Application of tazeostat in preparation of medicine for preventing and treating heart transplantation chronic rejection reaction

Abstract

The invention discloses application of tazeostat in preparation of a medicine for preventing and treating cardiac transplantation chronic rejection, and belongs to the technical field of biological medicine. The medicine reduces the trimethylation level of histone H3 around a B cell p21 gene at a lysine 27 site through epigenetic regulation, relieves epigenetic inhibition on the p21 gene to highly express p21 molecules, influences differentiation of the B cell to a hair growth center B cell and plasma cells, and further relieves antibody-mediated rejection reaction, so that the survival time of a graft is remarkably prolonged, and the survival rate of the graft is improved. The function of the transplanted heart is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of tazestatin in the preparation of drugs for the prevention and treatment of chronic rejection in heart transplantation. Background Technology

[0002] Heart transplantation is the ultimate treatment for end-stage heart disease and is widely used in clinical practice. However, long-term survival remains a major challenge for patients. Cardiac allograft vasculopathy (CAV) is one of the main manifestations of chronic rejection and has a significant impact on graft failure after transplantation. This pathological phenomenon is mainly caused by donor-specific antibodies (DSA) and antibody-mediated rejection (AMR).

[0003] Studies have shown that inhibiting B cell-mediated DSA production can improve the long-term survival rate of mouse heart transplants, indicating that regulating B cell function is a promising approach for treating chronic adenocarcinoma (CAV). Tazemetostat (Taz) is the world's first approved EZH2 methyltransferase inhibitor. It selectively inhibits EZH2 enzyme activity, blocks abnormal histone H3K27 methylation, restores tumor suppressor gene transcription, and induces tumor cell apoptosis. This drug has received conditional approval in China for the treatment of adult lymphoma. Studies have shown that tazemetostat can downregulate methylation-driven pro-inflammatory and pro-proliferative pathways in hematologic malignancies and has a broad-spectrum inhibitory effect on abnormal activation of the immune system. In contrast to the pathological characteristics of transplant rejection, tazemetostat is expected to exert a "triple" protective effect: ① inhibiting excessive T and B cell proliferation and reducing donor-specific antibody production; ② inducing apoptosis of allogeneic reactive lymphocytes and reducing persistent inflammatory infiltration within the graft; ③ blocking abnormal activation and migration of vascular endothelial and smooth muscle cells, significantly delaying intimal hyperplasia and luminal stenosis. Compared with traditional immunosuppressants, tazestat acts directly on the upstream nodes of the epigenetic-inflammation-proliferation axis, and has both immunomodulatory and vascular protective functions, providing a new and precise entry point for intervening in chronic rejection of heart transplants.

[0004] Existing technological shortcomings: (1) Inability to effectively suppress chronic rejection: Most existing immunosuppressants are designed for T cell-mediated rejection. Although they can control acute rejection, they lack specificity for graft vascular lesions in chronic rejection. (2) Although rituximab (anti-CD20 monoclonal antibody) developed for B cells has significant efficacy, its application outside of hospitals is limited by the high incidence of severe infusion reactions (including fever, chills, rash, hypotension, and even anaphylactic shock, ARDS, or death). Furthermore, its high cost makes it unsuitable for most heart transplant patients.

[0005] (3) Tazesta has shown great potential in the treatment of hematologic malignancies, but it has not yet been involved in studies on immune regulation related to heart transplant rejection. Summary of the Invention

[0006] This invention provides the use of tazexat or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and treatment of chronic rejection in heart transplantation.

[0007] In one embodiment of the present invention, the CAS No. of the tazexta is 1403254-99-8.

[0008] In one embodiment of the invention, tazestat or a pharmaceutically acceptable salt thereof is the sole active ingredient in the drug.

[0009] In one embodiment of the invention, the drug further contains a pharmaceutically acceptable carrier.

[0010] The present invention also provides a pharmaceutical composition for preventing chronic rejection after heart transplantation, the pharmaceutical composition containing tazetine or a pharmaceutically acceptable salt thereof.

[0011] In one embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of tazestra in the preparation of drugs for preventing chronic rejection in heart transplantation. It involves epigenetic regulation, reducing the trimethylation level of histone H3 around the p21 gene in B cells at lysine 27, relieving epigenetic inhibition of the p21 gene and resulting in high expression of p21 molecules. This affects the differentiation of B cells into germinal center B cells and plasma cells, thereby alleviating antibody-mediated rejection, significantly prolonging graft survival time, and improving the function of the transplanted heart. Attached Figure Description

[0013] Figure 1A-1GThe results of the animal experiments in Example 1 are shown, in which (A) tazestat (Ezh2i) was injected starting on day 7 post-surgery in a chronic rejection model of heart transplantation. Serum from recipient mice was collected at specific time points. On day 35, samples of transplanted organs, spleen, and bone marrow were collected for flow cytometry analysis. (B) Survival rate of transplanted organs after heart transplantation, n=5. (C) Mean fluorescence intensity of IgG1, n=5. (DE) H&E and Masson stained sections of heart transplant tissue (40x magnification). Scale bar is 20 micrometers. (F) Proportion of plasma cells (GC B cells) in spleen B cells. (G) CD45 in bone marrow and spleen. + The proportion of plasma cells (PCs) in the cells. ns = no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0014] Figure 2A-2C The results of flow cytometry analysis in Example 2 are shown in (A) for B cells isolated from the spleen of C57 mice, sorted using magnetic beads, and co-cultured in vitro with anti-CD40, IL-21, CpG-B, and anti-IgM antibodies. Tazesta was added to the cells as the experimental group, and tazesta and UC2288 were added to the cells as the treatment group. Analysis was performed by flow cytometry on day 5. (B) Representative contour plots and bar graphs show the EZH2 of mouse B220+ live cells in vitro. - P21 + and EZH2 + P21 - Cell percentage. (C) Representative contour plots and bar charts show mouse B220. + Percentage of GC B cells in vitro. Detailed Implementation

[0015] Example 1: Pharmacodynamic Validation of Tazesta 1. Experimental Design: Animal grouping: C57BL / 6 mice that received BALB / c→C57BL / 6 heart transplants were randomly divided into two groups. One group was given tazocine (1 mg / kg, dissolved in physiological saline, intraperitoneal injection, 4 times a week), and the other group was given an equal volume of physiological saline as a control.

