Novel peptides with hyaluronic acid degradation-delaying activity and their applications

Abstract

The peptide of the present invention has the activity to promote skin cell activation and can therefore be used for skin regeneration or improvement of skin condition. Furthermore, the peptide of the present invention has the activity to delay or suppress the degradation of hyaluronic acid by hyaluronidase and reactive oxygen species. When used together with hyaluronic acid fillers during their manufacture, it has the effect of extending the time it takes for hyaluronic acid fillers to be degraded in vivo, thereby improving the performance of the fillers.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the benefit of priority based on Korean Patent Application No. 10 - 2023 - 0050317, filed on April 17, 2023, and all the contents disclosed in the document of the Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a novel peptide having hyaluronic acid degradation - delaying activity and its use.

Background Art

[0003] The skin is composed of the epidermis, dermis, and subcutaneous tissue from the outside in. The epidermis consists of various cells, including keratinocytes (which are made of keratin), melanocytes (which form and secrete melanin), and Langerhans cells (immune-related cells). The dermis is composed of fibrous and matrix components. Collagen, as a fibrous component, provides strength and tension to the skin, protecting it and making up about 90% of the dermis. Collagen also plays an important role in wound healing, promoting collagen synthesis in damaged epithelium, which allows wounds to heal quickly and without scarring. Collagen is synthesized by the action of fibroblasts and broken down by collagenase and elastinase. As we age, the activity and number of fibroblasts decrease, the amount of collagen synthesized decreases, water is lost from skin cells, the structure of the stratum corneum changes, and the activity of collagenase increases, leading to an increase in the cross-linked form of collagen, resulting in a decrease in smoothness, moisture retention, and firmness. Besides age, external factors that cause skin aging include pollution, wind, temperature, and ultraviolet radiation. Interest in collagen synthesis-promoting substances is growing in order to prevent skin aging. Conventionally known collagen synthesis-promoting substances include vitamin C, retinoic acid, animal placenta-derived proteins, betulinic acid, and chlorella extract. However, these substances have limitations on usage due to safety issues such as irritation and redness when applied to the skin, or their effects are minimal, making it practically impossible to expect improvement in skin function or wound healing.

[0004] Fillers are substances used to increase and expand the volume of soft tissue by injecting them into the skin or tissue to improve wrinkles, skin depressions, and loss of skin and tissue volume that occur in the skin due to aging. Hyaluronic acid (HA) is a sugar that forms an essential component of skin and connective tissue, and 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 in the dermis, and consists of polysaccharides called acidic mucopolysaccharides such as hyaluronic acid and dermatan sulfate, and fibrous proteins such as collagen and elastin. Thus, hyaluronic acid is a component that naturally exists in various human tissues such as skin, joints, and mucous membranes of the eyes, and is easy to use, non-immunogenic, non-carcinogenic, and biocompatible, so it is widely used as the main material for fillers.

[0005] The body contains an enzyme called hyaluronidase, which breaks down hyaluronic acid. It is also known that hyaluronic acid is broken down by oxidation caused 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 break down and disappear. Conventionally, in order to extend the duration of hyaluronic acid fillers, a method of cross-linking hyaluronic acid using an appropriate cross-linking agent such as BDDE (1,4-Butanediol diglycidyl ether) has been used. When fillers are manufactured and used using hyaluronic acid that has been cross-linked in this way, the fillers are protected to some extent from the breakdown action by hyaluronidase in the body, and the duration of the fillers is extended. However, the actual effect is not satisfactory.

[0006] Korean Registered Patent No. 10-2369261 discloses a method for producing a core-shell structured hyaluronic acid gel by adding a shell to a core made of hyaluronic acid that has been primary crosslinked with a first crosslinking agent and hyaluronic acid that has been crosslinked with a second crosslinking agent to the core; Korean Registered Patent No. 10-2225971 discloses a method for producing a hyaluronic acid-based hydrogel using a peptide crosslinking agent; and Korean Registered Patent No. 10-2418096 discloses a method for orally administering collagen to maintain the efficacy of hyaluronic acid fillers.

