KR102460570B1 - anti-atopic composition which comprises mutant saccharomyces cerevisiae - Google Patents

Abstract

The present invention relates to a therapeutic agent for atopic dermatitis, psoriasis, and allergy containing aldehyde dehydrogenase, and aldehyde dehydrogenation promoting enzymes and variants thereof comprising a fusion protein or recombinant protein comprising an action site involved in aldehyde dehydrogenation. It relates to a treatment agent for atopic dermatitis, a treatment for psoriasis, and a treatment for allergies, which are contained as an active ingredient.

Description

Atopic inhibitory composition containing novel mutant yeast {anti-atopic composition which comprises mutant saccharomyces cerevisiae}

The present invention relates to an atopy inhibitor containing a novel mutant yeast. More particularly, the present invention relates to a composition for treating atopy, allergy or psoriasis, containing a novel mutant yeast that reduces IgE.
Further, the present invention relates to a therapeutic agent for atopic dermatitis, a therapeutic agent for psoriasis, and a therapeutic agent for allergy containing novel mutant yeast.

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Atopic dermatitis is a chronic recurrent inflammatory skin disease that causes itching. It is an excessive allergic reaction that can occur without antigen contact and an abnormal inflammatory reaction. Atopic dermatitis is a skin disease that reacts sensitively to food or inhalable substances, and is caused by environmental factors, genetic predisposition, immunological abnormalities, and abnormal skin barrier.

Environmental factors of atopic dermatosis include environmental pollution such as soot, an increase in the use of food additives, the use of carpets, beds, and sofas, and an increase in substances that cause allergies such as house dust mites. It seems that allergic reactions to antigens that are easy to invade due to the deterioration of skin defense function are involved, and environmental factors such as house dust mites and mold are involved as representative antigens.

The genetic influence on atopic dermatitis is confirmed by the fact that there is a family history of atopic dermatitis patients. It is known that a decrease in the skin barrier function occurs due to abnormalities in the filagrin gene in a significant number of atopic patients. As a cause of atopy, overexpression of receptors due to genetic predisposition is also pointed out. Therefore, atopic allergy, psoriasis, etc. are both hereditary and environmental diseases. These skin diseases are considered to be diseases in which severe pruritus (itching), dry skin, and dermatitis (eczema) are the main symptoms, and type 1 and type 4 allergic reactions are related.

Topical steroids and local immunomodulators (calcineurin inhibitors) have been developed for the treatment of atopic dermatitis, and antihistamines are often used to suppress itching. Treatment is also needed to avoid allergens, irritants, and stress that aggravate or cause skin symptoms.

On the other hand, interleukins are soluble proteins and are a type of cytokine, which is a substance that transmits signals between cells. Because it is a cytokine produced by lymphocytes, it is also called lymphokine, and because it is a signaling substance between leukocytes, it is called interleukin.

To date, more than 32 types of interleukins have already been known, and interleukins are known to be involved in the regulation of various bioactivity, in particular, the regulation of various immune responses by binding to a receptor on the surface of a target cell. Among them, interleukin 4 or interleukin 13 is known to be related to skin diseases such as atopy and allergic psoriasis.

Interleukin 4 (hereinafter, abbreviated as IL-4) secreted from T cells is a cytokine of about 20 kD consisting of 129 amino acids, and is an important cytokine that increases B-cell differentiation factors and B-cell proliferation factors, especially IgE. . IL-4 is initially secreted by basophils and additionally secreted by activated T-cells (TH2). It has been reported to contribute to B cell activation, IgE type conversion, and induction of Th2 differentiation. IL-4 production by Th2 cells is also accelerated by IL-4, and the receptor for IL-4 is a 130 kD protein that binds two cysteines, IL-4Rα+γb and IL-4Rα+IL-13Rα. receptors are present.

IL-4 has many biological functions, such as stimulating activated B cells and T cells, and differentiating B cells into plasma cells, and is an important regulator of humoral and adaptive immunity. It is also known that IL-4 induces class switching from B progenitor cells to B cells capable of producing IgE and IgG4, as well as promotes the production of MHC class II.

