WO2021167310A1 - Atopy inhibitory composition containing aldehyde dehydrogenase - Google Patents

Atopy inhibitory composition containing aldehyde dehydrogenase Download PDF

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WO2021167310A1
WO2021167310A1 PCT/KR2021/001935 KR2021001935W WO2021167310A1 WO 2021167310 A1 WO2021167310 A1 WO 2021167310A1 KR 2021001935 W KR2021001935 W KR 2021001935W WO 2021167310 A1 WO2021167310 A1 WO 2021167310A1
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composition
present
aldehyde dehydrogenase
aldehyde
aldh
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PCT/KR2021/001935
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French (fr)
Korean (ko)
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권흥택
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주식회사 피코엔텍
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • A23L33/145—Extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast

Definitions

  • the present invention relates to an atopic inhibitor containing aldehyde dehydrogenase. More particularly, the present invention relates to a composition for treating atopy, allergy or psoriasis, containing an aldehyde dehydrogenase that reduces IgE.
  • the present invention relates to a treatment for atopic dermatitis, treatment for psoriasis, and treatment for allergies, containing aldehyde dehydrogenases and their variants as active ingredients, including fusion proteins or recombinant proteins including an action site involved in aldehyde dehydrogenation. .
  • Atopic dermatitis is a chronic, recurrent inflammatory skin disease that causes itching, 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 an allergic reaction to an antigen that is easy to invade due to a decrease in skin defense function is involved, and environmental factors such as house dust mites and mold are involved as representative antigens.
  • 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 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.
  • 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.
  • 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.
  • 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-4R ⁇ + ⁇ b and IL-4R ⁇ +IL-13R ⁇ . 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, which is converted from B progenitor cells to B cells capable of producing IgE and IgG4, as well as promotes the production of MHC class II.
  • IL-4 acts synergistically with IL-13 and is the causative agent of IgE- or eosinophil-mediated inflammatory responses.
  • interleukin 13 is a protein composed of 122 amino acids, and the receptor for IL-13 is CD132 ( ⁇ c). IL-13 is functionally similar to IL-4 and is secreted from Th2 cells, dendritic cells, and mast cells. make it
  • the receptor for IL-13 consists of IL-13R ⁇ and IL-4R ⁇ , 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.
  • IL-13 inhibitors or IL-13 signaling blockers can be used as therapeutic agents for atopy, psoriasis, and allergies.
  • 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 inhibits IgE production, and thus can be used as a treatment for allergies, psoriasis, and atopic dermatitis.
  • the aldehyde dehydrogenation reaction is a reaction for changing acetaldehyde or various aldehydes present in human metabolites into organic acids that can be discharged as waste products.
  • the enzyme that promotes this aldehyde dehydrogenation reaction decomposes the aldehyde, which is toxic to cells, and relieves carbonyl stress applied to cells and tissues.
  • ALDH aldehyde dehydrogenases
  • 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.
  • 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 analog 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.
  • ALDH which is the subject of the present invention, may inhibit or may inhibit interleukin-4 or interleukin-13.
  • This prior art describes in detail mitochondrial aldehyde dehydrogenase-2 (ALDH2), a tetrameric protein consisting of four identical subunits encoded in the nuclear genome and transported 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.
  • ADH2 mitochondrial aldehyde dehydrogenase-2
  • ALDH2 related diseases and conditions include ischemic stress, chronic free-radical related diseases, acute free-radical related diseases, insensitivity to nitroglycerin (eg, in angina and heart failure), hypertension, diabetes. , osteoporosis, cancer, Fanconi anemia, Alzheimer's disease, Parkinson's disease, alcoholism, alcohol intolerance, alcohol addiction, alcohol abuse disease, alcohol intoxication, alcohol dependence, alcohol poisoning
  • modulators of ALDH2, agonists of ALDH2, or antagonists of ALDH2, including symptoms of alcohol consumption, and drug addiction include ischemic stress, chronic free-radical related diseases, acute free-radical related diseases, insensitivity to nitroglycerin (eg, in angina and heart failure), hypertension, diabetes. , osteoporosis, cancer, Fanconi anemia, Alzheimer's disease, Parkinson's disease, alcoholism, alcohol intolerance, alcohol addiction, alcohol abuse disease, alcohol intoxication, alcohol dependence, alcohol poisoning
  • Agonists of ALDH2 are also useful for reducing the 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.
  • 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.
  • 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.
  • the present inventors confirmed the novel fact that all or part of various ALDH fusion proteins and recombinant proteins containing an aldehyde dehydrogenation reaction promoting site inhibit IL-4 or IL-13, so that the ALDH-containing composition of the present application can be used as a pharmaceutical or New uses as functional foods have been invented.
  • the present inventors inhibit the secretion of IL-4 and IL-13 from Th2 cells by using the ALDH-containing composition, thereby reducing the IgE production of activated B lymphocytes, thereby reducing inflammation such as histamine produced by degranulation of mast cells.
  • the production of mediators was also reduced, confirming the fact that diseases caused by an 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 is excessively generated, it causes abnormalities in the immune system and IL-4 and IL- as one of the causes of autoimmune diseases.
  • ROS reactive oxygen species
  • 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 be the cause of various diseases.
  • the IL-4 or 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.
  • the primary object of the present invention is to contain all or a part of a fusion protein or recombinant protein (hereinafter abbreviated as ALDH) comprising an aldehyde dehydrogenase that inhibits IL-4 or IL-13 and a site that promotes a dehydrogenation reaction. It is to provide a therapeutic agent for immune diseases such as atopy, allergy, and psoriasis.
  • ALDH fusion protein or recombinant protein
  • the primary object of the present invention is to provide a composition containing, as an active ingredient, aldehyde dehydrogenation promoting enzymes and variants thereof, including fusion proteins or recombinant proteins comprising a site of action involved in aldehyde dehydrogenation reaction. .
  • Another object of the present invention is an immune response inhibitor or autoimmune disease containing aldehyde dehydrogenation promoting enzymes and variants thereof as active ingredients, including fusion proteins or recombinant proteins comprising a site involved in aldehyde dehydrogenation reaction.
  • aldehyde dehydrogenation promoting enzymes and variants thereof as active ingredients, including fusion proteins or recombinant proteins comprising a site involved in aldehyde dehydrogenation reaction.
  • Another object of the present invention is to provide a therapeutic agent for atopic dermatitis, psoriasis, and allergic dermatitis, which contains aldehyde dehydrogenases and variants thereof as active ingredients, including fusion proteins or recombinant proteins comprising a site of action involved in aldehyde dehydrogenation reaction will do
  • aldehyde dehydrogenase and its variants containing a fusion protein containing a part or all of the action site that promotes the aldehyde dehydrogenase reaction of the aldehyde dehydrogenase, recombinant protein containing It can be achieved by providing a therapeutic agent for atopic dermatitis, psoriasis, and allergic dermatitis.
  • the gist of the object and solution of the present invention as described above is a novel use of a known substance, ALDH, as an IL-4 or IL-13 inhibitor. Therefore, the ALDH that can be used as an IL-4 or IL-13 inhibitor of the present invention can include all of the various ALDHs, which are known substances exhibiting aldehyde dehydrogenation activity to date, and all of these are atopic dermatitis, psoriasis, and allergies of the present invention. It can be used as an active ingredient in a dermatitis treatment agent, and is included in the scope of the present invention.
  • KCTC International Depositary Organization
  • KCTC13925BP Saccharomyces cerevisiae KwonP-1
  • KCTC14122BP Saccharomyces cerevisiae KwonP-2
  • KCTC14123BP Saccharomyces cerevisiae KwonP-3
  • ALDH-producing strains were deposited, and the measurement test results for atopic, psoriasis, and allergic dermatitis treatments according to the IL-4 or IL-13 inhibitory effect of various known ALDHs were reviewed. It is disclosed through the specification of the application.
  • ALDH or a composition containing ALDH of the present invention exhibits a pharmaceutical effect for preventing and treating various immune diseases due to the inhibitory action of interleukin-4 or interleukin-13.
  • ALDH contained in the yeast extract according to the present invention showed immunoglobulin E (IgE) and total number of inflammatory cells and lymphocytes, monocytes, neutrophils, and eosinophils in the bronchoalveolar lavage fluid of asthma-induced mice in the in vivo experiment of FIG. It has the activity of inhibiting the increase and the secretion of airway mucus, and by reducing the 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 as a health food.
  • IgE immunoglobulin E
  • the ALDH-containing composition according to the present invention is effective in treating and preventing inflammatory reactions caused by oxidative stress such as inflammation, allergy, asthma, and the like.
  • ALDH is extracted from edible yeast, it has the advantage that the possibility of side effects is low even if it is taken.
  • 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
  • FIG. 2 is a graph showing changes in blood leukocytes (WBC) in atopy-induced control group and experimental group mice.
  • WBC blood leukocytes
  • DNCB-induced increase in the amount of leukocytes in the blood was reduced by 65% in the case of dexamethasone administration, and reduced by about 74% in the case of administration of the composition of the present invention (150mg/Kg).
  • FIG. 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 of the present invention (150mg/Kg) was administered.
  • FIG. 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.
  • FIG. 6 is a graph showing changes in IL-6 mRNA in atopy-induced control and experimental mice.
  • Example 1-1 Saccharomyces cerevisiae yeast fermentation process for preparing the composition of the present invention
  • the ALDH-containing Saccharomyces cerevisiae yeast seed (KCTC13925BP) was fermented for 24 hours in an incubator at 160 rpm and 30°C using YPD medium (a medium containing yeast extract, peptone, and glucose) in a 200 mL flask and cultured. and this 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).
