Use of indobufen in the preparation of a drug for non-alcoholic fatty liver

Abstract

The application discloses application of indobufen in preparation of non-alcoholic fatty liver disease drugs, and particularly relates to application of indobufen or a pharmaceutically acceptable derivative thereof in preparation of blood lipid-lowering drugs, drugs for reducing accumulation of body fat, and drugs for treating or preventing non-alcoholic fatty liver disease. The indobufen can be used for inhibiting lipid accumulation, inflammatory cell infiltration and liver fibrosis of the liver, can reduce levels of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in blood, has a significant slowing effect on non-alcoholic fatty liver, and can effectively reduce body weight and improve blood lipid levels, thereby opening up a new road for treating fatty liver disease, liver function damage, obesity and blood lipid abnormality and other metabolic diseases, and providing a new indication for clinical application of the indobufen, and opening up a potential and good application prospect of the indobufen in the field of liver disease and metabolic disorder treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of indobufen in preparation of a medicine for preventing, relieving and / or treating fatty liver disease and related metabolic diseases. BACKGROUND

[0002] Fatty liver disease refers to a pathological change of excessive accumulation of fat in liver cells caused by various reasons, and is a common liver pathological change rather than an independent disease. Fatty liver disease is generally divided into two categories of alcoholic fatty liver disease and non-alcoholic fatty liver disease (NAFLD), and non-alcoholic fatty liver disease refers to a chronic liver disease caused by excessive accumulation of fat in the liver without excessive alcohol drinking and other factors causing liver steatosis. NAFLD is usually related to metabolic disorders such as insulin resistance, type 2 diabetes, dyslipidemia or obesity, and is usually manifested as simple liver steatosis in the early stage. With the progression of the disease, about 20% of patients will develop non-alcoholic steatohepatitis, which will increase the risk of developing cirrhosis, liver failure and hepatocellular carcinoma. With the change of lifestyle and dietary structure, the prevalence of NAFLD is increasing year by year, affecting 25% of adults worldwide.

[0003] Indobufen is currently known to have the functions of anti-platelet, anticoagulation, anti-renal fibrosis, vasodilation, and inhibition of monocyte tissue factor (TF) expression and activity. It is mainly used in the clinic to treat coronary heart disease, ischemic cerebrovascular disease, and graft vessel patency, and is also used to treat type 2 diabetes, improve renal function, and resist renal fibrosis. The latest reports have found that indobufen or aspirin combined with clopidogrel / cell adhesion factor (CAM) or ticagrelor pretreatment can reduce inflammasome-mediated cerebral middle artery occlusion / reperfusion (MCAO / R) rats and oxygen-glucose deprivation / reperfusion (OGD / R PC12) cell pyroptosis through the NF-κB / NLRP3 signaling pathway. These findings suggest that the drug combination may be an effective strategy for the treatment of ischemic stroke. However, there is no report on the therapeutic effect of indobufen on NAFLD. SUMMARY

[0004] The purpose of the present application is to provide a new use of indobufen.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides the use of indobufen or a pharmaceutically acceptable derivative thereof in the preparation of a hypolipidemic drug.

[0007] Further, the hypolipidemic agent is used to inhibit the increase in blood lipid caused by high-fat diet, and further, the blood lipid is cholesterol and / or triglyceride.

[0008] The present application also provides the use of indobufen or a pharmaceutically acceptable derivative thereof in the preparation of a medicament for reducing body fat accumulation; further, the medicament for reducing body fat accumulation is used to inhibit the increase in body fat accumulation caused by high-fat diet, and in particular, for the prevention of overweight, obesity, and dyslipidemia.

[0009] The present application also provides the use of indobufen or a pharmaceutically acceptable derivative thereof in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease in a patient.

[0010] Further, the non-alcoholic fatty liver disease is one or more of simple hepatic steatosis, non-alcoholic steatohepatitis, fatty liver fibrosis, liver cirrhosis, liver failure, and hepatocellular carcinoma.

[0011] The patient has one or more conditions selected from the group consisting of insulin resistance, type 2 diabetes, dyslipidemia, overweight, and obesity.

[0012] Further, the non-alcoholic fatty liver disease is caused by high-fat diet.

