Novel peptide having hyaluronic acid degradation delaying activity and use thereof

Abstract

The peptide of the present invention has an activity of promoting skin cell activation, and can be used for skin regeneration or improvement of skin conditions. Furthermore, the peptide of the present invention has an activity of delaying or inhibiting the degradation of hyaluronic acid by hyaluronidase and reactive oxygen species, and thus, when used for preparing a hyaluronic acid filler, can prolong the degradation time of the hyaluronic acid filler in the body, thereby improving the performance of the filler.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of Korean Patent Application No. 10-2023-0050317, filed on April 17, 2023, with the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to a novel peptide with activity that delays the degradation of hyaluronic acid and its uses. Background Technology

[0004] The skin is composed of the epidermis, dermis, and subcutaneous tissue from the outside in. The epidermis is made up of various cells, such as keratinocytes composed of keratin, melanocytes that form and secrete melanin, and Langerhans cells, which are immune-related cells. The dermis is composed of fibrous and matrix components. Collagen, as a fibrous component, provides strength and tension to the skin and plays a protective role, making up about 90% of the dermis. Furthermore, collagen plays an important role in wound healing and promotes collagen synthesis in damaged epithelium, enabling wounds to repair quickly and without scars. Collagen is synthesized under the action of fibroblasts and degraded by collagenase and elastase. With age, the function and number of fibroblasts decrease, leading to a reduction in the synthesis of collagen as a fibrous component. This results in water loss from skin cells, changes in the structure of the stratum corneum, and increased activity of collagenase, leading to an increase in cross-linked collagen, thereby reducing the smoothness, hydration, and firmness of the skin. Besides aging, external factors contributing to skin aging include pollution, wind, temperature, and ultraviolet radiation. In an effort to prevent skin aging, there is growing interest in substances that promote collagen synthesis. Previously known collagen-boosting substances include vitamin C, retinoic acid, placental proteins, betulinic acid, and chlorella extract. However, their use is limited due to safety concerns such as irritation and redness when applied to the skin, or because their effects are minimal. Consequently, it is difficult to expect them to have any real impact on improving skin function or healing wounds.

[0005] Fillers are substances injected into the skin or tissue to increase and expand the volume of soft tissue, improving problems such as wrinkles, sagging skin, and loss of skin or tissue volume caused by aging. Hyaluronic acid (HA) is a sugar that is an important component of skin and connective tissue. It is a natural high-molecular-weight polysaccharide composed of repeating disaccharide units of N-acetylglucosamine and D-glucuronic acid. The dermis of the skin is filled with a macromolecular network structure called the extracellular matrix, which is composed of substances produced by fibroblasts and other cells in the dermis. These substances include polysaccharides called acidic mucopolysaccharides (such as hyaluronic acid and dermatan sulfate) and fibrinogens (such as collagen and elastin). Therefore, hyaluronic acid is a naturally occurring component in various tissues of the human body (such as skin, joints, and eye mucosa), and is widely used as a primary filler material due to its ease of use, lack of immunogenicity, non-carcinogenicity, and biocompatibility.

[0006] It is well known that the human body contains an enzyme called hyaluronidase, which breaks down hyaluronic acid. Hyaluronic acid can also be degraded by free radicals (reactive oxygen species) present in the body. Therefore, even if hyaluronic acid fillers are injected into the body, the hyaluronic acid will naturally decompose and disappear. To prolong the duration of hyaluronic acid fillers, a method has been adopted that uses a suitable cross-linking agent (such as 1,4-butanediol diglycidyl ether (BDDE)) to cross-link hyaluronic acid. In this way, when the filler is made by cross-linking hyaluronic acid using a cross-linking agent, the filler can be protected to some extent from the degradation by hyaluronidase in the body, thereby prolonging the duration of the filler. However, the effect is not ideal.

[0007] Korean Patent No. 10-2369261 discloses a method for preparing a core-shell structured hyaluronic acid gel, wherein the core is made of hyaluronic acid that has been cross-linked primarily by a first cross-linking agent, and the shell is added to the core containing cross-linked hyaluronic acid using a second cross-linking agent. Korean Patent No. 10-2225971 discloses a method for preparing hyaluronic acid-based hydrogels using a peptide cross-linking agent. Korean Patent No. 10-2418096 discloses a method for orally administering collagen to maintain the efficacy of hyaluronic acid fillers.

[0008] Despite numerous attempts using conventional techniques, a safe and effective technology to delay the degradation of hyaluronic acid fillers has yet to be developed.

[0009] [Existing Technical Documents]

[0010] [Patent Literature]

[0011] Korean Patent No. 10-2369261

[0012] Korean Patent No. 10-2225971

[0013] Korean Patent No. 10-2418096 Summary of the Invention

[0014] Technical issues

[0015] The present invention aims to solve the above-mentioned problems, and the purpose of the present invention is to provide a biocompatible peptide that has the activity of delaying the degradation of hyaluronic acid by hyaluronidase and reactive oxygen species, as well as the activity of promoting skin cell activation, while the peptide has relatively excellent stability even under acidic or high temperature conditions.

