Application of compound AZD5718 in preparation of medicine for preventing and / or treating cholestatic liver disease

Abstract

The invention discloses application of a compound AZD5718 in preparation of a medicine for preventing and / or treating cholestatic liver diseases, and belongs to the field of biological medicines. The compound AZD5718 can effectively improve hepatocyte damage and bile metabolism disorder caused by Mdr2 gene deletion, provides solid experimental data support for developing novel drugs for treating cholestatic liver diseases, is expected to provide differentiated therapeutic drug choices for clinic, and has important medical value and social significance.

Description

TECHNICAL FIELD

[0001] The application relates to application of compound AZD5718 in preparation of a medicine for preventing and / or treating cholestatic liver disease and belongs to the field of biological medicine. BACKGROUND

[0002] Cholestatic liver disease is a group of heterogeneous diseases characterized by impaired bile production or flow, the pathophysiological mechanism of which mainly involves bile acid excretion disorder, resulting in abnormal accumulation of hydrophobic bile acids in the liver and systemic circulation. Long-term cholestasis can induce hepatocyte necrosis and apoptosis, and further activate hepatic stellate cells, leading to liver fibrosis, and eventually progress to cirrhosis and even liver failure. The existing treatment scheme for cholestatic liver disease has significant limitations. First-line drug ursodeoxycholic acid (UDCA) has poor response to some cholestatic liver disease patients, while second-line drug obeticholic acid (OCA) has the risk of inducing severe skin itching and potential liver damage. In addition, the current treatment drugs generally lack disease-modifying ability to effectively prevent or reverse liver fibrosis, and there is still a large unmet clinical treatment demand for diseases such as PSC and refractory cholestatic drug-induced liver injury.

[0003] Compound AZD5718 (Atuliflapon, CAS No. 2041075-86-7) has the chemical name of (1R, 2R)-2-[4-(5-methyl-1H-pyrazol-3-yl)benzoyl]-N-(4-oxo-6,7-dihydro-5H-pyrazolo[1,5-a]pyrazin-3-yl)cyclohexane-1-carboxamide, a molecular formula of C 24 H 26 N6O3, and is a highly selective 5-lipoxygenase activating protein (ALOX5AP) inhibitor. The known common use of compound AZD5718 is currently focused on the field of treating cardiovascular diseases such as coronary artery disease, and no related report of its use in cholestatic liver disease has been found. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide application of compound AZD5718 in preparation of a medicine for preventing and / or treating cholestatic liver disease.

[0005] In order to solve the above technical problems, the technical scheme of the present application is as follows: Application of compound AZD5718 in preparation of a medicine for preventing and / or treating cholestatic liver disease.

[0006] Optionally, the medicine further comprises a pharmaceutically acceptable carrier.

[0007] Optionally, the dosage form of the medicine comprises one of a tablet, a capsule, a powder, an injection, a powder, a syrup, a pill, a mixture, and a granule.

[0008] The present application also provides a medicament for treating cholestatic liver disease, comprising an effective dose of compound AZD5718 and a pharmaceutically acceptable carrier.

[0009] The present application confirms the pharmacodynamic value of compound AZD5718 in treating cholestatic liver disease through Mdr2 gene knockout (Mdr2- / -) mouse model. The research results prove the application potential of the compound from the following key dimensions: first, in terms of histopathology, the Masson trichrome staining results show that compound AZD5718 can effectively inhibit bile duct damage and the accompanying fibrosis process. Second, in terms of serum biochemistry, compared with the model group, the alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) levels of the compound AZD5718 administration group showed a significant downward trend. In summary, compound AZD5718 can effectively improve liver cell damage and bile metabolism disorder caused by Mdr2 gene deletion, providing solid experimental data support for the development of new drugs for treating cholestatic liver disease, and has significant clinical translation prospects.