Excessive secretion of IL-4 causes an increase in IgE, resulting in an immediate onset hypersensitivity allergic reaction. IL-4 synergizes with IL-13 and is the causative agent of IgE or eosinophil-mediated inflammatory responses.

By the way, while there was no problem in the production of total T-cells and other immune responses in the IL-4 gene inactivated mice, it is noted that the production of Th2 cells and the production of IgE are decreased in such mice. It can be a treatment for atopy with alleviated side effects on the immune system. Therefore, various attempts are being made to develop IL-4 inhibitors as therapeutic agents for various immune diseases, and in particular, various attempts are being made to develop them as therapeutic agents for atopy, psoriasis, and urticaria.

On the other hand, interleukin 13 is a protein consisting of 122 amino acids, and the receptor for IL-13 is CD132 (γc). IL-13 is functionally similar to IL-4 and is secreted from Th2 cells, dendritic cells, and mast cells. make it

Since the receptor for IL-13 consists of IL-13Rα and IL-4Rα, the IL-13 receptor also responds to IL-4. The receptor for Il-13 is expressed in human B cells, but not in mouse B cells. IL-13 also differentiates monocytes into dendritic cells.

However, it has been reported that IL-13 gene inactive mice do not actively produce Th2 cells or IgE. Therefore, since the production of IgE is inhibited when IL-13 production is inhibited or its action is blocked, IL-13 inhibitors or IL-13 signaling blockers can be used as therapeutic agents for atopy, psoriasis, and allergies.

An allergic reaction caused by an external substance is an immune response that is sensitive to the quality without regard to the amount of an immune stimulant. Immune disease is an immune disease in which an immune response essential for maintaining human health is expressed in an excessive or harmful form.
The IL-4 or IL-13 inhibitor composition of the present invention suppresses IgE production, and thus can be used as a treatment for allergies, psoriasis, and atopic dermatitis. The composition of the present invention reduces IgE by inhibiting IL-4 or IL-13, thereby alleviating allergies, psoriasis, and atopic diseases.