[0016] Therefore, one object of the present invention is to provide a novel peptide that has the activity of promoting skin cell activation and delaying the degradation of hyaluronic acid.

[0017] Another object of the present invention is to provide a cosmetic composition for skin regeneration or improvement of skin condition, comprising peptides having the above-mentioned activity as active ingredients.

[0018] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of skin diseases, comprising a peptide having the above-mentioned activity as an active ingredient.

[0019] Another object of the present invention is to provide a composition for delaying the degradation of hyaluronic acid, comprising a peptide having the above-described activity as an active ingredient.

[0020] Another object of the present invention is to provide a filler composition comprising a peptide having the above-described activity and hyaluronic acid.

[0021] Technical solution

[0022] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0023] Another aspect of the present invention provides a cosmetic composition for skin regeneration or improvement of skin condition, comprising the peptide as an active ingredient.

[0024] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of skin diseases, comprising the peptide as an active ingredient.

[0025] Another aspect of the present invention provides a composition for delaying the degradation of hyaluronic acid, comprising the peptide as an active ingredient.

[0026] Another aspect of the present invention provides a filler composition comprising the peptide and hyaluronic acid.

[0027] The present invention will be described in detail below.

[0028] Peptides and their activities

[0029] According to one aspect of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1 is provided.

[0030] [Amino acid sequence of SEQ ID NO: 1]

[0031]

[0032] As used in this article, the term "peptide" refers to a linear molecule formed by amino acid residues linked together by peptide bonds.

[0033] The peptide containing the amino acid sequence of SEQ ID NO: 1 in this invention can be used directly without modification. However, within the range that does not affect the original activity of the peptide, such as the range that does not affect its activity of inhibiting or delaying the degradation of hyaluronic acid by hyaluronidase or reactive oxygen species, mutants or fragments of amino acids with different sequences by deletion, insertion, substitution or combination of amino acid residues can be used.

[0034] The peptides of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., without changing their activity.

[0035] The peptides of the present invention include peptides containing an amino acid sequence substantially identical to that of the peptide containing the amino acid sequence of SEQ ID NO: 1, and their variants or active fragments. A substantially identical amino acid sequence refers to an amino acid sequence with a sequence identity of 75% or more compared to the amino acid sequence of SEQ ID NO: 1, for example, a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more. Additionally, the peptide may further include a targeting sequence, a tag, labeled residues, or an amino acid sequence prepared for a specific purpose of increasing half-life or peptide stability.

[0036] The peptides of the present invention may be peptides in which a portion of the amino acid sequence is selected and modified with N-terminus and / or C-terminus to increase their activity. Such N-terminal and / or C-terminal modifications can significantly improve the stability of the peptides of the present invention, and, for example, can prolong the half-life of the peptides when administered in vivo. The term "stability" includes not only in vivo stability that protects the peptides of the present invention from attack by proteolytic enzymes in vivo, but also storage stability (e.g., storage stability at room temperature).

[0037] N-terminal modification can be a modification in which a protecting group selected from the group consisting of acetyl, fluorenemethyloxycarbonyl, formyl, palmitoyl, myristyl, stearoyl, and polyethylene glycol (PEG) binds to the N-terminus of the peptide. C-terminal modification can be, but is not limited to, modifications in which hydroxyl (-OH), amino (-NH2), azide (-NHNH2), etc., bind to the C-terminus of the peptide.

[0038] The peptides of the present invention can be prepared by various methods well known in the art to which this invention pertains. For example, the peptides of the present invention can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (see Merrifield, J. Amer. Chem. Soc. 85:2149-54(1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111(1984)) or liquid-phase synthesis techniques (US Patent No. 5,516,891).

[0039] The peptides of this invention have the activity of activating skin cells.

[0040] In one embodiment, the peptide of the present invention has activity that promotes the proliferation of fibroblasts or keratinocytes.

[0041] In one embodiment, the peptide of the present invention has activity that promotes the expression of collagen or hyaluronic acid.

[0042] In one embodiment, the peptide of the present invention has the activity of promoting the expression of collagen or hyaluronic acid in fibroblasts or keratinocytes.

[0043] In one embodiment, the peptide of the present invention has the activity of delaying or inhibiting the degradation of hyaluronic acid.

[0044] In one embodiment, the degradation of hyaluronic acid can be caused by hyaluronidase or by reactive oxygen species.

[0045] As described above, the peptide of the present invention has the activity of delaying or inhibiting the degradation of hyaluronic acid by hyaluronidase or reactive oxygen species; therefore, when used with fillers made of hyaluronic acid, the peptide has the effect of prolonging the presence time of the hyaluronic acid filler in vivo, thereby improving the performance of the filler.

[0046] Furthermore, the peptides of the present invention exhibit stability under pH and temperature variations. As shown in the examples described below, the peptides of the present invention exhibit the same stability in a pH range of 4 to 10 and a temperature range of 25°C to 50°C, thus demonstrating stability under pH and temperature variations and thus possessing excellent biological stability.

