JP6023328B2 - Composition for preventing or treating hyperlipidemia and related diseases comprising rebamipide as an active ingredient - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a pharmaceutical use of rebamipide used for preventing or treating hyperlipidemia and diseases related thereto, and more specifically, hyperlipidemia containing rebamipide as an active ingredient, and The present invention relates to a composition for preventing or treating a related disease.

  Modern people overdose saturated fats and cholesterol due to Western diets and lifestyle changes, and the demands of simplicity in pursuit of simplicity, which are obese, hyperlipidemic, arterial It is known to induce cardiovascular diseases such as sclerosis and myocardial infarction, and lipid peroxides cause degenerative processes in vivo, promote cancer and aging, and change or destroy biological membranes. . Blood cholesterol has been shown to be a risk factor for ischemic cerebrovascular disease and coronary artery disease, and free radicals have been shown to cause aging and chronic diseases such as cerebrovascular disease, cardiovascular disease, and cancer.

  In Korea, 48.1% of all fatalities are malignant neoplasms (cancer), cerebrovascular diseases, and heart diseases (including ischemic heart diseases and other heart diseases), which are the three leading causes of death, based on 2008. Although the mortality rate of cerebrovascular disease decreased compared with 1998, it can be confirmed from statistical results that heart disease (including ischemic heart disease and other heart diseases) has increased [National Statistical Office] Public of Korea: Annual Report on the Cause of Death Statistics 2008 (2010)].

  In addition, such diseases are closely related to changes in dietary habits. According to the results of the 2008 National Health and Nutrition Survey, obesity (19 years and over) 30.7%, hypertension ( (30 years and older) 26.8%, diabetes (30 years and older) 9.7%, hyperlipidemia (30 years and older) 10.9%, prevalence appeared, and for the last 10 years, hypertension and diabetes Studies have shown that obesity and hyperlipidemia increased but decreased.

  Hyperlipemia refers to a condition in which more fat component substances are present in the blood than is necessary, causing inflammation on the blood vessel wall, resulting in cardiovascular disease. Hyperlipidemia is a normal range of fasting serum lipids in which one or more serum lipids such as cholesterol, triglycerides, phospholipids and free fatty acids are in the normal range. Higher than -150 mg / dl neutral fat, 150-250 mg / dl phospholipid, 130-230 mg / dl cholesterol, and 5-10 mg / dl free fatty acids. When such hyperlipidemia is left untreated, there is a high possibility of causing complications with poor disease states such as hypertension, coronary sclerosis (angina, myocardial infarction), and cerebral arteriosclerosis (cerebral infarction).

  Currently, as a therapeutic agent for hyperlipidemia, a resin that binds to bile acids (for example, cholestyramine, cholesipol, etc.), an HMG-CoA reductase inhibitor (for example, lovastatin) that is a major enzyme in the process of cholesterol biosynthesis ), Fluvastatin, simvastatin, pravastatin) and the like are used clinically, but side effects may occur in long-term use. In particular, it is known that a statin series drug that is an HMG-CoA reductase inhibitor can cause liver damage and myopathy.

  In addition, blood cholesterol lowering agents currently on the market are largely classified into 3-hydroxy-3-methylglutayl-CoA (HMG-CoA) reductase inhibitors, bill-salt sequences, fibrates, and niconic acid derivatives. Among them, lovastatin, simvastatin, and pravastatin are the most well-known cholesterol-lowering agents that inhibit HMG-CoA reductase at the initial stage of cholesterol biosynthesis, but inhibit the synthesis of steroid hormones and dolichol. Have caused serious side effects (Havel, RL et al., Ann. Internal Med., 107: 609-615, 1987; Illingworth, DR and Bacon, S. Am. J. Cardioropy. 60: 33-42, 1987).

  Therefore, there is an urgent need for the development of a new therapeutic agent that is safer and suppresses lipid metabolism and has an excellent therapeutic effect on hyperlipidemia.

  Therefore, the present inventor has produced TNF-a, IL-6 and IL-1b, which are hyperlipidemia-induced inflammatory cytokines secreted by macrophages from rebamipide, which is currently used as a therapeutic agent for gastric ulcer. , Suppresses the generation of foam cells that form progenitor cells of arteriosclerotic plaque, and lowers blood lipids by lowering the total cholesterol, LDL cholesterol and triglyceride levels in the blood Increased expression of Treg cells that can control inflammatory responses, along with decreased Th17 expression that improves, improves fatty liver, suppresses B cell activity, and accelerates the production of arteriosclerotic plaques First we investigated what we can do, And completed the present invention by confirming that can be usefully employed as a therapeutic agent for diseases and disorders associated therewith.

  Accordingly, an object of the present invention is to provide a novel use of rebamipide having the effect of preventing or treating hyperlipidemia and diseases associated therewith.

  In order to achieve the object of the present invention as described above, the present invention is directed to the prevention or prevention of hyperlipidemia and related diseases comprising a rebamipide compound or a pharmaceutically acceptable salt thereof as an active ingredient. A therapeutic composition is provided.

  In one embodiment of the present invention, the rebamipide can suppress the production of TNF-a, IL-6, and IL-1b, which are hyperlipidemia-induced inflammatory cytokines secreted by macrophages.

  In one embodiment of the present invention, the rebamipide can not only suppress the generation of foam cells that form progenitor cells of arteriosclerotic plaques, but also accelerate the generation of arteriosclerotic lesions. It is possible to suppress the formation of arteriosclerotic lesions through decreased expression of Th17 and increased expression of Treg cells capable of controlling inflammatory responses.

  In one embodiment of the present invention, the rebamipide can improve blood lipids by lowering the total cholesterol, LDL cholesterol and neutral fat levels in the blood.

  In one embodiment of the present invention, the rebamipide may be included in the composition at a concentration of 0.1 μM to 10,000 μM.

  In one embodiment of the present invention, the hyperlipidemia may be hyperlipidemia derived from an autoimmune disease.

