WO2021162463A9

WO2021162463A9 - Composition for preventing, treating, and alleviating atopic dermatitis comprising flavone-resveratrol conjugate compound

Info

Publication number: WO2021162463A9
Application number: PCT/KR2021/001805
Authority: WO
Inventors: 신순영; 임융호; 이혜지; 정유정; 이경희
Original assignee: 주식회사 아제라바이오텍; 건국대학교 산학협력단
Priority date: 2020-02-14
Filing date: 2021-02-10
Publication date: 2021-09-30
Also published as: WO2021162463A1; KR102174194B1

Abstract

The present invention relates to a composition for preventing, treating, and alleviating atopic dermatitis, comprising AB1711, which is a flavone-resveratrol conjugate compound, or a salt thereof, wherein the composition can prevent, treat, and alleviate atopic dermatitis by inhibiting EGR-1, which is a transcription factor involved in the expression of POMC that is a precursor of various cytokines, chemokines, and beta-endorphins that cause atopic dermatitis.

Description

Composition for preventing, treating and improving atopic dermatitis comprising a flavone-resveratrol conjugate compound

The present invention relates to a composition for preventing, treating and improving atopic dermatitis comprising a flavone-resveratrol conjugate compound.

Human skin consists of three layers: the epidermis, dermis, and subcutaneous fat tissue. It acts as a protective barrier.

From the outside, the epidermal layer is divided into a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale. Cells constituting the epidermal layer include various cells such as keratinocytes, Langelhans cells, and Merkel cells, and the majority of these cells are keratinocytes. Keratinocytes proliferate in the basal layer, and as they differentiate, they rise to the stratum corneum and become corneocytes, forming the keratin of the skin, thereby protecting the skin from the harmful external environment.

Atopic dermatitis is a chronic recurrent skin inflammatory disease accompanied by severe itching as the skin barrier weakens and the skin becomes dry due to allergic symptoms caused by immune hypersensitivity reaction (J Clin Invest 2012;122:440). -447). When the skin barrier is damaged due to an inflammatory reaction, exposure to other antigens is easily made, and the vicious cycle of chronic inflammation, itching, and dry skin is repeated (N Engl J Med 2008;358:1483-1494). Although the cause of atopic dermatitis has not yet been clearly identified, it is considered that a variety of genetic, environmental, immunological, and skin structural factors act in a complex way (J Clin Invest 2004;113:651-657).

Thymic stromal lymphopoietin (TSLP) secreted by keratinocytes, interleukin-4 (IL-4) secreted by CD4+helper type 2 T-lymphocytes (Th2), and interleukin-4 (IL-4) secreted by keratinocytes and Interleukin-13 (IL-13) and interleukin-31 (IL-31) are greatly increased, and interferon-gamma (IFNγ), a cytokine secreted by Th1-lymphocytes, and tumor necrosis in chronic atopic dermatitis sites Tumor necrosis factor-alpha (TNFα) is increased (J Allergy Clin Immunol 2000;105:860-876). IL-4 and IL-33 activate various inflammatory cells such as monocytes/macrophages, dendritic cells, keratinocytes, eosinophils, basophils, and mast cells to generate interleukin-31 (IL-31).

TSLP is a cytokine secreted in large amounts from keratinocytes in an inflammatory environment of the skin. Interleukin-4 activates dendritic cells and CD4+helper type 2 T-lymphocytes (Th2), which are important for innate immunity response. It is a representative atopic cytokine that induces atopic dermatitis and promotes pathological progression by promoting the production of Th2 cytokines such as , interleukin-5, interleukin-13, and TNFα (Nature Immunol 2010;11:289-293). TSLP mediates the progression of chronic atopic dermatitis patients to complex asthma disease, and activates TRPA-1 positive-sensory neurons to induce severe itchiness (Cell. 2013;155:285-295). In fact, TSLP expression was increased in keratinocytes in the skin of atopic dermatitis patients, and spontaneous atopic-like dermatitis occurred in transgenic mice overexpressing TSLP (J Exp Med 2005:202; 541-549). In keratinocytes, the expression of TSLP is regulated by the transcription factor EGR-1.

Interleukin-31 secreted from Th2 lymphocytes is a representative cytokine that induces histamine-independent itch by directly stimulating sensory nerves or acting on keratinocytes to promote beta-endorphin production (Br J Dermatol). 2012;167:794-803). Interleukin-31 promotes the expression of beta-endorphin precursor pro-opiomelanocortin (POMC) gene in keratinocytes.

Atopic dermatitis is a chronic disease that cannot be treated well enough to be called an incurable disease, so once it is onset, it is a chronic disease that must be continuously managed and treated for several to several decades. Most of the existing commercialized first-line treatments were mainly steroid-based external treatments that relieve severe itchiness and suppress cytokine production due to hypersensitivity immune response. However, long-term use of steroids for infants and young children may cause various side effects such as weakened immunity and skin atrophy or growth retardation. In addition, immunosuppressants such as calcineurin-inhibiting cyclosporin, which are nonsteroidal agents, have been developed and commercialized. However, when used for a long period of time, various side effects such as cancer development due to weakening of biological immunity are caused, making it difficult to treat fundamental atopic dermatitis. (J Invest Dermatol 2007;127:808-816). Although topical moisturizers, antihistamines, and vitamin D derivatives are used, the improvement is known to be weak (Semin Cutan Med Surg 2011;30:118-126). Therefore, research to find a substance with relatively high safety and excellent therapeutic effect by replacing steroids with many side effects for infants and young children is being actively conducted at home and abroad.

The present inventors found that EGR-1, a transcription factor expressed in keratinocytes, is associated with cytokines (TSLP, interleukin-1β, interleukin-6, interleukin-17, interleukin-23) that induce hyperimmune responses in atopic dermatitis. It produces chemokines (CXCL1, CCL5) that attract inflammatory cells such as T-lymphocytes and mast cells to the lesion site, and stimulates skin sensory nerves to deliver itching to the brain, a precursor of beta-endorphin. It was found to be a key transcription factor regulating POMC gene expression, and a study was conducted on a substance that could have a therapeutic effect on atopic dermatitis by regulating the expression of EGR-1.

