Application of prasugrel in preparation of medicine for treating or preventing hepatic fibrosis

Abstract

The invention discloses a novel application of an anti-platelet drug prasugrel or a pharmaceutically acceptable salt thereof in preparation of a drug for treating and / or preventing hepatic fibrosis. The invention finds that prasugrel can inhibit expression levels of Asporin, alpha-SMA and Collagen I in hepatic fibrosis tissues, effectively inhibit activation of hepatic stellate cells and relieve fibrosis progress, so that the diameter of hepatic fibrosis portal vein is reduced, hepatic echo is weakened, the hepatic tissue structure is improved, and inflammatory cell infiltration and collagen deposition are reduced. Therefore, the prasugrel has the effect of preventing and / or treating the hepatic fibrosis, a brand new treatment strategy is provided for the hepatic fibrosis, and the new application of the prasugrel is developed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a new medical use of a thienopyridine anti-platelet drug prasugrel, especially to an application of the thienopyridine anti-platelet drug prasugrel in preparation of a drug for treating or preventing liver fibrosis. BACKGROUND

[0002] Liver fibrosis is a common pathological change caused by various chronic liver injuries (such as chronic hepatitis B, non-alcoholic fatty liver disease, etc.), which is characterized by excessive deposition of extracellular matrix and may progress to severe diseases such as liver cirrhosis and liver cancer. At present, the treatment of liver fibrosis is facing great challenges, although various potential therapies (such as stem cell exosomes, S100A11 inhibitors, GDF15 modified macrophages, etc.) have shown anti-fibrosis effects in research, but no effective drug has been approved for clinical treatment. The pathogenesis of liver fibrosis involves multiple factors, including activation of hepatic stellate cells, hypoxic environment, metabolic reprogramming and inflammatory response, which makes the development of drugs targeting specific targets particularly important.

[0003] Prasugrel is a thienopyridine P2Y 12 receptor antagonist, which is clinically used only for preventing platelet aggregation and thrombotic events in patients with acute coronary syndrome. So far, there is no basic research or clinical application report on prasugrel for preventing and treating liver fibrosis at home and abroad. SUMMARY

[0004] In view of the problems in the prior art, the present application provides a new medical use of a thienopyridine anti-platelet drug prasugrel, especially an application of the thienopyridine anti-platelet drug prasugrel in preparation of a drug for treating or preventing liver fibrosis.

[0005] To achieve the object of the present application, the following technical solutions are adopted in the present application: Firstly, the present application provides an application of prasugrel or a pharmaceutically acceptable salt thereof in preparation of a drug for treating and / or preventing liver fibrosis.

[0006] Prasugrel, with English name Prasugrel and CAS number: 150322-43-3, has a chemical structural formula as shown in Figure 1 .

[0007] Preferably, the liver fibrosis is caused by viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, cholestasis, autoimmune liver disease, drug / toxic liver injury or metabolic disease. Preferably, the drug inhibits Asporin protein expression.

[0008] Asporin (ASPN) is a member of the aspartate-rich albumin family, initially discovered in cartilage tissue, where it participates in bone mineralization and extracellular matrix remodeling. Asporin can promote fibrosis by regulating the TGF-β signaling pathway, extracellular matrix (ECM) deposition, and fibroblast activation.

[0009] Preferably, the drug inhibits the expression of hepatic α-SMA and Collagen I, suppresses hepatic stellate cell activation, and alleviates the progression of fibrosis.

[0010] α-SMA is mainly expressed in activated hepatic stellate cells (HSCs), which transform from a resting state into myofibroblast-like cells during liver injury. Elevated α-SMA expression indicates active progression of liver fibrosis and is a commonly used immunohistochemical marker for assessing the degree of liver fibrosis.

[0011] Activated hepatic stellate cells (HSCs) are the primary source of Collagen I. During liver injury, HSCs synthesize and secrete large amounts of Collagen I. Collagen I is a major component of the extracellular matrix (ECM), and its excessive deposition leads to disordered liver lobule structure, ultimately resulting in liver fibrosis and even cirrhosis. The expression level of Collagen I is positively correlated with the degree of liver fibrosis and is widely used for evaluating animal models of liver fibrosis and for research on therapeutic targets.

[0012] Preferably, the dosage of prasugrel or its pharmaceutically acceptable salt is 0.08–0.17 mg / kg / day based on body weight.

[0013] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0014] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, and lubricants. The combination of at least two is, for example, a combination of binders and excipients, a combination of binders and flavoring agents, a combination of binders and fillers, etc. Other combinations are also possible and will not be elaborated here.

[0015] Preferably, the drug dosage form is a tablet, capsule, granule, injection, oral liquid, pill, ointment, suspension, dispersant, syrup, suppository, gel, aerosol, or patch.

[0016] Secondly, the present invention also provides a pharmaceutical combination for treating and / or preventing liver fibrosis, comprising prasugrel or a pharmaceutically acceptable salt thereof and pharmaceutical excipients that can be used to treat liver fibrosis.