  • YPD medium a medium containing yeast extract, peptone, and glucose
  • Example 1-2 ALDH-containing yeast lysate preparation process
  • the centrifuged ALDH-containing yeast was frozen in a cryogenic freezer (CLN-52U, Nihon freezer, Japan) for 2 days, and then freeze-dried for 2 days in a freeze dryer (FDU-7006, Operon, Korea).
  • a freeze dryer FDU-7006, Operon, Korea.
  • PBS phosphate-buffered saline
  • 0.5 mm glass beads for cell disruption 11079105, Biospec 10 g was put into the bead homogenizer (Mixer Mill MM400, Retsch, Germany) to disrupt yeast over a total of 3 times for 2 minutes each.
  • 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).
  • Example 2-1 Preparation of atopy-induced test animals.
  • mice with an average weight of about 20 g were purchased from Samtaco Bio Korea.
  • DNCB mouse dermatitis inducer
  • Example 2-2 autopsy of animals
  • composition of the present invention was orally administered in the second week of induction and for 5 days, and autopsies were performed the next day.
  • 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.
  • Example 2-3 Analysis of blood IgE content in atopic mice
  • the 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 ALDH-containing composition of the present invention, IgE was reduced by about 40% or more compared to the atopy-induced experimental group.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • 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). Atopic therapeutically effective amount of the composition of the present invention was evaluated as 150 mg/Kg in a mouse animal test.
  • Example 2-4 Confirmation of the pharmacological effect of inhibiting interleukin-13 by the ALDH-containing composition of the present invention in atopic mice
  • Example 2-5 Hematology analysis of atopic mice
  • 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).
  • the more the eosinophil-type cells differentiate the more the bone marrow is biased toward an atopic state, which can cause allergic inflammatory reactions.
  • blood cells such as leukocytes, eosinophils, and neutrophils were analyzed in atopic-induced mice to confirm the pharmacological effect of the ALDH-containing yeast lysate of the present invention.
  • the analysis of blood cells was performed immediately after the autopsy of the blood contained in the heparin tube using a hemocytometer (Hemavet 950FS, Drew Scientific Inc, Korea).
  • 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 neutrophil (NE) content characteristically in allergic reactions such as atopy
  • 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
  • composition of the present invention was orally administered to Balb/c mice induced by atopy for 2 weeks with DNCB solution for 5 days.
  • 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 to quantify IL-6 protein and mRNA levels.
  • 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.
  • 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 (150mg/Kg), it was reduced by about 48%.
  • Example 3-1 Preparation of atopic dermatitis human cell model using TNF- ⁇ and IFN- ⁇
  • HaCaT cells which are human keratinocytes, were used as the cells.
  • 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 ALDH-containing composition of the present invention was treated at concentrations of 0.5, 1, and 2 ⁇ g/mL, and after 1 hour, inflammation was induced by treatment with 10 ng/mL of TNF- ⁇ and IFN- ⁇ .
  • DMEM Dolbicco's Modified Eagle's Medium, USA
  • FBS Fetal bovine serum
  • P/S Penicillin/Streptomycin
  • Inflammation was induced in HaCaT cells with TNF- ⁇ and IFN- ⁇ , and after 1 hour, the medium in a 6-well plate was transferred to an E-tube, and centrifugation was performed at 13,000 rpm and 4°C for 10 minutes. After that, only the supernatant was transferred to a new tube and used for the experiment.
  • Enzyme-Linked Immunosorbent Assay For the analysis, an IgE ELISA set (BD bioscience, US) was used. Capture Ab diluted in coating buffer was added to micro wells 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 added to each well and incubated for 30 minutes in dark conditions at room temperature. To terminate the reaction, 50 ⁇ L of Stop Solution was added to each well, and absorbance at 450 nm wavelength was measured with an ELISA reader (Multiskan sky, Thermo Fisher, USA).
  • the human keratinocyte HaCaTcell line was pretreated with the composition of the present invention at each concentration for 20 minutes, and TNF ⁇ /IFN ⁇ was added to the pretreated cells to induce an intracellular inflammatory state.
  • the expression level of IL-6 a major marker of atopic disease animal models, was measured. Compared to the IL6 expression level of the atopic-induced cell line, it was confirmed that the IL-6 expression level decreased by 0.03%, 14%, and 27% for each concentration in the cells treated with the composition of the present invention. (Fig. 8)
  • mice Female and male ICR mice (7 weeks old) were received and acclimatized for 7 days. During the acclimatization period, general symptoms were observed, and only healthy animals were used for the test. 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 the average body weight of about 20 g the day before oral administration.
  • 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.
  • KNTP National Toxicology Program
  • 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).
  • Symptoms were observed at least once a day from the date of acquisition to the day of autopsy for all animals in the test group, 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.
  • psoriasis treatment, atopic treatment, and allergy treatment containing aldehyde dehydrogenase (ALDH) of the present invention psoriasis treatment, atopic treatment, and allergy treatment containing aldehyde dehydrogenase (ALDH) of the present invention, pharmacological effect compared to control drug, administration method, therapeutically effective amount for disease model, acute toxicity
  • ADH aldehyde dehydrogenase

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Abstract

The present invention relates to a therapeutic agent for atopic dermatitis, psoriasis, and allergies containing aldehyde dehydrogenase, wherein the therapeutic agent for atopic dermatitis, psoriasis, and allergy contains, as active ingredients, aldehyde dehydrogenation accelerating enzymes and variants thereof, including a fusion protein or a recombinant protein comprising a site of action involved in aldehyde dehydrogenation.

Description

알데히드탈수소 효소를 함유하는 아토피 억제 조성물Atopy inhibitory composition containing aldehyde dehydrogenase
본 발명은 알데히드탈수소효소를 함유하는 아토피 억제제에 관한 것이다. 보다 상세하게는, 본 발명은 IgE를 감소시키는 알데히드탈수소 효소를 함유하는, 아토피, 알러지 또는 건선 치료제 조성물에 대한 것이다.The present invention relates to an atopic inhibitor containing aldehyde dehydrogenase. More particularly, the present invention relates to a composition for treating atopy, allergy or psoriasis, containing an aldehyde dehydrogenase that reduces IgE.
또한, 본 발명은, 알데히드탈수소반응에 간여하는 작용 부위를 포함하는 융합단백질 또는 재조합단백질을 포함하는 알데히드탈수소 효소들과 그 변이체들을 유효성분으로서 함유하는, 아토피 치료제, 건선 치료제, 알러지 치료제에 대한 것이다.In addition, the present invention relates to a treatment for atopic dermatitis, treatment for psoriasis, and treatment for allergies, containing aldehyde dehydrogenases and their variants as active ingredients, including fusion proteins or recombinant proteins including an action site involved in aldehyde dehydrogenation. .
아토피 피부염은 가려움증을 발생시키는 만성 재발성의 염증성 피부질환으로서, 항원의 접촉 없이도 발생할 수 있는 과잉 알러지 반응이며 비정상적인 염증 반응이다. 아토피 피부염은 음식물이나 흡입성 물질에 대한 과민하게 반응하는 피부 질환으로서, 환경적인 요인, 유전적 소인, 면역학적 이상 및 피부보호막의 이상 등이 발병 원인이다.Atopic dermatitis is a chronic, recurrent inflammatory skin disease that causes itching, 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.
아토피 피부병의 환경적 요인으로는 매연 등 환경 공해, 식품첨가물 사용의 증가, 카펫, 침대, 소파의 사용 및 집먼지 진드기 등의 알레르기를 일으키는 원인 물질의 증가 등이 있다. 피부 방어 기능의 저하로 인해 침입하기 쉬워진 항원에 대한 알레르기 반응이 관여하는 것으로 보이며 대표적인 항원으로는 집먼지진드기나 곰팡이 등의 환경 인자가 관여한다.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 an allergic reaction to an antigen that is easy to invade due to a decrease in skin defense function is involved, and environmental factors such as house dust mites and mold are involved as representative antigens.
아토피 피부염에 대한 유전적인 영향은 아토피피부염 환자들에서 가족력이 있다는 사실에서 확인된다. 상당수의 아토피 환자에서 필라그린 (filagrin) 유전자의 이상으로 피부장벽 기능의 저하가 발생하였다고 알려지고 있다. 아토피 발병 원인으로서, 유전적 소인으로 인한 수용체의 과다 발현도 지목된다. 따라서 아토피 알러지 건선등은 유전성 질환이면서도 환경적 질병이다. 이러한 피부질환들은 심한 소양증(가려움증)과 피부건조증, 피부염(습진)이 주요 증상이고 제1형 알러지와 제4형 알러지 반응이 관계되는 질환으로 여겨진다. 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 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.
현재까지 개발된 아토피 피부염의 치료제로는 국소스테로이드제, 국소면역조절제(칼시뉴린 억제제)가 치료제이고, 가려움증을 억제하기 위하여 항히스타민제도 흔히 사용된다. 또한 피부 증상을 악화시키거나 유발하는 알레르겐, 자극 물질, 스트레스를 피하도록 하는 치료가 필요하다.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.
한편 인터류킨은 가용성단백질로서 세포간 신호를 전달하는 물질인 사이토카인의 일종이다. 림프구가 생산하는 사이토카인이므로 림포카인(lymphokine)으로 불리기도 하고, 백혈구(leukocyte)들 간의 신호전달 물질이므로 인터류킨으로 지칭된다. 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.
