KR102050265B1 - Composition for treating or preventing of arthritis, fibrosis, colitis or transplant rejection comprising Lactobacillus Acidophilus - Google Patents

Abstract

The present invention relates to at least one prophylactic or therapeutic composition selected from the group consisting of arthritis, fibrosis, colitis and transplant rejection comprising Lactobacillus asidophilus as an active ingredient and various applications thereof. The composition of the present invention, when used alone, Lactobacillus asidophilus, effectively inhibits inflammatory cytokines, has Th17 / Treg control ability and inhibits oxidative stress and apoptosis, thereby preventing arthritis, fibrosis, colitis or transplant rejection. The therapeutic effect is excellent. In addition, when used in combination with the calcineurin inhibitor FK506 in the Lactobacillus asidophilus, T cells are regulated to suppress immune rejection in alloresponse, thereby preventing graft rejection and graft-versus-host disease (GVHD, The treatment for graft-versus-host disease is effective and can be useful for related industries.

Description

Composition for treating or preventing of arthritis, fibrosis, colitis or transplant rejection comprising Lactobacillus Acidophilus}

The present invention relates to at least one prophylactic or therapeutic composition selected from the group consisting of arthritis, fibrosis, colitis and transplant rejection comprising Lactobacillus asidophilus as an active ingredient and various applications thereof.

Although diseases due to immune hypersensitivity reactions are increasing all over the world, the root cause for the occurrence of these diseases is insufficient. Currently, a method for treating a disease caused by an excessive immune response is to alleviate or reduce various symptoms caused by the disease by administering an immunosuppressant alone or in combination. Immunosuppressants refer to a variety of substances used to reduce or block the host's ability to produce antibodies (a humoral immune response) or a cellular immune response to the action of an antigen. Such immunosuppressants may be useful for autoimmune diseases such as lupus, rheumatoid arthritis, and skin hypersensitivity reactions such as atopy and allergy as well as organ transplantation. Good immunosuppressants should be able to control the imbalance of the immune response, ensure the safety of the human body, and reduce the incidence of disease recurrence during long-term treatment (Wollenberg 2008).

Meanwhile, T cells are one of a group of cells that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the thymus of the human body and undergo a series of differentiation processes to differentiate into T cells with unique characteristics.T cells, which have completed differentiation, are largely divided into type 1 helper cells (Th1) and type 2 according to their function. It is divided into helper cells (Th2). Among them, the main function of Th1 cells is involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations are balanced with each other to prevent overactivation of each other.

Therefore, most of the immune diseases may be due to an imbalance between these two immune cells. For example, abnormally increased activity of Th1 cells may cause autoimmune diseases, and abnormal activity of Th2 cells may increase. It is known that immune diseases occur due to hypersensitivity reactions.

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

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

Unlike Treg cells, Th17 cells have been found to be involved in the forefront of inflammatory reactions seen in immune diseases, maximizing the signal of inflammatory responses and accelerating disease progression. Therefore, in the case of autoimmune diseases that are not controlled by Treg cells among autoimmune diseases, the development of therapeutic agents for autoimmune diseases and transplant rejection diseases that target the inhibition of Th17 cell activity has been highlighted.

Therefore, there is a need to develop stable and effective therapeutic agents for autoimmune diseases and transplant rejection diseases.

Haihua Li et al., Lactobacillus acidophilus alleviates the inflammatory response to enterotoxigenic Escherichia coli K88 via inhibition of the NF-κB and p38 mitogen-activated protein kinase signaling pathways in piglets, BMC Microbiol. 2016; 16: 273 (Nov. 10, 2016).

An object of the present invention is one selected from the group consisting of arthritis, fibrosis, colitis, and transplant rejection comprising Lactobacillus acidophilus as an active ingredient. Prophylactic or therapeutic pharmaceutical compositions can be provided.

In addition, it is an object of the present invention can provide at least one preventive or improved health functional food composition selected from the group consisting of arthritis, fibrosis, colitis and transplant rejection comprising Lactobacillus asidophilus as an active ingredient.

In addition, an object of the present invention can provide a pharmaceutical composition for the prevention and treatment of graft rejection response comprising Lactobacillus asidophilus or FK506 (tacrolimus, tacrolimus) as an active ingredient.

In addition, an object of the present invention can provide a health functional food composition for the prevention and improvement of transplant rejection response comprising Lactobacillus asidophilus or FK506 as an active ingredient.

In order to achieve the above object, the present invention is arthritis, fibrosis, colitis (colitis) and transplant rejection (transplant rejection) containing Lactobacillus acidophilus as an active ingredient Provided is one or more prophylactic or therapeutic pharmaceutical compositions selected from the group consisting of:

The present invention also provides at least one preventive or improved health functional food composition selected from the group consisting of arthritis, fibrosis, colitis and transplant rejection comprising Lactobacillus asidophilus as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating a transplant rejection response comprising Lactobacillus asidophilus or FK506 (tacrolimus, tacrolimus) as an active ingredient.