Therefore, it is an object of the present invention to target EGR-1, which can suppress the expression of cytokines causing atopic dermatitis and genes causing itch, and flavone-resveratrol, which is a low molecular weight compound for alleviating the occurrence and symptoms of atopic dermatitis. To provide a composition comprising the conjugate compound AB1711.

In order to achieve the above object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising a compound represented by the following formula (1), a pharmaceutically acceptable salt thereof, as an active ingredient.

[Correction 30.07.2021 under Rule 91]

The compound of Formula 1 is a flavone-resveratrol conjugate compound, as 2-(6-(4-methoxystyryl)-2,4-dimethoxyphenyl)-3-hydroxy-6-nitro-4H-chromen-4-one (AB1711). is named The material may be synthesized by the inventors of the present invention or may be derived from nature, but is not limited thereto, and the AB1711 compound synthesized according to the synthesis method described in Example 1 of the present invention may be used. The present inventors found that the AB1711 directly binds to the transcription factor EGR-1 and blocks the DNA-binding ability of EGR-1, thereby inhibiting the production of various cytokines and chemokines that cause atopic dermatitis, It was confirmed that it performs the function of suppressing expression.

That is, the compound AB1711 of Formula 1 of the present invention directly binds to EGR-1 and blocks the ability of EGR-1 to bind to the DNA sequence of the 5'-transcription regulatory region of the target gene, thereby preventing atopic dermatitis. TSLP, IL-1β, IL-6, IL-17, IL-23, which are cytokines that induce Since it has an effect of inhibiting the expression of POMC (Ppro-opiomelanocortin), a precursor of β-endorphin that transmits a signal to the brain, it may have therapeutic and symptom relief effects for atopic dermatitis. That is, since it has both a therapeutic effect of atopic dermatitis as well as an improvement effect that can prevent it, and further relieve symptoms, it can be used as an effective drug for the treatment of atopic dermatitis.

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of skin wrinkles or atopic dermatitis by administration of the composition of the present invention, and "treatment" refers to skin wrinkles or atopic dermatitis by the composition of the present invention. It refers to any action in which the symptoms caused by dermatitis are improved or changed to a beneficial effect. In addition, as used herein, the term "improvement" refers to any action in which the symptoms of skin wrinkles and atopic dermatitis suspected and affected individuals are improved or beneficial by using the composition.

The AB1711 compound of Formula 1 may be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed with a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.

The addition salt according to the present invention is prepared by a conventional method, that is, the compound is dissolved in a water-miscible organic solvent, for example, acetone, methanol, ethanol, or acetonitrile, and an equivalent or excess of an organic acid is added or an aqueous solution of an inorganic acid is added. It can be prepared by precipitation or crystallization, or by evaporation of the solvent or excess acid followed by suction filtration of the dried or precipitated salt.

The present invention may include within the scope of the present invention not only the compound or a pharmaceutically acceptable salt thereof, but also solvates, hydrates and stereoisomers having the same efficacy, which can be prepared therefrom.

The pharmaceutical composition comprising the AB1711 compound of the present invention or a pharmaceutically acceptable salt thereof may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition. At this time, the content of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof included in the composition is not particularly limited, but may be included in an amount of 0.001 to 50% by weight based on the total weight of the composition, preferably 0.1 to 10 It may be included in weight %.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents and suppositories It may have a dosage form, and may be oral or parenteral various dosage forms. In the case of formulation, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin. and the like. In addition, lubricants commonly used in addition to excipients may be included. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be formulated as a pharmaceutical or quasi-drug. “Drug” refers to a general drug, and is not limited to its formulation. "Quasi-drugs" refer to textiles, rubber products, or similar products used for the purpose of treating, alleviating, treating, or preventing diseases of humans or animals, which have weak effects on the human body or do not act directly on the human body, and are not instruments or machines. Products that are similar to those used for sterilization, insecticide and similar purposes for infection prevention, and are used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals; It refers to items that are not machines or devices, and items used for the purpose of pharmacologically affecting the structure and function of humans or animals, excluding those that are not instruments, machines, or devices, and includes external preparations for skin and personal care products.

In addition, the composition of the present invention can be administered in a pharmaceutically effective amount.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the individual type and severity, age, sex, and disease. It can be determined according to the type, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered simultaneously or sequentially with conventional therapeutic agents. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, the degree of disease, the drug form, the route and period of administration, and a suitable total daily amount may be determined by the treating physician within the scope of correct medical judgment, In general, an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, and more preferably 0.1 to 100 mg/kg may be administered once a day or divided into several times a day.

The composition is not particularly limited as long as it is an individual for the purpose of preventing, treating, and alleviating symptoms of atopic dermatitis, and it is applicable to both humans and animals. The mode of administration may include without limitation as long as it is a conventional method in the art, and a method through topical application, oral administration, etc. may be used, but is not limited thereto.

In order to achieve another object of the present invention, the present invention provides a health functional food composition for the prevention or improvement of atopic dermatitis, containing the compound represented by the formula (1) and a pharmaceutically acceptable salt thereof as an active ingredient.

When the composition of the present invention is included in health functional food, the composition may be added as it is or used together with other health functional food or health functional food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be suitably determined according to the purpose of use. In general, in the production of food or beverage, the composition of the present invention may be added in an amount of preferably 20 parts by weight or less, more preferably 10 parts by weight or less, based on the raw material, and is used for health control and hygiene purposes. In the case of long-term ingestion, the amount may be less than or equal to the above range.

There is no particular limitation on the type of health functional food that can contain the composition of the present invention, and there are various functional foods, gum, candy, jelly, beverage, tea, drink, alcoholic beverage and vitamin complex, and the like, and It may include all health functional foods, and may include feed or feed additives for animals. In addition, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin , alcohol, a carbonation agent used in carbonated beverages, and the like.

