Pharmaceutical composition for preventing or treating alcoholic and non-alcoholic fatty liver disease

Abstract

The present invention relates to a composition for preventing or treating fatty liver, which contains IF1 (ATPase inhibitory factor 1) as an active ingredient. The composition protects liver cells from alcohol toxicity, inhibits an increase in blood lipid levels and lipid accumulation in liver tissue caused by overnutrition, thereby reducing the formation of fatty liver, inhibits liver fibrosis and inflammatory responses, and increases the proportion of beneficial bacteria in the intestinal microbial community, and is therefore useful for the prevention and treatment of fatty liver.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating alcoholic and non-alcoholic fatty liver, which contains IF1 (ATPase inhibitory factor 1) as an active ingredient. [Background technology]

[0002] With the development of industry, the dietary habits of Korea are becoming more Westernized, and this, along with the decline in physical activity of modern people, is rapidly increasing the risk of various metabolic diseases due to an increase in the obese population and metabolic syndrome.Metabolic diseases are the cause of various other diseases, and at the same time, they are a factor that adversely affects various biological metabolisms in the human body, such as aging, weakened immune system function, and stress, and their seriousness is becoming greater.

[0003] Fatty liver disease is the most common liver disease, and generally refers to the excessive accumulation of fat in the liver, with neutral lipid content exceeding 5%. It can be classified into non-alcoholic fatty liver disease (NAFLD), which is primarily caused by metabolic syndromes such as diabetes, obesity, and hyperlipidemia, and alcoholic fatty liver disease, which is caused by alcohol intake, i.e., excessive drinking.

[0004] Alcoholic fatty liver is a condition in which excess fat accumulates in the liver due to fat synthesis promoted by continuous alcohol intake, and the expanded liver cells caused by this condition affect blood vessels and lymph nodes, causing abnormalities in nutritional supply and immune function. As a result, liver function declines and energy metabolism becomes unbalanced. In particular, depending on the level of alcohol intake, it can develop into hepatitis or cirrhosis, posing a great risk to the health and welfare of the public.

[0005] Nonalcoholic fatty liver occurs due to causes other than alcohol-induced fatty liver, and shows symptoms including inflammation, liver fibrosis, cirrhosis, etc. Nonalcoholic fatty liver can be divided into simple steatosis and nonalcoholic steatohepatitis (NASH), and NASH is a condition that is accompanied by fatty deposition, liver cell damage and inflammation, which can cause liver fibrosis, cirrhosis, and hepatocellular carcinoma.

[0006] Patients suffering from metabolic diseases such as obesity and diabetes have a high incidence of fatty liver (approximately 75% and 50%, respectively), which has attracted much attention. However, there is no precise mechanism of onset and no effective treatment or drug, and only basic exercise and dietary therapy is offered, so there is a growing demand for new treatments for fatty liver disease. Summary of the Invention [Problem to be solved by the invention]

[0007] Under the above circumstances, the present inventors have conducted research to find a substance that is safe for the human body and can treat fatty liver. As a result, they have confirmed that IF1, a human-derived protein, protects liver cells from alcohol toxicity, prevents the increase in blood lipid concentration and lipid accumulation in liver tissue caused by overnutrition, reduces the formation of fatty liver, inhibits liver fibrosis and inflammatory responses, and increases the proportion of beneficial bacteria in the intestinal microbial community, and is therefore effective in preventing and treating fatty liver disease, leading to the completion of the present invention.

[0008] Therefore, an object of the present invention is to provide a composition for preventing or treating alcoholic and non-alcoholic fatty liver, which comprises IF1, a human-derived protein, as an active ingredient. [Means for solving the problem]

[0009] To achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating alcoholic and non-alcoholic fatty liver, comprising IF1 as an active ingredient.

[0010] In this specification, alcoholic fatty liver disease refers to a state in which fat accumulates in liver cells due to excessive drinking. There are no particular symptoms in the early stages, but as inflammation progresses, symptoms such as weakness, fatigue, fever, nausea, and vomiting appear. If not treated appropriately, it progresses to alcoholic cirrhosis, and ascites, bleeding from esophageal varices, hepatic coma, etc. appear.

[0011] As used herein, nonalcoholic fatty liver disease (NAFLD) refers to a condition in which fat accumulates in liver cells, regardless of whether the patient drinks little or no alcohol. Rather than being a single disease, it includes various forms of liver disease, ranging from simple fatty liver without inflammation to nonalcoholic steatohepatitis (NASH) and cirrhosis.

[0012] The term "prevention" as used in the present invention means any action of inhibiting the progression or delaying the onset of alcoholic and non-alcoholic fatty liver disease by administering the pharmaceutical composition according to the present invention.