[0047] Peptide-containing compositions

[0048] In another aspect of the invention, the invention provides a composition for skin regeneration or improvement of skin condition, comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0049] In another aspect of the invention, the present invention provides a cosmetic composition for skin regeneration or improvement of skin condition, comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0050] The term “improvement” as used in this article refers to any behavior that can improve or benefit the symptoms of a disease or condition.

[0051] In this instruction manual, improving skin condition refers to the process of treating, reducing, or alleviating skin damage caused by internal or external factors and their effects.

[0052] In one implementation, improving skin condition can be achieved by increasing skin elasticity, improving skin wrinkles, increasing skin hydration, inhibiting skin aging, repairing skin damage caused by ultraviolet radiation, protecting skin from ultraviolet damage, or improving skin wounds.

[0053] In one embodiment, the cosmetic composition contains a peptide as an active ingredient comprising the amino acid sequence of SEQ ID NO: 1, which promotes the expression of collagen or hyaluronic acid in skin cells (e.g., fibroblasts or keratinocytes).

[0054] In this invention, "improving skin wrinkles," "promoting skin elasticity," "inhibiting skin aging," "repairing skin damage caused by ultraviolet radiation," "protecting skin from ultraviolet radiation," or "improving skin wounds" can be induced by any behavior that increases the total amount of collagen in the skin, including promoting collagen synthesis in skin cells. Skin cells can be, for example, fibroblasts or keratinocytes.

[0055] "Inhibiting skin aging" can mean inhibiting the decline of skin function, such as wrinkles, sagging skin, and loss of elasticity. Skin aging can also be photoaging, such as skin aging caused by ultraviolet radiation.

[0056] In addition to comprising a peptide containing the amino acid sequence of SEQ ID NO: 1, the cosmetic composition of the present invention may further comprise a cosmetically acceptable carrier.

[0057] Cosmetic compositions can be prepared into any formulation commonly prepared in the technical field to which this invention pertains, and can be topical skin formulations. For example, they can be formulated into solutions, suspensions, lotions, creams, gels, ointments, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, lotion foundations, wax foundations, and sprays, but are not limited thereto.

[0058] Cosmetic compositions can be prepared in various forms, such as solutions (e.g., toners, nourishing waters, nourishing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, masks, sprays, powders, hair tonics, hair creams, hair conditioners, shampoos, hair dyes, hair gels, hair aerosols, hair oils, and gels), sol-gels, emulsions, oils, waxes, aerosols, etc., but not limited to these.

[0059] The cosmetic compositions of the present invention may contain other additives, such as excipients and carriers, and can incorporate and blend commonly used ingredients that are mixed in ordinary skin cosmetics as needed.

[0060] When the dosage form of a cosmetic composition is a cream, lotion, or gel, animal oils, vegetable oils, waxes, paraffin wax, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc., can be used as carrier ingredients.

[0061] When the cosmetic composition is in the form of a powder or spray, it may contain lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder as a carrier component. In the case of a spray, it may also contain a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether, but is not limited thereto.

[0062] When the dosage form of a cosmetic composition is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component. Examples of such carrier components may include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol aliphatic esters, polyethylene glycol, or fatty acid esters of sorbitol.

[0063] When the dosage form of a cosmetic composition is a suspension, liquid diluents (e.g., water, ethanol, propylene glycol, etc.) can be used as carrier components; suspending agents (e.g., ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester, polyoxyethylene dehydrated sorbitol ester, etc.); microcrystalline cellulose, aluminum hydroxide, bentonite, agar, tragacanth gum, etc.

[0064] When the dosage form of a cosmetic composition is a cleanser containing surfactants, the carrier components may include fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurine, sarcosinates, fatty acid amide ether sulfates, alkyl amide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, etc.

[0065] When the cosmetic composition is in the form of a shampoo, the basic ingredients used to constitute the shampoo (such as thickeners, surfactants, viscosity modifiers, humectants, pH adjusters, preservatives, and essential oils) can be mixed with the peptides of the present invention. CDE can be used as a thickener; LES as an anionic surfactant and cocobetaine as an amphoteric surfactant can be used as surfactants; polyquaternium salts can be used as viscosity modifiers; glycerin can be used as a humectant; and citric acid or sodium hydroxide can be used as a pH adjuster. Grapefruit extract and the like can be used as preservatives. Furthermore, essential oils (e.g., cedarwood, peppermint, rosemary, etc.), silk amino acids, pentanol, or vitamin E can be added.

[0066] In addition to the peptides of this invention as active ingredients and the carrier components, the cosmetic composition may also contain ingredients commonly used in cosmetic compositions, such as conventional adjuvants (e.g., antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances), but is not limited thereto.

[0067] The cosmetic composition of the present invention may contain a cosmetically effective amount of a peptide, the peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0068] The effective amount of a cosmetic product refers to the amount of a cosmetic composition sufficient to achieve skin regeneration or improve skin condition.

[0069] The peptides of the present invention can be included in the above-mentioned cosmetic compositions at concentrations from 0.01 μM to 1000 μM. Specifically, the peptides of the present invention can be in concentrations of 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1... Concentrations of 5 μM to 200 μM, 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM are included in, but not limited to, the above cosmetic compositions.

