Medicine for improving non-alcoholic fatty liver disease

Abstract

The invention relates to the field of biological medicines, in particular to a medicine for improving non-alcoholic fatty liver diseases. The invention provides application of ML-SA1 to preparation of a medicine for treating a non-alcoholic fatty liver disease. The application accidentally finds that ML-SA1 can improve the non-alcoholic fatty liver disease, which is specifically embodied in reducing lipid droplet accumulation in hepatic cells, reducing liver weight, relieving hepatic cell vacuolation and hepatic tissue fibrosis, and improving related pathological indexes of the non-alcoholic fatty liver disease. The application also finds that the combination of ML-SA1 and other substances can more significantly improve the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a drug for improving non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the world. In the coming decades, NAFLD will become the leading cause of end-stage liver disease. The growth rate of total NAFLD cases ranges from 0% to 30% between 2016 and 2030. Among them, the total prevalence of NAFLD in Asia is 29.62%, which is more than 16 times that of primary liver cancer. NAFLD is a general term that covers a wide range of liver diseases, from nonalcoholic fatty liver (NAFL), liver fibrosis, cirrhosis to hepatocellular carcinoma (HCC). NAFLD begins with abnormal accumulation of lipid droplets in the liver, triggering lipotoxicity, endoplasmic reticulum stress and inflammatory response, and progresses to cirrhosis and even liver cancer. Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD, characterized by liver steatosis, cavitation and / or fibrosis.

[0003] Clinically, there are currently no approved drugs that directly target nonalcoholic fatty liver disease (NAFLD). Current treatments are designed to increase systemic insulin sensitivity, such as thiazolidinediones, rosiglitazone, and metformin, and / or reduce food intake, such as GLP-1 receptor agonists (exenatide and liraglutide)3,5-7. However, these treatments have unwanted off-target effects. For obesity and type 2 diabetes-associated NAFLD, insulin resistance in adipose tissue impedes insulin-mediated suppression of lipolysis, thereby increasing serum free fatty acid (FFA) levels. As lipid droplets accumulate, free fatty acids are metabolized by the liver and esterified into neutral triglycerides. However, excess saturated fatty acids exceed the liver's ability to esterify fatty acids, resulting in hepatocellular toxicity (i.e., lipotoxicity).

[0004] Therefore, there is a need to develop a drug that can directly target nonalcoholic fatty liver disease. Summary of the invention

[0005] In view of the above-mentioned disadvantage that there is no approved drug directly targeting non-alcoholic fatty liver disease in the prior art, the purpose of the present application is to provide a drug for improving non-alcoholic fatty liver disease, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose and other related purposes, the first aspect of the present application provides the use of ML-SA1 in preparing a drug for treating non-alcoholic fatty liver disease.

[0007] In some embodiments, the drug for treating non-alcoholic fatty liver disease includes at least one of the following functions:

[0008] 1) Reduce lipid droplet accumulation in hepatocytes;

[0009] 2) Reduce liver weight;

[0010] 3) Alleviate hepatocyte vacuolization;

[0011] 4) Alleviate liver fibrosis;

[0012] 5) Restore liver cell function;

[0013] 6) Relieve the symptoms of fatty liver;

[0014] 7) Improve the pathological indicators of non-alcoholic fatty liver disease.

[0015] In some embodiments, the pathological indicators include a combination of one or more of total cholesterol concentration in serum, cholesterol in low-density lipoprotein in serum, cholesterol concentration in high-density lipoprotein in serum, total cholesterol concentration in liver tissue, aspartate aminotransferase activity in liver tissue, and alanine aminotransferase activity in liver tissue.

[0016] The second aspect of the present application provides a composition comprising ML-SA1 and other substances, wherein the other substances include ML-SA5 and / or Ambroxol.

[0017] The third aspect of the present application provides the use of the aforementioned composition in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0018] The fourth aspect of the present application provides a drug for treating non-alcoholic fatty liver disease, comprising an effective dose of ML-SA1 in the aforementioned use or the aforementioned combination.

[0019] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0020] The fifth aspect of the present application provides a method for treating non-alcoholic fatty liver disease, which comprises administering the aforementioned drug to a subject.

[0021] In some embodiments, the subject is a mammal.

[0022] In some embodiments, the mammal is selected from the order rodent, artiodactyl, perissodactyl, lagomorph, or primate.