현재까지 32종 이상의 인터류킨이 이미 공지되어 있고, 인터류킨은 표적 세포 표면의 수용체와 결합하여 다양한 생체활성 조절, 특히 다양한 면역 반응 조절에 관여하는 것으로 알려지고 있다. 그 중에서 인터류킨4 또는 인터류킨13은 아토피, 알레르기 건선 등의 피부 질환과 관련이 있다고 알려지고 있다. 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.
T세포에서 분비되는 인터류킨4(이하, IL-4로 약칭함)는 129개의 아미노산으로 이루어지는 약20kD의 사이토카인으로서, B-세포 분화 인자 그리고 B세포 증식 인자, 특히 IgE를 증가시키는 중요한 사이토카인이다. IL-4는 최초로 호염구에서 분비되고 활성화된 T-세포(TH2)에서 추가적으로 분비된다. B세포 활성화, IgE 종류변환, Th2분화유도에 기여하는 것으로 보고되었다. Th2 세포에 의한 IL-4의 생산은 IL-4에 의해 빨라지기도 하며, IL-4의 수용체는 두개의 시스테인이 결합하는 130kD의 단백질이고, IL-4Rα+γｃ형과 IL-4Rα+IL-13Rα 수용체가 존재한다. 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-4는 활성화된 B세포와 T세포를 자극하거나, B세포를 형질세포로 분화시키는 등 많은 생물학적 기능을 갖고 있고, 체액성 면역과 후천성 면역의 중요한 조절인자이다. 또한 IL-4는 B 전구세포에서 IgE 및 IgG4 생산이 가능한 B세포로 전환되는 종류변환(class swithching)을 유도할 뿐만 아니라, MHC클래스 II의 생산을 촉진한다고 알려져 있다. 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, which is converted from B progenitor cells to B cells capable of producing IgE and IgG4, as well as promotes the production of MHC class II.
IL-4의 과량 분비는 IgE의 증가를 야기하여 즉시 발현되는 과민 알러지 반응을 일으킨다. IL-4는 IL-13과 상승 작용을 하고 IgE 또는 호산구 매개성 염증 반응의 원인 물질이다. Excessive secretion of IL-4 causes an increase in IgE, resulting in an immediate hypersensitivity allergic reaction. IL-4 acts synergistically with IL-13 and is the causative agent of IgE- or eosinophil-mediated inflammatory responses.
그런데, IL-4 유전자 불활성화 생쥐에서는 전체 T-세포의 생성 및 다른 면역 반응에는 문제가 없었으나, 그러한 생쥐에서는 Th2 세포의 생성과 IgE의 생성은 저하된다는 사실에 주목하면, IL-4 억제제는 면역계에 미치는 부작용이 완화된 아토피 치료제가 될 수 있다. 따라서 IL-4 억제제를 여러가지 면역질환 치료제로 개발하는 다양한 시도가 이루어지고 있으며, 특히 아토피, 건선, 두드러기 치료제로의 개발이 다양하게 시도되고 있다. 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 were reduced in such mice. It can be a treatment for atopic dermatitis with alleviated side effects on the immune system. Therefore, various attempts have been made to develop IL-4 inhibitors as therapeutic agents for various immune diseases, and in particular, various attempts have been made to develop them as therapeutic agents for atopy, psoriasis, and urticaria.
한편, 인터류킨13은 아미노산 122개로 이루어진 단백질로서 IL-13의 수용체는 CD132(γc)이다. IL-13은 IL-4와 기능적으로 유사하고 Th2세포, 수지상 세포, 비만세포에서 분비되며, Th2 세포의 생성 및 IgE 생성을 억제하여 B세포 증식분화, Th1을 제어하고, 알레르기, 천식 증상을 완화시킨다. On the other hand, interleukin 13 is a protein composed 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
IL-13에 대한 수용체는 IL-13Rα와 IL-4Rα로 구성되므로 IL-13 수용체는 IL-4에도 반응한다. Il-13에 대한 수용체는 사람의 B세포에서는 발현되지만, 생쥐의 B세포에서는 발현되지 아니한다. IL-13은 모노사이트를 수지상 세포로 분화시키도 한다.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.
그런데 IL-13 유전자 불활성 생쥐에서는 Th2 세포 또는 IgE를 활발하게 생산하지 못하는 것으로 보고되고 있다. 따라서, IL-13 생성을 저해하거나 작용을 차단하면 IgE의 생성이 억제되므로, IL-13 억제제나 IL-13 신호전달 차단제는 아토피나 건선, 알레르기 치료제로 사용될 수 있다. 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.
외부 물질에 의한 알러지 반응은 면역 자극물질의 양에 대해서는 무관하고 질에 대해서는 예민하게 반응하는 면역반응이고, 자가면역질환은 내부물질에 면역계가 반응하여 인체 일부 또는 전부를 공격하는 것이므로, 알러지나 자가면역질환은 인체 건강 유지에 반드시 필요한 면역 반응이 너무 과도하거나 해로운 형태로 발현되는 면역 질환이다. 본 발명의 IL-4 또는 IL-13 억제제 조성물은 IgE 생성을 억제하여, 알러지, 건선, 아토피 치료제로 사용이 가능하다. An allergic reaction caused by an external substance is an immune response that is sensitive to the quality and is irrelevant 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 inhibits IgE production, and thus can be used as a treatment for allergies, psoriasis, and atopic dermatitis.
한편, 알데히드탈수소 반응은 아세트알데히드나 인체 대사 산물에 존재하는 다양한 알데히드를 노폐물로 배출될 수 있는 유기산으로 변화시키는 반응이다. 이러한 알데히드탈수소 반응을 촉진하는 효소는 세포에 대한 독성을 나타내는 알데히드를 분해하여 세포와 조직에 가해지는 카보닐 스트레스를 완화시킨다. On the other hand, the aldehyde dehydrogenation reaction is a reaction for changing acetaldehyde or various aldehydes present in human metabolites into organic acids that can be discharged as waste products. The enzyme that promotes this aldehyde dehydrogenation reaction decomposes the aldehyde, which is toxic to cells, and relieves carbonyl stress applied to cells and tissues.
알데히드탈수소 반응에 개입하는 효소의 작용기를 함유하는 일련의 단백질들을 알데히드탈수소 효소(이하 ALDH라고 약칭함)라고 지칭하며 현재까지 약 20여종의 ALDH가 알려져있다. 따라서 본명세서에서 ALDH라는 용어의 범위에는 현재까지 알려진 20여종의 ALDH 전부와 알데히드탈수소 반응에 개입하는 효소의 탈수소 반응 작용기를 전부 또는 일부분을 포함하는 모든 융합단백질, 그리고 알데히드탈수소 반응에 개입하는 효소의 탈수소 반응 작용기의 일부분 또는 전부를 포함하는 모든 재조합 단백질을 모두 포함한다. 본 발명의 ALDH함유 조성물은 IL-4 또는 IL-13을 억제하여 IgE를 감소시켜, 알러지, 건선, 아토피 병증을 완화한다.A series of proteins containing functional groups of enzymes involved in aldehyde dehydrogenation are referred to as aldehyde dehydrogenases (hereinafter abbreviated as ALDH), and about 20 types of ALDH are known so far. Therefore, in the present specification, the scope of the term ALDH includes all of the 20 known types of ALDH, all fusion proteins containing all or part of the dehydrogenation functional groups of enzymes involved in aldehyde dehydrogenation, and enzymes involved in aldehyde dehydrogenation. All recombinant proteins containing some or all of the dehydrogenation functional groups are included. The ALDH-containing composition of the present invention reduces IgE by inhibiting IL-4 or IL-13, thereby alleviating allergies, psoriasis, and atopic diseases.
WO2016021847에서는 쌀 프롤라민(prolamin)을 활성성분으로 포함하는 것을 특징으로 하는 경구용 항(抗)아토피 조성물을 기재하고 있다. 이 문헌에 기재된 조성물은 항아토피 효능을 가질 뿐만 아니라 상용식품의 단백질을 활성성분으로 포함함으로써 안전성이 높아 광범위하게 이용될 수 있다. WO2008/044894에서는 대나무 추출물을 함유하는 아토피 억제 조성물을 공개하고 있다.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.
한편 국제공개번호 WO 2017/134525로 공개된 국제출원 제PCT/IB2017/000166호에서는 IL4Rα, TRPA1, 또는 F2RL1을 표적화하는 RNA 복합체를 사용한 아토피 피부염 및 천식의 치료제를 공개하고 있다. 그러나, 이러한 문헌들에서는 본원 발명의 요지인 ALDH함유 조성물이 인터류킨-4 또는 인터류킨-13의 발현을 억제하거나, 할 수도 있다는 가능성에 대한 어떠한 암시나 언급도 하지 아니하고 있다. 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.
