KR20130129496A - Composition for preventing or treating immune disease comprising metformin - Google Patents

Abstract

The present invention relates to a pharmaceutical use of a metformin compound used to prevent or treat immune diseases caused by abnormal immune responses, and more particularly, to a composition for preventing or treating immune diseases comprising a metformin compound as an active gradient. The metformin compound has excellent effects of inhibiting the differentiation into cytotoxic Th17 cells that generate and secrete inflammatory cytokine; enhancing activity of immunoregulatory T cells (Treg) that inhibit functions of abnormally activated immune cells and regulate inflammatory responses; and effectively maintaining and enhancing functions of the immunoregulatory T cells (Treg) through inhibition of plasticity of differentiation of the immunoregulatory T cells (Treg) into Th17 cells. Therefore, the metformin compound is useful as a pharmaceutical composition or an immune inhibitor capable of preventing or treating immune diseases caused by abnormal regulations of various immune responses, such as autoimmune diseases, inflammatory diseases, and transplant rejection disorders.

Description

Composition for preventing or treating immune disease comprising metformin as an active ingredient

The present invention relates to a pharmaceutical use of a metformin compound used for preventing or treating an immune disease caused by an abnormal immune response, and more particularly, to an immunological disease comprising a metformin compound as an active ingredient. It relates to a prophylactic or therapeutic composition.

Although diseases caused by immune hyperresponsiveness are increasing worldwide, fundamental causes for the occurrence of these diseases have not been sufficiently elucidated. Currently, as a method of treating diseases due to an excessive immune response, immunosuppressive agents are administered alone or in combination to alleviate or reduce various symptoms caused by the diseases. Immunosuppressants refer to a variety of substances used to reduce or block the host's ability to produce antibodies (a humoral immune response) or a cellular immune response to the action of an antigen. Such immunosuppressive agents may be useful for autoimmune diseases such as lupus, rheumatoid arthritis, and skin hypersensitivity reactions such as atopy and allergy as well as organ transplant field. Good immunosuppressants should be able to control the imbalance of the immune response, ensure the safety of the human body, and reduce the incidence of disease recurrence during long-term treatment (Wollenberg 2008).

Cyclosporin A and FK506, which are currently used as immunosuppressants, are compounds derived from natural materials with complex chemical structures. These compounds are uneconomical due to high cost in terms of raw material supply and may cause various side effects due to long-term administration. . Therefore, there is an urgent need to develop a new immunosuppressive agent capable of inducing low toxicity, immunity tolerance and economic production.

On the other hand, there are T cells as one of the cell groups which plays a central role in the immune system as a bio-defense system for 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 are caused by the imbalance between these two immune cells. For example, when the activity of Th1 cells abnormally increases, an autoimmune disease may occur and the activity of Th2 cells abnormally increases It is known that immune diseases are caused by hypersensitivity reactions.

Meanwhile, according to a recent study on the differentiation of Th1 cells, the existence of a new group of immunoregulatory T cells (Tregs), which can control Th1 cell activity, is known, and studies on the treatment of immune diseases using the same are emerging. In addition, since Treg cells inhibit the function of abnormally activated immune cells to control the inflammatory response, many experiments have been reported to treat immune diseases through the action of increasing the activity of Treg cells.

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, in the case of autoimmune diseases which are not controlled by Treg cells among autoimmune diseases, development of therapeutic agents for autoimmune diseases that target the inhibition of Th17 cell activity has been highlighted.

Currently used immunosuppressive drugs are immunosuppressants that block signal transduction pathways in T cells. These immunosuppressive agents are toxic, infection, lymphoma, diabetes, tremor, headache, diarrhea, high blood pressure, and nausea. There is a problem that side effects such as renal failure occur.

In addition to the treatment of immune diseases by inhibiting the activation of T cells, a treatment for controlling the amount of cytokines secreted from immune cells and a therapeutic method using antibodies targeting cytokines secreted from immune cells have been developed. There is. However, this method has to take a long time to apply to the patients after the actual clinical trials, the method using the antibody has a problem that too much cost in the antibody manufacturing process.

Therefore, there is an urgent need to develop a new immune disease treatment agent and treatment method which is inexpensive and has excellent therapeutic effect, and disclosed in Korean Laid-Open Patent Publication No. 2011-0052990 that can inhibit type 2 diabetes in metformin. Although the Korean Patent Application Publication No. 2009-0005513 discloses that metformin malonate has antidiabetic activity, it is not disclosed that metformin is used for the prevention and treatment of immune diseases.

