KR20140016848A - Composition comprising rebamipide for preventing or treating hyperlipemia and disease associated therewith - Google Patents

Abstract

The present invention relates to the pharmaceutical use of rebamipide used for preventing or treating hyperlipidemia and diseases therewith and more specifically, to a composition containing rebemipide as an active ingredient for preventing or treating hyperlipidemia and diseases associated therewith. The rebamipidie compound according to the present invention: suppresses the formation of TNF-a, IL-6, and IL-1b, which are inflammatory cytokines inducing hyperlipidemia and secreted by macrophages; inhibits the generation of foam cells forming atherosclerotic plaque precursor cells; improves blood lipid by reducing the levels of total cholesterol, LDL-cholesterol, and triglycerides in blood; improves a fatty liver; inhibits B cell activities; decreases the expression of Th17 accelerating atherosclerotic plaque formation; and increases the expression of Treg cells controlling the inflammatory response. Thus, the rebamipide compound can be useful as a pharmaceutical composition for preventing or treating hyperlipidemia and diseases associated therewith including atherosclerosis, heart failure, hypertensive cardiac diseases through various mechanisms. Also, the composition has no toxicity or side effect, thereby being safe to use for a long period and stable in the body.

Description

Composition comprising rebamipide for preventing or treating hyperlipemia and disease associated therewith}

The present invention relates to a pharmaceutical use of rebamipide used to prevent or treat hyperlipidemia and related diseases. Specifically, the present invention relates to the prevention of hyperlipidemia and related diseases including rebamipide as an active ingredient. Or to a therapeutic composition.

Modern people are consuming excessive amounts of saturated fat and cholesterol due to the demands of the western diet, lifestyle changes and simplicity, which lead to cardiovascular diseases such as obesity, hyperlipidemia, arteriosclerosis and myocardial infarction, and lipid peroxides. And the like are known to cause degenerative processes in vivo, promote cancer, 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 deaths, including malignant neoplasm (cancer), cerebrovascular disease, and heart disease (including ischemic heart disease and other heart diseases), which are the three major causes of death, were 48.1% in 2008. The mortality rate of vascular disease decreased, but the increase in heart disease (including ischemic heart disease and other heart diseases) can be confirmed by statistical results [National Statistical Office Republic of Korea: Annual Report on the Cause of Death Statistics 2008 (2010) )].

In addition, these diseases are closely related to dietary changes.The results of the 2008 National Health and Nutrition Survey indicate that chronic diseases are obese (over 19 years old), 30.7%, high blood pressure (over 30 years), 26.8%, and diabetes (30 years old). The prevalence rate was 9.7% and hyperlipidemia (over 30 years old) was 10.9%.

Hyperlipemia (hyperlipemia) refers to a condition in which a lot of fatty substances are present in the blood and accumulated on the walls of blood vessels, causing inflammation and consequently cardiovascular disease. Hyperlipidemia consists of 50-150 mg / dl of triglycerides, which are the normal ranges of serum lipids on fasting levels of serum lipids such as cholesterol, triglycerides, phospholipids and free fatty acids. / dl, cholesterol 130 ~ 230 mg / dl and free fatty acids 5 ~ 10mg / dl. If left untreated, hyperlipidemia is likely to cause serious complications such as hypertension, coronary atherosclerosis (angina, myocardial infarction), and cerebral atherosclerosis (cerebral infarction).

Resin that binds bile acids (e.g. cholestyramine, colesripol, etc.), HMG-CoA reductase inhibitors that are major enzymes in cholesterol biosynthesis processes (e.g., lovastatin, fluvastatin, Simvastatin, pravastatin] and the like are used in the clinic, there is a risk of side effects when taking long-term. In particular, statin drugs, which are inhibitors of HMG-CoA reductase, are known to cause liver damage and myopathy.

In addition, currently available cholesterol lowering agents are mainly 3-hydroxy-3-methylglutayl-CoA (HMG-CoA) reductase inhibitors, bile-salt sequestrants, fibrates and niconic acid derivatives. Of these, lovastatin, simvastatin and pravastatin are the most well known cholesterol-lowering agents that inhibit HMG-CoA reductase in the early stages of cholesterol biosynthesis, but also cause serious side effects by inhibiting the synthesis of steroid hormones and dolichol (Havel, RL et al., Ann.Internal Med., 107: 609-615, 1987; Illingworth, DR and Bacon, S. Am. J. Cardiolopy 60: 33-42, 1987).

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

The present inventors therefore inhibit the production of TNF-a, IL-6 and IL-1b, hyperlipidemia-induced inflammatory cytokines secreted from macrophages by rebamipide, currently used as a treatment for gastric ulcers; Inhibit the production of Foam cells forming Artherosclerotic plaque progenitor cells; Improves blood lipids by lowering total cholesterol, LDL-cholesterol and triglyceride levels in the blood; Improve fatty liver; Inhibit the activity of B cells; For the first time, it has been shown to reduce the expression of Th17, which accelerates the production of artherosclerotic plaques, and to increase the expression of Treg cells that can control the inflammatory response, thereby making it useful as a therapeutic agent for hyperlipidemia and related diseases. The present invention has been completed by confirming that it can be used.