[0023] Compared with the prior art, the beneficial effects of this application are:

[0024] 1. The present application unexpectedly discovered that ML-SA1 can improve non-alcoholic fatty liver disease, which is specifically reflected in reducing the accumulation of lipid droplets in hepatocytes, reducing liver weight, alleviating hepatocyte vacuolation and liver tissue fibrosis, and improving relevant pathological indicators of non-alcoholic fatty liver disease.

[0025] 2. The present application also unexpectedly discovered that the two drugs ML-SA5 and Ambroxol can significantly improve non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 To construct a mouse model of non-alcoholic fatty liver disease. Figure 1 a shows the weight changes of mice fed with normal diet and high-fat diet from 2 months of age to 12 months of age. Figure 1 b is the anatomy of liver tissue in high-fat-induced obese mice. Figure 1 c is a statistical chart of liver tissue weight. Figure 1 d is H&E, Masson and Oil Red O staining of liver tissue, white arrows indicate vacuoles in liver tissue, and black arrows indicate tissue fibrosis. ND: Normal diet; HFD: high-fat diet; PTM: peritubular myoid cells.

[0027] Figure 2 It is a liver cell that simulates a high-fat environment in vitro. Figure 2 a is Oil Red O staining of AML12 cell line. Figure 2 b is Figure 2 Statistics of the area of ​​lipid droplets in each cell in a. FFA: free fatty acid.

[0028] Figure 3 ML-SA1 improves liver cell lines in high-fat environment in vitro. Figure 3 a is Oil Red O staining of AML12 cell line. Figure 3 b is Figure 3 Statistics of the area of ​​lipid droplets in each cell in a. FFA: free fatty acid.

[0029] Figure 4 ML-SA1 improves non-alcoholic fatty liver disease in mice. Figure 4 a is the anatomical diagram of mouse liver tissue. Figure 4 b is the statistical chart of liver tissue weight. Figure 4c is H&E, Masson and Oil Red O staining of liver tissue. White arrows indicate vacuoles in liver tissue, and black arrows indicate tissue fibrosis.

[0030] Figure 5 Other related manifestations of ML-SA1 improving non-alcoholic fatty liver disease in mice. Figure 5 a is the cholesterol content in serum. Figure 5 b is the content of low-density lipoprotein cholesterol in serum. Figure 5 c is the content of high-density lipoprotein cholesterol in serum. Figure 5 d is the AST activity in liver tissue. Figure 5 e is the ATL activity in liver tissue. Figure 5 f is the total cholesterol content in liver tissue. HFD: high-fat diet; LDL-c: low-density lipoprotein cholesterol; HDL-c: high-density lipoprotein cholesterol; AST: aspartate aminotransferase; ATL: alanineaminotransferase.

[0031] Figure 6 To add two drugs to improve the effects of lysosomes on AML12 cells. Among them, Figure 6 a shows the effect of drug addition on lipid droplets of AML12 cells. Figure 6 b is Figure 6 a Statistics of lipid droplet area to total cell area. FFA: free fatty acid. DETAILED DESCRIPTION

[0032] In order to make the invention purpose, technical scheme and beneficial effect of the present application clearer, the present application is further described below in conjunction with the examples. It should be understood that the examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present application from the contents disclosed in this description.

[0033] After extensive exploration and research, the inventor of this application unexpectedly discovered a drug for improving non-alcoholic fatty liver disease, and completed this application on this basis.

[0034] On the one hand, the present application provides the use of ML-SA1 in preparing a drug for treating non-alcoholic fatty liver disease.

[0035] ML-SA1 acts as a selective agonist of TRPML, a member of the mucolipid subfamily of channels that release Ca 2+ Cation channels are important regulators of transport and autophagy-related events. Non-alcoholic fatty liver disease is one of the most common liver diseases in the world today, affecting 20% ​​to 30% of the general population. Multiple genetic and epigenetic factors contribute to the onset of NAFLD. Untreated NAFLD will turn into non-alcoholic steatohepatitis (NASH), which can develop into cirrhosis, which may seriously damage the function of the liver, and a small part may deteriorate into liver cancer. In some embodiments of the present application, the present application unexpectedly discovered that ML-SA1 can improve non-alcoholic fatty liver disease. Under the action of ML-SA1, the manifestations of obesity-induced non-alcoholic fatty liver disease (NAFLD) in mice can be reversed.

