KR102227522B1 - PHARMACEUTICAL COMPOSITION FOR USE IN PREVENTING OR TREATING INFLAMMATORY SKIN DISORDERS COMPRISING GHKWKYMVm - Google Patents

Abstract

The present invention relates to a peptide therapeutic capable of treating inflammatory skin diseases by controlling the invasion of immune cells and differentiation into microfibrous cells using GHKWKYMVm. The GHKWKYMVm peptide according to the present invention is effective in reducing the number of microglia fibroblasts and fibroblasts, reducing skin accumulation of macrophages, and reducing inflammatory cytokines levels, so it can be used for the treatment of inflammatory skin diseases including scleroderma. . In addition, the GHKWKYMVm peptide of the present invention exhibits a synergistic effect by recombining WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ ) and GHK (Glycyl-Histidyl-Lysine), so it has more effective inflammatory properties. Since it is possible to prevent or treat skin diseases, it can be usefully used in related medical, pharmaceutical, cosmetic and health functional food related businesses.

Description

Pharmaceutical composition for preventing or treating inflammatory skin diseases containing GHKWKYMVm peptide {PHARMACEUTICAL COMPOSITION FOR USE IN PREVENTING OR TREATING INFLAMMATORY SKIN DISORDERS COMPRISING GHKWKYMVm}

The present invention relates to a peptide therapeutic capable of treating inflammatory skin diseases by controlling the invasion of immune cells and differentiation into microfibrous cells using GHKWKYMVm.

Systemic sclerosis or scleroderma is a type of skin disease, causing extensive tissue fibrosis in various organs. Among them, prominent scleroderma and organ fibrosis are representative symptoms of scleroderma, accompanied by nonspecific increase in fibrous tissue and abnormal immune function.

Microglia fibroblasts, known as the main mechanism of action of scleroderma, become positive for α-smooth muscle actin (α-SMA) as fibroblasts differentiate, and cells such as collagen It has the property of hardening the skin fibrous tissue by excessively producing external matrix (ECM) components. The activation of these microglia fibroblasts is known to occur due to the action of immune cytokines. Among them, tumor necrosis factor alpha (TNF-α, Tumor Necrosis Factor alpha) and Interferon are representative, and chronic due to their action. It is known that immune cells such as macrophages infiltrate the tissues and induce the differentiation of fibroblasts during the process of inflammation.

Meanwhile, Formyl Peptide Receptor 2, a G-protein-coupled receptor, is involved in the activation of immune cells such as neutrophils, monocytes, and macrophages. Thus, it is known to play an important role in the inflammatory response. In particular, it regulates the inflammatory response by engaging in cytokine secretion and mononuclear immune cells in diseases such as bacterial infection. It is known that various ligands act in order to activate FPR2, and among them, the synthetic peptide WKYMVm (Trp-Lys-Tyr-Met -Val-D-Met-NH ₂ ) is known to have the highest reactivity.

WKYMVm is a synthetic peptide that acts through FPR2. WKYMVm plays an important role in inflammatory diseases by activating neutrophils and macrophages through FPR2 activation, and is known to be able to regulate as well as activate.

Varga J, Abraham D. Cuevas-Velazquez CL, Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest. 2007 Mar;117(3):557-67. Fuschiotti P. Current perspectives on the immunopathogenesis of systemic sclerosis. Immunotargets Ther. 2016 Apr 11;5:21-35. Le Y, Murphy PM, Wang JM. Formyl-peptide receptors revisited. Trends Immunol. 2002 Nov;23(11):541-8.

As a result of the present inventors' efforts to develop a new therapeutic agent for inflammatory skin diseases, WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ ) and GHK (Glycyl- Histidyl-Lysine) is recombined to produce GHKWKYMVm peptide, and when it is processed in a scleroderma model, which is a kind of inflammatory skin disease, GHKWKYMVm is compared to the existing WKYMVm peptide. The present invention was completed by confirming that the activity inhibitory ability and scleroderma treatment effect were more excellent.

