hepatocarcinoma cell Huh7 and breast cancer cell T47D were selected and seeded at a density of 1.0 x 10 6 The cells were seeded in 6-well plates at a density of 1.0 x 10 Figure 3Experimental results showed that CYM50308 could 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 H292 non-small cell lung cancer cells. The specific steps are as follows:

[0023] Non-small cell lung cancer H292 cells were selected and cultured at a concentration of 1.0 × 10⁻⁶. 6 Cells were seeded at a density of 1 / well in 6-well plates and cultured overnight at 37°C with 5% CO2. The following day, cells were treated with the protein synthesis inhibitor actinomycin (CHX, 10 μg / mL) or CHX (10 μg / mL) + CYM50308 (10 μM) for 0 h, 2 h, 4 h, 6 h, 8 h, and 12 h, respectively. Cells were collected and lysed, and proteins were extracted. PD-L1 protein expression in cells was detected by Western blotting. The experimental results were then statistically analyzed using ImageJ software, and statistical graphs were generated. The effect of CYM50308 on the stability of PD-L1 protein in non-small cell lung cancer cells H292 is shown in [the table below]. Figure 4 Experimental results showed that CYM50308 could significantly promote the degradation of PD-L1 protein and shorten the half-life of PD-L1 protein.

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

[0025] Peripheral blood mononuclear cells (PBMCs) were isolated from human blood samples using Ficoll separation solution. Freshly extracted PBMCs (1.5 × 10⁻⁶ cells) were then... 7 H292 cells (5 × 10⁻⁶ cells / mouse) were transfused via the tail vein into NSG mice to construct a humanized mouse model of the immune system. Five days after PBMC transfusion, H292 cells were injected at a rate of 5 × 10⁻⁶ cells / mouse into NSG mice. 6 Cells per mouse were subcutaneously injected into the axilla of NSG mice, and the tumors were allowed to grow to approximately 100 mm. 3 Mice were then randomly divided into a control group, a CYM50308 (10 mg / kg) administration group, and a PD-1 monoclonal antibody group (5 mg / kg). The CYM50308 administration group received 10 mg / kg CYM50308 via tail vein injection daily, while the PD-1 monoclonal antibody group received 5 mg / kg nivolumab twice weekly. Tumor growth was measured every two days using calipers, and tumor volume (TV) was calculated as 0.5 × tumor long axis × tumor short axis. 2Calculation. At the experimental endpoint, tumor tissue was dissected and CD8+ levels in the tumor were detected by flow cytometry. + T cell infiltration and PD-L1 expression levels. See details for specific effects. Figure 5 CYM50308 can significantly inhibit tumor growth, downregulate PD-L1 protein expression, and increase CD8+ expression in tumor tissue. + T-cell infiltration.

Claims

1. The application of CYM50308 in the preparation of antitumor drugs, wherein the chemical name of CYM50308 is (2Z,5Z)-5-((1-(2,4-difluorophenyl)-2,5-dimethyl-1H-pyrrolo-3-yl)methylene)-2-((2-methoxyethyl)imino)-3-methylthiazolidin-4-one, and the molecular formula is C 20 H 21 F2N3O2S, with a molecular weight of 405.46, is characterized by... Application in the preparation of drugs for treating liver cancer.

2. The application according to claim 1, characterized in that, The application described is that CYM50308 significantly reduces the expression of PD-L1 in tumor cells, effectively inhibits the growth of transplanted tumors, and increases the infiltration of cytotoxic T lymphocytes in tumor tissue.

3. The application according to claim 1, characterized in that, The drug is made from CYM50308 and pharmaceutically acceptable excipients, and is formulated as an injection.

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