Application of CD39 inhibitor or A2a receptor inhibitor in preparation of product for treating non-alcoholic steatohepatitis

Abstract

The invention discloses application of a CD39 inhibitor or an A2a receptor inhibitor in preparation of a product for treating non-alcoholic steatohepatitis, and belongs to the technical field of biological medicines. According to the application, a CD39 inhibitor or an A2a receptor inhibitor is applied to treatment of an NASH mouse model, and experiments find that the CD39 inhibitor ARL67156 and the A2a receptor inhibitor Istradafylline can both reduce senescence-related gene expression of NASH mice and effectively improve lipid droplet accumulation of the NASH mice, liver fibrosis and the metabolic function of the mice. According to the research of the invention, the CD39 inhibitor ARL67156 and the A2a receptor inhibitor Istradafylline can inhibit the CD39-A2a receptor pathway in a targeted manner, so that the killing ability of T cells on senescent hepatocytes can be enhanced, the accumulation of the senescent hepatocytes can be reduced, the liver fibrosis can be alleviated, and the medicinal value on the prevention and treatment of NASH can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the use of a CD39 inhibitor or an A2a receptor inhibitor in the preparation of a product for treating non-alcoholic steatohepatitis. Background Art

[0002] Non-alcoholic steatohepatitis (NASH) is a progressive subtype of non-alcoholic fatty liver disease (NAFLD). NASH is characterized by excessive fat accumulation in the liver accompanied by hepatocyte damage, inflammatory response, and liver fibrosis. Existing studies have shown that there is a significant accumulation of senescent cells in the liver of NASH patients. As the condition worsens, NASH can further progress to cirrhosis or even liver cancer, posing a serious health threat to patients. Approximately 25%-40% of adults globally suffer from NAFLD, and approximately 20% of them will develop into NASH. With the prevalence of obesity, diabetes, and metabolic syndrome, the incidence of NASH has been increasing year by year, and it has become an increasingly serious public health problem worldwide.

[0003] The pathological mechanism of NASH can generally be summarized into two main stages:

[0004] The first hit: Excessive fat deposition (steatosis) in the liver, which causes damage to hepatocytes and induces oxidative stress response;

[0005] The second hit: The inflammatory response triggered by oxidative stress further exacerbates hepatocyte damage, induces the occurrence of liver fibrosis, and forms a vicious cycle.

[0006] Currently, the treatment methods for non-alcoholic steatohepatitis mainly include lifestyle interventions (diet control, exercise) and drug therapies. Drug therapies include obeticholic acid, GLP-1 receptor agonists, vitamin E, and insulin sensitizers, etc. However, there are the following defects: Although obeticholic acid (FXR agonist) can partially improve liver fibrosis, its efficacy is limited and the side effects are obvious, such as problems like itching and increased LDL; The direct effect of GLP-1 receptor agonists on liver inflammation and fibrosis is weak, and it depends on blood glucose regulation. The treatment effect is not comprehensive enough, and the patient compliance is poor, and the drug needs to be injected for a long time; Although drugs such as vitamin E / pioglitazone can relieve oxidative stress or insulin resistance, they cannot prevent the progression of liver fibrosis, and long-term use may increase the cardiovascular risk. Therefore, how to adopt a more precise treatment strategy to relieve immunosuppression, improve the function of T cells, and eliminate senescent cells, so as to improve non-alcoholic steatohepatitis, has become a technical problem to be solved urgently. Summary of the Invention

[0007] The CD39 gene has a single nucleotide polymorphism (SNP) site - rs_10748643 in the population. Individuals with the AA phenotype have significantly decreased CD39 expression, and their proportion in the population is approximately 30%, and there are no obvious health problems. Therefore, blocking CD39 has good safety. Currently, many pharmaceutical companies are developing CD39 antibodies for the treatment of tumors, and many have passed safety sequencing. There are many related blocking antibodies and inhibitors for adenosine receptor A2a currently under development, mainly for the treatment of tumors. And one of the adenosine receptor A2a - istradefylline has been approved for marketing in Japan for the treatment of Parkinson's disease. Therefore, inhibiting or blocking CD39 / adenosine receptor A2a has good safety.