[0070] In another aspect of the invention, the invention provides a pharmaceutical composition for the prevention or treatment of skin diseases, comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0071] In one implementation, the skin condition can be a wound or scar on the skin.

[0072] In one embodiment, a peptide (containing the amino acid sequence of SEQ ID NO: 1) included as an active ingredient in the pharmaceutical composition can promote the expression of collagen or hyaluronic acid in skin cells (such as fibroblasts or keratinocytes).

[0073] The pharmaceutical compositions of the present invention may contain a therapeutically effective amount of the peptides of the present invention.

[0074] The term "therapeutic effective amount" refers to an amount sufficient to enable the peptide, which is the active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, for example, an amount sufficient to achieve the efficacy of treating or preventing skin diseases.

[0075] As used in this article, “prevention” means reducing the risk of developing a disease or condition, and refers to any action that inhibits or delays the onset of a disease by preventing the progression of the disease or one or more of its clinical symptoms.

[0076] As used in this article, the term "treatment" means to alleviate a disease or symptom, and includes any action that improves or beneficially alters the symptoms of a disease by stopping or slowing its progression or the progression of one or more of its clinical symptoms.

[0077] In this invention, prevention or treatment of skin diseases can be achieved by eliminating the cause of the skin disease or inhibiting its progression.

[0078] The pharmaceutical compositions of the present invention may contain a peptide comprising the amino acid sequence of SEQ ID NO: 1 and a pharmaceutically acceptable carrier.

[0079] The pharmaceutically acceptable carriers mentioned above are commonly used carriers in formulation preparation, including but not limited to lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc.

[0080] In addition to the above-mentioned components, the pharmaceutical composition of the present invention may also contain lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but is not limited thereto.

[0081] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: Pharmaceutical Science and Practice (19th edition, 1995, Williams & Wilkins).

[0082] The pharmaceutical compositions of the present invention can be administered via any suitable route for treating skin diseases, for example, orally or parenterally. In the case of parenterally administration, they can be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical application, transdermal application, etc. Because the pharmaceutical compositions of the present invention have activity in preventing or treating skin diseases, they can be applied topically, such as by applying to the skin.

[0083] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg or 1.0 μg to 1000 mg per day, and may be prescribed in various ways based on factors such as preparation method, administration method, patient age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness.

[0084] The pharmaceutical compositions of the present invention can be prepared in unit dose form, or can be formulated by placing them in a multi-dose container and using pharmaceutically acceptable carriers and / or excipients, a method readily performed by those skilled in the art to which this invention pertains. Specifically, the formulations can be in the form of solutions, suspensions, or emulsions in oil or aqueous media, or in the form of extracts, powders, granules, tablets, or capsules, and may additionally contain dispersants or stabilizers.

[0085] The pharmaceutical compositions of the present invention can be topical skin preparations. Topical skin preparations are formulations that can be applied externally to the skin. When the pharmaceutical compositions of the present invention are used as topical skin preparations, they can be applied to the skin, particularly to areas of skin where a skin disease has occurred. Topical skin preparations can be creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, medicated bandages, lotions, or combinations thereof. Where necessary, topical skin preparations may appropriately incorporate ingredients commonly used in topical skin preparations such as cosmetics or pharmaceuticals, such as aqueous components, oily components, powdered components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. Topical skin preparations may also contain, as appropriate, metal chelating agents such as disodium EDTA, trisodium EDTA, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannins, licorice extract, glycyrrhizin, various herbs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0086] Another aspect of the present invention provides a composition for delaying the degradation of hyaluronic acid, comprising a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0087] Another aspect of the present invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in delayed degradation of hyaluronic acid.

[0088] In one embodiment, the degradation of hyaluronic acid can be caused by hyaluronidase or by reactive oxygen species.

[0089] In this invention, "delayed" hyaluronic acid degradation is used as an equivalent meaning to "inhibit" or "prevent" hyaluronic acid degradation.

[0090] Another aspect of the present invention provides a filler composition comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 and hyaluronic acid.

[0091] In this invention, hyaluronic acid (HA) refers to a biopolymer represented by the following formula 1, wherein repeating units composed of N-acetyl-D-glucosamine and D-glucuronic acid are linearly connected.

[0092] [Formula 1]

[0093]

[0094] Hyaluronic acid is abundant in the vitreous fluid of the eye, the synovial fluid of joints, and the comb of roosters. Due to its excellent biocompatibility, it is widely used in the medical and medical device fields, such as ophthalmic surgical aids, joint function improvers, drug delivery materials, eye drops, and wrinkle-reducing agents used in cosmetics.

[0095] In one embodiment, the hyaluronic acid contained in the filler composition of the present invention may include, in addition to hyaluronic acid, its salts. Salts of hyaluronic acid include inorganic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, and cobalt hyaluronate, as well as organic salts such as tetrabutylammonium hyaluronate, but are not limited thereto.

[0096] In one embodiment, hyaluronic acid or a salt thereof may be hyaluronic acid cross-linked via a suitable cross-linking agent.

[0097] In one embodiment, the crosslinked hyaluronic acid can be a hyaluronic acid crosslinked with one or more crosslinking agents selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol (PEG), divinyl sulfone (DVS), polyethylene glycol diglycidyl ether (PEGDGE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and polytetramethylene glycol diglycidyl ether.