In another aspect, the present invention provides a health functional food composition for the prevention or improvement of graft rejection response comprising Lactobacillus asidophilus or FK506 as an active ingredient.

The composition of the present invention, when used alone, Lactobacillus asidophilus, effectively inhibits inflammatory cytokines, has Th17 / Treg control ability and inhibits oxidative stress and apoptosis, thereby preventing arthritis, fibrosis, colitis or transplant rejection. The therapeutic effect is excellent. In addition, when used in combination with the calcineurin inhibitor FK506 in the Lactobacillus asidophilus, T cells are regulated to suppress immune rejection in alloresponse, thereby preventing graft rejection and graft-versus-host disease (GVHD, The treatment for graft-versus-host disease is effective and can be useful for related industries.

1 is a diagram showing the results confirmed the TH17 / Treg regulating ability according to Lactobacillus acidophilus ( Lactobacillus acidophilus ) alone treatment.
Figure 2 is a diagram showing the effect of rheumatoid arthritis treatment according to Lactobacillus asidophilus alone treatment.
Figure 3 is a measure of the length, mouse weight and DAI (disease activity index) according to Lactobacillus asidophilus alone treatment.
Figure 4 is a diagram confirming the histological score (histological score) following treatment with Lactobacillus asidophilus alone.
5 is a diagram confirming the inhibition of inflammatory cytokine expression by treatment with Lactobacillus asidophilus alone.
Figure 6 is a diagram showing the results confirming the inhibition of IL-6, TNF-α and IL-1β mRNA expression by treatment with Lactobacillus asidophilus alone.
Figure 7 is a diagram confirming the Th17 / Treg regulating ability according to Lactobacillus asidophilus alone treatment.
8 is a diagram confirming the inhibition of oxidative stress and apoptosis according to Lactobacillus asidophilus alone treatment.
9 is a diagram confirming the treatment effect of fibrosis according to Lactobacillus asidophilus alone treatment.
10 is a diagram confirming the treatment effect of osteoarthritis according to Lactobacillus asidophilus alone treatment.
Figure 11 is a diagram confirming the T cell proliferation response by the treatment alone or in combination of Lactobacillus asidophilus and FK506 in human peripheral blood cells.
Fig. 12 shows the T cell proliferation response by Lactobacillus asidophilus and FK506 alone or in combination treatment in mouse cells.
Fig. 13 shows the results of confirming the expression of cytokines by single or combination treatment of Lactobacillus asidophilus and FK506 in human blood cells.
14 is a diagram evaluating Treg / Th17 control ability by Lactobacillus asidophilus and FK506 alone or in combination in liver transplantation patients.
15 is a diagram evaluating Treg related gene activity by Lactobacillus asidophilus and FK506 alone or in combination in liver transplantation patients.
16 is a diagram evaluating cytokine regulation ability by Lactobacillus asidophilus and FK506 alone or in combination in liver transplantation patients.
Figure 17 is a diagram confirming the therapeutic effect of either Lactobacillus asidophilus and FK506 alone or in combination in Acute Graft versus host disease (GVHD) animal model.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, the detailed description can be omitted for techniques well known to those skilled in the art. In addition, in describing the present invention, when it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. Also, the terminology used herein is a term used to properly express a preferred embodiment of the present invention, which may vary depending on a user, an operator's intention, or customs in the field to which the present invention belongs.

Therefore, the definition of the terms should be made based on the contents throughout the specification. Throughout the specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding other components unless specifically stated otherwise.

The present invention is one or more selected from the group consisting of arthritis, fibrosis, colitis, and transplant rejection comprising Lactobacillus acidophilus as an active ingredient Or it provides a pharmaceutical composition for treatment.

As used herein, the term " Lactobacillus Acidophilus " is a kind of Lactobacillus Lactobacillus, a kind of probiotic, a stick-shaped lactobacillus that is acid resistant to acid, and has an intestinal action and anticancer effect. It is known to act as a blood cholesterol lowering, vitamin B group synthesis.

As used herein, the term "transplantation rejection" refers to organ transplant rejection when the transplanted tissue is recognized as non-self by the recipient's immune system to attack and remove the transplanted organ. It is called. The most important factor involved in transplant rejection is the major histocompatibility complex (MHC), and the minor histocompatibility complex is also known to be involved. Rejection reactions involve both cellular and immune responses. In cell-mediated reactions, the recipient's lymphocytes meet the donor MHC of the transplant organ (CD4 T cell-type II MHC molecule, or CD8 T cell-type I MHC molecule). Activated T cells secrete cytokine, increase permeability of blood vessels, and invade monocytes such as macrophage. As a result, microvascular damage, tissue ischemia, graft damage and cell destruction occur.

The transplant rejection reaction is at least one transplant rejection reaction selected from the group consisting of cells, blood, tissues and organs, preferably bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney At least one selected from the group consisting of rejection of grafts and skin grafts, but is not limited thereto.

