Composition for preventing, ameliorating or treating inflammatory skin diseases comprising hyaluronic acid nanoparticles formed by electron beam irradiation

Abstract

The present invention relates to a composition for preventing, ameliorating or treating inflammatory skin diseases, comprising hyaluronic acid nanoparticles formed by electron beam irradiation and, more specifically, to a composition for preventing, ameliorating or treating inflammatory skin diseases, comprising hyaluronic acid nanoparticles that are prepared only by electron beam irradiation without a crosslinking agent, and thus exhibit excellent biocompatibility.

Description

Composition for preventing, improving or treating inflammatory skin diseases comprising hyaluronic acid nanoparticles formed by electron beam irradiation

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0048719, filed April 11, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for preventing, improving or treating inflammatory skin diseases, comprising hyaluronic acid nanoparticles formed by electron beam irradiation, and more particularly, to a composition for preventing, improving or treating inflammatory skin diseases, comprising hyaluronic acid nanoparticles having excellent biocompatibility and manufactured solely by electron beam irradiation without a crosslinking agent.

[0003]

[0004] Inflammatory skin disease is a skin disease accompanied by inflammatory reactions such as itching, swelling, erythema, and peeling due to various irritants that cause a series of inflammatory reactions within the skin epithelium.

[0005]

[0006] Among inflammatory skin diseases, psoriasis is a chronic inflammatory skin disease caused by abnormal proliferation of keratinocytes, abnormal immune response of various inflammatory cells, especially infiltrated T cells, resulting in abnormal proliferation and altered differentiation of keratinocytes. The exact etiology of psoriasis is unknown.

[0007]

[0008] Antigen stimulation induces the activation of innate immune cells, which in turn produces proinflammatory cytokines in the skin, leading to the differentiation of helper T1 and T17 cells, which are then activated and secrete various cytokines (IL-1, TNF, IL-8, IL-6, etc.). This stimulates keratinocytes in the skin, which rapidly proliferate and cause inflammation, leading to the accumulation of abnormal keratinocytes, such as dandruff, and psoriasis. IL-17 and IL-22 secreted by keratinocytes increase the production of proinflammatory cytokines and chemokines, promoting psoriasis inflammation. The secretion of IL-17 and IL-22 activates keratinocytes, and the activated keratinocytes produce many inflammatory cytokine / chemokine precursors, which induce the onset of psoriasis.

[0009]

[0010] Common treatments for psoriasis include topical medications applied directly to the skin, phototherapy using light, and systemic treatments such as immunosuppressants or steroids. Topical treatments include steroids, coal tar, anthralin, vitamin D3 and its analogs, and retinoids. These topical steroids can cause skin atrophy in the elderly and children with thin skin, induce steroid acne on the face, chest, and back, and suppress the pituitary-adrenal axis, leading to side effects such as growth retardation, iatrogenic Cushing's syndrome, osteoporosis, avascular necrosis, decreased immune response due to drug interactions, and growth retardation. Furthermore, steroids can induce resistance, which can affect subsequent steroid treatments. Phototherapy, which involves administering psoralen along with ultraviolet B or ultraviolet A light, has the disadvantage of accelerating skin aging and increasing the risk of skin cancer. For severe psoriasis, immunomodulators such as cyclosporine are administered, but careful monitoring is necessary as prolonged use can cause nephrotoxicity and hypertension. Thus, most existing psoriasis treatments, while effective in alleviating symptoms, do not provide a fundamental cure and are associated with numerous side effects.

[0011]

[0012] Therefore, there is a growing demand for new psoriasis treatments that have no side effects and are effective in alleviating skin inflammation.

[0013]

[0014] Accordingly, the inventor of the present invention has conducted repeated research to develop a new inflammatory skin disease treatment agent that is not only highly biocompatible and free of side effects but also can be manufactured using a simple method. As a result, the inventor has confirmed that hyaluronic acid nanoparticles manufactured using only electron beam irradiation are not only very effective in alleviating inflammatory reactions in the skin, but also can be manufactured using a very simple method without containing any ingredients that cause side effects, such as cross-linking agents, and has thus completed the present invention.

[0015]

[0016] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition, cosmetic composition and quasi-drug composition for preventing, improving or treating inflammatory skin diseases, which comprises as an active ingredient hyaluronic acid nanoparticles prepared by irradiating an aqueous solution of hyaluronic acid or its salt with an electron beam to form intermolecular and / or intramolecular crosslinking of hyaluronic acid.