In order to achieve another object of the present invention, the present invention provides a cosmetic composition for the prevention or improvement of atopic dermatitis, comprising the compound represented by Formula 1 and a cosmetically acceptable salt thereof as an active ingredient.

The cosmetic composition of the present invention may include, without limitation, commonly acceptable ingredients in addition to the active ingredients, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. have. The cosmetic composition according to the present invention includes solutions, external ointments, creams, foams, nourishing lotions, softening lotions, packs, soft water, emulsions, makeup bases, essences, soaps, liquid detergents, bathing agents, sunscreen creams, sun oils, suspensions, It may be prepared in formulations such as emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch and spray, but is not limited thereto.

In addition, the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and conventional ingredients include, for example, oil, water, surfactant, humectant, lower alcohol, A thickener, a chelating agent, a colorant, a preservative, a fragrance, etc. may be appropriately mixed, but the present invention is not limited thereto. The cosmetically acceptable carrier included in the cosmetic composition of the present invention may be included in various ways depending on the formulation.

When the compound AB1711 and its salts of the present invention are included in the health functional food, quasi-drugs and cosmetics to have a preventive or symptom relief (improvement) effect for atopic dermatitis, the content thereof is not limited thereto, but the total weight of the composition It may be included in the range of 0.001 to 5% by weight, and may be included in an amount greater than or equal to the minimum to exhibit efficacy for atopic dermatitis.

The present invention provides a composition for preventing, treating, and improving atopic dermatitis, including the AB1711 compound or a salt thereof, which is a flavone-resveratrol conjugate compound, and various inflammatory cytokines and chemokines that induce atopic dermatitis and beta- By inhibiting the DNA binding ability of EGR-1, a transcription factor involved in the expression of an endorphin precursor, it can have a preventive, therapeutic and ameliorating effect on atopic dermatitis.

1 is a schematic diagram of the synthesis process of the AB1711 compound of the present invention.

Figure 2 shows the results of analyzing the binding state of the AB1711 compound of the present invention and the EGR-1 protein by molecular docking method. (A) Binding of EGR-1 and AB1711 compound using LigPlot program (B) Three-dimensional image of binding of EGR-1 and AB1711 compound using PyMol program.

3 shows the results of analyzing the effect of the AB1711 compound of the present invention in blocking the DNA binding ability of EGR-1 by targeting the EGR-1 protein using the EMSA method. (A) Results of EMSA analysis of the EGR-1 binding DNA sequence position of the AB1711 compound and the EGR-1 protein (EGR1::DNA complex) and not (Free probe). (B) The result of quantitatively measuring the band intensity of the EGR-1 and DNA-bound complex using the ImagJ program

Figure 4 shows the results of confirming the fact that the AB1711 compound of the present invention does not induce EGR-1 expression non-specifically by immunoblot method. (A) Immunoblot method. (B) The result of quantitative measurement of the EGR-1 band intensity of the immunoblot using the ImagJ program.

5 shows the results of confirming that EGR-1 plays a role in regulating the expression of various cytokines and chemokines causing atopic dermatitis in an inflammatory environment using HaCaT cells in which expression of the transcription factor EGR-1 is knocked down. will be. (A, B) mRNA expression analysis using RT-PCR method. (C) The result of quantitative measurement of each gene mRNA band intensity in RT-PCR using the ImagJ program.

6 shows the results of confirming that the AB1711 compound of the present invention has the effect of inhibiting the expression of atopic dermatitis-induced cytokines and chemokines regulated by EGR-1 by targeting EGR-1. (A) Analysis of mRNA expression using RT-PCR method. (B) The result of quantitative measurement of each gene mRNA band intensity in RT-PCR using the ImagJ program.

7 shows the results of confirming that the AB1711 compound of the present invention has the effect of inhibiting the expression of the EGR-1 regulated POMC gene that causes itching in atopic dermatitis. (A, B) EGR-1 protein expression analysis using immunoblot method. (C, D) Result of quantitative measurement of POMC mRNA band intensity in RT-PCR using ImagJ program. (E) RT-PCR quantitative measurement of the POMC gene mRNA band intensity in Fig. 7D using the ImagJ program.

8 is a result confirming that when the AB1711 compound of the present invention is applied to the skin of mice induced atopic dermatitis by applying DNCB, an atopic-inducing drug, to the skin, it has an effect of improving the symptoms of atopic inflammation. (A) Schematic of the experimental schedule. (B) Atopic dermatitis symptom improvement effect by application of AB1711 compound. (C) Effect of reducing blood IgE by application of AB1711 compound

9 shows that the AB1711 compound of the present invention has the effect of reducing the epidermis and dermis thickness increased due to an inflammatory reaction in the skin of mice induced atopic dermatitis by applying DNCB, an atopic-inducing drug. (A) H&E staining of mouse skin tissue. (B) Measurement of the thickness of the stained epidermis and dermis in FIG. A.

Figure 10 confirms that the AB1711 compound of the present invention has the effect of reducing the infiltration of mast cells increased by the inflammatory response in the skin of mice induced atopic dermatitis by applying DNCB, an atopic-inducing drug. (A) Toluidine blue staining of rat skin tissue. (B) Measurement of mast cells stained with toluidine blue in FIG. A.

Hereinafter, examples will be given to describe the present specification in detail. However, the embodiments according to the present specification may be modified in various other forms, and the scope of the present specification is not to be construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those of ordinary skill in the art.

<실시예 1> AB1711 합성<Example 1> Synthesis of AB1711

The flavone-resveratrol conjugate compound AB1711 of the present invention is named as 2-(6-(4-methoxystyryl)-2,4-dimethoxyphenyl)-3-hydroxy-6-nitro-4H-chromen-4-one, It is synthesized according to the steps described. Hereinafter, the synthesis process of AB1711 was described in detail step by step.