The present inventors found that metformin compounds inhibit or reduce the activity of Th17, promote or increase the activity of Regulatory T cells (Treg), and variability in the differentiation of Regulatory T cells (Tregs) into Th17 cells. The present invention was completed by confirming that there is an activity of inhibiting plasticity, and it can be used as a therapeutic agent in various immune-related diseases.

Accordingly, an object of the present invention is a composition for preventing or treating immune diseases, including a metformin compound or a pharmaceutically acceptable salt thereof as an active ingredient to effectively prevent or treat immune diseases caused by abnormalities of various immune responses. To provide.

In order to achieve the above object, the present invention provides a composition for preventing or treating immune diseases, including a metformin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the invention, the composition may further comprise an AP-1 inhibitor.

In one embodiment of the present invention, the AP-1 inhibitor may be one selected from the group consisting of curcumin, tanshinone IIA, and SR11302.

In one embodiment of the present invention, the composition may inhibit or reduce the activity of Th17 and promote or increase the activity of Regulatory T cells (Treg).

In one embodiment of the present invention, the composition may have an activity of inhibiting the plasticity of differentiation of regulatory T cells (Treg) into Th17 cells.

In one embodiment of the present invention, the composition may further comprise myeloid derived suppressor cells (MDSC).

In one embodiment of the present invention, the composition may inhibit or reduce the activity of Th17 and promote or increase the activity of Regulatory T cells (Treg).

In one embodiment of the present invention, the immune disease is Rheumatoid Arthritis, Asthma, Dermititis, Psoriasis, Cystic Fibrosis, Late Lung Transplantation and Chronic Rejection ( Post transplantation late and chronic solid organ rejection, Multiple Sclerosis, Systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, Polymyositis, Scleroderma ), Addison disease, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Dieseses, Autoimmune Diabetes , Diabetic retinopathy, Rhinitis, Ischemia-reperfusion injury, Post-angioplasty restenosis, Chronic lung Chronic obstructive pulmonary diseases (COPD), Graves disease, Gastrointestinal allergies, Conjunctivitis, Atherosclerosis, Coronary artery disease, Angina , Cancer metastasis and small arterial disease, graft-versus-host disease.

The metformin compound according to the present invention has an excellent effect of inhibiting differentiation into cytotoxic Th17 cells that produce and secrete inflammatory cytokines, and inhibit the function of abnormally activated immune cells and control the inflammatory response. It has an excellent effect of enhancing the activity of immunoregulatory T cells (Treg) having the ability to control the function of regulatory T cells (Treg) through the activity of inhibiting the plasticity of the regulatory T cells (Treg) to Th17 cells. It can be effectively used as a pharmaceutical composition or immunosuppressant that can effectively prevent or treat immune diseases such as autoimmune diseases, inflammatory diseases and transplant rejection diseases caused by abnormal control of various immune responses due to its excellent effect of maintaining and strengthening. have.

1 is an embodiment of the present invention when treated with metformin alone in the Th17 cell-derived animal model, when treated with a combination of metformin and AP-1 inhibitors (curcumin, tanshinone IIA, SR11302) Th17 cells for each The analysis of FACs to determine whether differentiation of
Figure 2 is a result confirmed by flow cytometry (FACs) whether the metformin compound of the present invention inhibits Treg differentiation variability according to an embodiment of the present invention.
Figure 3 is the result of confirming the Th17 and Treg cell regulation effect by metformin and MDSC of the present invention by flow cytometry (FACs) according to an embodiment of the present invention.
Figure 4 is a result of observing the expression level of STAT3 and STAT5, the major factors of Th17 cells by metformin (metformin) according to an embodiment of the present invention.
Figure 5 is the result of measuring the expression level of IgG when treated with metformin after inducing inflammation with LPS and IL-21 according to an embodiment of the present invention.
Figure 6 is a graph showing the clinical evaluation of disease symptoms over time in the group treated with metformin in graft-versus-host disease animal model in one embodiment of the present invention.
Figure 7 is a result of measuring the expression of IL-17 and IFNr transplantation rejection disease T cell proliferation in the group treated with metformin in one embodiment of the present invention by ELISA.