Accordingly, the object of the present invention is to prevent inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (GHKWKYMVm (Gly-His-Lys-Trp-Lys-Tyr-Met-Val-D-Met-NH _{2 ))} Or to provide a therapeutic pharmaceutical composition.

Another object of the present invention is to provide a cosmetic composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a food composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a cosmetic composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a quasi-drug composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a food composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The GHKWKYMVm peptide according to the present invention is effective in reducing the number of microglia fibroblasts and fibroblasts, reducing skin accumulation of macrophages, and reducing inflammatory cytokines levels, so it can be used for the treatment of inflammatory skin diseases including scleroderma. . In addition, the GHKWKYMVm peptide of the present invention exhibits a synergistic effect by recombining WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ ) and GHK (Glycyl-Histidyl-Lysine), so it has more effective inflammatory properties. Since it is possible to prevent or treat skin diseases, it can be usefully used in related medical, pharmaceutical, cosmetic and health functional food related businesses.

1 is a skin sclerosis model, which is a kind of inflammatory skin disease, by treatment with WKYMVm at different concentrations (0.1uM, 1.0uM, 10uM), followed by hematoxylin & eosin staining and Masson trichrome staining. It is a diagram showing the results of measuring the tissue thickness and the extent of the collagen staining area (*p <0.05; ***p <0.005).
2 is a diagram showing the result of confirming the change in the numerical values of fibroblasts and microfibroblasts in skin tissue when WKYMVm is treated in a scleroderma model (*p <0.05; **p <0.01; ***p <0.005).
3 is a diagram showing the results of confirming changes in the level of inflammatory cytokines in the blood and macrophage infiltration in skin tissue when WKYMVm is treated in a scleroderma model (**p <0.01; ***p <0.005).
Figure 4 is a diagram showing the results of measuring the skin tissue thickness and the extent of the collagen-stained area after treatment with WKYMVm in a skin sclerosis model and a scleroderma model in which FPR2 expression is suppressed in normal mice (*p <0.05; ** p <0.01).
5 is a diagram showing the results of confirming the numerical change of fibroblasts and microfibroblasts in the skin tissue when WKYMVm is treated in the skin sclerosis model and the FPR2 expression suppressed skin sclerosis model of a normal mouse, respectively (*p <0.05).
6 is a diagram showing the results of confirming changes in the level of inflammatory cytokines in the blood and invasion of macrophages in skin tissues when treated with WKYMVm in a skin sclerosis model and a scleroderma model in which FPR2 expression is suppressed in normal mice (*p <0.05 ; ***p <0.005).
7 is a diagram showing the results of measuring the thickness of the skin tissue and the extent of the collagen-stained area through hematoxylin & eosin staining and Masson trichrome staining when GHKWKYMVm is treated in a scleroderma model. (*p <0.05; **p <0.01).
8 is a diagram showing the result of confirming the numerical change of fibroblasts and microfibroblasts in skin tissue when GHKWKYMVm is treated in a scleroderma model (*p <0.05; **p <0.01; ***p <0.005).
9 is a diagram showing the results of confirming changes in the level of inflammatory cytokines in the blood and macrophage infiltration in skin tissue when GHKWKYMVm is treated in a skin sclerosis model (*p <0.05; **p <0.01; ***p <0.005) .

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In the present specification, the term “skin” refers to all skin including hairs, nails and toenails of mammals including humans.

In the present invention, "inflammatory skin disease" is not limited thereto, but atopic dermatitis, allergic dermatitis, contact dermatitis, acne, seborrheic dermatitis, heat rash, urticaria, psoriasis, sclerosis, eczema, vitiligo, lupus, circular It means skin troubles or skin diseases that occur through various routes such as hair loss, preferably scleroderma or psoriasis, and more preferably scleroderma.