Korean Patent Publication No. 10-2011-0035313 Korean Patent Publication No. 10-2008-0024075 Korean Patent Publication No. 10-2002-0060115

It is therefore an object of the present invention to provide a novel use of rebamipide that has the effect of preventing or treating hyperlipidemia and related diseases.

In order to achieve the above object of the present invention, the present invention provides a composition for the prevention or treatment of hyperlipidemia and related diseases comprising a rebamipide compound or a pharmaceutically acceptable salt thereof as an active ingredient. .

In one embodiment of the invention, the rebamipide (rebamipide) can inhibit the production of TNF-a, IL-6 and IL-1b, hyperlipidemia-induced inflammatory cytokines secreted from macrophages.

In one embodiment of the present invention, the rebamipide may not only suppress the production of foam cells forming artherosclerotic plaque progenitor cells, but also inhibit the production of atherosclerotic plaques. Atherosclerosis plaque formation can be inhibited through reduced expression of Th17 to accelerate and increased expression of Treg cells that can control the inflammatory response.

In one embodiment of the invention, the rebamipide (rebamipide) can improve blood lipid by lowering the total cholesterol, LDL-cholesterol and triglyceride 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 10000 μM.

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

In one embodiment of the present invention, the disease associated with hyperlipidemia may be selected from the group consisting of arteriosclerosis, heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina pectoris, stroke and peripheral vascular disease.

The rebamipide compound according to the present invention inhibits the production of TNF-a, IL-6 and IL-1b, which are hyperlipidemic inflammatory cytokines secreted by macrophages; Inhibit the production of Foam cells forming Artherosclerotic plaque progenitor cells; Improves blood lipids by lowering total cholesterol, LDL-cholesterol and triglyceride levels in the blood; Improve fatty liver; Inhibit the activity of B cells; In addition to reducing the expression of Th17, which accelerates the production of atherosclerotic plaques, it can increase the expression of Treg cells that can control the inflammatory response. Therefore, the rebamipide compound may be usefully used as a pharmaceutical composition capable of preventing or treating hyperlipidemia and related arteriosclerosis, heart failure, hypertensive heart disease, etc. through various mechanisms as described above. In addition, there is no toxicity and side effects to the drug can be used safely even when taking long-term, has a stable effect on the body.

Figure 1 shows the amount of inflammatory cytokines secreted from macrophages following levamifeed treatment measured by ELISA analysis (Nil: LPS and levamifeed untreated group).
Figure 2 shows the results of MTT assay to determine the cytotoxicity of levamipid against macrophage RAW 264.7 (Nil: LPS and levamipid untreated group).
Figure 3 is a photograph showing the degree of foam cell formation according to the treatment of levamifeed by concentration (100, 250, 500, 1000μM) in the formation of oxLDL-induced foam cells using THP1 cells, which are human macrophage lines, through oil red O staining. .
Figure 4 is a photograph showing the degree of foam cell formation according to the combination of levamipid alone, simvastatin alone or levamipid and simvastatin in the formation of oxLDL-induced foam cells using THP1 cells, which are human macrophage lines, through oil red O staining. .
FIG. 5 is a graph showing the change in total serum cholesterol, LDL-cholesterol and triglyceride levels in mouse serum after oral administration of levamipid to a hyperlipidemic animal model mouse.
6 is a graph showing the measurement of AST and ALT activity levels in mouse serum after oral administration of levamipid to a hyperlipidemic animal model mouse.
Figure 7 is a photograph showing the degree of aorta branch in the aorta of the mouse after oral administration of levamipid to the hyperlipidemic animal model mouse through Oil red O staining.
8 is a graph showing the measurement of total IgG and IgG1 levels in mouse serum after oral administration of levamipid to a hyperlipidemic animal model mouse.
9 is a FACS analysis of the expression level of Th17 cells and Treg cells in spleen cells of mice after oral administration of levamipid to a hyperlipidemic animal model mouse.
FIG. 10 shows the expression level of Th17 cells and Treg cells in spleen fragments of mice after oral administration of levamipid to hyperlipidemic animal model mice through confocal microscopy.
FIG. 11 shows the mRNA expression level of MMP-9 in spleen cells of mice after oral administration of levamipid to hyperlipidemic animal model mice through RT-PCR.

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

The inventors have focused on rebamipide compounds to develop new therapeutics that are safe and have excellent lipid metabolism inhibition and hyperlipidemic effects. Rebamipide is an acute exacerbation of gastric ulcer, acute gastritis or chronic gastritis. A drug that has an excellent effect on the treatment of gastric mucosal injury, it is widely used as a therapeutic agent for peptic ulcer, and its chemical name is 2- (4-chlorobenzoylamino) -3- [2 (1H) -quinolinone-4-yl]. Propionic acid. The drug protects gastric mucosa by promoting PGE2 biosynthesis to increase mucus, promotes cell proliferation, and inhibits bacterial adhesion and invasion to gastric mucosa cells, especially in patients infected with Helicobacter pylori. Suppresses stomach inflammation.

However, there is no mention in the related art that rebamipide can be used for the treatment of hyperlipidemia and related diseases.