The present invention relates to a composition for preventing or treating immune diseases, including a metformin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention has an excellent effect of inhibiting differentiation into cytotoxic Th17 cells that produce and secrete inflammatory cytokines, and prevent the function of abnormally activated immune cells and control the inflammatory response. It has an excellent effect of enhancing the activity of immunoregulatory T cells (Treg), and effectively regulates its function as a regulatory T cell (Treg) through its activity of inhibiting the plasticity of regulatory T cells (Treg) into Th17 cells. It was first identified that it can be maintained and strengthened, and therefore, it is characterized by providing a metformin compound of the present invention as a composition for preventing or treating immune diseases.

The present inventors have focused on the metformin compound of the present invention while studying a new compound capable of preventing or treating immune diseases. Metformin has been known to have a therapeutic effect on diabetes. In addition, Korean Patent Publication No. 2009-0005513 discloses that there is an antidiabetic activity in metformin malonate, but there is no mention of the use of metformin in the prevention and treatment of immune diseases.

The immune system controls specific immune responses to autoantigens under normal conditions, and in some cases suppresses immune responses against external antigens. For example, the immune system responds to the fetus and to chronically infected microorganisms. Can be mentioned. These phenomena are known to be induced by clonal deletion, clone anergy and active control by immunoregulatory T cells (Tregs) as a mechanism by which antigen-specific immunotolerance can be induced. Investigation of some patients who accidentally acquired immunotolerance against or experimentally induced animal models showed that all three of these mechanisms are involved in graft-immunity tolerance. It is attracting attention as an important cell involved in controlling almost all immune responses of the living body such as autoimmune, tumoral immunity, infectious immune response as well as immune response.

In particular, immunoregulatory T cells, ie immunoregulatory T lymphocytes (Tregs), whose presence has recently been identified, can be largely divided into natural and adaptive Treg cells, and CD4 + CD25 + T cells, which are natural Tregs, are cells. Has been given immunosuppressive function when it is newly created in the thymus, and is present at a frequency of 5 to 10% of peripheral CD4 + T lymphocytes in normal individuals. Although the immunosuppressive mechanism of this cell has not yet been elucidated yet, it has recently been shown that the expression control gene of Foxp3 plays an important role in the differentiation and activity of this cell. In addition, peripheral T cells can be differentiated into cells exhibiting an immunosuppressive effect upon being stimulated by a self or external antigen under a specific environment, which is called an adaptive or inducible Treg and secretes IL-10 Tr1 that secretes TGF-beta, Th3 and CD8 Ts that secrete TGF-beta, and the like.

In addition, as described in the prior art, T cells are also differentiated into Th17 cells through differentiation in addition to Treg cells. Th17 cells are formed in the presence of TGF-β in common with Treg cells, but IL- for Treg cells. On the other hand, Th17 cells are characterized by differentiating in the presence of IL-6 with TGF-β and secreting IL-17.

Th17 cells, however, are characterized by cytotoxicity, which maximizes the signal of the inflammatory response and accelerates disease progression. Therefore, inhibition of differentiation or activity into Th17 cells is one of the methods for treating immune diseases.

In this regard, the present inventors examined whether the metformin compound has an effect of inhibiting Th17 cell differentiation, which secretes inflammatory cytokines. According to an embodiment of the present invention, Th17 is isolated from a mouse group by separating CD4 T cells. After differentiation into cells, 5 μM of curcumin, tanshinone IIA, and SR11302, which were AP-1 inhibitors, were treated with 5 uM, and curcumin, tanshinone IIA, and SR11302, which were AP-1 inhibitors, respectively, were treated. The differentiation of IL-17 was observed by flow cytometry (FACs) by treating 5uM and 5uM of metformin, a mTOR inhibitor, and as a result, cells expressing IL-17 were curcumin, an Ap-1 inhibitor. , But also inhibited by tanshinone IIA and SR11302, but the expression was significantly inhibited when treated with the Ap-1 inhibitor curcumin, tanshinone IIA, SR11302 and the mTOR inhibitor metformin. It was found (see Fig. 1).