[0017]

[0018] Another object of the present invention is to provide a use of hyaluronic acid nanoparticles prepared by forming intermolecular and / or intramolecular cross-linking of hyaluronic acid by irradiating an aqueous solution of hyaluronic acid or its salt with an electron beam for preparing a pharmaceutical composition for treating inflammatory skin diseases.

[0019]

[0020] In addition, another object of the present invention is to provide a method for treating an inflammatory skin disease, which comprises administering to a subject in need thereof an effective amount of a composition comprising hyaluronic acid nanoparticles as an active ingredient, which are prepared by irradiating an electron beam to an aqueous solution of hyaluronic acid or a salt thereof to form intermolecular and / or intramolecular cross-links of hyaluronic acid.

[0021]

[0022] In order to achieve the above-described object of the present invention, the present invention provides a pharmaceutical composition, a cosmetic composition, a quasi-drug composition and a food composition for preventing, improving or treating inflammatory skin diseases, which comprises as an active ingredient hyaluronic acid nanoparticles prepared by forming intermolecular and / or intramolecular cross-linking of hyaluronic acid by irradiating an electron beam to an aqueous solution of hyaluronic acid or a salt thereof.

[0023]

[0024] In order to achieve another object of the present invention, the present invention provides a use of hyaluronic acid nanoparticles prepared by forming intermolecular and / or intramolecular cross-linking of hyaluronic acid by irradiating an aqueous solution of hyaluronic acid or a salt thereof with an electron beam for preparing a pharmaceutical composition for treating inflammatory skin diseases.

[0025]

[0026] In addition, in order to achieve another object of the present invention, the present invention provides a method for treating an inflammatory skin disease, which comprises administering to a subject in need thereof an effective amount of a composition comprising hyaluronic acid nanoparticles as an active ingredient, which are prepared by irradiating an electron beam to an aqueous solution of hyaluronic acid or a salt thereof to form intermolecular and / or intramolecular cross-linking of hyaluronic acid.

[0027]

[0028] In addition, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for treating inflammatory skin diseases, which is composed of hyaluronic acid nanoparticles prepared by forming intermolecular and / or intramolecular cross-linking of hyaluronic acid by irradiating an electron beam to an aqueous solution of hyaluronic acid or a salt thereof.

[0029]

[0030] In addition, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for treating inflammatory skin diseases, which is essentially composed of hyaluronic acid nanoparticles prepared by irradiating an electron beam to an aqueous solution of hyaluronic acid or a salt thereof to form intermolecular and / or intramolecular cross-linking of hyaluronic acid.

[0031]

[0032] Hereinafter, the present invention will be described in detail.

[0033]

[0034] In this specification, the term “comprising” is used with the same meaning as “including” or “characterized by,” and does not exclude additional components or method steps, etc. that are not specifically mentioned in the composition or method according to the present invention. In addition, the term “consisting of” means excluding additional elements, steps, or components, etc. that are not separately described. The term “essentially consisting of” means that, in the scope of the composition or method, it may include materials or steps, etc. that do not substantially affect the basic characteristics thereof, in addition to the materials or steps described.

[0035]

[0036] In the present invention, the nanoparticle refers to a particle having a size of several to several hundred nanometers (nm, a material that is one billionth of a meter). In the present invention, the nanoparticle may be characterized by a particle size of 1 to 100 nm, but is not limited thereto. In the present invention, the nanoparticle is a concept that also includes nanogels or nanohydrogels, and hereinafter, nanoparticles, nanogels, or nanohydrogels are all used to mean nanoparticles according to the present invention.

[0037]

[0038] The above-described aqueous solution of hyaluronic acid or its salt may be characterized by being acidic. For the acidic aqueous solution, the content of hyaluronic acid in the aqueous solution of hyaluronic acid or its salt may be adjusted, or an acid such as HClO4, HCl, H2SO4, CH3COOH, etc. may be additionally included. When the aqueous solution of hyaluronic acid or its salt is acidic, or an acid is added to the aqueous solution of hyaluronic acid or its salt, the efficiency of nanoparticle synthesis may be increased compared to when it is not. In addition, the size of the nanoparticles can be unexpectedly controlled by adjusting the pH and adjusting the concentration of the added acid compound.

