KR101652721B1 - Composition for preventing or treating of inflammatory bowel disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an inflammatory bowel disease comprising any one selected from the group consisting of SMP30, an agonist thereof, and an agent for promoting the expression of SMP30, wherein the composition comprises a granulocyte colony stimulating factor and an inflammatory cytokine Expression can be suppressed, and intestinal inflammation can be suppressed.
In addition, the present invention can be effectively used as a biomarker composition capable of diagnosing inflammatory bowel disease. In addition, the screening method for the therapeutic agent for inflammatory bowel disease comprising the step of promoting the expression of SMP30 according to the present invention or the step of selecting a candidate drug that activates SMP30 can be used to specifically select a drug that promotes or activates SMP30 expression, Can be effectively developed.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating inflammatory bowel disease,

The present invention relates to a composition for the prophylaxis or treatment of inflammatory bowel disease including Crohn's disease, ulcerative colitis and inflammatory bowel disease.

SMP30 is a 34-kDa aged-labeled protein that is the first calcium-binding protein found in the liver of rats. The expression of SMP30 gradually increases from postnatal to 12 weeks of age and reaches its peak at 12 weeks of age. Thereafter, the expression level decreases with progressive aging, and apoptosis and necrosis of cells are prevented, It prevents aging.

SMP30 plays an important role in the biosynthesis of vitamin C in the body by acting as a gluconolactonase involved in the synthesis of vitamin C in the body. As a result, vitamin C, which is increased by SMP30, acts as an antioxidant in damaged cells and thus reduces apoptosis and necrosis of cells. It also protects cells from apoptosis and necrosis by maintaining intracellular and intracellular homeostasis of the signaling molecule, calcium ion, which plays an important role in apoptosis and necrosis of cells. In addition, SMP30 itself acts as an antioxidant protein that eliminates free radicals and radicals harmful to the living body, thereby preventing aging of the living body, apoptosis and necrosis of cells.

Recently, a number of studies have been carried out on various functions of SMP30. Among them, SMP30 deficiency is associated with apoptosis induced by cytokines (Non-Patent Document 1) and cell death induced by radioactivity of small intestine (Non-Patent Document 2) .

Inflammatory bowel disease (IBD) generally refers to ulcerative colitis and Crohn's disease. Ulcerative colitis causes inflammatory ulcers in the large intestine mucosa and diarrhea, intestinal bleeding, Symptoms include fever, anorexia and weight loss. About 3% to 13% of patients with ulcerative colitis are reported to progress to colorectal cancer. Although it has been studied by many researchers worldwide for decades, despite its efforts, the etiology, pathophysiology and effective treatment of the disease remain unclear.

The efficacy of SMP30 in the treatment of inflammatory bowel disease has not been reported yet.

1. Am J Pathol. 2002 161 (4): 1273-81. 2. Int J Mol Sci. Jun 2013; 14 (6): 11084-11095.

Accordingly, an object of the present invention is to provide a composition for preventing or treating inflammatory bowel disease, which comprises as an active ingredient any one selected from the group consisting of SMP30, an agonist thereof and an expression promoter thereof.

It is still another object of the present invention to provide a biomarker composition for the diagnosis of inflammatory bowel disease containing SMP30 as an active ingredient.

It is still another object of the present invention to provide a method for screening a therapeutic agent for inflammatory bowel disease comprising the step of selecting a candidate drug that promotes SMP30 expression or activates SMP30.

In order to achieve the above object, the present invention provides a composition for the prevention or treatment of inflammatory bowel disease comprising as an active ingredient any one selected from the group consisting of SMP30, an agonist thereof and an expression promoter thereof.

The present invention also provides a biomarker composition for diagnosing inflammatory bowel disease comprising SMP30 as an active ingredient.

In addition, the present invention provides a method for screening a therapeutic agent for inflammatory bowel disease comprising the step of promoting SMP30 expression or activating SMP30.

The SMP30, its agonist or its expression promoter according to the present invention inhibits the expression of interleukin (IL) -1 and IL-6, which are granulocyte colony stimulating factors and inflammatory cytokines, and the expression of anti-apoptotic protein Bcl-2 To inhibit intestinal inflammation, and thus can be usefully used as a composition for prevention, improvement and treatment of inflammatory bowel disease. In addition, it can be useful as a biomarker composition capable of diagnosing inflammatory bowel disease.

In addition, the screening method for the therapeutic agent for inflammatory bowel disease comprising the step of promoting SMP30 expression or activating SMP30 according to the present invention can be used to specifically select a drug that promotes or activates SMP30 expression, Can be effectively developed.