After CD4 T cells were isolated from mice and differentiated into Treg cells, differentiated Treg cells were cultured again under Th17 differentiation conditions. At this time, 5 μM of curcumin, tanshinone IIA, and SR11302, which were AP-1 inhibitors, were treated, and 5 μM of curcumin, tanshinone IIA, and SR11302, which were AP-1 inhibitors, were respectively 5 μM and mTOR inhibitor. The degree of differentiation of regulatory T cells (Tregs) into Th17 cells was examined by flow cytometry (FACs) by treatment with 5 uM of metformin, and as a result, AP-1 inhibitors tanshinone IIA and SR11302. When treated with metformin (metformin), compared to the group treated with AP-1 inhibitor tanshinone ⅡA (Tanshinone ⅡA) and SR11302 alone, it was confirmed that it is effective in overcoming Treg differentiation problem. That is, it has been shown that cells already differentiated into Treg cells are re-differentiated into Th17 cells (see FIG. 2).

In general, Treg cells are known to be used as immunosuppressants or cell therapeutic agents for immunomodulation because they have an activity of modulating an immune response as described above, but in the case of cell therapy using Treg, the plasticity of Treg Due to the cellular characteristics of the T), when the Treg cells are exposed to a pathological environment after being injected into the living body, they may be differentiated into pathogenic cells such as Th17 cells, which makes it difficult to use them as complete cell therapy.

Accordingly, in the present invention, the AP-1 inhibitors curcumin, tanshinone IIA, SR11302 together with metformin as a method for overcoming the problems caused by the plasticity of Treg cells. In this case, the conversion of Treg cells into pathological cells can be suppressed even in a pathological environment, thereby providing a more effective Treg-containing cell therapy.

In addition, when treated with metformin (MDformin) of the present invention and MDSC, a bone marrow-derived inhibitory cell, CD4 T cells from the mouse group of the present invention were examined to determine whether there is activity to modulate Th17 and regulatory T cells (Treg). Isolates were differentiated into Th17 or Treg cells for 3 days and then MDSCs were differentiated in bone marrow of normal mice. Each differentiated cell was further differentiated with MDSC for three more days, and IL-17 and Foxp3 secreted from Th17 cells and Treg were observed by flow cytometry (FACs). As a result, metformin showed Th17 / Treg with MDSC. It was confirmed that the ability to adjust at the same time (see Figure 3).

In order to investigate the role of metformin under conditions where STAT3 is activated, the present inventors treated IL-6 to total cells of the spleen of a normal mouse group and observed the expression of the activated transcription factor by WB. metformin) was able to selectively inhibit the expression of STAT3, a major transcription factor of Th17 cells (see FIG. 4).

In order to confirm whether metformin can inhibit IgG expression, we measured the amount of IgG expressed by treating LPS and IL-21 on total cells of the spleen of the normal mouse group for 3 days, respectively, by ELISA. As a result, metformin not only significantly suppressed the expression of basic IgG, but also significantly inhibited the expression of IgG increased by LPS and IL-21 (see FIG. 5).

The inventors of the present invention in order to determine whether the treatment of metformin can actually be treated with the improvement and prevention of graft-versus-host disease, according to an embodiment of the present invention first graft-versus-host disease After inducing the disease, the clinical scores of metformin and the survival rate of graft-versus-host disease were measured. As a result, mice receiving metformin were compared with the control graft-versus-host disease model. A lower clinical score was observed, metformin increased survival for graft-versus-host disease and, in particular, inhibited invasion of intestinal inflammatory cells (see FIG. 6).

In addition, the inventors performed a mixed lymphocyte reaction (MLR) to investigate whether the compound of the present invention has an activity of inhibiting the transplant rejection problem, which is a problem in transplantation, that is, CD4 + of a normal recipient. T cells were mixed and cultured with splenocytes from which allogeneic derived T cells were removed and splenocytes from which allogeneic derived T cells were removed, and then divided into groups treated with metformin compounds and untreated groups, and then cultured. The levels of cytokines and T cell proliferation in culture were observed.

As a result, metformin suppressed the rejection after transplantation of T cells in a concentration-dependent manner, and expression of inflammatory cytokines IL-17 and IFN-r was significantly suppressed (see FIG. 7).

Therefore, the present inventors have found that the metformin compound can effectively suppress the rejection after transplantation.

Therefore, the metformin compound of the present invention inhibits or reduces the differentiation of undifferentiated T cells into Th17 cells that secrete inflammatory cytokines, or the activity or amplification of regulatory T cells that can normally regulate excessive immune responses. It has an effect of promoting or increasing, and has an excellent effect of effectively maintaining and enhancing the function as a regulatory T cell (Treg) through the activity of inhibiting the plasticity of regulatory T cells (Treg) into Th17 cells, There is a characteristic that can prevent or treat immune diseases caused by abnormal control of the immune response.

