WO2018151442A1 - Composition for alleviation, prevention, or treatment of atopy - Google Patents

Abstract

The present invention provides: a pharmaceutical composition for alleviation, prevention, or treatment of an allergic disease; and a cosmetic composition for improvement of a skin condition. The composition of the present invention can effectively alleviate atopic dermatitis by inhibiting the expression of TSLP gene and protein.

Description

Compositions for improving, preventing or treating atopy

The present invention is made by the task number 201621018 under the support of the Ministry of Education of the Republic of Korea, the research management specialized organization of the project is (Foundation) Korea Research Foundation, the research project name is "university center research center project", the research title is "cell damage control material application System development ", the lead organization is Sogang University Industry-Academic Cooperation Foundation, and the research period is from Sept. 01, 2016 to Aug. 31, 2016.

This patent application claims priority to Korean Patent Application No. 10-2017-0021567 filed with the Korean Intellectual Property Office on February 17, 2017, the disclosure of which is incorporated herein by reference.

The present invention relates to a composition for improving, preventing or treating atopy.

Recently, patients suffering from skin diseases are increasing. In particular, allergic skin diseases such as atopic dermatitis and psoriasis are difficult to cure and cause limitations in the individual's social activities due to secondary skin damage caused by extreme itching and increased scratching behavior. It is a social problem because it leads to suicide.

Conventionally used drugs for treating atopic dermatitis include antihistamines, steroid hormones (cortisol, prednisolone, methylprednisolone, dexamethasone injections and ointments), and moisturizers. However, these drugs are not effective for the treatment of allergic dermatitis, especially in the case of steroid treatments, they show temporary efficacy, but in the long run, they cause enlargement of capillaries and thinning the skin, resulting in worse symptoms. In addition, if the patient using the steroid treatment is discontinued, it causes a more severe dermatitis symptom called steroid rebound, so it is urgent to develop an appropriate treatment for the treatment of allergic skin diseases.

Aryl hydrocarbon receptor (hereinafter referred to as 'AHR') is a receptor protein that is activated by binding to a substance such as dioxin and the like, and acts as a transcription factor. Most of the aryl hydrocarbon receptors that do not bind to the ligand are present in the cytoplasm, but when bound to the ligand, they move to the nucleus and act as transcription factors. These aryl hydrocarbon receptors are heterochromic with the aryl hydrocarbon receptor nuclear translocator (ARNT) in the nucleus, and the cytochrome P450 monooxygenase (CYP1A1, CYP1A2, CYP1B1). ), Glutathione-S-transferase (GSTs), NADPH / quinone oxidoreductase (NQO1), aldehyde dehydrogenase 3 gene expression It is known to be responsible for detoxification. In addition to dioxin and polychlorinated biphenyl (PCB), endogenous tryptophan derivatives, arachidonic acid metabolites, such as ITE [2- (1H-Indol-3-ylcarbonyl) -4-thiazolecarboxylic acid methyl ester] Arachidonic acid metabolites, heme metabolites, equilenin and indigoids are known ligands of AHR.

The present inventors have tried to develop a novel atopic treatment that can replace the conventional steroidal atopic treatment. As a result, when processing of ITE [2- (1 H -Indol- 3-ylcarbonyl) -4-thiazolecarboxylic acid] a ligand of the aryl hydrocarbon receptor, symptoms such as edema, hemorrhage, ulceration, inflammation, and atopic dermatitis by drying The present invention was completed by confirming the effect of mitigating.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for improving, preventing or treating allergic diseases.

Another object of the present invention to provide a cosmetic composition for improving the skin condition.

Still another object of the present invention is to provide a method for treating an allergic disease.

According to one aspect of the invention, the invention is to improve allergic diseases, prevention comprising the ITE [2- (1 H -Indol- 3-ylcarbonyl) -4-thiazolecarboxylic acid methyl ester] represented by the general formula (1) as an active ingredient Or provide a pharmaceutical composition for treatment.

[Formula 1]

The present inventors have tried to develop a novel atopic treatment that can replace the conventional steroidal atopic treatment. As a result, the treatment of ITE, which is a ligand of the aryl hydrocarbon receptor, confirmed the effect of alleviating symptoms such as edema, bleeding, ulceration, inflammation and drying caused by atopic dermatitis.

The pharmaceutical composition for improving, preventing or treating allergic diseases of the present invention includes the compound of Formula 1 as an active ingredient.

The compound of formula (I) is 2- (1 H -Indol-3- ylcarbonyl) -4-thiazolecarboxylic acid methyl ester (ITE).

