Biotin-based PD-1 / PD-L1 inhibitor and application thereof in tumor immunotherapy

Abstract

The invention discloses a biotin-based PD-1 / PD-L1 inhibitor and application thereof in tumor immunotherapy, relates to the field of pharmacy, and finds that biotin has a good inhibition effect on combination of PD-L1 protein and PD1 protein and can induce T cells to recognize PD-L1 high-expression tumor cells for the first time, and a new basis is provided for developing new anti-tumor application of biotin.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and particularly to the application of biotin in the preparation of anti-tumor drugs. Background Art

[0002] Biotin (CAS No.: 58-85-5) is an organic bicyclic heterocyclic compound composed of 2-oxohexahydro-1H-thieno[3,4-d]imidazole and has a valeric acid substituent attached to the tetrahydrothiophene ring. It functions as a cofactor, coenzyme, dietary supplement, human metabolite, Saccharomyces cerevisiae metabolite, Escherichia coli metabolite, mouse metabolite, cofactor, and essential metabolite. It is a member of biotin and vitamin B7. It is the conjugate acid of biotinylate. This biotin is a water-soluble enzyme cofactor that is present in trace amounts in every living cell. It mainly binds to proteins or polypeptides and is abundant in the liver, kidney, pancreas, yeast, and milk.

[0003] Programmed cell death protein 1 (PD1) is an immune checkpoint receptor that regulates T cell responses. Its ligand, programmed death ligand 1 (PD-L1), is usually overexpressed on the surface of tumor cells. When PD1 binds to PD-L1, T cell responses are inhibited, leading to tumor immune resistance. Checkpoint inhibitors that block the formation of the PD1 / PD-L1 complex have generated great interest in cancer immunotherapy.

[0004] Tumors have the characteristic of escaping immune attack, acting by hiding cancer cells from immune recognition, directly or indirectly interfering with immune effector cells, and protecting malignant cells from immune cytotoxicity. The recognition, locking, and destruction of tumor cells by the immune system consist of multiple consecutive steps, which is the theoretical basis of immunotherapy. Nowadays, immunotherapy is becoming a new method for the treatment of advanced liver cancer worldwide. Programmed death 1 (PD-1) and its ligand PD-L1 are currently widely studied checkpoints related to immune escape in liver cancer. In the liver cancer microenvironment, the expression of PD-L1 is significantly increased on the surfaces of tumor cells and surrounding stromal cells (tumor-associated macrophages, dendritic cells). PD-1 is a signaling pathway expressed on T cells, B cells, and NK cells. It can inhibit the activation of antigen-specific T cells by binding to ligands PD-L1 and PD-L2, downregulating the immune response of T cells in the tumor microenvironment, thus leading to tumor immune escape. Previous phase I and II clinical trials have demonstrated the good efficacy and safety of anti-PD-1 and anti-PD-L1 antibodies in the treatment of advanced liver cancer. Existing data show that blocking the PD-1 / PD-L1 signaling pathway has excellent anti-tumor activity and safety for the treatment of liver cancer. Currently, most studies on blocking the PD-1 / PD-L1 signaling pathway focus on antibody drugs. Although antibody drugs show excellent anti-tumor activity, the dangerous side effects caused by their immunogenicity cannot be ignored, such as immune pneumonia, immune hepatitis, immune enteritis, and even immune myocarditis. Therefore, the research and development of natural small molecule inhibitors have attracted much attention. Obtaining anti-tumor drugs from natural products is a hot field in tumor drug development at home and abroad. As a major country of natural products, China not only has huge natural product resources but also has a long history of natural product development and use. However, there are still no reports on the correlation between natural products and immunotherapy. Therefore, screening small molecule natural products to block the PD-1 / PD-L1 signaling pathway is a difficult problem that China urgently needs to solve at present. Small molecule natural products have low cost, high purity, no immunogenicity, and good stability. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides a biotin-based PD-1 / PD-L1 inhibitor and its application in tumor immunotherapy.

[0006] A PD-1 / PD-L1 inhibitor of the present invention, wherein the inhibitor is biotin, and the structural formula of biotin is as follows:

[0007]

[0008] The application of biotin of the present invention as a PD-1 / PD-L1 inhibitor in tumor immunotherapy.

[0009] Furthermore, the inhibitor is used alone or in the form of a pharmaceutical composition in tumor immunotherapy.

[0010] Furthermore, the application of biotin as a PD-1 / PD-L1 inhibitor in tumor immunotherapy, wherein the molar concentration of biotin is 100 nM to 10,000 nM.

[0011] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of biotin, as well as pharmaceutically acceptable excipients and / or carriers.

[0012] Furthermore, the pharmaceutical composition is an oral preparation, an injection or a topical administration preparation.

[0013] Furthermore, the oral preparation includes tablets, capsules, pills, granules; the injection includes injection solution, freeze-dried powder injection form for injection; the topical administration preparation includes dripping pills, sprays, patches.