[0010] Compared with the prior art, the present application has the following beneficial effects: 1. Based on the recognized cholestasis pathological model, Mdr2- / - mouse model, the present application confirms the therapeutic effect of compound AZD5718 on cholestatic liver disease from the dual dimensions of histopathology and serum biochemical indicators. The compound can effectively reduce liver cell damage, improve bile duct response and inhibit fibrogenesis, and functionally correct bile acid metabolism disorder. This synergistic effect on multiple pathological links provides a solid pharmacodynamic basis for clinical development.

[0011] 2. The chemical synthesis route of compound AZD5718 used in the present application is clear, the required precursor raw materials are easy to obtain, the preparation process is mature and has high controllability. This feature ensures the feasibility and cost advantage of large-scale production of drugs, and provides reliable material support for subsequent formulation research and development, clinical trials and future commercial production.

[0012] 3. The present application first discloses the application potential of compound AZD5718 in the field of cholestatic liver disease and liver fibrosis treatment, expanding its therapeutic indication range. The present application provides a potential solution to overcome the problems of insufficient response, obvious side effects or limited disease modification ability of existing drugs (such as UDCA, OCA), and is expected to provide differentiated treatment options for clinical practice, which has important medical value and social significance. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1Mdr2 - / - Masson's trichrome staining of the liver of mice with induced cholestatic liver disease.

[0014] Figure 2 Mdr2 - / - Graph of the proportion of positive areas of Masson's trichrome staining of the liver of mice with induced cholestatic liver disease.

[0015] Figure 3 Mdr2 - / - Graph of the changes in serum AST (left) and ALT (right) of mice with induced cholestatic liver disease.

[0016] Figure 4 Mdr2 - / - Graph of the changes in serum ALP of mice with induced cholestatic liver disease.

[0017] Figure 5 Mdr2 - / - Graph of the serum TBIL of mice with induced cholestatic liver disease. DETAILED DESCRIPTION

[0018] The application will be described in detail below with reference to the examples. It should be noted that the examples in the application and the features in the examples can be combined with each other without conflict.

[0019] Example 1 Compound AZD5718 for Mdr2 - / - Hepatoprotective effect of compound AZD5718 on mice with induced cholestatic liver disease: Experimental animals: Mdr2 - / - (FVB / NJ) mice, SPF level, 4 weeks old, male, purchased from Shanghai South Model Organism Technology Co., Ltd. Among them, Mdr2 - / - (FVB / NJ) mice were constructed using CRISPR / Cas9 technology, with FVB / NJ strain mice as background, and exon3-4 (encoding the key functional region) of Abcb4-201 transcript was designed as a strategy. Specific gRNA was designed in intron2 and intron4 regions to guide Cas9 nuclease to cut the target site, and NHEJ repair caused exon3-4 deletion, which caused reading frame shift and inactivated the protein function. The specific process is as follows: gRNA and Cas9 mRNA mixed solution was microinjected into fertilized eggs to obtain F0 generation, and positive F0 generation was identified by PCR+sequencing, then it was crossed with wild type to obtain F1 generation, and then Mdr2- / - (FVB / NJ) mice.

[0020] Drugs and reagents: AZD5718 (HY-122908, Shanghai Meixi Pharmaceutical Technology Co., Ltd.). Aspartate aminotransferase (AST) kit (C010-2-1), alanine aminotransferase (ALT) kit (C009-2-1), alkaline phosphatase (ALP) kit (A059-2-2), and total bilirubin (TBIL) kit (C019-1-1) were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0021] Main instruments: Precision electronic balance, Beijing Sartorius Scientific Instruments Co., Ltd.; Multifunctional microplate reader, ThermoFisher, USA; High-speed refrigerated centrifuge, ThermoFisher, USA; Inverted microscope, Zeiss, Germany.

[0022] Experimental methods: Experimental Grouping: The experiment was divided into two groups: the model group (Mdr2) - / - Mice, n=8; treatment group (Mdr2) - / - Mice + AZD5718, n=8).

