KR20230165499A - Use of 6-diazo-5-oxo-L-norleucine for treating non-alcoholic fatty liver diseases - Google Patents

Abstract

The present invention relates to the treatment of non-alcoholic fatty liver disease using a composition containing 6-diazo-5-oxo-L-norleucine (DON) as an active ingredient. ; NAFLD) for prevention or treatment.

Description

{Use of 6-diazo-5-oxo-L-norleucine for treating non-alcoholic fatty liver diseases}

The present invention relates to the treatment of non-alcoholic fatty liver disease using a composition containing 6-diazo-5-oxo-L-norleucine (DON) as an active ingredient. ; NAFLD) for prevention or treatment.

Fatty liver is defined as a case where the weight due to fat deposition in the liver is more than 5% of the weight of the liver. Non-alcoholic fatty liver disease (NAFLD) refers to a case where the cause of fatty liver is not caused by viruses, drugs, genetics, or alcohol, and is closely related to metabolic syndrome such as insulin resistance, obesity, high blood pressure, and dyslipidemia. It is a chronic liver disease known to be closely related and includes a series of processes ranging from simple steatosis in the liver to non-alcoholic steatohepatitis and cirrhosis. Worldwide, the prevalence of non-alcoholic fatty liver disease is reported to vary depending on the population group. In the West, it is known that non-alcoholic fatty liver disease occurs in about 20 to 30% of normal adults without any specific cause of liver disease, and in Korea, it is known to have non-alcoholic fatty liver disease. The prevalence of nonalcoholic fatty liver disease in adults is reported to be approximately 16 to 50%. In general, the prevalence of non-alcoholic fatty liver disease is known to increase in those who are obese, because insulin resistance caused by obesity is known to be an important cause of fat deposition in the liver. In the case of mild fatty liver, such as simple fat deposition, where there is only fat and no damage to liver cells, the prognosis is relatively good, but if left untreated, it can progress to cirrhosis, a serious liver disease, and 4-27% of patients with cirrhosis develop liver cancer. It is known that 30 to 40% of patients with cirrhosis die from complications of liver disease and cardiovascular disease after 10 years, so active management from the beginning is important.

To treat this non-alcoholic fatty liver disease, pioglitazone, vitamin E, and ursodeoxycholic acid (UDCA) are used, but they lack efficacy or may cause side effects during long-term administration. , To date, there is no approved drug for the treatment of non-alcoholic fatty liver disease.

Accordingly, the present inventors made intensive research efforts to discover a small molecule compound that can prevent or treat non-alcoholic fatty liver disease, and as a result, the small molecule compound 6-diazo-5-oxo-L-norleucine (6-diazo-5-oxo- It was confirmed that L-norleucine (DON) efficiently reduces fat accumulation and/or fibrosis in the liver in an animal model of non-alcoholic fatty liver disease, and the present invention was completed.

Clin. Gastroenterol. Hepatol., 2018, 16(4): 558-566, Antioxidants, 2018, 7(1): 12, BMC Gastroenterology, 2013, 13: 140.

One object of the present invention is to prepare a ratio comprising 6-diazo-5-oxo-L-norleucine (DON) or a pharmaceutically acceptable salt thereof as an active ingredient. To provide a pharmaceutical composition for preventing or treating alcoholic fatty liver disease (NAFLD).

Another object of the present invention includes administering a composition containing 6-diazo-5-oxo-L-norleucine or a pharmaceutically acceptable salt thereof as an active ingredient to an individual in need thereof. To provide a treatment method for non-alcoholic fatty liver disease.

Another object of the present invention is to provide a food composition for preventing or improving non-alcoholic fatty liver disease containing 6-diazo-5-oxo-L-norleucine or a foodologically acceptable salt thereof as an active ingredient. will be.

Another object of the present invention is to provide a feed composition for preventing or improving non-alcoholic fatty liver disease containing 6-diazo-5-oxo-L-norleucine or a feedologically acceptable salt thereof as an active ingredient. will be.

Hereinafter, the present invention will be described in more detail. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, it cannot be said that the scope of the present invention is limited by the specific description described below.

One aspect of the present invention is a ratio comprising 6-diazo-5-oxo-L-norleucine (DON) or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for preventing or treating alcoholic fatty liver disease (NAFLD) is provided.