한편, 국제공개번호 WO 2013/078371호로 공개된 존스 홉킨스 유니버시티의 국제출원번호 PCT/US2012/066340에서는 아세트알데히드의 축적으로 인한 에탄올 독성을 감소시키기 위한 방법들 및 조성물들을 공개하고 있다. 이건 선행기술에서 공개하는 방법은 알데히드 탈수소효소(ALDH) 슈퍼패밀리의 적어도 하나의 구성원의 발현, 양, 및/또는 활성을 증가시키는 화합물을 포함하는 조성물을 세포에 투여하여 알데히드로 인한 세포 독성을 감소시키는 것이다.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 by administering to a cell a composition comprising a compound that increases the expression, amount, and/or activity of at least one member of the aldehyde dehydrogenase (ALDH) superfamily to reduce aldehyde-induced cytotoxicity. will make it
이건 선행기술에서는 세포에서 하나 이상의 아세트알데히드 탈수소효소의 발현, 양, 또는 효소 활성을 조정하는 적어도 하나의 화합물인 유기 황 화합물 설포라판 (1-이소티오시아네이토-4R-(메틸설피닐)부탄), 설포라판의 유도체, 설포라판의 유사체의 유효량을 투여하는 것을 포함하는, 대상체에서 아세트알데히드의 피크 혈액 수준들을 감소시키고 아세트알데히드의 이화 속도를 증가시키기 위한 방법과 아세트알데히드 탈수소효소의 발현이 유전자들 ALDH2, ALDH1A, ALDH3A1, ALDH1B1, ALDH 1a1 또는 이들의 조합 중 하나 이상에 의해 인코딩되는 방법을 제시하고 있다. 그러나, 이건 선행기술은 본원 발명의 요지인 ALDH가 인터류킨-4 또는 인터류킨-13을 억제하거나, 할 수도 있다는 가능성에 대한 어떠한 암시나 언급도 하지 아니하고 있다. This is in the prior art 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 analog 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 does not make any suggestion or mention the possibility that ALDH, which is the subject of the present invention, may inhibit or may inhibit interleukin-4 or interleukin-13.
한편, 국제공개번호 WO 2014/160185로 공개된 더 보드 오브 트러스티스 오브 더 리랜드 스탠포드 쥬니어 유니버시티의 국제출원번호 PCT/US2014/025993에는 미토콘드리아 알데히드 탈수소효소-2 (ALDH2) 활성의 조절인자로서 기능하는 화합물들, 및 이러한 화합물들의 제조 방법들이 공개되어 있다. 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.
이건 선행기술에서는 미토콘드리아 알데히드 탈수소효소-2 (ALDH2)을 상세하게 기재하고 있으며, 핵 게놈에서 인코딩되어 미토콘드리아 내부로 이동하는 4개의 동일한 서브유닛으로 이루어지는 사합체 단백질이며, 각각은 500개의 아미노산 잔기들로 구성되고, 이러한 사합체는 이합체의 이합체로서, 이합체를 형성하는 단량체들 사이의 경계면은 사합체를 형성하는 2개의 이합체들 사이의 경계면과 상이하며, 각각의 서브유닛은 3개의 주요 부위, 즉 알데히드 탈수소 반응 촉매 부위, 조효소 또는 NAD+-결합 부위, 및 올리고머화 부위를 공개하고 있다.This prior art describes in detail mitochondrial aldehyde dehydrogenase-2 (ALDH2), a tetrameric protein consisting of four identical subunits encoded in the nuclear genome and transported 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.
이건 선행기술은 ALDH2와 관련된 질병 및 상태들에는 허혈성 스트레스, 만성 자유-라디칼 관련 질병들, 급성 자유-라디칼 관련질병들, 니트로글리세린에 대한 무감응성 (예컨대, 협심증 및 심장 마비에서), 고혈압, 당뇨병, 골다공증, 암, 판코니 빈혈, 알츠하이머 병, 파킨슨병, 알코올의존증(alcoholism), 알코올 불내성, 알코올 탐닉(alcoholaddiction), 알코올 남용 질환, 알코올 도취(alcohol intoxication), 알코올 의존, 알코올 중독(alcohol poisoning),알코올 소비 증상, 및 마약 중독이 포함하여, ALDH2의 조절인자, ALDH2의 작용제, 또는 ALDH2의 길항제를 개시한다. 또한 ALDH2의 작용제는 에탄올, 메탄올, 에틸렌 글리콜 모노메틸 에테르, 폴리비닐 클로라이드, 이종 알데히드 및 생체내 알데히드와 같은 화합물의 수준을 피험체에서 감소시키기에 유용하다. 또한 ALDH2의 작용제는 ALDH2의 알데히드 기질을 발생시키는 화합물을 섭취, 흡수 또는 흡입시 피험체에서 ALDH2의 알데히드 기질을 발생시키는 화합물의 수준을 감소시키기에 유용하다. ALDH2의 길항제는 암과 같은 질환을 치료 또는 억제하는데 유용한데, 여기서 ALDH2 길항제는 표준 암 치료에 대한 보조제로서 사용된다. 또한 ALDH2의 길항제는 알코올의존증 및 마약 중독을 치료 또는 억제하는데 유용하다는 사실을 공개하고 있다. 그러나, 이건 선행기술 역시 본원 발명의 요지인 ALDH가 인터류킨-4 또는 인터류킨-13을 억제하거나, 할 수도 있다는 가능성에 대한 어떠한 암시나 언급도 하지 아니하고 있다. This prior art states that ALDH2 related diseases and conditions include ischemic stress, chronic free-radical related diseases, acute free-radical related diseases, insensitivity to nitroglycerin (eg, in angina and heart failure), hypertension, diabetes. , 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 the 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 about the possibility that ALDH, which is the subject of the present invention, may inhibit or may inhibit interleukin-4 or interleukin-13.
한편, 국제공개번호 WO 2015/127137로 공개된 아비브 테라퓨틱스, 인크.의 국제출원번호 PCT/US2015/016703호는 암의 치료를 위한 폴리사이클릭 아미드와 결합하는 미토콘드리아 알데히드 탈수소효소 2(ALDH2) 및 그의 용도를 개시하고 있다. 이건 선행 기술은 미토콘드리아 알데히드 탈수소효소-2 (ALDH2)와 결합하는 하기 식 I의 화합물 및 즉, 암, 판코니 빈혈및 알코올-관련 장애를 갖는 환자를 치료하기 위한 상기 화합물의 사용 방법을 제공한다. 이건 선행기술 문헌도 본원 발명의 요지인 ALDH함유 조성물이 인터류킨-4 또는 인터류킨-13을 억제할 수도 있다는 가능성에 대한 어떠한 암시나 언급도 하지 아니하고 있다. 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 about 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. 이 문헌에는 19가지 종류의 ALDH가 생체외부로부터 체내로 공급되는 알데히드나 생체 내에서 생성되는 다양한 종류의 알데히드를 분해하는 역할을 하여 생체 독성 제거에 매우 중요한 역할을 수행하고, 인체 조직 세포에 강력한 산화 반응을 야기하는 알데히드의 작용을 저해하는 약물들에 대하여 상세하게 기재하고 있다. 이상의 선행 기술에서는 ALDH들이 IL-4 또는 IL-13 억제하여 면역조절제 용도를 갖는다는 것을 암시하거나 제시하지 못하였다.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.
본 발명자는 알데히드 탈수소 반응 촉진 부위를 포함하는 여러가지 ALDH 융합단백질, 재조합 단백질들 전부 또는 그들의 일부가, IL-4 또는 IL-13을 억제한다는 새로운 사실을 확인하여, 본 출원의 ALDH함유 조성물이 의약 또는 기능성 식품으로서의 새로운 용도를 발명하게 되었다. The present inventors confirmed the novel fact that all or part of various ALDH fusion proteins and recombinant proteins containing an aldehyde dehydrogenation reaction promoting site inhibit IL-4 or IL-13, so that the ALDH-containing composition of the present application can be used as a pharmaceutical or New uses as functional foods have been invented.
본 발명자는 ALDH함유 조성물을 사용하여, Th2 세포로 부터의 IL-4와 IL-13의 분비를 억제함으로써 그로 인해 활성화된 B림파구의 IgE 생성도 줄어들게 되어 비만세포가 탈과립하여 생산하는 히스타민 등의 염증 매개 물질의 생성도 줄어들어서, IgE 생성의 증가로 인한 질병들을 예방하거나 치료할 수 있다는 사실을 확인하였다. The present inventors inhibit the secretion of IL-4 and IL-13 from Th2 cells by using the ALDH-containing composition, thereby reducing the IgE production of activated B lymphocytes, thereby reducing inflammation such as histamine produced by degranulation of mast cells. The production of mediators was also reduced, confirming the fact that diseases caused by an increase in IgE production could be prevented or treated.
본 발명자는 세포의 대사과정에서 발생하는 활성산소종(reactive oxygen species, ROS)이 과도하게 생성될 경우, 면역계통의 이상을 유발하고, 자가면역질환의 발병 원인 중 하나로서 IL-4와 IL-13의 발현이 증가되고, 이로 인한 산화적 스트레스가 폐에서 평활근의 수축, 혈관의 투과성 증가, 약물 반응 수용체의 손상, 점액 분비의 증가, 염증성 매개자의 분비, 기도 상피 세포의 손상을 야기하여 아토피, 건선, 알러지성 피부염, 기관지 천식을 포함한 다양한 질병의 원인으로 작용한다는 사실도 확인하게 되었다.The present inventors found that when reactive oxygen species (ROS) generated in the metabolic process of cells is 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 be the cause of various diseases.
본 발명의 조성물의 IL-4와 IL-13 억제 실험 결과를 통하여, ALDH의 IL-4 또는 IL-13 억제 효과와 현재까지 보고된 IL-4 또는 IL-13의 인체 면역계에 미치는 효과에 의하여, ALDH의 면역조절제로서의 새로운 의약품 용도를 가능하게 한다. 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.
따라서 본 발명의 1차적인 목적은 IL-4 또는 IL-13 억제하는 알데히드탈수소 효소와 탈수소 반응을 촉진하는 부위를 포함하는 융합단백질 또는 재조합 단백질의 전부 또는 그의 일부(이하 ALDH라고 약칭함)를 함유하는, 아토피, 알러지, 건선 등의 면역질환 치료제를 제공하는 것이다. Therefore, the primary object of the present invention is to contain all or a part of a fusion protein or recombinant protein (hereinafter abbreviated as ALDH) comprising an aldehyde dehydrogenase that inhibits IL-4 or IL-13 and a site that promotes a dehydrogenation reaction. It is to provide a therapeutic agent for immune diseases such as atopy, allergy, and psoriasis.