  In one embodiment of the present invention, the diseases associated with hyperlipidemia are arteriosclerosis, heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina, stroke and peripheral blood vessels. It can be selected from the group consisting of diseases.

  The rebamipide compound according to the present invention suppresses the production of TNF-a, IL-6, and IL-1b, which are hyperlipidemia-induced inflammatory cytokines secreted by macrophages, and is a precursor of arteriosclerotic plaque. Suppresses the generation of foam cells that form cells, lowers total cholesterol, LDL cholesterol and triglyceride levels in the blood, improves blood lipids, improves fatty liver, and activates B cells Treg cells that can control the inflammatory response can be increased along with a decrease in Th17 expression that suppresses and accelerates the formation of arteriosclerotic lesions. Therefore, the rebamipide compound is useful as a pharmaceutical composition capable of preventing or treating hyperlipidemia and related arteriosclerosis, heart failure, hypertensive heart disease and the like through various mechanisms as described above. Can be used for In addition, since there are no toxicity and side effects to the drug, it can be used with confidence even when taken for a long period of time, and has a stable effect on the body.

The amount of inflammatory cytokine production secreted in macrophages by rebamipide treatment is shown by measurement through ELISA analysis (Nil: LPS and rebamipide untreated group). It is the result of having performed MTT assay in order to investigate the cytotoxicity of the rebamipide with respect to RAW264.7 which is a mouse | mouth macrophage cell (Nil: LPS and a rebamipide non-treatment group). In the formation of oxLDL-induced foam cells using THP1 cells, a human macrophage cell line, a photograph showing the degree of foam cell formation by treatment with rebamipide at different concentrations (100, 250, 500, 1000 μM) through Oil red O staining is there. In the formation of oxLDL-induced foam cells using THP1 cells, a human macrophage cell line, a photograph showing the extent of foam cells generated by rebamipide alone, simvastatin alone, or a combination of rebamipide and simvastatin through Oil red O staining is there. FIG. 2 is a graph showing changes in total serum cholesterol, LDL cholesterol and triglyceride concentration in mouse serum after oral administration of rebamipide to hyperlipidemic animal model mice. The graph shows the AST and ALT activity values in mouse serum after oral administration of rebamipide to hyperlipidemic animal model mice. It is the photograph which showed the aorta branch grade in the aorta of a mouse | mouth through Oil red O dyeing | staining after orally administering rebamipide to a hyperlipidemia animal model mouse. The graph shows the results obtained by orally administering rebamipide to a hyperlipidemic animal model mouse and then measuring the values of total IgG and IgG1 in the mouse serum. After oral administration of rebamipide to hyperlipidemic animal model mice, the expression levels of Th17 cells and Treg cells were analyzed by FACS in the spleen cells of the mice. After rebamipide was orally administered to a hyperlipidemic animal model mouse, the expression levels of Th17 cells and Treg cells were analyzed with a confocal microscope in the spleen fragments of the mice. After rebamipide was orally administered to a hyperlipidemic animal model mouse, mRNA expression level of MMP-9 was analyzed by RT-PCR in the spleen cells of the mouse.

  The present invention relates to a composition for preventing or treating hyperlipidemia and diseases associated therewith comprising a rebamipide compound or a pharmaceutically acceptable salt thereof as an active ingredient.

  The present inventor has focused on rebamipide compounds in order to develop a new therapeutic agent that is safe and suppresses lipid metabolism and has an excellent therapeutic effect on hyperlipidemia. Rebamipide is useful for gastric ulcer, acute gastritis, or chronic gastritis. It is a drug that has an excellent effect on the treatment of gastric mucosal damage due to acute deterioration, and is widely used as a therapeutic agent for peptic ulcer. ) -Quinolinon-4-yl] propionic acid. This drug promotes PGE2 biosynthesis and increases mucus to protect the gastric mucosa and promote cell proliferation, especially in patients infected with Helicobacter pylori bacteria. By suppressing adhesion and infiltration, it has the characteristic of suppressing gastric inflammation.

  However, there has been no mention of the contents that rebamipide can be used for the treatment of hyperlipidemia and related diseases.

  Therefore, in the present invention, the rebamipide compound can be used as a use for preventing or treating hyperlipidemia and diseases associated therewith, and in particular, TNF-a, which is a hyperlipidemia-induced inflammatory cytokine, Suppresses the production of IL-6 and IL-1b, suppresses the production of foam cells that form the precursor cells of arteriosclerotic plaques, and suppresses total cholesterol, LDL cholesterol and neutral fat in the blood Treg cells that can control the inflammatory response with decreased expression of Th17 that improves blood lipids by reducing the numerical value, improves fatty liver, suppresses B cell activity, and accelerates the formation of arteriosclerotic lesions Preventive or therapeutic effects of hyperlipidemia and related diseases through a mechanism to increase the expression of The fact that it can be induced was first investigated.

  In general, hyperlipidemia induces various complications while there are more fat component substances in the blood than necessary, but such complications include arteriosclerosis, angina, myocardial infarction. Because there are many very dangerous diseases such as, it is a situation where special prevention or treatment is required.

  Atherosclerosis is characterized by the accumulation of lipids in the endothelium, causing an inflammatory response and the formation of an atherosclerotic plaque, where the plaque narrows the artery and the narrowed artery is blocked by a thrombus Because it is easy to rupture, it can cause cardiovascular disease.

  Macrophages derived from mononuclear cells play an important role in such an early stage of arteriosclerosis. When the LDL concentration in the blood is too high due to hyperlipidemia, or LDL is deformed by oxidation, macrophages are oxidized LDL using a scavenger receptor (oxLDL: oxidized LDL). However, macrophages transfected with oxidized LDL are referred to as foam cells. In general, macrophages that have phagocytosed the pathogen migrate to other locations, but foam cells do not leave the site after the transfer of oxidized LDL, thus losing normal biological function and blood vessels. Smooth muscle is thickened through the formation of atherosclerotic plaques by penetrating into endothelial cells. Furthermore, it is known that when foam cells are trapped in the endothelium of an artery, they trigger a local inflammatory response.