[0098] In the filler composition of the present invention, relative to the total composition, it comprises SEQ ID NO: The content of the peptide in the amino acid sequence of 1 can be 0.001-0.5 wt%, 0.002-0.5 wt%, 0.003-0.5 wt%, 0.004-0.5 wt%, 0.005-0.5 wt%, 0.001-0.4 wt%, 0.002-0.4 wt%, 0.003-0.4 wt%, 0.004-0.4 wt%, 0.005-0.4 wt%, 0.001-0.3 wt%, 0.002-0.3 wt%, 0.003-0.3 wt%, 0.004-0.3 wt%, 0.005-0.3 wt%, 0.001-0.2 wt%, 0.002-0.2 wt%, 0.003-0.2 wt%, 0.004-0.2 wt%, 0.005-0.2 wt%, but is not limited to these.

[0099] In the filler composition of the present invention, the content of hyaluronic acid relative to the total composition may be 0.001-0.5% by weight, 0.002-0.4% by weight, 0.003-0.3% by weight, 0.004-0.2% by weight, or 0.005-0.1% by weight, but is not limited thereto.

[0100] In one embodiment, the filler composition of the present invention comprises a peptide of the amino acid sequence of SEQ ID NO: 1 and hyaluronic acid in a simple mixed state.

[0101] The term "filler" as used in this invention broadly refers to a material or composition intended to increase volume in areas of soft tissue defects, and also includes the meaning of soft tissue fillers or dermal fillers.

[0102] As used in this invention, the term "soft tissue" generally refers to tissues that connect to, support, or surround other structures and organs of the body. Such soft tissues include, for example, muscles, tendons, vocal cords, endothelial tissue, fibrous tissue, fat, blood vessels, nerves, and synovial tissue.

[0103] In one embodiment, the filler composition may optionally include, but is not limited to, buffers, preservatives, isotonic agents, antioxidants, emulsifiers, humectants, etc., and may include other pharmaceutically acceptable ingredients.

[0104] Buffers may include, but are not limited to, one or more of the following: citric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, acetic acid, diethylbarbituric acid, sodium acetate, tris(hydroxymethyl)methylaminopropanesulfonic acid (TAPS), 2-bis(2-hydroxyethyl)aminoacetic acid (Bicine), tris(hydroxymethyl)aminomethane (Tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (Tricine), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino]methanesulfonic acid (TES), and piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES).

[0105] Isotonic agents can be used without limitation, as long as they are used to prepare hyaluronic acid fillers and can be contained in a buffer solution. Sodium chloride is a preferred isotonic agent.

[0106] In one embodiment, the filler composition of the present invention may also contain other substances or combinations of substances that provide beneficial effects when administered to a subject, including but not limited to antioxidants, antipruritics, anti-cellulite agents, anti-scarring agents, anti-inflammatory agents, anesthetics, irritant relievers, vasoconstrictors, vasodilators, antihemorrhagic agents (such as hemostatic agents or antifibrinolytic agents), exfoliants, elastomers, antiacne agents, colorants, antipigmentation agents, or moisturizers.

[0107] In one embodiment, the filler composition can be used to improve wrinkles and tissue volume reduction in soft tissues selected from the group consisting of the neck, chest, buttocks, arms, armpits, hands, legs, and feet, or to treat wounds, scars, or stretch marks.

[0108] In one embodiment, the filler composition may be a cosmetic composition, and is preferably a cosmetic composition for improving wrinkles and tissue volume reduction in soft tissues selected from the group consisting of the neck, chest, buttocks, arms, armpits, hands, legs and feet.

[0109] In one embodiment, the filler composition may be a pharmaceutical composition, preferably a pharmaceutical composition for treating or preventing wounds, scars, or stretch marks.

[0110] When the filler composition is in the form of a cosmetic composition or a pharmaceutical composition, the technical content and features described in the above composition items for cosmetic compositions and pharmaceutical compositions can be applied as is to the filler composition of the present invention. Therefore, in order to avoid making the specification too complicated, they will not be described again.

[0111] The filler composition of the present invention can be applied in dosage forms suitable for the above-described uses, preferably in the form of an injection.

[0112] The filler compositions of the present invention can be sterilized and can be quantitatively filled, sealed and sterilized in suitable containers (such as syringes).

[0113] Uses of peptides

[0114] In another aspect of the invention, the invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in skin regeneration or improvement of skin condition.

[0115] In another aspect of the invention, the invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the preparation of cosmetics for skin regeneration or improvement of skin condition.

[0116] In another aspect of the invention, the invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the treatment of skin diseases.

[0117] In another aspect of the invention, the invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the preparation of a medicament for treating skin diseases.

[0118] In another aspect of the invention, the invention provides the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in delayed degradation of hyaluronic acid.

[0119] Since the technical content and features of the present invention described in the peptides and compositions of the present invention are applied in the same way to the uses of the peptides of the present invention, in order to avoid making the specification too complicated, these technical content and features will not be described again.