Transplant rejection reactions include graft-versus-host disease (GVHD), but are not limited to those associated with transplant rejection by treatment with Lactobacillus asidophilus of the present invention alone.

The arthritis includes osteoarthritis, rheumatoid arthritis, spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis, gout, bacterial arthritis, childhood rheumatoid arthritis, lupus, scleroderma, multiple sclerosis, fibromyalgia, multiple myositis, dermatitis, Behcet's disease, lighter syndrome, Lyme arthritis Or at least one member selected from the group consisting of adherent arthritis, fifty shoulders, tendon synovitis, elbow head positis, dequabain tendon lubricitis, recurrent rheumatism, rheumatoid polymyalgia, and adult Still's disease. This is not restrictive.

The colitis is inflammatory bowel disease, acute enteritis, bacterial colitis, bacterial dysentery, cholera, typhoid fever, traveler's diarrhea, viral colitis, gastric colitis, amoebic colitis, Crohn's disease, ulcerative colitis, ischemic colitis, Behcet colitis, drug-induced At least one selected from the group consisting of colitis, microscopic colitis, collagen colitis, lymphocytic colitis and radiation colitis, but is not limited thereto.

The composition has excellent regulation of Th17 / Treg cells and may inhibit or inhibit one or more inflammatory cytokines selected from the group consisting of IL-17, IL-1β, IL-6 IFN-γ and TNF-α. In addition, it is possible to increase or decrease the expression of IL-10 and Foxp3, and inhibits oxidative stress and apoptosis, the Lactobacillus asidophilus of the present invention is characterized by such excellent control ability.

Lactobacillus asidophilus of the present invention may include a fermentation broth obtained by culturing in a culture medium, concentrated fermentation broth, dried fermentation broth, culture filtrate, concentrated culture filtrate, or dried culture broth, and the strain It may be a containing or culture filtrate to remove the strain after culturing. In addition, the culture is not limited in its formulation, and may be, for example, a liquid, an emulsion, or a solid.

 As used herein, the term "culture" refers to growing microorganisms under appropriately artificially controlled environmental conditions.

The Lactobacillus asidophilus can be grown in a conventional medium, and the medium contains nutrients required by the microorganisms to be cultured, that is, the microorganisms to culture a specific microorganism. It may be added and mixed. The medium may also be referred to as an incubator or a culture medium, and is a concept that includes all natural, synthetic, or selective media. Lactobacillus asidophilus can be cultured according to a conventional culture method.

The medium used for the cultivation should meet the requirements of the particular strain in an appropriate manner while controlling the temperature, pH, etc. in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like. Carbon sources that can be used include mixed sugars of glucose and xylose as the main carbon source, and sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, coconut Oils such as oils and fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, suitable precursors to the culture medium may be used. The raw materials described above may be added batchwise, fed-batch or continuous in a suitable manner to the culture in the culture process, but is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

Lactobacillus asidophilus of the present invention can be appropriately adjusted according to the weight, age, sex, health status, diet, administration time, administration method and severity of the disease, for example, 1 to 1000 mg / kg It can be included as a range. Specifically, the Lactobacillus asidophilus is 10 to 1000 mg / kg, 50 to 1000 mg / kg, 100 to 1000 mg / kg, 300 to 1000 mg / kg, 1 to 700 mg / kg, 1 to 500 mg / kg, 1 to 300 mg / kg, 1 to 100 mg / kg or 100 to 500 mg / kg. It may be included in the range of 1 to 1000 mg / kg even when administered to a human.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. The adjuvant may be used without limitation as long as it is known in the art, but may further include, for example, Freund's complete adjuvant or incomplete adjuvant to increase its immunity.

The pharmaceutical composition according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical art. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. .

When formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient in the active ingredient, for example starch, calcium carbonate, sucrose, lactose, gelatin It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The pharmaceutical compositions can be formulated in a variety of oral or parenteral dosage forms.

Formulations for oral administration include, for example, tablets, pills, hard capsules, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These formulations may contain diluents (e.g., lactose, dextrose, water, etc.) in addition to the active ingredients. Cross, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid. Or disintegrant or boiling mixtures such as sodium salts thereof and / or absorbents, colorants, flavors and sweeteners. The formulations may be prepared by conventional mixing, granulating or coating methods.

Representative of parenteral formulations are also injectable preparations, which may include water, Ringer's solution, isotonic saline or suspensions as solvents for injectable preparations. Sterile fixed oils of the injectable preparations may be used as solvents or suspending media and any non-irritating fixed oil may be used for this purpose, including mono-, diglycerides.

In addition, the injectable preparation may use a fatty acid such as oleic acid.

The present invention also provides at least one preventive or improved health functional food composition selected from the group consisting of arthritis, fibrosis, colitis and transplant rejection comprising Lactobacillus asidophilus as an active ingredient.