  In the following Example 1 of the present invention, in order to observe the effect of rebamipide on macrophages, after treatment of mouse peritoneal macrophages with rebamipide and stimulation with LPS, hyperlipidemia-inducing factor It was confirmed that the production of certain TNF-a, IL-6 and IL-1b was effectively suppressed (see FIG. 1).

  Further, in Example 3 below, the effect of rebamipide on the generation of foam cells that form progenitor cells of arteriosclerotic lesions was observed, and as a result, vascular pathogenesis was dependent on rebamipide treatment concentration. It is shown that the generation of foam cells, which are cells, is suppressed (see FIG. 3), and in particular, the foam cell generation suppression effect is further superior to simvastatin, which is an existing drug for treating hyperlipidemia. (See FIG. 4).

  In addition, in Example 4 below of the present invention, the improvement effect of blood lipids by administration of rebamipide was observed in a hyperlipidemic animal model, and as a result, when rebamipide was orally administered to a hyperlipidemic animal model mouse, It was confirmed that the total cholesterol, LDL cholesterol and triglyceride concentration in the serum were effectively reduced (see FIG. 5).

  Further, in Example 5 below of the present invention, the effect of improving fatty liver by the administration of rebamipide was observed in a hyperlipidemic animal model, and as a result, when rebamipide was orally administered to a hyperlipidemic animal model mouse, Serum AST and ALT activities showed a significant decrease, confirming that rebamipide can suppress liver damage in a hyperlipidemic animal model (see FIG. 6).

  In Example 6 of the present invention described below, the effect of inhibiting the formation of arteriosclerotic lesions by administration of rebamipide was observed in a hyperlipidemic animal model, and as a result, rebamipide was orally administered to a hyperlipidemic animal model mouse. In the experimental group, the aorta branch visible in the aorta showed significantly less Oil red O positivity, confirming that rebamipide actually suppressed the formation of arteriosclerotic lesions (see FIG. 7).

  In Example 7 of the present invention described below, the immune response of B cell antibody by administration of rebamipide was investigated in a hyperlipidemic animal model, and as a result, rebamipide was orally administered to a hyperlipidemic animal model mouse. The values of IgG and IgG1 in the serum decreased, and it was confirmed that rebamipide can suppress B cell activity (see FIG. 8).

  In Example 8 of the present invention described below, a simultaneous regulatory reaction of Th17 cell suppression and Treg cell induction by administration of rebamipide was observed in a hyperlipidemic animal model, and as a result, rebamipide was applied to a hyperlipidemic animal model mouse. It was confirmed that the expression of Th17, which accelerates the formation of arteriosclerotic lesions, decreased in spleen cells, while the expression of Treg cells, which are immunoregulatory cells, increased. (See FIGS. 9 and 10).

As a reference, T cells are one of a group of cells that play a central role in the immune system as a biological defense system against various pathogens. T cells are generated in the thymus of the human body and differentiate into T cells having unique characteristics through a series of differentiation processes. The differentiated T cells are largely classified into type 1 helper cells (Th1) depending on their functions. It is divided into type 2 helper cells (Th2). Among them, the main functions of Th1 cells are involved in cell-mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, such two cell populations are mutually restrained so as not to activate each other. Maintain the balance of the immune system.
Therefore, many of the immune diseases can be attributed to such variation between the two immune cells. For example, if the activity of Th1 cells increases abnormally, the immune disease can occur, When the activity of Th2 cells increases abnormally, it is known that immune diseases due to hypersensitivity reactions occur.

  On the other hand, according to recent research results on the differentiation of Th1 cells, there is a new group that can regulate the activity of Th1 cells (Regulatory T cell, hereinafter simply referred to as “Treg”). Is known, and research on the treatment of immune diseases using this has been taken up, but Treg cells have the property of controlling the inflammatory response by suppressing the function of abnormally activated disease cells, Many experiments have been reported to treat immune diseases and inflammatory diseases through the action of increasing the activity of Treg cells.

  In addition to Treg cells, there is Th17 cells as another group produced in the differentiation process. Th17 cells are formed through a process similar to the differentiation of Treg cells in the differentiation process of undifferentiated T cells. Are known. That is, Treg cells and Th17 cells are commonly differentiated in the presence of TGF-β, whereas Treg cells do not require IL-6, whereas Th17 cells together with TGF-β. Differentiate in the presence of IL-6. Differentiated Th17 cells are characterized by secreting IL-17.

  Th17 cells, unlike Treg cells, have been shown to be involved in the forefront of the inflammatory response seen in immune diseases, maximizing inflammatory response signals and accelerating disease progression.

  Therefore, when simultaneous suppression of Th17 cell suppression and Treg cell induction is possible, it is possible to effectively suppress an excessive immune response or inflammatory response. In the case of rebamipide, Treg cells are simultaneously suppressed with Th17 cell expression suppression. Therefore, it was determined that excessive immune reaction or inflammatory reaction can be effectively suppressed.

  In Example 9 below, the expression level of MMP-9 mRNA by administration of rebamipide was investigated in a hyperlipidemic animal model. MMP-9, which degrades non-fibrillar collagen, is expressed in arteriosclerotic lesions and is known to play a major role in the pathophysiological mechanisms important for atherosclerotic lesion rupture. As a result, when rebamipide was orally administered to a hyperlipidemic animal model mouse, the expression level of MMP-9 in the spleen cells of the mouse was surely decreased as compared with the group that was orally administered simvastatin which is a positive symmetric group. It was confirmed (see FIG. 11). Thereby, the present inventor can effectively prevent the occurrence of cerebral infarction and myocardial infarction caused by the rupture of arteriosclerotic lesions by rebamipide effectively reducing the expression of MMP-9. It was judged.