According to one embodiment of the invention, the compound reduces the expression of thymic stromal lymphopoietin (TSP) gene or protein. The TSLP is a cytokine secreted from keratinocytes of the skin and stimulates the sensory nerves present in the subcutaneous to cause itching or dermatitis.

As used herein, the term “comprising as an active ingredient” means that the compound of Formula 1 contains an amount sufficient to achieve anti-allergic activity.

As used herein, the term "improvement" or "prevention" refers to any action that inhibits allergies or delays progression by administration of a composition of the present invention.

As used herein, the term “treatment” refers to the inhibition of the development of allergic diseases; Relief of allergic diseases; And elimination of allergic diseases.

As used herein, the term “allergy” refers to a variety of diseases, diseases or abnormalities caused by hypersensitivity to certain substances in the human body, ie, excessive reaction of the body's immune system to substances from outside. Allergic diseases to be applied to the compositions of the present invention are preferably formulation immediate hypersensitivity and formulation delayed hypersensitivity. Type I hypersensitivity reactions are bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria and anaphylatic shock, and type delayed hypersensitivity reactions are contact hypersensitivity, allergy Sexual contact dermatitis, bacterial allergies, fungal allergies, viral allergies, drug allergies, thyroiditis and allergic encephalitis. Type I immediate hypersensitivity is divided into two stages: the first phase is a Th1 cell response that produces IL-12 and IFN-γ that inhibits the release of IgE and IgG1 and increases the secretion of IgG2a by invasion of allergens. When the balance of Th2 cell responses that produce IL-4, IL-5, and IL-13 is inclined toward Th2, excessive immune responses of Th2 secrete IL-4 and IL-13, and B cells are produced by the effects. One IgE-specific antibody is attached to the surface of mast cells and basophils to prepare for allergic development. It is said to be sensitized to allergens. The second stage of the onset of allergy is divided into early and late reactions, and the initial reaction is an allergen reinvading the body to stimulate mast cells and induce a degranulation reaction, which is released by histamine, lipid metabolites, cytokines, etc. Expansion and the like is occurring, the late reaction is activated by the infiltration of neutrophils, eosinophils, macrophages, Th2 cells, basophils, etc. in the tissues, causing inflammation, causing atopic dermatitis, rhinitis, asthma.

According to one embodiment of the invention, the allergic disease is any one allergic disease selected from the group consisting of atopic dermatitis, contact dermatitis, urticaria, pruritus, edema and allergic dermatitis. .

According to another embodiment of the invention, the allergic disease is atopic dermatitis, pruritus or edema.

As used herein, the term “atopic dermatitis” is a skin eczema disease accompanied by chronic recurring severe itching, which is a kind of dermatitis.

As used herein, the term "pruritus" is itching, accompanied by diseases or conditions such as atopic dermatitis, contact dermatitis, urticaria, nodular benign, scabies, chronic simple thyroid, insect bites, eczema.

As used herein, the term "edema" is a condition in which excessive accumulation of moisture in tissues.

The composition of the present invention may be prepared as a pharmaceutical composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the compound of formula 1 of the present invention as described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the compounds described above.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and is preferably applied by parenteral administration.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. General dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg, but are not limited to adults.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

According to another aspect of the present invention, there is provided a cosmetic composition for improving skin conditions comprising ITE represented by the following formula (1) as an active ingredient.

[Formula 1]

According to one embodiment of the invention, the improvement of the skin condition is an improvement of edema, bleeding, ulceration, inflammation or dryness of the skin.

When atopic dermatitis develops, itching increases the scratching behavior, accompanied by symptoms such as swelling, bleeding, ulceration, inflammation, and dryness of the skin. These symptoms are exacerbated by repeated repetition of the scratching action.

The cosmetic composition of the present invention can effectively improve the condition of edema, bleeding, ulceration, inflammation or dryness of the skin.

When the composition of the present invention is made of a cosmetic composition, the composition of the present invention may include not only the compound of Formula 1, which is the active ingredient, but also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, and dissolutions. Conventional adjuvants such as agents, vitamins, pigments and flavorings, and carriers.

As the carrier, purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butylene glycol or propylene glycol), higher fatty acids (palmitylic acid or linolenic acid), fats and oils (wheat embryo oil, Camellia oil, jojoba oil, olive oil, squalene, sunflower oil, macadamia peanut oil, avocado oil, soybean hydrogenated lecithin or fatty acid glycerides), and the like. In addition, surfactants, bactericides, antioxidants, ultraviolet absorbers, anti-inflammatory agents and refreshing agents may be added as necessary.