In the present specification, the term “skin sclerosis” refers to autoimmune diseases that can affect skin, blood vessels, muscles, and organs, and may appear locally on the skin or affect other organs other than the skin. Type exists. Multiple systemic fibrosis forms, also referred to as systemic sclerosis, include two subtypes: limited and distributed, depending on the degree of association of the skin. The constrained type affects the limbs, while the scatter type also affects the body. In addition to frontal diseases, local forms exist for diseases including, for example, focal cutaneous sclerosis (morphea), which affects certain areas of the skin, often joints and muscles, but not internal organs. The main features of systemic sclerosis are prevalent microvascular damage and progressive fibrosis in the skin and internal organs. In general, the most obvious clinical symptoms are skin hardening and associated scars. The skin may become hard, red, or peel. Despite advances in managing complications that arise primarily from organ failure, there are still no cures or disease-specific treatments to date.

In the present specification, the term "prevention" refers to any action that suppresses or delays the onset of inflammatory skin diseases by administration of the pharmaceutical composition according to the present invention.

In the present specification, the term "treatment" refers to any action in which symptoms caused by inflammatory skin diseases are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.

The peptide as an active ingredient of the pharmaceutical composition according to the present invention consists of the amino acid sequence of GHKWKYMVm (Gly-His-Lys-Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ ) (SEQ ID NO: 1, hereinafter It is effective in the treatment of scleroderma, a kind of inflammatory skin disease, by reducing the number of microglia fibroblasts and fibroblasts, reducing skin accumulation of macrophages, and reducing inflammatory cytokines levels (referred to as “GHKWKYMVm peptide”).

The GHKWKYMVm peptide has a high skin transmittance due to its small molecular weight and size, and thus has excellent scleroderma treatment effect.

The GHKWKYMVm peptide according to the present invention can inhibit the production of inflammatory cytokines, and the inflammatory cytokines may be at least one selected from the group consisting of TNF-α, IFN-γ, IL-6 and IL-12, Preferably, it may be TNF-α or IFN-γ, but is not limited thereto.

Cytokines are known to exhibit various physiological activities in the body, and among them, inflammatory cytokines function to activate an inflammatory response in the body to stimulation. When inflammatory cytokines such as TNF-α and IFN-γ are excessively activated by an external stimulus exceeding the allowable value, the inflammatory reaction caused by this may lead to skin diseases such as scleroderma.

In one embodiment of the present invention, the result of measuring skin thickness and collagen accumulation through hematoxylin & eosin staining and Masson Trichrome staining by treating the GHKWKYMVm peptide in a mouse inducing scleroderma. , It was confirmed that skin thickness and collagen accumulation were significantly reduced compared to the case of treating only the WKYMVm peptide, so that scleroderma could be treated more effectively.

In addition, in another embodiment of the present invention, when the GHKWKYMVm peptide is treated in a mouse inducing scleroderma, the number of fibroblasts and microfibroblasts is further reduced compared to the case where the WKYMVm peptide is treated, and macrophages and inflammatory cytokines in the skin. It was confirmed that the secretion of TNF-α and IFN-γ was further reduced. Through the above results, the GHKWKYMVm peptide according to the present invention is synergistic with the recombination of WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ ) and GHK (Glycyl-Histidyl-Lysine). It was confirmed that it can be used to prevent or treat more effectively.

The pharmaceutical composition may be administered in the form of oral delivery or parenteral delivery. The peptide may be administered systemically or locally, and the administration may include oral administration and parenteral administration. When the peptide is administered as a pharmaceutical composition, it may be formulated with an appropriate amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form.

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, but are not limited thereto, and any suitable agent known in the art may be used. .

In addition, the pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injectable solutions.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, in the peptide and its fractions, Alternatively, it can be prepared by mixing gelatin or the like. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

As the liquid formulation for oral administration, a suspension, a liquid solution, an emulsion, a syrup, etc. may be used, and various excipients, such as a wetting agent, a sweetening agent, a fragrance, a preservative, etc., may be used in addition to water and liquid paraffin, which are simple diluents. have.

In the formulation for parenteral administration, a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried formulation, and a suppository may be used. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, and glycerogelatin may be used.

The pharmaceutical composition may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is preferable to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and this can be easily determined by a person skilled in the art.

In addition, the present invention provides a cosmetic composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example a symptom.