[0036] In the use provided in this application, the functions of the drug for treating non-alcoholic fatty liver disease are mainly reflected in the following aspects:

[0037] 1) Reduce lipid droplet accumulation in hepatocytes;

[0038] 2) Reduce liver weight;

[0039] 3) Alleviate hepatocyte vacuolization;

[0040] 4) Alleviate liver fibrosis;

[0041] 5) Restore liver cell function;

[0042] 6) Relieve the symptoms of fatty liver;

[0043] 7) Improve the pathological indicators of non-alcoholic fatty liver disease.

[0044] In the use provided in the present application, the pathological index includes a combination of one or more of the total cholesterol concentration in serum, the cholesterol in low-density lipoprotein in serum, the cholesterol concentration in high-density lipoprotein in serum, the total cholesterol concentration in liver tissue, the aspartate aminotransferase activity in liver tissue, and the alanine aminotransferase activity in liver tissue. In a specific embodiment of the present application, under the action of the ML-SA1 drug, the pathological indexes related to the symptoms of fatty liver disease have a significant degree of decrease.

[0045] Another aspect of the present application provides a composition, including ML-SA1 and other substances, wherein the other substances include ML-SA5 and / or Ambroxol. In some embodiments, the combination of ML-SA1 and other substances can more significantly improve non-alcoholic fatty liver disease.

[0046] In the composition provided by the present application, ML-SA5 is a potent TRPML1 cation channel agonist that can activate the overall activity of phagolysosomes in DMD muscle cells. ML-SA5 has anti-cancer activity and can inhibit tumor growth. The present application discloses for the first time that ML-SA5 can be used to treat non-alcoholic fatty liver disease.

[0047] In the composition provided by the present application, Ambroxol (NA-872) is an active metabolite of the precursor bromhexine, and has a strong expectorant effect. Ambroxol is a glucocerebrosidase (GCase) chaperone that can increase the activity of glucocerebrosidase. Ambroxol can induce pulmonary autophagy and has the potential to be used in the study of Parkinson's disease and neurological Gaucher disease. The present application discloses for the first time that Ambroxol can be used for the treatment of non-alcoholic fatty liver disease.

[0048] Another aspect of the present application provides the use of the aforementioned composition in preparing a drug for treating non-alcoholic fatty liver disease. In a specific embodiment of the present application, the composition can reduce the accumulation of lipid droplets in liver cell lines under a high-fat environment.

[0049] On the other hand, the present application provides a drug for treating non-alcoholic fatty liver disease, comprising an effective dose of ML-SA1 in the aforementioned use, or the aforementioned combination.

[0050] In some embodiments, the effective dose refers to the dose at which the drug can show its efficacy. Because the drug must be absorbed by the body at a certain dose before a certain drug concentration can be reached, and the drug effect can only appear when a certain drug concentration is reached. If the dose is too small, the effective concentration cannot be obtained in the body, and the drug cannot play its effective role. However, if the dose is too large and exceeds a certain limit, the effect of the drug may change qualitatively, and may produce different degrees of toxicity to the body. Therefore, in order to play the effective role of the drug while avoiding its adverse reactions, it is necessary to strictly control the dosage range of the drug.

[0051] The medicine provided in the present application also includes a pharmaceutically acceptable carrier or excipient.

[0052] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human.

[0053] "Pharmaceutically acceptable carriers or excipients" should be compatible with lysosomal regulatory factors, that is, they can be mixed with them without significantly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0054] The drugs provided in the present application can be adapted to any form of administration, which can be oral or parenteral administration, for example, can be pulmonary, nasal, rectal and / or intravenous injection, more specifically can be intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, oral, sublingual, nasal, transdermal, vaginal, oral or parenteral administration.

[0055] Those skilled in the art can select a suitable formulation form according to the mode of administration. For example, the formulation form suitable for oral administration may include but is not limited to pills, tablets, chewable tablets, capsules, granules, drops or syrups, etc. For another example, the formulation form suitable for parenteral administration may include but is not limited to solutions, suspensions, reconstituted dry preparations or sprays, etc. For another example, the formulation form suitable for rectal administration may generally be a suppository.

[0056] Another aspect of the present application provides a method for treating non-alcoholic fatty liver disease, comprising administering the aforementioned drug to a subject.

[0057] In the methods provided in the present application, the subject is a mammal, such as a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. Primates are, for example, monkeys, apes or Homo sapiens.

[0058] The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0059] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the art. These techniques have been fully described in existing literature.