[0007] Despite these various attempts with conventional technologies, a safe and effective technique for delaying the degradation of hyaluronic acid fillers has yet to be developed. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Korean Registered Patent Publication No. 10-2369261 [Patent Document 2] Korean Registered Patent Publication No. 10-2225971 [Patent Document 3] Korean Registered Patent Publication No. 10-2418096 [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention was proposed to solve the aforementioned problems, and aims to provide a biocompatible peptide that has activity to delay the degradation of hyaluronic acid by hyaluronic acid-degrading enzymes and reactive oxygen species, and activity to promote the activation of skin cells, while also exhibiting relatively good stability even in acids and at high temperatures.

[0010] Therefore, the object of the present invention is to provide a novel peptide having skin cell activation promoting activity and hyaluronic acid degradation delaying activity.

[0011] Another object of the present invention is to provide a cosmetic composition for skin regeneration or improvement of skin condition that contains the aforementioned active peptide as an active ingredient.

[0012] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of skin diseases, comprising the aforementioned peptide having the activity as an active ingredient.

[0013] Another object of the present invention is to provide a composition for delaying the degradation of hyaluronic acid, which contains the aforementioned active peptide as an active ingredient.

[0014] Another object of the present invention is to provide a filler composition comprising the peptide and hyaluronic acid having the aforementioned activity. [Means for solving the problem]

[0015] One aspect of the present invention is to provide a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0016] Another aspect of the present invention is to provide a cosmetic composition for skin regeneration or improvement of skin condition, comprising the peptide as an active ingredient.

[0017] Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of skin diseases, comprising the peptide as an active ingredient.

[0018] Another aspect of the present invention provides a composition for delaying the degradation of hyaluronic acid, comprising the peptide as an active ingredient.

[0019] Another object of the present invention is to provide a filler composition comprising the peptide and hyaluronic acid.

[0020] The present invention will be described in detail below.

[0021] Peptides and their activity According to one aspect of the present invention, there is provided a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1. [Amino acid sequence of SEQ ID NO: 1] KIWNLEPSRNGT (H2N-Lys-Ile-Trp-Asn-Leu-Glu-Pro-Ser-Arg-Asn-Gly-Thr-COOH)

[0022] As used herein, the term "peptide" means a linear molecule formed by linking amino acid residues together by peptide bonds.

[0023] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention may be used without modification, but within a range that does not affect the original activity of the peptide, for example, the activity of suppressing or delaying the degradation of hyaluronic acid by hyaluronidase or reactive oxygen species, amino acid mutants or fragments having different sequences due to deletion, insertion, substitution, or combinations thereof of amino acid residues may be used.

[0024] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. within a range that does not change its activity.

[0025] The peptide of the present invention includes peptides containing an amino acid sequence substantially identical to the peptide containing the amino acid sequence of SEQ ID NO: 1, and mutants or active fragments thereof. The substantially identical amino acid sequence means an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more with the amino acid sequence of SEQ ID NO: 1. Further, the peptide may additionally contain a targeting sequence, a tag, a labeled residue, a half-life, or an amino acid sequence produced for a specific purpose of increasing peptide stability.

[0026] The peptides of the present invention may have N-terminal and / or C-terminal modifications induced by selecting a portion of the amino acid sequence to increase their activity. Such N-terminal and / or C-terminal modifications can significantly improve the stability of the peptides of the present invention, for example, by increasing their half-life upon in vivo administration. The term "stability" includes not only in vivo stability that protects the peptides of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0027] The N-terminal modification may involve the attachment of a protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) to the N-terminus of the peptide. The C-terminal modification may involve the attachment of a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), etc., to the C-terminus of the peptide, but is not limited to these.

[0028] The peptides of the present invention can be produced by a variety of widely known methods in the art to which the present invention pertains. For example, the peptides of the present invention can be produced by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (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).

[0029] The peptide of the present invention has the activity to activate skin cells.

[0030] In one embodiment, the peptide of the present invention has the activity to promote the proliferation of fibroblasts or keratinocytes.

[0031] In one embodiment, the peptide of the present invention has the activity to promote the expression of collagen or hyaluronic acid.

[0032] In one embodiment, the peptide of the present invention has the activity to promote the expression of collagen or hyaluronic acid in fibroblasts or keratinocytes.

[0033] The peptide of the present invention has the activity to delay or inhibit the degradation of hyaluronic acid.

[0034] In one embodiment, the decomposition of hyaluronic acid may be by decomposition by hyaluronidase or by reactive oxygen species.

