KR101580389B1 - Composition for preventing or treating immune disease comprising metformin and coenzyme Q10 as active ingredients - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of immune diseases comprising metformin and coenzyme Q10, which are used for preventing or treating immune diseases caused by abnormality of immune response, as an active ingredient. The combination administration of metformin and coenzyme Q10 according to the present invention has an effect of inhibiting the expression of the inflammatory cytokines TNF-a, IL-6, IL-17, IFN-y and / Lt; RTI ID = 0.0 > osteoclast < / RTI > differentiation or inhibit the infiltration of inflammatory cells.
In addition, the composition of the present invention has an effect of improving weight loss caused by inflammatory bowel disease, has an effect of regulating the activity of IgG of pathologic B cells, inhibits the expression of inflammatory cytokines, inhibits the expression of Foxp3 + Treg Activity can be effectively utilized as a composition for preventing or treating various immune diseases such as inflammatory bowel disease, lupus, graft versus host disease, and the like.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating immunological diseases containing metformin and coenzyme Q10 as an active ingredient,

The present invention relates to a pharmaceutical composition for the treatment of immune diseases, which contains both metformin and coenzyme Q10 as active ingredients, as a novel therapeutic agent capable of effectively treating immune diseases caused by abnormality of immune response.

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 various substances that are used to reduce or block the ability of a host to make antibodies (humoral immune response) or the ability to cause a cellular immune response to the action of an antigen. Such immunosuppressants can be used not only in organ transplantation fields but also in autoimmune diseases such as rheumatoid arthritis and rheumatoid arthritis, and skin hypersensitivity reactions such as atopy and allergy. Superior immunosuppressants should be able to control the imbalance of the immune response, ensure human safety, and have a low incidence of disease recurrence during long-term treatment.

There are currently used immunosuppressants such as cyclosporin A and FK506. These compounds are derived from natural materials having complex chemical structures, which are uneconomical due to high costs in terms of raw material supply and demand, and various side effects may be caused due to long-term administration Which is a risk. Therefore, there is a desperate need to develop a new immunosuppressive agent which can induce low toxicity and induce immunity and economical 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 these, Th1 cells are mainly involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations maintain the balance of the immune system through mutual checks to prevent mutual activation 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, autoimmune diseases may occur and the activity of Th2 cells abnormally increases It is known that immune diseases are caused by hypersensitivity reactions.

On the other hand, recent studies on the differentiation of Th1 cells have revealed the existence of a new group of immunoregulatory T cells (Tregs) capable of regulating the activity of Th1 cells, , Treg cells have the property of suppressing the function of abnormally activated immune cells and controlling the inflammatory reaction. Therefore, many studies are being conducted to treat immune diseases through the action of increasing Treg cell activity.

In addition, T17 cells are known to be formed through a process similar to the differentiation of Treg cells in the differentiation process of undifferentiated T cells. In other words, the differentiation of Treg cells and Th17 cells is commonly performed in the presence of TGF-β, whereas IL-6 is not required for Treg cells, while IL-6 is present along with TGF- To differentiate. In addition, differentiated Th17 cells are characterized by secretion of IL-17.

Th17 cells, unlike Treg cells, are involved in the forefront of the inflammatory response seen in immune diseases, thereby maximizing the signal of the inflammatory response and accelerating the progression of the disease. Therefore, in the case of autoimmune diseases that are not controlled by Treg cells among autoimmune diseases, the development of therapeutic agents for autoimmune diseases targeting the inhibition of Th17 cell activation has been greatly emphasized.

Immunosuppressants that block the signal transduction pathway in T cells are the most commonly used therapeutic agents for immune diseases. Such immunosuppressants are toxic, infectious, lymphoid, diabetes, tremor, headache, diarrhea, hypertension, nausea , And a disorder of renal function.

In addition to the method of treating immune diseases through the method of inhibiting the activation of T cells, a method of regulating the amount of cytokine secreted from the immune cells and a method of using the antibody targeting the cytokine secreted from the immune cells have been developed . However, this method has a problem in that it takes much time to actually apply to patients through clinical experiments, and that the method using antibody is too expensive in the process of antibody production.

Therefore, it is urgent to develop a therapeutic agent and a treatment method for a new immune disease which has no side effects and is inexpensive and excellent in therapeutic effect.

Korea Patent Publication No. 2012-0017465 Korean Patent Publication No. 1995-7002117

Accordingly, the present invention has been accomplished by confirming that, when a combination of metformin and coenzyme Q10 is used as an effective treatment for an immune disease, the immune diseases can be effectively treated.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating an immunological disease comprising metformin and coenzyme Q10 as an active ingredient.

Another object of the present invention is to provide a method for treating immune diseases, which comprises administering Metformin and Coenzyme Q10 in combination to an immune disease-inducing individual.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating immune diseases comprising metformin and coenzyme Q10 as an active ingredient.

In one embodiment of the present invention, the composition may comprise 0.1 to 20 mg of metformin and 0.001 to 10 mg of coenzyme Q10.

In one embodiment of the present invention, the composition may reduce osteoclast differentiation or inhibit the infiltration of inflammatory cells.

In one embodiment of the present invention, the composition may reduce or inhibit the expression of inflammatory cytokines TNF-a, IL-6, IL-17, IFN-y and / or IL-lb.

In one embodiment of the present invention, the composition may have the effect of keeping the thickness of the small intestine and the length of the large intestine as normal.

In one embodiment of the present invention, the composition may promote or increase the activity of regulatory T cells (Treg) and / or regulatory B cells (Breg).

In one embodiment of the present invention, the composition can increase the amount of mitochondria reduced by inflammation and inhibit damage to the mitochondrial membrane.

In one embodiment of the present invention, the immunological disease may be selected from the group consisting of autoimmune diseases, inflammatory diseases, and transplantation rejection diseases of cells, tissues or organs.

In one embodiment of the invention, the immune disorder is selected from the group consisting of Behcet's disease, multiple myelitis or skin myositis, autoimmune hemodilution, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Atherosclerosis, neurodegenerative disease, meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes mellitus, eosinophilic epidermolysis epididymitis, glomerulonephritis, Myelodysplasia syndrome and ulcer are associated with a variety of conditions including, but not limited to, acute myelogenous leukemia, Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, arthritis, sarcoidosis, scleroderma, spondyloarthropathies, Sexually transmitted diseases.

In addition, the present invention provides a method for treating an immune disorder comprising administering Metformin and Coenzyme Q10 to a subject suffering from an immune disorder other than a human.

In one embodiment of the present invention, the metformin may be administered to an individual at the same time with 0.1 to 20 mg / kg of coenzyme Q10 and 0.001 to 10 mg / kg of Q10.

In one embodiment of the present invention, the administration can be carried out in the form of a combination of metformin and coenzyme Q10 together, or metformin and coenzyme Q10 can be administered separately at the same time.

Furthermore, the present invention provides a combination preparation for the treatment of immune diseases, comprising 0.1 to 20 mg of metformin and 0.001 to 10 mg of coenzyme Q10.

The combination administration of metformin and coenzyme Q10 according to the present invention has an effect of inhibiting the expression of the inflammatory cytokines TNF-a, IL-6, IL-17, IFN-y and / Lt; RTI ID = 0.0 > osteoclast < / RTI > differentiation or inhibit the infiltration of inflammatory cells.

In addition, the composition of the present invention has an effect of improving weight loss caused by inflammatory bowel disease, has an effect of regulating the activity of IgG of pathologic B cells, inhibits the expression of inflammatory cytokines, inhibits the expression of Foxp3 + Treg Activity can be effectively utilized as a composition for preventing or treating various immune diseases such as inflammatory bowel disease, lupus, graft versus host disease, and the like.