  Through these results, the present inventors have experimentally demonstrated that rebamipide is effective for the prevention and treatment of hyperlipidemia and related diseases through various mechanisms.

  Therefore, the composition of the present invention containing rebamipide or a pharmaceutically acceptable salt thereof as an active ingredient can be usefully used for the prevention or treatment of hyperlipidemia and diseases associated therewith.

  The rebamipide according to the present invention is a compound represented by the following chemical formula 1.

  The rebamipide compound according to the present invention can be used in the form of a flame, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid can be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, Contains glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid and aspartic acid. The inorganic acid is not limited to this, and includes hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

  The rebamipide compound according to the present invention may be one that is sold in the market, or one that is isolated from nature or manufactured by a chemical synthesis method known in the art. Good.

  In one embodiment of the present invention, the rebamipide is hyperlipidemic through a mechanism that suppresses the production of TNF-a, IL-6 and IL-1b, which are hyperlipidemia-induced inflammatory cytokines in macrophages. And having a preventive or therapeutic effect on diseases associated therewith.

  In another embodiment of the present invention, the rebamipide may be included in the composition of the present invention at a concentration of 0.1 to 10,000 μM.

  On the other hand, the composition for preventing or treating hyperlipidemia and diseases associated therewith provided by the present invention is preferably a pharmaceutical composition.

  In the present invention, “hyperlipidemia-related disease” means a disease induced or promoted by hyperlipidemia, such as arteriosclerosis, heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, Although it can be selected from the group consisting of myocardial infarction, angina pectoris, stroke and peripheral vascular disease, the type is not particularly limited.

In the present invention, “treatment” means, unless otherwise stated, the disease or disease to which the term is applied, or the symptoms or symptoms of any one or more of the disease or disease are reversed, alleviated, or the progression thereof. The term treatment as used herein refers to the act of treating when “treating” is defined as above. Thus, in mammals, “treatment” or “therapeutic therapy” for hyperlipidemia and related diseases includes one or more of the following.
(1) prevent the development of hyperlipidemia and related diseases,
(2) prevent the spread of hyperlipidemia and related diseases,
(3) alleviate hyperlipidemia and related diseases,
(4) prevent recurrence of hyperlipidemia and related diseases, and (5) alleviate symptoms of hyperlipidemia and related diseases.

  The pharmaceutical composition of the present invention can be produced using a pharmaceutically compatible and physiologically acceptable adjuvant in addition to the active ingredient rebamipide, Excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants or flavoring agents can be used.

  The pharmaceutical composition can be preferably formulated as a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to rebamipide which is an active ingredient for administration. .

  The pharmaceutical composition consists of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, injectable solutions, and the like. Also good. For example, for purification or formulation in capsule form, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. . If desired or necessary, suitable binders, lubricants, disintegrating agents and color formers may also be included as a mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate , Magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. The disintegrant is not limited to this, and includes starch, methylcellulose, agar, bentonite, xanthan gum and the like. In compositions formulated in liquid solutions, acceptable pharmaceutical carriers are those that are sterile and biocompatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, malt Dextrin solution, glycerol, ethanol and one or more of these components can be mixed and used, and if necessary, other usual additives such as antioxidants, buffers, bacteriostatic agents can be added. Also good. In addition, diluents, dispersants, surfactants, binders and lubricants can be added to injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or purified. You may formulate. Furthermore, as an appropriate method in the art, a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be used, and it can be preferably formulated according to each disease or by ingredient.

  In one embodiment of the present invention, the rebamipide of the present invention may be included in the composition at a concentration of 0.1 to 10000 μM, and the rebamipide of the present invention is about 0.1% relative to the total weight of the composition. It may be included at 1 to 95% by weight.

  The types of diseases for which the pharmaceutical composition of the present invention can exhibit a therapeutic effect include hyperlipidemia, arteriosclerosis, heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, The disease is selected from the group consisting of angina pectoris, stroke and peripheral vascular disease, but is not limited thereto.

  The pharmaceutical composition of the present invention may comprise rebamipide in a pharmaceutically effective amount with a pharmaceutically acceptable carrier.

  In the present invention, the “pharmacologically effective amount” refers to an amount of an active ingredient exhibiting a alleviating, suppressing, improving and / or completely ameliorating effect on a disease to be treated. The dose of rebamipide of the present invention varies in range depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, disease severity, and the like. For example, a therapeutically effective dose can be initially determined using in vitro analysis through cell culture. A therapeutically effective amount can be determined without undue experimentation in the art, and such information can be used to more accurately determine useful doses in humans.

  In one embodiment of the present invention, the pharmaceutically effective amount of the rebamipide compound or salt thereof according to the present invention is 0.5-100 mg / day / kg body weight, preferably 0.5-5 mg / day / kg body weight. It is.

  The present invention also provides use of a composition comprising the rebamipide as an active ingredient for the manufacture of a medicament for the prevention or treatment of hyperlipidemia and related diseases. The composition of the present invention containing rebamipide as an active ingredient can be used for the production of a medicament for the prevention or treatment of hyperlipidemia and related diseases.

  The present invention also provides a method for preventing or treating hyperlipidemia and diseases associated therewith comprising administering to a mammal a therapeutically effective amount of a pharmaceutical composition of the present invention.

  The term “mammal” as used herein refers to a mammal that is the subject of treatment, observation or experiment, preferably a human.

  The term “therapeutically effective amount” as used herein refers to an active ingredient or pharmaceutical that induces a biological or medical response in a tissue system, animal or human being considered by a researcher, veterinarian, doctor or other clinic. Means the amount of the therapeutic composition, which includes an amount that induces the alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration for the active ingredients of the present invention will vary depending on the desired effect. Accordingly, the optimum dosage to be administered can be easily determined by those skilled in the art, and includes the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, and the type of dosage form. And the patient's age, weight, general health, sex and diet, administration time, route of administration and secretion rate of the composition, duration of treatment, and drugs used at the same time.