[0060] The following oil red staining method is as follows: prepare the working solution. Before use, mix 6 parts of saturated oil red O dye solution with 4 parts of distilled water, place in a 60-70℃ water bath for 30 minutes, cool naturally and filter with qualitative filter paper to obtain oil red O working solution. For cells: aspirate the cell culture solution and slowly add PBS to the edge of the well plate to simply wash the cells. Add 4% paraformaldehyde fixative to fix at room temperature for 8-10 minutes, and rinse with PBS twice. For frozen sections: After the sections are taken out from -20℃, let them stand at room temperature for 5-10 minutes to return to room temperature. The staining steps are as follows: for cells, add a small amount of 60% isopropanol to the well plate to cover the cells for 15-20 seconds, aspirate 60% isopropanol, and dry the water slightly; add oil red O working solution to the well plate to cover the cells, stain at room temperature and avoid light for 30 minutes, and remove the staining solution; add 60% isopropanol to quickly differentiate for 3-5 seconds, wash with pure water 3 times, 5 minutes each time; add PBS to cover the cells and observe under a microscope. For frozen sections, the frozen sections restored to room temperature were gently immersed in Oil Red O working solution for 8-10 minutes (covered to avoid light); the sections were taken out, left for 3 seconds, and then immersed in two cylinders of 60% isopropanol for differentiation for 3-5 seconds; the sections were immersed in two cylinders of pure water for 10 seconds each time; the sections were immersed in hematoxylin staining solution to stain the cell nuclei, washed with water, blued and washed with water again, and after slightly drying the water, glycerol gelatin sealing agent was added to seal the sections.

[0061] The reagent information used is as follows: ML-SA1 (Cat. No.: S9926) was purchased from selleck, and ML-SA5 (Cat. No.: HY-152182) and Ambroxol (Cat. No.: HY-B1039) were purchased from MedChemExpress.

[0062] Example 1

[0063] Construction of model mice

[0064] In order to construct an obese mouse model, 2-month-old C57BL / 6 mice bearing mCherry-eGFP-LC3 were fed with a 60 kcal% fat high-fat diet (Deci Biotechnology (Wuxi) Co., Ltd., Catalog No.: HF60) until 12 months of age ( Figure 1a). mCherry-EGFP-LC3 mice were purchased from Shanghai Model Organisms Technology Co., Ltd. Mice fed a high-fat diet showed symptoms of obesity and non-alcoholic fatty liver disease, including increased liver tissue weight, accumulation of lipid droplets in hepatocytes, hepatocyte vacuolization, and liver tissue fibrosis ( Figure 1 b, 1c, 1d).

[0065] Example 2

[0066] All cells were mouse hepatocyte cell line AML12, purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-550. The cells exhibit typical hepatocyte characteristics, such as peroxisomes and bile duct-like structures. The AML12 (mouse liver 12) cell line was constructed from hepatocytes of transgenic mice carrying the human TGF-alpha gene (CD1 strain, MT42 line). AML12 cells retain the ability to express high levels of mRNA for serum (albumin, α-1 antitrypsin and transferrin) and gap junctions (connexins 26 and 32), and contain only isoenzyme 5 of lactate dehydrogenase.

[0067] The high-fat induction environment was as follows: 0.5 mM FFA (free fatty acid, OA:PA = 2:1, OA: oleic acid; PA: palmitic acid) was added to the normal culture medium (DMEM medium containing 10% FBS) and incubated at 37°C and 5% CO 2 After culturing for 8 hours, obvious lipid droplet accumulation could be observed in AML12 cells.

[0068] Oil red staining revealed that hepatocytes in a high-fat environment were accompanied by lipid droplet accumulation, and this phenotype was basically consistent with the in vivo situation in Example 1 ( Figure 2 a,2b).

[0069] Example 3

[0070] Based on Example 2, ML-SA1 was used to treat hepatic parenchymal cells AML12. The results showed that ML-SA1 significantly improved the accumulation of lipid droplets in hepatic parenchymal cells.

[0071] The specific operation was in accordance with the method of Example 2, except that: after pretreatment with high-fat medium for 8 hours, the medium was replaced with normal medium and 25uM ML-SA1 was added to continue culturing for 24 hours, and then the samples were collected and the intracellular lipid droplets were detected by oil red staining (the method was the same as in Example 2). The results showed that ML-SA1 incubation could significantly reduce the accumulation of lipid droplets in hepatocytes under high-fat environment ( Figure 3a and 3b). This indicates that the use of ML-SA1 in a high-fat environment can help hepatocytes metabolize intracellular lipid droplets.