이러한 본 발명의 1차적인 목적은 알데히드탈수소 반응에 간여하는 작용 부위를 포함하는 융합단백질 또는 재조합단백질을 포함하는 알데히드탈수소 반응 촉진 효소들과 그 변이체들을 유효성분으로서 함유하는, 조성물을 제공함으로써 달성된다. The primary object of the present invention is to provide a composition containing, as an active ingredient, aldehyde dehydrogenation promoting enzymes and variants thereof, including fusion proteins or recombinant proteins comprising a site of action involved in aldehyde dehydrogenation reaction. .
본 발명의 또 다른 목적은, 알데히드탈수소 반응에 간여하는 작용부위를 포함하는 융합단백질 또는 재조합단백질을 포함하는 알데히드탈수소 반응 촉진효소들과 그 변이체를 유효성분으로 함유하는, 면역반응 억제제 또는 자가면역질환 치료 및 예방을 위한 식품 조성물을 제공하는 것이다. Another object of the present invention is an immune response inhibitor or autoimmune disease containing aldehyde dehydrogenation promoting enzymes and variants thereof as active ingredients, including fusion proteins or recombinant proteins comprising a site involved in aldehyde dehydrogenation reaction. To provide a food composition for treatment and prevention.
본 발명의 또 다른 목적은, 알데히드탈수소 반응에 간여하는 작용 부위를 포함하는 융합단백질 또는 재조합단백질을 포함하는 알데히드탈수소 효소들과 그 변이체를 유효성분으로 함유하는, 아토피, 건선, 알러지 피부염 치료제를 제공하는 것이다.Another object of the present invention is to provide a therapeutic agent for atopic dermatitis, psoriasis, and allergic dermatitis, which contains aldehyde dehydrogenases and variants thereof as active ingredients, including fusion proteins or recombinant proteins comprising a site of action involved in aldehyde dehydrogenation reaction will do
전술한 본 발명의 목적들은, 알데히드탈수소 효소의 알데히드 탈수소 반응을 촉진하는 작용 부위 일부 또는 전부를 함유하는 융합단백질, 재조합단백질을 포함하는 알데히드탈수소 효소와 그 변이체들(이하 ALDH라고 약칭함)을 함유하는, 아토피, 건선, 알러지 피부염 치료제를 제공함으로써 달성될 수 있다.The above-described objects of the present invention, aldehyde dehydrogenase and its variants (hereinafter abbreviated as ALDH) containing a fusion protein containing a part or all of the action site that promotes the aldehyde dehydrogenase reaction of the aldehyde dehydrogenase, recombinant protein containing It can be achieved by providing a therapeutic agent for atopic dermatitis, psoriasis, and allergic dermatitis.
이상과 같은 본 발명의 목적과 해결 수단의 요지는, 공지의 물질인 ALDH의 IL-4 또는 IL-13 억제제로서의 새로운 용도이다. 따라서 본 발명의 IL-4 또는 IL-13 억제제로 사용될 수 있는 ALDH에는, 현재까지 알데히드탈수소 반응 활성을 나타내는 공지된 물질인 다양한 모든 ALDH가 포함될 수 있기 때문에 이들은 모두가 본 발명의 아토피, 건선, 알러지 피부염 치료제 의 유효성분으로 사용될 수 있고, 본 발명의 권리범위에 포함된다.The gist of the object and solution of the present invention as described above is a novel use of a known substance, ALDH, as an IL-4 or IL-13 inhibitor. Therefore, the ALDH that can be used as an IL-4 or IL-13 inhibitor of the present invention can include all of the various ALDHs, which are known substances exhibiting aldehyde dehydrogenation activity to date, and all of these are atopic dermatitis, psoriasis, and allergies of the present invention. It can be used as an active ingredient in a dermatitis treatment agent, and is included in the scope of the present invention.
또한 이러한 IL-4 또는 IL-13 억제 활성을 나타내는 ALDH를 생산하는 균주를 제공하기 위하여, 본 발명자들은 부다페스트 조약에 따라 국제 기탁기관(KCTC)에 기탁번호 KCTC13925BP( Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2), KCTC14123BP( Saccharomyces cerevisiae KwonP-3) ALDH 생산 균주를 기탁하였고, 여러가지 공지된 ALDH의 IL-4 또는 IL-13 억제 효과에 따른 아토피, 건선, 알러지 피부염 치료제 측정 시험 결과를 본 출원의 명세서를 통하여 공개한다. In addition, in order to provide a strain producing ALDH exhibiting such IL-4 or IL-13 inhibitory activity, the present inventors have deposited with the International Depositary Organization (KCTC) in accordance with the Budapest Treaty, deposit number KCTC13925BP ( Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2), KCTC14123BP ( Saccharomyces cerevisiae KwonP-3) ALDH-producing strains were deposited, and the measurement test results for atopic, psoriasis, and allergic dermatitis treatments according to the IL-4 or IL-13 inhibitory effect of various known ALDHs were reviewed. It is disclosed through the specification of the application.
본 발명의 ALDH 또는 ALDH를 함유하는 조성물은 인터류킨-4 또는 인터류킨-13의 억제 작용에 따른 다양한 면역 질환들에 대한 예방 및 치료하는 약학적 효과를 나타낸다. ALDH or a composition containing ALDH of the present invention exhibits a pharmaceutical effect for preventing and treating various immune diseases due to the inhibitory action of interleukin-4 or interleukin-13.
본 발명에 따른 효모추출물에 함유된 ALDH는 도 1의 생체내 실험(in vivo)에서 천식 유발 마우스의 기관지 폐포 세척액에서 면역글로불린 E(IgE) 및 전체 염증 세포수 및 림파구, 단핵구, 호중구, 호산구의 증가를 억제시키는 활성, 기도 점액분비를 억제하는 활성을 가지고, Th2 사이토카인인 IL-4와 IL-13 및 기도 염증 을 감소시킴으로써, 아토피, 건선, 알러지 피부염 치료제 및 건강식품으로도 사용될 수 있다. ALDH contained in the yeast extract according to the present invention showed immunoglobulin E (IgE) and total number of inflammatory cells and lymphocytes, monocytes, neutrophils, and eosinophils in the bronchoalveolar lavage fluid of asthma-induced mice in the in vivo experiment of FIG. It has the activity of inhibiting the increase and the secretion of airway mucus, and by reducing the 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 as a health food.
따라서 본 발명에 따른 ALDH함유 조성물은 염증, 알레르기, 천식 등과 같은 산화 스트레스에 의한 염증반응의 치료 및 예방에 효과적이다. 특히, ALDH는 식용 효모에서 추출한 것이므로 복용하여도 부작용이 발생할 가능성이 낮다는 장점이 있다.Therefore, the ALDH-containing composition according to the present invention is effective in treating and preventing inflammatory reactions caused by oxidative stress such as inflammation, allergy, asthma, and the like. In particular, since ALDH is extracted from edible yeast, it has the advantage that the possibility of side effects is low even if it is taken.
도 1은 아토피 유발 대조군 및 실험군 마우스에서의 혈중 IgE의 변화를 나타낸 그래프이다. A: CON, B: DNCB로 아토피 유발군, C: 덱사메타손 투여군, D: 본 발명의 조성물 50mg/kg, E: 본 발명의 조성물 150mg/kg, F: 본 조성물 300 mg/kg 투여군1 is a graph showing changes in blood IgE in atopic-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
도 2는 아토피 유발 대조군 및 실험군 마우스에서의 혈중 백혈구(WBC)변화를 나타낸 그래프이다. DNCB유도로 증가된 혈중 백혈구 양을, 덱사메타손 투여의 경우 65% 감소시키고, 본 발명의 조성물 투여(150mg/Kg)의 경우 약 74% 감소시켰다. 2 is a graph showing changes in blood leukocytes (WBC) in atopy-induced control group and experimental group mice. DNCB-induced increase in the amount of leukocytes in the blood was reduced by 65% in the case of dexamethasone administration, and reduced by about 74% in the case of administration of the composition of the present invention (150mg/Kg).
도 3은 아토피 유발 대조군 및 실험군 마우스에서의 호산구(Eosinophil)의 변화를 나타낸 그래프이다. DNCB에 의한 아토피 유도로 증가된 혈중 에오시노필이 덱사메타손을 투여하는 경우 60%감소되었고, 본 발명의 조성물(150mg/Kg)을 투여하면 약 55% 감소되었다. 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 of the present invention (150mg/Kg) was administered.
도 4는 아토피 유발 대조군 및 실험군 마우스에서의 혈중 호중구(neutrophil) 변화를 나타낸 그래프이다. DNCB 유도로 증가된 혈중 호중구를 덱사메타손 투여로 35% 감소시켰고, 본 발명의 조성물 투여(150mg/Kg)를 통해 약 46% 감소시켰다. 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).
도 5은 아토피 유발 대조군 및 실험군 마우스에서의 IL-6 변화를 나타낸 그래프이다. 5 is a graph showing changes in IL-6 in atopy-induced control group and experimental group mice.
도 6은 아토피 유발 대조군 및 실험군 마우스에서의 IL-6 mRNA변화를 나타낸 그래프이다. 6 is a graph showing changes in IL-6 mRNA in atopy-induced control and experimental mice.
