Application of CPI-203 in preparation of product for preventing and treating cardiac transplantation chronic rejection

Abstract

The invention discloses application of CPI-203 in preparation of a product for preventing and treating cardiac transplantation chronic rejection, and belongs to the technical field of biological medicine. The CPI-203 disclosed by the invention can be applied to preparation of medicines for preventing and treating chronic rejection of heart transplantation. Meanwhile, according to the experimental result, the CPI-203 can obviously prolong the survival time of the graft and reduce fibrosis and M2 cell infiltration, and the safety is superior to that of a traditional scheme. The strategy not only provides an innovative therapy for heart transplantation chronic rejection, but also opens up a new direction for transplantation of other organs and treatment of fibrosis diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of CPI-203 in the preparation of products for preventing and treating chronic rejection of heart transplantation. Background Art

[0002] Heart transplantation is an effective means for treating end-stage heart failure, but postoperative chronic rejection (CR) remains the main problem affecting the long-term survival of the graft. The pathological features of CR include graft vascular lesions and interstitial fibrosis, ultimately leading to graft failure. Currently, the treatment methods for CR are limited, and there is an urgent need to develop new intervention strategies.

[0003] Studies have shown that macrophage polarization plays a key role in CR, and among them, M2 macrophages exacerbate rejection by secreting anti-inflammatory factors (such as IL-10, TGF-β) and promoting fibrosis. In the prior art, the application of the BET family protein inhibitor CPI-203 in heart transplantation rejection has not been explored.

[0004] Defects of the Prior Art:

[0005] 1. There is currently a lack of specific therapeutic drugs for macrophage polarization: The pathological features of CR are closely related to the infiltration of M2 macrophages, but the prior art does not involve specific intervention in macrophage polarization;

[0006] 2. It is unable to effectively inhibit chronic rejection (CR): Although existing immunosuppressive agents (such as CTLA4-Ig) can control acute rejection, they lack specificity for the graft vascular lesions and fibrosis in CR;

[0007] 3. The research on CPI-203 in the field of oncology does not involve the immune regulation mechanism of heart transplantation;

[0008] 4. The treatment means are single and the synergistic effect is insufficient: Existing programs are mostly single drugs and cannot synergistically extend the survival of the graft through multiple targets. Summary of the Invention

[0009] The core object of the present invention is to provide the application of the BET inhibitor CPI-203 in the preparation of drugs for preventing and treating chronic rejection of heart transplantation. By specifically inhibiting macrophage polarization (especially M2 type), it reduces macrophage infiltration and fibrosis progression in chronic rejection (CR) after heart transplantation, thereby significantly extending the survival time of the graft and improving the function of the transplanted heart.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] In the first aspect, the present invention provides the application of CPI-203 in the preparation of products for preventing and treating chronic rejection of heart transplantation.

[0012] Furthermore, the product is a drug for preventing and treating chronic rejection after heart transplantation.

[0013] Furthermore, the drug further comprises one or more pharmaceutically acceptable carriers.

[0014] Furthermore, the dosage form of the drug is selected from injection, solution, emulsion, suspension, drop, powder, granule, tablet or sustained-release preparation.

[0015] In a second aspect, the present invention also provides an application of a composition containing CPI-203 in the preparation of a product for preventing and treating chronic rejection after heart transplantation.

[0016] Furthermore, the composition containing CPI-203 is a pharmaceutical composition.

[0017] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0018] Furthermore, the dosage form of the pharmaceutical composition is selected from injection, solution, emulsion, suspension, drop, powder, granule, tablet or sustained-release preparation.

[0019] In a third aspect, the present invention also provides an agent for inhibiting chronic rejection after heart transplantation, and the agent contains CPI-203.

[0020] Targeting mechanism: ① Inhibiting BRD4 function: CPI-203 competitively binds to the bromodomains (BD1 and BD2) of BET proteins (especially BRD4), blocks its binding to acetylated histones, and thus inhibits macrophage gene transcription; ② Downregulating the proportion of M2 macrophages: By inhibiting the activity of BRD4-dependent super-enhancers (SEs), reducing the expression of key genes of M2 macrophages (such as ARG1, CD206, TGF-β); ③ Regulating the immune microenvironment: Reducing the secretion of profibrotic factors (such as TGF-β, IL-10) by M2 macrophages, and inhibiting intimal hyperplasia and interstitial fibrosis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Precisely regulating the immune microenvironment: By targeting M2 macrophages, reducing the secretion of profibrotic factors (TGF-β, IL-10), and inhibiting the progression of CR from the root cause;

[0023] Avoiding extensive immunosuppression: Compared with traditional immunosuppressants (such as hormones), CPI-203 does not interfere with T cell function and reduces the risk of infection;

[0024] Time window advantage: Administering drugs during the critical period of CR to maximize the curative effect;

[0025] Synergistic effect: When combined with CTLA4-Ig, it enhances the therapeutic effect through the dual mechanisms of "acute rejection control + chronic rejection blockade".

