KR20230052792A - Composition for preventing or treating liver fibrosis comprising beta-guanidinopropionic acid - Google Patents

Abstract

The present invention relates to a composition including beta-guanidinopropionic acid and a use for preventing or treating liver fibrosis. The beta-guanidinopropionic acid reduces the expression of hydroxyproline and alpha-smooth muscle actin proteins in liver tissue.

Description

Composition for preventing or treating liver fibrosis comprising beta-guanidinopropionic acid {Composition for preventing or treating liver fibrosis comprising beta-guanidinopropionic acid}

The present invention relates to a composition containing beta-guanidinopropionic acid (β-GPA) as an active ingredient, which is excellent in preventing or treating liver fibrosis.

Fibrosis is a state in which normal control is impossible during the wound healing process after tissue is damaged by various stresses (infection, chemical stimulation, radiation, etc.) in the human body, and is particularly common in chronic diseases. However, the mechanism is very complex and has not been fully elucidated to date.

When the liver is stimulated by various harmful environmental factors such as alcohol and viruses, hepatic stellate cells are activated by various cytokines including TGF-β (transforming growth factor beta) secreted from Kupffer cells. The secreted TGF-β promotes collagen synthesis, accumulates extracellular matrix, causes hepatic fibrosis by continuously accumulated collagen, and affects not only hepatic stellate cells themselves but also surrounding hepatocytes, resulting in EMT (epithelial to mesenchymal known to cause a transition. Since liver cirrhosis is eventually induced through the continuous process of hepatic fibrosis, understanding and studying the process of hepatic fibrosis is the most basic step in solving all diseases that can cause liver cirrhosis.

In general, liver fibrosis is known to be reversible, composed of thin fibrils, and without nodule formation, unlike liver cirrhosis. If the fibrosis process continues repeatedly, crosslinking between ECM (extra cellular matrix) increases, leading to irreversible liver cirrhosis with nodules.

Liver fibrosis progresses further and cirrhosis occurs. When liver cell necrosis occurs for some reason, liver cell regeneration and fibrous tissue increase, and when this process continues repeatedly for a long time, liver cirrhosis occurs. Liver cirrhosis formed by liver nodules or nodules regenerated by persistent or repetitive diffuse liver parenchymal damage, fibrous tissue growth, and regeneration of hepatocytes is a chronic disease pathologically accompanied by necrosis, inflammation, and fibrosis. Ultimately, it progresses to diseases such as liver cirrhosis complications and liver cancer, leading to death. In particular, because there are no subjective symptoms in the early stages and it is discovered only after considerable progress, research is being actively conducted to develop methods for rapidly diagnosing and treating liver fibrosis.

As a substance that inhibits liver fibrosis, penicillamine, 16,16-dimethyl prostiglidin E2, biphenyl dimethyldicarboxylic acid, colchicine, glucocorticoid, malotilate, gamma-interferon, pentoxifylline ( pentoxifylline), pyridine-2,4-dicarboxylic-diethylamide, pyridine-2,4-dicarboxylic-di(2-methoxyethyl)amide, etc. have been developed, but their action is weak when applied clinically. or severe side effects, currently there is no treatment for liver fibrosis that is used clinically.

β-Guanidinopropionic acid (β-GPA), also referred to as guanidinopropionic acid, beta-guanidinopropionic acid, or N-(aminoiminomethyl)-beta-alanine, is a creatine analogue. Animal (rat, monkey, hamster) studies show that acidic guanidine derivatives, such as β-GPA, can alleviate hyperglycemia in animal models of non-insulin-dependent diabetes. Therefore, it is sometimes used as a dietary supplement in diabetic patients to control blood sugar levels. β-GPA is a white crystalline powder that is highly soluble (>50 mg/mL) in water.

Korean Patent Registration No. 190257 discloses a pharmaceutical composition for the treatment of metabolic diseases containing 3-guanidinopropionic acid, and Korean Patent Publication No. 2018-0059446 discloses β-guanidinopropionic acid with improved characteristics. Pharmaceutically acceptable salts of and uses thereof are disclosed.

