A medicament for improving non-alcoholic fatty liver disease

Abstract

The application relates to the field of biological medicine, in particular to a medicine for improving non-alcoholic fatty liver disease. The application provides the use of ML-SA1 in the preparation of a medicine for treating non-alcoholic fatty liver disease. It is found that ML-SA1 can improve non-alcoholic fatty liver disease, specifically, can reduce the accumulation of lipid droplets in hepatocytes, reduce the weight of the liver, relieve hepatocyte vacuolization and liver tissue fibrosis, and improve the related pathological indexes of non-alcoholic fatty liver disease. It is also found that the combination of ML-SA1 and other substances can more significantly improve non-alcoholic fatty liver disease.

Description

Technical Field

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

[0002] Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. In the coming decades, NAFLD is projected to become a leading cause of end-stage liver disease. The overall incidence of NAFLD is projected to increase by 0% to 30% between 2016 and 2030. In Asia, the overall prevalence of NAFLD is 29.62%, more than 16 times that of primary liver cancer. NAFLD is a general term encompassing a wide range of liver diseases, from nonalcoholic fatty liver (NAFL), liver fibrosis, cirrhosis to hepatocellular carcinoma (HCC). NAFLD begins with the abnormal accumulation of lipid droplets in the liver, triggering lipotoxicity, endoplasmic reticulum stress, and inflammatory responses, progressing to cirrhosis and even liver cancer. Nonalcoholic steatohepatitis (NASH) is a late-stage form of NAFLD characterized by hepatic steatosis, vacuolation, and / or fibrosis.

[0003] Clinically, there are currently no approved drugs directly targeting non-alcoholic fatty liver disease (NAFLD). Current treatments involve increasing systemic insulin sensitivity, such as with thiazolidinediones, rosiglitazone, metformin, and / or reducing food intake, such as with GLP-1 receptor agonists (exenatide and liraglutide) 3,5-7. However, these treatments have unwanted off-target effects. In NAFLD associated with obesity and type 2 diabetes, insulin resistance in adipose tissue hinders insulin-mediated lipolysis inhibition, 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, excessive saturated fatty acids exceed the liver's capacity to esterify fatty acids, leading to hepatotoxicity (i.e., lipotoxicity).

[0004] Therefore, there is a need to develop a drug that can directly target non-alcoholic fatty liver disease. Summary of the Invention

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

[0006] To achieve the above and other related objectives, the first aspect of this application provides the use of ML-SA1 in the preparation of a medicament 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 the accumulation of lipid droplets in hepatocytes;

[0009] 2) Reduce liver weight;

[0010] 3) Alleviates hepatocyte vacuolation;

[0011] 4) Relieves liver fibrosis;

[0012] 5) Restore liver cell function;

[0013] 6) Relieves symptoms of fatty liver;

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

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

[0016] A second aspect of this application provides a composition comprising ML-SA1 and other substances, said other substances including ML-SA5 and / or Ambroxol.

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

[0018] The fourth aspect of this application provides a medicament for treating non-alcoholic fatty liver disease, comprising an effective dose of ML-SA1 or the aforementioned composition as described above.

[0019] In some implementations, the drug also includes a pharmaceutically acceptable carrier or excipient.

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

[0021] In some implementations, the object is a mammal.

[0022] In some implementations, the mammal is selected from rodents, even-toed ungulates, perissodactyls, lagomorphs, or primates.

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

[0024] 1. This application unexpectedly discovered that ML-SA1 can improve non-alcoholic fatty liver disease, specifically by reducing lipid droplet accumulation 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. This application also unexpectedly discovered that ML-SA5 and Ambroxol can significantly improve non-alcoholic fatty liver disease. Attached Figure Description

[0026] Figure 1 To construct a mouse model of non-alcoholic fatty liver disease. Among other things, Figure 1 a represents the weight changes of mice from 2 months of age when they were fed a normal diet and a high-fat diet, respectively, until they were 12 months of age. Figure 1 b is an anatomical diagram of liver tissue in high-fat induced obese mice. Figure 1 c is a statistical chart of liver tissue weight. Figure 1 d represents H&E, Masson, and Oil Red O staining of liver tissue. White arrows indicate vacuoles within the liver tissue, and black arrows indicate tissue fibrosis. ND: Normal diet; HFD: High-fat diet; PTM: Peritubular myoid cells.

