Composition for preventing or treating non-alcoholic fatty liver disease comprising miRNA204 inhibitor

Abstract

The present invention relates to a composition comprising a miR204 inhibitor and the use thereof for the prevention or treatment of non-alcoholic fatty liver disease.

Description

Technology Field

[0001] The present invention relates to a composition for the prevention or treatment of non-alcoholic fatty liver disease comprising a miRNA204 inhibitor. Background Technology

[0003] Fatty liver disease, also known as fatty liver, is a disease that causes liver damage due to the abnormal accumulation of fat (such as triglycerides) in liver cells. The initial pathological stage of fatty liver disease is simple fatty liver, characterized only by fat deposition in liver cells; it is known that the condition subsequently progresses to fatty liver hepatitis (including liver fibrosis), as well as to cirrhosis or hepatocellular carcinoma. Generally, causes of fat deposition in the liver include alcohol consumption, obesity, diabetes, lipid metabolism disorders, medications (steroids, tetracycline, etc.), Cushing's syndrome, poisoning (such as yellow phosphorus), and severe nutritional deficiencies.

[0004] Non-alcoholic fatty liver disease (NAFLD) is a modern disease with a rapidly increasing mortality rate worldwide. It is characterized by the absence of distinct symptoms even when fat accumulates chronically; if left untreated, it is an early-stage disease that can progress to liver cirrhosis and liver cancer, and currently, there are no specialized treatments available for it.

[0005] Pharmacotherapy for non-alcoholic fatty liver disease (NAFLD) utilizes drugs that treat or inhibit the progression of the disease by acting on mechanisms that exacerbate it, such as insulin resistance, oxidative stress, apoptosis, and inflammatory cytokines. Among these, diabetes medications are known to improve fatty liver conditions in addition to their blood glucose-lowering effects by addressing the common pathophysiological mechanisms of fatty liver development.

[0006] U.S. Patent Publication No. 20160122762 relates to a method for treating arteriosclerosis and discloses the use of a micro-RNA-204-5p inhibitor for treating arteriosclerosis, Korean Registered Patent No. 2142791 discloses the use of a miR-204 inhibitor for treating osteoarthritis, and Korean Registered Patent No. 2242639 discloses a biomarker miRNA4668-5p for diagnosing liver fibrosis.

[0007] However, no composition for the prevention or treatment of non-alcoholic fatty liver disease comprising the miRNA204 inhibitor of the present invention has been reported. Prior art literature

[0009] U.S. Published Patent No. 20160122762, Korean Registered Patent No. 2142791, Korean Registered Patent No. 2242639 The problem to be solved

[0011] The present invention was derived from the above-mentioned needs, and the inventors completed the invention by confirming the effect of a composition containing a miRNA204 inhibitor as an active ingredient on improving or treating non-alcoholic fatty liver disease. means of solving the problem

[0013] To solve the above problem, the present invention provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising the miR204 inhibitor of SEQ ID NO. 1 as an active ingredient.

[0014] In addition, the present invention provides a food composition for preventing or improving non-alcoholic fatty liver disease comprising the miR204 inhibitor of SEQ ID NO. 1 as an active ingredient.

[0015] In one example of the present invention, the non-alcoholic fatty liver disease is characterized by being induced by liver inflammation and fat accumulation.

[0016] In another example of the present invention, the pharmaceutical or food composition may include additional ingredients effective for non-alcoholic fatty liver disease.

[0017] The present invention provides a method for preventing or treating non-alcoholic fatty liver disease in individuals other than humans, comprising the step of administering the miR204 inhibitor of SEQ ID NO. 1. Effects of the invention

[0019] The composition containing the miR204 inhibitor of the present invention has the effect of reducing fat accumulation and inflammation in liver tissue, so it can be usefully used for the prevention or treatment of non-alcoholic fatty liver disease. Brief explanation of the drawing

[0021] Figure 1 shows the change in body weight after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. *p<0.05 vs. WT-HFD+miR204-I. Figure 2 shows an abdominal photograph after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. Figure 3 shows the abdominal fat mass after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. #p<0.05 vs. WT-HFD+miR204-I Fig. 4 shows liver tissue fat accumulation after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. Fig. 5 shows changes in macrophage markers (brown) after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. Fig. 6 shows miR204 expression after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. *p<0.05 vs. WT-ND+miR204-I; #p<0.05 vs. WT-HFD+miR204-I Specific details for implementing the invention

[0022] Preferred embodiments of the present invention will be described in detail below. In addition, many specific details, such as specific components, are illustrated in the following description; however, these are provided merely to aid in a more comprehensive understanding of the present invention, and it will be obvious to those skilled in the art that the present invention can be practiced without such specific details. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations are omitted if it is determined that such detailed descriptions may unnecessarily obscure the essence of the present invention.

[0024] In the present invention, the terms "prevention" or "protection" mean suppressing or delaying the occurrence of a disease from its cause.

[0025] In this specification, the term “treatment” means stopping the progression of damage by suppressing the progression and / or worsening of symptoms without complete healing, or improving some or all of the symptoms to guide them toward healing.