[0013] The term "treatment" as used herein means any action that reverses or beneficially alters alcoholic and non-alcoholic fatty liver disease by administration of a pharmaceutical composition according to the present invention.

[0014] IF1 (ATPase inhibitory factor 1) is a major protein that is expressed in cells and binds to ATPase present in the mitochondrial membrane, interfering with its rotational movement, thereby interfering with ATP synthesis / decomposition via the electron transport chain, thereby affecting intracellular energy regulation and mitochondrial homeostasis. The action of IF1 has been reported to have a positive effect on the improvement of various diseases, including breast cancer and degenerative nervous system diseases, but its effect on fatty liver disease is not yet known.

[0015] In the present invention, the IF1 may be a protein comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment of the present invention, the IF1 sequence of SEQ ID NO: 1 is used.

[0016] [Table 1]

[0017] According to a specific example of the present invention, when hepatocytes are pretreated with IF1 and then treated with alcohol, the production of reactive oxygen species is significantly reduced and the hepatocytes are protected compared to when they are treated with alcohol alone (Figure 1).

[0018] Furthermore, when IF1 was administered to mice with induced non-alcoholic fatty liver, the activities of both ALT and AST, indicators of liver damage, were significantly reduced (Figure 2), indicating that IF1 protects liver cells from liver damage caused not only by alcohol but also by lipid accumulation.

[0019] IF1 reduced blood total cholesterol and free fatty acid levels in mice with induced NAFLD (Figure 3), reduced lipid accumulation in liver tissue (Figure 4), and suppressed excessive collagen accumulation and immune cell infiltration in liver tissue, improving liver fibrosis and inflammatory responses (Figure 5).

[0020] When simple fatty liver progresses to nonalcoholic steatohepatitis (NASH), liver fibrosis and inflammatory responses appear. The present invention improves such liver fibrosis and inflammatory responses, and is therefore useful for treating nonalcoholic steatohepatitis.

[0021] Therefore, the pharmaceutical composition according to the present invention is characterized in that IF protects liver cells from alcohol toxicity, reduces the formation of fatty liver, suppresses liver fibrosis and immune response, and increases the proportion of beneficial intestinal bacteria, thereby exhibiting therapeutic effects on both alcoholic and non-alcoholic fatty liver and improving symptoms that appear throughout the progression of non-alcoholic fatty liver.

[0022] The pharmaceutical compositions of the present invention may be prepared in a form further comprising a suitable carrier, excipient or diluent that is conventionally used in the manufacture of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier.

[0023] Specifically, the pharmaceutical compositions can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions by conventional methods.

[0024] In the present invention, the carrier, excipient and diluent that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.When formulating, it is prepared by using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.

[0025] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0026] Oral liquid preparations include suspensions, liquids for internal use, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc., in addition to water and liquid paraffin, which are frequently used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0027] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oleaginous suspension, or a sterile injectable preparation. The suspension may be formulated by using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents according to techniques known in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Acceptable vehicles and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Sterile fixed oils are also usually used as a solvent or suspending medium. For this purpose, any non-volatile oil with low irritation may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in injectable preparations, as are pharma-ceutically acceptable natural oils (e.g., olive oil or castor oil), especially their polyoxyethylated versions.

[0028] Parenteral administration of the pharmaceutical compositions according to the invention is particularly useful when the desired treatment involves an area or organ easily accessible by topical application. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.

[0029] The content of the active ingredient in the pharmaceutical composition of the present invention is not particularly limited, but may be 0.0001 to 50% by weight, more preferably 0.01 to 10% by weight, based on the total weight of the final composition.

[0030] Another aspect of the present invention provides a method for treating alcoholic and non-alcoholic fatty liver disease, comprising the step of administering the composition for preventing or treating alcoholic and non-alcoholic fatty liver disease to an individual in need of such treatment.

[0031] Since the treatment method of the present invention uses the pharmaceutical composition for preventing or treating alcoholic and non-alcoholic fatty liver as described above, the description common to both of them will be omitted to avoid overcomplicating this specification.

[0032] The method of administration of the pharmaceutical composition can be oral or parenteral, as described above, and the dosage of the pharmaceutical composition can be determined by a treating specialist. Effect of the Invention

[0033] IF1, a human-derived protein, protects liver cells from alcohol toxicity, suppresses increases in blood lipid concentrations and lipid accumulation in liver tissue caused by overnutrition, thereby reducing the formation of fatty liver, suppresses liver fibrosis and inflammatory responses, and increases the proportion of beneficial bacteria in the intestinal microbial community, making it useful for the prevention and treatment of fatty liver disease. [Brief description of the drawings]