Therefore, in the present invention, rebamipide compounds may be used for the prevention or treatment of hyperlipidemia and related diseases, and in particular, the production of hyperlipidemia-induced inflammatory cytokines TNF-a, IL-6 and IL-1b. Inhibit; Inhibit the production of Foam cells forming Artherosclerotic plaque progenitor cells; Improves blood lipids by lowering total cholesterol, LDL-cholesterol and triglyceride levels in the blood; Improve fatty liver; Inhibit the activity of B cells; For the first time, a mechanism for reducing the expression of Th17, which accelerates the formation of atherosclerotic plaques, and increasing the expression of Treg cells that can control the inflammatory response can lead to the prevention or treatment of hyperlipidemia and related diseases. It was.

In general, hyperlipidemia causes a lot of complications due to the presence of more fatty substances in the blood than necessary, and these complications are very dangerous diseases such as arteriosclerosis, angina pectoris, and myocardial infarction. There is a situation.

Atherosclerosis is characterized by the accumulation of lipids in the endothelium and inflammatory reactions leading to the formation of atherosclerotic plaques. Plaques narrow the arteries and the narrowed arteries can easily rupture if clogged by blood clots, leading to cardiovascular disease.

An important role in this early stage of atherosclerosis is macrophages derived from monocytes. When hyperlipidemia causes high LDL levels in the blood or when LDL is modified for oxidation, macrophages use scavenger receptors to introduce oxidized LDL (oxLDL). Macrophages are called foam cells. In general, pathogen-induced macrophages migrate to other places, but since foam cells do not leave their place after the introduction of oxidized LDL, they lose their normal biological function, penetrate into vascular endothelial cells and become atherosclerotic. thickens smooth muscle through plaque) formation. Moreover, foam cells are known to trigger a local inflammatory response when trapped in the artery's endothelium.

In Example 1 of the present invention, in order to examine the effect of rebamipide on macrophages, when treated with LPS after treatment of levamipids in mouse peritoneal macrophages, TNF-a which is a hyperlipidemic factor , It was confirmed that effectively inhibit the production of IL-6 and IL-1b (see Fig. 1).

In addition, in Example 3 below, the effect of levamifeed on the production of foam cells forming artherosclerotic plaque progenitor cells was examined, and as a result, levamifeed treatment concentration In addition, foam cell production, which is an vascular pathogen cell, was suppressed (see FIG. 3), and in particular, it was confirmed that foam cell production was superior to the conventional hyperlipidemic drug simvastatin (Simvastatin). See FIG. 4).

In addition, in Example 4 of the present invention, the blood lipid improvement effect of the administration of levamipid to the hyperlipidemic animal model was examined, and as a result, total or cholesterol in serum when levamipid was orally administered to the hyperlipidemic animal model mouse , LDL-cholesterol and triglyceride concentration was confirmed that the effective reduction (see Figure 5).

In addition, in Example 5 of the present invention, the effect of fatty liver improvement according to the administration of levamipid to the hyperlipidemic animal model was examined. It was shown to be significantly reduced, it was confirmed that levamifeed can suppress liver damage in hyperlipidemic animal model (see FIG. 6).

In addition, in Example 6 of the present invention, the effect of inhibiting arteriosclerosis formation according to the administration of levamipid to the hyperlipidemic animal model was examined, and as a result, the aorta in the experimental group orally administered levamipid to the hyperlipidemic animal model mouse It was confirmed that the aorta branch showed significantly less oil red O positive, and levamifeed actually inhibited the formation of atherosclerotic plaques (see FIG. 7).

In addition, in Example 7 of the present invention, the B-cell antibody immune response was investigated in the hyperlipidemic animal model according to the administration of levamipid, and as a result, when oral administration of levamipid to the hyperlipidemic animal model mouse was performed, It was shown that the number is reduced, it was confirmed that the activity of B cells by levamifeed (see Figure 8).

In addition, in Example 8 of the present invention, we examined the Th17 cell inhibition and Treg cell induction co-regulation response according to the administration of levamipid to the hyperlipidemic animal model, and as a result, oral administration of levamipid to the hyperlipidemic animal model mouse In one case, the expression of Th17, which accelerates the production of artherosclerotic plaque, was shown to be reduced in the splenocytes, whereas the expression of Treg cells, which are immunoregulatory cells, was increased (see FIGS. 9 and 10). .

For reference, T cells are one of cell groups that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the thymus of the human body, and undergo a series of differentiation processes, resulting in differentiation into T cells with inherent characteristics. The differentiated T cells are largely classified into type 1 (Th1) Auxiliary cells (Th2). Among them, the main function of Th1 cells is involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations are balanced with each other so that they are not activated with each other.

Therefore, most of the immune diseases can be attributed to the imbalance between these two immune cells, for example, when an abnormal increase in the activity of Th1 cells may cause an immune disease, an abnormal increase in the activity of Th2 cells It is known that immune diseases occur due to hypersensitivity reactions.

On the other hand, according to recent research on the differentiation of Th1 cells, the existence of a new group that regulates the activity of Th1 cells (regulatory T cells, hereinafter abbreviated as 'Treg') is known. Researches on the treatment of immune diseases have emerged. Treg cells are characterized by controlling the inflammatory response by inhibiting the function of abnormally activated immune cells. Many experiments have been reported to treat the disease.