[0035] As described above, the peptide of the present invention has the activity to delay or suppress the degradation of hyaluronic acid by hyaluronic acid-degrading enzymes or reactive oxygen species. Therefore, when used together with fillers made from hyaluronic acid, it exhibits the effect of extending the period in which the hyaluronic acid filler exists in the body and improving the performance of the filler.

[0036] Furthermore, the peptide of the present invention has pH and temperature stability. As demonstrated in the embodiments described later, the peptide of the present invention exhibits the same stability in the pH range of 4 to 10 and the temperature range of 25°C to 50°C, and is stable to pH and temperature changes, thus having superior biological stability.

[0037] composition containing peptides In another aspect of the present invention, the present 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.

[0038] In another aspect of the present 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.

[0039] In this specification, the term “improvement” means any action that causes the symptoms of a disease or disorder to improve or benefit from.

[0040] In this specification, improvement of skin condition comprehensively means the process or effect of treating, reducing, or alleviating skin damage induced by intrinsic or extrinsic factors of the skin.

[0041] In one example, the improvement of the skin condition may be an increase in skin elasticity, an improvement in skin wrinkles, an increase in skin moisture, an inhibition of skin aging, a recovery from skin damage caused by ultraviolet rays, protection of the skin from ultraviolet rays, or an improvement in skin wounds.

[0042] In one embodiment, the peptide containing the amino acid sequence of SEQ ID NO: 1, which is included as an active ingredient in the cosmetic composition, promotes the expression of collagen or hyaluronic acid in skin cells, such as fibroblasts or keratinocytes.

[0043] In the present invention, the "improvement of skin wrinkles," "enhancement of skin elasticity," "inhibition of skin aging," "recovery from skin damage caused by ultraviolet rays," "protection of the skin from ultraviolet rays," or "improvement of skin wounds" can be induced by any action that increases the total amount of collagen in the skin, including promoting collagen synthesis in skin cells. The skin cells may be, for example, fibroblasts or keratinocytes.

[0044] The aforementioned "suppression of skin aging" may mean suppressing the decline in skin function, such as wrinkles, sagging skin, and loss of elasticity. The aforementioned skin aging may be photoaging, for example, skin aging caused by ultraviolet rays.

[0045] The cosmetic composition of the present invention may further contain a cosmetically acceptable carrier in addition to the peptide containing the amino acid sequence of SEQ ID NO: 1.

[0046] The cosmetic composition may be manufactured in any dosage form commonly produced in the art to which the present invention belongs, and may be a topical skin preparation. For example, it may be formulated in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto.

[0047] The aforementioned cosmetic composition may be manufactured in various forms such as solutions, sol-gels, emulsions, oils, waxes, and aerosols, including softening lotions, nourishing lotions, nourishing creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, packs, sprays, powders, hair tonics, hair creams, hair lotions, hair shampoos, hair rinses, hair conditioners, hair sprays, hair aerosols, pomades, and gels, but is not limited thereto.

[0048] The cosmetic composition of the present invention may contain excipients, carriers, and other additives, and it is possible to apply and incorporate as needed any common ingredients that are typically used in general skin cosmetics.

[0049] If the dosage form of the cosmetic composition is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as the carrier component.

[0050] When the dosage form of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, propellants such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be included, but are not limited thereto.

[0051] When the dosage form of the cosmetic composition is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as the carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan may be used.

[0052] If the dosage form of the cosmetic composition is a suspension, the carrier component may be a liquid diluent such as water, ethanol, or propylene glycol, a suspension agent such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester, or polyoxyethylene sorbitan ester, or microcrystalline cellulose, aluminum metahydroxyl, bentonite, aga, or tragacanth.

[0053] If the dosage form of the cosmetic composition is a surfactant-containing cleanser, then the carrier component may be an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinate monoester, isethionic acid, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

[0054] If the dosage form of the cosmetic composition is a hair shampoo, the peptide of the present invention may be mixed with base components for forming the shampoo, such as a thickener, surfactant, viscosity modifier, humectant, pH adjuster, preservative, and essential oil. As the thickener, CDE may be used; as the surfactant, LES, an anionic surfactant, and cocobetaine, an amphoteric surfactant, may be used; as the viscosity modifier, polyquartz may be used; as the humectant, glycerin may be used; and as the pH adjuster, citric acid and sodium hydroxide may be used. As the preservative, grapefruit extract may be used, and in addition, essential oils such as cedarwood, peppermint, and rosemary, as well as silk amino acids, pentaol, or vitamin E may be added.