1 단계 : 화합물 II 합성Step 1: Synthesis of Compound II

2.24 g (20 mmol) of resveratrol was slowly added to 20 mL of a 10% aqueous NaOH solution in an ice bath at a temperature of 2-4° C., followed by stirring. When the sample in the reaction flask was dissolved into a clear green solution, an excess of dimethyl sulfate (10 mL, 140 mmol) was slowly added. The mixture was stirred for 15 hours, and ethyl acetate was extracted from the reaction mixture. The organic layer was washed twice with saturated sodium hydrogen carbonate solution (sat'd NaHCO ₃ , 100 mL). The organic layer was separated _{, mixed with magnesium sulfate (MgSO 4} ), dried, and filtered. The solvent was removed from the filtrate under reduced pressure. The resultant obtained in a syrup state was used in the next reaction in a crude state. After dissolving 2.7 g (10 mmol) of trans-Trimethoxystilbene in 20 mL of N,N'-dimethylformamide (DMF), POCl ₃ (2 mL) was slowly added in an ice bath. After removing the ice bath, the reaction mixture was stirred at 50˚C for about two hours, and then cooled slowly at room temperature. Thereafter, 400 mL of ice water was prepared, and the reaction mixture was slowly added thereto, followed by stirring. When a precipitate was formed, the mixture was stirred for an additional 1 to 2 hours, and the precipitate was filtered under reduced pressure. The solid compound washed sufficiently with water was purified by recrystallization by adding hot ethanol to obtain compound II in a yield of 45%.

2 단계 : 화합물 III 합성Step 2: Synthesis of Compound III

2 mmol (596 mg) of compound II synthesized in step 1 and 2.1 mmol (380 mg) of 2-hydroxy-5-nitroacetophenone were added to 20 mL of ethanol, and 2 mL of aq.KOH (50 w/v%) was added and dissolved, followed by stirring with heating for 8 hours using a reflux device. The reaction mixture cooled to room temperature was added to 100 mL of ice water, and the entire reaction mixture was acidified to pH 3-4 using 3N HCl to harvest an orange solid precipitate. The precipitate was separated by filtration under reduced pressure, thoroughly washed with distilled water, and dried to obtain compound III (m.p: 189-192 °C) in 90% yield.

3 단계 : 화합물 IV (AB1711) 합성Step 3: Synthesis of Compound IV (AB1711)

1 mmol Compound III (461 mg) was dissolved in a 3:2 ratio of 10 mL methanol-tetrahydrofuran (THF) mixed solution. After adjusting the temperature in the reaction flask to 3~4 ^o C using an ice bath device, add 1 mL of 16% NaOH to dissolve all the compounds. After 10 minutes _{, add 2 mL of 35% H 2} O ₂ and basic pH 8 or higher. kept in solution. After the reaction mixture was stirred at room temperature for 10 hours, 100 mL of ice water was added. Then, the precipitate was harvested by acidification with 3N HCl (pH 3-4). The solid precipitate was filtered under reduced pressure, washed with water, recrystallized from a methanol-ethyl acetate mixed solution, and 50% yield of Compound IV (2-(6-(4-methoxystyryl)-2,4-dimethoxyphenyl)-3-hydroxy- 6-nitro-4H-chromen-4-one, mp: 238-240˚C) was obtained and named AB1711.

<실시예 2> AB1711 및 EGR-1의 결합 분자 도킹 (Molecular docking)<Example 2> Binding molecule docking of AB1711 and EGR-1 (Molecular docking)

<2.1> 실험 방법 <2.1> Experimental method

To determine the binding between EGR-1 and AB1711, Autodock vina program [J. Comp. Chem. 31: 455 (2010)] and Sybyl program (Tripos, St. Louis, MO) were used to perform in silico docking experiments. Several types of tertiary structures of EGR-1 are registered in the protein data bank (pdb). One of these, 5n14.pdb, has a structure determined by NMR spectroscopy and contains only 25 amino acid residues. 4x9j.pdb and 4r2a.pdb are structures determined by X-ray crystallography. Since the latter contains slightly more amino acid residues than the former, 4r2a.pdb was used in this experiment [Genes Dev. 28: 2304 (2014)]. This structure contains amino acid residues between E335 - D423, and includes ZnF1 (338 - 362), ZnF2 (368 - 390), and ZnF3 (396 - 418) between E335 - D423. Since 4r2a.pdb does not contain a ligand, the binding site was determined using the MOLCAD module included in the Sybyl program: F377, S378, H382, T385, H386, T389, R407. The size (62, 56, 70) and center (-4.421, -10.84, -19.227) of the docking box were determined to include the determined binding site, and an in silico docking experiment was performed with the Autodock vina program. Because the Sybyl program uses a flexible docking method, it was difficult to determine whether EGR-1 and AB1711 were combined. For the docking results, the LigPlot program provided by the European Bioinformatics Institute was used [Protein. 1999;37:228], 3D images are from PyMOL software (The PyMOL Molecular Graphics System, Version 1.0r1, Schr

dinger, LLC). AB1711, (E)-2-(2-(4-methoxystyryl)-4,6-dimethoxyphenyl)-3-hydroxy-6-nitro-4H-chromen-4-one, is the stilbene moiety of 3-hydroxyflavone and resveratrol. contains Since the tertiary structure containing these two is not known, the energy minimization method is used in the Sybyl program using the tertiary structure of 2',3'-dimethoxy-3-hydroxyflavone registered as CID 688713 in PubChem as a template. Thus, the tertiary structure was determined and used in this experiment.

<2.2> 실험 결과 <2.2> Experimental results

To obtain apo-protein of EGR-1, oligonucleotides included in 4r2a.pdb were removed using Sybyl program. Nine protein-ligand complexes were obtained from docking using Autodock vina. As a result, the binding energy of the obtained protein-ligand complexes was between -6.3 and -5.0 kcal/mol. The complex with the lowest energy was selected and analyzed by LigPlot (FIG. 2A). As a result, AB1711 compound showed hydrophobic interaction with 10 amino acid residues (R357, H358, R360, I361, G364, K366, R375, F377, R379), and hydrogen-bond (H) with two amino acid residues (S378, H382). -bond) was formed. The hydroxyl group of S378 formed 1-position oxygen of the flavonol moiety, and the nitrogen of the imidazole group of H382 formed 6-position oxygen and H-bond). A three-dimensional image (3D image) of the binding complex between EGR-1 and AB1711 compound was obtained using the PyMOL program, and the result of binding of the AB1711 compound to ZnF2 and ZnF3 of EGR-1 was confirmed (FIG. 2B).