In addition to the Treg cells, another group made during the differentiation is Th17 cells, which are known to be formed through a process similar to the differentiation of Treg cells during the differentiation of undifferentiated T cells. That is, differentiation of Treg cells and Th17 cells is performed in the presence of TGF-β in common but does not require IL-6 in Treg cells, whereas IL-6 is present in combination with TGF-β in Th17 cells. Differentiate in situations In addition, differentiated Th17 cells are characterized by the secretion of IL-17.

Unlike Treg cells, Th17 cells have been found to be involved in the forefront of inflammatory reactions seen in immune diseases, maximizing the signal of inflammatory responses and accelerating disease progression.

Therefore, when Th17 cell suppression and Treg cell induction are co-regulated, excessive immune response or inflammatory response can be effectively suppressed. In the case of levamifeed, Treg cell expression can be enhanced simultaneously with inhibition of Th17 cell expression. It was determined that the immune or inflammatory response can be effectively suppressed.

In addition, in Example 9 of the present invention, the mRNA expression level of MMP-9 according to the administration of levamifeed to the hyperlipidemic animal model was investigated. MMP-9, which degrades non-fibrillar collagen, is expressed in atherosclerotic plaques and is known to play a major role in pathophysiological mechanisms in atherosclerotic plaque rupture. As a result, when oral administration of levamipid to the hyperlipidemic animal model mice, it was confirmed that the expression level of MMP-9 in the spleen cells of the mouse was significantly reduced in the ratio of simvastatin orally administered group, which is a positive control group (Fig. 11). Reference). Accordingly, the present inventors determined that levamifeed effectively reduces the expression of MMP-9, thereby more effectively preventing the occurrence of cerebral infarction or myocardial infarction caused by atherosclerotic plaque rupture.

Through these results, the present inventors have experimentally demonstrated that levamipid is effective in 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 related diseases.

The levamipid according to the present invention may be a compound represented by the following Chemical Formula 1.

≪ Formula 1 >

The rebamipide compound according to the present invention may be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The rebamipide compound according to the present invention may be commercially available, or may be one that is separated from nature or manufactured by chemical synthesis known in the art.

In one embodiment of the invention, the rebamipide (rebamipide) of hyperlipidemia and related diseases through a mechanism that inhibits the production of hyperlipidemia-induced inflammatory cytokines TNF-a, IL-6 and IL-1b in macrophages It may have a prophylactic or therapeutic effect.

In another embodiment of the present invention, the rebamipide (rebamipide) may be included in the composition of the present invention at a concentration of 0.1 to 10000μM.

Meanwhile, the composition for preventing or treating hyperlipidemia and related diseases provided by the present invention is preferably a pharmaceutical composition.

In the present invention, "hyperlipidemia related disease" refers to a disease caused or promoted by hyperlipidemia, for example, arteriosclerosis, heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina pectoris, stroke and peripheral vascular disease It may be selected from the group consisting of, but is not particularly limited in kind.

In the present invention, "treatment ", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term treatment as used herein refers to the act of treating when "treating" is defined as above. Thus, in mammals, "treatment" or "therapy" of hyperlipidemia and related diseases may include one or more of the following:

(1) arrest the development of hyperlipidemia and related diseases,

(2) prevent the spread of hyperlipidemia and related diseases,

(3) relieve hyperlipidemia and related diseases,

(4) prevent the recurrence of hyperlipidemia and related diseases, and

(5) alleviating the symptoms of hyperlipidemia and related diseases

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant, in addition to rebamipide, which is an active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, Coatings, swelling agents, lubricants, lubricants, or flavoring agents can be used.

In addition, the pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to rebamipide as an active ingredient for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, natural sugars such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, rebamipide of the present invention may be included in the composition at a concentration of 0.1 to 10000 μM, and also rebamipide of the present invention is 0.1 to 95 with respect to the total weight of the composition It may be included in weight percent.

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

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

In the present invention, the "pharmaceutically effective amount" refers to an amount of an active ingredient that exhibits an alleviating, suppressing, improving and / or curative effect on a disease to be treated. The dose of rebamipide of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, administration method and severity of disease of the patient. For example, therapeutically effective dosages can be initially determined using in vitro assays through cell culture. It will be possible to determine the amount effective for treatment without undue experimentation in the art, and this information can be used to more accurately determine the useful dose 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 Can be.

The present invention also provides the 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 comprising rebamipide as an active ingredient may be used for the manufacture of a medicament for preventing or treating hyperlipidemia and related diseases.

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

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

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief 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 the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like.

The present invention also provides a health functional food for the prevention or improvement of hyperlipidemia and related diseases comprising rebamipide as an active ingredient.

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

In the present invention, the term "health functional food" refers to a food prepared and processed by using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, and the nutritional control on the structure and function of the human body Or for the purpose of obtaining a beneficial effect for health use such as physiological action.

The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards.

Examples of the food items included in the above food additives include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets is a conventional method of granulating a mixture of rebamipide, an active ingredient of the present invention with an excipient, a binder, a disintegrant and other additives, and then a lubricant and the like. Compression molding, or the mixture may be directly compression molded. In addition, the health functional food in the form of tablets may contain a mating agent and the like as necessary.