In addition to containing the active ingredient, the food composition of the present invention may contain various flavors, natural carbohydrates, and the like as additional components, as in general food compositions.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The aforementioned flavoring agents can advantageously be used natural flavoring agents (tautin), stevia extracts (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice creams, alcoholic beverages, vitamin complexes, and health supplements. There is this.

In addition to the active ingredient, the food composition may contain various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain a fruit flesh for producing natural fruit juice and fruit juice beverage and vegetable beverage.

The functional food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., for the purpose of preventing or improving one or more selected from the group consisting of arthritis, fibrosis, colitis and transplant rejection. have. In the present invention, the "health functional food composition" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act. Ingestion is intended to obtain useful effects for health purposes such as nutrient control or physiological action. The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards. Examples of the items listed in the 'Food Additive Revolution' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high color pigment and guar gum; Mixed preparations, such as a sodium L- glutamate preparation, an addition of an alkali, a preservative preparation, and a tar coloring agent, etc. are mentioned. For example, the health functional food in the form of tablets may be granulated in a conventional manner by mixing the active ingredient of the present invention with excipients, binders, disintegrants and other additives, and then compression-molded with a lubricant or the like, or The mixture can be compression molded directly. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary. Hard capsules among the health functional foods in the form of capsules may be prepared by filling a conventional hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and the soft capsule agent may include the active ingredient of the present invention as an additive such as an excipient. It can be prepared by mixing the mixture with a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary. The health functional food in the form of a cyclic form can be prepared by molding a mixture of the active ingredient and the excipient, the binder, the disintegrant, etc. of the present invention by a known method, and can be avoided with sucrose or other epidermis, if necessary, Alternatively, the surface may be coated with a material such as starch or talc. The health functional food in the form of granules may be prepared by granulating a mixture of an excipient, a binder, a disintegrant, and the like of the active ingredient of the present invention in a known manner, and may contain a flavoring agent, a coagulant, etc. as necessary. Can be.

The present invention provides a pharmaceutical composition for preventing or treating a transplant rejection response comprising Lactobacillus asidophilus or FK506 (tacrolimus, tacrolimus) as an active ingredient.

The Lactobacillus acidophyllus or FK506 may be used in combination with a weight ratio of 20: 1 to 100: 1, and synergism by combination treatment when Lactobacillus asidophilus is used in combination with a weight ratio of less than 20 and more than 100 The effect may be ineffective, but is not limited thereto in order to achieve the purpose of preventing and treating transplant rejection.

The term "FK-506" is isolated from Streptomyces tsukubaensis , and structurally different from cyclosporin, but similar in mechanism of action to suppress T-lymphocytes, indicating an immunosuppressive function. Has a potency of 10-100 times that of cyclosporin.

The composition has excellent regulation of Th17 / Treg cells and may inhibit or inhibit one or more inflammatory cytokines selected from the group consisting of IL-17, IL-1β, IL-6 IFN-γ and TNF-α. In addition, the expression of IL-10 and Foxp3 can be increased, and when the Lactobacillus asidophilus of the present invention is treated alone or in combination with FK506, the above regulation is performed in a model in vitro or in vivo where allogeneic reactions are induced. It has excellent features.

Since the pharmaceutical composition includes the above-described pharmaceutical formulation, the content overlapping with the composition of the present invention described above is omitted in order to avoid excessive complexity of the present specification by the description of the overlapping content.

In another aspect, the present invention provides a health functional food composition for the prevention or improvement of graft rejection response comprising Lactobacillus asidophilus or FK506 (tacrolimus, tacrolimus) as an active ingredient.

Since the food composition includes the above-described food formulation, the description overlapping the composition of the present invention is omitted to avoid excessive complexity of the present specification by the description of the overlapping description.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are merely to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Lactobacillus asidophilus ( Lactobacillus acidophilus ) TH17 / alone Check Treg Control

Th17 cells induce inflammatory cytokine IL-17 and Treg cells induce anti-inflammatory cytokine IL-10. Upon treatment with Lactobacillus acidophilus of the present invention alone, TH17 / Treg regulation was confirmed.

Specifically, the Lactobacillus asidophilus used in the present invention was purchased from CTC Bio, and isolated from splenocytes of C57BL / 6 mice, the Lactobacillus asidophilus 1, under anti-CD3 (0.5 ug / ml) stimulation, 10 or 100ug / ml was treated and incubated for 72 hours. The number of Treg cells in the culture of these cells was analyzed by flow cytometry. In addition, the amounts of IL-17 and IL-10 were examined by ELISA method. The results are shown in FIG.

As shown in Figure 1a, it was confirmed that the number of Treg cells increased through the treatment of Lactobacillus asidophilus alone.

 As shown in Figure 1b, when treated with Lactobacillus asidophilus alone, IL-17 was suppressed while IL-10 was confirmed to increase.