The cosmetic composition may be formulated by a conventional method, and specifically, the cosmetic composition may be prepared in the form of a general emulsion formulation and solubilization formulation. For example, a lotion such as a flexible lotion or a nutritional lotion; Emulsions such as facial lotion and body lotion; Creams such as nourishing cream, moisture cream, and eye cream; essence; Cosmetic ointment; spray; Gel; pack; Sunscreen; Makeup base; Foundations such as liquid type, solid type or spray type; powder; Makeup removers such as cleansing cream, cleansing lotion, and cleansing oil; Alternatively, it may be formulated with a detergent such as a cleansing foam, soap, or body wash, but is not limited thereto.

In each formulation, the cosmetic composition may be appropriately blended with other components within a range that does not impair the object according to the present invention depending on the type of formulation or purpose of use, in addition to the essential components in each formulation.

The cosmetic composition may contain a generally acceptable carrier, and for example, oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately blended, but are limited thereto. no.

The acceptable carrier may vary depending on the formulation. For example, when formulated as an ointment, paste, cream or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or these Mixtures can be used

When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and in the case of spray, chlorofluorohydrogen It may further include a propellant such as carbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil can be used, and in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan can be used.

When the cosmetic composition or external preparation is formulated as a suspension, a liquid diluent such as water, ethanol or propylene glycol as a carrier component, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester. Suspension agents, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like may be used.

When the cosmetic composition is formulated with soap, as a carrier component, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, isethionate, a lanolin derivative, an aliphatic alcohol, vegetable oil, glycerol, sugar, etc. Can be used.

The cosmetic composition is a fatty substance, an organic solvent, a solubilizer, a thickener, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, and a fragrance, which are commonly used in the industry depending on the quality or function of the final product. , Surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, commonly used in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetology or dermatology, such as any other ingredients.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

In addition, the present invention provides a quasi-drug composition for preventing or improving inflammatory skin diseases including a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In the present specification, the term "quasi-drug" refers to items that are less effective than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals, for example, according to the Pharmaceutical Affairs Act. Quasi-drugs exclude items used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases of humans or animals, and products with minor or no direct action on the human body.

The quasi-drug composition of the present invention may be prepared in a formulation selected from the group consisting of a body cleanser, foam, mask, ointment, cream, lotion, essence, and spray, but is not limited thereto.

In addition, the present invention provides a food composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The "food" of the present invention can be prepared in all forms such as functional food, nutritional supplement, health food, and food additives. For example, as a health food, the GHKWKYMVm peptide of the present invention may be prepared in the form of tea, juice, and drink to be consumed, or granulated, encapsulated, and powdered to be consumed. In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods thereof (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat and processed foods thereof (eg, ham, sausage, corn beef, etc.). , Breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food , Frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.), and the like, can be prepared by adding the GHKWKYMVm peptide of the present invention.

When the peptide consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention is used in the form of a food composition, the food composition can be used as it is, added to other foods, or used together with other foods or food ingredients, which is a conventional method. It can be used accordingly. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

The food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. As the natural carbohydrates described above, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. . The ratio of the natural carbohydrate is generally about 0.01 to 0.4 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols. , Carbonated beverages, and the like. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The ratio of these additives is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples. The statistical significance between each group was analyzed through Student's t-test.

Example One. WKYMVm ( Trp - Lys - Tyr -Met-Val-D-Met-NH ₂ ) Confirmation of treatment effect of inflammatory skin disease according to treatment

1-1. In an animal model of scleroderma WKYMVm ( Trp - Lys - Tyr -Met-Val-D-Met-NH ₂ ) Confirmation of reduction in skin thickness and collagen accumulation according to treatment

In order to determine whether the WKYMVm peptide has a therapeutic effect on inflammatory skin diseases and at which concentration it is most effective, changes according to the treatment of each concentration of WKYMVm in the skin sclerosis animal model were confirmed. At this time, the WKYMVm peptide was synthesized by Synpeptide (Sanghai, China) through a chemical peptide synthesis method through a general peptide bond between amino acids.