[0017] Preferably, the pharmaceutical composition contains 0.001-99 wt% prasugrel or a pharmaceutically acceptable salt thereof.

[0018] Finally, the present invention also provides the use of prasugrel or a pharmaceutically acceptable salt thereof in the preparation of a reagent for in vitro inhibition of Asporin protein.

[0019] Beneficial effects of this invention: This invention, through animal experiments, found that prasugrel significantly improves liver tissue structure and reduces fibrosis-related indicators under both preventative and therapeutic intervention modes. Prasugrel inhibits the expression levels of Asporin, α-SMA, and Collagen I in fibrotic liver tissue, effectively suppressing hepatic stellate cell activation and mitigating fibrosis progression. Prasugrel can reduce the diameter of the portal vein and decrease liver echogenicity in fibrotic liver tissue, improve liver tissue structure, and reduce inflammatory cell infiltration and collagen deposition, indicating that prasugrel has a preventative and / or therapeutic effect on liver fibrosis. This provides a novel treatment strategy for liver fibrosis and develops a new application for prasugrel. Attached Figure Description

[0020] Figure 1 The chemical structural formula of prasugrel; Figure 2 The diagram shows the screening method and results for Asporin inhibitors. A is a schematic diagram of high-throughput screening of 3196 FDA-approved drugs based on LX2 cells with stable EGFP-ASPN expression; B is the Western blot validation results. Figure 3 The results show the therapeutic effect of prasugrel in a mouse model of liver fibrosis. In the figure, A is a schematic diagram of the experimental design, B is the Western blot result, C is the ultrasound imaging result, D is a photograph of the liver appearance, the lower part of E is the HE and Masson staining result, and the upper part of E and F are the immunohistochemical and immunofluorescence staining results. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0022] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0023] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0024] The following are some of the sources of reagents and consumables in the examples: Prasugrel was purchased from Selleck Biotechnology Co., Ltd. (S1258), carbon tetrachloride (CCl4) was purchased from MedChemExpress Shanghai Co., Ltd. (HY-Y0298; MCE; Shanghai, China), and C57BL / 6 mice were purchased from Changsheng Biotechnology Co., Ltd. (Liaoning, China).

[0025] Example 1: Screening of Asporin Inhibitors The human ASPN gene was fused with enhanced green fluorescent protein (EGFP) to construct an EGFP-ASPN expression plasmid, which was then transfected into the human hepatic stellate cell line LX2 to establish a cell model expressing the EGFP-ASPN fusion protein. These cells were seeded in 384-well plates, and a small molecule compound library consisting of 3196 FDA-approved drugs was added to each well. After 24 hours of drug treatment, changes in intracellular EGFP fluorescence intensity were detected using a fluorescence microplate reader. Based on the decrease in fluorescence signal, candidate drugs that may inhibit asporin expression or stability were initially obtained. The screening results were further validated using Western blot experiments (see schematic diagram of the asporin inhibitor screening method). Figure 2 A).

[0026] See results Figure 2 The results showed that one of the candidate compounds, prasugrel, significantly downregulated asporin protein expression. Figure 2 (B) suggests that it has the potential to be used as a potential inhibitor of Asporin.

[0027] Example 2: Therapeutic effect of prasugrel in a mouse model of liver fibrosis Experimental Modeling, Drug Administration, and Methods: Experimental animals were male C57BL / 6 mice (8-10 weeks old, approximately 20-25g in weight), provided by Changsheng Biotechnology (Liaoning, China). All animals were housed under specific pathogen-free (SPF) conditions with a 12h / 12h light / dark cycle, a temperature of 23±3°C, and a relative humidity of 30-70%, with free access to food and water. Control group: fed a normal diet and intraperitoneally injected with an equal volume of corn oil (carbon tetrachloride solvent); Model group: fed a high-fat diet (HFD) for 28 days, and intraperitoneally injected with carbon tetrachloride (10%, 2 ml / kg, dissolved in olive oil) on days 16, 19, 23, and 26 to simulate metabolic and toxin-related liver injury; Prasugrel group: in addition to the modeling treatment, prasugrel was administered by gavage at a dose of 2 mg / kg / day, every two days for two consecutive weeks, divided into preventative (days 1-42) and therapeutic (days 28-42) interventions. See the experimental design diagram. Figure 3 A.

[0028] 2. Detection Method After treatment, liver ultrasound was used for non-invasive assessment of fatty and fibrotic liver damage. Mice were anesthetized with intraperitoneal injection of Avertin (250 mg / kg), and a suitable amount of coupling gel was applied to the anterior chest. The probe was placed approximately 1 cm to the left of the axillary midline, with a probe frequency of 30 MHz. The probe was slowly moved until 3–4 branches of the left portal vein were observed. B-mode images were acquired using a modified short-axis view to visualize the upper lobe of the liver and the renal cortex; in addition, B-mode and M-mode images of the portal vein were simultaneously acquired in the long-axis view (Vevo®2000 system).