[0027] Figure 2 This was designed to simulate hepatocytes in an in vitro environment simulating a high-fat environment. Among them, Figure 2 a is Oil Red O staining of the AML12 cell line. Figure 2 b is Figure 2 Statistics on the area of ​​lipid droplets in each cell in figure a. FFA: free fatty acid.

[0028] Figure 3 This study aimed to improve hepatocyte cell lines in vitro under high-lipid conditions using ML-SA1. Among them, Figure 3 a is Oil Red O staining of the AML12 cell line. Figure 3 b is Figure 3 Statistics on the area of ​​lipid droplets in each cell in figure a. FFA: free fatty acid.

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

[0030] Figure 5 ML-SA1 improves other related characteristics of non-alcoholic fatty liver disease in mice. Figure 5 'a' represents the cholesterol content in serum. Figure 5 b represents the cholesterol content in low-density lipoprotein in serum. Figure 5 c represents the cholesterol content in high-density lipoprotein in serum. Figure 5 d represents AST activity in liver tissue. Figure 5 e represents ATL activity in liver tissue. Figure 5 f represents 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: alanine aminotransferase.

[0031] Figure 6 To investigate the effects of two drugs that improve lysosomal function on AML12 cells. Among them, Figure 6 a represents the effect of drug addition on lipid droplets in AML12 cells. Figure 6 b is Figure 6 The statistical representation of the area of ​​lipid droplets in a cell relative to the total cell area. FFA: free fatty acid. Detailed Implementation

[0032] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0033] The inventors of this application, through extensive research and exploration, unexpectedly discovered a drug for improving non-alcoholic fatty liver disease, and based on this discovery, completed this application.

[0034] This application provides, in one aspect, the use of ML-SA1 in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0035] ML-SA1 is a selective TRPML agonist. TRPML is a member of the channel mucolipid subfamily and is a type of channel mucolipid that releases Ca2+. 2+ Cation channels are important regulators of transport and autophagy-related events. Non-alcoholic fatty liver disease (NAFLD) 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 pathogenesis of NAFLD. Untreated NAFLD progresses to non-alcoholic steatohepatitis (NASH), which can develop into cirrhosis, potentially severely impairing liver function, and in a small percentage, may even progress to liver cancer. In some embodiments of this application, it was 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] The functions of the drug for treating non-alcoholic fatty liver disease provided in this application are mainly reflected in the following aspects:

[0037] 1) Reduce the accumulation of lipid droplets in hepatocytes;

[0038] 2) Reduce liver weight;

[0039] 3) Alleviates hepatocyte vacuolation;

[0040] 4) Relieves liver fibrosis;

[0041] 5) Restore liver cell function;

[0042] 6) Relieves symptoms of fatty liver;

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

[0044] In the uses provided in this application, the pathological indicators include one or more combinations of serum total cholesterol concentration, serum low-density lipoprotein cholesterol concentration, serum high-density lipoprotein cholesterol concentration, liver tissue total cholesterol concentration, liver tissue aspartate aminotransferase activity, and liver tissue alanine aminotransferase activity. In a specific embodiment of this application, under the action of ML-SA1 drug, the pathological indicators related to fatty liver disease symptoms all decreased to a significant extent.

[0045] This application also provides a composition comprising ML-SA1 and other substances, including 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 in this application, ML-SA5 is a potent TRPML1 cation channel agonist that activates the overall activity of phagolysosomes in DMD muscle cells. ML-SA5 also possesses anticancer activity and can inhibit tumor growth. This application discloses for the first time that ML-SA5 can be used for the treatment of non-alcoholic fatty liver disease.

[0047] The composition provided in this application includes Ambroxol (NA-872), an active metabolite of the precursor bromhexine, which possesses potent expectorant activity. Ambroxol is a GCase chaperone, increasing GCase activity. Ambroxol can induce pulmonary autophagy and has potential applications in Parkinson's disease and neurogenic Gaucher disease research. This application discloses for the first time that Ambroxol can be used to treat non-alcoholic fatty liver disease.

[0048] This application also provides the use of the aforementioned composition in the preparation of a medicament for treating non-alcoholic fatty liver disease. In one specific embodiment of this application, the presence of the composition can reduce the accumulation of lipid droplets in hepatocyte lines under high-fat conditions.