[규칙 제91조에 의한 정정 06.03.2021]　
[Correction by Rule 91 06.03.2021]
[규칙 제91조에 의한 정정 06.03.2021]　
[Correction by Rule 91 06.03.2021]
이하, 본 발명의 알데히드탈수소효소를 함유하는 아토피, 건선, 알러지 피부염 치료제의 유효성분 제조 과정, 분리 정제 과정, 약리효과와 대조 실험, 유효량 측정, 투여제형과 투여방법독성에 대한 구체적인 실시예를 설명한다. 그러나 이러한 실시예들은 본 발명의 예시를 위한 것으로서 본 발명의 권리범위를 이들로만 한정하고자 하는 것은 아니다. Hereinafter, the production process of the active ingredient of the treatment agent for atopic dermatitis, psoriasis, and allergic dermatitis containing the aldehyde dehydrogenase of the present invention, separation and purification process, pharmacological effect and control experiment, effective amount measurement, dosage form and administration method Toxicity will be described. do. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
[실시예 1] 본 발명의 ALDH 함유 효모 용해물 준비 [Example 1] Preparation of ALDH-containing yeast lysate of the present invention
실시예1-1: 본 발명의 조성물 준비를 위한 사카로마이세스세레비지에 효모 발효 과정 Example 1-1: Saccharomyces cerevisiae yeast fermentation process for preparing the composition of the present invention
ALDH 함유 사카로마이세스세레비지에 효모의 종균(KCTC13925BP)을 200 mL flask에 YPD 배지(효모추출물, 펩톤, 글루코즈 함유 배지)를 사용하여 160 rpm, 30℃ 조건의 인큐베이터에서 24시간 동안 발효하여 배양했으며, 본 배양은 5L 발효기 (Marado-05D-PS, CNS, Korea)을 통하여 72시간 동안 진행하였다. 배양 종료 후 고속원심분리기(Supra R22, Hanil, Korea)를 이용하여 효모를 원심분리하였다.The ALDH-containing Saccharomyces cerevisiae yeast seed (KCTC13925BP) was fermented for 24 hours in an incubator at 160 rpm and 30°C using YPD medium (a medium containing yeast extract, peptone, and glucose) in a 200 mL flask and cultured. and this 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).
실시예1-2: ALDH 함유 효모 용해물 준비 과정Example 1-2: ALDH-containing yeast lysate preparation process
원심분리된 ALDH 함유 효모를 초저온냉동고(CLN-52U, Nihon freezer, Japan)에서 2일간 냉동시킨 후 동결건조기(FDU-7006, Operon, Korea)로 2일간 동결건조를 진행하였다. 동결건조된 효모 파우더 3 g에 단백질분해효소억제제(A32955, Thermo fisher, USA)를 넣은 50 mL 인산완충식염수(phosphate-buffered saline, PBS)에 녹인 후, 0.5 mm 세포파쇄용유리구슬 (11079105, Biospec) 10 g을 넣고 비드호모나게이저(Mixer Mill MM400, Retsch, Germany)로 2분씩 총 3번에 걸쳐 효모 파쇄시켰다. 고속원심분리기(Supra R22, Hanil, Korea)를 이용하여 원심분리한 후 상등액만 분리하여 동결건조기(FDU-7006, Operon, Korea)로 2일간 동결건조를 진행하였다. The centrifuged ALDH-containing yeast was frozen in a cryogenic freezer (CLN-52U, Nihon freezer, Japan) for 2 days, and then freeze-dried for 2 days in a freeze dryer (FDU-7006, Operon, Korea). 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) to disrupt yeast over a total of 3 times for 2 minutes each. 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).
[실시예 2] 본 발명의 ALDH 함유 효모 용해물 조성물의 아토피 억제 효과 [Example 2] Atopy inhibitory effect of ALDH-containing yeast lysate composition of the present invention
실시예 2-1. 아토피 유발 시험 동물의 제작. Example 2-1. Preparation of atopy-induced test animals.
평균 체중 20g 내외의 7주령 암컷 BALB/c 마우스를 ㈜샘타코바이오코리아에서 구입하였다. 마우스 피부염 유도제 DNCB(1-Chloro-2,4-dinitrobenzene; 마우스 피부염 유도제, Junsei) 40 mg을 acetone과 olive oil의 비율이 4:1로 혼합된 용액에 녹여 1% DNCB solution을 만들어 유도 1주차에 아토피 유발을 위한 시험에 사용하였다. 유도 1주차 첫 날, 마우스의 등 부분의 털을 1.5 cm 2의 면적으로 깎은 후, 1% DNCB solution을 1주일 중 총 4일간 한 마리에 100 μL 씩 뿌려주고 부드러운 브러쉬로 고루 도포하였다.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 the test for atopy induction. On the first day of the first 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.
유도 2주차에는 DNCB 80 mg을 acetone과 olive oil이 4:1의 비율로 혼합된 용액에 녹여 2% DNCB solution을 만들었다. 2주차 2% DNCB solution을 120 μL 씩 1주차와 동일한 방식으로 도포했다. (A.F. Hamad. et al., Bangladesh J Pharmacol, 12:147-150, 2017) 아토피 피부염 유도 2주차 시점부터 본 발명의 ALDH함유 효모 추출물 분말을 생리식염수에 녹여 마우스에게 경구 투여를 총 5일간 진행하였다. 본 발명의 조성물을 50mg/kg/day, 150mg/kg/day, 300mg/kg/day의 양으로 경구 투여하였다. 대조군에는 덱사메타손(Dexamethasone)을 투여하였다. 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, 120 μL each. (AF 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.
실시예 2-2. 동물의 부검 Example 2-2. autopsy of animals
유도 2주 차, 5일간 본 발명의 조성물을 경구 투여하고 다음 날 부검하였다. 한 그룹에 총 5마리가 있으며 5마리 전부 혈액, 등 피부, 비장을 적출하였으며, 등 피부 조직과 비장은 2등분하여 하나는 cytokine의 분비량을 확인하기 위한 용도로, 나머지 하나는 유전자 발현을 확인하는 용도로 사용하기 위해 -80℃에 보관하였다. 혈액은 혈구분석을 위해 채취한 혈액 중 일부를 해파린 튜브로 1.5 mL E-tube에 옮겨 담고, 나머지 혈액은 혈청의 IgE 분비량을 분석하기 위해 1.5 mL E-tube에 보관하였다. The composition of the present invention was orally administered in the second week of induction and for 5 days, and autopsies were 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.
실시예 2-3. 아토피 유발 마우스에서 혈중 IgE 함량 분석 Example 2-3. Analysis of blood IgE content in atopic mice
시험 동물에서 채취한 혈액은 1.5 mL E-tube 담아 12,000 rpm, 4℃ 조건에서 20분간 원심분리를 진행하였다. 그 후, 상층액만 분리하여 실험에 사용하였다. IgE의 정량은 Enzyme-Linked Immunosorbent Assay (ELISA)방법으로 수행 되었으며, IgE ELISA set (BD bioscience)를 활용하여 정량 분석을 진행하였다. 반응 진행 후, 최종 450nm 파장에서의 흡광도를 ELISA reader (Multiskan sky, Thermo Fisher, USA)로 측정한 결과는 도 1에 나타내었다. 본 발명의 ALDH함유 조성물 투여 그룹은 아토피가 유도된 실험군 대비 약 40% 이상의 IgE가 감소하였다. (도 3) 아토피 유발 그룹(DNCB) 대비 본 발명의 조성물 투여군(150mg/Kg)에서 IgE가 38% 감소 하였고, 본 발명의 조성물 투여군(300mg/Kg)에서는 IgE가 45% 감소하였다. 본 발명의 조성물의 아토피 치료 유효량은 마우스 동물 시험에서 150mg/Kg으로 평가되었다. The 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 ALDH-containing 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). Atopic therapeutically effective amount of the composition of the present invention was evaluated as 150 mg/Kg in a mouse animal test.
실시예 2-4. 아토피 유발 마우스에서 본 발명의 ALDH함유 조성물에 의한 인터류킨-13 억제 약리 효과 확인Example 2-4. Confirmation of the pharmacological effect of inhibiting interleukin-13 by the ALDH-containing composition of the present invention in atopic mice
혈중 인터류킨-13의 정량은 각 Enzyme-Linked Immunosorbent Assay (ELISA)방법으로 수행하였다. 마이크로 웰(Micro well) 플레이트에 완충액에 희석한 Capture 항체를 넣고 4 ℃에서 16시간 방치 한후, 워싱(washing)한 후 시험 물질을 넣고 반을 시켜 최종 450 nm 파장에서의 흡광도를 ELISA reader (Multiskan sky, Thermo Fisher, USA)로 측정하여 혈중 인터류킨-13 mRNA의 양을 확인하였다. 채취한 마우스 등 피부 조직에서 RNA를 추출하여 인터류킨-13에 대한 실시간 중합 효소 반응을 수행하였다. 본 발명의 ALDH함유 효모 용해물에 의한 인터류킨-13 mRNA의 감소를 확인하였다.(도 7). 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 ℃ 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. It was confirmed that interleukin-13 mRNA was decreased by the ALDH-containing yeast lysate of the present invention (FIG. 7).