However, the effect of the beta-guanidinopropioic acid of the present invention on liver fibrosis has not been reported.

Korean Registered Patent No. 190257 Korean Patent Publication No. 2018-0059446

The present invention was derived from the above needs, and the present inventors completed the present invention by confirming the effect of improving or treating liver fibrosis of a composition containing beta-guanidinopropioic acid as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis comprising beta-guanidinopropionic acid (β-GPA) as an active ingredient.

In one embodiment of the present invention, the beta-guanidinopropionic acid reduces the expression of hydroxyproline and alpha-smooth muscle actin proteins in liver tissue, and the beta-guanidinopropionic acid is an interleukin 17 and interferon gamma, and the beta-guanidinopropionic acid reduces the distribution of CD11b+Ly6C+ monocytes.

In addition, the present invention provides a food composition for preventing or improving liver fibrosis comprising beta-guanidinopropionic acid (β-GPA) as an active ingredient.

In another embodiment of the present invention, the present invention provides a method for preventing or treating liver fibrosis in a non-human subject comprising administering beta-guanidinopropionic acid (β-GPA).

The composition containing beta-guanidinopropionic acid (β-GPA) of the present invention is effective in expressing hydroxyproline and alpha-smooth muscle actin proteins in liver tissue, interleukin 17 and interferon Since it has an effect on reducing the expression of gamma and the distribution of CD11b+Ly6C+ monocytes, it can be usefully used for preventing or treating liver fibrosis.

1 shows the structure of β-guanidinopropioic acid according to an embodiment of the present invention.
2 shows an experimental protocol for a high-fat, high-fructose diet-based steatohepatitis/hepatic fibrosis animal model according to an embodiment of the present invention.
FIG. 3 shows a graph of weight change by β-guanidinopropioic acid in an animal model of steatohepatitis/hepatic fibrosis according to an embodiment of the present invention.
4 shows changes in food intake by β-guanidinopropioic acid in an animal model of steatohepatitis/hepatic fibrosis according to an embodiment of the present invention.
5 shows the results of measuring liver tissue hydroxyproline by β-guanidinopropioic acid in an animal model of steatohepatitis/hepatic fibrosis according to an embodiment of the present invention.
6 shows the results of measuring alpha-smooth muscle actin protein expression in liver tissue by β-guanidinopropioic acid in an animal model of steatohepatitis/hepatic fibrosis according to an embodiment of the present invention.
7 shows the distribution of memory/naive CD4+, CD8+ T cells in liver tissue by β-guanidinopropioic acid in an animal model of steatohepatitis/hepatic fibrosis according to an embodiment of the present invention. will be.
8 is a distribution of T cells expressing liver tissue interleukin 17 (IL-17) and interferon gamma (IFN-γ) by β-guanidinopropioic acid in an animal model of steatohepatitis/hepatic fibrosis according to an embodiment of the present invention. is shown.
9 shows the distribution of monocytes in the liver by β-guanidinopropioic acid in an animal model of steatohepatitis/hepatic fibrosis according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

In the present invention, the term "prevention" or "prevention" means suppressing or delaying the occurrence of a disease from its cause.

As used herein, "treatment" means to stop the progression of damage by suppressing the progression and/or deterioration of symptoms even if not completely cured, or to improve some or all of the symptoms and lead them in the direction of healing.

In the present invention, the term "improvement" refers to all activities that improve or beneficially change symptoms.

The present invention provides a pharmaceutical composition for preventing or treating liver fibrosis comprising beta-guanidinopropionic acid (β-GPA) as an active ingredient.

In one embodiment of the present invention, the beta-guanidinopropionic acid reduces the expression of hydroxyproline and alpha-smooth muscle actin proteins in liver tissue, and the beta-guanidinopropionic acid is an interleukin 17 and interferon gamma, and the beta-guanidinopropionic acid reduces the distribution of CD11b+Ly6C+ monocytes.