FIG. 1 shows the result of measuring the arthritis index and the degree of onset of arthritis in these experimental groups by treating Enbrel, which is treated with metformin and coenzyme Q10 alone or in combination, and arthritis medicines, in a collagen-induced arthritis-inducing animal model.
FIG. 2 shows results of histological examination of H & E stain after sacrificing the arthritis animal model divided into five experimental groups and then dividing the joint cells.
FIG. 3A shows the result of measuring serum IgG after sacrificing an animal model of arthritis divided into five experimental groups.
FIG. 3B shows the results of measurement of immature B cells, mature B cells, and Breg cells in spleen cells after sacrificing an animal model of arthritis divided into five experimental groups.
FIG. 4 shows the results of measurement of Th1, Th17 and Treg cells in spleen cells after sacrificing an animal model of arthritis divided into five experimental groups.
FIG. 5 shows the results of enzyme-linked immunosorbent assay (ELISA) for the expression of IL-6, IL-1b and TNF-a in spleen cells after sacrificing an animal model of arthritis divided into five experimental groups.
FIG. 6 shows results of osteoclast differentiation experiments in which M-CSF and RANKL were stimulated in mouse bone marrow cells, and metformin and coenzyme Q10 were treated alone or in combination.
FIG. 7 shows the result of measuring the arthritis index and the degree of onset of arthritis in these experimental groups by treating Enbrel treated with metformin and coenzyme Q10 alone or in combination and arthritis treating medicines in SKG arthritis-inducing animal model.
FIG. 8 shows the result of histological examination with H & E staining after sacrificing the SKG arthritis animal model divided into five experimental groups and then removing the articular cells.
FIG. 9 shows the result of measuring serum IgG after sacrificing an SKG arthritis animal model divided into five experimental groups.
FIG. 10 shows the results of measurement of immature B cells and mature B cells in spleen cells after sacrificing an SKG arthritis animal model divided into five experimental groups.
FIG. 11 shows the results of measurement of Th1, Th2, Th17 and Treg cells in spleen cells after sacrificing an SKG arthritis animal model divided into five experimental groups.
FIG. 12A is a graph showing the results of analysis of the transplant rejection disease T-cell proliferative response in the group treated with metformin alone or in combination with metformin and coenzyme Q10.
FIG. 12B shows the results of measurement of expression patterns of IFN-y and IL-17 by flow cytometry and ELISA in the group treated with metformin alone or in combination with metformin and coenzyme Q10.
FIG. 12C shows the results of measurement of regulatory T cell activity by metformin alone or in combination with metformin and coenzyme Q10 by flow cytometry.
FIG. 13 shows the results of observing weight change, DAI score and histological changes of experimental animals when metformin alone or metformin and coenzyme Q10 were administered to the inflammatory bowel disease induced animal model group (IBD mouse model group).
FIG. 14 shows the expression levels of IL-17, IL-6 and TNF-a in normal mouse group not affected by inflammatory bowel disease, IBD mouse group as an inflammatory bowel disease-induced animal model, metformin and coenzyme Q10- .
Fig. 15 shows the results of measurement of the expression amount of VEGF, which is involved in the stimulation of neovascularization and promotion of inflammation, in combination with metformin alone or metformin and coenzyme Q10 in an HT-29 colon cancer cell line by ELISA.
FIG. 16 shows the results of measuring the cytotoxicity of coQ10 alone, metformin alone, coQ10 and metformin in various concentrations.
FIG. 17 shows the results of measuring whether or not the effect of inhibiting the T cell (Th1, Th17) activity, which is an inflammatory disease caused by the combination treatment of metformin and Coenzyme Q10.
Fig. 18 shows the results of analysis by PCR and ELISA for the effect of inhibiting the activity of T cells, an inflammatory disease caused by the combination of metformin and coenzyme Q10.
FIG. 19 shows the results of flow cytometry and PCR analysis to determine whether the effect of metformin and coenzyme Q10 treatment on regulatory T cell activity is enhanced.
FIG. 20 shows the results of ELISA analysis of the effect of inhibiting IgG expression in B cells by treatment with metformin and coenzyme Q10.
FIG. 21 shows the results of confirming the effect of Breg enhancement by treatment with metformin and Coenzyme Q10 using flow cytometry.
FIG. 22A shows the results of confirming the inhibitory effect of inflammatory cytokines IL-6, IL-1b, and TNF-a on the expression of metopamine and coenzyme Q10 in combination.
FIG. 22B shows the results of confirming the effect of suppressing the expression of inflammatory cytokines IL-17 and TNF-a by combination treatment with metformin and coenzyme Q10.
FIG. 23A shows the results of confirming the changes in the membrane potential of mitochondria by treatment with metformin and Coenzyme Q10.
FIG. 23B shows the result of confirming the amount of mitochondria by co-treatment with metformin and coenzyme Q10.
24A is a result of confirming the change in mitochondrial membrane potential by treatment with metformin and coenzyme Q10 in a rheumatoid arthritis model.
FIG. 24B shows the results of confirming the amount of mitochondria by the combination of metformin and coenzyme Q10 in a rheumatoid arthritis model.

The present invention relates to a pharmaceutical composition for preventing or treating immunological diseases comprising metformin and coenzyme Q10 as an active ingredient.

The inventors of the present invention have found that when combined with metformin and coenzyme Q10 (coenzyme Q10), they can effectively treat various immune diseases as compared with the respective groups, The present invention has been completed.

In particular, among these active ingredients contained in the composition of the present invention, metformin is a compound used as a therapeutic agent for diabetes mellitus in the past, but has not been studied to be used as a therapeutic agent for an immunological disease. Coenzyme Q10 is known as an antioxidant, It is not known that it can be used as an effective therapeutic agent for immune diseases.

Even if these drugs are known as different therapeutic agents, when they are used as a combination agent, mixing of different drugs may generate unexpected chemical reactants and cause various side effects and cytotoxicity. For drug treatments, the pharmacological effect and the toxicity evaluation should be verified through experiments.

In this respect, the present invention is directed to a method for treating a variety of immune diseases such as rheumatoid arthritis, inflammatory bowel disease, colon cancer, and mitochondrial dysfunction syndrome without causing toxicity to the cells, And it was proved through experimentation that it was not induced.

According to one embodiment of the present invention, when metformin and coenzyme Q10 are treated together in an arthritis-induced mouse model, it is confirmed that osteoclast differentiation is reduced and inflammatory cell infiltration is effectively inhibited and thus, it is effective for rheumatoid arthritis And it can be used as a composition for preventing or treating graft-versus-host disease by inhibiting the transplantation rejection of T cells and decreasing the expression of IFN-γ and IL-17, which are inflammatory cytokines, and increasing the expression of Fox3 + Treg Respectively.

The present inventors have found that when the metformin compound or a pharmaceutically acceptable salt thereof is co-administered with Coenzyme Q10 (coenzyme Q10), the body weight of the inflammatory bowel disease-inducing animal model is effectively improved and the neovascularization is accelerated in the HT-29 colon cancer cell line And inflammatory bowel disease (IBD) by effectively inhibiting VEGF that is involved in inflammation promotion.

In addition, the present inventors have demonstrated that when combined with metformin and coenzyme Q10 (coenzyme Q10), they exhibit an effect of regulating IgG production and can be used as a composition for preventing or treating lupus.

In addition, when combined with metformin and coenzyme Q10 (coenzyme Q10), the amount of mitochondria reduced during the inflammatory process is increased, and mitochondrial membrane damage is suppressed, thereby preventing or treating mitochondrial-related syndromes And the composition can be utilized as a composition.

Accordingly, it was found that the combination of the metformin compound and coenzyme Q10 (coenzyme Q10) according to the present invention can be used as a composition for preventing or treating various immune diseases, and the following experiment was conducted.

 According to one embodiment of the present invention, the arthritic index and disease severity of the SKG arthritis-induced animal model group and the collagen-induced arthritis animal model group were significantly improved by the single administration of metformin and coenzyme alone, (See Figs. 1 and 7).

According to another embodiment of the present invention, the histological examination of the collagen-induced arthritis animal model group and the SKG arthritis-induced animal model group showed that the disruption of joint and cartilage was improved by the single administration of metformin and coenzyme, The effect was remarkably improved when the combination was administered as compared with the single administration (see FIGS. 2 and 8).

According to another embodiment of the present invention, serologic tests were performed in an animal model of collagen-induced arthritis and a model of SKG arthritis-induced animal model. As a result, the expression of IgG in serum was inhibited by metformin and coenzyme alone, (See Figs. 3a and 9). ≪ tb > < TABLE >

According to one embodiment of the present invention, the expression of T cells in the collagen-induced arthritis animal model group and the SKG arthritis-induced animal model group showed that the activity of Th1 and Th17, which are pathogenic cells, decreased by the administration of metformin and coenzyme alone, Cells were increased, and the effect was remarkably improved as compared with the single administration when they were combined (see FIGS. 4 and 11).

Therefore, the inventors of the present invention have shown that the combined administration of metformin compound and coenzyme Q10 can effectively inhibit arthritis caused by various mechanisms.

In addition, the present inventors have also examined the regulatory effect of metformin and coenzyme Q10 combination treatment on alloresponse in the transplant rejection reaction. As a result, the combination treatment of metformin and coenzyme Q10 resulted in a rejection of transplantation by T cells IFN-y, and IL-17, which are inflammatory cytokines, were significantly reduced and the activity of Foxp3 + Treg cells was increased (see Fig. 12).

Therefore, the inventors of the present invention have found that co-administration of metformin compound and coenzyme Q10 can effectively inhibit graft-versus-host disease.

The present inventors measured weight and DAI in an animal model for inducing inflammatory bowel disease. As a result, weight was decreased by metformin alone, and DAI score for measuring inflammatory growth disease was improved, and coenzyme Q10 was added (Fig. 13). ≪ tb > < TABLE >

According to one embodiment of the present invention, measurement of VEGF involved in the promotion of neovascularization and promotion of inflammation in HT-29 colon cancer cell line showed that the amount of VEGF expression was decreased by administration of metformin alone, and when coenzyme Q10 was further administered It was observed that the effect was remarkably improved as compared with the single administration (see Fig. 15).