[0013] Further, indobufen or a pharmaceutically acceptable derivative thereof is the main effective component of the medicament.

[0014] Further, indobufen or a pharmaceutically acceptable derivative thereof is the only effective component of the medicament.

[0015] Further, the non-alcoholic fatty liver disease is non-alcoholic simple fatty liver, non-alcoholic steatohepatitis (NASH), liver fibrosis, or liver cirrhosis.

[0016] Preferably, in any of the above uses, the medicament is a composition containing indobufen or a pharmaceutically acceptable derivative thereof, indobufen or a pharmaceutically acceptable derivative thereof is the main effective component or the only effective component of the medicament. Preferably, the above composition is a medicament prepared by taking indobufen as the active ingredient and adding pharmaceutically acceptable excipients.

[0017] The pharmaceutically acceptable excipients according to the present application refer to various conventional excipients required during the preparation of different dosage forms, such as diluents, binders, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorption materials, etc. to prepare any commonly used oral preparation by conventional preparation methods, for example, granules, powders, tablets, capsules, pills, oral liquids, decoctions, dripping pills, etc.

[0018] The present application first shows through pharmacological experiments that indobufen can be used to effectively treat liver diseases and related metabolic diseases, slow down liver lipid accumulation, inflammatory response and fibrosis, has a significant slowing effect on non-alcoholic fatty liver, and can reduce the levels of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in blood, reduce the body weight of the body, and improve blood lipid levels, thereby opening up a potential and good application prospect for treating fatty liver disease and metabolic disorders. At the same time, indobufen has been applied to the clinical treatment of coronary heart disease, ischemic cerebrovascular disease, and patent vessel patency, and has small side effects and relatively low cost compared with the development of new drugs. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings are used to provide an understanding of the technical solutions of the present application, and constitute a part of the specification, and together with the embodiments of the present application, are used to explain the technical solutions of the present application, and do not constitute a limitation on the technical solutions of the present application.

[0020] Figure 1 A high-content imaging analysis diagram of liver cell nile red staining after administration of indobufen.

[0021] Figure 2 A graph of the changes in the body weight and body weight gain of mice after administration of indobufen (**, P<0.01).

[0022] Figure 3 A graph of the detection results of the liver weight of mice after administration of indobufen (**, P<0.01).

[0023] Figure 4 A graph of the changes in the TG and TC content in the serum of mice after administration of indobufen (*, P<0.05;

[0024] **, P<0.01).

[0025] Figure 5 A graph of the HE and oil red O staining results of the liver tissue of mice after administration of indobufen.

[0026] Figure 6 A graph of the CD11b immunofluorescence staining results of the liver tissue of mice after administration of indobufen.

[0027] Figure 7 A graph of the PSR staining results of the liver tissue of mice after administration of indobufen.

[0028] Figure 8 A graph of the changes in the AST and ALT content in the serum of mice after administration of indobufen (*, P<0.05; **, P<0.01). DETAILED DESCRIPTION

[0029] For the purposes of the present application, the technical solutions and advantages will be more clearly apparent in the following detailed description of embodiments of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other in any manner without conflict.

[0030] 1. Experimental animals and feeding

[0031] Experimental animals: 8-10 weeks old, body weight of 21.0-25.0 g male C57BL / 6 mice were selected as experimental objects.

[0032] Feeding environment: SPF level experimental animal center, SPF level mouse feed was purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0033] Feeding conditions: room temperature was between 22-24℃, humidity was between 40-70%, light and dark alternating lighting time was 12h, and free water and food were taken.

[0034] 2. Experimental cells and schemes

[0035] Experimental cells: human liver cell line L02 (human normal liver cells) was selected as the experimental object. Human liver cell line L02 was purchased from the Chinese Academy of Sciences Cell Bank.

[0036] Culture of human liver cell line L02: in DMEM high glucose culture medium (containing 10% fetal bovine serum

[0037] +1% double antibody, hereinafter referred to as culture medium), 37℃, 5% CO2 environment.

[0038] Experimental steps:

[0039] (1) Plate cells: observe the state of the cells, select cells with good state and vitality, digest, centrifuge and resuspend the cells, count the cells, and then dilute the cells according to the cell density of 8000 cells per well and uniformly plate them in a high-content cell imaging microwell plate.