In addition, in the composition according to one embodiment of the present invention, the composition may include additional ingredients useful for preventing or treating liver fibrosis, and the additional ingredients include all ingredients known to be effective in liver fibrosis, including compounds and natural products. can include

The composition of the present invention may be prepared in any form of composition by further including appropriate carriers, excipients and diluents commonly used in the preparation of compositions, preferably in the form of pharmaceutical compositions, health functional food compositions or cosmetic compositions. It may be, but is not limited thereto.

The pharmaceutical composition of the present invention may be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, and powders , tablets, capsules, injections and solutions are more preferred. Such formulation may be performed by a method commonly performed in the pharmaceutical field, and may be preferably formulated according to each disease or component using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

Carriers, excipients and diluents that may be included in the 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, mineral oil, and the like.

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

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 or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to water and liquid paraffin, which are commonly used simple diluents. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous agents and suspending agents. As the base of the suppository, witepsol, macrogol, tween, cacao butter, laurin paper, glycerogeratin and the like may be used.

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

In addition, the present invention provides a food composition for preventing or improving liver fibrosis comprising beta-guanidinopropionic acid (β-GPA) as an active ingredient.

Health functional foods to which the composition can be added include, for example, various general foods, beverages, gum, tea, vitamin complexes, and the like.

In addition, the composition may be added to food or beverages for the purpose of preventing diseases. At this time, the amount of the extract in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, and the health drink composition may be added at a rate of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 g there is.

The health functional beverage composition of the present invention is not particularly limited in other components other than containing the extract as an essential component in the indicated ratio, and may contain additional components such as various flavoring agents or natural carbohydrates like conventional beverages. there is. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of flavoring agents other than those described above include natural flavoring agents such as thaumatin and stevia extracts such as rebaudioside A and glycyrrhizin; and synthetic flavoring agents such as saccharin, aspartame, and the like can advantageously be 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 extract of the present invention is various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective Colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, the extracts of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. At this time, the proportion of the additive is not particularly important, but 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.

In another embodiment of the present invention, the present invention provides a method for preventing or treating liver fibrosis in a non-human subject comprising administering beta-guanidinopropionic acid (β-GPA).

The advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments will complete the disclosure of the present invention and allow common knowledge in the art to which the present invention belongs. It is provided to fully inform the holder of the scope of the invention, and the present invention is only defined by the scope of the claims.

<Example: Experimental method>

A total of 12 6-week-old C57BL/6J male mice were fed either a normal diet or a high-fat, high-fructose diet (Rodent Diets With 40 kcal% Fat (Palm Oil), 20 kcal% Fructose and 2% Cholesterol; D09100310 Saeron Bio). A high-fat, high-fructose diet was used to induce liver fibrosis caused by steatohepatitis.

When the mice were 21 weeks old, the normal diet group and the high-fat, high-fructose diet group were treated with beta-Guanidinopropanoic acid (cat no: BD9459; BLD pharmatech Ltd.) at a concentration of 2% by weight in drinking water, respectively, and drank twice a week. The barrel was changed so that it could be freely drunk (Fig. 2). After a total of 12 weeks of 2% beta-Guanidinopropanoic acid in drinking water, mice were necropsied and blood and liver tissue were collected. As a result, the administration period of the high-fat, high-fructose diet for the induction of hepatic fibrosis by steatohepatitis was 27 weeks, and 2% beta-Guanidinopropanoic acid was administered for a total of 12 weeks to determine the antifibrotic effect (FIG. 2).

The groups used in the experiment were normal diet (normal diet-DW), normal diet + drinking water containing 2 wt% beta-Guanidinopropanoic acid (normal diet-β-GPA), and high-fat, high-fructose diet (high-fat/high-fructose diet-DW). , a high-fat high-fructose diet + beta-Guanidinopropanoic acid-containing drinking water (high-fat/high-fructose diet-β-GPA) was administered (Fig. 3).