[0055] The components included in the cosmetic composition may, but are not limited to, components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, in addition to the peptide and carrier component of the present invention as active ingredients.

[0056] The cosmetic composition of the present invention may contain a cosmetically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0057] The aforementioned cosmetically effective amount means an amount sufficient to achieve the effect of skin regeneration or improvement of skin condition of the cosmetic composition.

[0058] The peptide of the present invention may be included in the aforementioned cosmetic composition at a concentration of 0.01 μM to 1000 μM. Specifically, the peptide of the present invention may be included 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. The substance may be included in concentrations of μM, 0.1μM to 500μM, 0.1μM to 300μM, 0.1μM to 200μM; 1μM to 800μM, 1μM to 700μM, 1μM to 600μM, 1μM to 500μM, 1μM to 300μM, 1μM to 200μM; 5μM to 800μM, 5μM to 700μM, 5μM to 600μM, 5μM to 500μM, 5μM to 300μM, or 5μM to 200μM, but is not limited to these.

[0059] In another aspect of the present invention, the present 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.

[0060] In one example, the skin disease may be a skin wound or a skin scar.

[0061] In one embodiment, the peptide containing the amino acid sequence of SEQ ID NO: 1, which is included as an active ingredient in the pharmaceutical composition, promotes the expression of collagen or hyaluronic acid in skin cells, such as fibroblasts or keratinocytes.

[0062] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of the peptide of the present invention.

[0063] The term "therapeutically effective amount" refers to an amount sufficient to achieve the activity or efficacy of the peptide, which is the active ingredient of the pharmaceutical composition of the present invention, for example, an amount sufficient to achieve the efficacy of treating or preventing a skin disease.

[0064] In this specification, the term “prevention” means reducing the risk of contracting a disease or disability, and includes all actions that suppress or delay the onset of a disease by preventing the progression of a disease or one or more of its clinical symptoms.

[0065] In this specification, the term “treatment” means alleviating a disease or disorder, and includes all actions that improve or modify the symptoms of a disease by preventing or reducing the progression of the disease or one or more of its clinical symptoms.

[0066] In the present invention, prevention or treatment of a skin disease may involve eliminating the cause of the skin disease or suppressing its progression.

[0067] The pharmaceutical composition of the present invention may include a peptide containing the amino acid sequence of SEQ ID NO: 1 and a pharmaceutically acceptable carrier.

[0068] The aforementioned pharmaceutically acceptable carriers include, but are not limited to, those commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil.

[0069] The pharmaceutical composition of the present invention may further contain, but is not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspension agents, preservatives, and the like, in addition to the above-mentioned components.

[0070] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy (19th ed., 1995, Williams & Wilkins).

[0071] The pharmaceutical composition of the present invention may be administered via any suitable route for treating skin diseases, for example, orally or parenterally. In the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, or transdermal administration. Since the pharmaceutical composition of the present invention has prophylactic or therapeutic activity for skin diseases, it can be applied by topical administration such as application to the skin.

[0072] The dosage of the pharmaceutical composition may be 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, but is not limited thereto. It can be prescribed in various ways depending on factors such as the formulation method, administration method, the patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity.

[0073] The pharmaceutical compositions of the present invention may be manufactured in unit volume form or encapsulated in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains. The dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may further contain a dispersant or stabilizer.

[0074] The pharmaceutical composition of the present invention may be a topical skin preparation. The topical skin preparation is a formulation that can be applied to the outside of the skin, and when the pharmaceutical composition of the present invention is used as a topical skin preparation, it may be applied to the skin, specifically to a skin area where a skin disease has occurred. The topical skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof, and the topical skin preparation may be appropriately formulated with ingredients commonly used in topical skin preparations such as cosmetics and pharmaceuticals, for example, aqueous components, oily components, powder components, alcohols, humectants, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof as needed. The aforementioned topical skin preparation may also contain, as appropriate, chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; drugs such as caffeine, tannins, licorice extract, glabridin, various herbal medicines, tocopherol acetate, glycyrrhizin, tranexamic acid, and their derivatives or salts; and sugars such as vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0075] In another aspect of the present invention, a composition for delaying hyaluronic acid degradation is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0076] In another aspect of the present invention, the invention provides an application for delaying hyaluronic acid degradation of a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0077] In one embodiment, the decomposition of hyaluronic acid may be by decomposition by hyaluronidase or by reactive oxygen species.