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WO2016021847 describes an oral anti-atopic composition comprising rice prolamin as an active ingredient. The composition described in this document not only has anti-atopic efficacy, but also has high safety and can be widely used by including the protein of commercial food as an active ingredient. WO2008/044894 discloses an atopy-inhibiting composition containing a bamboo extract. Meanwhile, International Application No. PCT/IB2017/000166 published as International Publication No. WO 2017/134525 discloses a therapeutic agent for atopic dermatitis and asthma using an RNA complex targeting IL4Rα, TRPA1, or F2RL1. However, in these documents, there is no suggestion or mention of the possibility that the ALDH-containing composition, which is the subject of the present invention, may inhibit the expression of interleukin-4 or interleukin-13. Meanwhile, International Application No. PCT/US2012/066340 of Johns Hopkins University published as International Publication No. WO 2013/078371 discloses methods and compositions for reducing ethanol toxicity due to accumulation of acetaldehyde. This is a method disclosed in the prior art that reduces aldehyde-induced cytotoxicity by administering to cells a composition comprising a compound that increases the expression, amount, and/or activity of at least one member of the aldehyde dehydrogenase (ALDH) superfamily will make it It is known in the prior art that the organosulfur compound sulforaphane (1-isothiocyanato-4R-(methylsulfinyl)butane) which is at least one compound that modulates the expression, amount, or enzymatic activity of one or more acetaldehyde dehydrogenases in a cell. A method for reducing peak blood levels of acetaldehyde and increasing the rate of catabolism of acetaldehyde in a subject, comprising administering an effective amount of a derivative of sulforaphane, an analogue of sulforaphane, wherein the expression of acetaldehyde dehydrogenase genes ALDH2, provided is a method encoded by one or more of ALDH1A, ALDH3A1, ALDH1B1, ALDH 1a1, or a combination thereof. However, this prior art also does not make any suggestion or mention of the possibility that ALDH, which is the subject of the present invention, may inhibit or may inhibit interleukin-4 or interleukin-13. On the other hand, in International Application No. PCT/US2014/025993 of The Board of Trusts of the Leland Stanford Junior University, published as International Publication No. WO 2014/160185, mitochondrial aldehyde dehydrogenase-2 (ALDH2) functions as a regulator of activity. Compounds, and methods of making such compounds, are disclosed. This prior art describes in detail mitochondrial aldehyde dehydrogenase-2 (ALDH2), a tetrameric protein encoded in the nuclear genome and composed of four identical subunits that migrate into the mitochondria, each of 500 amino acid residues. wherein the tetramer is a dimer of a dimer, wherein the interface between the monomers forming the dimer is different from the interface between the two dimers forming the tetramer, and each subunit has three major sites, i.e., an aldehyde Dehydrogenation catalytic sites, coenzyme or NAD+-binding sites, and oligomerization sites are disclosed. This prior art states that diseases and conditions associated with ALDH2 include ischemic stress, chronic free-radical related diseases, acute free-radical related diseases, insensitivity to nitroglycerin (eg, in angina pectoris and heart failure), hypertension, diabetes mellitus. , osteoporosis, cancer, Fanconi anemia, Alzheimer's disease, Parkinson's disease, alcoholism, alcohol intolerance, alcohol addiction, alcohol abuse disease, alcohol intoxication, alcohol dependence, alcohol poisoning Disclosed are modulators of ALDH2, agonists of ALDH2, or antagonists of ALDH2, including symptoms of alcohol consumption, and drug addiction. Agonists of ALDH2 are also useful for reducing levels of compounds such as ethanol, methanol, ethylene glycol monomethyl ether, polyvinyl chloride, heterogeneous aldehydes and aldehydes in vivo in a subject. An agonist of ALDH2 is also useful for reducing the level of a compound that generates an aldehyde substrate of ALDH2 in a subject upon ingestion, absorption, or inhalation of a compound that generates an aldehyde substrate of ALDH2. Antagonists of ALDH2 are useful for treating or inhibiting diseases such as cancer, where ALDH2 antagonists are used as an adjuvant to standard cancer treatment. It has also been disclosed that antagonists of ALDH2 are useful in treating or suppressing alcoholism and drug addiction. However, this prior art also does not make any suggestion or mention of the possibility that ALDH, which is the subject of the present invention, may inhibit or may inhibit interleukin-4 or interleukin-13. Meanwhile, International Application No. PCT/US2015/016703 of Aviv Therapeutics, Inc., published as International Publication No. WO 2015/127137, relates to mitochondrial aldehyde dehydrogenase 2 (ALDH2) binding to polycyclic amide for the treatment of cancer. and uses thereof. This prior art provides compounds of formula I that bind mitochondrial aldehyde dehydrogenase-2 (ALDH2) and methods of using said compounds for treating patients with cancer, Fanconi's anemia and alcohol-related disorders. This prior art document does not make any suggestion or mention the possibility that the composition containing ALDH, which is the subject of the present invention, may also inhibit interleukin-4 or interleukin-13.

Aldehyde Dehydrogenase Inhibitors: a Comprehensive Review of the Pharmacology, Mechanism of Action, Substrate Specificity, and Clinical Application, by The American Society for Pharmacology and Experimental Therapeutics Pharmacol Rev. 64:520-539, 2012. In this document, 19 types of ALDH play a very important role in eliminating biotoxicity by decomposing various types of aldehydes produced in the body or aldehydes supplied from outside the body. and drugs that inhibit the action of aldehydes that cause a strong oxidative reaction in human tissue cells are described in detail. The above prior art did not suggest or suggest that ALDHs have immunomodulatory use by inhibiting IL-4 or IL-13.