[0016] Dosage schedule: Begin administration on day 7 post-transplant and continue until day 35.

[0017] Observation indicators: graft survival rate, pathological changes (H&E staining, Masson staining), DSA levels (flow cytometry), and the ratio of GC-B cells to plasma cells (flow cytometry) at different time points after transplantation (7, 14, 21, 28, 35 days). Figure 1A ).

[0018] 2. Experimental steps: (1) Select 8-10 week old C57BL / 6 mice and acclimatize them for 1 week.

[0019] (2) Follow the standard heterotopic heart transplantation method, the method is the same as above.

[0020] (3) Starting from the 7th day after transplantation, mice in the tazestat group were injected intraperitoneally with tazestat (1 mg / kg) 4 times a week; mice in the control group were injected intraperitoneally with an equal volume of physiological saline.

[0021] (4) Blood was collected from the tail vein on days 7, 14, 21, 28 and 35 after transplantation, and serum was separated for DSA detection.

[0022] (5) Some mice were sacrificed, and their spleens and bone marrow were removed. Single-cell suspensions were prepared for flow cytometry analysis of the ratio of GC B cells and plasma cells.

[0023] (6) The transplanted heart was removed, fixed in 4% paraformaldehyde, and subjected to H&E staining and Masson staining to assess pathological changes.

[0024] (7) The methods for DSA detection, flow cytometry analysis and pathological evaluation are the same as above.

[0025] result: (1) The survival time of transplanted hearts in mice treated with tazestat was significantly prolonged, with a median survival time more than 30 days longer than that in the control group. Figure 1B ).

[0026] (2) On day 35 post-transplantation, the serum DSA level in mice treated with tazestat was significantly reduced, and the IgG1 MFI value was reduced by more than 70% compared with the control group. Figure 1C ).

[0027] (3) Pathological sections showed that the vascular fibrosis area of ​​the transplanted heart in mice treated with tazestat was reduced by more than 60%, and the degree of lymphocyte infiltration was reduced. Figure 1D -E).

[0028] (4) Flow cytometry showed that the proportions of GC-B cells and plasma cells in the spleen of mice treated with tazestat were significantly reduced, decreasing by more than 60% and 50%, respectively. Figure 1F -G).

[0029] Example 2: Application of P21 inhibitor UC2288 in combination with tazetine 1. Experimental Design: Cell culture: CD19 isolated from mouse spleen+ B cells were cultured in a medium containing anti-CD40, IL-21, CpG-B, and anti-IgM antibodies to simulate the activation environment of GC B cells.

[0030] Drug treatment: Tazexat (1 µM) and P21 inhibitor UC2288 (10 µM) were added to cultured B cells, either alone or in combination.

[0031] Observation indicators: After 5 days of culture, the proportion of GC B cells (GL7) was detected by flow cytometry. + FAS + The expression levels of IgG, IgD, and CD138, as well as cell viability ( Figure 2A ).

[0032] 2. Experimental Procedure (1) Single-cell suspensions were prepared from mouse spleens, and B cells were sorted using Thermo Fisher MagniSort™ Mouse B cellEnrichment Kit. Cells with a purity of >95% were collected.

[0033] (2) The sorted B cells were seeded at a density of 1×10^6 / ml in 96-well U-bottom culture plates. Anti-CD40 antibody (1µg / ml), IL-21 (100ng / ml), CpG-B (2.5µg / ml) and anti-IgM antibody (5µg / ml) were added to simulate the GC B cell activation environment.

[0034] (3) Add tazostat (1µM) and UC2288 (10µM) to the culture medium respectively, and set up single treatment group and combined treatment group. The control group only added culture medium.

[0035] (4) After 5 days of culture, cells were collected and the expression levels of EZH2 and P21 in the cells were detected by flow cytometry. Dead cells were labeled with the fluorescent dye Zombie Aqa and GC B cells were labeled with the fluorescent antibodies anti-FAS and anti-GL7. The proportion and cell viability of GC B cells were detected by flow cytometry using a SONY ID7000.

[0036] 3. Results: (1) Treatment with tazestat and UC2288 can inhibit the expression of EZH2 and P21 in B cells, respectively (Figure 2B).

[0037] (2) In B cells treated with tazestat alone, the proportion of GC B cells decreased significantly. In B cells treated with a combination of tazestat and UC2288, the proportion of GC B cells recovered. Figure 2C ).

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of tazestat or its pharmaceutically acceptable salts in the preparation of drugs for the prevention and treatment of chronic rejection in heart transplantation.

2. The application according to claim 1, characterized in that, The drug contains tazetine or its pharmaceutically acceptable salt as the sole active ingredient.

3. The application according to claim 2, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

4. A pharmaceutical composition for preventing and treating chronic rejection after heart transplantation, characterized in that, The pharmaceutical composition contains tazetine or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition also contains a pharmaceutically acceptable carrier.

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