[0026] Mechanism transparency: The role of the BRD4-ARG1 axis in CR is clarified for the first time, providing a theoretical basis for subsequent drug development.

[0027] Target scalability: ARG1 can be used as an independent target for the development of other anti-fibrotic therapies.

[0028] By targeting and inhibiting BRD4-dependent M2 macrophage polarization, combined with an optimized dosing strategy and mechanism research, the present invention solves the core problem of insufficient control of chronic rejection in the prior art. Experimental data show that CPI-203 can significantly prolong the graft survival time, reduce fibrosis and M2 cell infiltration, and has better safety than traditional regimens. This strategy not only provides an innovative therapy for chronic rejection after heart transplantation, but also opens up a new direction for the treatment of other organ transplants and fibrotic diseases. Brief Description of the Drawings

[0029] Figure 1 Results of the expression of M2 macrophage markers in Example 1 (wherein, Figure A shows the relative expression levels of M2 macrophage markers Arg-1, MRC1, Chi3l3, and Fizz1; Figures B-C are immunoblotting images).

[0030] Figure 2 Results of the animal experiment in Example 2 (wherein, Figure A is the experimental design flow chart; Figure B is the graft survival time graph; Figures C-D are the results of HE staining, Sirus Red staining, and VVG staining of the graft).

[0031] Figure 3 Results of the flow cytometry analysis in Example 3 (wherein, Figure A is the experimental design flow chart; Figure B shows the change in the expression level of M2 macrophage markers. Compared with the control group, the M2 macrophage markers Arg-1 and TGF-β are significantly decreased; Figures C-D are the flow cytometry analysis results).

[0032] Figure 4 (wherein, Figures A-F are the results of bioinformatics analysis graphs, showing the association between macrophage super-enhancers and M2 macrophage marker genes. Arg1, MRC1, and TGFβ1 are related to super-enhancers (SE), and there are many overlapping genes between M2 macrophage marker genes and super-enhancer-related genes; Figures G-H are ChIPseq peak graphs, showing that the M2 macrophage marker gene Arg1 is related to the SE enhancer). Detailed Description of the Invention

[0033] To better illustrate the present invention, specific embodiments are listed below. Obviously, the described embodiments are only a part of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts all fall within the scope of protection of the present invention.

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] Basic information of the compound

[0036] Name: CPI-203

[0037] Molecular weight: 399.90

[0038] Molecular formula: C 19 H 18 ClN 5 OS

[0039] CAS No.: 1446144-04-2

[0040] Structural formula:

[0041]

[0042] Example 1 In vitro experiment on the inhibition of M2 macrophage polarization by CPI-203

[0043] 1. Experimental design

[0044] Verify that CPI-203 inhibits IL-4-induced M2 macrophage polarization by inhibiting BRD4 in vitro, and detect the expression of related markers by qPCR and Western blot.

[0045] Mechanism of action: BRD4 regulates the transcription of genes such as ARG1 and CD206 by binding to the super enhancer (SE) of M2 macrophages; CPI-203 competitively inhibits the binding of BRD4 to acetylated histones, blocks the activity of SE, and thus inhibits M2 polarization.

[0046] 2. Experimental steps

[0047] ① Cell culture: Isolate primary macrophages from mouse bone marrow, culture them in DMEM medium containing 10% FBS, and add M-CSF (20 ng / mL) to induce differentiation for 7 days.

[0048] ②Polarization induction: The differentiated macrophages were divided into two groups and induced in DMEM medium containing 10% FBS. The control group was stimulated with IL-4 (20 ng / mL) for 48 hours to induce M2 polarization; the CPI-203 group was treated with IL-4 (20 ng / mL) + CPI-203 (500 nM) for 48 hours.

[0049] ③Sample collection: Cells were collected and RNA and proteins were extracted.

[0050] ④qPCR detection: The mRNA expression of M2 markers (Arg1, CD206, Chi3l3, Fizz1) was detected using the SYBR Green method.

[0051] ⑤Western blot: The expression level of ARG1 protein was detected.

[0052] 3. Experimental results

[0053] ①The mRNA expression levels of Arg1, CD206, Chi3l3, and Fizz1 in the CPI-203 group were decreased compared with the control group (p < 0.001).

[0054] ②The expression level of ARG1 protein in the CPI-203 group was significantly decreased (p < 0.001).

[0055] 4. Experimental conclusion

[0056] CPI-203 significantly blocks M2 macrophage polarization by inhibiting BRD4.

[0057] Example 2 Animal experiment on CPI-203 prolonging the survival time of cardiac grafts

[0058] 1. Experimental design

[0059] In a chronic rejection model of heart transplantation, the effects of CPI-203 on the survival time and pathological damage of grafts were verified.