FIG. 1 is a graph showing mortality (A), weight loss (B), hemorrhage (C) and diarrhea (D) of SMP30 deficient mice (SMP30 ^{Y / -} ) induced by colitis and normal mice.
FIG. 2 is an image comparing colon tissues of normal mice with SMP30 deficient mice (SMP30 ^{Y / -} ) induced with colitis.
FIG. 3 is a graph showing the expression of inflammatory cytokines of SMP30 deficient mice (SMP30 ^{Y / -} ) induced by colitis and normal mice.
FIG. 4 is a graph showing the expression of colitis-induced SMP30 deficient mice (SMP30 ^{Y / -} ) and normal mouse anti-apoptotic protein Bcl-2.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention found that when SMP30 deficient mice are infected with colitis, the expression of granulocyte colony stimulating factor and inflammatory cytokines such as interleukin (IL) -1 and IL-6 is increased, (B-cell lymphoma-2, Bcl-2), which is an apoptosis-suppressing protein, is found in the intestinal inflammation, and thus the prevention, improvement and treatment of inflammatory bowel disease Confirming the function of the SMP30, thereby completing the present invention.

Accordingly, the present invention provides a composition for the prevention or treatment of inflammatory bowel disease comprising as an active ingredient any one selected from the group consisting of SMP30, an agonist thereof and an expression promoter thereof.

The inflammatory bowel disease is any one or more selected from the group consisting of Crohn's disease, ulcerative colitis, and inflammatory colitis, but is not limited thereto.

The composition is provided as a pharmaceutical composition or a health food composition.

In one embodiment of the present invention, a pharmaceutical composition for preventing or treating inflammatory bowel disease, which contains, as an active ingredient, any one selected from the group consisting of SMP30, an agonist thereof and an expression promoter thereof, May be contained in an amount of 0.01 to 90 parts by weight, 0.1 to 90 parts by weight, 1 to 90 parts by weight, or 10 to 90 parts by weight, based on the total weight of the composition, The condition of the patient, the type of disease, and the degree of progression.

The pharmaceutical composition may be selected from the group consisting of carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, lubricants, flavors, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, The above-mentioned adjuvant may be further included.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The amount of SMP30, an agonist thereof and an expression promoting agent thereof, which are effective ingredients of the pharmaceutical composition according to the present invention, may vary depending on the age, sex, weight and disease of the patient, but is preferably 0.001 to 100 mg / kg, / kg can be administered once to several times per day.

In addition, the dose of SMP30, its agonist and its expression promoter according to the present invention may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intratracheal, intrauterine or intracerebroventricular injections.

In another embodiment of the present invention, there can be provided a food composition for preventing or ameliorating inflammatory bowel disease, which contains, as an active ingredient, any one selected from the group consisting of SMP30, an agonist thereof and an expression promoter thereof.

The food composition may be provided in the form of granules, tablets, capsules, syrups or beverages and may be used together with other food or food additives other than the active ingredient SMP30, its agonist and its expression promoter, Can be used. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of SMP30, its agonist and its expression promoter contained in the above food composition can be used in accordance with the effective dose of the above pharmaceutical composition, but it can be used for health and hygiene purposes or for long-term intake It 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 exceeding the above range.

There is no particular limitation on the kind of the food composition. Examples of the food composition include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

The present invention also provides a biomarker composition for diagnosing inflammatory bowel disease comprising SMP30 as an active ingredient.

The present invention also provides a method for screening a therapeutic agent for inflammatory bowel disease comprising the step of selecting a candidate drug that promotes SMP30 expression or activates SMP30.

In one embodiment of the present invention, the screening method comprises the steps of treating a candidate drug with a sample suspected of having inflammatory bowel disease; And analyzing the expression or activity of SMP30 in the sample treated with the candidate drug.

Expression of the SMP30 can be confirmed through protein confirmation experiments such as Western blotting, immunoprecipitation (IP), enzyme-linked immunoabsorbent assays (ELISA), and immunohistochemistry (IHC) The activity of SMP30 can be ascertained through measurement of the absorbance change of p-nitrophenol by free acid generated by SMP30 activity using lactone as a substrate, but not limited thereto .