Therefore, the inventors of the present invention have found that co-administration of metformin compound and coenzyme Q10 can effectively inhibit inflammatory growth disease.

Unlike other cell organelles, mitochondria are known to have unique gene transcription and translation systems that produce proteins and tRNAs that are essential for the regulation and maintenance of mitochondrial function.

Mitochondrial dysfunction reduces ATP production and increases the production of active oxygen such as superoxide, peroxynitrite, hydroxyl radical, peroxidized water, destroys the homeostasis of calcium, . Lipid peroxidation by active oxygen is known to be an important cause of central nervous system damage in degenerative and ischemic diseases.

Mitochondrial dysfunction is known to play an important role in leading to apoptosis in various cells. In various cells including neurons, a decrease in mitochondrial membrane potential, that is, a sign of mitochondrial dysfunction, causes cell death through nuclear DNA degradation.

In addition, it is known that mitochondrial dysfunction causes the development and aggravation of various diseases related to aging process and aging (important chronic diseases such as cancer and diabetes, functional abnormalities of important organs such as circulatory, respiratory, internal organs, and degenerative neurological diseases) . However, specific studies such as how to induce the disease by what mechanism and how to treat it are insignificant.

Therefore, the present inventors have found that Th17 / IL-17 cytokine induces mutation of mitochondrial function in the mechanism of promoting inflammation of the cell and increasing the pathology, thereby exacerbating immune diseases. At the same time, it was found that treatment with metformin and coenzyme Q10 combination prevented mitochondrial function mutation.

According to one embodiment of the present invention, JC-1 staining was performed to examine changes in mitochondrial membrane potential by coadministration of metformin and coenzyme Q10. As a result, when metformin and coenzyme Q10 were individually treated, the change in mitochondrial membrane potential was increased However, when the combination treatment was carried out, it was found that the change in mitochondrial membrane potential was most increased and red fluorescence was more increased than when each treatment was performed alone (see FIG. 23A).

According to one embodiment of the present invention, the amount of mitochondria treated with the combination of metformin and coenzyme Q10 was examined. As a result, the amount of mitochondria was reduced by IL-17 in the group treated with 10 ng of IL-17, , And that the amount of mitochondria decreased in the group treated with metformin and coenzyme Q10 was significantly higher than that in the group treated with metformin and coenzyme Q10 (See FIG. 23B).

Therefore, the inventors of the present invention have found that the combined administration of metformin compound and coenzyme Q10 can effectively inhibit mitochondrial-related syndrome.

Therefore, the present invention can provide a composition for the prevention or treatment of immune diseases comprising a metformin compound and coenzyme Q10 as an active ingredient, wherein the metformin compound and the coenzyme Q10 compound are each in the form of a salt, preferably a pharmaceutically acceptable salt form . 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 metformin compound and coenzyme Q10 according to the present invention may be isolated from nature or prepared by chemical synthesis methods known in the art.

The pharmaceutical composition for the prevention or treatment of immune diseases according to the present invention may contain only metformin compound and coenzyme Q10 as an active ingredient but may also contain vitamin C, D, retinoid, retinoic acid, EGCG, catechin, tannin, polyphenol, UDCA, pyrimidine, quinolones, quercetin compounds, and the like can be further used.

In the present invention, the immunological disease refers to a disease in which components of the mammalian immune system cause, mediate, or contribute to a pathological condition of a 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, including Lupus; Inflammatory diseases including inflammatory bowel disease; And transplantation rejection diseases of cells, tissues or organs including graft-versus-host disease.

One of the most important properties of all normal individuals is that they do not react harmlessly to the antigenic substances that make up the self whereas the ability to recognize and react to non-self antigens Lt; / RTI > Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

However, when problems arise in inducing or maintaining such self-tolerance, an immune response to self-antigen occurs, thereby attacking the tissue of self. The disease caused by this process is called autoimmune disease .

In addition, inflammatory diseases include TNF-α, IL-1 (interleukin-1), IL-1 (interleukin-1 and IL-2) secreted from immune cells such as macrophages by overexpression of the human immune system due to harmful stimuli, Inflammatory substances such as inflammatory cytokines such as IL-1, 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, mucinemias, malignant anemia, acute myelogenous leukemia, acute myasthenia gravis, psoriasis, 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) Reducing 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 . A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat symptoms of an immune disease.

The pharmaceutically effective amount of the metformin compound and the coenzyme Q10 according to the present invention is 0.1 to 100 mg / day / kg body weight, preferably 0.1 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 one embodiment of the present invention, metformin and coenzyme Q10 were mixed with each other at a weight ratio of 1: 1 in a weight ratio of 1: 1 to a 300-g body weight mouse. Metformin and coenzyme Q10 1 mg and 0.1 mg, respectively, in a weight ratio of 10: 1.

Therefore, the amount of the metformin compound and the coenzyme Q10 (coenzyme Q10) that can be used for preparing the composition for treating an immunological disease provided by the present invention is 0.1 to 20 mg of metformin and 0.001 to 10 mg of coenzyme Q10 , And preferably Metformin: Coenzyme Q10 contained in the composition may be used in a weight ratio of 10: 1 to 1: 1.

If it is contained outside this range, that is, when it is used in an amount smaller than the minimum content range of the two drugs, the therapeutic effect is insufficient, and when the content exceeds the maximum content range, a superior therapeutic effect can not be obtained, . Therefore, when a mixture of Metformin: Coenzyme Q10 at a weight ratio of 10: 1 to 1: 1 is used in the above-described range, it is possible to produce a complex which can maintain its activity for a long time in the most stable form.

Also, the pharmaceutically acceptable hereinabove is physiologically acceptable and refers to a composition which, when administered to a human, does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, or the like. 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 immunological diseases according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the dose of the active ingredient may be appropriately determined depending on the route of administration, The composition of the present invention can be appropriately selected according to various factors such as body weight and the severity of the patient and the composition for preventing or treating immune diseases according to the present invention can be used in combination with known compounds having the effect of preventing, Lt; / RTI >

Therefore, the present invention can provide a medicament for the prophylaxis or treatment of an immunological disease comprising a metformin compound or a salt thereof and a composition containing coenzyme Q10 as an active ingredient, and further, the present invention relates to a metformin compound or a salt thereof, Salt and coenzyme Q10 as an active ingredient.

The present invention also relates to a method for the treatment of Foxp3-expressing Treg cells in vitro, comprising the step of treating the undifferentiated T cells with a compound represented by formula 1 of the present invention or a pharmaceutically acceptable salt thereof and coenzyme Q10 Lt; RTI ID = 0.0 > differentiation and proliferation. ≪ / RTI >

Furthermore, the composition for preventing or treating immunological diseases according to the present invention is excellent in the activity or amplification effect of immunoregulatory T cells (Treg) and is excellent in the effect of inhibiting Th1 cell differentiation which is a pathogenic cell that produces inflammatory cytokine There is no toxicity and side effects to the drug, so it can be used safely even when taken for a long time, and it is stable against the body.

Accordingly, the present invention can provide a food composition capable of improving or preventing the symptoms of an immunological disease containing a metformin compound or a salt thereof and Coenzyme Q10 as an active ingredient, and the composition for food according to the present invention can be used for immunization It can be easily utilized as a food which is effective for improving or preventing the symptom of a disease, for example, as a raw material, an additive, a food additive, a functional food or a drink of food.

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

Foods to which the composition for food according to the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complex, and functional food. 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 refers to a food group imparted with added value to function and express the function of the food by using physical, biochemical, biotechnological techniques, etc., or to control the biological defense rhythm of the food composition, Means a food which has been designed and processed so as to sufficiently express the body's control function with respect 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 beverage is a collective term for drinking thirst or for enjoying a taste, and includes a functional beverage. The beverage is not particularly limited as long as it contains a composition for improving or preventing symptoms of the immunological disease as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient 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 food composition of the present invention, the amount of the composition according to the present invention may be 0.001% by weight to 90% by weight, preferably 0.1% by weight to 40% by weight, And may be contained in a ratio of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml in the case of beverage. However, in the case of long-term intake for health and hygiene purposes or for health control purposes May be less than the above range, and since the active ingredient has no problem in terms of safety, it can be used in an amount of more than the above range, so it is not limited to the above range.

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>

Metformin and  Coenzyme Q10 &Lt; RTI ID = 0.0 &gt; Rheumatism  Arthritis treatment effect

The present inventors produced a collagen-induced arthritis mouse model to examine whether the administration of metformin and Coenzyme Q10 in combination was actually effective in treating rheumatoid arthritis and conducted the following experiment. Each mouse used in the experiment was weighed (body weight) 300 g.