Another aspect of the present invention includes administering a composition containing 6-diazo-5-oxo-L-norleucine or a pharmaceutically acceptable salt thereof as an active ingredient to an individual in need thereof, Provides a treatment method for non-alcoholic fatty liver disease.

“6-Diazo-5-oxo-L-norleucine” of the present invention is (S)-2-amino-6-diazo-5-oxohexanoic acid ((S)- represented by the following formula 1: It is a compound with the IUPAC name of 2-amino-6-diazo-5-oxohexanoic acid), and is a type of non-standard amino acid isolated from Streptomyces in soil samples in Peru. It is known to have anticancer activity due to the diazo structure it contains, and has inhibitory activity against various enzymes such as carbamoyl phosphate synthase (CAD), PRPP amidotransferase, and NAD synthase. Confirmed. However, nothing is yet known about its preventive or therapeutic effects on non-alcoholic fatty liver disease.

[Formula 1]

The present invention confirms that the 6-diazo-5-oxo-L-norleucine has the effect of reducing steatosis and intralobular inflammation in liver tissue and suppressing fibrosis in an animal test model inducing non-alcoholic fatty liver disease. It is based on the first discovery that it can be used to treat non-alcoholic fatty liver disease.

The 6-diazo-5-oxo-L-norleucine can be synthesized through a known synthetic method, or a commercially available compound can be purchased and used, but is not limited thereto.

6-Diazo-5-oxo-L-norleucine, a compound contained as an active ingredient in the pharmaceutical composition of the present invention, may exist in the form of a pharmaceutically acceptable salt. As the salt, an acid salt formed from a pharmaceutically acceptable free acid is useful. The term "pharmaceutically acceptable salt" of the present invention refers to a concentration that has an effective effect that is relatively non-toxic and harmless to patients, and any organic or organic salt of the compound where side effects due to the salt do not reduce the beneficial efficacy of the compound. It means inorganic addition salt.

Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water can be heated, and the mixture can then be evaporated to dryness, or the precipitated salt can be suction filtered.

At this time, organic acids and inorganic acids can be used as free acids. Hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as inorganic acids, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, and maleic acid can be used as organic acids. (maleic acid), succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid. (gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. can be used. , but is not limited to these.

Additionally, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare metal salts, especially sodium, potassium, or calcium salts, but is not limited to these. Additionally, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (e.g., silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention include salts of acidic or basic groups that may be present in the compounds, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium, and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, and hydrogen phosphate. , dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, etc., and the preparation of salts known in the art. It can be manufactured through a method.

The salt of the compound is a pharmaceutically acceptable salt, and any salt that exhibits pharmacological activity equivalent to that of the compound, for example, inhibits aggregation and/or hyperphosphorylation of tau protein, can be used without limitation.

In addition, the compound includes, without limitation, not only its pharmaceutically acceptable salts, but also solvates such as possible hydrates that can be prepared therefrom, and all possible stereoisomers. Solvates and stereoisomers of the above compounds can be prepared using methods known in the art.

Furthermore, the compounds may be prepared in crystalline or amorphous form and, when prepared in crystalline form, may be optionally hydrated or solvated. In the present invention, not only stoichiometric hydrates of the above compounds but also compounds containing various amounts of water may be included. Solvates of the compounds according to the invention include both stoichiometric and non-stoichiometric solvates.

For example, the composition of the present invention may further include a pharmaceutically acceptable carrier, diluent, or excipient, and may be formulated into powders, granules, tablets, capsules, suspensions, emulsions, etc. according to conventional methods to suit each purpose of use. It can be formulated and used in a variety of forms, including oral formulations such as syrup and aerosol, and injections of sterile injectable solutions. It can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. . Examples of suitable carriers, excipients or diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, Examples include cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. Formulated by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used.

Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. The basis of suppositories is Wethepsol, Macrogol, and Twin 61. Cacao, laurel, glycerogeratin, etc. can be used. Meanwhile, injectables may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

The formulation may be prepared by conventional mixing, granulating or coating methods and may contain the active ingredient in the range of about 0.1 to 75% by weight, preferably about 1 to 50% by weight. A unit dosage form for a mammal weighing about 50 to 70 kg contains about 10 to 200 mg of active ingredient.