Hard capsules among the health functional foods in the form of capsules may be prepared by filling a mixture of additives such as rebamipide, an active ingredient of the present invention, in a conventional hard capsule with an excipient, and the like. (Rebamipide) may be prepared by mixing a mixture of additives such as excipients with 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, if necessary.

The health functional food in the form of a cyclic form can be prepared by molding a mixture of an excipient, a binder, a disintegrant, and the like of rebamipide, which is an active ingredient of the present invention. Other coatings may be applied, or the surface may be coated with materials such as starch, talc.

The health functional food in the form of granules may be prepared by granulation of a mixture of rebamipide, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method. And a copper may be contained.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

On macrophages Levamifeed  Inhibitory Effects of Inflammatory Cytokines on Treatment

In order to determine the effect of levamipid on macrophages, the present inventors pretreated levamifeed in mouse macrophages and stimulated with LPS (lipopolysaccharide) to secrete inflammatory cytokines TNF-a (tumor necrosis factor). -alpha), IL-6 (interleukin-6) and IL-1β (interleukin-1β) production was evaluated by ELISA analysis.

<1-1> Cell culture

Macrophage lines of mice, RAW 264.7 cells, were obtained from the Korea Cell Line Bank (KCLB), and used DMEM (Dulbeccos Modified Eagle Medium) containing 10% FBS and 1% penicillin-streptomycin for cell culture. Cells were cultured in 37, 5% CO ₂ conditions.

<1-2> Effect lever US feed on the production of inflammatory cytokines (TNF -a, IL -6 and IL -1 β)

First, pretreatment with levamifeed in RAW 264.7 cells at a concentration of 20, 100 μg / ml, and 1 hour after treatment with LPS (lipopolysaccharide) at a concentration of 100 ng / ml, incubated for 48 hours in a 37 ° C incubator, inducing an inflammatory response in macrophages. It was.

In order to measure the amount of cytokines produced (TNF-α, IL-6 and IL-1β), the supernatants of the cultured cells were collected and purified by ELISA for TNF-α, IL-6 and IL-1β. The degree of production was investigated separately. Monoclonal anti-TNF-α, anti-IL-6, and anti-IL-1β were reacted overnight at 4 ° C. at 1 μg / mL in a 96-well plate, followed by nonspecific binding with blocking solution (1% BSA / PBST). Blocked. TNF-α recombinant, IL-6 recombinant, IL-1β recombinant were used as a standard by diluting 1/2 successively, the cell culture supernatant was added and reacted at room temperature for 2 hours. Thereafter, biotinylated anti-TNF-α, biotinylated anti-IL-6, and biotinylated anti-IL-1β were reacted at room temperature for 2 hours, washed four times, and then diluted by adding the ExtraAvidin-Alkaline Phosphatase conjugate, followed by 2 at room temperature. The reaction was carried out for a time. Thereafter, PNPP / DEA solution was added and developed, and then absorbance was measured at 405 nm.

As a result, as shown in Figure 1, when only LPS without pretreatment of levamifeed, the production of TNF-α and IL-6 was significantly increased compared to the control (normal cells), but levamifeed In the experimental group pretreated with it was confirmed that the concentration of TNF-α and IL-6 production is reduced depending on the concentration. In addition, when only LPS was treated without pretreatment of levamifeed, the production of IL-1β was not different from that of the control group (normal cells), but the production of levamifeed at 100 μg / ml was reduced. appear. Nil represents an untreated group as a negative control.

Through these results, it was determined that levamifeed may be useful for preventing or treating hyperlipidemia and related diseases through a mechanism that inhibits the inflammatory response of macrophages, which play an important role in hyperlipidemia and atherosclerosis.

< Example  2>

Rebamipide  Determination of Toxicity to Cells

MTT assay was performed to determine the cytotoxicity of levamifeed against mouse macrophage RAW 264.7.

This method measures the conversion of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) to formazan. The 96 well plate contains 1 × 10 ⁴ cells / well. RAW 264.7 cells were dispensed and levamifeed was treated for 18 hours by concentration (20, 100, 500, 1000, 5000 μM). 100 μl of MTT solution was added to each well for 4 hours at 37 ° C., 5% CO 2 incubator, and then the change in absorbance at 570 nm was measured using a microplate reader (VERSAmax, Molecular Devices, USA).

As a result, as shown in Figure 2, it was found that even at effective concentrations of 20μM, 100μM and more effective effective cytotoxicity is low, basically does not affect the survival rate of the cells. This fact suggests that the inhibitory effect of levamipid in macrophages is inherent to levamifeed, not merely to inhibit the production of inflammatory mediators (TNF-α, IL-6 and IL-1β) by cell death. It is an effect.

< Example  3>

Rebamipide  According to treatment Vascular pathogen  Inhibitory effect

In this experiment, we examined whether levamifeed can actually inhibit the formation of vascular pathogen cells. The 'angiogenic cell' refers to a foam cell that forms atherosclerosis (Artherosclerotic plaque) progenitor cells that induce atherosclerosis, and is a cell that causes atherosclerosis in the present invention. Pathogen cells were randomly named.