<1-1> Evaluation of arthritis index and degree of onset

To prepare an arthritic mouse model, an animal model of rheumatoid arthritis was prepared by injecting 100 ug of type II collagen into normal DBA1 / J mouse group and mixing with CFA on the tail epidermis (first immunization) The drug was administered. The drug was administered five times per week. The coenzyme Q10 alone was treated with 0.1 mg / mouse (0.1 mg / 300 g), metformin alone was treated with 0.1 mg / mouse (0.1 mg / 300 g), metformin 0.1 mg / (0.1 mg / 300 g) and coenzyme Q10 0.1 mg / mice (0.1 mg / 300 g) were jointly administered to the arthritis mouse model. The administration of the drug was oral feeding. Enbrel, an anti-arthritic drug currently marketed as a positive control, was used and injected 5 times a week at a dose of 100 ug / mouse per dose.

The arthritic index was assessed by 4 observers who did not know the contents of this experiment and evaluated the severity of joint inflammation three times a week for up to 6 weeks. The mean value divided by 3 was used as the score. The scores and criteria for arthritis evaluation are as follows.

-Evaluation standard-

0 point: No edema or swelling.

1 point: mild swelling and redness limited to the foot or ankle joint

2 points: Mild swelling and redness from ankle joint to metatarsal

3 points: Moderate edema and redness from ankle joint to foot

4 points: with edema and redness across the entire ankle

The best arthritis index for a mouse is 4, so the highest disease index for a mouse is 16.

The arthritis index and the degree of onset of the arthritis indices after the treatment of the arthritis after the induction of arthritis were 5.74 in the Enbrel treated group and 4 in the coenzyme Q10 alone group and the metformin alone group respectively , And in the group treated with metformin and coenzyme Q10, the arthritis index was significantly reduced to 1.6. Therefore, the inventors of the present invention found that the combined treatment of metformin and coenzyme Q10 significantly improved the treatment effect of arthritis (see Fig. 1), compared with the treatment with each of them and the treatment with Enbrel.

<1-2> Histological examination

The mice of each experimental group used in the experiment of <1-1> were induced to arthritis and treated with each drug. After 42 days, the mice were euthanized and the hind paws of the mice were fixed with 10% formalin The fixed tissue was washed with water to remove excess amount of fixative reagent and the tissue was dehydrated using graded alcohol. After removing the calcite from the bone, the paraffin, which is not mixed with water, was treated with tissue. The paraffin dissolved in the organic solvent benzene was permeated into the dehydrated tissue and then treated with high temperature (60 ° C) To be completely penetrated. The tissue was then cooled at low temperature, then made into a solid state, and cut to the appropriate size (7 mu m). The tissue sections were then placed on a glue-treated glass slide and the tissue paraffin was removed with xylene organic solvent. Since most of the dye is an aqueous solution, the slides with tissues are treated with a low concentration of alcohol solution in a high concentration of alcohol to perform hydration, primary staining with an aqueous solution of hematoxylin, secondary staining with eosin . At first, they were dyed in a differentiation process in which the degree of staining of an excessively stained specimen was appropriately adjusted with an alcohol solution.

As shown in Fig. 2, as shown in Fig. 2, joints of rheumatoid arthritis mice not treated with any drug had infiltration of many immune cells, pannus formation, cartilage destruction and bone erosion Respectively. On the other hand, in the group treated with metformin and coenzyme Q10 alone, the degree of destruction of joints and cartilage was improved, but in the group administered with metformin and coenzyme Q10, the degree of joint inflammation was remarkably higher than in the group treated with metformin and coenzyme Q10 alone Respectively.

<1-3> Serologic test

Serum was obtained from each mouse, and the following experiment was performed on the mouse experimental groups used in the <1-2> experiment.

To investigate the extent of B cell activation, total IgG and IgG2a antibodies specific for serum total IgG and CII were assayed by enzyme linked immunosorbent assay (ELISA). In general, when arthritis develops, it is known that the total IgG in serum is increased and IgG2a is specifically increased. Therefore, total IgG and antibody specific IgG and IgG2a were measured by sandwich ELISA. After reacting each monoclonal anti-mouse IgG and CII in 96 well plate at room temperature for 1 hour, non-specific binding was blocked with blocking solution (1% BSA / PBST). Mouse contorl serum was serially diluted 1/2-fold and used as a standard. Cell culture supernatant was added and reacted at room temperature for 1 hour. After incubation for 1 hour at room temperature with anti-mouse IgG-HRP and anti-mouse IgG2a-HRP, the cells were washed four times and then developed with TMB system and absorbance was measured at 450 nm.

Serum levels of total IgG, which is an indicator of arthritis disease, were suppressed in the group treated with metformin and coenzyme Q10 alone compared with those without arthritis. And the expression of IgG2a with a total IgG and Th1 response specific to the test compound was also inhibited. However, in the group treated with metformin and coenzyme Q10, it was found that the expression of total IgG and IgG2a having a Th1 response was further inhibited compared to the group treated with metformin and coenzyme Q10 alone (see FIG. 3A) ).

<1-4> B cell expression

In order to analyze the levels of immature B (immature B) and mature B (Mature B) cells in spleen cells after immunoprecipitation, spleen cells were isolated from each mouse group and immunoprecipitated with anti Anti-CD1 FITC and anti-CD1d PE were used to analyze Breg cell levels. These materials were incubated at 4 ° C And then analyzed for the number of cells using FACs caliber.

As a result, the number of immature B cells and Breg cells known to have immunomodulatory ability was significantly higher in metformin and coenzyme Q10-treated groups than in other experimental groups, and the number of Breg cells was more than doubled Respectively. In addition, mature B cells showed a decrease in the number of cells in the combination of metformin and coenzyme Q10 in combination with the other experimental groups (see FIG. 3B).

Therefore, the inventors of the present invention have found that a combination comprising metformin and coenzyme Q10 together can increase the number of immature B cells and Breg cells that have immunomodulating ability and effectively treat arthritis with enhanced immunoregulatory ability Furthermore, it was found that the number of mature B cells is decreased and the number of immature B cells is increased, thereby inhibiting the differentiation and maturation of B cells.

<1-5> Analysis of effect on T cell expression

In <1-4>, the effect of metformin and coenzyme Q10 on B cells was analyzed, and the effect on T cells was also analyzed. In order to analyze the number of Th1, Th17 and Treg cells in each mouse spleen cell, anti-CD4-percp (using Th1, Th17 group) and anti-CD4 -percp and anti-CD25-APC (using Treg) were treated with spleen cells and reacted at 4 ° C for 30 minutes. After permeabilization of the cells, anti-IL-17-PE, anti-IFNr- -Foxp3-PE fluorescent Ab.

As a result of analysis of each cell using the flow cytometry after the reaction, the number of Th1 and Th17 cells, which are pathologic cells, in the group administered with metformin and coenzyme Q10 in comparison with the control group (splenocytes of arthritis-induced mice Fold, whereas Treg cells with immunoregulatory ability increased 1.5-fold (see FIG. 4).

<1-6> Influence on the production of inflammatory cytokines

The present inventors conducted the following experiment to determine whether the combination of metformin and coenzyme Q10 can inhibit inflammatory cytokines that are actually causing diseases and causing diseases.

Sanroque mice were mice in which the production of germinal centers was increased by the increase of TFH cells due to overexpression of ICOS and B cells were overactivated, that is, autologous antibodies Mice with increased production and the autoimmune disease is maintained in a pathological state.

The present inventors treated 0.5 ug / ml of anti-CD3 and 1 ug / ml of anti-CD28 capable of stimulating T cells to the spleen cells obtained from the Sanroque mouse prepared as described above, wherein 0.5 ug / ml of anti-CD3 was 2 Hour pretreatment. The metformin treated group was treated with 0.2 mM of metformin, the coenzyme Q10 treated group was treated with 1 uM of coenzyme Q10, the combination treatment of metformin and coenzyme Q10 was treated with 0.2 mM of metformin and 1 uM of coenzyme Q10 simultaneously, Lt; / RTI &gt;

In addition, as a stimulant capable of stimulating B cells, 100 ng / ml of LPS was treated with spleen cells, and a group treated with metformin and coenzyme Q10 alone or a combination thereof was cultured for 2 days. In each group, 6, IL-1b, and TNF-a were measured by enzyme-linked immunosorbent assay (ELISA).

As a result, as shown in Fig. 5, the production of TNF-a, IL-1b and IL-6, which are inflammatory cytokines, in the splenocytes of mice in which the autoimmune disease was maintained in pathological state was treated with metformin and coenzyme Q10 alone It was found that there was an effect of remarkably suppressing the combination treatment group compared to the control group.