  The present invention also provides a health functional food for the prevention or amelioration of hyperlipidemia and diseases associated therewith containing rebamipide as an active ingredient.

  The health functional food of the present invention can be produced and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing or improving hyperlipidemia and diseases associated therewith.

  In the present invention, “health functional food” refers to food manufactured and processed using ingredients and ingredients having functionality useful to the human body according to Law No. 6727 on health functional food, and the structure and function of the human body. It is taken for the purpose of obtaining useful effects for health use such as regulating nutrients or physiological effects.

  The health functional food of the present invention can contain ordinary food additives, and whether or not it is suitable as a food additive is determined by the Food Additives Approved by the Food and Drug Safety Agency unless otherwise specified. Judged according to the standards and standards related to the item by general rules and general test methods.

  Examples of the items listed in the above-mentioned “Food Additives” include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, cinnamic acid; amber pigment, licorice extract, crystalline cellulose, Takahashi pigment And natural additives such as guar gum; mixed preparations such as sodium L-glutamate preparations, noodles-added alkaline agents, preservative preparations, tar pigment preparations, and the like.

  For example, a health functional food in the form of a tablet is obtained by granulating a mixture of rebamipide, which is an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives by a conventional method, The mixture can be compression molded or the mixture can be directly compression molded. Moreover, the health functional food in the form of a tablet may contain an orthodontic agent or the like as necessary.

  Hard capsules in health foods in the form of capsules can be manufactured by filling ordinary hard capsules with a mixture of rebamipide, which is the active ingredient of the present invention, and additives such as excipients. Capsules can be produced by filling a mixture of rebamipide and additives such as excipients into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, and the like as necessary.

  Pill-form health function food can be prepared by molding a mixture of rebamipide, which is the active ingredient of the present invention, with excipients, binders, disintegrants, etc. by a known method, If necessary, it can be made with sucrose or other skin preparations, or the surface may be coated with a substance such as starch or talc.

  The health functional food in the form of granules can be produced in a granular form by a known method by mixing a mixture of rebamipide, which is the active ingredient of the present invention, with excipients, binders, disintegrants, etc. Depending on the case, flavoring agents, orthodontic agents, etc. may be included.

  The health functional foods include beverages, meats, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice creams, alcoholic beverages, vitamin complexes, health supplements, etc. It may be.

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

<Example 1>
Inhibitory effect of rebamipide treatment on macrophage cells In order to examine the effect of rebamipide on macrophages, the present inventors pretreated mouse macrophages with rebamipide and then stimulated with LPS (lipopolysaccharide) The degree of production of the inflammatory cytokines TNF-a (tumor necrosis factor-alpha), IL-6 (interleukin-6) and IL-1β (interleukin-1β) secreted from macrophages was evaluated through ELISA analysis.

<1-1> Cell Culture RAW264.7 cells, a macrophage cell line of mice, were distributed from the Korea Cell Line Bank (KCLB), and 10% FBS and 1% penicillin-streptomycin were used for cell culture. The DMEM (Dulbecos Modified Eagle Medium) medium containing was used. The cells were cultured at 37 ° C. with 5% CO ₂ .

<1-2> Effect of rebamipide on the production amount of inflammatory cytokines (TNF-a, IL-6 and IL-1β) First, RAW264.7 cells were pretreated with rebamipide at a concentration of 20,100 μg / ml, One hour later, LPS was treated at a concentration of 100 ng / ml, and an inflammatory reaction was induced in macrophages while culturing in a 37 ° C. incubator for 48 hours.

  In order to measure the amount of cytokines produced (TNF-α, IL-6 and IL-1β), the upper layer fluid of the cultured cells was collected and TNF-α, IL-6 and IL were collected using ELISA. The degree of formation of -1β was investigated. Monoclonal anti-TNF-α, anti-IL-6, and anti-IL-1β were each reacted at 1 μg / mL at 4 ° C. overnight in a 96-well plate, and the blocking solution after the reaction (1% Non-specific binding was blocked with BSA / PBST). TNF-α recombinant, IL-6 recombinant, and IL-1β recombinant were serially diluted in half and used as a standard, and the cell culture upper layer solution was added and allowed to react at room temperature for 2 hours. Thereafter, biotinylated anti-TNF-α, biotinylated anti-IL-6, biotinylated anti-IL-1β were reacted at room temperature for 2 hours, washed 4 times, and then added with ExtraAvidin-Alkaline Phosphate concentrate. And allowed to react for 2 hours at room temperature. Thereafter, the PNPP / DEA solution was added for color development, and the absorbance was measured at a wavelength of 405 nm.

  As a result, as shown in FIG. 1, when only LPS was treated without pretreatment with rebamipide, the amount of TNF-α and IL-6 produced was significantly increased compared to the control group (normal cells). However, in the experimental group pretreated with rebamipide, it was confirmed that the production amounts of TNF-α and IL-6 decreased in a concentration-dependent manner. In addition, when only LPS was treated without pretreatment with rebamipide, the amount of IL-1β produced was not different from that of the control group (normal cells), but in the experimental group treated with rebamipide at a concentration of 100 μg / ml. , Appeared as the production amount decreased. Nil represents an untreated group as a negative control group.

  Through these results, rebamipide prevents or treats hyperlipidemia and related diseases through a mechanism that suppresses macrophage inflammatory response, which plays an important role in the early stages of hyperlipidemia and arteriosclerosis. It was judged useful for treatment.

<Example 2>
Measurement of rebamipide toxicity to cells To examine the cytotoxicity of rebamipide against RAW264.7, a macrophage of mice, MTT assay was performed.

This method measures the conversion of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) to formazan, and in a 96-well plate, 1 × 10 ⁴ cells / well. RAW264.7 cells were separated and rebamipide was treated at different concentrations (20, 100, 500, 1000, 5000 μM) for 18 hours. After adding 100 μl of MTT solution per well and reacting at 37 ° C. in a 5% CO ₂ incubator for 4 hours, the absorbance was measured at 570 nm using a microplate reader (VERSAmax, Molecular Devices, USA). Changes were measured.