<3-1> Rebamipide  According to treatment Vascular pathogen  Inhibitory effect

For this experiment, THP1 cells, human macrophages, were treated with 160nM concentration of PMA (phorbol 12'-myristate 13'-acetate) to activate the cells, followed by treatment of PAF (platelet-activating factor) with 10ug / ml Treatment of feed by concentration (100, 250, 500, 1000 μM) was performed to determine the degree of foam cells forming Artherosclerotic plaque precursor cells that induce atherosclerosis. Oil Red O solution (Sigma aldrich- Sigma Aldrich) staining.

As a result, as shown in Figure 3, it was confirmed that the foam cell (Foam cell) cells, which are vascular pathogen cells depending on the level of levamifeed treatment is suppressed.

<3-2> Compared to existing hyperlipidemic drugs Rebamipide Vascular pathogen  Comparison of inhibitory effect

The inhibitory effect of levamifeed on vascular pathogen cells was compared with simvastatin, which is currently used as a hypolipidemic drug (hyperlipidemic drug).

For this experiment, THP1 cells, human macrophages, were treated with 160nM concentration of PMA (phorbol 12'-myristate 13'-acetate) to activate the cells, followed by treatment of PAF (platelet-activating factor) with 10ug / ml The degree of foam cell formation to form atherosclerotic plaque progenitor cells that induced atherosclerosis by treating feed (1000 μM) or simvastatin (10 μM), respectively, was measured by Oil Red O solution staining.

As a result, as shown in Figure 4, the experimental group treated with levamipid alone was confirmed that less foam cells (Foam cell) is formed than the experimental group treated with simvastatin alone, which is in the existing hyperlipidemic drug simvastatin 20% more effective.

< Example  4>

Hyperlipidemia in animal models Rebamipide  Blood lipid improvement effect by administration

In this experiment, in order to measure the blood lipid improvement effect of levamipid in vivo, the total cholesterol and LDL-cholesterol and triglyceride levels in the serum of mice were measured after oral administration of levamipid to the hyperlipidemic animal model. .

Specifically, 8-week-old Apoe ko mice (mouse with apolipoprotein E gene removed) were given oral administration of 100 mg / kg of levamifeed, followed by Western diet, and sacrificed at 8 weeks after Western diet. Cholesterol, LDL-cholesterol and triglyceride levels were measured. The positive control group was orally administered 25 mg / kg of simvastatin, which is currently used as a lipid lowering agent, in place of levamipid. Total-cholesterol calculations used the general total-cholesterol calculation (LDL = T-CHO- (0.2 * TG) -HDL, T-CHO = HDL + (0.2 * TG) + LDL).

As a result, as shown in Figure 5, oral administration of levamipid to the hyperlipidemic animal model mice, it was confirmed that the serum total-cholesterol, LDL-cholesterol and triglyceride concentrations effectively reduced, especially simvastatin (Simvastatin, a positive control) It was found that LDL-cholesterol and triglyceride concentrations were lowered more effectively in the administration group.

< Example  5>

Hyperlipidemia in animal models Rebamipide  Fatty liver improvement effect by administration

In this study, in order to measure the fatty liver improvement effect of levamifeed in vivo, serum AST and ALT levels of mice were measured after oral administration of levamifeed to a hyperlipidemic animal model.

When the structure and function of the cell membrane are destroyed, the enzymes AST (Aspartateamino-transferase) and ALT (Alanine aminotransferase), which are widely present in the liver cytoplasm, are leaked into the blood. In this study, serum AST and ALT levels were measured to verify whether levamifeed is effective in preventing fatty liver and improving fatty liver.

Specifically, 8-week-old Apoe ko mice (mouse with apolipoprotein E gene removed) were given oral administration of 100 mg / kg of levamifeed, followed by Western diet, and sacrificed at 8 weeks after Western diet. ALT values were measured. The positive control group was orally administered 25 mg / kg of simvastatin, which is currently used as a lipid lowering agent, in place of levamipid. AST and ALT activity was measured by quantitative kit reagent (Youngdong Pharmaceutical, Korea). 1.0 mL of AST and ALT substrate solution were warmed for 2 minutes in a 37 tank, and then 0.2 mL of plasma was added and reacted for 30 minutes in a 37 ℃ water bath. After 30 minutes, 1.0 mL of the color development reagent was added, and the mixture was allowed to stand at room temperature for 20 minutes. Then, 10.0 mL of 0.4 N NaOH was added and absorbance was measured at 505 nm. AST and ALT standard solution (2mM pyruvate) was developed in the same manner as the above method by measuring the absorbance and extrapolated to the standard curve to calculate the activity of the sample.

As a result, as shown in Figure 6, oral administration of levamipid to the hyperlipidemic animal model mice was confirmed that significantly reduced serum AST and ALT activity. This suggests that levamifeed has the effect of suppressing liver damage as well as improving blood lipid in hyperlipidemic animal model.

< Example  6>

Hyperlipidemia in animal models Rebamipide  Atherosclerotic plaque according to administration Artherosclerotic plaque ) Inhibitory effect on formation

In this experiment, in order to measure the inhibitory effect of levamipids on arteriosclerosis formation in vivo, we examined the aorta branches of mice after oral administration of levamipids in a hyperlipidemic animal model. The aorta branch corresponds to the formation of atherosclerotic plaques during atherosclerosis.