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> induced colitis SMP30  Deficient mice ( SMP30 ^{Y / -} Clinical Symptoms of Colitis in Korea

In order to induce acute colitis, SMP30 deficient mice (SMP30 ^{Y / -} ) and male mice (C57BL / 6 model) of 8 to 11 weeks of age were treated with 3% dextran sodium sulfate (DSS, MP Biomedicals, Irvine, Calif.). The clinical symptoms of colitis such as rectal bleeding and diarrhea were observed daily during the experiment and the severity of symptoms was assessed with a grade of 0 to 4 and the survival rate was analyzed using a log-rank test.

As a result, it was confirmed that the mortality due to colitis of SMP30 deficient mice (SMP30 ^{Y / -} ) was higher than that of normal mice as shown in A of FIG. Also, like B, SMP30 deficient mice (SMP30 ^{Y / -} ) lost weight more severely than normal mice (SMP30 ^{Y / +} ). In addition, like C and D, SMP30 deficient mice (SMP30 ^{Y / -} ) showed clinical symptoms of colitis, including rectal bleeding and diarrhea, more severe than normal mice (SMP30 ^{Y / +} ).

< Example  2> Colitis induced SMP30  Deficient mice ( SMP30 ^{Y / -} ) Colon Inflammation Comparison

The activity of the mice of Example 1 significantly decreased until the end of the experimental period of 14 days and euthanized when the body weight loss was 20% or more. The colon was then removed and a portion of the transverse colon was treated with 10% formalin solution and fixed with paraffin. The colon tissue fixed with paraffin was then stained with immunostaining using hematoxylin-eosin.

As a result, as shown in Fig. 2, in the colon tissues of SMP30 deficient mice (SMP30 ^{Y / -} ) suffering from colitis, more severe tissue damage, cryptic architecture and infiltration of inflammatory cells than normal mice (SMP30 ^{Y /} Were observed.

< Example  3> Colitis induced SMP30  Deficient mice ( SMP30 ^{Y / -} ) Inflammatory Cytokine pro-inflammatory cytokine ) Expression observation

In order to observe the expression of G-CSF and interleukin-1α and interleukin-6, a sandwich enzyme reaction was performed with a multiple enzyme-linked immunosorbent assay array (Multi-analyte ELISA Array, MEM-004A, SABiosciences Oiagen, Inc., Valencia, CA) Immunosorption was performed. Multiple enzyme-linked immunosorbent assays First, the three colon tissues of each group of mice were dissolved in a protein lysis solution, and each lysate was incubated with capture antibody for 2 hours at room temperature. After incubation, the cells were washed three times, and then the sample was incubated with the antibody for detecting biotin at room temperature for 1 hour. After washing three times after incubation, the sample was incubated with Avidin-horseradish peroxidase conjugate for 30 minutes in the dark. After adding the stop solution, the absorbance was measured at a wavelength of 450 nm using a spectrophotometer.

Samples were prepared by the sandwich enzyme-linked immunosorbing method and IL-6 (IL-6) was prepared by using a murine IL-6-ELISA kit (DuoSet murine IL-6 ELISA Kit, DY406, R & D Systems, Wiesbaden, Germany) ) Were measured.

As a result, inflammatory cytokine interleukins Interleukin (IL) -1α, IL-6 and granulocyte colony stimulating factor (G-CSF) were significantly increased in tissues of colitis-induced SMP30 deficient mice (SMP30 ^{Y /} .

IL-6 expression was significantly increased in both normal (SMP30 ^{Y / +} ) and SMP30 deficient mice (SMP30 ^{Y / -} ), but increased in SMP30 deficient mice (SMP30 ^{Y / -} ).

< Example  4> Colitis induced SMP30  Deficient mice ( SMP30 ^{Y / -} ) &Lt; / RTI &gt; anti-apoptotic protein Bcl -2 expression

In order to observe the expression of Bcl-2, an anti-apoptotic protein, proteins were isolated from normal mouse (SMP30 ^{Y / +} ) and SMP30 deficient mice (SMP30 ^{Y / -} ) and treated with 10% sodium dodecylsulfite polyacrylate Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to the gel and western blotting was performed with Bcl-2 antibody. In order to confirm whether the same amount of protein was compared, western blotting was performed with β-actin antibody.

As a result, the expression of Bcl-2 was significantly decreased in the tissues of SMP30 deficient mice (SMP30 ^{Y / -} ) compared with normal mouse (SMP30 ^{Y / +} ) tissues as shown in Fig.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (1)

SMP30 deficient mice were treated with water containing dextran sodium sulfate to induce acute colitis, thereby increasing the expression of granulocyte colony stimulating factors and inflammatory cytokines such as interleukin (IL) -1 and IL-6, and the anti-apoptotic protein Bcl -2 expression and induce intestinal inflammation more than normal mice.

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