KR102615692B1 - Composition for preventing or treating liver fibrosis comprising beta-guanidinopropionic acid - Google Patents

Abstract

The present invention relates to a composition containing beta-guanidinopropionic acid (β-GPA) and its use for preventing or treating liver fibrosis.

Description

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 has excellent effects in preventing or treating liver fibrosis.

Fibrosis is a condition in which normal control is not possible during the wound healing process after tissues in the human body are damaged by various stresses (infection, chemical stimulation, radiation, etc.). It is especially common in chronic diseases. The mechanism through which this happens is so complex that it has not been fully elucidated to this day.

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

In general, liver fibrosis, unlike cirrhosis, is known to be reversible, composed of thin fibrils, and without nodule formation. Normal recovery may be possible if the cause of liver damage is eliminated, but this liver If the fibrosis process continues repeatedly, crosslinking between ECM (extra cellular matrix) increases and progresses to irreversible cirrhosis with nodules.

Liver fibrosis progresses further, resulting in cirrhosis. When hepatocyte necrosis occurs for some reason, liver cell regeneration and fibrous tissue increase, and when this process continues repeatedly for a long time, cirrhosis occurs. Liver cirrhosis, which is formed by persistent or repetitive diffuse liver parenchymal damage, increased fibrous tissue, and regenerated liver nodules or nodules through hepatocyte regeneration, is a chronic disease pathologically accompanied by necrosis, inflammation, and fibrosis. Ultimately, it progresses to diseases such as liver cirrhosis and liver cancer, leading to death. In particular, because there are no noticeable symptoms in the early stages and it is only discovered when the disease has progressed considerably, research is being actively conducted to develop methods for quickly diagnosing and treating liver fibrosis.

Substances that inhibit liver fibrosis include penicillamine, 16,16-dimethyl prostigladin E2, biphenyl dimethyldicarboxylic acid, colchicine, glucocorticoids, malotilate, gamma-interferon, and pentoxifylline ( pentoxifylline), pyridine-2,4-dicarboxylic-diethylamide, and pyridine-2,4-dicarboxylic-di(2-methoxyethyl)amide have been developed, but their effectiveness is weak when applied clinically. There are currently no treatments for liver fibrosis in clinical use due to severe side effects.

β-Guanidinopropionic acid (β-GPA), also referred to as guanidinopropionic acid, beta-guanidinopropionic acid, or N-(aminoiminomethyl)-beta-alanine, is a creatine analog. Animal studies (rats, monkeys, hamsters) 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 in water (>50 mg/ml).

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

However, there has been no report on the effect of beta-guanidinopropioic acid of the present invention on liver fibrosis.

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

The present invention was derived from the above-mentioned 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 containing beta-guanidinopropionic acid (β-GPA) as an active ingredient.

In one example 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 reduces interleukin 17 and interferon gamma, and the beta-guanidinopropionic acid reduces the distribution of CD11b+Ly6C+ monocytes.

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

In another example of the present invention, the present invention provides a method for preventing or treating liver fibrosis in subjects other than humans, including the step of administering beta-guanidinopropionic acid (β-GPA).

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

Figure 1 shows the structure of β-guanidinopropioic acid according to one embodiment of the present invention.
Figure 2 shows an experimental protocol for a high-fat, high-fructose diet-based steatohepatitis/liver fibrosis animal model according to an embodiment of the present invention.
Figure 3 shows a graph of body weight change caused by β-guanidinopropioic acid in an animal model of steatohepatitis/liver fibrosis according to an embodiment of the present invention.
Figure 4 shows changes in food intake caused by β-guanidinopropioic acid in a steatohepatitis/liver fibrosis animal model according to an embodiment of the present invention.
Figure 5 shows the results of measuring liver tissue hydroxyproline by β-guanidinopropioic acid in an animal model of steatohepatitis/liver fibrosis according to an embodiment of the present invention.
Figure 6 shows the results of measuring liver tissue alpha-smooth muscle actin protein expression by β-guanidinopropioic acid in a steatohepatitis/liver fibrosis animal model according to an embodiment of the present invention.
Figure 7 shows the distribution of memory/naive CD4+, CD8+T cells in liver tissue by β-guanidinopropioic acid in an animal model of steatohepatitis/liver fibrosis according to an embodiment of the present invention. will be.
Figure 8 shows the distribution of T cells expressing interleukin 17 (IL-17) and interferon gamma (IFN-γ) in liver tissue by β-guanidinopropioic acid in an animal model of steatohepatitis/liver fibrosis according to an embodiment of the present invention. It represents.
Figure 9 shows the distribution of intrahepatic monocytes by β-guanidinopropioic acid in a steatohepatitis/liver fibrosis animal model 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, but this is provided to facilitate a more general understanding of the present invention, and it is known 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. Additionally, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist 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.

In this specification, “treatment” means stopping the progression of damage by suppressing the progression and/or worsening of symptoms even if not completely cured, or improving some or all of the symptoms and leading toward healing.

In the present invention, the term “improvement” refers to any action that improves or beneficially changes symptoms.

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

In one example 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 reduces 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 contain 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. It can be included.