At this time, the composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level refers to the patient's health condition, Factors including the type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the medical field. You can.

The preferred dosage of the compound of the present invention varies depending on the patient's condition and weight, degree of disease, form of drug, route and period of administration, but can be appropriately selected by a person skilled in the art. However, for desirable effects, it is recommended to administer the compound of the present invention at 0.0001 to 100 mg/kg (body weight) per day, preferably 0.001 to 100 mg/kg (body weight). Administration can be administered once a day or in divided doses via oral or parenteral routes.

For example, the dosage may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., so the above dosage does not limit the scope of the present invention in any way.

Additionally, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response regulators for the prevention and treatment of non-alcoholic fatty liver disease. For the purposes of the present invention, the pharmaceutical composition according to the present invention may be administered in combination with one or more additional drugs commonly used in the treatment of non-alcoholic fatty liver disease.

When the pharmaceutical composition according to the present invention is administered for the treatment of non-alcoholic fatty liver disease, it can be administered in combination and/or in combination with one or more ingredients and/or drugs effective in treating or preventing non-alcoholic fatty liver disease. there is. At this time, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, taking into account all of the above-mentioned factors, and this can be easily determined by a person skilled in the art.

Specifically, the combination and/or combination administration involves administering 6-diazo-5-oxo-L-norleucine or a pharmaceutically acceptable salt thereof of the present invention and one or more additional drugs simultaneously, sequentially, or in reverse order, respectively. It can be administered simultaneously in a combination of appropriate effective amounts, or stored in separate containers and administered together simultaneously, sequentially, or in reverse order.

Examples of ingredients and/or drugs effective in the treatment or prevention of non-alcoholic fatty liver disease include TZDs (Thiazolidinediones), vitamin E, metformin, statins, UDCA (ursodeoxycholic acid), unsaturated fatty acids such as omega 3, etc. Polyunsaturated fatty acids, Angiotensin receptor blockers, Pentoxifylline, Glucagon-like peptide 1 receptor agonists, DPP-4 inhibitors (Dipeptidyl peptidase 4 inhibitors), SGLT2 Inhibitors (sodium/glucose cotransporter 2 inhibitors), OCA (Obeticholic acid), Elafibranor, Telmisartan, etc., but are not particularly limited, and are known in the art for the treatment of non-alcoholic fatty liver disease Alternatively, any ingredient and/or drug with a preventive effect may be used without limitation.

As the term of the present invention, "non-alcoholic fatty liver disease" is a disease caused by excessive fat accumulation in the liver without a clear cause such as alcohol intake, metabolic (dysfunction) associated fatty liver disease ; It is also called MAFLD). This largely includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), which involves liver inflammation. Non-alcoholic fatty liver disease is less dangerous than non-alcoholic steatohepatitis and usually does not progress to non-alcoholic steatohepatitis or cirrhosis. If non-alcoholic fatty liver disease progresses to a level of non-alcoholic steatohepatitis accompanied by inflammation or fibrosis, symptoms can be reversibly alleviated through diet, exercise, weight loss, and/or bariatric surgery, and further, a healthy liver. Recovery can be expected, but if non-alcoholic steatohepatitis accompanied by cirrhosis and/or cancer has progressed, recovery is difficult except for transplantation. The non-alcoholic fatty liver disease may be caused by obesity or type 2 diabetes, and other causes include being overweight; metabolic syndrome, defined as three or more medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum HDL cholesterol; diet high in fructose; and old age.

For example, non-alcoholic fatty liver diseases that can be prevented or treated by administering the pharmaceutical composition according to the present invention include simple steatosis in the liver, non-alcoholic fatty liver, steatohepatitis, non-alcoholic steatohepatitis, and cirrhosis. ), or it may be liver cancer. Specifically, the non-alcoholic steatohepatitis may be accompanied by one or more symptoms selected from the group consisting of inflammation, fibrosis, cirrhosis, and cancer, but is not limited thereto. Alternatively, the non-alcoholic steatohepatitis may cause complications of liver failure and/or cardiovascular disease, but is not limited thereto.