[0008] CD39 is expressed in both immune cells and non-immune cells, and is significantly upregulated in the livers of aging and non-alcoholic steatohepatitis mice. CD39 is an extracellular ATP / ADP hydrolase that can hydrolyze ATP and ADP into AMP, and then generate adenosine (ADO) through CD73. Adenosine is an important immunosuppressant that significantly inhibits the killing function of CD8 T cells through adenosine receptor A2a. Studies have shown that CD8 T cells play an important role in the clearance of senescent cells, and impaired function will lead to the accumulation of senescent cells, and the accumulation of senescent cells in the liver is closely related to non-alcoholic steatohepatitis. Therefore, CD39 and adenosine receptor A2a are potential drug targets in the prevention and treatment of non-alcoholic steatohepatitis.

[0009] In view of the problems existing in the prior art, the present invention provides a treatment plan targeting the CD39-adenosine receptor A2a receptor pathway, which relates to the application of a CD39 inhibitor or an A2a receptor inhibitor in the preparation of a product for the treatment of non-alcoholic fatty hepatitis. By inhibiting CD39 or adenosine receptor A2a, immunosuppression is relieved, and the killing function of T cells is restored, thereby clearing senescent hepatocytes and slowing down the course of non-alcoholic steatohepatitis.

[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 a CD39 inhibitor or an inhibitor of the downstream action target of the CD39 catalytic product in the preparation of a product for the treatment of non-alcoholic fatty hepatitis, wherein the inhibitor of the downstream action target of the CD39 catalytic product includes an A2a receptor inhibitor.

[0012] Further, the CD39 inhibitor is ARL67156.

[0013] Further, the A2a receptor inhibitor is Istradefylline.

[0014] Further, the product is a drug for treating non-alcoholic steatohepatitis.

[0015] Further, the drug further comprises one or more pharmaceutically acceptable carriers.

[0016] Further, the CD39 inhibitor or A2a receptor inhibitor is administered by injection, and the injection method includes one or more of intraperitoneal injection, intramuscular injection, subcutaneous injection, and intravenous injection.

[0017] In a second aspect, the present invention provides the use of a composition containing a CD39 inhibitor or an A2a receptor inhibitor in the preparation of a product for treating non-alcoholic steatohepatitis.

[0018] Further, the composition containing a CD39 inhibitor or an A2a receptor inhibitor is a pharmaceutical composition.

[0019] Further, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0020] In a third aspect, the present invention further provides a drug for treating non-alcoholic steatohepatitis, which contains one of a CD39 inhibitor or an A2a receptor inhibitor.

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

[0022] The present invention targets the CD39-A2a receptor pathway, relieves immunosuppression, improves the killing function of T cells, thereby clearing senescent cells and improving the condition of non-alcoholic steatohepatitis.

[0023] The present invention discovers through the following research that targeting and inhibiting the CD39-adenosine-A2a receptor pathway by a CD39 inhibitor (ARL67156) and an A2a receptor inhibitor (Istradefylline) can enhance the killing ability of T cells on senescent hepatocytes, reduce the accumulation of senescent hepatocytes, alleviate liver fibrosis, and has medicinal value for the prevention and treatment of NASH. Description of the Drawings

[0024] Figure 1 It is the analysis result of the gene expression level of the mice in the NASH-Istradefylline group in Example 1;

[0025] Figure 2 It is the HE staining experimental result of the mice in the NASH-Istradefylline group in Example 1;

[0026] Figure 3Results of Sirius red staining of mice in the NASH-Istradefylline group in Example 1;

[0027] Figure 4 Results of ELISA experiments on mice in the NASH-Istradefylline group in Example 1;

[0028] Figure 5 Results of analysis of gene expression levels of mice in the NASH-ARL67156 group in Example 1;

[0029] Figure 6 Results of HE staining experiments on mice in the NASH-ARL67156 group in Example 1;

[0030] Figure 7 Results of Sirius red staining of mice in the NASH-ARL67156 group in Example 1;

[0031] Figure 8 Results of ELISA experiments on mice in the NASH-ARL67156 group in Example 1. Detailed implementation manners

[0032] For better illustration of the present invention, the following examples are specifically listed. Obviously, the described examples are only a part of the present invention, rather than all the examples. Based on the examples in the present invention, other examples obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0033] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples.