[0026] In this invention, the term “improvement” refers to any act in which symptoms are alleviated or beneficially altered.

[0027] The present invention provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising the miR204 inhibitor of SEQ ID NO. 1 as an active ingredient.

[0028] In one example of the present invention, the non-alcoholic fatty liver disease is characterized by being induced by liver inflammation and fat accumulation.

[0029] In addition, in a composition according to one embodiment of the present invention, the composition may include additional components useful for the prevention or treatment of non-alcoholic fatty liver disease, and the additional components may include all components known to be effective for non-alcoholic fatty liver disease, including compounds and natural products.

[0030] The composition of the present invention may be prepared in any form, including a suitable carrier, excipient, and diluent commonly used in the preparation of the composition, and preferably in the form of a pharmaceutical composition or a health functional food composition, but is not limited thereto.

[0031] The pharmaceutical composition of the present invention may be formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions, and powders, tablets, capsules, injectables, and liquids are more preferred. Such formulation may be carried out by methods conventionally practiced in the field of pharmaceuticals, and may be preferably formulated according to each disease or component using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

[0032] Carriers, excipients, and diluents that may be included in the above pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, etc.

[0033] When formulating, it may be prepared by additionally using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0034] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients.

[0035] Liquid formulations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous agents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Bases for suppositories may include witepsol, macrogol, tween, cacao gel, laurin gel, and glycerogelatin.

[0036] The preferred dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. However, for a desired effect, the pharmaceutical composition of the present invention may be included in an amount of 0.01 to 99.9% by weight per day, preferably 0.1 to 99% by weight. The daily dosage may be about 0.1 to 1,000 mg / kg, preferably 100 to 300 mg / kg.

[0037] In addition, the present invention provides a food composition for preventing or improving non-alcoholic fatty liver disease comprising the miR204 inhibitor of SEQ ID NO. 1 as an active ingredient.

[0038] Health functional foods to which the above composition can be added include, for example, various general foods, beverages, chewing gum, tea, vitamin complexes, etc.

[0039] In addition, the above composition may be added to food or beverages for the purpose of preventing disease. At this time, the amount of the extract in the food or beverage may be added in an amount of 0.01 to 15 weight% of the total weight of the food, and the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 g.

[0040] The health functional beverage composition of the present invention contains the extract as an essential component in the indicated proportions, but there are no special restrictions on other components, and it may contain additional components such as various flavoring agents or natural carbohydrates, as in conventional beverages. Examples of the natural carbohydrates described above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those described above, natural flavoring agents such as thaumatin, stevia extract, e.g., rebaudioside A, glycyrrhizin, etc.; and synthetic flavoring agents, e.g., saccharin, aspartame, etc., may be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 g of the composition of the present invention. In addition to the above, the extracts of the present invention may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the extracts of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. At this time, although the ratio of additives is not particularly important, it is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

[0041] In another example of the present invention, the present invention provides a method for preventing or treating non-alcoholic fatty liver disease in individuals other than humans, comprising the step of administering the miR204 inhibitor of SEQ ID NO. 1.

[0043] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0045] Example 1. In vivo miR204 inhibition

[0046] miR204 inhibitor (5'-AGG ATG ACA AAG GGA-3'; miR204-I; SEQ ID NO. 1) was ordered and administered subcutaneously via an ALZET Osmotic pump (Cat. 1004) at a dose of 0.5 mg / kg / day for 4 weeks to inhibit the expression of miR204.

[0048] Example 2. Establishment of a mouse model of non-alcoholic fatty liver disease

[0049] Ten 6-week-old wild-type mice were purchased from Orient Bio Co., Ltd. and reared according to the regulations of the Preclinical Center at Chungnam National University Hospital. A 1 kg high-fat diet (fat 60%) was purchased from Dooyul Biotech and used.

[0050] The weight of the mice was recorded every 2 days while feeding them a normal diet and a high-fat diet. After 12 weeks, each mouse was anesthetized using 25% urethane, cardiac blood samples, abdominal fat samples (fat mass measurement), and liver tissue samples were obtained; half of the liver was fixed in a 4% paraformaldehyde (PFA) solution, and the other half was stored in liquid nitrogen.

[0052] Example 3. Detection of miR204 expression in liver tissue (qPCR)