<1-7> Inhibitory effect of osteoclast differentiation

To investigate the effects of metformin and coenzyme Q10 in the osteoclast differentiation process, mouse bone marrow cells were obtained and the bone marrow cells were induced to differentiate in the presence of MCSF (macrophage colony-stimulating factor) and soluble RANKL (Sugatani et al 2003, J. Cell. Biochem., 90, 59-67). Bone marrow cells were prepared from the femur and tibia of 6-week-old DBA / 1J mice and cultured in M-CSF (30 ng / ml) in an 8-chamber chamber slide (3 x 10 ⁵ cells / well; Nalge Nunc International, Naperville, IL) (30 ng / ml) and RANKL (30 ng / ml) were added to the adherent osteoclast precursor cells. After 3 days, the non-adherent cells including lymphocytes were removed, ; Strathmann, Hamburg, Germany) for an additional 4 days to obtain osteoclasts. At this time, the cell culture medium was changed once while M-CSF and RANKL were added. Cells were fixed and TRAP (tartrate-resistant acid phosphatase) was stained using a staining kit (sigma) according to the manufacturer's protocol. Microscopic examination of each chamber (40 magnification) showed that TRAP-positive polynuclear cells containing at least three nuclei were counted as osteoclasts (Sugatani et al., 2003, J. Cell. Biochem., 90, 59-67) .

In order to determine whether the drugs used in the present invention, metformin and coenzyme Q10, induced bone marrow cells differentiated from bone marrow cells, bone marrow cells (1 × 10 ⁵ cells / well) . Then, 10 uM of metformin and coenzyme Q10 were added to the cells and incubated for 48 hours. Cells were cultured for 48 hours after co-administration of 10 uM of metformin and 10 uM of coenzyme Q10.

The surviving cells were then stained with the water-soluble tetrazolium salt WST-8 (Cell Counting Kit-8, Donjindo Laboratories, Kumamoto, Japan) according to the manufacturer's protocol. In addition, the cytotoxicity of the differentiated osteoclasts was determined by differentiating bone marrow cells into osteoclasts as described above, treating metformin and coenzyme Q10 at the concentrations described above in the presence of M-CSF and RANKL, Respectively. The cells were then stained for TRAP and counted for TRAP positive polynuclear cells.

Since osteoclasts are known to play a significant role in bone destruction of joints, the effects of metformin and coenzyme Q10 administration on osteoclast differentiation of bone marrow macrophages were confirmed.

As a result, the formation of TRAP, MMP, and b3 integrin-induced multinuclear osteoclasts induced by M-CSF and RANKL was significantly reduced in osteoclast differentiation factors such as TRAP, MMP, and b3 integrin were all significantly decreased (see FIG. 6). Therefore, the inventors of the present invention have found that a combination preparation containing metformin and coenzyme Q10 together can be useful for the treatment of osteopathy through inhibition of osteoclast differentiation.

<1-8> SKG  Evaluation of arthritis index and incidence of arthritis in animal models

The present inventors prepared a mouse model of SKG arthritis as an animal model of arthritis produced in Example <1-1> and another disease model. SKG mice have a genetic defect of T cell immune system, and T cell-induced chronic autoimmune disease It is known to induce ...

Therefore, the inventors injected Zymosan (2 mg / kg) intraperitoneally into 8-week-old SKG mice lacking the 70 kD zeta-associated protein (Zap-70) To produce SKG arthritis-induced mice. Then, the present inventors treated the arthritis-induced mice with 0.1 mg / mouse (0.1 mg / 300 g) of coenzyme Q10 five times a week, and the other group treated the metformin with 0.1 mg / mice (0.1 mg / , Another group received metformin and coenzyme Q10 in combination, and metformin and coenzyme Q10 were orally administered in an amount of 0.1 mg / mouse (0.1 mg / 300 g), respectively. In addition, Enbrel, a therapeutic agent for arthritis, was used as a positive control, and injection was performed five times a week in an animal model of rheumatoid arthritis.

The arthritis index and incidence of the SKG arthritis mouse model were evaluated as follows. The arthritis index of the Enbrel treated group was 4, the coenzyme Q10 treated group was 5.6, and the metformin treated group was 6.3. The combination of metformin and coenzyme Q10 in combination with metformin and coenzyme Q10 showed that the combination of metformin and coenzyme Q10 (combination) of the present invention has an excellent therapeutic effect on arthritis and thus can be used as a novel arthritis treatment agent capable of replacing enbrel (See FIG. 7).

<1-9> SKG  Histological examination of arthritis mouse model

As shown in Table 1, SKG arthritis-induced animal models prepared by the method of Example <1-8> were subjected to the following experiment on the mice treated with drugs. After 56 days of induction of arthritis, mice were euthanized, and the hind paws of the mice were fixed with 10% formalin. After washing the fixed tissues with water to remove excessive amounts of fixing reagents, the tissues were treated with alcohol and dehydrated using graded alcohol. After removing the calcite from the bone, paraffin mixed with moisture was buried. The paraffin dissolved in benzene, an organic solvent, was impregnated into dehydrated tissues and then treated with pure paraffin in a liquid state at a high temperature (60 ° C) to completely infiltrate. The tissue-containing paraffin was allowed to cool to a solid state at low temperatures and then trimmed to the appropriate size. Joint sections (7 μm) were prepared, tissue was placed on a glue-treated glass slide, and tissue paraffin was removed with an organic solvent such as xylene. Since most of the dye is an aqueous solution, the slides with tissues are treated with a low concentration of alcohol solution in a high concentration of alcohol and hydrated. Primary staining is performed with an aqueous solution of hematoxylin and secondary staining is performed with eosin. Was stained with a differentiation process in which the degree of staining of the excessively stained specimen was suitably adjusted with an alcohol solution.

Histologic examination showed that the joints of SKG arthritis mice were infiltrated by many immune cells and had pannus formation, cartilage destruction and bone erosion. On the other hand, the degree of destruction of joints and cartilage was improved in the group treated with metformin and coenzyme Q10 alone. In the group administered with metformin and coenzyme Q10 according to the present invention, the group treated with metformin and coenzyme Q10 alone was significantly inhibited, Compared with the Enbrel treatment group, and was more than twice as effective as the metopamine and coenzyme Q10 alone treatment groups (see FIG. 8 ).

<1-10> SKG  Serological studies in arthritis animal models

The SKG arthritis-induced group of mice prepared according to the method of Example <1-8> was subjected to euthanasia after the induction of arthritis from the mice treated with the respective drugs as shown in Table 1, and then the mice were euthanized to obtain serum.

An enzyme-linked immunosorbent assay (ELISA) was performed to measure the amount of total IgG in the blood. Generally, when arthritis is induced, it is known that the total IgG is increased and IgG2a is specifically increased. For this purpose, total IgG and antibody specific IgG and IgG2a were measured by sandwich ELISA. After incubation for 1 hour at room temperature with each monoclonal anti-mouse IgG and CII in a 96-well plate, non-specific binding was blocked with the blocking solution (1% BSA / PBST). Mouse contorl serum was serially diluted 1/2-fold and used as a standard. Cell culture supernatant was added and reacted at room temperature for 1 hour. Then, anti-mouse IgG-HRP and anti-mouse IgG2a-HRP were reacted at room temperature for 1 hour, washed 4 times after the reaction, developed with TMB system, and absorbance was measured at 450 nm wavelength.

As a result of the serologic test as described above, the expression of total IgG in the serum, which is an activity index of arthritic disease, was suppressed in the group treated with metformin and coenzyme Q10 alone, and metformin and coenzyme Q10 alone (Fig. 9). In the group treated with metformin and coenzyme Q10, the inhibitory effect of total IgG on the serum was more excellent than that of the group treated with Enbrel (see Fig. 9).

<1-11> SKG  Effect of B cell differentiation and expression on arthritis animal model

After splenocytes were isolated from the mouse models used in the experiments of Examples <1-10>, immature B (immature B) cells and mature B (Mature B) cells in the splenocytes after drug treatment were counted For analysis, anti-CD20 Percp, anti-CD5 FITC and anti-CD1d PE were used to analyze the number of Breg cells. The cells were allowed to react for 30 minutes and analyzed using FACs caliber.

As a result, immature B cells and Breg cells, which are known to have immunomodulatory ability, showed similar numbers of cells in the group treated with Metformin and Coenzyme Q10 alone and in the group treated with Enbrel, while mature B cells showed metformin and coenzyme Q10 Compared to the control group. On the other hand, in the group treated with metformin and coenzyme Q10, the number of immature B cells and Breg cells was increased more than that of the group treated with these drugs alone, and the number of mature B cells was the combination of metformin and coenzyme Q10 (See Fig. 10).

Thus, these results demonstrate that combined administration of metformin and coenzyme Q10 or a combination of metformin and coenzyme Q10 can effectively control the inflammatory immune response of B cells in an animal model of arthritis that develops due to genetic defects of the T cell immune system I could.

<1-12> SKG  Arthritis in animal models Metformin and  Coenzyme Q10  T cell expression by co-administration

In order to analyze the number of Th1 and Th17 cells in the splenocytes of each mouse, the anti-SKG anti-tumor agent was administered to each mouse as shown in Table 1 above, using the SKG arthritis-inducing animal model prepared in Example <1-8> CD4-percp was used. To investigate the number of Treg cells, anti-CD4-percp and anti-CD25-APC were incubated at 4 ° C for 30 minutes. After permeabilization, anti-IL- And Ab anti-Foxp3-PE fluorescence Ab, respectively.