Specifically, the pharmaceutical composition of the present invention may exhibit a reducing effect on body weight gain. Furthermore, the pharmaceutical composition of the present invention can reduce non-alcoholic fatty liver activity score (NAFLD activity score (NAS)). Specifically, the pharmaceutical composition of the present invention can reduce steatosis and/or lobular inflammation scores. In addition, the pharmaceutical composition of the present invention can reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), or both, which are indicators of non-alcoholic fatty liver disease. .

The term "prevention" of the present invention refers to all actions that inhibit or delay the occurrence, spread, and recurrence of non-alcoholic fatty liver disease by administering the pharmaceutical composition of the present invention, and the term "treatment" refers to the use of the pharmaceutical composition of the present invention. It refers to any action in which the symptoms of the disease are improved or changed to a beneficial effect by administration.

In one specific embodiment of the present invention, the treatment includes reducing or alleviating the signs of non-alcoholic fatty liver disease, reducing the extent of the disease, delaying or slowing the disease, palliating or stabilizing the disease state, and other beneficial results, but are not limited thereto.

The term "individual" of the present invention refers to an individual suspected of having non-alcoholic fatty liver disease, who has developed or can develop non-alcoholic fatty liver disease, including monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, including humans. This refers to all animals including cats, dogs, mice, rats, rabbits or guinea pigs, and the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to these individuals. Additionally, the pharmaceutical composition of the present invention can exhibit a synergistic effect when administered in combination with existing therapeutic agents.

The term "administration" of the present invention means providing a predetermined substance to a patient by any suitable method, and the administration route of the composition of the present invention can be administered through any general route as long as it can reach the target tissue. there is. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonaryly, or rectally, but is not limited thereto. Additionally, the pharmaceutical composition of the present invention may be administered by any device that allows the active agent to move to target cells. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. Injections include aqueous solvents such as physiological saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). It can be manufactured using stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulphite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, and agents to prevent microbial growth. It may contain pharmaceutical carriers such as preservatives (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

For example, the composition of the present invention may further include a pharmaceutically acceptable carrier, diluent, or excipient, and may be formulated into powders, granules, tablets, capsules, suspensions, emulsions, etc. according to conventional methods to suit each purpose of use. It can be formulated and used in a variety of forms, including oral formulations such as syrup and aerosol, and injections of sterile injectable solutions. It can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. . Examples of suitable carriers, excipients or diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, Examples include cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. Formulated by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used.

Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. The basis of suppositories is Wethepsol, Macrogol, and Twin 61. Cacao, laurel, glycerogeratin, etc. can be used. Meanwhile, injectables may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

The formulation may be prepared by conventional mixing, granulating or coating methods and may contain the active ingredient in the range of about 0.1 to 75% by weight, preferably about 1 to 50% by weight. A unit dosage form for a mammal weighing about 50 to 70 kg contains about 10 to 200 mg of active ingredient.

At this time, the composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level refers to the patient's health condition, Factors including the type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the medical field. You can. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

For example, the dosage may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., so the above dosage does not limit the scope of the present invention in any way.

The preferred dosage of the compound of the present invention varies depending on the patient's condition and weight, degree of disease, form of drug, route and period of administration, but can be appropriately selected by a person skilled in the art. However, for desirable effects, it is recommended to administer the compound of the present invention at 0.0001 to 100 mg/kg (body weight) per day, preferably 0.001 to 100 mg/kg (body weight). Administration can be administered once a day or in divided doses via oral or parenteral routes.

Another aspect of the present invention provides a food composition for preventing or improving non-alcoholic fatty liver disease, comprising 6-diazo-5-oxo-L-norleucine or a foodologically acceptable salt thereof as an active ingredient.

The “6-diazo-5-oxo-L-norleucine”, “non-alcoholic fatty liver disease”, and “prevention” are as described above.

“Improvement” in the present invention refers to any action that improves or benefits the suspected disease and the symptoms of the subject of the invention using a composition containing trametinib or a foodologically acceptable salt thereof.

The food composition according to the present invention includes the form of pills, powders, granules, precipitates, tablets, capsules, or liquids. Foods to which the composition of the present invention can be added include, for example, various foods, such as , beverages, gum, tea, vitamin complexes, health supplements, etc.

Except for the inclusion of trametinib as an essential ingredient that can be included in the food composition of the present invention, there are no restrictions on other ingredients, and like ordinary foods, it can contain various herbal extracts, food supplements, or natural carbohydrates as additional ingredients.