[0039]

[0040] In a preferred embodiment of the present invention, the pH of the aqueous solution of hyaluronic acid or a salt thereof may be less than 7. For example, the pH of the aqueous solution of hyaluronic acid or a salt thereof may be 0.1 to 6, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 2, 0.1 to 1, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, or 5 to 6.

[0041]

[0042] In the present invention, the hyaluronic acid is a major component of the extracellular matrix, which is widely distributed in the interstitium of connective tissues and forms an adhesive and elastic solution under physiological conditions, thereby mechanically protecting tissues such as the iris and retina, vascular endothelial cells, and epithelial cells, and providing a buffering function. In the present invention, the hyaluronic acid is not limited in terms of physical properties, shape, size, etc., and it is preferably sterilized. In addition, the hyaluronic acid may have a weight average molecular weight of 1 to 5000 kDa, and preferably a weight average molecular weight of 3 to 2000 kDa. In addition, the hyaluronic acid may be used in the form of a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt includes a sodium salt, a potassium salt, a calcium salt, and the like, and preferably includes a sodium salt. Additionally, derivatives of hyaluronic acid may also be used, such derivatives being specifically hyaluronic acid that has been chemically modified by cross-linking, sulfation, esterification or amino acid linkage.

[0043]

[0044] In the method for manufacturing hyaluronic acid nanoparticles provided by the present invention, the concentration of the hyaluronic acid aqueous solution may be 0.05 to 20% (w / w), and preferably 1 to 10% (w / v).

[0045]

[0046] The above aqueous solution is characterized in that it does not contain a crosslinking agent or an organic solvent.

[0047]

[0048] After irradiating the electron beam, additional steps of dialysis and freeze-drying are included.

[0049]

[0050] When the irradiation dose of the electron beam is changed, the size of the hyaluronic acid nanoparticles can be controlled, and specifically, when the irradiation dose of the electron beam is increased, the size of the hyaluronic acid nanoparticles can be reduced.

[0051]

[0052] The electron beam to be investigated above can be irradiated at a dose of 0.5 to 500 kGy, preferably 10 to 400 kGy, and most preferably 50 to 350 kGy.

[0053]

[0054] In the present invention, the total irradiation energy intensity of the electron beam may be characterized as being 0.1 MeV to 20 MeV. Preferably, the total irradiation energy intensity of the electron beam may be 0.5 MeV to 10 MeV, and most preferably 1.0 MeV to 5 MeV.

[0055]

[0056] The hyaluronic acid nanoparticles included in the composition of the present invention may be hyaluronic acid nanoparticles having an average diameter of 100 nm or less, preferably hyaluronic acid nanoparticles having an average diameter of 50 nm or less, more preferably hyaluronic acid nanoparticles having an average diameter of 30 nm or less, and most preferably hyaluronic acid nanoparticles having an average diameter of 10 nm or less.

[0057]

[0058] In the present invention, the nanoparticles may be characterized as being hydrogels having swelling properties.

[0059]

[0060] In the present invention, the inflammatory skin disease is not particularly limited in type, but may be selected from the group consisting of psoriasis, contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, contact urticaria syndrome, autosensitization dermatitis, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, and eczema, and preferably may be psoriasis.

[0061]

[0062] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0063]

[0064] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. In addition, the pharmaceutical composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient. The pharmaceutical composition of the present invention may include 0.1 to 99.9 wt% of hyaluronic acid prepared by the method of the present invention, and 99.9% to 0.1 wt% of the carrier.

[0065]

[0066] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0067]

[0068] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0069]

[0070] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable preparations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.

[0071]

[0072] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0073]

[0074] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.

[0075]

[0076] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0077]

[0078] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, administration route, etc. of the pharmaceutical composition. A person skilled in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0079]

[0080] The 'effective amount' of the present invention refers to an amount that, when administered to an individual, exhibits an effect of improving, treating, detecting, diagnosing, or inhibiting or reducing an inflammatory skin disease. The 'individual' may be an animal, preferably a mammal, particularly an animal including a human, and may also be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect.

[0081]

[0082] The ingredients included in the cosmetic composition of the present invention include, in addition to the above-mentioned effective ingredients, ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0083]

[0084] The cosmetic composition of the present invention can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it can be manufactured into the formulation of a flexible toner (skin), a nourishing toner (milk lotion), a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

[0085]

[0086] Preferably, the cosmetic composition of the present invention may be characterized as being a W / O cream, O / W cream, O / W essence, and hydrogel formulation, and most preferably, a hydrogel formulation, but is not limited thereto.