[0023] Drug dissolution and dosage: AZD5718 is dissolved in 1% DMSO, 2% Tween 80 and 97% physiological saline (v:v:v) and administered by intraperitoneal injection at a dose of 6 mg / kg / day.

[0024] Experimental Procedure: For the treatment group, AZD5718 was administered via intraperitoneal injection at a dose of 6 mg / kg / day; for the model group, equal doses of 1% DMSO, 2% Tween 80, and 97% saline were administered intraperitoneally; all treatments were administered continuously for four weeks. Forty-eight hours after the last injection, mice were anesthetized with isoflurane, blood was collected by enucleation, and the mice were euthanized by cervical dislocation, and liver tissue and serum were collected.

[0025] Masson's trichrome staining: Liver paraffin sections were dewaxed and hydrated, then dewaxed in two separate tubs of xylene for 13 minutes each. Next, they were treated with a gradient of ethanol solutions (100 vol% in two tubs, 95 vol%, 90 vol%, 80 vol%, and 70 vol% in two tubs) for 4 minutes each, followed by rinsing with distilled water for 4 minutes. Ponceau S-Acid Fuchsin was then used to stain the cytoplasm and muscle fibers red. Phosphomolybdic acid solution was then used for color separation to wash away non-specific red dye from collagen fibers. Aniline blue staining was then applied to specifically mark the collagen fibers as blue. After brief differentiation with 0.5% glacial acetic acid, the sections were rapidly dehydrated in a gradient of ethanol solutions (70 vol% to 100 vol% in two tubs, 3 minutes per tub), cleared with xylene, and mounted with neutral resin. Images were acquired and observed using an inverted microscope. Under the microscope, collagen fibers appeared blue, while muscle fibers and cytoplasm appeared red. The area of ​​the positive region for Masson's trichrome staining was calculated and analyzed using ImageJ software (see results). Figure 2 —The area occupied by collagen fibers stained blue in liver tissue after Masson's trichrome staining reflects the severity of liver fibrosis. Comparisons between two groups were analyzed using t-tests (Graph Pad Prism 10.0, San Diego, CA, USA). Data are expressed as mean ± standard deviation. P <0.05 indicates a significant difference compared to the model group.

[0026] Experimental results: See Figure 1 and Figure 2 Masson's trichrome staining results showed that the model group (Mdr2) - / - Significant periportal and peribiliary fibrosis was observed in the liver of mice, with abnormal deposition and proliferation of collagen fibers along the damaged bile ducts. After intervention with AZD5718, the area of ​​collagen deposition in the portal vein region and peribiliary area of ​​the treated mice was significantly reduced, the fibrous septa became thinner and fewer, and liver fibrosis was improved.

[0027] Example 2 AZD5718 vs Mdr2 - / - Hepatoprotective effect of induced cholestatic liver disease in mice: Laboratory animal: Mdr2 - / - (FVB / NJ) mice, SPF grade, 4 weeks old, male, purchased from Shanghai Southern Model Biotechnology Co., Ltd. Among them, Mdr2... - / -The (FVB / NJ) mouse strain was constructed using CRISPR / Cas9 technology. Based on the FVB / NJ mouse line, a design strategy targeting exon 3-4 (encoding the critical functional region) of the Abcb4-201 transcript was employed. Specific gRNAs were designed in the intron 2 and intron 4 regions to guide Cas9 nuclease cleavage of the target sites. NHEJ repair resulted in exon 3-4 deletion, inducing a reading frame shift and inactivating the protein. The specific procedure involved preparing a mixture of gRNA and Cas9 mRNA, microinjecting it into fertilized eggs to obtain the F0 generation. Positive F0 generations were identified by PCR and sequencing and then mated with wild-type mice to obtain the F1 generation. Finally, Mdr2 mice were obtained through heterozygous crosses. - / - (FVB / NJ) mice.