As the surfactant, polyoxyethylene, hardened castor oil, polyoxyethylene, oleyl ether, monooleate polyoxyethylene, polyoxyethylene, glyceryl monostearate, monostearate sorbitan, monooleate polyoxyethylene, sorbitan, Sucrose fatty acid ester, hexaglycerol monolauric acid, polyoxyethylene reduced lanolin, POE, glyceryl pyroglutamic acid, isostearic acid, diester, N-acetylglutamine, isostearyl ester and the like can be used.

Examples of the fungicides include hydroxy thiol, trichromic acid, chlorohexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zinc pyrithaone, and the like.

As the antioxidant, any of butylhydroxyanisole, molten acid, molten acid propyl and erythorbic acid can be used.

Examples of the ultraviolet absorber include benzophenones such as dihydroxy benzophenone, melanin, ethyl paraaminobenzoate, 2-ethylhexyl ester of paradimethylaminobenzoic acid, synoxite and 2-ethylhexyl ester of paramethoxy cinnamic acid, and 2- (2- Hydroxy-5-methylphenyl) benzotriazole, urokanoic acid, metal oxide fine particles and the like can be used.

Examples of the anti-inflammatory agent include dipotassium glytilinate, allantoin, and the like, and a refreshing agent may be red pepper tink, 1-menthol, or the like.

According to another aspect of the present invention, the present invention provides a method for alleviating or treating an allergic disease, comprising administering to a subject a therapeutically effective amount of a composition comprising an ITE represented by Formula 1 as an active ingredient. to provide.

As used herein, the term “administration” or “administer” refers to administering a therapeutically effective amount of a composition of the invention directly to a subject (an individual) in need thereof so that the same amount is formed in the subject's body. Say that.

A "therapeutically effective amount" of a composition means a content of the composition that is sufficient to provide a therapeutic or prophylactic effect to a subject to which the composition is to be administered, and includes a "prophylactically effective amount". Also, as used herein, the term "subject" includes, without limitation, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

The method for treating the allergic disease of the present invention is the same as the composition comprising the above-described compound of Formula 1 as an active ingredient and the target disease of the present invention, so that the description thereof is omitted in order to avoid excessive complexity of the present specification. do.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for improving, preventing or treating allergic diseases and a cosmetic composition for improving skin condition.

(b) The composition of the present invention can effectively improve atopic dermatitis by inhibiting the expression of TSLP genes and proteins.

Figure 1 shows the result that the increased TSLP mRNA expression by TNFα or flagellin in HaCaT dermal keratinocytes is reduced by ITE.

FIG. 2 shows the result that TS increased TSLP protein expression by TNFα or flagellin in HaCaT dermal keratinocytes.

Figure 3 shows the results of the symptoms of atopic dermatitis alleviated by ITE in an atopic animal model. Dexamethasone (Dex) means a positive control.

4 shows atopic dermatitis severity results.

5a to 5h show the results of inhibiting the expression of TSLP, IL-4, IL-10, IL-13, IL-22, IL-25, IL-31 and IL-33 by ITE in skin tissue of an atopic animal model, respectively. Indicates. Dexamethasone (Dex) means a positive control.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Throughout this specification, unless otherwise indicated, “%” used to indicate the concentration of a particular substance is solid / solid (weight / weight)%, solid / liquid (weight / volume)%, and Liquid / Liquid is (volume / volume)%

Example 1 Atopic Dermatitis Improvement Effect at the Cell Level

Cell culture

Human keratinocytes, HaCaT dermal keratinocytes, contained Dulbecco's Modification of Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units / ml penicillin and 100 µg / ml streptomycin The medium was incubated in a 37 ° C. incubator maintaining proper humidity and 5% CO ₂ . To examine the expression of the thymic stromal lymphopoietin (TSLP) gene, TNFα (50 ng / ml) or flagellin (10 ng / ml) were passaged to 12-well plates in medium. After 1 hour, the same medium was treated with ITE (1 μM) and incubated again for 1 hour.