[0087]

[0088] When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0089]

[0090] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0091]

[0092] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0093]

[0094] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0095]

[0096] When the formulation of the present invention is a surfactant-containing cleansing agent, a fatty alcohol sulfate, a fatty alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkyl amidobetaine, a fatty alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a lanolin derivative, or an ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0097]

[0098] In the present invention, the above “quasi-drug” refers to products that are used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases of humans or animals, and have a milder effect than pharmaceutical products. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products other than those used for pharmaceutical purposes, and include products used for treating or preventing diseases of humans or animals, products that have a mild effect on the human body or do not directly affect it, etc.

[0099]

[0100] The 'treatment' of the present invention comprehensively refers to improving symptoms caused by the inflammatory skin disease, which may include curing, substantially preventing, or improving the condition of the inflammatory skin disease, and includes, but is not limited to, alleviating, curing, or preventing one or most of the symptoms resulting from the disease.

[0101]

[0102] In the present invention, when the hyaluronic acid nanoparticles are used as additives, they may be added as is or used in combination with other over-the-counter drugs or over-the-counter drug ingredients, and may be used appropriately according to conventional methods. The mixing ratio with other ingredients used together may be appropriately determined depending on the intended use (e.g., preventive, improvement, or therapeutic treatment).

[0103]

[0104] Examples of the above-mentioned pharmaceutical compositions include, but are not limited to, scalp and hair care compositions such as shampoos and rinses, body cleansers, foams, soaps, masks, ointments, creams, lotions, essences, sprays, etc.

[0105]

[0106] The present invention also provides a food composition for preventing or improving inflammatory skin diseases, which comprises hyaluronic acid nanoparticles as an active ingredient, which are prepared by irradiating an electron beam to an aqueous solution of hyaluronic acid or a salt thereof to form intermolecular and / or intramolecular cross-linking of hyaluronic acid.

[0107]

[0108] The above food composition includes all forms, including functional foods, nutritional supplements, health foods, and food additives. The above types can be manufactured in various forms using conventional methods known in the art.

[0109]

[0110] For example, as a health food, the food composition of the present invention can be manufactured and consumed in the form of tea, juice, or drink, or can be granulated, encapsulated, or powdered for consumption. Furthermore, the food composition of the present invention can be manufactured in the form of a composition by mixing it with a known substance or active ingredient known to have an anti-inflammatory effect.

[0111]

[0112] In addition, functional foods can be produced by adding the food composition of the present invention to beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and their processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.).

[0113]

[0114] The preferred content of the food composition according to the present invention is not limited thereto, but may preferably be 0.01 to 50 wt% of the total weight of the final manufactured food. In order to use the food composition of the present invention in the form of a food additive, it may be manufactured and used in the form of a powder or concentrate.

[0115]

[0116] Hyaluronic acid nanoparticles manufactured solely by electron beam irradiation exhibit high biocompatibility, skin permeability, and excellent anti-inflammatory activity, and thus can be very useful in the development of drugs to prevent, improve, or treat inflammatory skin diseases.

[0117]

[0118] Figure 1 is a diagram illustrating the experimental schedule and experimental groups for evaluating the psoriasis alleviation effect of each test substance after inducing psoriasis in mice.

[0119]

[0120] Figure 2 shows the results of visual observation of the skin after the experimental schedule in a psoriasis animal model was completed.

[0121]

[0122] Figure 3 shows the results of microscopic observation of skin tissue after H&E staining after the experimental schedule in a psoriasis animal model was completed.

[0123]

[0124] Figure 4 shows the results of microscopic observation after the experimental schedule in a psoriasis animal model was completed and the skin was stained with Ki67 antibody.

[0125]

[0126] Figure 5 is a graph showing the number of Ki67 positive cells quantified after tissue staining of the skin with Ki67 antibody after the experimental schedule in a psoriasis animal model was completed.

[0127]

[0128] Figure 6 shows the results of microscopic observation after the experimental schedule in a psoriasis animal model was completed and the skin was stained with IL-17A antibody.

[0129]

[0130] Figure 7 is a graph showing the quantification of the stained area after tissue staining of the skin with IL-17A antibody after the experimental schedule in a psoriasis animal model was completed.

[0131]

[0132] Figure 8 is a diagram showing an experimental schedule for evaluating the psoriasis prevention effect of oral administration of nano hyaluronic acid (nHA) after manufacturing a psoriasis animal model.