First, in an animal model, a Bleomycin anticancer drug, known to induce scleroderma, is injected into the back skin of a mouse at a concentration of 1 mg/ml each day by subcutaneous injection, and then WKYMVm peptide is added at a concentration of 0.1 μM to 10 μM every day. Treated each. After that, after collecting the skin tissue of the animal model, the change in skin thickness was confirmed through hematoxylin & eosin staining, and the Masson Trichrome (Masson Trichrome, which is known to be stained specifically blue with collagen). ) The staining was performed to check the degree of collagen accumulation in the skin tissue. The results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that the skin thickness increased in the animal model treated with bleomycin only (BLM), whereas when WKYMVm was treated with 1.0uM, the degree of increase in skin thickness was significantly reduced ( BLM+Wm 1.0uM). In the case of the degree of collagen accumulation, the intensity and range of the blue-stained area increased under bleomycin treatment conditions, but when WKYMVm was treated with 1.0 uM, the intensity and range of the stained area were the smallest. Through the above results, it was confirmed that scleroderma can be improved when WKYMVm according to the present invention is treated in a scleroderma model, and in subsequent experiments, WKYMVm was tested at a concentration of 1.0 uM.

1-2. In an animal model of scleroderma WKYMVm According to the treatment Fibroblast , Microfibroblasts Checking the numerical change

To confirm whether the WKYMVm peptide reduces fibroblasts and microfibroblasts, which are specifically increased in scleroderma, fibroblast counts and microfibroblast counts were confirmed using immunohistochemistry. Specifically, staining was performed using Vimentin, a specific marker for fibroblasts, and α-SMA, a specific marker for microfibroblasts. The number of cells that were double-positive for α-SMA was confirmed. The results are shown in FIG. 2.

As shown in FIG. 2, it was confirmed that the number of cells positive for vimentin or α-SMA and DAPI was increased in the scleroderma model compared to the mouse model in which the disease was not induced (BLM). However, in the animal model treated with WKYMVm 1.0uM, where tissue thickness and collagen accumulation were reduced, the number of vimentin-positive fibroblasts and α-SMA-positive microfibroblasts decreased compared to the scleroderma model without peptide treatment. It was confirmed (BLM+Wm). Through the above results, it was confirmed that WKYMVm according to the present invention reduces the number of fibroblasts and microfibroblasts in scleroderma, thereby improving scleroderma.

1-3. In an animal model of scleroderma WKYMVm Confirmation of reduction in macrophage count and inflammatory cytokine secretion following treatment

To confirm that WKYMVm peptide decreases the level of macrophages known to increase in skin accumulation in scleroderma and the secretion of inflammatory cytokines TNF-α and IFN-γ, the level of macrophage marker CD68 positive cells and enzyme immunity. Increased cytokine levels in the blood were confirmed in a scleroderma model using a measurement method (ELISA, Emzyme-linked immunosorbent assay). The results are shown in FIG. 3.

As shown in FIG. 3, the skin sclerosis model increased the number of CD68-positive cells compared to the general mouse model, and it was confirmed that TNF-α and IFN-γ levels were also increased through the enzyme immunoassay (BLM). Here, in the scleroderma model treated with WKYMVm 1.0uM, the number of CD68-positive cells decreased compared to the scleroderma model not treated with the peptide, and TNF-α and IFN-γ levels were also relatively decreased (BLM+Wm). . Through the above results, it was confirmed that WKYMVm according to the present invention can improve scleroderma by reducing the accumulation of macrophages in the skin and secretion of immune cytokines in scleroderma.

1-4. To WKYMVm For the identification of signal transmission in the treatment of scleroderma caused by FPR2 Confirmation of skin sclerosis treatment in Knockout mice

To confirm whether the WKYMVm peptide mediates FPR2 (Formyl Peptide Receptor 2) in the treatment of scleroderma, after inducing scleroderma in mice with suppressed FPR2 expression, it was confirmed whether it has a therapeutic effect by WKYMVm. Specifically, in the same manner as in Example 1-1, bleomycin was injected daily to a mouse in which FPR2 expression was inhibited in the same manner as in Example 1-1 at a concentration of 1 mg/ml, and after treatment with WKYMVm peptide 1.0uM, an animal model Hematoxylin & eosin staining and tricolor staining were performed by collecting the skin tissue of. The results are shown in FIG. 4.