[0034] The CDA-HFD feed formula of the present invention is shown in Table 1 below.

[0035] Table 1 CDA-HFD feed formula

[0036]

[0037]

[0038]

[0039] Example 1

[0040] 1. Experimental method

[0041] Eight-week-old female C57BL / 6N mice (Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into 4 groups: ① Normal diet group (Normal); ② NASH model group (NASH); ③ NASH-ARL67156 treatment group (NASH-ARL); ④ NASH-Istradefylline treatment group (NASH-Istradefylline), with 5 mice in each group.

[0042] Among them, the mice in the normal diet group were fed with normal feed produced by Beijing Huafukang Biotechnology Co., Ltd. for 10 weeks, the mice in the NASH model group were fed with CDA-HFD feed produced by Beijing Huafukang Biotechnology Co., Ltd. for 10 weeks, the mice in the NASH-ARL67156 treatment group were fed with CDA-HFD feed produced by Beijing Huafukang Biotechnology Co., Ltd. for 10 weeks, and the mice in the NASH-Istradefylline treatment group were fed with CDA-HFD feed produced by Beijing Huafukang Biotechnology Co., Ltd. for 10 weeks.

[0043] The mice in the NASH-ARL67156 treatment group started intraperitoneal injection of ARL67156 (2 mg / kg, MCE, soluble in PBS), 200 μl each time, once every two days, for a total of 14 injections at the 6th week (at this time, the model was successfully constructed by detecting common indicators in the art. The criteria for successful model construction included obvious hepatic steatosis, significant fibrosis in the liver, significant increase in indicators such as AST and ALT in plasma, and up-regulation of senescence-related genes such as P16 and P21 due to the accumulation of senescent cells in the NASH pathogenesis, etc.). The required organs were obtained by perfusion 24 hours after the last injection; the mice in the NASH-Istradefylline treatment group started intraperitoneal injection of Istradefylline (1 mg / kg, MCE), 200 μl each time, once every two days, for a total of 14 injections at the 6th week. The required organs were obtained by perfusion 24 hours after the last injection; at the same time, the mice in the normal diet group and the NASH model group were intraperitoneally injected with PBS, 200 μl each time, once every two days, for a total of 14 injections. The required organs were obtained by perfusion 24 hours after the last injection.

[0044] Analysis of lipid droplet accumulation and liver fibrosis: A small piece of liver was fixed in 20% neutral buffered formalin and then embedded in paraffin. The paraffin-embedded liver tissue was cut into thin sections and stained with standard hematoxylin and eosin (H&E) and Sirius red. The stained tissue sections were observed under a microscope with bright-field illumination.

[0045] Metabolic function assay: Blood was collected from mice by cardiac puncture and left at room temperature for 15 minutes. To obtain serum, the samples were centrifuged at 3,400 g for 15 minutes at 4°C. Any severely hemolyzed serum samples were excluded from further analysis. Assays were performed using AST (E-BC-K236-M), ALT (E-BC-K235-M), and PGE2 (E-EL-0034) kits, all from Elabscience.

[0046] qPCR: Among them, the PCR reaction reagents were purchased from TaKaRa (RR820A), and the PCR reaction system and reaction program are shown in Table 2-3 below:

[0047] Table 2 PCR reaction system

[0048]

[0049] Table 3 PCR reaction program

[0050]

[0051] The primer sequences used for the PCR amplification are shown in Table 4 below:

[0052] Table 4 PCR amplification primer sequences

[0053]

[0054] 2. Experimental results

[0055] 2.1 Istradefylline can reduce the expression of senescence-related genes in NASH mice

[0056] The qPCR results ( Figure 1 ) showed that compared with the normal diet group, the expression of senescence-related genes in the NASH model group was significantly increased, while Istradefylline treatment could significantly improve the increased gene expression, where, *p < 0.05, **p < 0.01.