[0120] Beneficial effects

[0121] The peptides of the present invention have the activity of promoting skin cell activation, and therefore can be used for skin regeneration or improving skin condition. Furthermore, the peptides of the present invention also have the activity of delaying or inhibiting the degradation of hyaluronic acid by hyaluronidase and reactive oxygen species; therefore, when used in the preparation of hyaluronic acid fillers, the peptides of the present invention have the following effect: improving the performance of the filler by prolonging the degradation time of the hyaluronic acid filler in vivo.

[0122] However, the effects of the present invention are not limited to those mentioned above, and those skilled in the art will be able to clearly understand other unmentioned effects based on the description below. Attached Figure Description

[0123] Figure 1 The experimental results demonstrate the effect of the peptides of the present invention on promoting the proliferation of NIH3T3 cells (mouse fibroblasts) and HaCaT cells (human keratinocyte lineage).

[0124] Figure 2 The experimental results demonstrate the effect of the peptides of the present invention on promoting the expression of collagen and hyaluronic acid (HA) in NIH3T3 cells (mouse fibroblasts) and HaCaT cells (human keratinocyte lineage).

[0125] Figure 3 These are experimental results, which demonstrate the effect of hyaluronidase degradation of hyaluronic acid in the peptide-delayed hyaluronic acid solution of the present invention.

[0126] Figure 4 These are experimental results, which demonstrate the effect of hyaluronidase degradation of hyaluronic acid in the peptide-delayed BDDE crosslinked hyaluronic acid filler of the present invention.

[0127] Figure 5 The experimental results demonstrate the effect of the peptide-delayed hyaluronic acid solution of the present invention on the degradation of hyaluronic acid by reactive oxygen species (hydrogen peroxide).

[0128] Figure 6 The experimental results demonstrate the effect of reactive oxygen species (hydrogen peroxide) on the degradation of hyaluronic acid in the peptide-delayed BDDE crosslinked hyaluronic acid filler of the present invention.

[0129] Figure 7a The experimental results show that the peptides of the present invention are stable even at high temperatures of 50°C.

[0130] Figure 7b The experimental results show that the peptides of the present invention are stable even in a pH range of 4 to 10.

[0131] Figure 8 This is a schematic diagram illustrating the effect of peptide-delayed degradation of hyaluronidase on hyaluronidase according to the present invention. Detailed Implementation

[0132] The invention will be described in detail below by way of examples. However, the following examples are intended to specifically illustrate the invention, and the scope of the invention is not limited to these examples.

[0133] Preparation Example 1: Preparation of Peptides and Peptide Complexes

[0134] Peptides with the amino acid sequence SEQ ID NO: 1, as shown in Table 1, were synthesized using an automated peptide synthesizer (CEM Liberty, CEM Corporation, USA). These synthesized peptides were purified using C18 reversed-phase high-performance liquid chromatography (HPLC) (Thermo Fisher Scientific, USA). The chromatographic column used was an ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA).

[0135] [Table 1]

[0136]

[0137] The efficacy of the peptide with SEQ ID NO: 1 prepared above was evaluated through the following experiments.

[0138] Experimental Example 1: Cell Proliferation Analysis

[0139] The effect of the peptide with SEQ ID NO: 1 prepared in Preparation Example 1 above on cell proliferation was evaluated.

[0140] NIH3T3 cells (mouse fibroblasts) and HaCaT cells (human keratinocyte line) were used at a ratio of 1×10⁻⁶. 4Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours. Cells were washed once with serum-free DMEM medium. Different amounts of peptide were added to 200 μL of serum-free DMEM medium to prepare peptide medium solutions with concentrations of 1.6 μM, 8 μM, 40 μM, 200 μM, and 1000 μM, and the washed cells were treated with the prepared peptide medium solutions. The treated cells were then cultured in a CO2 incubator at 37°C for 72 hours, washed twice with PBS, and MTT (Sigma, catalog number: M2003, USA) solution was aliquoted into each well to a final concentration of 0.5 mg / mL. After incubation in the dark, the cells were cultured in a CO2 incubator at 37°C for 4 hours. The absorbance was measured at 540 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA).

[0141] like Figure 1 The experimental results show that when treated with peptides, a significant effect on promoting cell proliferation was observed in both skin cell lines (NIH3T3 cells and HaCaT cells).

[0142] Experiment Example 2: Analysis of the effects on promoting collagen and hyaluronic acid (HA) expression

[0143] The effect of the peptide with SEQ ID NO: 1 prepared in Preparation Example 1 above on the expression of collagen and hyaluronic acid (HA) was confirmed.

[0144] NIH3T3 cells (mouse fibroblasts) and HaCaT cells (human keratinocyte line) were used at a ratio of 1×10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 24-well plates and cultured for 24 hours. Cells were washed once with serum-free DMEM medium. Different amounts of peptide were added to 500 μL of serum-free DMEM medium to prepare peptide medium solutions at concentrations of 50 μM, 100 μM, and 200 μM, and the washed cells were treated with these solutions. As positive controls, 100 nM TGF-β1 was used in NIH3T3 cells and 100 nM IGF-1 was used in HaCaT cells. The treated cells were then cultured in a CO2 incubator at 37°C for 72 hours. The cell culture medium was collected, centrifuged, and the supernatant was transferred to test tubes. The supernatant was analyzed by ELISA using a Collagen ELISA kit (Abcam, catalog number: ab210579, UK) and a HA ELISA kit (ECHELON Biosciences, catalog number: K-1200, USA), respectively.