However, the present inventors confirmed the novel fact that the novel mutant yeast of the present invention inhibits IL-4 or IL-13, and the composition of the present application has invented a new use as a medicine or a functional food.

The present inventors use a novel mutant yeast composition to suppress the secretion of IL-4 and IL-13 from Th2 cells, thereby reducing the IgE production of activated B lymphocytes, and histamine produced by degranulation of mast cells. The production of inflammatory mediators was also reduced, confirming the fact that diseases caused by the increase in IgE production could be prevented or treated.

The present inventors found that when reactive oxygen species (ROS) generated in the metabolic process of cells are excessively generated, it causes abnormalities in the immune system and IL-4 and IL- as one of the causes of autoimmune diseases. The expression of 13 is increased, and the resulting oxidative stress causes contraction of smooth muscle in the lungs, increased vascular permeability, damage to drug-responsive receptors, increased secretion of mucus, secretion of inflammatory mediators, and damage to airway epithelial cells, leading to atopy, psoriasis, and psoriasis. , allergic dermatitis, and bronchial asthma were confirmed to act as a cause of various diseases.

According to the IL-4 and IL-13 inhibition test results of the composition of the present invention, the IL-4 or IL-13 inhibitory effect of ALDH and the IL-4 or IL-13 effect on the human immune system reported to date, It enables new pharmaceutical uses of ALDH as immunomodulators.

Therefore, a primary object of the present invention is to provide a therapeutic agent for immune diseases such as atopy, allergy, psoriasis, containing a novel mutant yeast that inhibits IL-4 or IL-13.

The primary object of the present invention is achieved by providing a novel mutant yeast composition of the present invention.

Another object of the present invention is to provide a food composition for the treatment and prevention of immune response inhibitors or autoimmune diseases, containing the novel mutant yeast of the present invention as an active ingredient.

Another object of the present invention is to provide a therapeutic agent for atopic dermatitis, psoriasis, and allergic dermatitis containing the novel mutant yeast of the present invention.

The above objects of the present invention can be achieved by providing a therapeutic agent for atopic dermatitis, psoriasis, and allergic dermatitis containing the novel mutant yeast of the present invention.

The gist of the object and solution of the present invention as described above is a novel use of the novel mutant yeast of the present invention as an IL-4 or IL-13 inhibitor. Therefore, the novel mutant yeast of the present invention can be used as an IL-4 or IL-13 inhibitor of the present invention, and can be used as an active ingredient for atopic, psoriasis, and allergic dermatitis treatment agents of the present invention, and is included in the scope of the present invention do.

In addition, the novel mutant yeast of the present invention exhibiting such IL-4 or IL-13 inhibitory activity, the present inventors, in accordance with the Budapest Treaty, deposit number KCTC13925BP ( Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces ) to the International Depositary Organization (KCTC) cerevisiae KwonP-2) and KCTC14123BP ( Saccharomyces cerevisiae KwonP-3) were deposited, and the test results for atopic, psoriasis, and allergic dermatitis treatment according to various known IL-4 or IL-13 inhibitory effects were disclosed through the specification of the present application. do.

The composition containing the novel mutant yeast of the present invention exhibits a pharmaceutical effect for preventing and treating various immune diseases according to the inhibitory action of interleukin-4 or interleukin-13.

The yeast extract according to the present invention inhibits the increase in immunoglobulin E (IgE) and total inflammatory cell number and lymphocytes, monocytes, neutrophils, and eosinophils in the bronchoalveolar lavage fluid of asthma-induced mice in the in vivo experiment of FIG. It has activity, inhibiting airway mucus secretion, and by reducing Th2 cytokines IL-4 and IL-13 and airway inflammation, it can be used as a therapeutic agent for atopic dermatitis, psoriasis, allergic dermatitis, and health food.

Therefore, the composition according to the present invention is effective for the treatment and prevention of inflammatory reactions caused by oxidative stress such as inflammation, allergy, asthma, and the like. In particular, the novel mutant yeast of the present invention has the advantage that the possibility of side effects occurring even when taken is low.