Example 2 Effect of Rheumatoid Arthritis Treatment by Lactobacillus asidophilus Treatment

In order to confirm the therapeutic effect of an animal model induced by rheumatoid arthritis by treatment with Lactobacillus asidophilus alone, the following experiment was performed. Specifically, arthritis by injecting type II collagen 100ug / CFA (Complete Freund's Adjuvant) into the DBA1 / J mouse subcutaneously and reinjecting type II collagen 100ug / IFA (incomplete Freund's Adjuvant) subcutaneously 2 weeks after induction. This induced mouse was prepared. Fifteen days after arthritis induction, Lactobacillus asidophilus (400 mg / kg) was orally administered. Then, arthritis evaluation was performed twice for 6 days.

Three weeks after the first inoculation, four observers, who did not know the contents of the experiment, evaluated the severity of the joint inflammation three times a week and observed it until seven weeks. At this time, arthritis evaluation was averaged by dividing the scores of the three legs except the legs to which CII / CFA was administered at the second dose per dividing according to the following scale, divided by three, and then obtained by three observers in each animal model. The average value divided by summing was used. The scores and criteria according to arthritis evaluation are as follows.

-Evaluation standard-

0 points: There is no edema or swelling.

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

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

3 points: moderate swelling and redness from the ankle joint to the ankle bone

4 points: swelling and redness from ankle to leg

The best arthritis index per head is 4, so the best disease index per mouse is 16.

As shown in FIG. 2, the arthritis index and induction were significantly reduced in comparison with control mice following the oral administration of Lactobacillus asidophilus after arthritis induction.

Example 3 Efficacy of Colitis Treated with Lactobacillus Ashidophilus Alone

In order to confirm the therapeutic effect of colitis-induced animal model according to Lactobacillus asidophilus alone treatment, the following experiment was performed.

3-1. Long-length, Mouse Weight, and DAI (Disease Activity Index) Measurement Results of Lactobacillus asidophilus alone

As shown in FIG. 3A, C57BL / 6 mice (male, 8 weeks old, obtained from Orient Bio) were fed with 3% DSS (Dextran sulfate sodium) for 5 days to induce colitis, and after 3 days after lactobacillus Ashdophylus (400 mg / kg) was administered orally daily. Mice were weighed daily during the experiment, and bowel lengths were measured at the expense of mice on day 10 after DSS feeding. Then, colitis symptoms were confirmed by measuring the activity activity index (DAI). The results are shown in FIG.

As shown in Figure 3b, as a result of confirming the weight of the mouse, it can be seen that the weight loss in the Lactobacillus asidophilus alone injection group significantly recovered. In addition, as a result of comparing the intestinal length of the mouse, as shown in Figure 3c, it can be seen that the intestinal length is recovered in the Lactobacillus asidophilus alone injection group as compared to the vehicle injection group. As a result of comparison with the activity activity index (DAI), as shown in FIG. 3D, it can be seen that the DAI is reduced in the Lactobacillus asidophilus alone injection group.

3-2. Confirmation of histological score according to Lactobacillus asidophilus alone treatment

Lactobacillus asidophilus alone (DSS + L. acidophilus ), DSS + Vehicle treated group and the control group (WT) in the colitis animal model of Example 3-1, histological The index was confirmed. The results are shown in FIG.

As shown in FIG. 4, it was confirmed that the histologic index was significantly lower in the Lactobacillus asidophilus-treated group (DSS + L. acidophilus ).

3-3. Inhibition of Inflammatory Cytokine Expression by Lactobacillus asidophilus Treatment

In the colitis animal model of Example 3-1, Lactobacillus asidophilus treatment group (DSS + L. acidophilus ), DSS + Vehicle treatment group and the control group (WT), the intestinal tissue staining and inflammatory cytokine IL- 6, TNF-α and IL-1β expression changes were analyzed. The results are shown in FIG.

As shown in FIGS. 5A and 5B, it was confirmed that IL-6, TNF-α, and IL-1β expression was significantly decreased in the Lactobacillus asidophilus alone injection group compared with the DSS + Vehicle treatment group.

3-4. Inhibition of IL-6, TNF-α and IL-1β mRNA Expression by Lactobacillus asidophilus Treatment

To investigate the effect of Lactobacillus asidophilus on inflammatory cytokine regulation, we applied the HT-29 cell line. Stimulated with LPS (Lipopolysaccharide) (100 ng / ml) and incubated for 48 hours by treating Lactobacillus asidophilus with each concentration (10 ㎍ / ml and 50 ㎍ / ml). IL-6, TNF-α and IL-1β mRNA expression in these cells were analyzed by real-time PCR and the results are shown in FIG. 6.

As shown in Figure 6, it was confirmed that the expression of IL-6, TNF-α and IL-1β mRNA is significantly reduced through Lactobacillus asidophilus alone stimulation.

3-5. Confirmation of Th17 / Treg Regulation by Lactobacillus asidophyllus Treatment

In the colitis animal model of Example 3-1, Lactobacillus asidophilus treatment group (DSS + L. acidophilus ), DSS + Vehicle treatment group and the control group (WT), and after staining the intestinal tissue, Th17 / Treg Expression of IL-17, Foxp3 (forkhead box P3) and IL-10 according to cell regulation was confirmed. The results are shown in FIG.