[0145] like Figure 2The experimental results show that treatment with peptides at concentrations ranging from 50 μM to 200 μM significantly increased collagen expression and hyaluronic acid (HA) expression in NIH3T3 and HaCaT cells. These results indicate that the peptides of this invention stimulate skin cells, thereby promoting the production of the extracellular matrix (ECM).

[0146] Experimental Example 3: Analysis of the effect of delaying the degradation of hyaluronidase on hyaluronic acid (HA)

[0147] The effect of the peptide with SEQ ID NO: 1 prepared in Preparation Example 1 above on delaying the degradation of hyaluronic acid by hyaluronidase was evaluated.

[0148] 1. Evaluation of delayed degradation of hyaluronic acid in hyaluronic acid solution

[0149] Hyaluronic acid was dissolved in PBS to a concentration of 1.5% by weight, hydrated, and 10 g of the solution was aliquoted into each conical tube. Peptide solution was added to the aliquoted hyaluronic acid solutions to achieve final peptide concentrations of 0 ppm (no peptide added), 100 ppm, 1000 ppm, and 5000 ppm. 2 mL of hyaluronidase (Hadase, Sigma-Aldrich, derived from bovine testis) was added to each conical tube. Viscosities were measured at 0 and 30 minutes using a rheometer (single shear stress) and compared with peptide concentrations. As described above, the 1.5% by weight hyaluronic acid solutions containing each peptide concentration were treated with hyaluronidase, and the enzyme reaction was maintained for 30 minutes to degrade the hyaluronic acid. After the degradation reaction, the viscosity of the hyaluronic acid solution was measured to confirm the degradation-delaying effect. Figure 3 As shown, the experimental results confirmed that the degradation of hyaluronic acid was delayed with increasing peptide concentration. Since the difference in degradation delay efficacy between 1000 ppm and 5000 ppm was not significant, subsequent experiments were conducted at a concentration of 1000 ppm.

[0150] 2. Evaluation of hyaluronic acid degradation in delayed crosslinking fillers

[0151] 1,4-Butanediol diglycidyl ether (BDDE) crosslinked hyaluronic acid fillers, with or without the peptide from Preparation Example 1, were prepared, and Congo red and hyaluronidase (HAdase) were prepared as absorption dyes. 20 μL of 1 mM Congo red was added to 1.5 mL Eppendorf tubes, and 100 μL of peptide-free filler (control filler) and peptide-containing filler were added to the absorption dyes, respectively. Each sample was treated with 350 μL of 0.15 mg / mL (10⁸ units) hyaluronidase. Each tube was incubated at 37°C, and 100 μL of sample supernatant was collected at 6 and 24 hours and transferred to 96-well plates. Absorbance was measured at 500 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA). As described above, BDDE cross-linked hyaluronic acid filler samples with or without peptides were prepared, and the degree of degradation of the hyaluronic acid filler by hyaluronidase was compared based on the presence or absence of peptides. The degree of degradation of the hyaluronic acid filler was measured based on the change in absorbance caused by dye diffusion. That is, the principle of increased sample fluidity due to hyaluronic acid degradation by the degrading enzyme is utilized. Figure 4 As shown, the experimental results confirmed that after 24 hours of treatment with hyaluronidase, the degradation delay was improved by more than 15% due to the peptides contained in the sample.

[0152] Experiment Example 4: Analysis of the effect of delaying the degradation of hyaluronic acid (HA) by reactive oxygen species

[0153] The effect of the peptide with SEQ ID NO: 1 prepared in Preparation Example 1 above on delaying the degradation of hyaluronic acid by reactive oxygen species was evaluated.

[0154] 1. Evaluation of delayed degradation of hyaluronic acid in hyaluronic acid solution

[0155] Hyaluronic acid was dissolved in PBS to a concentration of 1.5% by weight, hydrated, and 10 g of the solution was aliquoted into each conical tube. Peptide solution was added to the aliquoted hyaluronic acid solutions to achieve final peptide concentrations of 0 ppm (no peptide added), 100 ppm, 1000 ppm, and 5000 ppm. 2 mL of 500 mM hydrogen peroxide (H₂O₂) was added to each conical tube. Samples were taken at 0, 30, 60, and 180 minutes using a rheometer (single shear stress) to measure viscosity, and the viscosity values ​​were compared with the peptide concentrations. As described above, the 1.5% by weight hyaluronic acid solutions containing various peptide concentrations were treated with hydrogen peroxide to maintain the reactive oxygen species-induced degradation reaction for 60 minutes and 180 minutes, thereby degrading the hyaluronic acid. After the degradation reaction, the viscosity of each hyaluronic acid solution was measured to confirm its degradation-delaying effect. Figure 5As shown, the experimental results confirm that the degradation of hyaluronic acid is delayed with increasing peptide concentration.