실시예 2-5. 아토피 유발 마우스의 혈구 분석 Example 2-5. Hematology analysis of atopic mice
아토피, 천식을 포함하는 알레르기성 반응에서 호산성, 호중구성 혈구등의 조직내 침윤 과정은 특징적인 현상이다 (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). 특히, 호산구 형태의 세포가 더 많이 분화 할수록 골수가 아토피 상태로 편향 되어 있어 알레르기 염증반응을 유발 할 수 있다. 본 발명에서는 아토피를 유도한 마우스에서의 백혈구, 호산구, 호중구등의 혈구 분석을 시행하여 본 발명의 ALDH함유 효모 용해물의 약리 효과를 확인하였다. 혈구의 분석은 해파린 튜브를 이용해 담아놓은 혈액을 부검이 끝난 즉시 혈구분석기(Hemavet 950FS, Drew Scientific Inc, Korea)를 활용하여 실시하였다. 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, blood cells such as leukocytes, eosinophils, and neutrophils were analyzed in atopic-induced mice to confirm the pharmacological effect of the ALDH-containing yeast lysate of the present invention. The analysis of blood cells was performed immediately after the autopsy of the blood contained in the heparin tube using a hemocytometer (Hemavet 950FS, Drew Scientific Inc, Korea).
DNCB유도로 증가된 혈중 백혈구(WBC)를 덱사메타손 (dexamethasone; Dex.)투여의 경우 백혈구를 65% 감소시켰으며, 본 발명의 조성물 150mg/kg 투여의 경우 혈중 백혈구를 약 74%까지 감소시켰다(도 2). 혈중 호중구(NE)함량은 dexamethasone투여의 경우 35% 감소시키고, 본 발명의 조성물 150mg/kg 투여의 경우 호중구를 약 46% 감소 시켰으며(도 4), 아토피와 같은 알러지 반응에서 특징적으로 타나타는 혈중 호산구(EO) 함량을 덱사메타손 투여의 경우 호산구를 60% 감소시키고, 본 발명의 조성물 150mg/kg 투여의 경우 약 호산구를 55% 까지 감소시킴으로서(도 3), 본 발명의 ALDH함유 조성물이 대조군 대비 유사하거나 우수한 약리 효과를 나타내었다. In the case of dexamethasone; 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 neutrophil (NE) content characteristically 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.
실시예 2-6 아토피 유발 마우스와 본 발명의 조성물 투여군의 IL-6 변화 분석 Example 2-6 Analysis of IL-6 changes in atopic-induced mice and the group administered with the composition of the present invention
DNCB 용액으로 2주간 아토피를 유도한 Balb/c 마우스에 5일간 본 발명의 조성물을 경구 투여하였다. 혈구분석은 serum, ELISA와 RT-PCR은 피부조직을 시료로 사용하였으며, 덱사메타손을 positive control로 사용하였다. 혈구분석및 피부 조직의 IL-6 cyokine의 발현 정도를 측정하였다. 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.
마우스 피부조직 내 IL-6 발현량을 ELISA assay와 realtime PCR로 분석하여, IL-6의 단백질 및 mRNA level 을 정량 하였다. 분석 결과 덱사메타손 투여 군 대비 본 발명의 조성물 투여군에서 동등 또는 우위의 효과를 확인하였다. DNCB유도로 증가된 피부 조직 내 IL-6함량을 덱사메타손 투여의 경우 50 % 감소시켰고, 본 발명의 조성물 투여(150mg/Kg)의 경우 약 48 % 감소시켰다.(도 5) DNCB유도로 증가된 피부 조직 내 IL-6 mRNA level 분석 결과 덱사메타손 투여의 경우 34% 감소시켰고, 본 발명의 조성물 투여(150mg/Kg)의 경우 약 77% 감소시켰다. (도 6) IL-6 expression levels in mouse skin tissues were analyzed by ELISA assay and real-time PCR to quantify IL-6 protein and mRNA levels. 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 (150mg/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 (150mg/Kg) was reduced by about 77%. (Fig. 6)
[실시예 3][Example 3]
실시예 3-1 : TNF-α와 IFN-γ를 이용한 아토피 피부염 인간 세포 모델 제작Example 3-1: Preparation of atopic dermatitis human cell model using TNF-α and IFN-γ
세포는 인간 각질형성세포인 HaCaT 세포를 사용하였다. 배지는 DMEM (Dolbicco’s Modified Eagle’s Medium, USA)에 10% 소태아혈청(Fetal bovine serum: FBS)과 + 1% P/S (Penicillin/Streptomycin:P/S)를 첨가한 배지를 사용하였으며, seeding하고 23시간 후 본 발명의 ALDH함유 조성물을 0.5, 1, 2 μg/mL의 농도로 처리하고 1시간 후 10 ng/mL의 TNF-α와 IFN-γ를 처리하여 염증을 유도하였다.HaCaT cells, which are human keratinocytes, were used as the cells. 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 ALDH-containing composition of the present invention was treated at concentrations 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.
실시예 3-2 : 결과분석Example 3-2: Result Analysis
(1) 아토피 피부염 세포 모델의 cytokine 변화 분석(1) Analysis of cytokine changes in atopic dermatitis cell model
HaCaT cell에 TNF-α와 IFN-γ로 염증을 유도하고 1시간 뒤 6-well plate의 배지를 E-tube에 옮겨담고, 13,000 rpm, 4℃ 조건에서 10분간 원심분리를 진행하였다. 이후 상층액만 새로운 튜브로 옮겨 실험에 사용했다. Inflammation was induced in HaCaT cells with TNF-α and IFN-γ, and after 1 hour, the medium in a 6-well plate was transferred to an E-tube, and centrifugation was performed at 13,000 rpm and 4°C for 10 minutes. After that, only the supernatant was transferred to a new tube and used for the experiment.
Enzyme-Linked Immunosorbent Assay (ELISA) : 분석을 위해 IgE ELISA set (BD bioscience, US)를 사용하였으며 micro well에 coating Buffer에 희석한 capture Ab를 넣고 4 ℃에서 overnight 하였다. 3번의 washing 후 각 well에 assay diluent를 넣고 실온에서 incubation 하고, 3번의 washing 후 각 well에 assay diluent로 희석한 standard, sample을 넣고 실온에서 incubation 하였다. 5번의 washing 후 각 well에 working detector를 넣고 실온에서 incubation하고 7번의 washing 후 각 well에 substrate solution을 넣고 실온의 암조건에서 30분간 incubation 진행하였다. 반응 종료를 위해 각 well에 Stop Solution 50 μL을 넣고 450 nm 파장에서의 흡광도를 ELISA reader (Multiskan sky, Thermo Fisher, USA)로 측정하였다.Enzyme-Linked Immunosorbent Assay (ELISA): For the analysis, an IgE ELISA set (BD bioscience, US) was used. Capture Ab diluted in coating buffer was added to micro wells 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 added to each well and incubated for 30 minutes in dark conditions at room temperature. To terminate the reaction, 50 μL of Stop Solution was added to each well, and absorbance at 450 nm wavelength was measured with an ELISA reader (Multiskan sky, Thermo Fisher, USA).
(2) 아토피 피부염에 대한 본 발명의 효모 추출물 분말의 in vitro efficacy (2) In vitro efficacy of yeast extract powder of the present invention for atopic dermatitis
Human keratinocyte HaCaTcell line에 본 발명의 조성물을 각 농도에 따라 20분 동안 전 처리하고 이렇게 전처린된 세포에 TNFα/IFNγ를가하여 세포내 염증 상태를 유발 시켰다. 아토피 질병 동물 모델의 주요 marker인 IL-6 발현정도를 측정하였다. 아토피 유도 세포주의 IL6 발현량대비, 본 발명의 조성물이 처리된 세포에서 각 농도별로 0.03%, 14%, 27%의 IL-6 발현량 감소를 확인하였다. (도 8)The human keratinocyte HaCaTcell line was pretreated with the composition of the present invention at each concentration for 20 minutes, and TNFα/IFNγ was added to the pretreated cells to induce an intracellular inflammatory state. The expression level of IL-6, a major marker of atopic disease animal models, was measured. Compared to the IL6 expression level of the atopic-induced cell line, it was confirmed that the IL-6 expression level decreased by 0.03%, 14%, and 27% for each concentration in the cells treated with the composition of the present invention. (Fig. 8)
[ 실시예 4] 본 발명의 조성물의 단회 투여 독성 시험 [ Example 4] Single dose toxicity test of the composition of the present invention
실시예 4-1. 실험 동물의 준비Example 4-1. Preparation of laboratory animals
실험동물은 암컷, 수컷 ICR 마우스(7 주령)를 분양받아 7일간 순화시켰으며 순화 기간 중 일반증상을 관찰하여 건강한 동물만 시험에 사용하였다. 사료와 물은 자유 섭취시켰고 경구투여 전날 평균 체중 약 20g을 기준으로 각 군별 암,수 5수씩 총 10수가 되도록 군 분리를 진행하였다.As experimental animals, female and male ICR mice (7 weeks old) were received and acclimatized for 7 days. During the acclimatization period, general symptoms were observed, and only healthy animals were used for the test. 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 the average body weight of about 20 g the day before oral administration.
실시예 4-2 시험 물질의 투여 Example 4-2 Administration of test substance
시험 물질은 본 발명의 ALDH 함유 효모 용해물의 함량을 기준으로 실험동물의 투여용량이 각 0, 750, 3000, 5000mg/Kg이 되도록 생리식염수에 녹여 제조하였다. 투여 용량의 기준은 식약처의 Korea national Toxicology Program(KNTP) 독성 시험 매뉴얼에 따랐다. KNTP 매뉴얼에서 가이드하는 적용 최대 용량 5000mg/Kg을 본 실험의 최대 농도로 적용하였다. 각 군별로 준비된 시료를 시험동물에 대하여 각 1회 경구투여를 실시 하였으며, 정상군(G1)의 경우 생리식염수를 투여하였다. 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).