<실시예 3> AB1711에 의한 EGR-1::DNA 결합 억제 효과 확인<Example 3> Confirmation of EGR-1::DNA binding inhibitory effect by AB1711

EMSA (Electrophoretic Mobility) to confirm whether the binding of AB1711 and EGR-1 predicted using the in silico molecular modeling method in [Example 2] inhibits EGR-1 binding to DNA at the actual protein level Shift Assay) was used for analysis.

<3.1> 실험 방법 <3.1> Experimental method

<3.1.1> Sf21 곤충세포에 사람 EGR-1 단백질 과발현<3.1.1> Human EGR-1 protein overexpression in Sf21 insect cells

For the EGR-1 protein used in the EMSA experiment, a cell extract in which human EGR-1 protein was overexpressed in Sf21 insect cells was used. To construct a recombinant baculovirus expressing EGR-1, the flashBAC Baculovirus expression system (Mirus Bio, Madison, WI, USA) was purchased and used. IPLB-Sf21 insect cells purchased from Clontech Company (Mountain View, CA, USA) were cultured in Grace Insect Medium (Gibco Company, Grand Island, NY, USA) with 10% fetal bovine serum (Hyclone Company, Logan, UT, USA). ) was incubated. The coding DNA sequence of EGR-1 in the EGR-1 expression plasmid (pcDNA3.1zeo/EGR1) was cut and isolated with Hind III and Bgl II restriction enzymes, and then transferred to the pOET1 transfer plasmid (Mirus Bio company; Madison, WI, USA). inserted to construct a pOET1/EGR1 plasmid. In ^{IPLB-Sf21 cells cultured at a density of 1.5x10 6} cells/well, 100 ng of transfer vector (pOET/EGR1), 100 ng viral DNA (flashBAC™, Mirus Bio) and 1.2 μl of TransIT®-Insect Transfection Reagent (Mirus Bio) ) was added. After culturing the cell culture plate at 28° C. for 5 days, it was harvested, and finally an Sf21 cell extract in which EGR-1 was mass-produced was obtained, and used for the following EMSA experiment.

<3.1.2> EMSA (Electrophoretic Mobility Shift Assay) <3.1.2> EMSA (Electrophoretic Mobility Shift Assay)

Protein and DNA binding analysis was performed using the LightShift chemiluminescence EMSA kit from ThermoFisher Scientific (Waltham, MA, USA) according to the manufacturer's instructions. EGR-1 binding biotin-nucleotide probe (5'-biotin-AGA GTG TGT CTC CTT CGC ACA CAT C-3'; hereinafter referred to as 'EGR-1 binding probe') is available from Macrogen (Macrogen, Seoul, Korea). It was synthesized and used.

11 μg Salmon sperm DNA and AB1711 were added to the EGR-1 containing Sf21 cell extract (3 μg) obtained using the above recombinant Baculovirus and reacted for 5 minutes. 50 fmol EGR-1 binding probe was added and reacted for 20 minutes. After electrophoresis of the reaction sample on a non-denaturing 6% polyacrylamide gel, the protein was attached to the nitrocellulose membrane using a Trans-Blot SD Semi-Dry Transfer Cell (BIO-RAD, Hercules, CA, USA). After blocking the nitrocellulose membrane, HRP (horseradish peroxidase) enzyme-conjugated streptavidin was added and reacted for 15 minutes. After washing the protein not bound to the nitrocellulose membrane, it was printed on an X-ray film using an enhanced chemiluminescence (ECL) detection system (ThermoFisher Scientific). The relative amount of the EGR-1::DNA complex was measured using the ImageJ program (National Institutes of Health, Bethesda, MD, USA).

<3.2> AB1711 화합물의 EGR-1과 EGR-1 결합 probe 결합능 억제 효과<3.2> Inhibitory effect of AB1711 compound on EGR-1 and EGR-1 binding probe binding ability

As a result of the above measurement, the AB1711 compound of the present invention inhibited the binding ability of EGR-1 and the EGR-1 binding probe in a concentration-dependent manner ( FIG. 3A ). The amount of EGR-1 and DNA-binding complex was quantitatively measured using the ImageJ program. As a result of quantification using ImageJ program, when 1.5 μL of 50mM AB1711 was added, the complex formation of EGR-1 and EGR-1 binding probe was reduced by about 51.2% compared to the control group, and when 3 μL was added, the complex formation decreased by about 96.5% compared to the control group became (Fig. 3B). Therefore, it was confirmed that the AB1711 compound of the present invention binds to EGR-1 and blocks the binding of EGR-1 to the target DNA.

<실시예 4> AB1711에 의한 EGR-1 발현에 대한 영향 분석<Example 4> Analysis of the effect on EGR-1 expression by AB1711

<4.1> 실험방법<4.1> Experimental method

<4.1.1> 세포배양<4.1.1> Cell culture

HaCaT cells, a human dermal keratinocyte line for use in the experiment, were purchased from Cell Lines Service GmbH (Eppelheim, Germany) and cultured in DMEM containing 10% fetal bovine serum (Gibco BRL, USA) and antibiotics (Gibco BRL, USA). was used to incubate. The culture vessel used 75T-flask and 6-well plate, and cultured in a 37°C incubator supplied with 5% CO2. The culture medium was changed every 3 to 4 days, and when the cells were excessively proliferated, subculture was performed.

<4.1.2> 면역블롯법<4.1.2> Immunoblot method

Changes in EGR-1 expression were measured by immunoblotting. Harvested HaCaT cells in cell lysis buffer (20 mM Tris-HCl (pH 7.5), 1% NP-40, 400 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NP40, 1 mM NaF, 1 mM phenylmethylsulfide) phonylfluoride), and the extracted protein samples (20 μg each) were electrophoresed by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech Inc). After the primary antibody (Santa Cruz, CA, USA) binding to EGR-1 was reacted, the secondary antibody to which horseradish peroxidase (HRP) was bound was reacted. After washing the antibody-bound nitrocellulose membrane, the protein band reacted with the antibody was printed on an X-ray film using LumiGLO peroxidase chemiluminescent substrate (SeraCare, Milford, MA, USA).