[0072] Example 4

[0073] ML-SA1 alleviates the phenotype of non-alcoholic fatty liver disease induced by high-fat diet in mice at the histological level.

[0074] In vivo experiments were conducted by feeding high-fat diet to induce obesity in mCherry-eGFP-LC3 mice in Example 1, and intraperitoneally injecting 2 mg / kg ML-SA1 or solvent group (10% DMSO, 40% PEG300, and 50% PBS) as a control every day. After 6 weeks of continuous administration, the mouse livers were collected and tested.

[0075] The results showed that ML-SA1 could significantly reduce the symptoms of fatty liver and liver weight in mice ( Figure 4 a and 4b). At the histological level, the accumulation of lipid droplets in hepatocytes, hepatocyte vacuolization, and liver tissue fibrosis in liver tissue sections of ML-SA1-treated mice were significantly alleviated ( Figure 4 c).

[0076] Example 5

[0077] ML-SA1 alleviates the symptoms of non-alcoholic fatty liver disease induced by high-fat diet in mice at a physiological level.

[0078] Based on the results of Example 4, other pathological indicators of fatty liver disease symptoms in mice treated with ML-SA1 and in the control group were further detected in mice induced to be obese by high-fat diet, including serum total cholesterol concentration ( Figure 5 a), serum low-density lipoprotein cholesterol ( Figure 5 b), serum high-density lipoprotein cholesterol concentration ( Figure 5 c), total cholesterol concentration in liver tissue ( Figure 5 d), AST activity in liver tissue ( Figure 5 e), ALT activity in liver tissue ( Figure 5 f). The results showed that the pathological indicators related to the symptoms of fatty liver disease were significantly reduced under the improvement of ML-SA1. ML-SA improved the fat accumulation in liver tissue and also improved the overall pathological indicators of mice caused by fatty liver disease.

[0079] Example 6

[0080] Other drugs alleviate the phenotype of hepatocytes under high lipid environment

[0081] The operation was carried out in accordance with Example 3, except that two other drugs were added to the supporting cell phagocytosis in vitro model: 25uM ML-SA5 and 5uM Ambroxol. The lipid droplets were detected by oil red staining (the method was the same as that in Example 2). The results showed that the three drugs added could reduce the accumulation of lipid droplets in the hepatocytes under high-fat environment ( Figure 6 ).

[0082] In summary, the present application unexpectedly discovered that ML-SA1 can improve non-alcoholic fatty liver disease, which is specifically reflected in reducing lipid droplet accumulation in hepatocytes, reducing liver weight, relieving hepatocyte vacuolation and liver tissue fibrosis, and improving relevant pathological indicators of non-alcoholic fatty liver disease. The present application also discovered that the combination of ML-SA1 and other substances can more significantly improve non-alcoholic fatty liver disease.

[0083] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present application.

Claims

1. Use of ML-SA1 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that The drug for treating non-alcoholic fatty liver disease includes at least one of the following functions: 1) Reduce lipid droplet accumulation in hepatocytes; 2) Reduce liver weight; 3) Alleviate hepatocyte vacuolization; 4) Alleviate liver fibrosis; 5) Restore liver cell function; 6) Relieve the symptoms of fatty liver; 7) Improve the pathological indicators of non-alcoholic fatty liver disease.

3. The use according to claim 2, characterized in that The pathological indicators include one or more combinations of total cholesterol concentration in serum, cholesterol in low-density lipoprotein in serum, cholesterol concentration in high-density lipoprotein in serum, total cholesterol concentration in liver tissue, aspartate aminotransferase activity in liver tissue and alanine aminotransferase activity in liver tissue.

4. A composition comprising ML-SA1 and other substances, wherein the other substances comprise ML-SA5 and / or Ambroxol.

5. Use of the composition as claimed in claim 4 in preparing a medicament for treating non-alcoholic fatty liver disease.

6. A drug for treating non-alcoholic fatty liver disease, comprising an effective dose of ML-SA1 for use according to any one of claims 1 to 3, or the composition according to claim 4.

7. The drug according to claim 6, characterized in that It also includes pharmaceutically acceptable carriers or excipients.

8. A method for treating non-alcoholic fatty liver disease, characterized in that: The method comprises administering the medicament according to any one of claims 6 to 7 to a subject.

9. The method according to claim 8, characterized in that The subject is a mammal.

10. The method according to claim 9, characterized in that The mammal is selected from the group consisting of rodents, artiodactyls, perissodactyls, lagomorphs, or primates.

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