As shown in FIG. 7, the expression of Foxp3 and IL-10 was significantly increased while the expression of IL-17 was decreased in the Lactobacillus asidophilus alone treatment group compared to the DSS + Vehicle treatment group. Could.

3-6. Inhibition of oxidative stress and apoptosis following Lactobacillus asidophilus treatment alone

Lactobacillus asidophilus alone treatment group (DSS + L.acidophilus ), DSS + Vehicle treatment group and control group (WT) in the colitis animal model of Example 3-1, 8-OHG (oxidative) by securing the intestinal tissue Damage markers and TUNEL (apoptosis) were stained to check the expression level. The results are shown in FIG.

As shown in FIG. 8A, it was confirmed that 8-OHG, which is a major oxidation product and an oxidative stress marker, was significantly inhibited in the Lactobacillus asidophilus alone treatment group, thereby confirming the presence of an oxidative stress inhibitory effect.

In addition, as shown in FIG. 8B, it was confirmed that the amount of TUNNEL cell production was inhibited in the Lactobacillus asidophilus alone-treated group, thereby confirming the superior apoptosis inhibitory effect.

Example 4 Confirmation of Fibrosis Treatment Effect According to Lactobacillus asidophilus Treatment

Lactobacillus asidophilus alone (DSS + L.acidophilus ), DSS + Vehicle treated group and control group (WT) were set up and C57BL / 6 mice (male, 8 weeks old) were fed with DSS for 4 days to induce colitis. . Three days after DSS, Lactobacillus asidophilus (400 mg / kg) was orally administered daily. Ten days after DSS feeding, mice were sacrificed, intestinal tissues secured, Masson's trichrome staining to confirm fibrosis, and α-smooth muscle actin (α-SMA) and Type I collagen (Col-I). The results are shown in FIG.

As shown in FIG. 9a, it was confirmed that fibrosis was inhibited in the Lactobacillus asidophilus alone treatment group.

In addition, as shown in FIGS. 9B and 9C, the number of activated myofibroblasts in the Lactobacillus asidophilus-treated group was significantly reduced compared to the other groups, and the deposition of type I collagen was reduced. Deposition is confirmed to be effective in treating fibrosis.

Example 5 Confirmation of Osteoarthritis Treatment Effect According to Lactobacillus Asidophilus Treatment

To produce an osteoarthritis animal model, a 5-week-old male Wistar rat, 200-250 g, was bred at 12-hour light-dark cycles at a temperature of 21-22 and fed with sterilized water and feed. . Then, osteoarthritis was induced by administering monosodium iodoacetate (MIA, Sigma, ST. Louis, MO) at a dose of 3 mg / 50 μl to the right knee of the rat to induce osteoarthritis. MIA was administered by dissolving in physiological saline. Then, after oral administration of Lactobacillus asidophilus (500 mg / kg), pain was measured, which is an indicator for evaluating the effect on the behavior of osteoarthritis-induced animals, and the femur and tibia of rats were measured. Tibia was collected and analyzed for cartilage destruction by Indian ink staining.

Specifically, the pain was measured using a dynamic plantat aesthsiometer (Ugo Basile, Comerio, Italy), and the method of measuring the pain of a machine was placed on a measuring machine by placing a mesh plate on top of a rat in a acrylic animal frame. After inserting, the needle was inserted into the right foot in which the drug was injected. After stabbing, the machine automatically measured the time it took to release the foot (s, seconds) and how much weight it took off (g). The results are shown in FIG.

As shown in Figure 10a, the behavioral experiments (paw withdrawal latency (s); paw withdrawal threshold (g); and weight on right hind rimb (%)) was confirmed, Lactobacillus acidophyl 5 days after osteoarthritis induction It was confirmed that the degree of pain response was significantly recovered in the Ruth alone treatment group.

As shown in Figure 10b, the degree of cartilage destruction was confirmed that the Lactobacillus asidophilus treated group recovered similarly to normal.

As shown in FIG. 10C, the volume of cartilage confirmed in FIG. 10B was confirmed. As a result, the volume of cartilage was increased in the Lactobacillus asidophilus treated group, confirming that the cartilage damage was alleviated.

Example 6 Confirmation of T Cell Proliferation Response by Lactobacillus Ashidophilus and FK506 alone or in Combination Treatment

7-1. Confirmation of T Cell Proliferation Response in Mouse Cells

In order to confirm the T cell proliferation response by Lactobacillus asidophilus and FK506 alone or in combination in mouse cells, mixed leukocyte reaction (MLR) and Carboxyfluorescein succinimidyl ester (CFSE) staining were performed. As a control group, there was an untreated syngenic group and an untreated allogenic group. In addition, 100 and 200 μg / ml Lactobacillus acidophylls or 1, 5 and 20 μg / ml FK506 were treated with the allogeneic group alone. In addition, 100 or 200 μg / ml of Lactobacillus asidophilus was treated in combination with 1, 5 and 20 μg / ml of FK506. After 4 days of treatment, the cells were cultured according to the 3H-thymidine incorporation method to observe the T cell proliferation reaction and confirmed the alloresponse. The results are shown in FIG.