[0029] Mice were euthanized by cervical dislocation, and their livers were harvested and photographed. Proteins were extracted from a portion of the liver tissue, and the expression level of asporin was detected using Western blot. A portion of the liver tissue was fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin; sections were then cut to a thickness of 4 μm.

[0030] HE staining: Sections were dewaxed to water, stained with hematoxylin for 5 min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for 10-15 s, rinsed with running water, stained with 0.5% eosin for 30 s, dehydrated with graded alcohols, cleared with xylene, and mounted with neutral resin for assessing liver tissue structure and inflammatory cell infiltration.

[0031] Masson staining: Following the instructions of the kit (Solarbio, Beijing, China), hematoxylin staining of the nucleus, acid fuchsin staining of the cytoplasm, and tryptophan blue staining of collagen fibers were performed sequentially to assess the degree of collagen deposition in liver tissue.

[0032] Immunohistochemistry (IHC): Sections were subjected to high-temperature antigen retrieval with citrate buffer (pH 6.0), followed by incubation with 3% hydrogen peroxide at room temperature for 10 min to block endogenous peroxidase, and then blocking with 10% goat serum at room temperature for 30 min. Primary antibodies were added: anti-Collagen I (Proteintech, 14695-1-AP), anti-α-SMA (Abconal Inc., BF9212), and anti-Asporin (Affinity, DF13642), and incubated overnight at 4°C. The following day, after washing with PBS, HRP-labeled secondary antibody was added and incubated at room temperature for 30 min. DAB staining was performed, and the nuclei were counterstained with hematoxylin. The sections were then dehydrated and mounted for detection of fibrosis-related protein expression.

[0033] Immunofluorescence (IF): After antigen retrieval and blocking with goat serum, sections were incubated overnight at 4 °C with the above primary antibody, washed with PBS, and then incubated at room temperature for 1 h with fluorescently labeled secondary antibody. Nuclei were stained with DAPI for 5 min, and the sections were mounted with anti-fluorescence quenching. The sections were observed and photographed under a fluorescence microscope to assess the localization and expression changes of α-SMA and Collagen I.

[0034] 3. Experimental Results The experimental results are shown in Figure 3 , Figure 3 B shows the results of Western blot, which indicate that the expression level of Asporin in the liver tissue of mice in the model group was significantly increased, while prasugrel intervention could significantly downregulate its expression. Figure 3 C represents the ultrasound imaging results. In the model group, the liver parenchyma showed increased echogenicity and portal vein dilation, indicating liver tissue remodeling and portal hypertension. In the prasugrel intervention group, the above abnormalities were significantly reduced, as shown by a decrease in portal vein diameter and decreased liver echogenicity. Figure 3 Photograph D shows the appearance of the liver, indicating that the liver surface of mice treated with prasugrel was smoother and the number of nodules was reduced. Figure 3 The lower half of E shows the results of HE and Masson staining, suggesting that prasugrel can improve liver tissue structure and reduce inflammatory cell infiltration and collagen deposition. Figure 3 upper part of E and Figure 3 F represents the results of immunohistochemistry and immunofluorescence staining, showing that prasugrel intervention can reduce the expression of α-SMA and CollagenI in the liver of model mice, indicating that it can effectively inhibit the activation of hepatic stellate cells and alleviate the progression of fibrosis.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of prasugrel or a pharmaceutically acceptable salt thereof in the preparation of drugs for the treatment and / or prevention of liver fibrosis.

2. The application according to claim 1, characterized in that, The liver fibrosis is caused by viral hepatitis, alcoholic liver disease, non-alcoholic steatohepatitis, cholestasis, autoimmune liver disease, drug / toxic liver injury, or metabolic diseases.

3. The application according to claim 1, characterized in that, The drug inhibits the expression of Asporin protein.

4. The application according to claim 1, characterized in that, The drug inhibits the expression of hepatic α-SMA and Collagen I, suppresses hepatic stellate cell activation, and reduces the progression of fibrosis.

5. The application according to claim 1, characterized in that, The dosage of prasugrel or its pharmaceutically acceptable saline form is 0.08–0.17 mg / kg / day based on body weight.

6. The application according to any one of claims 1-5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application according to any one of claims 1-5, characterized in that, The drug dosage forms are tablets, capsules, granules, injections, oral liquids, pills, ointments, suspensions, dispersants, syrups, suppositories, gels, aerosols, or patches.

8. A pharmaceutical combination for treating and / or preventing liver fibrosis, comprising prasugrel or a pharmaceutically acceptable salt thereof and pharmaceutical excipients that can be used to treat liver fibrosis.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition contains 0.001-99 wt% prasugrel or a pharmaceutically acceptable salt thereof.

10. Use of prasugrel or a pharmaceutically acceptable salt thereof in the preparation of reagents for the in vitro inhibition of Asporin protein.

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