In addition, the food auxiliary additives include food auxiliary additives common in the art, such as flavoring agents, flavors, colorants, fillers, stabilizers, etc.

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above-mentioned flavoring agents, natural flavoring agents (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination.

In the present invention, the food composition includes health functional foods and health supplements.

The above-mentioned health functional food (functional food) is the same term as food for special health use (FoSHU), and is a food manufactured and/or processed using raw materials or ingredients with functional properties useful to the human body. In other words, it refers to foods with high medical effects that have been processed to efficiently perform bioregulatory functions in addition to supplying nutrients. Here, “function” means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. As mentioned above, foods that contain ingredients useful to the human body and whose function and safety have been recognized by the Ministry of Food and Drug Safety are called health functional foods. Other foods that have not been approved by the Ministry of Food and Drug Safety but are considered to have beneficial effects on the human body are called health functional foods. It is classified as a (supplementary) food. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Additionally, the food formulation can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various forms, and unlike general drugs, it is made from food as a raw material, so it has the advantage of not having side effects that may occur when taking the drug for a long time, and is highly portable.

Another aspect of the present invention provides a feed composition for preventing or improving non-alcoholic fatty liver disease, comprising 6-diazo-5-oxo-L-norleucine or a feedologically acceptable salt thereof as an active ingredient. .

The “6-diazo-5-oxo-L-norleucine”, “non-alcoholic fatty liver disease”, “prevention”, and “improvement” are as described above.

As used herein, the term "feed" means any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals.

The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, oils and fats, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more types.

The feed of the present invention may be powder feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, raw feed, etc., but is not limited thereto.

The feed composition of the present invention may include binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, and the feed composition may include feed additives. To increase utility, there may be amino acids, vitamins, enzymes, flavors, non-protein nitrogen compounds, silicate agents, buffers, extractants, oligosaccharides, etc. added to feed. In addition, feed mixtures, etc. may be additionally included, but are not limited thereto.

The pharmaceutical composition containing 6-diazo-5-oxo-L-norleucine (DON) of the present invention as an active ingredient not only reduces body weight gain but also reduces liver Because it can inhibit fat accumulation and fibrosis, it can be useful in preventing or treating non-alcoholic fatty liver disease.

Figure 1 is a diagram schematically showing the manufacturing and testing schedule of an animal testing model according to an embodiment of the present invention.
Figure 2 is a diagram showing body weight change over time in an animal test model according to an embodiment of the present invention.
Figure 3 is a diagram showing the rate of change in body weight over time in an animal test model according to an embodiment of the present invention.
Figure 4 is a diagram showing the results of H&E staining of liver tissue recovered from an animal test model according to an embodiment of the present invention.
Figure 5 is a diagram showing the steatosis and lobular inflammation scores of liver tissue recovered from an animal test model according to an embodiment of the present invention.
Figure 6 is a diagram showing the results of Masson's trichrome staining of liver tissue recovered from an animal test model according to an embodiment of the present invention.
Figure 7 is a diagram showing the blood ALT (alanine aminotransferase) level of an animal test model according to an embodiment of the present invention.
Figure 8 is a diagram showing the blood AST (aspartate aminotransferase) level in an animal test model according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples.

Preparation Example 1: Preparation of non-alcoholic fatty liver disease animal test model

7-week-old C57BL/6 male mice (Nara Biotech Co., Ltd.) were purchased, acclimatized for 7 days, and then selected as healthy animals. During the experiment period, the breeding environment was maintained at a temperature of 21 to 25°C, relative humidity of 45 to 65%, and lighting time of 12 hours, and feed and water were provided ad libitum.