<4.2> 실험결과<4.2> Experimental results

HaCaT cells of an appropriate passage number were transferred to a 6-well plate, and when they proliferated more than 90% of the culture vessel area, 50 μM and 100 μM concentrations of AB1711 were treated. After 30 minutes, TNFα at a concentration of 5 ng/ml was treated, and cells were harvested after culturing for an additional hour. The amount of EGR-1 expression was analyzed by performing an immunoblot method (FIG. 4A). As a result of quantitative measurement using the ImageJ program, as shown in FIG. 4B, AB1711 slightly increased the expression of EGR-1 increased by TNFα to a statistically insignificant degree.

From the experimental results of <Example 3> and <Example 4>, AB1711 has no effect on the cellular neurotransmitter system that non-specifically affects EGR-1 expression, and specifically only the DNA binding ability of EGR-1. It was confirmed that it was selectively blocked.

<실시예 5> EGR-1에 의한 아토피 피부염 유발 사이토카인 유전자 발현 조절 <Example 5> Atopic dermatitis-induced cytokine gene expression regulation by EGR-1

TNFα secreted from various stromal cells and inflammatory cells in the skin inflammatory environment stimulates keratinocytes to secrete inflammatory cytokines and chemokines such as TSLP, IL-1β, IL-6, CXCL1, CCL2, and CCL5 to activate Th2 lymphocytes. Atopic dermatitis worsens by attracting various inflammatory immune cells to the inflamed area.

In order to examine the gene types of atopic dermatitis-induced inflammatory cytokines and chemokines regulated by EGR-1, the target of AB1711 of the present invention, analysis using reverse transcription-polymerase chain reaction (RT-PCR) did.

<5.1> 실험방법<5.1> Experimental method

<5.1.1> short hairpin RNA(shRNA)를 이용한 EGR-1 knockdown 세포주<5.1.1> EGR-1 knockdown cell line using short hairpin RNA (shRNA)

For this experiment, Scrambled shRNA-injected HaCaT control cells (HaCaT/shCT) and EGR-1 knockdown HaCaT cells (HaCaT/shEgr1) cells were used (J Biol Chem 2011; 286:26860-26872).

<5.1.2> 역전사 중합효소 연쇄반응(Reverse Transcription-Polymerase Chain Reaction; RT-PCR)<5.1.2> Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA extraction for measuring the amount of gene mRNA was extracted using TRIzol RNA Isolation Reagents purchased from TRIzol Life Technologies Korea. For 0.5 μg of total RNA, complementary DNA (cDNA) was synthesized using reverse transcription using the iScript cDNA synthesis kit purchased from Bio-Rad, Hercules, CA, USA) according to the method recommended by the manufacturer. PCR was performed with 0.0125 μg of double-stranded cDNA. Primer bases for gene amplification were synthesized by Macrogen (Seoul, Korea), and the primer sequences are shown in Table 1 below. As a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, was used. PCR denatures DNA at 95°C for 5 minutes, and then at 94°C for 1 minute; 2 min at 65°C; and a cycle of reacting at 70° C. for 1 minute was repeated 30 times. The final PCR product was confirmed by electrophoresis on a 1% agarose gel and staining with EtBr (ethidium bromide). For the PCR product of each sample, the amount of GAPDH was normalized, the control was set to “1”, and the relative change was converted. GAPDH and gene PCR bands were quantified using ImageJ program.

<5.2> 실험 결과<5.2> Experimental results

EGR-1 expression was increased when HaCaT/shCT control cells were treated with TNFα, but it was confirmed that EGR-1 expression by TNFα was significantly reduced in HaCaT/shEgr1 cells expressing shEgr1 compared to control cells ( 5A). As a result, it was confirmed that EGR-1 expression was knocked down in HaCaT/shEgr1 cells.

After HaCaT/shCT and HaCaT/shEgr1 cells were treated with TNFα, inflammatory cytokine and chemokine mRNA expression levels were analyzed by RT-PCR (FIG. 5B). As a result of quantitative measurement using the ImageJ program, there was no difference in CXCL10, CCL2, and GAPDH mRNA expression levels between the two cells, but the TSLP, IL-1β, IL-6, CXCL1, CCL5 mRNA expression levels were higher in HaCaT/ It was confirmed that statistically significant reduction in shEgr1 cells (FIG. 5C). Therefore, EGR-1 promoted the production of TSLP, IL-1β, IL-6, CXCL1, and CCL5, which were secreted from keratinocytes to induce atopic dermatitis and promote pathological progression.

Through the results of the above examples, it was confirmed that EGR-1 plays an important role in Th2 lymphocyte activity and influx of inflammatory cells in an inflammatory environment.

<실시예 6> 본 발명의 AB1711에 의한 EGR-1 조절 아토피 피부염 유발 사이토카인 유전자 발현 억제 효과 <Example 6> EGR-1 regulated atopic dermatitis-induced cytokine gene expression inhibitory effect by AB1711 of the present invention

<6.1> 실험방법<6.1> Experimental method

Whether or not the AB1711 compound of the present invention targets EGR-1 and suppresses the expression of inflammatory cytokines and chemokines causing atopic dermatitis regulated by EGR-1 was analyzed. Under the condition that EGR-1 expression is induced by treatment with TNFα in keratinocytes, the AB1711 compound of the present invention is In order to confirm whether the expression of atopic dermatitis-induced cytokines and chemokines produced by TNFα is inhibited, the reverse transcription-polymerase chain reaction (RT-PCR) described in [Example 5] is used. and analyzed.