[0049] This application also provides a medicament for treating non-alcoholic fatty liver disease, comprising an effective dose of ML-SA1 used in the foregoing purposes, or the foregoing composition.

[0050] In some implementations, the effective dose refers to the dose at which a drug can exert its therapeutic effect. This is because a drug needs to be absorbed by the body in a certain dose to reach a specific drug concentration, and only when a certain drug concentration is reached can the drug exert its effect. If the dose is too small, an effective concentration cannot be achieved in the body, and the drug cannot exert its effective effect. However, if the dose is too large, exceeding a certain limit, the drug's effect may undergo a qualitative change, potentially causing varying degrees of toxicity to the body. Therefore, to achieve the effective effect of a drug while avoiding adverse reactions, it is essential to strictly control the dosage range.

[0051] The drug provided in this application also includes pharmaceutically acceptable carriers or excipients.

[0052] "Pharmaceutical acceptable" means that when the molecular basis and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions.

[0053] A pharmaceutically acceptable carrier or excipient should be compatible with lysosomal regulatory factors, meaning it can be mixed with them without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum 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, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

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

[0055] Those skilled in the art can select appropriate formulations based on the route of administration. For example, formulations suitable for oral administration may include, but are not limited to, pills, tablets, chewable tablets, capsules, granules, drops, or syrups. For another example, formulations suitable for parenteral administration may include, but are not limited to, solutions, suspensions, rehydrated dry formulations, or sprays. For yet another example, suppositories are typically suitable for rectal administration.

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

[0057] The method provided in this application targets mammals, such as rodents, even-toed ungulates, perissodactyls, lagomorphs, and primates. Examples of primates include monkeys, apes, or Homo sapiens.

[0058] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0059] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0060] The Oil Red staining method used below is as follows: Prepare the working solution by thoroughly mixing 6 parts saturated Oil Red O staining solution with 4 parts distilled water before use. Incubate at 60-70℃ for 30 minutes, allow to cool naturally, and then filter through qualitative filter paper to obtain the Oil Red O working solution. For cells: Discard the cell culture medium and slowly add PBS to the edge of the wells for simple washing. Add 4% paraformaldehyde fixative and fix at room temperature for 8-10 minutes, then rinse twice with PBS. For frozen sections: After removing the sections from -20℃, allow them to stand at room temperature for 5-10 minutes to reach room temperature. The staining steps are as follows: For cells, add a small amount of 60% isopropanol to the wells to cover the cells for 15-20 seconds, discard the 60% isopropanol, and allow to dry slightly. Add Oil Red O working solution to the wells to cover the cells, stain at room temperature in the dark for 30 minutes, and remove the staining solution. Add 60% isopropanol to rapidly differentiate for 3-5 seconds, wash three times with pure water for 5 minutes each time, and then cover the cells with PBS and observe under a microscope. For frozen sections, after restoring to room temperature, gently immerse the frozen sections in Oil Red O working solution for 8-10 minutes (cover and protect from light); remove the sections, let them stand for 3 seconds, and then immerse them in two separate tanks of 60% isopropanol for differentiation for 3-5 seconds; immerse the sections in two separate tanks of pure water for 10 seconds each time; immerse the sections in hematoxylin staining solution to stain the cell nuclei, wash with water, return to blue and wash with water again, air dry slightly, and then add glycerol gelatin mounting medium to mount the sections.

[0061] The reagents used below are as follows: ML-SA1 (catalog number: S9926) was purchased from Selleck, and ML-SA5 (catalog number: HY-152182) and Ambroxol (catalog number: HY-B1039) were purchased from MedChemExpress.

[0062] Example 1

[0063] Building a mouse model

[0064] To construct an obese mouse model, 2-month-old mCherry-eGFP-LC3 C57BL / 6 mice were fed a high-fat diet (60 kcal% fat intake, Daiz Biotechnology (Wuxi) Co., Ltd., catalog number: HF60) until 12 months of age. Figure 1a). mCherry-EGFP-LC3 mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd. Mice fed a high-fat diet exhibited obesity and symptoms of non-alcoholic fatty liver disease, including increased liver weight, accumulation of lipid droplets in hepatocytes, hepatocyte vacuolation, and liver fibrosis. Figure 1 b, 1c, 1d).