[0040] (2) Drug pretreatment: after the plated cells grow adherently for 12h, 20μM indomethacin is added to the culture medium (0.5% bovine serum albumin) for pretreatment of the cells.

[0041] (3) Palmitic acid (PA) / oleic acid (OA) induced lipid droplet accumulation: after 8h of drug pretreatment, palmitic acid and oleic acid are added to the culture medium, and the concentration of PA in the culture medium is 0.5mM PA, the concentration of OA is 1.0mM OA, and the concentration of indomethacin is 20μM, and the cells are treated to induce the production of lipid droplets.

[0042] (4) Staining: After 18h of induction treatment, the cell waste liquid was discarded, and the PBS was washed twice. The paraffin specimen was fixed with 4% paraformaldehyde at room temperature for 30min, and then the fixed liquid was discarded. The PBS was washed twice, and Nile red was used for staining for 5min. The staining liquid was discarded, and the PBS was washed twice. Hoechst was used for staining for 5min, and the PBS was washed twice.

[0043] (5) The high-content cell imaging system was used for on-machine detection.

[0044] (6) Data analysis and plotting.

[0045] 3. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC) level detection

[0046] After the mice were anesthetized, blood was taken through the orbital plexus, and the blood sample was placed at room temperature for 30min, and then serum was separated by centrifugation. The serum was used for detecting alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC) levels by using a blood biochemical instrument.

[0047] 4. Pathological detection

[0048] The main operation procedures for preparing paraffin specimen sections are as follows: trimming the liver → processing the embedding frame → water flushing → dehydration → transparency → embedding → sectioning → spreading the section → drying or baking for standby.

[0049] The main steps of hematoxylin-eosin (H&E) staining, CD11b staining, oil red O (Oil red O) staining and Sirius red (PSR) staining used in the embodiments of the present application are described as follows:

[0050] The main steps of hematoxylin-eosin (H&E) staining are as follows: the paraffin specimen section is deparaffinized to water → hematoxylin is used for staining the cell nucleus → eosin is used for staining the cytoplasm → dehydration and mounting → microscopic examination, and image acquisition and analysis.

[0051] The main steps of CD11b staining are as follows: after the paraffin section is baked, deparaffinized, hydrated and repaired, the mouse liver section is first blocked with 10% goat serum, and then the primary antibody (Boster, BM3925) is used for incubation at 4℃ overnight. After incubation, the PBS is used for washing, and then the secondary antibody (goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Thermo Fisher Scientific, A-11011, Massachusetts, USA)) is used for incubation at 37℃ for 1h. Finally, 4', 6-diamidino-2-phenylindole (DAPI) is used for staining the nucleus.

[0052] The main steps of Oil red O staining are as follows: after drying the section, wash it in 50% ethanol, then apply Oil red O ethanol dyeing solution, differentiate in 50% ethanol, terminate differentiation in tap water, re-stain the nucleus with hematoxylin, return blue in tap water, and seal the section with glycerol gelatin.

[0053] The main steps of Sirius red (PSR) staining are as follows: deparaffin the paraffin section to water, drop dye with Sirius red staining solution, slightly wash with running water to remove the dyeing solution on the surface of the section, dye the cell nucleus with Mayer's hematoxylin staining solution, wash with running water, and seal the section after dehydration.

[0054] Example 1 Effect of indobufen treatment on fat accumulation in hepatocytes

[0055] The hepatocytes were divided into two groups, PA / OA group and PA / OA+indobufen group. After the cells adhered for 12 h, 20 μM indobufen was added to the culture medium of the PA / OA+indobufen group, and the drug was pretreated for 8 h; then 0.2 mM PA and 0.4 mM OA were added to the culture medium, and the treatment was performed for 18 h. The PA / OA group treated with simple PA / OA was used as a control group. After the treatment of the PA / OA group and the PA / OA+indobufen group, Nile red staining was performed, and high-throughput high-content cell imaging system was used for imaging analysis.