As a result of analysis of each cell number using the effervescence, the combination of metformin and coenzyme Q10 significantly decreased the number of cells of Th1 and Th17, and the number of Treg cells was remarkably increased (See FIG. 11).

< Example  2>

Metformin and  Coenzyme Q10  Therapeutic effect of graft rejection disease by combination administration

<2-1> T cell rejection after transplantation and IFN -γ, IL -17 expression inhibition assay

The present inventors conducted the following experiments to confirm whether the combined administration of metformin and coenzyme Q10 can alleviate symptoms of graft rejection disease and treat them.

First, in vitro (In vitro) in 96well round bottom each per well 2x10 ⁵ normal grantor in the plate CD4 + T cells and 2x10 be examined by ^{the five} radiation woman of (Balb / c, responder) (same type isotype) or donors ( C57BL / 6, stimulator, homozygous) derived T cells were added to the cells, followed by mixed culture. At this time, each test group in which metformin was not treated or metopamine (200 μM, 1 mM) alone, metopamine (200 μM, 1 mM) and coenzyme Q10 (2 μM) were treated for 4 days and the cultured cells In order to confirm the T cell proliferation response, ELISA was performed on the culture medium, and the expression of IL-17 and IFNr was examined by flow cytometry.

As a result, metformin inhibited the post-transplant rejection of T cells in a concentration-dependent manner and significantly inhibited the expression of IL-17 and IFN-r. On the other hand, when combined with metformin and coenzyme Q10, they inhibited rejection of T cells after transplantation and also suppressed the expression of IL-17 and IFN-r. In the combination treatment with metformin alone (Fig. 12 (a) and Fig. 12 (b)).

<2-2> Regulatory T cells ( Treg  Cell) differentiation pattern observation

Furthermore, in order to confirm whether the combination administration of metformin and coenzyme Q10 promotes the activity of regulatory T cells (Tregs) having immunomodulating ability, the present inventors examined the cells used in Example <2-1> Treg cells were analyzed by flow cytometry.

As a result, the combination of metformin and coenzyme Q10 showed a greater increase in the number of Foxp3 + Treg cells compared to metopamine alone. Therefore, these results show that treatment of metformin and coenzyme Q10 together with them alone can treat immune diseases more effectively by promoting immunoregulatory ability of Treg cells (see FIG. 12C).

As observed in Examples <2-1> and <2-2>, the combined treatment of metformin and coenzyme Q10 inhibited T cell alloresponse, decreased the expression of inflammatory cytokines, and regulated T As a result, it was found that transplant rejection such as graft-versus-host disease can be effectively treated by increasing the activity of the cell (Treg).

< Example  3>

Metformin and  Coenzyme Q10  Therapeutic effect of inflammatory bowel disease by combination administration

<3-1> Production of inflammatory bowel disease animal model

In order to confirm whether co-administration of metformin and coenzyme Q10 can effectively treat inflammatory bowel disease, an inflammatory bowel disease-induced animal model was first prepared. C57BL / 6 (H-2kb) mice were used as test animals. To prepare an inflammatory bowel disease-induced animal model, mice were treated with 3.5% dextran sulfate sodium (DSS) It was a model for inducing intestinal disease.

<3-2> Metformin and  Coenzyme Q10  Therapeutic effect of inflammatory bowel disease by combination administration

In order to observe the therapeutic effect of inflammatory bowel disease by the combination of metformin and Coenzyme Q10, the present inventors administered IBD mouse model, which is an inflammatory bowel disease induced animal model prepared by the method of Example <3-1> (1 mg / kg), metformin (1 mg / kg) and coenzyme Q10 (20 mg / kg), and each drug was orally administered once a day.

Weights of the three animal groups were measured daily, and the body weight of the control group decreased rapidly after 5 days. Metformin alone showed a slight decrease compared with the control group. Metformin and coenzyme Q10 Showed remarkable improvement in weight loss and remarkable improvement in weight loss. Thus, it was found that the combination treatment of metformin and coenzyme Q10 is effective in improving symptoms of inflammatory bowel disease (see FIG. 13).

In addition, the DAI score, which can be used to measure inflammatory growth disease, was analyzed. As a result, the disease index was 3.8 in the group of mice with IBD disease control (untreated group), the disease index was 2 in metformin alone group, And coenzyme Q10, the disease index was 0.6, indicating that the therapeutic effect of the co-treatment was very remarkable (see FIG. 13).

In addition, H & E staining was performed directly on the lethal mouse to confirm the histological findings. As a result, compared to the animal model of inflammatory growth disease (IBD), metformin and coenzyme Q10 It was found that the villi remained intact (see Fig. 13).

<3-3> Metformin and  Coenzyme Q10  Analysis of inhibitory effect of inflammatory cytokines upon combination administration

The present inventors have examined the inhibitory effect of inflammatory cytokines by these drugs in order to examine the therapeutic effect of inflammatory bowel disease by combination treatment of metformin and coenzyme Q10 or combination administration.

 For this purpose, animals were killed in three groups of mouse animal models: normal mouse group not infected with inflammatory bowel disease, IBD mouse group as an inflammatory bowel disease-induced animal model, metformin and coenzyme Q10 combination group of the present invention, Mice from each group were harvested and fixed in 10% neutral buffered formalin. Subsequently, paraffin was blasted and the intestinal tissue was sectioned to 5 uM thickness and attached to the slides. Before the staining, xanthrene was passed through the deparaffinization process and ethanol was allowed to flow from high concentration to low concentration.

In order to investigate the expression of IL-17, IL-6 and TNF-a, 10% normal goat serum was applied for 30 minutes to block nonspecific reactions. Anti-mouse IL-17, anti-mouse IL- -mouse TNF-a ab was applied to the tissues and incubated overnight at 4 ° C. They were then reacted with HRP for 2 hours and then observed under a microscope.

IL-17, IL-6 and TNF-a were detected in the animal model group. As a result, IL-17, IL-6 and TNF-a, which are inflammatory cytokines, The amount of gene expression of IL-17, IL-6 and TNF-a was remarkably decreased in the combination of metformin and coenzyme Q10 of the present invention, and the expression level of these inflammatory cytokines And the disease can be treated by greatly reducing the expression (see Fig. 14).

<3-4> In colorectal cancer cell lines Metformin and  Coenzyme Q10  Analysis of inhibitory effect of inflammatory cytokines upon combination administration

In order to confirm the therapeutic effect of combination therapy of drugs in colorectal cancer as well as inflammatory bowel disease, HT-29 colon cancer cell line was treated with 1 mg of metformin alone and 1 mg of metformin and 50 μM of coenzyme Q10 The expression level of VEGF was analyzed in the group.

As a result, as shown in Fig. 15, it was confirmed that the expression of VEGF, which is involved in the stimulation of neovascularization and promotion of inflammation, is more excellent in the suppression effect in the group treated with the combination treatment than the metformin alone treatment. Specifically, the hVEGF expression level in the HT-29 colon cancer cell line, which had not been treated with the drug at all, was about 5,800 pg / ml, whereas in the metformin alone treatment group, the expression level was about 5,200 pg / ml, and metformin plus coenzyme Q10 The amount of hVEGF expression was about 3,950 pg / ml, indicating that VEGF production was significantly reduced.

< Example  4>

in vitro Evaluation of efficacy of pharmacological effect by drug combination treatment

<4-1> Cytotoxicity check

MTT assays were performed to determine if the combination of coenzyme Q10 (coenzyme Q10; coQ10) and metformin caused cytotoxicity in cells.

To this end, first, the spleen cells of the mouse C57bl6 penicillin (100IU / ㎖), streptomycin (100㎍ / ㎖), 5% FBS into a RPMI medium containing (fetal bovine serum), 37 ℃ in 5% CO ₂ conditions Lt; / RTI &gt; For the MTT assay, mouse spleen cells were divided into 96-well plates at a density of 2 × 10 ⁵ cells / well and co-treated with 1 uM, ⁵ uM, and 20 uM coenzyme Q10, The cells were cultured for 72 hours in divided doses of 0.2 mM, 1 mM and 5 mM of metformin, and co-administered with coenzyme Q10 and metformin at various concentrations. After culturing for 72 hours, MTT solution (0.5% 3-4, 5-dimethyl thiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide) was added and incubated for 4 hours. Absorbance was measured at 540 nm using an enzyme-linked immunospecific assay (ELISA).

As a result, it was found that no cytotoxicity was observed in the group treated with coenzyme Q10 alone, the group treated with metformin alone, and the group administered with coenzyme Q10 and metformin (see FIG. 16). Therefore, even when coenzyme Q10 and metformin are treated in combination, they do not cause toxicity to cells, and thus they can be used together as a stable therapeutic agent in the body.

For reference, even though a single drug is used for each disease treatment, if two or more different drugs are treated in combination, the toxicity is not caused to the cells when administered alone depending on the mutual chemical reaction between these drugs It can be said that coenzyme Q10 and metformin treatment do not cause cytotoxicity through actual experiments.