  As a result, as shown in FIG. 2, it was found that the cytotoxicity was low even at effective effective concentrations of 20 μM, 100 μM and higher, and basically the cell viability was not affected. . Such a fact is that rebamipide suppresses the production of inflammatory mediators (TNF-α, IL-6 and IL-1β) due to cell death, but rebamipide does not suppress the inflammatory response in macrophages. It means that it is an inherent effect of.

<Example 3>
Inhibition of vascular pathogenic cells by treatment with rebamipide In this experiment, when rebamipide was processed into vascular pathogenic cells, it was observed whether the formation of vascular pathogenic cells could actually be suppressed. The “vascular pathogenic cell” means a foam cell that forms a progenitor cell of an arteriosclerotic lesion that induces arteriosclerosis, and is a cell that causes arteriosclerosis. Named arbitrarily.

<3-1> Inhibitory effect of vascular pathogenic cells by treatment with rebamipide For this experiment, THP1 cells, a human macrophage cell line, were treated with 160 nM concentration of PMA (phorbol 12′-myristate 13′-acetate). After activating the cells, PAF (platelet-activating factor) is treated with 10 ug / ml, and rebamipide is treated at different concentrations (100, 250, 500, 1000 μM) to induce arteriosclerosis. The degree of formation of foam cells forming the sac was measured through Oil Red O solution (Sigma aldrich-Sigma Aldrich) staining.

  As a result, as shown in FIG. 3, it was confirmed that foam cells, which are vascular pathogenic cells, were suppressed depending on the rebamipide treatment concentration.

<3-2> Comparison of the inhibitory effect of rebamipide on vascular pathogenic cells compared to existing drugs for treating hyperlipidemia The inhibitory effect of rebamipide on vascular pathogenic cells is currently on the market. Compared to simvastatin, which is used as.

  For this experiment, THP1 cells, a human macrophage cell line, were treated with 160 nM concentration of PMA (phorbol 12′-myristate 13′-acetate) to activate the cells, and then PAF (platelet-activating factor) was applied. Simultaneously with the treatment of 10 ug / ml, rebamipide (1000 μM) or simvastatin (10 μM) was treated, respectively, and the degree of generation of foam cells forming the progenitor cells of arteriosclerotic lesions inducing arteriosclerosis was measured through Oil Red O solution staining. did.

  As a result, as shown in FIG. 4, it can be confirmed that the experimental group treated with rebamipide alone formed fewer foam cells than the experimental group treated with simvastatin alone, The effect was increased by 20% compared to simvastatin, which is a drug for treating hyperlipidemia.

<Example 4>
In this experiment, a certain amount of rebamipide was orally administered to a hyperlipidemic animal model in order to measure the effect of rebamipide on blood lipids in vivo. Thereafter, total cholesterol, LDL cholesterol and triglyceride values in the serum of the mice were measured.

  Specifically, 8-week-old Apoe ko mice (mice from which the apolipoprotein E gene was removed) were orally administered with rebamipide 100 mg / kg while feeding Western diet, then sacrificed when 8 weeks after Western diet diet. The serum was measured for total cholesterol, LDL cholesterol and triglyceride values. In the positive control group, 25 mg / kg of simvastatin currently used as a lipid lowering agent in the city was orally administered instead of rebamipide. The total cholesterol calculation used the general total cholesterol calculation method (LDL = T-CHO- (0.2 * TG) -HDL, T-CHO = HDL + (0.2 * TG) + LDL).

  As a result, as shown in FIG. 5, when rebamipide is orally administered to a hyperlipidemic animal model mouse, it can be confirmed that the total cholesterol, LDL cholesterol and triglyceride concentration in the serum are effectively reduced. In particular, it was found that LDL cholesterol and triglyceride concentrations were more effectively reduced in the simvastatin administration group, which is a positive symmetric group.

<Example 5>
Improvement of fatty liver by administration of rebamipide in hyperlipidemia animal model In this experiment, in order to measure the improvement effect of rebamipide on fatty liver in vivo, a certain amount of rebamipide was orally administered to hyperlipidemia animal model. After that, AST and ALT values in the serum of mice were measured.

  If the structure and function of the cell membrane are destroyed, AST (Apartate amino-transferase) and ALT (Aline aminotransferase), which are widely present in the liver cytoplasm, are discharged into the blood, so the AST and ALT values in the blood are It is frequently used as an indicator of liver damage. In this experiment, in order to verify whether rebamipide is effective in preventing fatty liver and improving fatty liver, AST and ALT values in serum were measured.

  Specifically, 8-week-old Apoe ko mice (mouse from which the apolipoprotein E gene was removed) were orally administered 100 mg / kg of rebamipide while feeding Western diet, and then sacrificed when 8 strains were obtained after Western diet. AST and ALT values were measured with serum. In the positive control group, 25 mg / kg of simvastatin currently used as a lipid lowering agent in the city was orally administered instead of rebamipide. The activity of AST and ALT was measured using a kit reagent for quantification (Yongdong Pharmaceutical, Korea). After heating AST and ALT substrate solution 1.0mL in a 37 water tank for 2 minutes, 0.2mL of plasma was put and it was made to react for 30 minutes in a 37 degreeC water tank. After 30 minutes, 1.0 mL of a coloring reagent was added and allowed to stand at room temperature for 20 minutes, and then 10.0 mL of 0.4N NaOH was added and the absorbance was measured at 505 nm. After the AST and ALT standard solution (2 mM pyruvate) were colored in the same manner as in the above method, the absorbance was measured and extrapolated to a standard curve to calculate the activity of the sample.

  As a result, as shown in FIG. 6, when rebamipide was orally administered to a hyperlipidemic animal model mouse, it was confirmed that serum AST and ALT activities were significantly reduced. Thus, it was possible to infer that rebamipide has an activity to suppress liver damage as well as blood lipid improvement in an animal model of hyperlipidemia.