In the present invention, the activity means that all the mechanisms of the regulatory T cells (Treg), ie, Treg cells including both natural and adaptive Treg cells, are promoted or promoted in vivo. In other words, the immunomodulatory action such as an immunosuppressive reaction is promoted or promoted so that the immune response in vivo is maintained in a normal state.

In addition, the amplification (expansion) refers to the differentiation and proliferation of undifferentiated T cells into regulatory T cells, 'differentiation' is a phenomenon that the structure or function of each other during the division and growth of cells, that is, the organism Cells or tissues are changed in form or function in order to perform a given task, and 'proliferation' refers to the division of cells and homogeneous ones, which usually increase the number of cells in the body of a multicellular organism. It says to go.

Therefore, the present invention can provide a composition for preventing or treating immune diseases, including a metformin compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the metformin compound may be a compound represented by Formula 1 below.

≪ Formula 1 >

In addition, the compound represented by Formula 1 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 compounds according to the invention can be used that are isolated from nature or prepared by chemical synthesis known in the art.

In the present invention, the immune disease means a disease in which components of the mammalian immune system cause, mediate or otherwise contribute to the pathology of the mammal. In addition, the stimulation or discontinuation of the immune response may include all diseases that have a compensatory effect on the progress of the disease, and the present invention may include diseases caused by an irritable immune response. Examples of such immune disorders include, but are not limited to, autoimmune diseases; Inflammatory disease; And transplant rejection of cells, tissues, or organs.

In addition, one of the most important properties of all normal individuals is that they do not react negatively to the antigenic material that constitutes the self, while non-self antigens are recognized and reacted to remove I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

However, when there is a problem in inducing or maintaining such self-tolerance, an immune response occurs to autoantigens, which causes the attack of one's own tissue. The disease caused by this process is called an autoimmune disease. .

Inflammatory diseases are also referred to as TNF-α (tumor necrosis factor-α) and IL-1 (interleukin- 1), refers to a disease caused by inflammatory substances (inflammatory cytokines) such as IL-6, prostagladin, luecotriene or nitric oxide (NO).

On the other hand, in order to successfully transplant the organ, the recipient's immune rejection response to the transplanted cells and organs must be overcome. The main mediator of transplantation immune rejection is T cell, which is expressed in the graft. Major histocompatibility complex (MHC) is recognized by T cell receptor (T cell receptor) A reaction occurs. Immunosuppressive agents have been used to reduce these transplant immune rejection responses. A common goal of these immunosuppressive agents is to inhibit T cell-mediated immune responses to grafts. Recently, regulatory T cells have been used to suppress immune responses, Methods to treat refractory diseases have been attempted.

In addition, the immunological diseases of the present invention include, but are not limited to, Behcet's disease, multiple myositis / dermatomyositis, autoimmune hemodilution, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, eosinophilic epidermolysis, epididymitis, glomerulonephritis, autoimmune thrombocytopenia, autoimmune thrombocytopenia, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, mucinous anemia, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, , Ulcerative colitis, and the like.

Therefore, the composition according to the present invention can be used as a pharmaceutical composition capable of preventing or treating immune diseases.

The term treatment refers to reversing, alleviating, inhibiting, or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder, unless otherwise stated, As used herein, the term " treatment " refers to an act of treating when the treatment is defined as above. The therapeutic or therapeutic treatment of an immune disorder in a mammal therefore may include one or more of the following:

(1) inhibiting the growth of the immune system, i.e., inhibiting its development,

(2) preventing the spread of immune diseases, i.e., preventing metastasis,

(3) alleviates immune diseases.

(4) preventing the recurrence of immune disorders, and

(5) palliating symptoms of immune disorders

The composition for the prophylactic or therapeutic treatment of immune diseases according to the present invention may comprise a pharmaceutically effective amount of a compound represented by the formula (I) or a salt thereof alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents . The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of an immune disease.

The pharmaceutically effective amount of the metformin compound or salt thereof according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmacologically effective amount may be appropriately changed depending on the severity of the immune disease symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

In addition, "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing or treating immune diseases according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is determined by the route of administration, age, sex, It may be appropriately selected according to various factors such as the weight and the severity of the patient, and the composition for preventing or treating an immune disease according to the present invention is combined with a known compound having the effect of preventing, ameliorating or treating the symptoms of an immune disease. May be administered.