<4-2> Metformin and  Coenzyme Q10  By combination treatment An inflammatory disease  T cell activity inhibitory effect

To examine whether the combination of metformin and coenzyme Q10 treatment inhibits the activity of T cells (Th1, Th17), an inflammatory disease, CD4 + T cells isolated from mouse spleen cells were divided into 1 × 10 5 cells and Th17 To stimulate with the cell differentiation condition, 5X10 &lt; ⁵ &gt; Cells were treated with 0.5 ug / ml of anti-CD3 and 1 ug / ml of anti-CD28, respectively, and metformin and coenzyme Q10 were cultured alone or in combination for 3 days. Then, each cell was obtained. Then, anti-CD4-percp was reacted for 30 minutes at 4 ° C in order to analyze Th1 and Th17 cell numbers and cell activity. After the reaction, the cells were permeabilized, and anti-IL-17-PE, anti-IFNr- &Lt; / RTI &gt; After the reaction, FACs caliber was used to analyze the expression of Th1 cells, CD4 + IFN-r +, and Th17 cells, CD4 + IL-17 +.

In the present invention, the suppression yield is expressed by plotting the expression of Th17 and Th1 cells in Th0 state as 100% and converting the remaining results into a graph.

As a result, the control group and each drug treatment group were compared in the Th0 condition in which the anti-CD3 0.5 ug / ml antibody was pretreated for 2 hours and stimulated with 1 ug / ml of anti-CD28. As a result, metformin and coenzyme Q10- (Fig. 17). These results indicate that Th1 and Th17, which are inflammatory cytokines, are significantly reduced compared with the group treated with Q10.

<4-3> Autoimmune arthritis  main An etiologic person IL -17 and IL -17 related Marker  observe

Splenocytes were obtained from normal mouse spleen cells and inflammatory cell activated disease mice (which treated the inflammatory stimulants as described above), and 0.5 μg / ml of anti CD3 (plate coating 2 h ) + anti-CD28 1 ug / ml and LPS 100 ng / ml, and the group treated with metformin and coenzyme Q10 alone or in combination was cultured for 3 days, and then RNA was isolated from the cells according to a method known in the art PCR was performed using a primer capable of amplifying IL-17, CCL20, RORgt and b-actin. The volume of the reactant for RT-PCR was 25 μL, and the composition of the solution , 2.5 l of 10x reaction buffer, 0.5 mM dNTP and each primer pair having the sequence shown in Table 2 below were used and the amplification was carried out using a dual-bay thermal cycler system instrument.

As a result, it was found that the expression level of IL-17 was significantly reduced in the group treated with the combination of metformin and coenzyme Q10 of the present invention, as compared with the group treated with metformin and coenzyme Q10, , IL-17 expression was not decreased, but the expression of IL-17 was significantly decreased in the treated group.

In addition, the expression of CCL20 used as a Th17 marker and RORrt, a transcription factor of Th17 cells, was significantly higher than that of the group treated with metformin and coenzyme Q10, respectively, in diseased cells. It was found that the treatment with these drugs was effective even in the normal state, but the effect in the disease state was more remarkable and the therapeutic effect could be derived. In particular, when the two treatment groups were used in combination, the RORrt Expression was nearly suppressed to almost 100%.

This means that the co-administration of metformin and coenzyme Q10 of the present invention can effectively inhibit the expression of Th17 cells, and the ELISA results of the supernatant of cells obtained from normal mouse spleen cells and inflammatory cell active mice The expression of IL-17 was significantly reduced in the group treated with the combination of metformin and coenzyme Q10 of the present invention (see Fig. 18) than the group treated with metformin and coenzyme Q10, respectively.

<4-4> Metformin and  Coenzyme Q10  Analysis of regulatory T cell enhancement by combination treatment

The following experiment was carried out to confirm that the combined treatment of metformin and coenzyme Q10 increases the number of regulatory T cells and increases the activity.

In normal and diseased cells used in <4-3>, anti-CD3 0.5 μg / ml (plate coating 2 h) + anti-CD28 1 μg / ml treatment simultaneously treated metformin and coenzyme Q10 alone or in combination for 3 days Each cell line was cultured and each cell was harvested and analyzed for the number of Treg cells. For cell analysis, anti-CD4-percp and anti-CD25 APC were reacted at 4 ° C for 30 minutes, -Foxp3-PE fluorescence Ab, the expression of Treg cells, CD4 + and CD25highFoxp3 + cells were analyzed using a flow cytometer, and then the expression of Th0 Treg cells was determined as 100%, and the remaining results were converted into graphs.

As a result, in the group treated with the combination of metformin and coenzyme Q10 of the present invention, the expression of immunoregulatory Treg cells in the diseased cells was markedly increased, and the expression of Foxp3 was also increased at the mRNA level . On the other hand, the expression of Foxp3, a marker of Treg cells, in the group treated with metformin and coenzyme Q10 alone in the diseased state was decreased rather than the control group (disease-induced drug-untreated cell group).

Therefore, the inventors of the present invention found that, in the case of Treg cell activation, when the metformin and coenzyme Q10 alone were treated in the diseased state, the expression of Foxp3 was reduced compared to the control without these substances, And the expression of Foxp3 was increased (see Fig. 19).

<4-5> Metformin and  Coenzyme Q10  B cell activity by combination treatment and IgG  Analysis of inhibition effect

When co-administered with metformin and coenzyme Q10, the effect of inhibiting IgG expression in B cells was confirmed.

For this purpose, B cells were isolated from the lymph node of immunological disease mice, treated with 100 ng / ml of LPS and induced to express IgG1. Metformin alone or together with metformin and coenzyme Q10 were treated with LPS and IgG1 And IgG3 expression patterns were analyzed.

IgG1 and IgG3 expression levels were analyzed by ELISA after culturing the cells for 3 days after the drug treatment. As a result, the expression level of IgG1 was also decreased when metformin and coenzyme Q10 were treated, respectively. However, Coenzyme Q10 was more effective in combination treatment.

On the other hand, unlike the expression pattern of IgG1, the expression of IgG3 was increased when the metformin and coenzyme Q10 were treated respectively, but the amount of IgG3 expression was significantly decreased when the combination treatment was performed (see FIG. 20).

These results indicate that the combined treatment of metformin and coenzyme Q10 is effective in inhibiting B-cell apoptosis in B-cell autoimmune diseases. In addition, in the treatment of immune diseases caused by B- Can be usefully used as a therapeutic agent.

<4-6> Metformin and  Coenzyme Q10  By combination treatment Breg  Promotion effect

To investigate the activity of regulatory B cells (Breg), splenocytes were isolated from mice. These cells were treated with metformin and coenzyme Q10 alone or with metformin and coenzyme Q10 together with 100 ng / ml of LPS and cultured for 3 days Respectively. After incubation, the cells were washed with FACS buffer for flow cytometry and reacted with anti-mouse B220 APC, anti mouse IgD FITC, anti mouse IgM PE and anti-mouse CD138 Percp ab for 30 min at 4 ° C . After washing with a FACS buffer, the cells were analyzed by flow joins after reading using a flow cytometer.

As a result, Breg cells and Naive B cells having immunomodulating function were significantly increased in the combination treatment with metformin and coenzyme Q10 than in the case of metformin and coenzyme Q10 alone. Plasma B cells involved in antibody production were administered alone (See Fig. 21).

Therefore, the present inventors confirmed that co-administration of metformin and coenzyme Q 10 alone is more effective in controlling immunity through regulation of B cell activity.

<4-7> Metformin and  Coenzyme Q10  Analysis of inhibitory effect of inflammatory cytokine by combination treatment (1)

The inventors of the present invention conducted the following experiment in order to investigate whether treatment with metformin and Coenzyme Q10 in combination inhibits inflammatory cytokines.

First, to activate B cells in mouse spleen cells, 100 ng / ml of LPS, metformin and coenzyme Q10 alone treatment, metformin and coenzyme Q10 treatment were combined and cultured for 3 days. The expression of IL-6, IL-1b, and TNF-a in the supernatant of the cultured medium was confirmed by ELISA analysis.

For the experiment, the capture ab of each of IL-6, IL-1b and TNF-a was placed on a 96-well plate overnight and incubated overnight (blocking) for 2 hours . Standard and samples were reacted at room temperature for 2 hours after the treatment. After 3 times of wash buffer, reaction was carried out at room temperature for 2 hours after detection ab treatment. After incubation for 2 hours with AP, the cells were developed with PNPP and the values were determined by ELISA reader.

As a result, the expression levels of IL-6, IL-1b and TNF-a, which are inflammatory cytokines, decreased slightly when metformin and coenzyme Q10 were treated alone, but when metformin and coenzyme Q10 were co- (See Fig. 22A).

<4-8> Metformin and  Coenzyme Q10  Analysis of inflammatory cytokine inhibitory effect by combination treatment (2)

To activate T cells in mouse dLN cells, anti-CD3 0.5 μg / ml (plate coating 2 h), metformin and coenzyme Q10 alone, and 3 days of incubation for 3 days were used to obtain supernatant The expression of TNF-a and IL-17 was examined by ELISA analysis.