<Example 6>
Inhibition of formation of arteriosclerotic lesions by administration of rebamipide in hyperlipidemic animal models In this experiment, rebamipide was applied to hyperlipidemic animal models in order to measure the inhibitory effect of rebamipide on the formation of arteriosclerotic lesions in vivo. After a fixed amount of oral administration, the mouse aorta branch was observed through staining. Aorta branch is where an arteriosclerotic lesion is formed when arteriosclerosis occurs.

  Specifically, 8-week-old Apoe ko mice (mice from which the apolipoprotein E gene was removed) were orally administered with 100 mg / kg of rebamipide while feeding Western diet, and then sacrificed when 8 strains were obtained after Western diet. Then, after obtaining the whole aorta (Aorta) and passing through a fixing process, Oil red O staining was performed. In the positive control group, 25 mg / kg of simvastatin currently used as a lipid lowering agent in the city was orally administered instead of rebamipide.

  As a result, as shown in FIG. 7, in the case of the negative control group (hyperlipidemic animal model mouse), the aorta branch was mostly expressed as Oil red O, whereas the hyperlipidemic animal model mouse In the experimental group to which rebamipide was orally administered, it was confirmed that the aorta branch seen in the aorta was significantly less positive for Oil red O. Especially, compared with the positive control group (simvastatin oral administration group), the aorta branch was It was found that there were fewer Oil red O positive parts.

<Example 7>
Investigation of immune response of B cell antibody by administration of rebamipide to hyperlipidemia animal model In this experiment, in order to observe the effect of rebamipide on the immune response of B cell antibody in vivo, a hyperlipidemia animal model was used. After a certain amount of rebamipide was orally administered to the mice, the values of total IgG and IgG1 in the mouse serum were measured.

  Specifically, 8-week-old Apoe ko mice (mouse from which the apolipoprotein E gene was removed) were orally administered 100 mg / kg of rebamipide while feeding Western diet, and then sacrificed when 8 strains were obtained after Western diet. Serum total IgG and IgG1 values were measured through ELISA analysis. In the positive control group, 25 mg / kg of simvastatin currently used as a lipid lowering agent in the city was orally administered instead of rebamipide. The total IgG and IgG1 concentrations in the serum were measured according to the manufacturer's instructions using ELISA kit (Betyl). That is, after performing an Overnight reaction with a 96-well plate plate on which Capture was spread, the serum was diluted at 1: 1000, reacted for 1 hour, then reacted with Total IgG, IgG1 detection ab for 1 hour, and HRP was added. The color was developed with TMB solution and the absorbance was read.

  As a result, as shown in FIG. 8, when rebamipide was orally administered to a hyperlipidemic animal model mouse, the values of IgG and IgG1 in the serum decreased, and rebamipide inhibited B cell activity. I was able to confirm that.

<Example 8>
In this experiment, in order to observe the effect of rebamipide on Th17 / Treg cells in vivo, we examined the effect of rebamipide on Th17 / Treg cells in vivo. After a certain amount of rebamipide was orally administered to the model, the expression levels of Th17 cells and Treg cells were examined in mouse spleen cells.

  Specifically, 8-week-old Apoe ko mice (mouse from which the apolipoprotein E gene was removed) were orally administered 100 mg / kg of rebamipide while feeding Western diet, and then sacrificed when 8 strains were obtained after Western diet. Then, the presence or absence of Th17 cell expression was analyzed by measuring the IL-17 expression level of CD4 + cells in the spleen fragment, and the presence or absence of Treg cells was analyzed by measuring Foxp3 expression level of CD4 + CD25 + cells. Expression analysis of Th17 cells and Treg was performed through FACS through staining and confocal microscopy. On the other hand, in the positive control group, 25 mg / kg of simvastatin currently used as a lipid lowering agent in the city was orally administered instead of rebamipide.

  For reference, since differentiated Th17 cells are characterized by secreting IL-17, confirmation of IL-17 expression is an indicator that can measure the activity or amplification of Th17 cells. Foxp3 is a transcriptional regulatory factor mainly present in immunoregulatory T cells (Regulatory T cells, Treg) derived from the thymus and present in cells having CD4 + CD25 + labeled antigen. The function is low in reactivity to antigen during antigen recognition to T cells expressing Foxp3, and at the same time potentially autoimmune among CD4 + CD25-T cells that have not differentiated from Foxp3 and express Foxp3. T cells that can be induced have a role as suppressor T cells that suppress IL-2 production and cell division. Foxp3 affects not only IL-2 but also NFAT, a transcription factor, on CD25-T cells through regulatory T cells that express Foxp3 and cell-cell contact. It has been revealed that it functions to suppress transcriptional regulation of IL-4, IFN- and the like. Therefore, in the case of T cells expressing Foxp3 that acts as described above, it is applied to treat immune diseases through the action of suppressing or regulating the immune response, and also expresses Foxp3 present in humans. The number of CD4 + T cell self-antigen-specific T cells (self-antigen specific T cell clone) is increased through treatment with high concentrations of IL-2 cytokine and combination treatment with anti-CD3 and anti-CD28 antibodies, Attempts have been made to apply it as a treatment method. Therefore, Foxp3 expression confirmation serves as an indicator that can measure the activity or amplification of Treg cells.

<8-1> Analysis of FACS stain FACS staining was performed on mouse spleen cells after lethality of the mouse. Th17 cells were reacted with anti-CD4 antibody for 4 to 30 minutes first, then treated with Cytofix / Cytoperm for 30 minutes, washed, and then treated with anti-IL-17 antibody for 30 minutes. After reading with FACScalibur, analysis was performed using Flow jo program. Treg cells were treated with anti-CD4 and anti-CD25 antibodies for 30 minutes, then treated with Treg-specific Cytofix / Cytoperm for 30 minutes, washed, and then reacted with anti-Foxp3 antibody for 30 minutes. After reading on a FACScalibur, it was analyzed using Flow jo program.