1 is a graph showing changes in blood IgE in atopy-induced control group and experimental group mice. A: CON, B: atopy-induced group with DNCB, C: dexamethasone administration group, D: composition of the present invention 50 mg/kg, E: composition of the present invention 150 mg/kg, F: group administered with composition 300 mg/kg
2 is a graph showing changes in blood leukocytes (WBC) in atopy-induced control group and experimental group mice. The DNCB-induced increase in the amount of leukocytes in the blood was reduced by 65% when administered with dexamethasone, and reduced by about 74% when administered with the composition of the present invention (150 mg/Kg).
3 is a graph showing changes in eosinophils in atopy-induced control and experimental mice. The increase in blood eosinophil due to atopy induction by DNCB was reduced by 60% when dexamethasone was administered, and decreased by about 55% when the composition (150mg/Kg) of the present invention was administered.
4 is a graph showing changes in blood neutrophils in atopic-induced control and experimental mice. Blood neutrophils increased by DNCB induction were reduced by 35% by administration of dexamethasone, and reduced by about 46% by administration of the composition of the present invention (150mg/Kg).
5 is a graph showing changes in IL-6 in atopy-induced control group and experimental group mice.
6 is a graph showing changes in IL-6 mRNA in atopy-induced control group and experimental group mice.
7 is a graph showing changes in IL-13 mRNA in atopy-induced control group and experimental group mice. A: CON, B: atopy-induced group with DNCB, C: dexamethasone administration group, D: 50 mg/kg of the composition of the present invention, E: 150 mg/kg of the composition of the present invention, F: 300 mg/kg of the present composition.
8 is a graph showing changes in IL-6 in atopic dermatitis human cell model using TNF-α and IFN-γ.

Hereinafter, the production process of the active ingredient of the therapeutic agent for atopic dermatitis, psoriasis, and allergic dermatitis containing the novel mutant yeast of the present invention, separation and purification process, pharmacological effect and control experiment, effective amount measurement, dosage form and administration method Toxicity Explain. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Lysate preparation of novel mutant yeast of the present invention

Example 1-1: Saccharomyces cerevisiae yeast fermentation process for preparing the composition of the present invention

The seed strain (KCTC13925BP) of the novel mutant yeast of the present invention was fermented and cultured for 24 hours in an incubator at 160 rpm and 30° C. using YPD medium (yeast extract, peptone, and glucose-containing medium) in a 200 mL flask. The culture was carried out for 72 hours through a 5L fermenter (Marado-05D-PS, CNS, Korea). After completion of the culture, the yeast was centrifuged using a high-speed centrifuge (Supra R22, Hanil, Korea).

Example 1-2: Preparation of novel mutant lysate of the present invention

The centrifuged novel mutant yeast of the present invention was frozen in a cryogenic freezer (CLN-52U, Nihon freezer, Japan) for 2 days, and then freeze-dried in a freeze dryer (FDU-7006, Operon, Korea) for 2 days. After dissolving 3 g of lyophilized yeast powder in 50 mL phosphate-buffered saline (PBS) containing a protease inhibitor (A32955, Thermo fisher, USA), 0.5 mm glass beads for cell disruption (11079105, Biospec) 10 g was put into the bead homogenizer (Mixer Mill MM400, Retsch, Germany) for 2 minutes a total of 3 times to disrupt the yeast. After centrifugation using a high-speed centrifuge (Supra R22, Hanil, Korea), only the supernatant was separated and freeze-dried for 2 days with a freeze dryer (FDU-7006, Operon, Korea).

Atopy inhibitory effect of the novel mutant yeast lysate composition of the present invention

Example 2-1. Preparation of atopy-induced test animals.