Gene expression analysis

RNA was extracted using RNAiso Plus (TAKARA, Japan). cDNA was synthesized using cDNA synthesis kit (TAKARA, JAPAN) and polymerization was performed using the synthesized cDNA and Sybrgreen kit (Enzynomics, Korea). The sequences of the primers used for PCR are shown in Table 1:

- Target genes Sequence (5 '-> 3') SEQ ID NO: One Human GAPDH Forward, CAC CCA CTC CTC CAC CTT TGA C One Reverse, GTC CAC CAC CCT GTT GCT GTA G 2 2 Human TSLP Forward, AAT CCA GAG CCT AAC CTT CAA TC 3 Reverse, GTA GCA TTT ATC TGA GTT TCC GAA TA 4

The human skin epidermal keratinocytes, HaCaT, were treated with TNFα or flagellin, respectively, to induce TSLP expression. Treatment of ITE, an endogenous ligand in vivo of AHR, resulted in a decrease in all mRNA expression of TSLP (FIG. 1).

Protein expression analysis

HaCaT treated with TNFα or flagellin, and ITE was refrigerated with 1 × PBS, harvested, and then added with 1 × protease inhibitor to IP 150 [10% glycerol, 0.5 mM EDTA, 25 mM Tris-HCl (pH 6.8). ), 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue] was added to boil for 5 minutes, placed on ice and subjected to SDS-PAGE. The protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA), placed in 5% skim milk / PBST, blocked for 1 hour, and washed with 1 × PBST. TSLP (Abcam, UK) and β-actin (Santa cruz, USA) antibodies were reacted for 12 hours in 4 stirrers using the primary antibody, and washed with 1 × PBST. Secondary antibodies, anti-mouse-HRP and anti-rabbit-HRP (1: 10,000, GenDEPOT, USA) were treated for 1 hour, washed with 1 × PBST, and then with an enhanced chemiluminescence (ECL) solution (DoGEN, Korea). Reaction for 1 minute and developed by Super RX film (Fuji, Japan) to confirm the expression of the protein.

The human skin epidermal keratinocytes, HaCaT, were treated with TNFα or flagellin, respectively, to induce TSLP expression. Treatment of ITE, an endogenous ligand in vivo of AHR, resulted in a decrease in the expression of all TSLP proteins (FIG. 2).

From the above results, ITE, an active ingredient of the present invention, suppresses the expression of TSLP, a cytokine that causes itching or dermatitis by stimulating the sensory nerves of the skin at the cellular level, thereby improving skin condition due to allergic skin diseases or allergies. It turned out that it can be usefully used as a composition for.

Example 2: Atopic Dermatitis Improvement Effect in Animal Models

Laboratory animals

BALB / c 6 week old female mice were used. 100 μl of 0.15% DNFB (1-Fluoro-2,4-dinitrobenzene) was applied to the abdomen to induce sensitization. After 7 days, 100 μl of 0.15% DNFB was applied to the back every 16 days until 16 days. ITE (100 μM), dexamethasone (200 μM) and PBS were applied daily from 7 days to 16 days.

Visual inspection of the skin condition

After removing the dorsal hair of the mouse, atopy dermatitis was induced by applying DNFB (1-Fluoro-2,4-dinitrobenzene). Thereafter, ITE and a positive control group dexamethasone were treated to check the skin condition. When treated with DNFB, it was confirmed that atopic dermatitis symptoms such as skin edema, bleeding, ulceration, inflammation, and dryness were induced (FIG. 3). On the other hand, it was confirmed that the treatment of ITE has an effect of alleviating symptoms similar to the treatment of dexamethasone, a positive control group (FIG. 3).

Atopic Dermatitis Severity Assessment

On the 16th day after DNFB was applied to mice and the like, severity was evaluated in non-testers. The severity is zero for none of the four symptoms of (i) erythema / bleeding, (ii) edema, (iii) abrasions / drows, and (iv) wounds / dryness; Normal) and 3 points (serious) were measured, and a total of 12 points were calculated. As a result, symptoms such as bleeding, edema, and wounds occurred when only PBS, a negative control group, were applied, and atopic symptoms such as bleeding, soreness, and wounds were alleviated when ITE and positive control group dexamethasone were applied (FIG. 4).