As shown in FIG. 11A, T cell proliferation was not inhibited in the allogenic group, but inhibited T cell proliferation in the Lactobacillus asidophilus alone treatment group, and Lactobacillus asidophilus and FK506. It was also confirmed that the combination treatment of significantly inhibited the proliferation of allogenic T cells.

In addition, as shown in FIG. 11B, T cell proliferation was not inhibited in the untreated allogenic group, but CD4-positive T cells were inhibited in the combination treatment group of Lactobacillus asidophilus and FK506 than each treatment group alone. It was confirmed.

7-2. Confirmation of T Cell Proliferation in Human Peripheral Blood Cells (PBMC)

Mixed lymphocyte reaction (MLR) and CFSE to identify T cell proliferative responses by Lactobacillus asidophilus and FK506 alone or in combination in human Peripheral blood mononuclear cells (PBMC). Carboxyfluorescein succinimidyl ester was performed. As a control group, there was an untreated syngenic group and an untreated allogenic group. In addition, the allotype group was treated with 100 or 200 μg / ml Lactobacillus asidophilus or 1, 5 or 20 μg / ml of FK506 alone. In addition, each 1, 5 or 20μg / ml FK506 was treated with 200μg / ml of Lactobacillus asidophilus in combination, and T cell proliferation of cells cultured by 3H-thymidine incorporation method was observed to confirm the homologous reaction. . The results are shown in FIG.

As shown in FIG. 12, it was confirmed that T cell proliferation was not inhibited in the untreated allogeneic group, and significantly inhibited T cell proliferation in the Lactobacillus asidophilus treatment group alone. In addition, it was confirmed that the combination treatment of Lactobacillus asidophilus and FK506 significantly inhibited T cell proliferation and specifically inhibited more than the treatment alone.

Example 7 Expression of Cytokines in Human Peripheral Blood Cells (PBMC) by Lactobacillus Asidophyllus and FK506, either alone or in combination

Confirmation of Anti-inflammatory Neuroprotective-Related Cytokines IL-10 and Inflammatory Cytokines IL-17, IFN-γ Expression in Human Peripheral Blood Cells (PBMC), either alone or in combination with Lactobacillus asidophilus and FK506 For the control, an untreated allotype group and an untreated allotype group were placed as controls. Allogeneic groups were treated with 5 and 500 μg / ml Lactobacillus asidophilus or 1, 5 and 20 μg / ml of FK506 alone. In addition, FK506 at 1, 5 and 20 μg / ml each; And 100 μg / ml Lactobacillus asidophilus in combination. After incubation for 3 days after treatment, ELISA According to the method, the inhibitory effect of IL-17, IFN-γ and IL-10 on the cultured cells was confirmed. The results are shown in FIG.

As shown in FIGS. 13A and 13B, it was confirmed that the activities of IL-17 and IFN-γ were inhibited in the Lactobacillus asidophilus and Fk506 combination treatment groups than the Lactobacillus asidophilus and the Fk506 treatment groups alone.

In addition, as shown in FIG. 13C, it was confirmed that IL-10 expression was increased in the Lactobacillus asidophilus alone group and significantly increased its expression in the combination treatment group.

Example 8 Evaluation of Treg / Th17 Control Capacity by In Vitro Liver Transplantation Patients with Lactobacillus asidophilus and FK506

In vitro liver transplant patients were treated with 100 μg / ml Lactobacillus asidophilus and 5 nM FK506 alone. In addition, each 5 nM of FK506 and 100 μg / ml Lactobacillus asidophilus were treated in combination. Thereafter, each group was stimulated with anti-CD4 (0.5 μg / ml) and Treg / Th17 regulation was evaluated. The result is shown in FIG.

As can be seen in FIG. 14, Th17 cell differentiation inhibited the expression of Il-17 and inhibited the expression of Il-17 in the Lactobacillus asidophilus and FK506 combination treatment groups, and increased Treg cell activity to express Il-10. It was confirmed that Treg / Th17 was effectively controlled.

Example 9 Evaluation of Treg Related Gene Activity by In Vitro Liver Transplantation Patients by Lactobacillus Asidophyllus and FK506

In vitro liver transplant patients 1, 5, 20 μg / ml Lactobacillus asidophilus and 1, 5, 20 μg / ml of FK506 were treated alone. In addition, 1, 5 and 20 μg / ml of FK506 and 200 μg / ml of Lactobacillus asidophilus were treated in combination. Afterwards, each group was stimulated with anti-CD3 (0.5 μg / ml) and mRNA expression levels of Foxp3 and IL-10, which are Treg-related genes, were confirmed in each group. The result is shown in FIG.