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

The pharmaceutical composition of the present invention can be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods. , tablets, capsules, injections and liquid formulations are more preferable. This formulation can be performed by a method commonly performed in the pharmaceutical field, and can be preferably formulated according to each disease or ingredient using the 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, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it can 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 ( It is prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous agents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween, cacao, laurin, glycerogeratin, etc. can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person 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.

Additionally, the present invention provides a food composition for preventing or improving liver fibrosis containing 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, etc.

Additionally, the composition can 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 can be added at 0.01 to 15% by weight of the total weight of the food, and the health drink composition can 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 drink composition of the present invention has no particular restrictions on other ingredients other than containing the extract as an essential ingredient in the indicated ratio, and may contain additional ingredients such as various flavoring agents or natural carbohydrates like a conventional beverage. there is. Examples of the above-mentioned 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. Flavoring agents other than those mentioned above include natural flavoring agent thaumatin, stevia extract, such as rebaudioside A, glycyrrhizin, etc.; and synthetic flavoring agents such as saccharin, aspartame, etc. can be advantageously used. The proportion of natural carbohydrates 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 contains various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, and protective properties. It may contain colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the extracts of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients can be used independently or in combination. At this time, the ratio of the additive is not very 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 example of the present invention, the present invention provides a method for preventing or treating liver fibrosis in subjects other than humans, including the step of administering beta-guanidinopropionic acid (β-GPA).

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments are merely intended to ensure that the disclosure of the present invention is complete and that common knowledge in the technical field to which the present invention pertains is not limited. It is provided to fully inform those who have 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 administered 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 due to steatohepatitis.

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

The groups used in the experiment were normal diet (normal diet-DW), normal diet + drinking water containing 2% by weight beta-Guanidinopropanoic acid (normal diet-β-GPA), and high-fat/high-fructose diet (high-fat/high-fructose diet-DW). , the group was divided into two groups administered a high-fat, high-fructose diet + drinking water containing beta-Guanidinopropanoic acid (high-fat/high-fructose diet - β-GPA) (Figure 3).

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

After digesting the mouse liver tissue with collagenase, centrifugation was performed using 40% Percoll (GE Healthcare, Buckingham, UK). After treating the cell pellet with red blood cell removal solution, 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>

Significant weight loss was observed due to beta-Guanidinopropanoic acid in both the normal diet and high-fat and high-fructose diet groups (Figure 3).

Beta-Guanidinopropanoic acid did not affect mouse feed intake in both the normal diet and high-fat and high-fructose diet groups (Figure 4).

beta-Guanidinopropanoic acid induced a significant decrease in liver tissue hydroxyproline in the high-fat, high-fructose diet group (Figure 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 liver fibrosis, was significantly decreased in the beta-Guanidinopropanoic acid treatment group (Figure 6).

Compared to the normal diet, the high-fat and high-fructose diet group showed an increase in liver tissue memory CD4+ and CD8+ T cells, while a decrease in naive CD4+ and CD8+ T cells (Figure 7). These results indicate that treatment with beta-Guanidinopropanoic acid induces a decrease in liver tissue memory CD4+ and CD8+ T cells and an increase in naïve CD4+ and CD8+ T cells, contributing to the reduction of intrahepatic inflammatory response.

When treated with a high-fat, high-fructose diet, the expression levels of interleukin 17 and interferon gamma increased in intrahepatic CD4+ T cells, and when treated with beta-Guanidinopropanoic acid, the expression levels of interleukin 17 and interferon gamma were significantly decreased in intrahepatic CD4+ T cells (Figure 8). This result suggests that beta-Guanidinopropanoic acid reduced inflammatory cytokines (interleukin 17, interferon gamma) that induce intrahepatic inflammatory response, resulting in the reduction of fibrosis shown in Figures 5 and 6.

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

Claims (11)

Pharmaceutical composition for preventing or treating liver fibrosis containing beta-guanidinopropionic acid (β-GPA) as an active ingredient The pharmaceutical composition for preventing or treating liver fibrosis according to claim 1, wherein the liver fibrosis is induced by liver inflammation. The pharmaceutical composition for preventing or treating liver fibrosis according to claim 1, wherein the beta-guanidinopropionic acid reduces the expression of hydroxyproline and alpha-smooth muscle actin proteins in liver tissue. The pharmaceutical composition for preventing or treating liver fibrosis according to claim 1, wherein the beta-guanidinopropionic acid reduces the expression of interleukin 17 and interferon gamma. The pharmaceutical composition for preventing or treating liver fibrosis according to claim 1, wherein the beta-guanidinopropionic acid reduces the distribution of CD11b+Ly6C+ monocytes. 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 6, wherein the liver fibrosis is induced by liver inflammation. The food composition for preventing or improving liver fibrosis according to claim 6, wherein the beta-guanidinopropionic acid reduces the expression of hydroxyproline and alpha-smooth muscle actin proteins in liver tissue. The food composition for preventing or improving liver fibrosis according to claim 6, 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 6, wherein the beta-guanidinopropionic acid reduces the distribution of CD11b+Ly6C+ monocytes. Method for preventing or treating liver fibrosis in subjects other than humans comprising administering beta-guanidinopropionic acid (β-GPA)