실시예 4-3. 관찰 및 부검 Example 4-3. Observation and autopsy
모든 시험군의 동물에 대하여 입수일부터 부검일까지 매일 1회 이상 증상관찰을 실시하였으며, 경구투여 후 7일 동안 증상을 관찰하였다. 증상 관찰 종류 후, 부검을 진행하였고 부검 시 육안으로 각 장기에 대한 변화를 관찰하였다. Symptoms were observed at least once a day from the date of acquisition to the day of autopsy for all animals in the test group, 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.
마우스를 사용하여 본 발명의 ALDH 함유 조성물의 단회투여독성 시험을 실시한 결과, ALDH 효모 추출물 5000mg/kg까지의 농도에서 7일간 사망예를 관찰할 수 없었으며, 체중증가, 사료 섭취량 등의 특이점을 발견할 수 없었다. 또한 관찰 종료 후 진행한 부검 결과에서도 특이한 소견은 발견되지 아니하였다.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 Also, no unusual findings were found in the autopsy results after the observation was completed.
이상의 실시예에서, 본 발명의 알데히드탈수소 효소(ALDH)를 함유하는 건선 치료제, 아토피 치료제, 알러지 치료제의 제조 방법, 대조약물 대비 약리효과, 투여방법, 질병 모델에 대한 치료적 유효량, 급성독성에 대한 대표적인 사례들을 각 도면과 시험 결과들을 통하여 상세하게 설명하였지만, 이들은 본 발명의 하나의 실시 사례에 불과하다. In the above examples, psoriasis treatment, atopic treatment, and allergy treatment containing aldehyde dehydrogenase (ALDH) of the present invention, pharmacological effect compared to control drug, administration method, therapeutically effective amount for disease model, acute toxicity Representative examples have been described in detail through respective drawings and test results, but these are merely exemplary examples of the present invention.
따라서 이 기술 분야에서 통상의 지식을 가진 자라면 전술한 본 발명의 실시 사례로부터 다양한 변형 및 본 발명과 균등한 또 다른 실시예를 용이하게 도출할 수 있다. 그러므로 다음의 특허청구범위에 기재된 본 발명의 기술적 요지를 실현하는 어떠한 변형된 형태의 알데히드탈수소 효소를 함유하는 인터류킨4, 인터류킨13 억제에 따른 아토피 치료제, 건선 치료제, 알러지 치료제라도, 본 발명의 법률적 보호범위에 속한다.Therefore, those skilled 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, even if it is a therapeutic agent for atopic dermatitis, psoriasis, or allergy treatment according to interleukin 4 or interleukin 13 inhibition containing any modified form of aldehyde dehydrogenase that realizes the technical gist of the present invention described in the following claims, the legal provisions of the present invention belong to the scope of protection.
[수탁번호][Accession Number]
기탁기관명 : 한국생명공학연구원Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC13925BPAccession number: KCTC13925BP
수탁일자 : 20190822Deposit date: 20190822
[규칙 제91조에 의한 정정 06.03.2021]　
Figure WO-DOC-PAGE-1
[Correction by Rule 91 06.03.2021]
Figure WO-DOC-PAGE-1
기탁기관명 : 한국생명공학연구원Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC14122BPAccession number: KCTC14122BP
수탁일자 : 20200130Deposit date: 20200130
[규칙 제91조에 의한 정정 06.03.2021]　
Figure WO-DOC-PAGE-2
[Correction by Rule 91 06.03.2021]
Figure WO-DOC-PAGE-2
기탁기관명 : 한국생명공학연구원Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC14123BPAccession number: KCTC14123BP
수탁일자 : 20200130Deposit date: 20200130
[규칙 제91조에 의한 정정 06.03.2021]　
Figure WO-DOC-PAGE-3
[Correction by Rule 91 06.03.2021]
Figure WO-DOC-PAGE-3

Claims (9)

  1. 알데히드탈수소 효소를 함유하는 아토피 예방 또는 치료용 약학조성물.A pharmaceutical composition for preventing or treating atopy containing aldehyde dehydrogenase.
  2. 제1항에 있어서, 상기 알데히드탈수소 효소가, 알데히드탈수소 반응에 간여하는 작용부위를 포함하는 융합단백질 또는 재조합단백질을 포함하는 것임을 특징으로 하는, 아토피 예방 또는 치료용 약학조성물.The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 1, wherein the aldehyde dehydrogenase comprises a fusion protein or a recombinant protein comprising a site involved in the aldehyde dehydrogenation reaction.
  3. 제1항 또는 제2항에 있어서, 상기 알데히드탈수소 효소가 KCTC13925BP ( Saccharomyces cerevisiae KwonP-1), KCTC14122BP( Saccharomyces cerevisiae KwonP-2) 그리고 KCTC14123BP( Saccharomyces cerevisiae KwonP-3)로 이루어진 군에서 선택되는 어느 하나의 균주 또는 이들의 혼합물에서 분리된 것임을 특징으로 하는, 아토피 예방 또는 치료용 조성물. According to claim 1 or 2, wherein the aldehyde dehydrogenase KCTC13925BP ( Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2) and KCTC14123BP ( Saccharomyces cerevisiae KwonP-3) any one selected from the group consisting of A composition for preventing or treating atopy, characterized in that it is isolated from a strain or a mixture thereof.
  4. 알데히드탈수소 효소를 함유하는 건선 예방 또는 치료용 조성물.A composition for preventing or treating psoriasis containing an aldehyde dehydrogenase.
  5. 제4항에 있어서 상기 알데히드탈수소 효소가, 알데히드탈수소 반응에 간여하는 작용부위를 포함하는 융합단백질 또는 재조합단백질을 포함하는 것임을 특징으로 하는, 건선 예방 또는 치료용 조성물.[Claim 5] The composition for preventing or treating psoriasis according to claim 4, wherein the aldehyde dehydrogenase comprises a fusion protein or a recombinant protein comprising a site involved in the aldehyde dehydrogenation reaction.
  6. 제4항 또는 제5항에 있어서, 상기 알데히드탈수소 효소가 KCTC13925BP ( Saccharomyces cerevisiae KwonP-1), KCTC14122BP( Saccharomyces cerevisiae KwonP-2) 그리고 KCTC14123BP( Saccharomyces cerevisiae KwonP-3)로 이루어진 군에서 선택되는 어느 하나의 균주 또는 이들의 혼합물에서 분리된 것임을 특징으로 하는, 건선 예방 또는 치료용 조성물.According to claim 4 or 5, wherein the aldehyde dehydrogenase KCTC13925BP ( Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2) and KCTC14123BP ( Saccharomyces cerevisiae KwonP-3) any one selected from the group consisting of A composition for preventing or treating psoriasis, characterized in that it is isolated from a strain or a mixture thereof.
  7. 알데히드탈수소 효소를 함유하는 알러지 치료용 조성물.A composition for treating allergies containing an aldehyde dehydrogenase.
  8. 제7항에 있어서 상기 알데히드탈수소 효소가, 알데히드탈수소 반응에 간여하는 작용부위를 포함하는 융합단백질 또는 재조합단백질을 포함하는 것임을 특징으로 하는, 알러지 치료용 조성물.The composition for treating allergy according to claim 7, wherein the aldehyde dehydrogenase comprises a fusion protein or a recombinant protein comprising a site involved in the aldehyde dehydrogenation reaction.
  9. 제7항 또는 제8항에 있어서, 상기 알데히드탈수소 효소가 KCTC13925BP ( Saccharomyces cerevisiae KwonP-1), KCTC14122BP( Saccharomyces cerevisiae KwonP-2) 그리고 KCTC14123BP( Saccharomyces cerevisiae KwonP-3)로 이루어진 군에서 선택되는 어느 하나의 균주 추출물 또는 이들의 혼합물에서 분리된 것임을 특징으로 하는, 알러지 치료용 According to claim 7 or 8, wherein the aldehyde dehydrogenase KCTC13925BP ( Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2) and KCTC14123BP ( Saccharomyces cerevisiae KwonP-3) any one selected from the group consisting of For the treatment of allergy, characterized in that it is isolated from the strain extract or a mixture thereof
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011158689A1 (en) * 2010-06-19 2011-12-22 天野エンザイム株式会社 Agent for reducing acetaldehyde in oral cavity
JP2013247939A (en) * 2012-06-04 2013-12-12 Kiso Machi New yeast, and pharmaceuticals or foods and drinks containing the same
KR20180003344A (en) * 2016-06-30 2018-01-09 (주)아모레퍼시픽 Anti-inflammatory composition comprising extracellular vesicles derived from yeast

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011158689A1 (en) * 2010-06-19 2011-12-22 天野エンザイム株式会社 Agent for reducing acetaldehyde in oral cavity
JP2013247939A (en) * 2012-06-04 2013-12-12 Kiso Machi New yeast, and pharmaceuticals or foods and drinks containing the same
KR20180003344A (en) * 2016-06-30 2018-01-09 (주)아모레퍼시픽 Anti-inflammatory composition comprising extracellular vesicles derived from yeast

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Study on the functional role of acetaldehyde dehydrogenase in atopic dermatitis for the development of biomarker", FINAL REPORT: HEALTH TECHNOLOGY R&D PROJECT, 11 November 2015 (2015-11-11), Korea, pages 1 - 78, XP009529965 *
BAZEWICZ CHRISTOPHER G.: "Aldehyde dehydrogenase in regulatory T‐cell development, immunity and cancer", IMMUNOLOGY, vol. 156, 1 January 2018 (2018-01-01), pages 47 - 55, XP055840160 *

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