Accordingly, the present invention provides a medicament for the prophylaxis or treatment of immune diseases, which comprises a composition containing a metformin compound or a salt thereof as an active ingredient. The present invention further provides a medformin compound or a salt thereof, As a component of the composition for immunosuppression.

The present invention also provides a method for reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating undifferentiated T cells with a compound represented by Formula 1 of the present invention or a salt thereof. It can be provided, the reduction or inhibition of the differentiation of the undifferentiated T cells into Th17 cells can be achieved through the inhibition of the production of IL-17 cytokines.

In addition, the present invention provides for the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising the step of treating undifferentiated T cells with a compound represented by Formula 1 of the present invention or a pharmaceutically acceptable salt thereof. It is possible to provide a method of reducing or suppressing.

In addition, the present invention, the differentiation and proliferation of Foxp3 expressing Foxp3 in vitro, comprising the step of treating a compound represented by the formula (1) of the present invention or a pharmaceutically acceptable salt thereof to undifferentiated T cells It can provide a method to promote the.

The method for reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro and the method for activating regulatory T cells in the above-described method according to the present invention are as described above. The compound may be directly treated with the medium for culturing cells, or the composition containing the compound of the present invention as an active ingredient may be treated with the culture medium. In addition, at this time, the concentration of the compound of the present invention that can be treated in the culture medium may be treated so that the final concentration is 0.5μM ~ 20μM, in one embodiment of the present invention the cell so that the metformin concentration (metformin) is the final 5μM Was added to the culture medium.

Furthermore, the composition for the prevention or treatment of immune diseases according to the present invention is excellent in the activity or amplification effect of immune regulatory T cells (Treg), and excellent in inhibiting the differentiation of Th17 cells, which are pathogenic cells producing inflammatory cytokines. In addition, there is no toxicity and side effects for the drug can be used safely even when taking a long time, and has a stable characteristic for the body.

Therefore, the present invention can provide a composition for food that can improve or prevent the symptoms of immune diseases containing a metformin compound (metformin) compounds or salts thereof as an active ingredient, the food composition according to the present invention It can be easily utilized as foods that are effective for improvement or prevention, for example, main ingredients, side ingredients, food additives, functional foods or beverages of food.

Herein, the term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be directly eaten through a certain processing step, , Food additives, functional foods and beverages.

Foods to which the food composition according to the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, and the like. In addition, in the present invention, the food may include special nutritive foods (e.g., crude oil, spirits, baby food, etc.), meat products, fish meat products, tofu, mackerel, noodles (Such as soy sauce, soybean paste, kochujang, mixed potatoes), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, milk products (eg, fermented milk, cheese, But are not limited to, pickled foods (various kinds of kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages and the like) and natural seasonings (e.g. The food, beverage or food additive may be prepared by a conventional production method.

In addition, the functional food is a biological defense rhythm control, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body regulatory function related to the living body, specifically, it may be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In addition, in the present invention, the drink refers to a generic term for drinking to quench thirst or enjoy a taste and includes a functional drink. The beverage contains, as an essential ingredient, a composition for improving or preventing the symptoms of the immune disease as an essential ingredient, and there are no special limitations on the other ingredients, and as a further beverage, contains various flavors or natural carbohydrates as additional ingredients. can do.

Furthermore, in addition to the above-described foods, the food containing the food composition for improving or preventing the symptoms of the immunological diseases according to the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, And carbonating agents used in fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, And these components can be used independently or in combination.

In the food containing the composition for food of the present invention, the amount of the composition according to the present invention may comprise from 0.001% to 90% by weight of the total food weight, preferably from 0.1% to 40% by weight In the case of a beverage, it may be included in a ratio of 0.001g to 2g, preferably 0.01g to 0.1g based on 100ml, in the case of long-term intake for health and hygiene purposes or health control purposes It may be less than the above range, and the active ingredient is not limited to the above range because it may be used in an amount above the above range because there is no problem in terms of safety.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

In metformin  by Th17 Cell inhibitory activity

In order to confirm whether the metformin of the present invention has activity to inhibit Th17 cells, CD4 T cells of DBA1 / J normal mice were isolated, and CD3 0.5ug / ml, CD28 1ug / The cells were differentiated into Th17 cells under the conditions of ml, 20ng / ml of IL-6, 2ng / ml of TGF-b, IFNr mAb, and IL-4 mAb. At the same time, 5 μM of curcumin, tanshinone IIA, and SR11302 were treated with AP-1 inhibitors, and 5 μM with curcumin, tanshinone IIA, and SR11302, respectively. Phosphorus metformin 5uM was treated together and the degree of differentiation of IL-17 was observed by flow cytometry (FACs).