[0014] The present invention has the following beneficial effects:

[0015] The present invention discovers for the first time that biotin has a good inhibitory effect on the binding of PD-1 and PD-L1 in vitro, and can significantly induce early apoptosis of tumor cells. The above results indicate a new use of biotin in the preparation of anti-tumor drugs, and biotin is an already marketed drug, which can be directly applied to humans without the need for further clinical safety assessment, and can quickly apply biotin to treat tumors, greatly saving time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a detection graph of the inhibitory activity of biotin at different concentrations on PD-1 / PD-L1 of the present invention; A represents the molecular structural formula of biotin; B represents the comparison of the inhibitory rate of the activity of PD-1 / PD-L1 protein by biotin at different concentrations (1 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM) and the standard product. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] In order to make the purposes, technical solutions and advantages of the embodiments of the present invention clearer, the spirit of the content disclosed by the present invention will be described in detail below. After any person skilled in the art in the technical field understands the embodiments of the content of the present invention, the techniques taught by the content of the present invention can be changed and modified, and it does not depart from the spirit and scope of the content of the present invention.

[0018] The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but are not intended to limit the present invention.

[0019] Embodiment

[0020] The reagents used in this example were the HTRF Human PD1 / PDL1 Binding Kit from Cisbio, the compounds were purchased from TargetMol, and the 384-well plates were ProxiPlate shallow-well non-transparent microplates.

[0021] Take out the HTRF kit from the -80 °C refrigerator and thaw it at room temperature. 4572 natural compounds were dissolved in DMSO in 58 96-well plates (30 μL, 10 mM). 20 dissolved compounds were mixed in one well, with 20 μL of the mixture in each well. Take out the 384-well plates and add 2 μL of the mixed liquid medicine, 4 μL of Tag1-PDL1 protein, 4 μL of Tag2-PD1 protein, 5 μL of Anti-Tag1 EuCryptate reagent, and 5 μL of Anti-Tag2 XL665 antibody to each well. Incubate at room temperature for 1 hour and measure the fluorescence value of the compounds on a microplate reader. The wavelengths are 620 nm and 665 nm, and F = 665 / 620 * 10000. The lower the fluorescence value, the better the inhibitory effect of the mixture on PD-1 / PD-L1. Compared with the control group, a mixture was preliminarily screened out. The natural compounds corresponding to this mixture were capsaicin, amphotericin, metronidazole, polymyxin B sulfate, theophylline, biotin, bepotastine besilate, penicillin V potassium, glimepiride, miconazole, mosapride, fusicoccin, diethyl trisulfide, chlortetracycline hydrochloride, 2-thiouracil, 4-aminobenzoic acid, cinchonidine, ouabain octahydrate, nonamide, ethyl pyrimidine. Thiouracil, 4-aminobenzoic acid, cinchonidine, ouabain octahydrate, nonanediamide, artemether.

[0022] Further screen the active compounds and set up a concentration gradient. Dilute the above compounds to 1 nM, 10 nM, 100 nM, 1000 nM, and 10000 nM respectively, and the standard product (BMS202) was also diluted to 1 nM, 10 nM, 100 nM, 1000 nM, and 10000 nM. Add 2 μL of the liquid medicine, 4 μL of Tag1-PDL1 protein, 4 μL of Tag2-PD1 protein, 5 μL of Anti-Tag1 Eu Cryptate reagent, and 5 μL of Anti-Tag2 XL665 antibody to each well of the diluted compound microplate. Incubate at room temperature for 1 hour and measure the fluorescence value of the compounds on a microplate reader. The calculation method is the same as above.

[0023] Figure 1The results showed that the inhibitory effects of 100 nM and 1000 nM biotin on PD-1 / PD-L1 protein were significantly different compared with the control group. However, the inhibition rate of biotin on PD-1 / PD-L1 protein was higher at a concentration of 10000 nM. Secondly, the half inhibitory concentration (IC50) value was further calculated using the concentration gradient inhibition rate. The results showed that the IC50 value of biotin acting on PD-1 / PD-L1 protein was 58.42 nM / ml.

[0024] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A PD-1 / PD-L1 inhibitor, characterized in that The inhibitor is biotin, and the structural formula of biotin is as follows:

2. Use of biotin as claimed in claim 1 as a PD-1 / PD-L1 inhibitor in tumor immunotherapy.

3. The use according to claim 2, characterized in that The inhibitor is used alone or in the form of a pharmaceutical composition in tumor immunotherapy.

4. The use according to claim 2 or 3, characterized in that The biotin is used as a PD-1 / PD-L1 inhibitor in tumor immunotherapy, and the molar concentration of biotin is 100nM to 10000nM.

5. The use according to claim 3, characterized in that The pharmaceutical composition comprises a therapeutically effective amount of biotin and a pharmaceutically acceptable auxiliary material and / or carrier.

6. The use according to claim 5, characterized in that The pharmaceutical composition is an oral preparation, an injection or a topical administration preparation.

7. The use according to claim 6, characterized in that The oral preparations include tablets, capsules, pills, and granules; the injections include injection solutions and freeze-dried powder injections; and the topical administration agents include pellets, sprays, and patches.

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