As shown in Figure 4, it was confirmed that the skin thickness was increased through hematoxylin & eosin staining of mice in which FPR2 expression was suppressed and scleroderma was induced, and the collagen portion stained in blue through the tricolor staining of the skin was increased. Confirmed. In addition, when WKYMVm treatment in a model that induces sclerosis in normal mice, the skin thickness decreases due to WKYMVm and the area of staining of collagen is reduced, while WKYMVm treatment in a skin sclerosis model using FPR2 expression suppression mice results in a decrease in skin thickness. It was confirmed that the treatment effect by WKYMVm was not significantly reduced compared to normal mice, and the stained area of collagen was not significantly reduced. Through the above results, it was confirmed that WKYMVm improves skin sclerosis through FPR2.

1-5. FPR2 Expression suppression To WKYMVm by Fibroblasts and Microfibroblasts Checking the impact on numerical changes

In the same manner as in Example 1-2, using a mouse model of scleroderma in which the expression of FPR2 was suppressed, changes in the numerical values of microfibroblasts by WKYMVm were confirmed. The results are shown in FIG. 5.

As shown in FIG. 5, in the model in which skin sclerosis was induced in normal mice, the number of microfibroblasts and fibroblasts decreased when WKYMVm was treated, but microfibroblasts were treated with the WKYMVm peptide in the FPR2-inhibited scleroderma mouse model. It was confirmed that the number of fibroblasts and fibroblasts did not decrease significantly. Through the above results, it was confirmed that FPR2 has an effect on the reduction in the number of fibroblasts and microfibroblasts by WKYMVm in scleroderma.

1-6. FPR2 Expression suppression To WKYMVm On the reduction of macrophage count and inflammatory cytokine secretion

In the same manner as in Example 1-3, using a scleroderma model in which FPR2 expression was suppressed, changes in macrophage levels and decreased secretion of inflammatory cytokines were confirmed by WKYMVm. The results are shown in FIG. 6.

As shown in Figure 6, it was confirmed that the skin accumulation level of macrophages and the secretion of inflammatory cytokines were reduced when WKYMVm treatment in the model inducing scleroderma in normal mice, but in the mice with suppressed FPR2 expression, despite WKYMVm treatment. It was confirmed that the level of skin accumulation of macrophages due to scleroderma did not decrease significantly, and the secretion of inflammatory cytokines did not decrease significantly. Through the above results, it was confirmed that the accumulation of macrophages in the skin and secretion of inflammatory cytokines in skin sclerosis caused by WKYMVm were affected by FPR2.

Example 2. In an animal model of scleroderma GHKWKYMVm Peptide Confirmation of reduction in skin thickness and collagen accumulation according to treatment

Following confirming the therapeutic effect of WKYMVm on scleroderma in Example 1, WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ ) and GHK (Glycyl-L-Histidyl-L-Lysine) To confirm the use of the GHKWKYMVm peptide (Gly-His-Lys-Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ ) of the present invention formed by recombining . The GHKWKYMVm peptide was synthesized by Synpeptide (Sanghai, China) through a chemical peptide synthesis method through a general peptide bond between amino acids.

Specifically, 1.0 μM GHKWKYMVm and 1.0 μM WKYMVm were injected into the skin at a daily dose of 100 μl along with bleomycin. After 3 weeks, the skin tissue was collected, and then skin thickness and collagen accumulation were measured through Hematoxylin & Eosin staining and Masson Trichrome staining, and the results are shown in FIG. 7.

As shown in FIG. 7, skin thickness decreased more in skin tissue treated with 1.0 μM GHKWKYMVm with bleomycin (BLM+GHKWm) than skin tissue treated with 1.0 μM WKYMVm with bleomycin (BLM+Wm), and collagen It was confirmed that the accumulation also decreased further. Through the above results, it was confirmed that the recombinant GHKWKYMVm peptide can more effectively improve scleroderma compared to the case of treating only the WKYMVm peptide of Example 1 to which GHK is not bound.