Seven-week-old female BALB/c mice with an average weight of about 20 g were purchased from Samtaco Bio Korea. Dissolve 40 mg of mouse dermatitis inducer DNCB (1-Chloro-2,4-dinitrobenzene; mouse dermatitis inducer, Junsei) in a solution in which the ratio of acetone and olive oil is 4:1 to make 1% DNCB solution. It was used in a test for atopy induction. On the first day of the 1st week of induction, the hair on the back of the mouse was cut with an area of 1.5 cm ² After shaving, 100 μL of 1% DNCB solution was sprayed on each animal for a total of 4 days out of 1 week, and evenly applied with a soft brush.

In the second week of induction, 80 mg of DNCB was dissolved in a solution in which acetone and olive oil were mixed in a ratio of 4:1 to prepare a 2% DNCB solution. Week 2 2% DNCB solution was applied in the same manner as in week 1 at 120 μL each. (A.F. Hamad. et al., Bangladesh J Pharmacol, 12:147-150, 2017) From the 2nd week of atopic dermatitis induction, the yeast extract powder containing ALDH of the present invention was dissolved in physiological saline and oral administration was performed to mice for a total of 5 days. . The composition of the present invention was orally administered in an amount of 50 mg/kg/day, 150 mg/kg/day, and 300 mg/kg/day. To the control group, dexamethasone was administered.

Example 2-2. autopsy of animals

The composition of the present invention was orally administered in the 2nd week of induction and for 5 days, and autopsy was performed the next day. There are a total of 5 animals in one group, and blood, back skin, and spleen were all extracted from 5 animals. The back skin tissue and spleen were divided into two, one for checking the secretion of cytokine and the other for checking gene expression. It was stored at -80°C for use. For blood, some of the blood collected for hemocytometry was transferred to a 1.5 mL E-tube with a heparinized tube, and the rest of the blood was stored in a 1.5 mL E-tube for analysis of serum IgE secretion.

Example 2-3. Analysis of blood IgE content in atopic mice

Blood collected from the test animals was placed in a 1.5 mL E-tube and centrifuged for 20 minutes at 12,000 rpm and 4°C. After that, only the supernatant was separated and used for the experiment. Quantification of IgE was performed by the Enzyme-Linked Immunosorbent Assay (ELISA) method, and quantitative analysis was performed using the IgE ELISA set (BD bioscience). After the reaction, the absorbance at the final 450 nm wavelength was measured with an ELISA reader (Multiskan sky, Thermo Fisher, USA), and the results are shown in FIG. 1 . In the group administered with the novel mutant yeast composition of the present invention, IgE was reduced by about 40% or more compared to the atopy-induced experimental group. (FIG. 3) Compared to the atopy-inducing group (DNCB), IgE was reduced by 38% in the group administered with the composition of the present invention (150 mg/Kg), and IgE was reduced by 45% in the group administered with the composition of the present invention (300 mg/Kg). The effective amount of the composition of the present invention for treating atopic dermatitis was evaluated as 150 mg/Kg in a mouse animal test.

Example 2-4. Confirmation of interleukin-13 inhibitory pharmacological effect by the novel mutant yeast composition of the present invention in atopic mice

Quantification of interleukin-13 in blood was performed by each Enzyme-Linked Immunosorbent Assay (ELISA) method. After adding the Capture antibody diluted in buffer to a micro-well plate and leaving it at 4 °C for 16 hours, after washing, add the test substance and halve the absorbance at the final 450 nm wavelength with an ELISA reader (Multiskan sky) , Thermo Fisher, USA) to determine the amount of interleukin-13 mRNA in the blood. A real-time polymerase reaction was performed on interleukin-13 by extracting RNA from the skin tissue, such as a mouse. The reduction of interleukin-13 mRNA by the novel mutant yeast lysate of the present invention was confirmed (FIG. 7).