Mice acclimatized for 1 week were fed a non-fat diet, a high-fat diet (HFD), and a choline deficient defined amino acid (CDAA) diet containing 0.1% methionine. , Research diet: A06071302) was provided for 7 weeks, and from the 7th week onwards, 0.1 mg/kg of DON was administered orally (p.o.) or subcutaneously (s.c.) to the CDAA-treated mice every day. The animal model production and experiment schedule is shown in Figure 1, and the composition and treatment conditions of each experimental group and control group are shown in detail in Table 1 below.

diet medication population Control group (CTL) standard diet 200 μl vehicle (H ₂ O) Subcutaneous injection 2 animals CDAA CDAA Research diet
A06071302 200 μl vehicle (H ₂ O) Subcutaneous injection 8 animals 0.1 mg/kg DON-SC. 0.025 mg/back/day DON Subcutaneous injection 8 animals 0.1 mg/kg DON-PO 0.025 mg/back/day DON oral- 8 animals

Example 1: Weight loss effect following DON administration

The actual weight of the control and experimental group mice was measured and shown in Figure 2, and the weight change rate calculated from this is shown in Figure 3. The weight change rate was expressed as a percentage of the change in mouse body weight every 3 days during the DON administration period based on the day before administration. As shown in Figure 2, compared to the control group, both CDAA diet groups were found to have lower body weight regardless of whether or not DON was administered. Additionally, as shown in Figure 3, the increased weight gain rate in the CDAA administered group decreased in the DON administered group.

Example 2: H&E staining

At 14 weeks after DON administration, liver tissue obtained from the experimental animals was stained with H&E and observed under a microscope, and the results are shown in Figure 4. Furthermore, from the H&E staining results for the liver tissue section, the steatosis and lobular inflammation scores, which are factors constituting the non-alcoholic fatty liver activity score (NAFLD activity score; NAS), were calculated and the results were obtained. It is shown in 5. Specifically, the steatosis score is 0 if the area occupied by fat in liver tissue is less than 5%, 1 if it is 5 to 33%, 2 if it is 34 to 66%, and 3 if it is more than 66%. My inflammation score counts inflammation foci per 200× field and gives 0 points for the absence of inflammation foci, 1 point for less than 2 inflammation foci, 2 points for 2 to 4 foci, and 3 points for more than 4 inflammation foci. Scoring was performed in the same manner as the pathology interpretation criteria.

As shown in Figure 5, the steatosis and intralobular inflammation scores increased by the CDAA diet were significantly reduced in the experimental group administered DON by subcutaneous injection or oral administration.

Example 3: Masson tricolor staining

In order to confirm the effect of DON on fibrosis of liver tissue, liver tissue obtained from the experimental animals at 14 weeks after DON administration was observed under a microscope using Masson's trichrome staining, and the results are shown in Figure 6 shown in As shown in Figure 6, it was confirmed that the production of collagen, which appears purple in the CDAA diet group, and the fibrosis induced thereby were all reduced in the DON-treated group.

Example 4: Biochemical analysis of mouse serum

At 14 weeks after DON administration, blood was collected from the experimental animals and blood ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels were analyzed through biochemical analysis, and the results are shown in Figures 7 and 8, respectively. As shown in Figures 7 and 8, the increased blood ALT and AST levels in the CDAA diet group were all significantly reduced in the DON-treated group.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (9)

Non-alcoholic fatty liver disease containing 6-diazo-5-oxo-L-norleucine (DON) or a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutical composition for preventing or treating fatty liver disease (NAFLD). According to paragraph 1,
The non-alcoholic fatty liver disease includes simple steatosis in the liver, non-alcoholic fatty liver (NAFL), steatohepatitis, non-alcoholic steatohepatitis (NASH), and liver cirrhosis ( hepatocirrhosis), or liver cancer, a pharmaceutical composition. According to paragraph 2,
A pharmaceutical composition, wherein the non-alcoholic steatohepatitis is accompanied by one or more symptoms selected from the group consisting of inflammation, fibrosis, cirrhosis, and cancer. According to paragraph 1,
A pharmaceutical composition, wherein the composition exhibits a reducing effect on body weight gain. According to paragraph 1,
The composition is a pharmaceutical composition that reduces non-alcoholic fatty liver activity score (NAFLD activity score (NAS)). According to paragraph 1,
The composition is a pharmaceutical composition that reduces the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), or both. A food composition for preventing or improving non-alcoholic fatty liver disease, comprising 6-diazo-5-oxo-L-norleucine or a foodologically acceptable salt thereof as an active ingredient. In clause 7,
A food composition wherein the composition is a health functional food or health supplement. A feed composition for preventing or improving non-alcoholic fatty liver disease, comprising 6-diazo-5-oxo-L-norleucine or a feed-chemically acceptable salt thereof as an active ingredient.