[0056] Table 1 Experiment for verifying the effect of indobufen treatment on fat accumulation in hepatocytes

[0057]

[0058] Figure 1 Figure 1 shows the high-content imaging analysis of Nile red staining of hepatocytes after indobufen administration, wherein the white highlighted area is the Nile red positive cytoplasmic region, and the gray color is the nucleus. Figure 1 Figure 2 shows that compared with the PA / OA group, the relative fluorescence intensity of fat droplets in the PA / OA+indobufen group was significantly reduced, Figure 1 Figure 3 shows that the number of cells was basically unchanged. It is indicated that indobufen can significantly inhibit the lipid accumulation in hepatocytes, and no toxic side effects are observed.

[0059] Example 2 Effect of indobufen on non-alcoholic fatty liver disease induced by high-fat high-cholesterol diet

[0060] C57 male mice (a total of 34) were randomly divided into 2 groups (17 in each group, HFHC-control group and HFHC-indobufen group respectively), after feeding with high-fat high-cholesterol diet for 4 weeks, the two groups of mice were given drugs, the HFHC-indobufen group was given indobufen (1wt% carboxymethylcellulose sodium solution containing indobufen) at a dose of 120mg / kg / d, the HFHC-control group was given blank drug (1wt% carboxymethylcellulose sodium solution) with the same volume of indobufen, the frequency of administration was once a day, and the high-fat high-cholesterol diet was continued at the same time.

[0061] After 12 weeks of administration, the body weight and liver weight of the two groups of mice were measured, and the body weight gain and liver weight / body weight ratio were calculated; the serum of the two groups of mice was taken, and the contents of ALT, AST, TG and TC in the serum were measured; and the liver tissue was pathologically detected.

[0062] Figure 2 For the body weight detection results of the HFHC-indobufen group of mice and the body weight gain calculation results, compared with the HFHC-control group, after giving indobufen drug gavage, the increase of body weight of mice induced by high-fat high-cholesterol diet can be significantly inhibited, which shows that indobufen has inhibitory effect on individual fat accumulation and obesity. Figure 3 For the liver weight detection results of mice, the liver weight of the HFHC-indobufen group of mice was significantly lower than that of the HFHC-control group.

[0063] Figure 4 For the TG and TC content detection results of mouse serum, the lipid content in the serum of the HFHC-indobufen group of mice was significantly lower than that of the HFHC-control group.

[0064] Figure 5 For the HE and oil red O staining results of mouse liver tissue, it can be seen that the fatty degeneration and lipid deposition of the liver of the HFHC-indobufen group of mice are significantly reduced. These results show that indobufen has obvious inhibitory effect on liver lipid deposition induced by high-fat diet.

[0065] Figure 6 For the CD11b immunofluorescence staining results of mouse liver tissue, it can be seen that the number of CD11b positive cells in the liver of the HFHC-indobufen group of mice is significantly lower than that of the HFHC-control group, which shows that giving indobufen can significantly inhibit the infiltration of inflammatory cells in the liver, inhibit liver inflammation and inflammation-related diseases.

[0066] Figure 7 For the Sirius red (PSR) staining results of mouse liver tissue, it can be seen that the Sirius red positive area in the liver of the HFHC-indobufen group of mice is significantly less than that of the HFHC-control group, which shows that giving indobufen can significantly inhibit the production of collagen fibers in the liver and inhibit liver fibrosis.

[0067] Figure 8 For the results of serum ALT and AST detection of mice, when hepatocytes are damaged, ALT and AST are released into the blood, resulting in an increase in the content of both in serum. Figure 8 The results in Table 6 show that the content of ALT and AST in serum of mice in the HFHC-indoprofen group is significantly lower than that in the HFHC-control group, indicating that indoprofen has the effect of inhibiting liver damage and protecting liver function.

[0068] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. Use of indobufen in the manufacture of a medicament for the prevention and / or treatment of non-alcoholic fatty liver disease in a patient.

2. Use according to claim 1, characterized in that: The patient has one or more conditions selected from the group consisting of insulin resistance, type 2 diabetes, dyslipidemia and obesity.

3. Use according to claim 1, characterized in that: The non-alcoholic fatty liver disease is caused by a high-fat diet.

4. The use according to any one of claims 1 to 3, characterized in that: Indobufen as the sole active ingredient of the medicament.

5. Use according to claim 4, characterized in that: Indobufen as the sole active ingredient of the medicament.

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