Use of a cd39 inhibitor or a2a receptor inhibitor in the manufacture of a product for the treatment of nonalcoholic steatohepatitis

Abstract

The application discloses application of a CD39 inhibitor or an A2a receptor inhibitor in preparation of a product for treating non-alcoholic fatty hepatitis, and belongs to the technical field of biological medicines.The CD39 inhibitor or the A2a receptor inhibitor is used in the treatment of a NASH mouse model, and experiments find that the CD39 inhibitor ARL67156 and the A2a receptor inhibitor Istradefylline can reduce the expression of aging-related genes of NASH mice, and effectively improve lipid droplet accumulation, liver fibrosis and metabolic function of the mice.It is known from the research of the application that the CD39 inhibitor ARL67156 and the A2a receptor inhibitor Istradefylline target inhibition of a CD39-adenosine-A2a receptor pathway, can enhance the killing ability of T cells to aging liver cells, reduce the accumulation of the aging liver cells, and reduce liver fibrosis, and has medicinal value for prevention and treatment of NASH.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a CD39 inhibitor or an A2a receptor inhibitor in preparation of a product for treating non-alcoholic steatohepatitis. BACKGROUND

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

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

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

[0005] Second hit: inflammation induced by oxidative stress further exacerbates hepatocyte damage and induces the occurrence of liver fibrosis, forming a vicious cycle.

[0006] Current treatment methods for non-alcoholic steatohepatitis mainly include lifestyle intervention (diet control, exercise) and drug therapy, and drug therapy includes obeticholic acid, GLP-1 receptor agonists, vitamin E, and insulin sensitizers, etc., but has the following defects: although obeticholic acid (FXR agonist) can partially improve liver fibrosis, the therapeutic effect is limited and the side effects are obvious, such as itching, LDL elevation, etc.; GLP-1 receptor agonists have weak direct effects on liver inflammation and fibrosis, and are dependent on blood glucose regulation, and 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 vitamin E / pioglitazone can alleviate oxidative stress or insulin resistance, it cannot prevent the progression of liver fibrosis, and long-term use may increase the risk of cardiovascular disease. Therefore, how to use more precise treatment strategies to relieve immune suppression, improve T cell function, and eliminate senescent cells, and thus improve non-alcoholic steatohepatitis, has become a technical problem to be solved. SUMMARY

[0007] The CD39 gene has a single nucleotide polymorphism (SNP) site in the human population--rs_10748643, the AA phenotype of which has significantly reduced CD39 expression, and the proportion thereof in the human population is about 30%, and there is no obvious health problem. Therefore, blocking CD39 has good safety, and many drug development companies are currently 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 being developed, mainly for the treatment of tumors. And one of the adenosine receptors 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 immune cells and non-immune cells, and is significantly up-regulated in the liver of mice with aging and non-alcoholic fatty liver disease. CD39 is an extracellular ATP / ADP hydrolysis enzyme 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 their dysfunction can lead to the accumulation of senescent cells, and the accumulation of senescent cells in the liver is closely related to non-alcoholic fatty liver disease. Therefore, CD39 and adenosine receptor A2a are potential drug targets for the prevention and treatment of non-alcoholic fatty liver disease.

[0009] To solve the problems in the prior art, the present application provides a treatment scheme targeting the CD39-adenosine receptor A2a receptor pathway, and relates to the use of a CD39 inhibitor or an A2a receptor inhibitor in the preparation of a product for treating non-alcoholic fatty liver disease. By inhibiting CD39 or adenosine receptor A2a to relieve immunosuppression, the killing function of T cells is restored, thereby eliminating senescent liver cells and slowing the course of non-alcoholic fatty liver disease.

[0010] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0011] In a first aspect, the present application provides the use of a CD39 inhibitor or a downstream target inhibitor of a catalytic product of CD39 in the preparation of a product for treating non-alcoholic fatty liver disease, wherein the downstream target inhibitor of the catalytic product of CD39 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 medicine for treating non-alcoholic steatohepatitis.

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

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

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

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

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

[0020] In a third aspect, the present application also provides a medicine for treating non-alcoholic steatohepatitis, wherein the medicine comprises one of a CD39 inhibitor or an A2a receptor inhibitor.

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

[0022] The present application targets the CD39-A2a receptor pathway, removes immune suppression, and improves the killing function of T cells, thereby eliminating senescent cells and improving the symptoms of non-alcoholic steatohepatitis.

[0023] The present application finds that by targeting and inhibiting the CD39-adenosine-A2a receptor pathway through a CD39 inhibitor (ARL67156) and an A2a receptor inhibitor (Istradefylline), the killing ability of T cells on senescent hepatocytes can be enhanced, the accumulation of senescent hepatocytes can be reduced, and liver fibrosis can be reduced, and the prevention and treatment of NASH has medicinal value. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The results of gene expression level analysis of mice in the NASH-Istradefylline group in Example 1;

[0025] Figure 2 The results of HE staining experiments of mice in the NASH-Istradefylline group in Example 1;

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

[0027] Figure 4 Results of ELISA experiment of NASH-ARL67156 group mice in Example 1;

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

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

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

[0031] Figure 8 Results of ELISA experiment of NASH-ARL67156 group mice in Example 1. DETAILED DESCRIPTION

[0032] In order to make the present application better understood, specific examples are listed as follows. Obviously, the described examples are only a part of the present application, but not all the examples. Based on the examples in the present application, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0033] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

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

[0035] Table 1 CDA-HFD feed formula

[0036]

[0037]

[0038]

[0039] Example 1

[0040] 1. Experimental method

[0041] 8-week-old female C57BL / 6N mice (Beijing Huafukang Bioscience Co., Ltd.) were randomly divided into 4 groups: ① normal diet group (Normal); ② NASH modeling group (NASH); ③ NASH-ARL67156 treatment group (NASH-ARL); ④ NASH-Istradefylline treatment group (NASH-Istradefylline), 5 in each group.