Example 2-5. Hematology analysis of atopic mice

In allergic reactions including atopic dermatitis and asthma, eosinophilic and neutrophilic infiltration in tissues is a characteristic phenomenon (Horn BR, Robin ED, Theodore J, Van Kessel A. Total eosinophil counts in the management of bronchial asthma) (N Engl J Med 1975;292:1152-5). In particular, the more the eosinophil-type cells differentiate, the more the bone marrow is biased toward an atopic state, which can cause allergic inflammatory reactions. In the present invention, the pharmacological effect of the novel mutant yeast lysate of the present invention was confirmed by performing blood cell analysis of leukocytes, eosinophils, and neutrophils in atopic-induced mice. Blood cells were analyzed immediately after the autopsy of the blood contained in a heparin tube using a hematocyte analyzer (Hemavet 950FS, Drew Scientific Inc, Korea).

In the case of dexamethasone (Dex.) administration, the DNCB-induced increase in blood leukocytes (WBC) was reduced by 65%, and in the case of administration of 150 mg/kg of the composition of the present invention, the leukocytes in the blood were reduced by about 74% (Fig. 2). The neutrophil (NE) content in blood was reduced by 35% in the case of dexamethasone administration, and by about 46% in the case of administration of 150 mg/kg of the composition of the present invention (FIG. 4), the blood neutrophils characteristically appearing in allergic reactions such as atopy By reducing the eosinophil (EO) content by 60% in the case of administration of dexamethasone, and by reducing about eosinophils by 55% in the case of administration of 150 mg/kg of the composition of the present invention (FIG. 3), the ALDH-containing composition of the present invention is similar to that of the control group or showed excellent pharmacological effects.

Example 2-6 Analysis of IL-6 changes in atopic-induced mice and the group administered with the composition of the present invention

The composition of the present invention was orally administered to Balb/c mice induced by atopy for 2 weeks with DNCB solution for 5 days. For blood cell analysis, skin tissue was used as a sample for serum, ELISA and RT-PCR, and dexamethasone was used as a positive control. The expression level of IL-6 cyokine in the skin tissue was measured by hemocytometry.

IL-6 expression levels in mouse skin tissues were analyzed by ELISA assay and real-time PCR, and IL-6 protein and mRNA levels were quantified. As a result of the analysis, the effect of equal or superiority was confirmed in the group administered with the composition of the present invention compared to the group administered with dexamethasone. In the case of dexamethasone administration, the IL-6 content in the skin tissue increased by DNCB induction was reduced by 50%, and in the case of administration of the composition of the present invention (150 mg/Kg), it was reduced by about 48%. (FIG. 5) Skin increased by DNCB induction As a result of analysis of intra-tissue IL-6 mRNA level, dexamethasone administration was reduced by 34%, and administration of the composition of the present invention (150 mg/Kg) was reduced by about 77%. (Fig. 6)

Example 3-1: Preparation of atopic dermatitis human cell model using TNF-α and IFN-γ

For atopic dermatitis human cells, HaCaT cells, which are human keratinocytes, were used. The medium was DMEM (Dolbicco's Modified Eagle's Medium, USA) in which 10% Fetal bovine serum (FBS) and + 1% P/S (Penicillin/Streptomycin: P/S) were added, seeding and After 23 hours, the novel mutant yeast composition of the present invention was treated at a concentration of 0.5, 1, and 2 μg/mL, and after 1 hour, inflammation was induced by treatment with 10 ng/mL of TNF-α and IFN-γ.

Example 3-2: Result Analysis

(1) Analysis of cytokine changes in atopic dermatitis cell model

Inflammation was induced in HaCaT cells with TNF-α and IFN-γ, and after 1 hour, the medium from the 6-well plate was transferred to an E-tube, and centrifugation was performed at 13,000 rpm and 4° C. for 10 minutes. Afterwards, only the supernatant was transferred to a new tube and used for the experiment.

Enzyme-Linked Immunosorbent Assay (ELISA): For the analysis, an IgE ELISA set (BD bioscience, US) was used. Capture Ab diluted in a coating buffer was added to a micro well and incubated at 4°C overnight. After washing 3 times, put assay diluent into each well and incubate at room temperature. After washing 3 times, standard and sample diluted with assay diluent were added to each well and incubated at room temperature. After washing 5 times, a working detector was put into each well and incubated at room temperature. After washing 7 times, the substrate solution was put into each well and incubated for 30 minutes at room temperature in the dark. To terminate the reaction, 50 μl of Stop Solution was put into each well, and absorbance at a wavelength of 450 nm was measured with an ELISA reader (Multiskan sky, Thermo Fisher, USA).