Example 3. In an animal model of scleroderma GHKWKYMVm Peptide According to the treatment Fibroblasts and Microfibroblasts Numerical change check

In order to confirm whether the GHKWKYMVm peptide according to the present invention can further reduce fibroblasts and microfibroblasts than WKYMVm, fibroblasts and microfibroblasts were identified using immunohistochemistry. Specifically, the skin tissue collected in the animal experiment conducted in FIG. 7 was stained using an antibody against Vimentin, a specific marker for fibroblasts, and α-SMA, a specific marker for microfibroblasts. , The number of cells that are double-positive for DAPI and vimentin or α-SMA, which can be stained in the cell nucleus to identify cells, was confirmed through immunostaining. The results are shown in FIG. 8.

As shown in FIG. 8, the skin sclerosis animal model (BLM) induced by injection of bleomycin showed an increase in the number of cells positive for vimentin or α-SMA and DAPI compared to the control animal model (PBS) in which the disease was not induced. In the animal model (BLM+GHKWm) in which 1.0 μM GHKWKYMVm was injected with bleomycin, the number of fibroblasts positive for vimentin and the number of microfibroblasts positive for α-SMA was a scleroderma model treated with WKYMVm peptide (BLM). +Wm). Through the above results, the recombined GHKWKYMVm peptide further reduces the number of fibroblasts and microfibroblasts in skin sclerosis compared to the case where only the WKYMVm peptide of Example 1 in which GHK is not bound, and through this, the number of fibroblasts and microfibroblasts is more effectively reduced. It was confirmed that it can be improved.

Example 4. In an animal model of scleroderma GHKWKYMVm Peptide Confirmation of reduction in secretion of macrophages and inflammatory cytokines following treatment

In order to confirm whether the GHKWKYMVm peptide according to the present invention can further reduce the secretion of macrophages and inflammatory cytokines TNF-α and IFN-γ in the skin compared to the WKYMVm peptide, the number of CD68 positive cells, which is a macrophage marker, was measured using an immunostaining method. The analysis was performed and the increased cytokine levels in the blood were confirmed in the skin sclerosis model using an enzyme immunoassay (ELISA, Emzyme-linked immunosorbent assay). The results are shown in FIG. 9.

As shown in FIG. 9, the skin sclerosis mouse model (BLM) also increased the number of CD68-positive cells and TNF-α and IFN-γ levels through enzyme immunoassay compared to the control mouse model (PBS) in which the disease was not induced. It was confirmed that the skin sclerosis model treated with 1.0 μM GHKWKYMVm (BLM+GHKWm) further reduced the number of CD68-positive cells in the skin tissue compared to the skin sclerosis model treated with 1.0 μM WKYMVm (BLM+Wm), and inflammatory in the blood. It was also confirmed that the levels of cytokines TNF-α and IFN-γ were further reduced. Through the above results, the recombinant GHKWKYMVm peptide reduces the accumulation of macrophages in the skin and secretion of immune cytokines in skin sclerosis compared to the case of treating only the WKYMVm peptide of Example 1 in which GHK is not bound to more effectively reduce sclerosis. It was confirmed that it can be improved.

As described above, when the GHKWKYMVm peptide according to the present invention is treated in an animal model of sclerosis, the number of microfibroblasts and fibroblasts is further reduced compared to the case where only WKYMVm peptide is treated, and skin accumulation of macrophages by the peptide is reduced. It was confirmed that the secretion of inflammatory cytokines including, was further reduced, and through this, skin thickness and collagen accumulation were also significantly reduced. That is, the GHKWKYMVm peptide according to the present invention exhibits a synergistic effect by recombining WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH ₂ ) and GHK (Glycyl-Histidyl-Lysine), and thus autoimmune diseases It was confirmed that it is very effective in reducing autoimmunity by reducing inflammation in scleroderma, which is a kind of scleroderma, and accordingly, it was confirmed that it can be usefully used as a therapeutic agent for inflammatory skin diseases including skin sclerosis.