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

[0043] The NASH-ARL67156 treatment group mice started intraperitoneal injection of ARL67156 (2 mg / kg, MCE, PBS soluble) at 6 weeks (at this time, the model has been successfully constructed by detecting common indicators in the field, and the criteria for successful model construction include obvious liver steatosis, significant fibrosis in the liver, significant increase in AST, ALT and other indicators in the plasma, and since the pathogenesis of NASH includes the accumulation of senescent cells, the expression of P16, P21 and other senescence-related genes will also be up-regulated, etc.) 200ul / time, every two days, a total of 14 times, 24 hours after the last injection, perfusion and take the required organs; The NASH-Istradefylline treatment group mice started intraperitoneal injection of Istradefylline (1 mg / kg, MCE) at 6 weeks, 200ul / time, every two days, a total of 14 times, 24 hours after the last injection, perfusion and take the required organs; At the same time, the normal diet group and the NASH modeling group mice were injected with PBS intraperitoneally, 200ul / time, every two days, a total of 14 times, 24 hours after the last injection, perfusion and take the required organs.

[0044] Lipid droplet accumulation and liver fibrosis analysis: 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 subjected to standard hematoxylin and eosin (H&E) and Sirius red staining. The stained tissue sections were observed under a microscope under 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, samples were centrifuged at 3,400g for 15 minutes at 4°C. Any serum sample with severe hemolysis was excluded from further analysis. Assay was performed with AST (E-BC-K236-M), ALT (E-BC-K235-M), PGE2 (E-EL-0034) kits, all from Elabscience.

[0046] qPCR: PCR reaction reagent was purchased from TaKaRa (RR820A), PCR reaction system and reaction program were shown in Table 2-3 as follows:

[0047] Table 2 PCR reaction system

[0048]

[0049] Table 3 PCR reaction program

[0050]

[0051] The primer sequences used in the PCR amplification were shown in Table 4 as follows:

[0052] Table 4 PCR amplification primer sequence

[0053]

[0054] 2. Experimental results

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

[0056] qPCR results( Figure 1 ) showed that the expression of aging-related genes in the NASH model group was significantly increased compared with the normal diet group, and Istradefylline treatment can significantly improve the increase of gene expression, wherein *p<0.05, **p<0.01.

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

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

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

[0060] The Sirius red staining results ( Figure 3 ) show that the liver fibrosis of the NASH model group is significantly higher than that of the normal diet group, and the Istradefylline treatment can reduce the symptoms of liver fibrosis and improve the liver structure.

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

[0062] The Elisa results ( Figure 4 ) show that the AST, ALT, PGE2 and other indicators of the NASH model group are higher than those of the normal diet group, and the Istradefylline treatment can improve these indicators, where *p<0.05, **p<0.01.

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

[0064] The qPCR results ( Figure 5 ) show that the expression of aging-related genes in the NASH model group is significantly higher than that in the normal diet group, and the ARL67156 treatment can significantly improve the increase in gene expression, where *p<0.05, **p<0.01.

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

[0066] The HE experimental results ( Figure 6 ) show that the accumulation of lipid droplets in the NASH model group is significantly increased compared with the normal diet group, and the ARL67156 treatment can reduce the accumulation of lipid droplets. At the same time, ARL67156 has no effect on the changes in the lungs.

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

[0068] The Sirius red staining results ( Figure 7 ) show that the liver fibrosis of the NASH model group is significantly higher than that of the normal diet group, and the ARL67156 treatment can reduce the symptoms of liver fibrosis and improve the liver structure.

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

[0070] The Elisa results ( Figure 8)indicate that the AST, ALT, PGE2 and other indicators of the NASH modeling group are increased compared with the normal diet group, and the ARL67156 treatment can improve these indicators, wherein, *p<0.05, **p<0.01.

[0071] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application defined by the claims.

Claims

1. Use of a CD39 inhibitor in the manufacture of a medicament for the treatment of nonalcoholic steatohepatitis, characterized in that, The CD39 inhibitor is ARL67156.

2. Use according to claim 1, characterized in that, The medicament also includes one or more pharmaceutically acceptable carriers.

3. Use according to claim 1, characterized in that, The medicament is administered by injection, including one or more of intraperitoneal injection, intramuscular injection, subcutaneous injection, and intravenous injection.

4. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment of nonalcoholic steatohepatitis, characterized in that, The pharmaceutical composition includes the CD39 inhibitor ARL67156.

5. Use according to claim 4, characterized in that, The pharmaceutical composition also includes one or more pharmaceutically acceptable carriers.

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