[0053] Liver tissue stored in liquid nitrogen was removed, dissolved by treatment with 2 mL of Trizol reagent (Invitrogen, USA), and then ground using a homogenizer. 1 mL of the supernatant was separated by centrifugation (12,000 g, 10 min) and transferred to a new tube. 200 µL of chloroform was added to the solution. The tube was shaken vigorously at least 10 times and stored at room temperature for 5 minutes. Only the top clear solution from the three-layer solution obtained by centrifugation (12,000 g, 15 min, 4°C) was transferred to a new tube. 500 µL of 100% isopropanol was added to the solution. Subsequently, the RNA precipitate obtained by centrifugation (12,000 g, 10 min, 4°C) was washed with 1 mL of 75% ethanol. The RNA precipitate obtained by centrifugation (7,500 g, 5 min, 4℃) was dried at room temperature for 20-30 minutes. The precipitate was dissolved in 50 µl of RNase-Free water. After quantifying the RNA concentration using a nanodrop, the miRNA cDNA was obtained from an amount of RNA equivalent to 1 µg using a miScript II RT kit (Cat. #218161, Qiagen, Hilden, Germany). Subsequently, real-time quantitative PCR (realtime qPCR) was performed using a miScript SYBR Green PCR kit (Cat. #218073, Qiagen). RNA-U6 small Nuclear 1 (RNU6) was used as a loading control, and the forward primers for RNU6 and miR204 (RNU6, 5'-GCA AAT TCG TGA AGC GTT CC-3'; miR204, 5'-CGC TTC CCT TTG TCA TCC TA-3') were ordered and used. For the reverse primers, the Universal primers (5'-GCG AGC ACA GAA TTA ATA CGA C-3') included in the SYBR green kit were used, and equal amounts were mixed for each.

[0055] Example 4. Histological analysis

[0056] - Hematoxylin & Eosin staining

[0057] After sufficient fixation in 4% PFA solution for at least 16 hours, blocks were prepared by infiltrating with paraffin. The blocks were sectioned onto slides to a thickness of 4 µm. The paraffin was dissolved by immersion in xylene solution for 15 minutes, and then conditions conducive to staining were gradually established by increasing the water density in the order of 100% ethanol -> 90% ethanol -> 80% ethanol -> 70% ethanol -> PBS. After staining in hematoxylin solution for 10 seconds, the blocks were immersed in eosin solution for 10 seconds and dehydrated in the order of PBS -> 70% ethanol -> 80% ethanol -> 90% ethanol -> 100% ethanol -> xylene. Subsequently, the blocks were mounted and photographed under a microscope.

[0059] - CD68 (macrophage) DAB staining

[0060] After sufficient fixation in 4% PFA solution for at least 16 hours, blocks were prepared by infiltrating with paraffin. Sections were made on slides to a thickness of 4 µm. The paraffin was dissolved by immersion in xylene solution for 15 minutes, and then conditions conducive to staining were gradually established by increasing the water density in the order of 100% ethanol -> 90% ethanol -> 80% ethanol -> 70% ethanol -> PBS. The antigens were restored by placing the sample in 1 mM sodium citrate solution and boiling at 100°C for 20 minutes. Subsequently, the sample was blocked using 1% bovine serum albumin, and the primary antibody was diluted 1:500 in the same solution and reacted at 4°C for 16 hours. The following day, the secondary antibody was diluted 1:500 and reacted at room temperature for 1 hour, after which it was stained with DAB solution; the brown-stained areas were observed as positive using a light microscope.

[0062] <Test Example: Result>

[0063] Figure 1 shows the change in body weight after administration of the miR204 inhibitor SEQ ID NO. 1 to mice fed a high-fat diet according to one embodiment of the present invention. A high-fat diet was administered for 12 weeks, and the miR204 inhibitor SEQ ID NO. 1 was injected for 4 weeks prior to the end of the study. The miR204 inhibitor SEQ ID NO. 1 did not affect body weight in the normal diet group, but it reduced the increased body weight of the mice fed a high-fat diet to a statistically significant level.

[0064] FIGS. 2 and 3 show the abdominal fat mass or fat weight after administration of the miR204 inhibitor SEQ ID NO. 1 to mice fed a high-fat diet according to one embodiment of the present invention. The miR204 inhibitor SEQ ID NO. 1 did not affect the abdominal fat mass in the normal diet group, but reduced the increased abdominal fat mass in mice fed a high-fat diet to a statistically significant level.

[0065] FIG. 4 shows the accumulation of fat in liver tissue after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. The miR204 inhibitor SEQ ID NO. 1 significantly reduced fat accumulation in the liver tissue of mice fed a high-fat diet.

[0066] Figure 5 shows the change in macrophage markers (brown) after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. The miR204 inhibitor SEQ ID NO. 1 significantly reduced the expression of CD68, a macrophage indicator, in the liver tissue of mice fed a high-fat diet.

[0067] Figure 6 shows the expression of miR204 after administration of the miR204 inhibitor SEQ ID NO. 1 in mice fed a high-fat diet according to one embodiment of the present invention. The expression of miR204 was increased in the livers of mice fed a high-fat diet, and the miR204 inhibitor SEQ ID NO. 1 reduced the expression of miR204 to a statistically significant level in both the normal diet group and the high-fat diet mice.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease induced by liver inflammation and fat accumulation, comprising the miR204 inhibitor of SEQ ID NO. 1 as an active ingredient. Claim 2 delete Claim 3 A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, characterized in that, in claim 1, the pharmaceutical composition comprises an additional component effective against non-alcoholic fatty liver disease. Claim 4 A food composition for improving non-alcoholic fatty liver disease induced by liver inflammation and fat accumulation, comprising the miR204 inhibitor of SEQ ID NO. 1 as an active ingredient. Claim 5 delete Claim 6 delete Claim 7 delete

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