[0133]

[0134] Figure 9 is a drawing showing the visual observation of the psoriasis prevention effect of oral administration of nHA in a psoriasis animal model.

[0135]

[0136] Figure 10 is a diagram showing an experimental schedule for evaluating the psoriasis treatment effect according to oral administration of nHA after producing a psoriasis animal model.

[0137]

[0138] Figure 11 is a drawing showing the visual observation of the psoriasis treatment effect of oral administration of nHA in a psoriasis animal model.

[0139]

[0140] Figure 12 is a diagram showing an experimental schedule for evaluating the atopic dermatitis treatment effect according to oral administration of nHA after producing an atopic dermatitis animal model.

[0141]

[0142] Figure 13 is a drawing showing the visual observation of the atopic dermatitis treatment effect according to oral administration of nHA in an atopic dermatitis animal model.

[0143]

[0144] Figure 14 is a diagram showing an experimental schedule for evaluating the atopic dermatitis treatment effect according to the percutaneous administration of nHA after producing an atopic dermatitis animal model.

[0145]

[0146] Figure 15 is a drawing showing the visual observation of the atopic dermatitis treatment effect of nHA percutaneous administration in an atopic dermatitis animal model.

[0147]

[0148] Figures 16a to 16c show the results of observing the atopic dermatitis treatment effect (skin thickness) and general toxicity (body weight and spleen weight) according to the transdermal administration of nHA in an atopic dermatitis animal model.

[0149]

[0150] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0151]

[0152] Example 1: Preparation of nano hyaluronic acid

[0153] After receiving 100 mL of DIW using a mess cylinder, the DIW was stirred using a mechanical stirrer. 5 g of oligo hyaluronic acid (OHA, 6 kDa) was weighed, added 1 g at a time, and stirred. The pH was checked to be 2.0 using pH test paper (Merck pH0~6.0). The electron beam irradiation conditions were 5.0 Me and 100 kGy, and the reaction vessel was shaken from side to side to prepare a uniform sample. The electron beam was irradiated three times for a total of 300 kGy.

[0154]

[0155] To verify the ease of storage of the manufactured nano hyaluronic acid (nHA), the nano hyaluronic acid aqueous solution was freeze-dried at -80°C to produce nano hyaluronic acid powder. The manufactured nano hyaluronic acid powder was dissolved in triple-distilled water to produce a 5% nano hyaluronic acid aqueous solution for particle size measurement and drug administration. Dynamic light scattering measurements were performed on the manufactured nano hyaluronic acid particles. The average particle size of the nano hyaluronic acid in the aqueous solution immediately after manufacture was ~5 nm.

[0156]

[0157] Example 2: Preparation of an animal model of psoriasis and evaluation of its efficacy in alleviating psoriasis (transdermal)

[0158] Five-week-old C57BL / 6J male mice were purchased, and the purchased animal models were received, managed, and tested in compliance with the requirements established by the Animal Care and Animal Experiment Ethics Committee of Kyungpook National University (Approval No. 2023-0444). The animal models were provided with standard food and water in sufficient quantities on a 12-hour cycle, and were raised and managed while maintaining appropriate humidity and temperature.

[0159] The purchased mice were used at 6 weeks of age after a 7-day rest period. The mice's backs were shaved using a depilator. Once sufficient hair was removed, depilatory cream was applied to the remaining hair in the shaved area. After 5 minutes, the mice were wiped off and gently rinsed under running water. After hair removal, the mice were rested for one day before starting psoriasis-inducing treatment.

[0160]

[0161] To induce psoriasis, Aldara cream (5% imiquimod, IMQ 3M) 62.5 mg was administered daily for 4 days, and the treatment area was protected with a bandage. For a comparative study with clinically used psoriasis treatments, the steroid treatment Dermovate (GlaxoSmithKline, Brentford, United Kingdom) was used.

[0162]

[0163] The drugs used in each experimental group were administered 4 hours after Aldara cream (IMQ) treatment. The OHA group was administered 100 μl of 5% hyaluronic acid aqueous solution, the Dermovate group was administered 50 mg of Dermovate (DMT), the nHA powder group was administered 100 μl of 5% nano hyaluronic acid aqueous solution with nano hyaluronic acid powder dissolved in it, and the nHA solution group was administered 100 μl of 5% nano hyaluronic acid aqueous solution. In the case of the aqueous solution, the gauze pad on the bandage was soaked in it for treatment and covered over the Aldara cream treatment area. Each treatment drug was administered daily for 4 days in the same manner (Fig. 1).