[0028] Drugs and reagents: AZD5718 (HY-122908, Shanghai Meixi Pharmaceutical Technology Co., Ltd.). Aspartate aminotransferase (AST) kit (C010-2-1), alanine aminotransferase (ALT) kit (C009-2-1), alkaline phosphatase (ALP) kit (A059-2-2), and total bilirubin (TBIL) kit (C019-1-1) were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0029] Main instruments: Precision electronic balance, Beijing Sartorius Scientific Instruments Co., Ltd.; Multifunctional microplate reader, ThermoFisher, USA; High-speed refrigerated centrifuge, ThermoFisher, USA; Inverted microscope, Zeiss, Germany.

[0030] Experimental methods: Experimental Grouping: The experiment was divided into two groups: the model group (Mdr2) - / - Mice, n=8; treatment group (Mdr2) - / - Mice + AZD5718, n=8).

[0031] Drug dissolution and dosage: AZD5718 was dissolved in 1% DMSO, 2% Tween 80 and 97% physiological saline (v:v:v) and administered via intraperitoneal injection at a dose of 6 mg / kg·d.

[0032] Experimental Procedure: For the treatment group, AZD5718 was administered intraperitoneally at a dose of 6 mg / kg / day. For the model group, equal doses of 1% DMSO, 2% Tween 80, and 97% saline were administered intraperitoneally. All treatments were administered continuously for four weeks. Forty-eight hours after the last injection, mice were anesthetized with isoflurane, blood was collected by enucleation, and the mice were euthanized by cervical dislocation. Plasma samples were placed on ice for 1.5 hours, then centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was collected, and serum biochemical levels of AST, ALT, ALP, and TBIL were measured using the corresponding kits described above. Comparisons between the two groups were analyzed using t-tests (Graph Pad Prism 10.0, San Diego, CA, USA). Data are expressed as mean ± standard deviation. P <0.05 indicates a significant difference compared to the model group.

[0033] Experimental results: AST (aspartate aminotransferase) and ALT (alanine aminotransferase) are key indicators reflecting hepatocellular damage. Figure 3 The results showed that, compared with the model group, the treatment group (Mdr2) - / - The serum AST and ALT levels of AZD5718 mice were significantly reduced, indicating that AZD5718 can effectively alleviate hepatocyte damage and improve hepatocyte function.

[0034] ALP (alkaline phosphatase) is mainly expressed in hepatocyte membranes and bile duct epithelial cells. Elevated serum ALP levels often indicate bile duct obstruction or bile excretion dysfunction. Figure 4 Experimental results showed that after treatment with AZD5718, Mdr2 - / - The serum ALP level in mice was significantly lower than that in the model group, indicating that AZD5718 helps improve bile duct function and alleviate cholestatic damage.

[0035] TBIL (total bilirubin) levels reflect the status of bilirubin metabolism and excretion. Figure 5 The experimental results showed that the serum TBIL level in the treatment group mice was significantly lower than that in the model group, suggesting that AZD5718 can promote bilirubin metabolism and alleviate bile excretion disorders.

[0036] In summary, AZD5718 can significantly reduce Mdr2 - / - The abnormally elevated levels of AST, ALT, ALP, and TBIL in the serum of model mice systematically improved hepatocellular damage, bile duct dysfunction, and bile metabolism disorders, and inhibited the progression of liver fibrosis, providing clear biochemical pharmacodynamic evidence for their application in the preparation of drugs for the prevention and / or treatment of cholestatic liver disease.

[0037] The above embodiments should be understood as being used only to illustrate the present invention more clearly, and not to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims.

Claims

1. The use of compound AZD5718 in the preparation of drugs for the prevention and / or treatment of cholestatic liver disease.

2. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

3. The application according to claim 1, characterized in that, The dosage form of the drug includes one of the following: tablets, capsules, powders, injections, granules, syrups, pills, mixtures, and granules.

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