Expression Assessment of Atopic Dermatitis-Related Genes

RNA was extracted from the skin tissue and TSLP, IL-4 (interleukin-4), IL-10 (interleukin-10), IL-13 (interleukin-13), and IL-22 (interleukin-22) in the same manner as in Example 1 ), IL-25 (interleukin-25), IL-31 (interleukin-31) and IL-33 (interleukin-33) expression was examined by PCR. The sequences of the primers used for PCR are shown in Table 2:

- Target genes Sequence (5 '-> 3') SEQ ID NO: One Mouse GAPDH Forward, AGG TCG GTG TGA ACG GAT TTG 5 Reverse, TGT AGA CCA TGT AGT TGA GG TCA 6 2 Mouse TSLP Forward, AGC TTG TCT CCT GAA AAT CGA G 7 Reverse, AGG TTT GAT TCA GGC AGA TG TT 8 3 Mouse IL-4 Forward, GGT CTC AAC CCC CAG CTA GT 9 Reverse, GCC GAT GAT CTC TCT CAA GTG AT 10 4 Mouse IL-10 Forward, GCT CTT ACT GAC TGG CAT GAG 11 Reverse, CGC AGC TCT AGG AGC ATG TG 12 5 Mouse IL-13 Forward, CCT GGC TCT TGC TTG CCT T 13 Reverse, GGT CTT GTG TGA TGT TGC TCA 14 6 Mouse IL-22 Forward, ATG AGT TTT TCC CTT ATG GGG GAC 15 Reverse, GCT GGA AGT TGG ACA CCT CAA 16 7 Mouse IL-25 Forward, ACA GGG ACT TGA ATC GGG TC 17 Reverse, TGG TAA AGT GGG ACG GAG TTG 18 8 Mouse IL-31 Forward, TCA GCA GAC GAA TCA ATA CAG C 19 Reverse, TCG CTC AAC ACT TTG ACT TTC T 20 9 Mouse IL-33 Forward, GCT GCA GAA GGG AGA AAT CAC G 21 Reverse, GGA GTT GGA ATA CTT CAT TCT AGG TCT CAT 22

As a result, similarly to Example 1, the expression of TSLP in the mouse was suppressed and the expression of IL-4, IL-10, IL-13, IL-22, IL-25, IL-31 and IL-33 was also inhibited, respectively. It was confirmed that (Figs. 5a to 5h).

From the above results, ITE, an active ingredient of the present invention, alleviates atopic symptoms in an animal model causing atopic dermatitis and suppresses the expression of cytokines causing atopic dermatitis. It turned out that it can be usefully used as a composition for.

Sequence Listing 1st and 2nd Sequence are forward and reverse primers for human GAPDH gene detection, respectively, Sequence Listing 3rd and 4th Sequence are forward and reverse primers for human TSLP gene detection, respectively. SEQ ID NOs: 5 and 6 are forward and reverse primers for detecting mouse GAPDH genes, respectively, and SEQ ID NOs. 7 and 8 are forward and reverse primers for detecting mouse TSLP genes, respectively. SEQ ID NOs: 9 and 10 are forward and reverse primers for detecting mouse IL-4 gene, respectively, and SEQ ID NO: 11 and 12 are forward and reverse primers for detecting mouse IL-10 gene, respectively. SEQ ID NO: 13 and 14 are forward and reverse primers for the detection of mouse IL-13 gene, respectively, SEQ ID NO: 15 and 16 sequences are forward and reverse primers for IL-22 gene detection, respectively. SEQ ID NO: 17 and 18 are forward and reverse primers for the detection of mouse IL-25, respectively, SEQ ID NO: 19 and 20 are forward and reverse primers for the detection of mouse IL-31 gene, respectively List 21 and 22 are forward and reverse primers for the detection of mouse IL-33 gene, respectively.

Claims (6)

To ITE [2- (1 H -Indol- 3-ylcarbonyl) -4-thiazolecarboxylic acid methyl ester] to improvement of allergic diseases comprising, as an active ingredient for prevention or treatment a pharmaceutical composition represented by the general formula (1): [Formula 1]

. The method of claim 1, wherein the allergic disease is any one of allergic diseases selected from the group consisting of atopic dermatitis, contact dermatitis, urticaria, pruritus, edema and allergic dermatitis. Pharmaceutical composition. The pharmaceutical composition of claim 1, wherein the compound reduces expression of a thymic stromal lymphopoietin (TSLP) gene or protein. To a cosmetic for improving skin conditions comprising the ITE [2- (1 H -Indol- 3-ylcarbonyl) -4-thiazolecarboxylic acid methyl ester] represented by the general formula (1) as an active ingredient composition: [Formula 1]

. The cosmetic composition according to claim 4, wherein the improvement of the skin condition is an improvement of edema, bleeding, ulceration, inflammation or dryness of the skin. An allergic disease, comprising administering a composition comprising an ITE [2- (1 H -Indol- 3-ylcarbonyl) -4-thiazolecarboxylic acid methyl ester] represented by the general formula (1) as an effective ingredient to a subject (subject) How to relieve or treat: [Formula 1]

.

Images