[0164]

[0165] After 4 days of Aldara cream and drug treatment, the skin condition of each experimental group was visually assessed and tissue staining was performed to evaluate the relief effect.

[0166]

[0167] (1) Visual evaluation of psoriasis relief effect

[0168] In the IMQ and OHA treatment groups, redness and keratinization were clearly observed with the naked eye, and in the clinically used Dermovate and nHA treatment group for evaluating the therapeutic effect, the symptoms were significantly alleviated, confirming a skin condition similar to that of the control group (Fig. 2).

[0169]

[0170] (2) H&E staining of skin tissue

[0171] In the IMQ and OHA treatment groups, a marked increase in epidermal thickness was observed, and a large number of inflammatory cells were observed, suggesting psoriasis induction and inflammation. In contrast, in the Dermovate and nHA treatment groups, although the epidermis was thicker than in the control group, no findings suggestive of inflammation were observed. This suggests that hyaluronic acid nanoparticles may alleviate inflammatory cell-mediated inflammatory responses (Fig. 3).

[0172]

[0173] (3) Ki67 antibody tissue staining

[0174] Tissue staining was performed using the Ki67 antibody, a marker overexpressed during cell division. Significant Ki67 expression was observed in the IMQ, OHA, and Dermovate treatment groups compared to the control group. In contrast, some Ki67 expression was observed in the nHA treatment group, but the expression level was significantly lower than in the other experimental groups. This suggests that nHA can alleviate epidermal tissue damage seen in psoriasis symptoms and regulate the epidermal tissue division cycle to a near-normal state (Figs. 4 and 5).

[0175]

[0176] (4) IL-17A antibody tissue staining

[0177] Tissue staining was performed using an antibody against IL-17, a representative inflammatory cytokine. Significant Ki67 expression was observed in the IMQ, OHA, and Dermovate treatment groups compared to the control group. In contrast, very low levels of IL-17 expression were observed in the nHA treatment group. This suggests that nHA can alleviate the IL-17 cytokine protein-mediated inflammatory response and is associated with the modulation of the activity of Th17 immune cells, which produce and release the cytokine (Figs. 6 and 7).

[0178]

[0179] Example 3: Preparation of an animal model of psoriasis and evaluation of its efficacy in alleviating psoriasis (oral)

[0180] After removing the hair on the back of the mice, Aldara cream (Imiquimod 50 mg / g) was applied 5 times to induce psoriasis. The mice were divided into a control group (n = 3) and an experimental group (n = 3). The control group was administered PBS orally, and the experimental group was administered nHA solution (50 mg / 200 uL) orally twice a day, in the morning and evening, for 7 days, and changes in body weight and skin were observed (Fig. 8).

[0181]

[0182] As a result, it was confirmed that in the group that was orally administered PBS, psoriasis was induced a lot on the skin, but in the group that was orally administered nHA, the skin was maintained in a smooth state with almost no scabs (Fig. 9).

[0183]

[0184] If the previous nHA psoriasis animal experiment was to see the inhibitory effect of nHA on the induction of psoriasis by orally administering nHA during the process of inducing psoriasis, this experiment aimed to confirm whether psoriasis could be cured by orally administering nHA after inducing psoriasis. Aldara cream was continuously applied to the skin of the back of mice for 4 days to induce psoriasis, and then nHA was orally administered twice a day for 5 days to verify the psoriasis improvement effect. The dose of nHA administered orally was 70 mg / 200 uL per mouse (Fig. 10).

[0185]

[0186] Psoriasis symptoms began to appear in all models starting on day 5, after four consecutive Aldara cream treatments. However, in the experimental group administered orally with nHA, symptoms gradually improved, with the skin returning to its original state around day 8 or 9. In contrast, in the control group administered orally with PBS, symptoms continued to worsen (Fig. 11).

[0187]

[0188] Example 4: Evaluation of in vivo atopic dermatitis relief efficacy (oral)

[0189] An atopic animal model was developed by shaving the backs of Balb / C mice and continuously treating them with a 1% solution of 1-chloro-2,4-dinitrobenzene (DNCB) for 7 days to induce atopic symptoms. A 0.5% DNCB solution was administered three times at 2-day intervals to maintain the symptoms. After 7 days of DNCB treatment, nHA was orally administered twice daily at a dose of 100 mg / 200 uL, starting on the 7th day, to observe the improvement in atopic symptoms. The control group received an equal amount of PBS orally (Fig. 12).