Weekly weight (Fig. 3) and food intake (Fig. 4) were measured, and alpha-smooth muscle actin protein expression (Fig. 6) and the amount of hydroxyproline in liver tissue (Fig. 5) were evaluated to evaluate the degree of liver fibrosis. ) was evaluated. In addition, for quantitative evaluation of the intrahepatic inflammatory response, the distribution and activity of immune cells present in the liver were evaluated using flow cytometry (FIG. 7).

After digestion of mouse liver tissue with collagenase, centrifugation was performed using 40% Percoll (GE Healthcare, Buckingham, UK). After processing the red blood cell removal solution for the cell pellet, it was diluted with Phosphate-buffered saline and centrifuged to obtain mononuclear cells present in the liver. Flow cytometry was performed by reacting these cells with antibodies labeled with various types of fluorescent substances.

Antibodies used for flow cytometry: anti-CD3-PerCP-Cy5.5, anti-CD3-PE-Cy7, anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD62L-APC, anti -CD44-FITC, anti-CD11b-PerCP-Cy5.5, anti-Ly6C-FITC, anti-IFN-γ-APC, anti-IL-17A-APC, fixable viability dye-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA)

<Test Example: Results>

In both the normal diet and high-fat, high-fructose diet groups, significant weight loss was shown by beta-Guanidinopropanoic acid (FIG. 3).

In both the normal diet and high-fat, high-fructose diet groups, beta-Guanidinopropanoic acid did not affect mouse feed intake (FIG. 4).

Beta-Guanidinopropanoic acid induced a significant decrease in liver tissue hydroxyproline in the high-fat, high-fructose diet group (FIG. 5). This result suggests that beta-Guanidinopropanoic acid has the function of inhibiting liver fibrosis.

The expression of alpha-smooth muscle actin, a protein representing the degree of hepatic fibrosis, was significantly decreased in the beta-Guanidinopropanoic acid-treated group (FIG. 6).

Compared to the normal diet, in the group treated with the high-fat, high-fructose diet, liver tissue memory CD4+ and CD8+ T cells increased, whereas non-contact CD4+ and CD8+ T cells decreased (FIG. 7). These results indicate that treatment with beta-Guanidinopropanoic acid contributes to the reduction of the hepatic inflammatory response by inducing a decrease in hepatic tissue memory CD4+ and CD8+ T cells and an increase in non-contact CD4+ and CD8+ T cells.

Treatment with a high-fat, high-fructose diet increased the expression of interleukin 17 and interferon gamma in intrahepatic CD4+ T cells, and treatment with beta-Guanidinopropanoic acid markedly decreased the expression of interleukin 17 and interferon gamma in intrahepatic CD4+ T cells (Fig. 8). This result suggests that beta-Guanidinopropanoic acid reduced inflammatory cytokines (interleukin 17, interferon gamma) inducing an intrahepatic inflammatory response, resulting in a reduction in fibrosis shown in FIGS. 5 and 6 .

Monocytes expressing CD11b+Ly6C+ tended to increase in steatohepatitis/hepatic fibrosis caused by the high-fat, high-fructose diet, and beta-Guanidinopropanoic acid reduced the distribution of CD11b+Ly6C+ monocytes (FIG. 9). This suggests that it may have contributed to reducing the inflammatory response in the liver and inhibiting fibrosis.

Claims (5)

Food composition for preventing or improving liver fibrosis containing beta-guanidinopropionic acid (β-GPA) as an active ingredient The food composition for preventing or improving liver fibrosis according to claim 1, wherein the liver fibrosis is induced by liver inflammation. The food composition for preventing or improving liver fibrosis according to claim 1, wherein the beta-guanidinopropionic acid reduces the expression of hydroxyproline and alpha-smooth muscle actin in liver tissue. The food composition for preventing or improving liver fibrosis according to claim 1, wherein the beta-guanidinopropionic acid reduces the expression of interleukin 17 and interferon gamma. The food composition for preventing or improving liver fibrosis according to claim 1, wherein the beta-guanidinopropionic acid reduces the distribution of CD11b+Ly6C+ monocytes.

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