As a result, cells expressing IL-17 were also inhibited by curcumin, tanshinone IIA, and SR11302, which are Ap-1 inhibitors, but curcumin and tanshinone IIA, which are Ap-1 inhibitors. When SR11302 and mTOR inhibitor metformin were treated together, the expression was significantly inhibited (see FIG. 1).

< Example  2>

Metformin Treg  Differentiation Inhibition Activity Analysis

The present inventors isolated CD4 T cells from DBA1 / J normal mouse group to determine whether the metformin of the present invention can inhibit the plasticity of differentiation from regulatory T cells (Treg) to pathogenic cells. Treg cells were differentiated under the conditions of CD3 0.5ug / ml, CD28 1ug / ml, TGF-b 10ng / ml, IFNr mAb, IL-4 mAb. After 3 days of differentiation, Th17 cell differentiation conditions were treated, and at the same time, 5 μM of AP-1 inhibitors curcumin, tanshinone IIA, and SR11302 were treated, and AP-1 inhibitors curcumin and tansinone IIA. (Tanshinone IIA) and SR11302 were treated with 5uM and mTOR inhibitor metformin 5uM, respectively, and the degree of differentiation of regulatory T cells (Treg) into Th17 cells was observed by flow cytometry (FACs).

As a result, when tanshinone IIA and SR11302 were treated with metformin, it was confirmed that the expression of Foxp3, an immunoregulatory cell, was significantly increased despite the Th17 cell differentiation condition (see FIG. 2).

It is possible to inhibit the differentiation of regulatory T cells (Treg) when tanshinone IIA and SR11302 are treated with metformin, thereby inhibiting Ap-1 inhibitors (Tanshinone IIA, SR11302) and mTOR. Inhibitors (metformin) can be used as a combination therapy.

< Example  3>

Metformin and  Myeloid-derived suppressor cells MDSC On by Th17 , Regulatory T cells ( Treg Regulated activity analysis

The present inventors, when treated with metformin of the present invention and MDSC, a bone marrow-derived suppressor cell, to determine whether there is activity to modulate Th17 and regulatory T cells (Treg), CD4 of DBA1 / J normal mouse group T cells were isolated. The isolated cells were differentiated for 3 days into Th17 or Treg cells and also differentiated MDSC in bone marrow of normal mice. Each differentiated cell was further differentiated with MDSC for 3 days, and then IL-17 and Foxp3 secreted from Th17 cells and Tregs were observed by flow cytometry (FACs).

As a result, MDSC, a myeloid-derived suppressor cell, decreased the expression of IL-17 under Th17 differentiation conditions, but increased the expression of Foxp3, an immunoregulatory cell under Treg conditions. In particular, when treated with metformin (MDformin) and MDSC together, the expression of IL-17 was significantly suppressed, and Foxp3 gene was significantly increased (see FIG. 3).

Therefore, metformin has the ability to control Th17 / Treg simultaneously with MDSC, and this result is significant in that it can co-regulate pathogenic and immune regulatory cells.

< Example  4>

In metformin  Signal transduction control

To investigate the role of metformin under conditions where STAT3 is active, the present inventors treated IL-6 10ng / ml and IL-2 10ng / ml with total cells of the spleen of DBA1 / J normal mice for 30 minutes. At this time, the expression of the activated transcription factor was observed by WB.

As a result, metformin inhibited both phosphorylated STAT3 705 and 727, and also inhibited the expression of phosphorylated ERK. On the other hand, the inhibitory effect on STAT5 is not so large, it is thought that the metformin of the present invention can selectively inhibit the expression of STAT3, a major transcription factor of Th17 cells (see FIG. 4).

< Example  5>

In metformin  by IgG  Expression Inhibition Activity Assay

In order to confirm whether metformin can inhibit IgG expression, we express LPS 100ng / ml and IL-21 10ng / ml for 3 days in total cells of the spleen of DBA1 / J normal mice. The amount of IgG obtained was measured by ELISA.

As a result, it was confirmed that metformin not only significantly suppressed the expression of basic IgG, but also significantly suppressed the expression of IgG increased by LPS and IL-21 (see FIG. 5).