[0065] Example 2

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

[0067] The high-lipid induction environment was as follows: 0.5 mM FFA (free fatty acid, prepared by OA:PA = 2:1, where OA: oleic acid and PA: palmitic acid) was added to normal culture medium (DMEM medium containing 10% FBS) and cultured at 37°C in 5% CO2 for 8 hours. Obvious lipid droplet accumulation was observed in AML12 cells.

[0068] Oil Red staining revealed lipid droplet accumulation in hepatocytes under high-lipid conditions, a phenotype largely consistent with the in vivo findings in Example 1. Figure 2 (a, 2b).

[0069] Example 3

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

[0071] The specific procedure was the same as in Example 2, except that after pretreatment with high-fat culture medium for 8 hours, the medium was replaced with normal culture medium and 25 μM ML-SA1 was added for further 24 hours of culture. Samples were then collected, and intracellular lipid droplets were detected using Oil Red staining (the method was the same as in Example 2). The results showed that ML-SA1 incubation significantly reduced the accumulation of lipid droplets in hepatocytes under high-fat conditions. Figure 3 (a and 3b). This indicates that using ML-SA1 in a high-fat environment can help hepatocytes metabolize intracellular lipid droplets.

[0072] Example 4

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

[0074] In vivo experiments were conducted using mCherry-eGFP-LC3 mice that were induced to be obese by a high-fat diet as described in Example 1. Control mice were administered 2 mg / kg ML-SA1 or a solvent group (10% DMSO, 40% PEG300, and 50% PBS) via intraperitoneal injection daily. Mice livers were harvested and analyzed after 6 weeks of continuous administration.

[0075] The results showed that ML-SA1 significantly reduced the symptoms of fatty liver in mice and reduced liver weight. Figure 4 (a and 4b). At the histological level, ML-SA1-treated mouse liver tissue sections showed significant relief of features such as lipid droplet accumulation in hepatocytes, hepatocyte vacuolation, and liver fibrosis. Figure 4 c).

[0076] Example 5

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

[0078] Based on the results of Example 4, other pathological indicators of fatty liver disease symptoms in mice with high-fat diet-induced obesity treated with ML-SA1 and in the control group were further examined, including serum total cholesterol concentration (…). Figure 5 a) Cholesterol in serum low-density lipoprotein (LDL) Figure 5 b) Cholesterol concentration in high-density lipoprotein in serum ( Figure 5 c) Total cholesterol concentration in liver tissue ( Figure 5 d), AST activity in liver tissue ( Figure 5 e), Alanine aminotransferase activity in liver tissue ( Figure 5 f). The results showed that the pathological indicators related to fatty liver disease symptoms decreased significantly under the improvement of ML-SA1. ML-SA1 improved the accumulation of fat 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 conditions.

[0081] The procedure was the same as in Example 3, except that two additional drugs, 25 μM ML-SA5 and 5 μM Ambroxol, were added to the in vitro model of supporting cell phagocytosis. The lipid droplet count was determined by Oil Red staining (method as in Example 2). The results showed that all three drugs reduced the accumulation of lipid droplets in hepatocytes under high-lipid conditions. Figure 6 ).

[0082] In summary, this application unexpectedly discovered that ML-SA1 can improve non-alcoholic fatty liver disease (NAFLD), specifically by reducing lipid droplet accumulation in hepatocytes, reducing liver weight, alleviating hepatocyte vacuolation and liver fibrosis, and improving related pathological indicators of NAFLD. This application also found that the combined use of ML-SA1 and other substances can more significantly improve NAFLD.

[0083] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. Use of a composition in the preparation of a medicament for treating non-alcoholic fatty liver disease, said composition comprising ML-SA1 and / or other substances selected from ML-SA5 and / or Ambroxol, wherein the concentration of ML-SA1 used is 2 mg / kg.

2. The use as described in claim 1, characterized in that, The medication for treating non-alcoholic fatty liver disease includes at least one of the following functions: 1) Reduce the accumulation of lipid droplets in hepatocytes; 2) Reduce liver weight; 3) Alleviates hepatocyte vacuolation; 4) Relieves liver fibrosis; 5) Restore liver cell function; 6) Relieves symptoms of fatty liver; 7) Improve the pathological indicators of non-alcoholic fatty liver disease.

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

4. A medicament for treating non-alcoholic fatty liver disease, comprising an effective dose of the composition used as described in any one of claims 1 to 3.

5. The drug as described in claim 4, characterized in that, It also includes pharmaceutically acceptable carriers or excipients.

Images