<6.2> 실험결과<6.2> Experimental results

HaCaT/shEgr1 cells in which the keratinocytes, HaCaT/shCT and EGR-1 expression were knocked down, were pre-treated with AB1711 at concentrations of 50 μM and 100 μM, and then treated with TNFα after 30 minutes. It was confirmed that the expression of inflammatory cytokines and chemokines, which are EGR-1 targets, produced by TNFα stimulation, is reduced by AB1711 of the present invention ( FIG. 6A ). As a result of quantitative measurement using the ImageJ program, the amount of GAPDH did not change, but the amount of TSLP, IL-1β, IL-6, CXCL1, CXCL1 CCL5 mRNA increased by TNFα stimulation was significantly reduced in a AB1711 concentration-dependent manner (Fig. 6B). ).

Therefore, it was confirmed that the AB1711 compound of the present invention could effectively block the expression of various cytokines and chemokine genes involved in the occurrence and worsening of atopic dermatitis by targeting EGR-1 of keratinocytes.

<실시예 7> 본 발명의 AB1711에 의한 아토피 피부염 가려움증 억제 효과<Example 7> Atopic dermatitis itch inhibitory effect by AB1711 of the present invention

Beta-endorphin, similar to morphine, when secreted in the brain, is well known as a happy hormone that relieves mental stress, prevents human aging, destroys cancer cells, and strengthens memory. However, beta-endorphin produced in keratinocytes of the skin acts as a neurotransmitter that stimulates skin sensory nerves and makes itching feel independent of histamine (J Inv Dermatol 2007;127:2228-2235). Interleukin-31 is known to increase the gene expression of POMC, a precursor of beta-endorphin (Br J Dermatol 2012; 167:794-803). In order to examine whether the AB1711 compound of the present invention has an effect of blocking the itch of atopic dermatitis, the POMC gene expression inhibition effect was analyzed.

<7.1> 실험방법<7.1> Experimental method

<7.1.1> RT-PCR<7.1.1> RT-PCR

RT-PCR was performed as described in <5.1.2> above. The POMC primer sequences are shown in Table 2 below. As a control group, GAPDH, an anti-susceptibility gene, was used.

<7.2> 실험결과<7.2> Experimental results

When HaCaT cells were stimulated with interleukin-31, it was confirmed that EGR-1 protein expression gradually increased after 40 minutes of treatment ( FIG. 7A ). HaCaT/shCT and HaCaT/shEgr1 cells were used to determine whether POMC gene expression by interleukin-31 is regulated by EGR-1 ( FIG. 7B ). No increase in POMC mRNA expression by interleukin-31 was observed in HaCaT/shEgr1 cells ( FIG. 7C ). Therefore, it was confirmed that EGR-1 is a transcription factor that regulates POMC mRNA expression induced by interleukin-31.

Whether the AB1711 compound of the present invention targeting EGR-1 had the effect of inhibiting POMC mRNA expression by interleukin-31 was analyzed using RT-PCR. HaCaT cells were pre-treated with AB1711 at concentrations of 50 μM and 100 μM, stimulated with interleukin-31 1 hour later, harvested 24 hours after treatment, and analyzed for changes in POMC mRNA expression using RT-PCR. As a result, AB1711 reduced POMC mRNA expression increased by interleukin-31 in a concentration-dependent manner in a statistically significant manner ( FIG. 7D ).

Therefore, it was confirmed that the AB1711 compound of the present invention effectively blocks the expression of the POMC gene, which causes the itch of atopic dermatitis by targeting EGR-1 of keratinocytes.

<실시예 8> 동물모델에서 본 발명의 AB1711에 의한 아토피 피부염 증상 완화 효과<Example 8> Atopic dermatitis symptom relief effect by AB1711 of the present invention in an animal model

<8.1> 아토피 피부염 동물모델 제작 및 AB1711 도포<8.1> Manufacture of atopic dermatitis animal model and application of AB1711

7-week-old male BALB/c mice were purchased from Orient Bio (Orient Co., Seongnam, Korea) and acclimatized for 1 week in an animal laboratory where temperature 20±2℃, humidity 50±10%, and light-dark cycle were maintained for 12 hours. After treatment, (1) normal group (n=3), (2) atopic dermatitis induced DNCB+Vehicle treatment group (n=3), (3) DNCB+AB1711 treatment group (n=7) were divided into 3 groups. So the experiment was carried out.

After hair removal on the back of the mouse, it was left for 24 hours to allow the micro-wounds to heal naturally. To increase skin sensitivity, 4% SDS is applied, followed by 1% DNCB (2,4-dinitrochlorobenzene, Sigma Chemical Co., St. Louis, MO) with a mixed solution of acetone and olive oil (3:1, v/v). , USA) was prepared, and 100 μL was applied to the back of the mouse once a day for 3 days. The positive control group induced atopic dermatitis by applying 100 μL of 4% SDS and 0.5% DNCB once a day, 5 times a week to the same site for 2 weeks after a rest period of 4 days ( FIG. 8A ). The experimental group applied 100 μL of 4% SDS and 0.5% DNCB in the same way as the control group, and after about 2-3 hours, an ointment containing 5% AB1711 was prepared in the same area to observe the symptom relief effect. The control group applied only the ointment without AB1711. <Table 3> shows the ingredients used to prepare the experimental ointment.

<8.2> 아토피 유발 마우스에서 AB1711의 피부 조직학적 증상 완화 효과<8.2> Effect of AB1711 on Alleviating Skin Histological Symptoms in Atopic-Induced Mice

At the end of the experiment (Day 22), all mice were sacrificed, and gross skin clinical signs were observed. In the mouse skin applied only with DNCB, a lot of keratin scabs were generated throughout the area due to the inflammatory reaction, but the mice coated with AB1711 of the present invention showed a significant improvement in overall skin condition compared to the control group as the scab disappeared in a time-dependent manner ( Figure 8B).

<8.3> AB1711의 혈중 IgE 함량 감소 효과<8.3> Effect of AB1711 on Reducing IgE Content in Blood

At the end of the experiment (Day 22), blood was collected from the mouse orbital vein. Blood was centrifuged at 13,000 rpm and 4° C. for 10 minutes to separate serum. Total IgE content in mouse serum was measured and quantified using an ELISA kit purchased from BioLegend (San Diego, CA, USA) according to the method provided by the company.