Therefore, metformin is thought to inhibit the expression of IgG expressed in B cells.

< Example  6>

Metformin Graft-versus-host disease  Analysis of effects on animal models

In order to determine whether metformin can treat these diseases in diseases caused by immunorejection, such as graft-versus-host disease, the present inventors have reported clinical scores and metformin-induced grafts in graft-versus-host disease animal models. Survival of major host disease was measured. To this end, we first developed a Graft-versus-Host Disease (GvHD) model, using BALB / c (H-2k / d) mice as recipients and C57BL / 6 (H-2kb) as donors. Using mice, bone marrow cells isolated from donor mouse C57BL / 6 (H-2k / b) were irradiated with 800cGy total body irradiation (TBI) on the host on the day of bone marrow transplantation. Bone marrow transplantation was performed by injecting ⁵ × 10 ⁶ ) and spleen cells (8 × 10 ⁶ ) into the tail vein. Splenocytes were treated for 2 hours at a concentration of 5 μM metformin.

As a result, as the time passed, the mice receiving metformin showed lower clinical evaluation scores than the control graft-versus-host disease model, and metformin survived the graft-versus-host disease. In particular, it was observed that the inhibition of intestinal inflammatory cells invasion (see FIG. 6).

Therefore, the present inventors found that metformin has an effect of controlling the symptoms of graft-versus-host disease, and it was found that the immune rejection disease can be prevented or treated through this activity.

< Example  7>

Inhibitory activity analysis for rejection after transplantation

To determine if metformin can inhibit rejection after transplantation, 2 × 10 ⁵ normal recipients (Balb / c, responder) CD4 + T cells per well in 96well round bottom plate in vitro And T cell removal splenocytes derived from recipient (homologous) or donor (C57BL / 6, stimulator, allogeneic) irradiated with 2x10 ⁵ radiation were mixed and cultured. At this time, after treatment with alloform reaction without metformin (metformin) or incubated together for 4 days, T cell proliferation in cultured cells and the expression of IL-17 and IFNr in ELISA were investigated in cultured cells.

As a result, metformin inhibited the rejection after transplantation of T cells in a concentration-dependent manner, and significantly inhibited the expression of IL-17 and IFN-r (see FIG. 7).

Therefore, it was found that metformin could effectively suppress the rejection after T cell transplantation.

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 (8)

A composition for preventing or treating immune diseases, including a metformin compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient;
[Formula 1]

. The method of claim 1,
The composition is a composition for the prevention or treatment of immune diseases, characterized in that it further comprises an AP-1 inhibitor. 3. The method of claim 2,
The AP-1 inhibitor is curcumin (curcumin), tanshinone IIA (Tanshinone IIA) and SR11302 selected from the group consisting of one of the composition for the prevention or treatment of immune diseases. The method of claim 3,
The composition inhibits or reduces the activity of Th17, and promotes or increases the activity of Regulatory T cells (Treg), the composition for preventing or treating immune diseases. The method of claim 3,
The composition is a composition for the prevention or treatment of immune diseases, characterized in that it has the activity of inhibiting the differentiation (plasticity) of the regulatory T cells (Treg) into Th17 cells. The method of claim 1,
The composition is a composition for the prevention or treatment of immune diseases, characterized in that it further comprises myeloid derived suppressor cells (MDSC). The method according to claim 6,
The composition inhibits or reduces the activity of Th17, and promotes or increases the activity of Regulatory T cells (Treg), the composition for preventing or treating immune diseases. 8. The method according to any one of claims 1 to 7,
The immune diseases include Rheumatoid Arthritis, Asthma, Dermititis, Psoriasis, Cystic Fibrosis, Post transplantation late and chronic solid organ rejection Multiple Sclerosis, Systemic Lupus erythematosus, Sjogren Syndrome, Hashimoto thyroiditis, Polymyositis, Scleroderma, Addison disease, Vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Dieseses, Autoimmune Diabetes, Diabetic retinopathy, Rhinitis Rhiitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive heart disease pulmonary diseases (COPD), Graves disease, Gastrointestinal allergies, Conjunctivitis, Atherosclerosis, Coronary artery disease, Angina, Cancer metastasis and small artery disease , Graft-versus-host disease (graft-versus-host disease) composition for the prevention or treatment of immune diseases, characterized in that selected from the group consisting of.

Images