(2) In-vitro of the yeast extract powder of the present invention for atopic dermatitis efficacy

The human keratinocyte HaCaTcell line was pre-treated with the composition of the present invention at each concentration for 20 minutes, and TNF-α and IFN-γ were added to the pretreated cells to induce an intracellular inflammatory state. The expression level of IL-6, a major marker in an atopic disease animal model, was measured. In the cells treated with the composition of the present invention compared to the IL-6 expression level of the atopic-induced cell line, it was confirmed that the IL-6 expression level was decreased by 0.03%, 14%, and 27% for each concentration. (Fig. 8)

Single dose toxicity test of the composition of the present invention

Example 4-1. Preparation of laboratory animals

As experimental animals, female and male ICR mice (7 weeks old) were received and acclimatized for 7 days, and only healthy animals were used for the test by observing general symptoms during the acclimatization period. Feed and water were ingested ad libitum, and group separation was performed so that there were 5 males and 5 females in each group based on an average body weight of about 20 g the day before oral administration.

Example 4-2 Administration of test substance

The test substance was prepared by dissolving in physiological saline so that the doses of the test animals were 0, 750, 3000, and 5000 mg/Kg, respectively, based on the content of the ALDH-containing yeast lysate of the present invention. The standard of administration dose was in accordance with the Korea National Toxicology Program (KNTP) toxicity test manual of the Ministry of Food and Drug Safety. The maximum application dose of 5000 mg/Kg guided by the KNTP manual was applied as the maximum concentration in this experiment. Samples prepared for each group were orally administered once to each test animal, and physiological saline was administered to the normal group (G1).

Example 4-3. Observation and autopsy

For all the animals in the test group, symptoms were observed at least once a day from the date of acquisition to the day of autopsy, and symptoms were observed for 7 days after oral administration. After the type of symptom observation, an autopsy was performed, and changes in each organ were visually observed at the time of autopsy.

As a result of a single dose toxicity test of the ALDH-containing composition of the present invention using mice, no deaths were observed for 7 days at a concentration of ALDH yeast extract up to 5000 mg/kg, and peculiarities such as weight gain and feed intake were found. couldn't In addition, no unusual findings were found in the autopsy results after the observation was completed.

In the above implementation, the novel mutant yeast-containing psoriasis treatment agent, atopic treatment agent, and allergy treatment method, pharmacological effect compared to control drug, administration method, therapeutically effective amount for a disease model, and a representative case of acute toxicity Although described in detail through each drawing and test results, these are only one embodiment of the present invention.

Therefore, those of ordinary skill in the art can easily derive various modifications and other embodiments equivalent to the present invention from the above-described embodiments of the present invention. Therefore, in any modified form of the novel mutant yeast of the present invention that realizes the technical gist of the present invention described in the following claims, the therapeutic agent for atopy, psoriasis, and allergy according to the inhibition of interleukin 4 and interleukin 13, It belongs to the scope of legal protection of the invention.

Korea Institute of Bioscience and Biotechnology KCTC13925BP 20190822 Korea Institute of Bioscience and Biotechnology KCTC14122BP 20200130 Korea Institute of Bioscience and Biotechnology KCTC14123BP 20200130

Claims (9)

A mutant yeast strain Saccharomyces cerevisiae KCTC13925BP, characterized in that it comprises a, atopy prevention or treatment pharmaceutical composition.
delete delete A composition for preventing or treating psoriasis, comprising KCTC13925BP in the mutant yeast strain Saccharomyces cerevisiae.
delete delete A mutant yeast strain Saccharomyces cerevisiae, characterized in that it comprises KCTC13925BP, an allergy treatment composition.
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