[0190]

[0191] As a result, atopic symptoms reached their peak in both the control and experimental groups by Days 8 and 9, but a clear difference was observed from Day 10. In particular, as of Days 13 and 14, it was confirmed that atopic symptoms were significantly improved in animals orally administered nHA compared to the control group (Fig. 13).

[0192]

[0193] Example 5: In vivo evaluation of atopic dermatitis relief efficacy (transdermal)

[0194] Atopic dermatitis was induced in BALB / C mice by topically applying 1% DNCB once a day for 1 week (7 times in total), followed by 0.5% DNCB three times a week from days 8 to 14. nHA was dissolved in distilled water at a 5% concentration and topically applied to the backs of the atopic animal models. Skin thickness was measured three times a week (Fig. 14).

[0195]

[0196] From the 4th day of DNCB treatment, an increase in skin thickness was observed compared to the untreated DNCB control group, and differences in skin thickness were observed until the 15th day. The nHA treatment group showed improvement in skin condition and a decrease in skin thickness compared to the DNCB treatment group (Figs. 15 to 16c). No rapid changes in animal body weight were observed due to nHA administration, and the increased spleen weight due to DNCB administration tended to be recovered by nHA administration (Figs. 16a to 16c).

[0197]

[0198] Hyaluronic acid nanoparticles manufactured solely through electron beam irradiation exhibit high biocompatibility, skin permeability, and excellent anti-inflammatory activity, and thus can be very useful in the prevention, improvement, or development of therapeutic agents for inflammatory skin diseases, and thus have high potential for industrial application.

Claims

1. A pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising hyaluronic acid nanoparticles as an active ingredient, which are manufactured by irradiating an electron beam to an aqueous solution of hyaluronic acid or its salt to form intermolecular and / or intramolecular cross-linking of hyaluronic acid.

2. A pharmaceutical composition according to claim 1, characterized in that the aqueous solution of hyaluronic acid or its salt is acidic.

3. A pharmaceutical composition according to claim 1, wherein the aqueous solution of hyaluronic acid or its salt has a concentration of 0.1 to 10% (w / w).

4. A pharmaceutical composition according to claim 1, characterized in that the electron beam is irradiated at a dose of 10 to 400 kGy.

5. A pharmaceutical composition according to claim 1, wherein the hyaluronic acid nanoparticles have an average diameter of 100 nm or less.

6. A pharmaceutical composition according to claim 1, characterized in that the inflammatory skin disease is selected from the group consisting of psoriasis, contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, contact urticaria syndrome, autosensitization dermatitis, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, and eczema.

7. A cosmetic composition for preventing or improving inflammatory skin diseases, comprising hyaluronic acid nanoparticles as an active ingredient, which are manufactured by irradiating an electron beam to an aqueous solution of hyaluronic acid or its salt to form intermolecular and / or intramolecular cross-linking of hyaluronic acid.

8. A pharmaceutical composition for preventing or improving inflammatory skin diseases, comprising hyaluronic acid nanoparticles as an active ingredient, which are manufactured by irradiating an aqueous solution of hyaluronic acid or its salt with an electron beam to form intermolecular and / or intramolecular cross-linking of hyaluronic acid.

9. A food composition for preventing or improving inflammatory skin diseases, comprising hyaluronic acid nanoparticles as an active ingredient, which are manufactured by irradiating an electron beam to an aqueous solution of hyaluronic acid or its salt to form intermolecular and / or intramolecular cross-linking of hyaluronic acid.

10. A food composition according to claim 9, characterized in that the food composition is a health functional food.

11. Use of hyaluronic acid nanoparticles produced by irradiating an aqueous solution of hyaluronic acid or its salt with an electron beam to form intermolecular and / or intramolecular cross-linking of hyaluronic acid for producing a pharmaceutical composition for preventing or treating inflammatory skin diseases.

12. A method for treating an inflammatory skin disease, comprising administering to a subject in need thereof an effective amount of a composition comprising hyaluronic acid nanoparticles as an active ingredient, the composition being prepared by irradiating an electron beam to an aqueous solution of hyaluronic acid or a salt thereof to form intermolecular and / or intramolecular cross-linking of hyaluronic acid.

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