[0060] Mechanism of action: The vascular lesions and fibrosis of CR enter an accelerated phase 3 - 4 weeks after transplantation. Administering CPI-203 at this stage can block the pathological process driven by M2 macrophages.

[0061] 2. Experimental procedures

[0062] ①Construction of animal model:

[0063] Donor: BALB / c mice; Recipient: C57BL / 6 mice.

[0064] Perform abdominal ectopic heart transplantation with end-to-side anastomosis of the ascending aorta and the abdominal aorta, and end-to-side anastomosis of the pulmonary artery and the inferior vena cava; on the 0th and 2nd days after the operation, inject CTLA4-Ig (0.25 mg) intraperitoneally to establish a chronic rejection model.

[0065] ② Grouping and administration:

[0066] Control group: The established chronic rejection model was used as the control group, that is, only inject CTLA4-Ig after the operation (n = 6);

[0067] CPI-203 group: Take the mice with the established chronic rejection model. Inject CPI-203 once a day in the third week after abdominal ectopic heart transplantation and once every two days in the fourth week (the dosage for each injection is 25 mg / kg, n = 9);

[0068] ③ Observation indicators:

[0069] Palpate the graft pulsation daily to monitor the situation and record the survival time;

[0070] Euthanize the mice on the 30th day after the operation, take the grafts for HE staining, Sirus Red staining and VVG staining to evaluate vascular lesions and fibrosis.

[0071] 3. Experimental results

[0072] ① Survival time:

[0073] The survival time of the graft in the control group was 35 days;

[0074] The survival time of the graft in the CPI-203 group was long-term survival (p < 0.001);

[0075] ② Pathological analysis:

[0076] Control group: There was significant intimal hyperplasia and a relatively large proportion of fibrosis area;

[0077] CPI-203 group: The intimal hyperplasia was reduced and the fibrosis area decreased (p < 0.01);

[0078] 4. Experimental conclusion

[0079] Although CTLA4-Ig can extend the survival time to 35 days, it cannot effectively inhibit the infiltration of M2 macrophages and fibrosis; CPI-203 significantly reduces the polarization of M2 macrophages by targeting and inhibiting BRD4, resulting in long-term survival of the graft and significantly reduced pathological damage; CPI-203 significantly extends the survival time of the graft and reduces the pathological damage of chronic rejection.

[0080] Example 3 Flow cytometry analysis of CPI-203 reducing the infiltration of M2 macrophages in the graft

[0081] 1. Experimental design

[0082] Verify the inhibitory effect of CPI-203 on the infiltration of M2 macrophages (CD206+) in the graft by flow cytometry.

[0083] 2. Experimental procedures

[0084] ① Sample preparation: Take the transplanted heart tissues of the control group and the CPI-203 group 30 days after surgery, and prepare single-cell suspensions after digestion with collagenase I.

[0085] ② Flow cytometry staining:

[0086] Antibody labeling: CD68 (macrophage marker), CD206 (M2 marker);

[0087] Detect the proportion of CD68+CD206+ cells using a FACSCanto II flow cytometer.

[0088] ③ qPCR detection:

[0089] Detect the mRNA expression of M2 markers (Arg1, TGF-β) of mouse macrophages using the SYBR Green method.

[0090] ④ Data analysis:

[0091] Analyze the percentage and absolute count of CD206+ macrophages using FlowJo software.

[0092] 3. Experimental results

[0093] Proportion of CD206+ cells:

[0094] Control group: The proportion of CD206+ cells was 27.3%;

[0095] CPI-203 group: The proportion decreased to 5.85% (p<0.001).

[0096] M2 markers:

[0097] The relative expression levels of Arg1 and TGF-β in the CPI-203 group were significantly lower than those in the control group.

[0098] 4. Experimental conclusion

[0099] CPI-203 significantly reduces the infiltration of M2 macrophages in the graft, verifying its targeted regulatory effect.

[0100] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of CPI-203 in the preparation of products for preventing and treating chronic rejection of heart transplantation.

2. The use according to claim 1, characterized in that: The product is a drug for preventing and treating chronic rejection of heart transplantation.

3. The use according to claim 2, characterized in that: The medicine further comprises one or more pharmaceutically acceptable carriers.

4. The use according to claim 2, characterized in that: The dosage form of the drug is selected from injection, solution, emulsion, suspension, drops, powder, granules, tablets or sustained-release preparations.

5. Use of a composition containing CPI-203 in the preparation of a product for preventing and treating chronic rejection of heart transplantation.

6. The use according to claim 5, characterized in that: The composition containing CPI-203 is a pharmaceutical composition.

7. The use according to claim 6, characterized in that: The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

8. The use according to claim 6, characterized in that: The dosage form of the pharmaceutical composition is selected from injection, solution, emulsion, suspension, drops, powder, granules, tablets or sustained-release preparations.

9. A drug for inhibiting chronic rejection of heart transplantation, characterized in that: The medicament contains CPI-203.

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