[0034] [Figure 1] The toxicity protection effect of IF1 treatment in hepatic cell lines was confirmed by its inhibitory effect on the production of reactive oxygen species (ROS). [Diagram 2] These are the results of confirming changes in blood liver enzyme levels after administration of IF1 to mice with induced fatty liver: A-ALT (asparate aminotransferase); and B-AST (alanine aminotransferase) measurement results. [Diagram 3] After administering IF1 to mice with fatty liver induction, the changes in blood lipid levels were examined: A - cholesterol; B - FFA (free fatty acid); and C - atherosclerosis index. [Figure 4] This is the result of administering IF1 to mice with induced fatty liver, and then confirming the degree of fat accumulation in the liver tissue. [Diagram 5] The results showed that IF1 was administered to mice with induced fatty liver, and the degree of improvement in liver fibrosis and inflammatory responses was confirmed in liver tissue. [Figure 6] The results showed that after administration of IF1 to fatty liver-induced mice, changes in the intestinal microbial community were confirmed: A-Changes in the proportion of intestinal microbial community; B-Akkermansia muciniphila, the proportion of which was specifically changed by IF; and C-Lactobacillus reuteri, the proportion of which was specifically changed by IF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Hereinafter, one or more specific examples will be described in more detail based on examples. However, these examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples.

[0036] Example 1: Confirmation of protective effect of IF1 against alcohol toxicity in hepatocytes To investigate the protective effect of IF1 against alcohol toxicity, we treated liver cells with alcohol, a representative substance that is toxic to liver cells, and measured the level of reactive oxygen species produced with and without IF treatment. It is known that alcohol promotes the production of reactive oxygen species during the metabolic process, and the reactive oxygen species produced act as a toxin, inducing cell damage, while also accelerating the inflammatory response and fibrosis process in liver tissue.

[0037] HepG2 liver cell line was seeded in 8-well chamber slides and cultured at 37℃ and 5% CO2 incubator, and pretreated with control substance (PBS) and IF1 for 6 hours. Then, 1M ethanol was treated for 18 hours to induce the production of reactive oxygen species. After washing the cells twice with HBSS, they were treated with 25uM DCF-DA (Dichlorodihydrofluorescein Diacetate) at 37℃ for 1 hour to stain the reactive oxygen species. After staining was completed, the cells were washed twice with HBSS and observed under a confocal microscope while immersed in HBSS buffer containing 25mM HEPES.

[0038] As a result, it was confirmed that the experimental group pretreated with IF1 and then treated with alcohol had weaker fluorescence intensity compared to the experimental group treated with alcohol only, indicating that IF1 suppresses the production of reactive oxygen species caused by alcohol in hepatocytes (Figure 1).

[0039] Example 2: Confirmation of fatty liver improvement effect by IF1 administration in mice with non-alcoholic fatty liver induction 2-1. Creation of a mouse model of non-alcoholic fatty liver disease Five-week-old C57BL / 6J male mice were provided by Orient Bio (Seongnam, Korea), and after a one-week adaptation period, fatty liver induction was carried out. The mice were kept in a constant environment of 18-24°C and 50-60% humidity, and were fed ad libitum during both the adaptation and fatty liver induction periods.

[0040] After the one-week adaptation period, the mice were fed with Research Diet's D09100310 product for 34 weeks to induce non-alcoholic fatty liver. The detailed ingredients of the product are shown in Table 2 below.

[0041] [Table 2]

[0042] 2-2: Administration of IF1 to mice with nonalcoholic fatty liver disease Mice with NAFLD were divided into two groups and reared for 9 weeks under the following conditions: IF1 protein was prepared by cloning the IF1 coding sequence containing a GST tag into Escherichia coli, and then isolating and purifying the recombinant protein. The GST-tagged protein was used as a control. Control group: GST protein 7.5 mg / kg BW intraperitoneal injection, n=7 Experimental group: IF1 protein 7.5 mg / kg BW intraperitoneal injection, n=9

[0043] 2-3: Analysis of liver enzyme values ​​(ALT / AST) and lipid concentrations After 43 weeks, when all experiments were completed, the mice were weighed and then sacrificed. Specifically, the mice were anesthetized, and blood was collected using an insulin syringe, and the collected blood was immediately centrifuged at 2,500 rpm for 30 minutes at 4°C to separate / obtain serum. The remaining tissues were then separated to measure mass and length, and fixed in a 10% formaldehyde solution and stored at -80°C for subsequent analysis.

[0044] At the end of the experiment, the body weight of the control group (GST-administered group) was 45.8±3.9g and that of the experimental group (IF1-administered group) was 42.4±2.6g, representing a decrease of approximately 7.5% in body weight in the experimental group. The weight of liver tissue was 3.12±0.5g in the control group (GST-administered group) and 2.65±0.6g in the experimental group (IF1-administered group), representing a decrease of approximately 1.5% in the experimental group.