As can be seen in Figure 15, it was confirmed that the mRNA expression of Foxp3 and IL-10 was high in the Lactobacillus asidophilus and FK506 combination treatment group. In addition, it was confirmed that the Lactobacillus asidophilus alone treatment group had higher mRNA expression than the FK506 alone treatment group.

<Example 10> Evaluation of cytokine regulation by Lactobacillus asidophilus and FK506 alone or in combination in liver transplantation patients

In vitro liver transplantation was treated with 1, 5 nM Lactobacillus asidophilus and 1, 5 nM FK506 alone. In addition, 1, 5 nM FK506 and 200 μg / ml Lactobacillus asidophilus were treated in combination. Thereafter, each group was stimulated with anti-CD3 (0.5 μg / ml), and mRNA levels of IL-10 and IL-10 were confirmed in each group. The result is shown in FIG.

As can be seen in Figure 16, it was confirmed that IL-17 significantly inhibited and increased IL-10 in the Lactobacillus asidophilus and FK506 combination treatment group. In addition, it was confirmed that the Lactobacillus asidophilus alone treatment group was superior in cytokine control ability than the FK506 alone treatment group.

Example 11 Confirmation of Therapeutic Effect by Lactobacillus Asidophyllus and FK506 in a Cute Graft versus Host Disease (GVHD) Animal Model

In order to construct a Graft versus host disease (GVHD) animal model, the recipient mouse Balb / c (H-2k / d) was subjected to systemic irradiation (TBI) 700cGy and the donor mouse C57BL / 6 (H-2k). Hematopoietic stem cells and splenocytes were isolated from the femur and tibia of / b), and hematopoietic stem cells 5x106 and splenocytes 1x107 were injected into the tail vein of the recipient mouse Balb / c (H-2k / d) for bone marrow transplantation. After aGVHD development, each drug was orally administered to analyze disease control efficacy.

0, 4, 7, 11, 14, 18 and 21 days after GVHD development of the animal model, each treated with 400μg / kg Lactobacillus asidophilus or 10μg / kg FK506 alone. In addition, 400 mg / kg of Lactobacillus asidophilus and 10 mg / kg FK506 were co-treated and orally administered, and then the disease level of disease-induced mice was measured. The results are shown in FIG.

As shown in FIG. 17, it was confirmed that the Lactobacillus asidophilus-treated group had lower disease activity than the FK506-only group and the Vehicle group. In addition, it was confirmed that the disease activity in the combination treatment group was lower than the other treatment alone, conclusively, GVHD treatment effect was confirmed.

Claims (18)

Lactobacillus acidophilus ( Lactobacillus Acidophilus ) as an active ingredient comprising a pharmaceutical composition for the prevention or treatment of transplant rejection (transplant rejection). delete delete The method of claim 1,
The transplant rejection reaction is a composition, characterized in that at least one transplant rejection reaction selected from the group consisting of cells, blood, tissues and organs. The method of claim 1,
The transplant rejection reaction is a composition characterized in that at least one selected from the group consisting of bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney transplantation and skin graft rejection. The method of claim 4, wherein
The transplant rejection reaction composition comprising graft-versus-host disease (GVHD). The method of claim 1,
The composition is characterized in that inhibiting or inhibiting one or more inflammatory cytokines selected from the group consisting of IL-17, IL-1β, IL-6, IFN-γ and TNF-α. The method of claim 1,
The composition is characterized in that for increasing the expression of IL-10 and Foxp3. The method of claim 1,
Wherein said composition inhibits or inhibits oxidative stress or apoptosis. A health functional food composition for preventing or improving a transplant rejection reaction comprising Lactobacillus asidophyllus as an active ingredient. A pharmaceutical composition for preventing or treating transplant rejection, comprising Lactobacillus asidophyllus and FK506 (tacrolimus, tacrolimus) as an active ingredient. The method of claim 11,
The Lactobacillus asidophilus and FK506 is characterized in that the composition is administered in combination in a weight ratio of 20: 1 to 100: 1. The method of claim 11,
The transplant rejection reaction is a composition, characterized in that at least one transplant rejection reaction selected from the group consisting of cells, blood, tissues and organs. The method of claim 11,
The transplant rejection reaction is a composition characterized in that at least one selected from the group consisting of bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney transplantation and skin graft rejection. The method of claim 13,
The transplant rejection reaction composition comprising graft-versus-host disease (GVHD). The method of claim 11,
The composition is characterized in that inhibiting or inhibiting one or more inflammatory cytokines selected from the group consisting of IL-17, IL-1β, IL-6, IFN-γ and TNF-α. The method of claim 11,
The composition is characterized in that for increasing the expression of IL-10 and Foxp3. A health functional food composition for preventing or improving transplant rejection, comprising Lactobacillus asidophilus and FK506 as an active ingredient.

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