[0156] 2. Evaluation of hyaluronic acid degradation in delayed crosslinking fillers

[0157] 1,4-Butanediol diglycidyl ether (BDDE) crosslinked hyaluronic acid fillers, with or without the peptide from Preparation Example 1, were prepared, and Congo red and hyaluronidase (HAdase) were prepared as absorption dyes. 20 μL of 1 mM Congo red was added to each 1.5 mL Eppendorf tube, and 100 μL of peptide-free filler (control filler) and peptide-containing filler were added to the absorption dyes, respectively. Each sample was treated with 350 μL of 500 mM hydrogen peroxide (H2O2). Each tube was placed in an incubator at 37°C, and 100 μL of sample supernatant was collected at 1 h, 3 h, 6 h, 24 h, 48 h, and 72 h, and transferred to 96-well plates. Absorbance was measured at 500 nm using a ELISA reader (SpectraMax iD3, Molecular devices, USA). As described above, BDDE cross-linked hyaluronic acid filler samples with or without peptides were prepared, and the degree of degradation by reactive oxygen species was compared based on the presence or absence of peptides. The degree of degradation of the hyaluronic acid filler was measured by the change in absorbance due to dye diffusion. That is, the principle that the sample's fluidity increases when hyaluronic acid is degraded by reactive oxygen species, thus increasing dye diffusion, is utilized. Figure 6 As shown, the experimental results indicate that after treatment with hydrogen peroxide (reactive oxygen species) for 24 hours, the degradation delay was improved by more than 50% due to the presence of peptides in the samples. In samples treated for 48 hours, most of the hyaluronic acid in peptide-free fillers was degraded, while the degradation delay was confirmed to be 85% when peptides were present.

[0158] Experimental Example 5: Evaluation of pH and thermal stability of peptides

[0159] The pH stability and thermal stability of the peptide with SEQ ID NO: 1 prepared in Preparation Example 1 above were evaluated.

[0160] pH stability was evaluated as follows. A diluted solution of the peptide from Preparation Example 1 at a concentration of 1000 ppm was prepared, and the initial pH of the solution was measured (pH = 3.21). The pH of the solution was titrated to pH 4, pH 7, and pH 10 by adding 0.5 M NaOH. Each sample was filtered through a 0.22 μm filter. The area values ​​were compared with pH 7 using HPLC.

[0161] Thermal stability was evaluated as follows. A diluted solution of the peptide from Preparation Example 1 at a concentration of 1000 ppm was prepared, aliquoted into 50 mL conical tubes, and placed in incubators at 25°C and 50°C. After 72 hours, 1 mL of each sample was filtered through a 0.22 μm filter. The area values ​​at 25°C were determined by HPLC, with pH 7 as a baseline.

[0162] like Figure 7a and 7b As shown, the experimental results confirmed that the peptide in Preparation Example 1 was stable even at a high temperature of 50°C and within a pH range of 4 to 10.

[0163] Although representative embodiments of this application have been described above as examples, the scope of this application is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate modifications within the scope of the claims of this application.

Claims

1. A peptide comprising the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, wherein the peptide has the activity of delaying the degradation of hyaluronic acid.

3. The peptide according to claim 1, wherein the peptide has the activity of promoting the proliferation of fibroblasts or keratinocytes; or the activity of promoting the expression of collagen or hyaluronic acid.

4. A composition for skin regeneration or improvement of skin condition, comprising the peptide according to claim 1 as an active ingredient.

5. A cosmetic composition for skin regeneration or improvement of skin condition, comprising the peptide according to claim 1 as an active ingredient.

6. The cosmetic composition for improving skin condition according to claim 5, wherein improving skin condition means improving skin elasticity, improving skin wrinkles, improving skin hydration, inhibiting skin aging, repairing skin damage caused by ultraviolet radiation, protecting skin from ultraviolet damage, or improving skin wounds.

7. A pharmaceutical composition for the prevention or treatment of skin diseases, comprising the peptide according to claim 1 as an active ingredient.

8. The pharmaceutical composition according to claim 7, wherein the dermatitis is a wound or scar on the skin.

9. A composition for delaying the degradation of hyaluronic acid, comprising the peptide according to claim 1 as an active ingredient.

10. The pharmaceutical composition according to claim 9, wherein the degradation of hyaluronic acid is achieved by hyaluronidase degradation or by reactive oxygen species degradation.

11. A filler composition comprising the peptide and hyaluronic acid according to claim 1.

12. The filler composition according to claim 11, wherein the hyaluronic acid is cross-linked hyaluronic acid.

13. The filler composition of claim 12, wherein the crosslinked hyaluronic acid is crosslinked with one or more crosslinking agents selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol (PEG), divinyl sulfone (DVS), polyethylene glycol diglycidyl ether (PEGDGE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and polytetramethylene glycol diglycidyl ether.

14. The filler composition of claim 11, wherein the filler composition is used to improve wrinkles and tissue volume reduction in soft tissue selected from the group consisting of the neck, chest, buttocks, arms, armpits, hands, legs and feet, or to treat wounds, scars or stretch marks.

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