  As a result, as shown in FIG. 9, when rebamipide was orally administered to a hyperlipidemic animal model mouse, the expression of Th17 that accelerates the formation of arteriosclerotic lesions was shown to decrease in spleen cells. On the other hand, it was confirmed that the expression of Treg cells, which are immunoregulatory cells, increased. In particular, the expression of Th17 was further reduced in the rebamipide administration group than in the simvastatin administration group, which is a positive symmetry group.

<8-2> Analysis of confocal microscope The confocal microscope analysis method is a method of killing the mouse, embedding the OCT compound using a spleen fragment of the mouse, and then rapidly cooling with liquefied nitrogen. Was attached to the slide with a thickness of 7 m using a frozen section machine. The sections were fixed with acetone and blocked nonspecific reaction with 10% normal chlorine serum for 30 minutes. Th17 cells utilized PE-labeled anti-CD4 and FITC-labeled anti-IL-17 antibodies. Treg cells utilized PE-labeled anti-CD4, FITC-labeled anti-Foxp3 and APC-labeled anti-CD25 antibodies. Stained tissues were analyzed with a confocal microscope (LSM 510 Meta. Zeiss, Gottingen, Germany).

  As a result, as shown in FIG. 10, when rebamipide was orally administered to a hyperlipidemic animal model mouse, the expression of Th17 that accelerates the formation of arteriosclerotic lesions was shown to decrease in spleen cells. On the other hand, it was confirmed that the expression of Treg cells, which are immunoregulatory cells, increased.

<Example 9>
Analysis of expression level of MMP-9 by administration of rebamipide to hyperlipidemia animal model In this experiment, the expression level of MMP-9 mRNA in hyperlipidemia animal model mice was investigated. MMP-9 (matrix metalloproteinase-9), which degrades non-fibrillar collagen, is expressed in arteriosclerotic lesions and may play a major role in pathophysiological mechanisms important for rupture of arteriosclerotic lesions. Are known.

  Specifically, 8-week-old Apoe ko mice (mice from which the apolipoprotein E gene has been removed) were orally administered with rebamipide 20 mg / kg and 100 mg / kg while feeding Western diet, respectively, and then 8 strains were fed after Western diet. At the time of sacrifice, spleen cells were obtained. The spleen cells thus obtained were treated with 25 ng / ml of PMA and 250 ng / ml of Ionomycin for 4 hours, and then the expression of MMP-9 was investigated by RT-PCR using the RNA of the cells. . RT-PCR is performed using an ABI PCR machine with LightCycler FastStart DNAmaster SYBR green I (Takara) fluorescing dye. After mixing 10 μl of DNAmaster SYBR green I (Takara) and 1 μl of Taqman probe (Applied biosystems), the final volume was made 20 μl with distilled water. The reaction conditions were 95 ° C. for 10 minutes, 95 ° C. for 10 seconds and 64 ° C. for 30 seconds for a total of 50 times. The cycle threshold (Ct) value was analyzed, and the mRNA expression level of MMP-9 was expressed by the partner quantification with the β-actin mRNA expression level, which is a house keeping gene. Primers used in the RT-PCR were Forward: 5'-CTG TCC AGA GTA CAG CCT-3 ', Reverse: 5'-GAG GTA TAG TGG GAC ACA TAG TGG-3'. On the other hand, in the positive control group, 25 mg / kg of simvastatin currently used as a lipid lowering agent in the city was orally administered instead of rebamipide.

  As a result, as shown in FIG. 11, when rebamipide was orally administered to a hyperlipidemic animal model mouse, the expression level of MMP-9 in the spleen cells of the mouse was reliably reduced as compared with the group to which simvastatin was orally administered. I was able to confirm that.

  Therefore, cerebral infarction caused by rupture of arteriosclerotic foci by rebamipide effectively reducing the expression of MMP-9, which plays a major role in the pathophysiological mechanism in rupture of arteriosclerotic foci, through such results. And the occurrence of myocardial infarction can be more effectively prevented.

  So far, the present invention has been studied focusing on its preferred embodiments. Those skilled in the art to which the present invention pertains can understand that the present invention can be embodied in a modified form without departing from the essential characteristics of the present invention. Accordingly, the disclosed embodiments should be considered in an illustrative, not a limiting sense. The scope of the present invention is shown not in the above description but in the claims, and all differences within the equivalent scope should be construed as being included in the present invention.

  The rebamipide compound according to the present invention is usefully used as a pharmaceutical composition capable of preventing or treating hyperlipidemia and related arteriosclerosis, heart failure, hypertensive heart disease and the like through various mechanisms. can do.

Claims (6)

Rebamipide (rebamipide) compound or composition for preventing or treating hyperlipidemia comprising a pharmaceutically acceptable salt thereof as an active ingredient. 2. The hyperlipidemia according to claim 1, wherein the rebamipide suppresses the production of TNF-a, IL-6, and IL-1b, which are hyperlipidemia-induced inflammatory cytokines secreted by macrophages. A composition for preventing or treating symptom . The rebamipide suppresses the formation of arteriosclerotic lesions by reducing the expression of Th17 that accelerates the generation of arteriosclerotic lesions while suppressing the generation of foam cells that form the precursor cells of arteriosclerotic plaques. a composition for preventing or treating hyperlipidemia according to claim 1, wherein the. Wherein rebamipide is, by total cholesterol in the blood, LDL cholesterol and triglyceride numerical lower, a composition for preventing or treating hyperlipidemia according to claim 1, characterized in that to improve blood lipid . Wherein rebamipide is a composition for preventing or treating hyperlipidemia according to claim 1, characterized in that in a concentration of 0.1μM~10000μM the composition. The hyperlipidemia is a composition for preventing or treating hyperlipidemia according to any one of claims 1 to 5, characterized in that hyperlipidemia from autoimmune diseases.