Specifically, 8-week-old Apoe ko mice (mouse with apolipoprotein E gene removed) were given oral administration of 100 mg / kg of levamifeed, followed by a Western diet, and sacrificed at 8 weeks after the Western diet. ) Was fixed and oil red O staining was performed. The positive control group was orally administered 25 mg / kg of simvastatin, which is currently used as a lipid lowering agent, in place of levamipid.

As a result, as shown in FIG. 7, the aorta branch was mostly oil red O-positive in the negative control group (hyperlipidemic animal model mouse), whereas the aorta branch seen in the aorta in the experimental group orally administered levamipid to the hyperlipidemic animal model mouse. It was confirmed that the oil red O positive was significantly less, and in particular, compared to the positive control group (simvastatin oral administration group), the aorta branch was found to have less oil red O positive portion.

< Example  7>

Hyperlipidemia in animal models Rebamipide  B cell antibody immune response following administration Research

In this study, to examine the effect of levamifeed on the B-cell antibody immune response in vivo, total IgG and IgG1 levels were measured in mouse serum after oral administration of levamifeed to a hyperlipidemic animal model.

Specifically, eight-week-old Apoe ko mice (mouse with apolipoprotein E gene removed) were fed orally with 100 mg / kg of levamifeed while feeding a Western diet and sacrificed at 8 weeks post-Western diet. And IgG1 levels were measured by ELISA analysis. The positive control group was orally administered 25 mg / kg of simvastatin, which is currently used as a lipid lowering agent, instead of levamipid. Serum total IgG and IgG1 concentrations were measured according to the manufacturer's instructions using an ELISA kit (bethyl). In other words, put the Capture on a 96well plate plate and react overnight for 1 hour after diluting the serum at 1: 1000 and reacting for 1 hour with Total IgG, IgG1 detection ab. After adding HRP, it developed with TMB solution. The absorbance was read.

As a result, as shown in Figure 8, oral administration of levamipid to the hyperlipidemic animal model mice showed a decrease in the level of IgG and IgG1 in the serum, it was confirmed that the B cell activity is inhibited by levamifeed .

< Example  8>

Hyperlipidemia in animal models Rebamipide  According to administration Th17  Cell inhibition and Treg  Cell-induced co-regulation

In this experiment, we investigated the expression of Th17 and Treg cells in mouse splenocytes after oral administration of levamipid to a hyperlipidemic animal model. .

Specifically, 8-week-old Apoe ko mice (mouse with apolipoprotein E gene removed) were fed orally with 100 mg / kg of levamifeed while feeding the Western diet, and sacrificed at 4 weeks after the Western diet. The expression of Th17 cells was analyzed by measuring IL-17 expression levels of cells, and the expression of Treg cells was analyzed by measuring Foxp3 expression levels of CD4 + CD25 + cells. Expression analysis of Th17 cells and Tregs was performed via FACS and confocal microscopy via staining. Meanwhile, the positive control group orally administered 25 mg / kg of simvastatin, which is currently used as a lipid lowering agent, instead of levamipid.

For reference, since differentiated Th17 cells are characterized by secreting IL-17, confirming the expression of IL-17 is an indicator for measuring the activity or amplification of Th17 cells. In addition, Foxp3 is mainly present in regulatory T cells (Tregs) derived from the thymus, and is a transcriptional factor present in cells with CD4 + CD25 + labeled antigens, whose function is Foxp3. IL-recognized T cells, which have low reactivity to the antigen upon recognition of T-expressing T cells, and potentially CD4 + CD25- T cells that do not express Foxp3 differentiated from the thymus, may cause autoimmunity. It has a role as a suppressor T cell that inhibits the production of -2 and cell division. Foxp3 is not only affected by IL-2, but also by the transcription factor NFAT on CD25- T cells through regulatory T cells expressing Foxp3 and through cell-cell contact. It has been found to function to suppress transcriptional regulation of the back. Therefore, in the case of T cells expressing Foxp3 having such a function, it is applied to the treatment of immune diseases through the action of inhibiting or regulating an immune response, and also of CD4 + T cells expressing Foxp3 present in humans. To apply self-antigen specific T cell clones to cell therapy by increasing their number through high concentration of IL-2 cytokine and combination treatment with anti-CD3 and anti-CD28 antibodies There have been attempts. Therefore, confirming the expression of Foxp3 is an indicator to measure the activity or amplification of Treg cells.

<8-1> FACS stain  analysis

FACS staining was performed on splenocytes of mice after lethal mice. Th17 cells were first reacted with anti-CD4 antibody for 30 minutes at 4, followed by 30 minutes reaction with Cytofix / Cytoperm, followed by 30 minutes reaction with anti-IL-17 antibody, followed by reading with FACScalibur and flow. The analysis was performed using the jo program. Treg cells were treated with anti-CD4 and anti-CD25 antibodies for 30 minutes and then reacted with Treg-specific Cytofix / Cytoperm for 30 minutes.Then, washed and then reacted with anti-Foxp3 antibody for 30 minutes and read with FACScalibur. Analysis was performed using the Flow jo program.