Although the present invention has been described as a preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the gist and scope of the invention. In addition, the appended claims include such modifications or variations that fall within the gist of the present invention.

Hereinafter, examples of preparations of pharmaceutical compositions and food compositions for preventing or treating inflammatory skin diseases comprising the GHKWKYMVm peptide according to the present invention as an active ingredient will be described, but are not intended to limit the present invention.

Formulation example 1. Pharmaceutical preparation

1-1. Powdery Produce

GHKWKYMVm peptide 20 mg

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

GHKWKYMVm peptide 10 mg

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

1-3. Preparation of capsules

GHKWKYMVm peptide 10 mg

3 mg of crystalline cellulose

14.8 mg lactose

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

1-4. Preparation of injections

GHKWKYMVm peptide 10 mg

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO ₄ 2H ₂ O 26 mg

It is prepared with the above ingredients per ampoule (2 ml) according to a conventional injection preparation method.

1-5. Liquid Produce

GHKWKYMVm peptide 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of the liquid formulation, add and dissolve each component in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, add purified water, adjust the total to 100 ml, and fill in a brown bottle. It is sterilized to prepare a liquid formulation.

Formulation example 2. Food formulation

2-1. Manufacture of health food

GHKWKYMVm peptide 100 mg

The right amount of vitamin mixture

Vitamin A acetate 70 μg

1.0 mg of vitamin E

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

Vitamin B12 0.2 μg

10 mg of vitamin C

Biotin 10 μg

1.7 mg of nicotinic acid amide

50 μg folic acid

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

90 mg of potassium citrate

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for health food, but it may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. , To prepare granules, and can be used for preparing a health food composition according to a conventional method.

2-2. Manufacturing of health drinks

GHKWKYMVm peptide 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

19.75 g of iron lactate

Zinc oxide 3.5 g

3.5 g of nicotinic acid amide

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g of vitamin B2

Water quantity

After mixing the above ingredients according to the general health drink manufacturing method, stirring and heating the mixture for about 1 hour at 85°C, the resulting solution is filtered and obtained in a sterilized 2 ℓ container, sealed and sterilized, and then stored in a refrigerator. It is used to manufacture the health beverage composition of the invention.

The composition ratio is composed of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

<110> Pusan National University Industry-University Cooperation Foundation <120> PHARMACEUTICAL COMPOSITION FOR USE IN PREVENTING OR TREATING INFLAMMATORY SKIN DISORDERS COMPRISING GHKWKYMVm <130> PNU1-377P-1 <150> KR 10-2018-0082398 <151> 2018-07-16 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> GHKWKYMVm peptide <220> <221> MOD_RES <222> (9) <223> D-Met <400> 1 Gly His Lys Trp Lys Tyr Met Val Met 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> WKYMVm peptide <220> <221> MOD_RES <222> (6) <223> D-Met <400> 2 Trp Lys Tyr Met Val Met 1 5

Claims (8)

A pharmaceutical composition for preventing or treating inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
The method of claim 1, wherein the inflammatory skin disease is selected from the group consisting of atopic dermatitis, allergic dermatitis, contact dermatitis, acne, seborrheic dermatitis, heat rash, urticaria, psoriasis, scleroderma, eczema, vitiligo, lupus, and alopecia areata. Pharmaceutical composition for preventing or treating inflammatory skin diseases, characterized in that at least one disease.
The pharmaceutical composition for preventing or treating inflammatory skin diseases according to claim 1, wherein the peptide inhibits the production of inflammatory cytokines.
The pharmaceutical composition for preventing or treating inflammatory skin diseases according to claim 3, wherein the inflammatory cytokine is at least one selected from the group consisting of TNF-α, IFN-γ, IL-6 and IL-12.
The pharmaceutical composition for preventing or treating inflammatory skin diseases according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
A cosmetic composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
Quasi-drug composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
Food composition for preventing or improving inflammatory skin diseases comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

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