[0057] 2.2 Istradefylline can improve lipid droplet accumulation in NASH mice

[0058] The HE experimental results ( Figure 2 ) showed that compared with the normal diet group, lipid droplet accumulation in the NASH model group was significantly increased, and Istradefylline treatment could reduce lipid droplet accumulation. At the same time, Istradefylline had no effect on the changes in the lungs. The results proved that inhibiting the A2a receptor could significantly improve lipid droplet accumulation in NASH.

[0059] 2.3 Istradefylline can improve hepatic fibrosis in NASH mice

[0060] Results of Sirius red staining ( Figure 3 ) showed that compared with the normal diet group, hepatic fibrosis in the NASH model group was obvious, while treatment with Istradefylline could relieve the symptoms of hepatic fibrosis and improve hepatic structure.

[0061] 2.4 Istradefylline can improve the metabolic function of NASH mice

[0062] Results of Elisa ( Figure 4 ) showed that compared with the normal diet group, the levels of indicators such as AST, ALT, and PGE2 in the NASH model group increased, while treatment with Istradefylline could improve these indicators, where *p<0.05, **p<0.01.

[0063] 2.5 ARL67156 can reduce the expression of senescence-related genes in NASH mice

[0064] Results of qPCR ( Figure 5 ) showed that compared with the normal diet group, the expression of senescence-related genes in the NASH model group increased significantly, while treatment with ARL67156 could significantly improve the increased gene expression, where *p<0.05, **p<0.01.

[0065] 2.6 ARL67156 can improve lipid droplet accumulation in NASH mice

[0066] Results of HE experiment ( Figure 6 ) showed that compared with the normal diet group, lipid droplet accumulation in the NASH model group increased significantly, and treatment with ARL67156 could reduce lipid droplet accumulation. At the same time, ARL67156 had no effect on the changes in the lungs.

[0067] 2.7 ARL67156 can improve hepatic fibrosis in NASH mice

[0068] Results of Sirius red staining ( Figure 7 ) showed that compared with the normal diet group, hepatic fibrosis in the NASH model group was obvious, while treatment with ARL67156 could relieve the symptoms of hepatic fibrosis and improve hepatic structure.

[0069] 2.8 ARL67156 can improve the metabolic function of NASH mice

[0070] Results of Elisa ( Figure 8)It shows that compared with the normal diet group, the indicators such as AST, ALT, and PGE2 in the NASH model group increased, while treatment with ARL67156 could improve these indicators, where, *p<0.05, **p<0.01.

[0071] 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 should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a CD39 inhibitor or an inhibitor of a downstream target of CD39 catalytic product in the preparation of a product for treating non-alcoholic steatohepatitis, characterized in that, The downstream acting target inhibitor of the CD39 catalytic product includes an A2a receptor inhibitor.

2. The application according to claim 1, characterized in that, The CD39 inhibitor is ARL67156.

3. The application according to claim 1, characterized in that, The A2a receptor inhibitor is Istradefylline.

4. The application according to claim 1, characterized in that, The product is a drug for treating non-alcoholic steatohepatitis.

5. The application according to claim 5, characterized in that, The drug further comprises one or more pharmaceutically acceptable carriers.

6. The application according to claim 1, characterized in that, The CD39 inhibitor or A2a receptor inhibitor is administered by injection, and the injection method includes one or more of intraperitoneal injection, intramuscular injection, subcutaneous injection, and intravenous injection.

7. Use of a composition containing a CD39 inhibitor or an A2a receptor inhibitor in the preparation of a product for treating non-alcoholic steatohepatitis.

8. The application according to claim 7, wherein The composition containing a CD39 inhibitor or an A2a receptor inhibitor is a pharmaceutical composition.

9. The application according to claim 8, characterized in that, The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

10. A drug for treating non-alcoholic steatohepatitis, characterized in that, The drug contains one of a CD39 inhibitor or an A2a receptor inhibitor.

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