As a result, as shown in Figure 9, oral administration of levamipid to the hyperlipidemic animal model mice, it was shown that the expression of Th17 to accelerate the production of artherosclerotic plaque in the splenocytes, while immunoregulatory cells It was confirmed that the expression of phosphorus Treg cells was increased. In particular, the expression of Th17 was further reduced in the levamifeed group than in the positive control group, Simvastatin.

<8-2> Confocal microscope  analysis

Confocal microscopy analysis method using the frozen slicer after killing the mouse and embedding the OCT compound using the spleen fragment of the mouse using a frozen slicer. Attached to the slide in thickness. The sections were fixed with acetone and blocked for 30 minutes of nonspecific reaction with 10% normal goat serum. Th17 cells used PE-labeled anti-CD4 and FITC-labeled anti-IL-17 antibodies. Treg cells used PE-labeled anti-CD4, FITC-labeled anti-Foxp3 and APC-labeled anti-CD25 antibodies. Stained tissue was analyzed by confocal microscopy (LSM 510 Meta. Zeiss, Go? Tingen, Germany).

As a result, as shown in Figure 10, oral administration of levamipid to the hyperlipidemic animal model mice, it was shown that the expression of Th17 to accelerate the production of artherosclerotic plaque in the splenocytes, while immunoregulatory cells It was confirmed that the expression of phosphorus Treg cells was increased.

< Example  9>

Hyperlipidemia in animal models Rebamipide  According to administration MMP -9 Expression Analysis

In this experiment, mRNA expression level of MMP-9 in hyperlipidemic animal model mouse was investigated. Matrix metalloproteinase-9 (MMP-9), which degrades non-fibrillar collagen, is expressed in atherosclerotic plaques and is known to play a major role in pathophysiological mechanisms in atherosclerotic plaque rupture.

Specifically, 8-week-old Apoe ko mice (mouse with apolipoprotein E gene removed) were fed orally with levamifeed 20 mg / kg and 100 mg / kg, respectively, and sacrificed at 8 weeks after Western diet. Splenocytes were obtained. The splenocytes thus obtained were treated with PMA 25ng / ml and Ionomycin 250ng / ml for 4 hours, and then the expression of MMP-9 in the RNA of the cells was examined by RT-PCR. RT-PCR was performed using an ABI PCR machine with a LightCycler FastStart DNAmaster SYBR green I (Takara) fluorescent dye, and the reaction compound for this was 1 μl of 1 μg of synthesized cDNA in 1 μl, LightCycler FastStart DNAmaster SYBR green After mixing 10 μl of I (Takara) and 1 μl of Taqman probe (Applied biosystems), the final volume was used to make 20 μl of distilled water. The reaction conditions were reacted 50 times at 95 degreeC for 10 minutes, 95 degreeC 10 second, and 64 degreeC 30 second. The cycle threshold (Ct) values were analyzed to express the mRNA expression level of MMP-9 as a relative quantitative expression of the mRNA expression level of β-actin, a house keeping gene. As primers used in the RT-PCR, Forward: 5'- CTG TCC AGA GTA CAG CCT-3 ', Reverse: 5'-GAG GTA TAG TGG GAC ACA TAG TGG-3' was used. Meanwhile, the positive control group orally administered 25 mg / kg of simvastatin, which is currently used as a lipid lowering agent, instead of levamipid.

As a result, as shown in Figure 11, when oral administration of levamipid to the hyperlipidemic animal model mice, it was confirmed that the expression of MMP-9 in the spleen cells of the mouse significantly reduced in the ratio of simvastatin orally administered group. .

These results suggest that levamifeed effectively reduces the expression of MMP-9, which plays a major role in pathophysiological mechanisms in atherosclerotic rupture, further reducing the incidence of cerebral infarction or myocardial infarction caused by atherosclerotic rupture. It could be effectively prevented.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

A composition for preventing or treating hyperlipidemia and related diseases comprising a rebamipide compound or a pharmaceutically acceptable salt thereof as an active ingredient. The method of claim 1,
The levamipid is a composition for preventing or treating hyperlipidemia and related diseases, characterized in that it inhibits the production of hyperlipidemia-induced inflammatory cytokines TNF-a, IL-6 and IL-1b secreted by macrophages. The method of claim 1,
The levamipid inhibits the formation of atherosclerotic plaques by reducing the expression of Th17 which accelerates the production of atherosclerotic plaques as well as inhibiting the production of foam cells forming artherosclerotic plaque progenitor cells. Composition for the prevention or treatment of hyperlipidemia and related diseases. The method of claim 1,
The levamipid is a composition for the prevention or treatment of hyperlipidemia and related diseases, characterized in that it improves blood lipids by lowering the total cholesterol, LDL-cholesterol and triglyceride levels in the blood. The method of claim 1,
The levamifeed is a composition for the prevention or treatment of hyperlipidemia and related diseases, characterized in that it is contained in a concentration of 0.1μM to 10000μM. The method according to any one of claims 1 to 5,
The hyperlipidemia is a composition for preventing or treating hyperlipidemia and related diseases, characterized in that the hyperlipidemia derived from autoimmune diseases. The method according to any one of claims 1 to 5,
Prevention of hyperlipidemia and related diseases is selected from the group consisting of arteriosclerosis, heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina pectoris, stroke and peripheral vascular disease. Therapeutic composition.

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