For the experiment, the present inventors coated each TNF-a and IL-17 capture ab on a 96-well plate overnight, overnight and then reacted for 2 hours with blocking buffer. Standard and samples were reacted at room temperature for 2 hours after treatment. After washing three times with wash buffer, they were subjected to detection ab treatment and reacted at room temperature for 2 hours. After incubation for 2 hours with AP, the cells were developed with PNPP and the values were determined by ELISA reader.

As a result, the expression levels of TNF-a and IL-17, which are inflammatory cytokines, were similar to those of the control group treated with metformin and coenzyme Q10 alone, but metformin and coenzyme Q10 were treated by the method of the present invention It was found that the expression levels of TNF-a and IL-17 were remarkably suppressed (see Fig. 22B).

< Example  5>

Metformin and  Coenzyme Q10  Identification of Mitochondrial Damage Controlled by Combined Treatment

<5-1> Metformin and  Coenzyme Q10  Investigation of Potential Changes in Mitochondria by Combined Treatment

The present inventors performed JC-1 staining to examine the changes in the membrane potential of mitochondria by co-administration of metformin and coenzyme Q10.

JC-1 200mM was diluted to 100 nM in DMEM medium at the time of staining with synovial cells (FLS) surrounding the joints, and then added at 10ug / ml and incubated at 37 ° C for 15 minutes. After washing with PBS for 10 minutes after the DMEM medium was changed, the cells were observed under a fluorescence microscope.

The JC-1 staining is an experimental method mainly used for studying the membrane potential change of mitochondria. In the normal cells, JC-1 dye accumulates red fluorescence in mitochondria, whereas in apotosis, (green) fluorescence.

Fluorescence microscopy revealed that mitochondrial membrane potential changes in the control group without any treatment were well maintained and red fluorescence was well expressed. However, mitochondrial damage by Th17 or cytokine IL-17 under IL-17 treatment Green fluorescence was increased due to induction.

However, when methamphetamine and coenzyme Q10 were treated alone, mitochondrial membrane potential changes were increased, but when they were used together, mitochondrial membrane potential was most significantly changed, and red fluorescence was more increased (See FIG. 23A).

<5-2> Metformin and  Coenzyme Q10  Investigation of Amount of Mitochondria by Combined Treatment

Mitotracker / a-tubulin staining was performed to determine whether there was a change in the amount of mitochondria induced by co-administration of metformin and coenzyme Q10.

First, mitotracker was diluted to 100 nM in DMEM medium and added to a synovial cell (FLS) plate surrounding the joints. After that, incubation was carried out at a temperature of 37 캜 for 15 minutes and PBS washing was performed.

 Acetone and methanol were diluted at a ratio of 1: 1 and fixed for 10 minutes, followed by PBS washing for 15 minutes. Blocking with 10% normal goat serum was performed. After 30 minutes, the cells were incubated at 4 ° C overnight. After incubation, the plate was washed with PBS and then stained with DAPI (1: 500).

As a result, it was found that IL-17 treated with 10 ng of IL-17 reduced the amount of mitochondria due to damage by IL-17, but the amount of reduced mitochondria was increased in the group treated with metformin and coenzyme Q10, and metformin and coenzyme Q10 was significantly increased in the group treated with metformin and coenzyme Q10 than in the group treated with metformin and coenzyme Q10 (see FIG. 23B).

<5-3> Rheumatoid arthritis  In the model Metformin and  Coenzyme Q10  Investigation of Potential Changes in Mitochondria by Combined Treatment

The present inventors conducted the following experiment to determine the change in mitochondrial membrane potential by the combination treatment of metformin and coenzyme Q10 in a rheumatoid arthritis model.

Mice were injected with 100 ug of type II collagen into CFA to induce a rheumatoid arthritis animal model. Five times a week coenzyme Q10 alone treatment group was administered 0.1 mg / mouse, metformin alone 0.1 mg / mouse in the treatment group, and metformin plus coenzyme Q10 in combination with metformin and coenzyme Q10 were orally administered in an amount of 0.1 mg / mouse, respectively.

Sputum cells were obtained after sacrificing the rheumatoid arthritis animal model prepared as described above, and the membrane potentials of mitochondria were destroyed by treating H ₂ O ₂ in the cells of each treatment group. Each treatment group was subjected to JC-1 staining. After the culture supernatant was discarded, the cells were inoculated with PBS, and then JC-1 200 mM was diluted to 100 nM and added at 10 ug / ml. Lt; / RTI &gt; for 15 minutes.

After the cell centrifugation, fluorescence microscopy revealed that red fluorescence was almost absent in the rheumatoid arthritis mouse group, and green fluorescence was mostly present. In the group treated with metformin and coenzyme Q10 alone, red fluorescence . In the group treated with the combination of metformin and coenzyme Q10 of the present invention, it was found that red fluorescence significantly increased as compared with the group treated with metformin and coenzyme Q10 alone (see FIG. 24A).

Therefore, the combination treatment of metformin and coenzyme Q10 of the present invention or the treatment of the combination agent containing them clearly showed that the mitochondrial membrane potential was remarkably increased due to the disease induction and the mitochondrial membrane potential change was maintained well.

<5-4> Rheumatoid arthritis  In the model Metformin and  Coenzyme Q10  Investigation of Amount of Mitochondria by Combined Treatment

The present inventors obtained spleen fragments at the sacrifice of the model of rheumatoid arthritis induced by the method of Example <5-3>. The spleen fragments were obtained from the rheumatoid arthritis-induced animal model group, the metformin treated group, the coenzyme Q10 Optimal cutting temperature compound (OCT compound) was embedded in the spleen tissues of the treated group and metformin and coenzyme Q10-treated group. The tissues were rapidly cooled in the liquid nitrogen, Lt; / RTI &gt; The sections were then fixed with acetone and 10% normal goat serum was applied to block nonspecific reactions for 30 minutes. CoxIV assay was performed overnight at 4 ° C with primary antibody CD4 alexa 488 (1: 200), CoxIV (1: 200, rabbit / 2nd anti-rabbit PE 1: 500) diluted in PBS , The next day in PBS solution, and the stained tissue was analyzed by confocal microscopy.

As a result, the amount of mitochondria was similar to that of the rheumatoid arthritis-induced animal model group, which was not treated in the group treated with metformin, so that the amount of mitochondria was not increased by metformin alone treatment. However, the amount of mitochondria was increased in the coenzyme Q10-treated group, and it was found that the amount of mitochondria was significantly increased in the group treated with coenzyme Q10 of metformin of the present invention (see FIG. 24B).

Therefore, it was found that the combination treatment of metformin and coenzyme Q10 of the present invention can effectively treat various diseases caused by a decrease in mitochondrial function.

The present invention has been described with reference to the preferred embodiments. 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 (14)

1. A pharmaceutical composition for the prevention or treatment of immune diseases comprising metformin and coenzyme Q10 as effective ingredients, wherein the metformin: coenzyme Q10 contained in the composition is in a weight ratio of 10: 1 to 1: 1 Wherein said immune disease is cancer, diabetes or a degenerative neurological disease caused by arthritis, graft rejection disease, inflammatory growth disease or dysfunction of mitochondria. The method according to claim 1,
Wherein said composition comprises 0.1 to 20 mg of metformin and 0.001 to 10 mg of coenzyme Q10. The method according to claim 1,
Wherein said composition reduces osteoclast differentiation or inhibits the infiltration of inflammatory cells. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; The method according to claim 1,
Wherein said composition reduces or inhibits the expression of inflammatory cytokines TNF-a, IL-6, IL-17, IFN-y or IL-lb. The method according to claim 1,
Wherein the composition has an effect of maintaining the thickness of the small intestine and the length of the large intestine in a steady state. The method according to claim 1,
Wherein said composition promotes or increases the activity of regulatory T cells (Treg) or regulatory B cells (Breg). The method according to claim 1,
Wherein said composition increases the amount of mitochondria reduced by inflammation and inhibits damage to the mitochondrial membrane. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; delete delete delete A method for treating an immune disorder, comprising administering to a subject suffering from an immune disorder other than a human a combination of metformin and coenzyme Q10, wherein the metformin: coenzyme Q10 is administered in a ratio of 10: 1 to 1: 1 Wherein the immunological disease is cancer, diabetes, or degenerative neurological disease caused by arthritis, graft rejection disease, inflammatory growth disease or a decrease in function of mitochondria. 12. The method of claim 11,
Wherein the metformin is administered to an individual at a dose of 0.1 to 20 mg / kg and the coenzyme Q10 is administered to an individual at a dose of 0.001 to 10 mg / kg. 13. The method of claim 12,
Wherein said administration is administered in the form of a combination of metformin and coenzyme Q10 together, or metformin and coenzyme Q10 are administered separately at the same time. delete

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