It is known that the blood concentration of IgE mediating allergic diseases is increased in mice induced by atopic dermatitis (Journal of Investigative Dermatology 2009;129:31-40). The blood IgE concentration of the DNCB-applied control group in which atopic dermatitis was induced was 1084±217 ng/mL, and the blood IgE concentration of the experimental group coated with AB1711-containing ointment was 448±188 ng/mL ( FIG. 8C ).

Therefore, it was confirmed that AB1711 has the effect of reducing the blood IgE concentration.

<8.4> 피부 조직학적 분석<8.4> Skin histological analysis

In atopic dermatitis, as the proliferation of keratinocytes increases due to repeated inflammatory reactions, an imbalance occurs in the differentiation process into keratin, the thickness of the skin increases, and it is known that major immune cells infiltrate into the tissue to promote the inflammatory response. (Journal of Investigative Dermatology 2009;129:31-40). Therefore, histopathological changes caused by AB1711 of the present invention were observed.

<8.4.1> H&E 염색<8.4.1> H&E staining

After extracting the experimental mouse skin and fixing the tissue in 100% acetone solution, a paraffin block was prepared. After the paraffin block was cut to a thickness of 5 μm, hematoxylin/eosin staining was performed. As a result, it was observed that the skin thickness was significantly increased in the tissues of the positive control group (DNCB) induced with atopic dermatitis compared to the negative control group, and in the tissues of the experimental group to which the ointment containing AB1711 (DNCB+AB1711) was applied. It was observed that the thickness of the skin tissue was significantly reduced compared to the positive control group (FIG. 9A).

<8.4.2> AB1711의 표피와 진피 두께 감소 효과 정량적 측정<8.4.2> Quantitative measurement of epidermal and dermal thickness reduction effect of AB1711

Changes in the thickness of the epidermis and dermis were quantitatively measured using H&E-stained skin tissue. In the untreated negative control (Naive Control) tissue, the skin thickness was 27.8±2.17 μm in the epidermis and 314±21.9 μm in the dermis. It was 94.5 μm, but it was confirmed that the epidermal layer was 41.4±6.27 μm and the dermal layer was 360±63.2 μm in the tissue of the experimental group to which the ointment containing AB1711 (DNCB+AB1711) was applied (FIG. 9B).

Based on the above results, it was confirmed that AB1711 of the present invention can alleviate skin inflammation symptoms by inhibiting the proliferation of skin keratinocytes.

<8.4.3> AB1711의 비만세포 침윤(mast cell infiltration) 억제 효과 <8.4.3> Inhibitory effect of AB1711 on mast cell infiltration

In order to observe the change in the number of mast cells present in the skin between the epidermis and the muscle tissue, the paraffin block was stained with toluidin blue (TB). As a result, it was observed that mast cell infiltration was significantly increased in the tissues of the positive control group (DNCB) induced with atopic dermatitis compared to the negative control group (Naive Control), and the experimental group tissue to which the ointment containing AB1711 (DNCB+AB1711) was applied. It was observed that mast cell infiltration was significantly reduced compared to the positive control group (FIG. 10A).

<8.4.4> AB1711의 비만세포 침윤 억제 효과 정량적 측정<8.4.4> Quantitative measurement of the inhibitory effect of AB1711 on mast cell invasion

The number of infiltrating mast cells in the TB-stained tissue of [FIG. 10A] was measured. As a result, the number of infiltrated mast cells in the untreated negative control (Naive Control) tissue ^{was 23±6 per 2.5 cm 2} and 113±8 in the atopic dermatitis-induced positive control (DNCB) tissue. In contrast, in the tissue of the experimental group to which the ointment containing AB1711 (DNCB+AB1711) was applied, it was confirmed that there were 60±9 ( FIG. 10B ).

That is, it was confirmed that the application of the AB1711 of the present invention can alleviate the symptoms of skin inflammation by reducing the number of mast cells infiltrating into the skin inflammation site.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated by the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims

[Correction 30.07.2021 under Rule 91]
A pharmaceutical composition for preventing or treating atopic dermatitis, comprising a compound represented by the following formula (1), a pharmaceutically acceptable salt thereof, as an active ingredient.
[Formula 1]
According to claim 1,

The compound and a pharmaceutically acceptable salt thereof, a composition characterized in that it inhibits DNA binding of EGR-1.
According to claim 1,

The compound and a pharmaceutically acceptable salt thereof are a composition, characterized in that it inhibits the expression of atopy-induced inflammatory cytokines including TSLP, IL-1β and IL-6.
According to claim 1,

The compound and a pharmaceutically acceptable salt thereof are a composition, characterized in that it inhibits the expression of atopy-induced inflammatory chemokines, including CXCL1 and CCL5.
According to claim 1,

The compound and a pharmaceutically acceptable salt thereof, a composition characterized in that it inhibits POMC (Ppro-opiomelanocortin) gene expression, which is a precursor of β-endorphin.
A pharmaceutical for the prevention, improvement or treatment of atopic dermatitis comprising the compound represented by Formula 1 of claim 1, or a pharmaceutically acceptable salt thereof, as an active ingredient.
A quasi-drug for preventing or improving atopic dermatitis comprising the compound represented by Formula 1 of claim 1, or a pharmaceutically acceptable salt thereof, as an active ingredient.
[Correction 30.07.2021 under Rule 91]
A health functional food composition for preventing or improving atopic dermatitis, comprising a compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof, as an active ingredient.
[Formula 1]
[Correction 30.07.2021 under Rule 91]
A cosmetic composition for preventing or improving atopic dermatitis, comprising a compound represented by the following formula (1), a cosmetically acceptable salt thereof, as an active ingredient.
[Formula 1]
10. The composition of any one of claims 1, 8 and 9, wherein the concentration of the compound, or a pharmaceutically acceptable salt thereof, is 0.001 to 5% by weight.