[0045] The liver damage indicator enzyme activity (ALT / AST) was measured from the collected serum, and it was confirmed that both serum ALT and AST were significantly reduced in the experimental group (IF1 administration group) compared to the control group (GST administration group) (Figure 2).

[0046] In addition, analysis of blood lipid levels showed that total cholesterol and free fatty acid were both significantly reduced in the experimental group (IF1 administration group). The atherogenic index (AI) (low-density lipoprotein cholesterol (LDLc) / high-density lipoprotein cholesterol (HDLc)), which is used as a cardiovascular biomarker, was 1.60±0.4 / 1.63±0.3, confirming that there was no significant difference between the control group and the experimental group (Figure 3).

[0047] 2-4. Analysis of changes in liver tissue The mouse liver tissue obtained in Example 2-3 was analyzed for structure through H&E (Haematoxylin and Eosin) staining and Masson's trichrome staining. H&E was used to analyze cell structure because it stains cell nuclei blue and extracellular matrix and intracellular structures red. Masson's trichrome staining stains collagen fibers blue, cytoplasm red, and cell nuclei dark brown, so it was used to analyze the degree of collagen accumulation, which is the cause of liver fibrosis induced by fatty liver disease, and immune cell infiltration, which is the cause of inflammation.

[0048] The staining results confirmed that lipid accumulation in liver tissue was significantly reduced in the experimental group (IF1-administered group) compared to the control group (GST-administered group) (Figure 4).In addition, Masson's Trichrome staining confirmed that the excessive collagen accumulation (arrow) and immune cell infiltration phenomenon (star) seen in the control group (GST-administered group) were significantly reduced by 19% in the experimental group (IF1-administered group), indicating that IF1 improves liver fibrosis and inflammatory responses (Figure 5).

[0049] 2-5. Analysis of liver fibrosis indices We confirmed the expression changes of the genes for type I collagen α1 (Col1a1), type V collagen α1 (Col5a1), periostin, and lysyl oxidase (LOX), which are major biomarkers of liver fibrosis.

[0050] The results showed that the expression of liver fibrosis biomarkers was reduced in the experimental group (IF1-administered group) compared to the control group (GST-administered group), indicating that IF1 improves liver fibrosis by regulating it at the genetic level (Table 3).

[0051] [Table 3]

[0052] To further verify the effect of IF1, Gal-3 was specifically stained to observe and quantify its expression level. As a result, it was confirmed that the accumulation of type I collagen α1 (Col1a1) and galectin (galectin-3, Gal-3) was significantly reduced by 13% and 12%, respectively, in the experimental group (IF1 administration group) compared to the control group (GST administration group), verifying the effect of IF1 in improving liver fibrosis at the molecular level.

[0053] 2-6. Analysis of changes in intestinal microorganisms It is widely known that changes in the intestinal microbial community and maintenance of its function play a major role in metabolic diseases, including non-alcoholic fatty liver disease. In particular, the effects of intestinal microbes on the development and fibrosis of non-alcoholic fatty liver disease were confirmed.

[0054] Feces from mice in which fatty liver was induced by the method of Example 2-2 were collected, genomic DNA was extracted, and 16s rRNA of intestinal microorganisms was obtained. The obtained genetic information was used for observing the composition ratio and degree of change of each subgroup in the microbial community by next generation sequencing (NGS).

[0055] As a result, it was confirmed that the proportions of Deferribacteres, Bacteroidetes, Firmicutes, and Verrucomicrobia changed in the experimental group (IF1-administered group) compared to the control group (GST-administered group) (Figure 6A). The microorganisms that increased significantly among the changes in the entire gut microbiota were the intestinal beneficial bacteria Akkermansia muciniphila and Lactobacillus reuteri (Figures 6B and 6C). Akkermansia muciniphila is a representative beneficial bacterium that feeds on the intestinal mucin layer, secretes short-chain fatty acids (SCFA), reduces inflammation, and improves metabolic syndrome and obesity. Lactobacillus reuteri is a beneficial bacterium that produces lactic acid and exhibits antibacterial effects.

Claims

1. A pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis (NASH), comprising IF1 (ATPase inhibitor factor 1) as an active ingredient.

2. The pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis (NASH) according to claim 1, wherein the composition protects hepatocytes from toxicity caused by alcohol.

3. The pharmaceutical composition for preventing or treating nonalcoholic steatohepatitis (NASH) according to claim 1, wherein the composition suppresses an increase in blood lipid levels.

4. The pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis (NASH) according to claim 1, wherein the composition inhibits lipid accumulation in liver tissue.

5. The pharmaceutical composition for preventing or treating nonalcoholic steatohepatitis (NASH) according to claim 1, wherein the composition suppresses liver fibrosis.

6. The pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis (NASH) according to claim 1, further comprising a pharma- ceutical acceptable carrier, excipient or diluent.

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