[0078] In this invention, the term "delay" in hyaluronic acid degradation is used interchangeably with "inhibition" or "suppression" of hyaluronic acid degradation.

[0079] In another aspect of the present invention, a filler composition comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 and hyaluronic acid is provided.

[0080] In the present invention, hyaluronic acid (HA) refers to a biopolymer represented by the following chemical formula 1, in which repeating units consisting of N-acetyl-D-glucosamine and D-glucuronic acid are linearly linked.

[0081] [ka]

[0082] Hyaluronic acid is abundant in the vitreous fluid of the eye, synovial fluid of joints, and cockscomb, and possesses excellent biocompatibility. Therefore, it is widely used in medical and medical device applications, as well as in cosmetics, as an ophthalmic surgical aid, joint function improver, drug delivery substance, eye drops, and wrinkle reducer.

[0083] In one embodiment, the filler composition of the present invention may include hyaluronic acid in addition to hyaluronic acid itself, including its salts. These hyaluronic acid salts include, but are not limited to, 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.

[0084] In one embodiment, the hyaluronic acid or salt thereof may be hyaluronic acid crosslinked with an appropriate crosslinking agent.

[0085] In one embodiment, the crosslinked hyaluronic acid may be crosslinked with one or more crosslinking agents selected from the group consisting of BDDE (1,4-butane diol diglycidyl ether), PEG (polyethylene glycol), DVS (divinyl sulfone), PEGDGE (Poly(ethylene glycol) diglycidyl ether), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, poly(propylene glycol) diglycidyl ether, and poly(tetramethylene glycol) diglycidyl ether.

[0086] In the filler composition of the present invention, the content of the peptide containing the amino acid sequence of SEQ ID NO: 1 is, relative to the total composition, 0.001-0.5% by weight, 0.002-0.5% by weight, 0.003-0.5% by weight, 0.004-0.5% by weight, 0.005-0.5% by weight, 0.001-0.4% by weight, 0.002-0.4% by weight, 0.003-0.4% by weight, and 0.004-0.4% by weight. The percentage may be, but is not limited to, a certain amount, 0.005-0.4% by weight, 0.001-0.3% by weight, 0.002-0.3% by weight, 0.003-0.3% by weight, 0.004-0.3% by weight, 0.005-0.3% by weight, 0.001-0.2% by weight, 0.002-0.2% by weight, 0.003-0.2% by weight, 0.004-0.2% by weight, or 0.005-0.2% by weight.

[0087] In the filler composition of the present invention, the hyaluronic acid content may be, but is not limited to, 0.001 to 0.5% by weight, 0.002 to 0.4% by weight, 0.003 to 0.3% by weight, 0.004 to 0.2% by weight, or 0.005 to 0.1% by weight, relative to the total composition.

[0088] In one embodiment, the filler composition of the present invention may be in a simply mixed state with the peptide containing the amino acid sequence of SEQ ID NO: 1.

[0089] In this invention, the term "filler" is used to broadly refer to a material or composition designed to add volume to areas lacking soft tissue, and includes the meanings of soft tissue filler or dermal filler.

[0090] In the present invention, the term "soft tissue" as used generally refers to tissue that connects, supports, or surrounds other structures and organs of the body. Such soft tissues include, for example, muscles, tendons, vocal cords, intima, fibrous tissue, fat, blood vessels, nerves, and synovial tissue.

[0091] In one embodiment, the filler composition may selectively contain, but is not limited to, buffering agents, preservatives, isotonic agents, antioxidants, emulsifiers, wetting agents, and other pharmaceutically acceptable components.

[0092] The buffering agent may include, but is not limited to, one or more selected from the group consisting of citric acid, monohydrogen phosphate, dihydrogen phosphate, acetic acid, diethyl barbituric acid, sodium acetate, tris(hydroxymethyl)methylamino)propanesulfonic acid (TAPS), 2-bis(2-hydroxyethyl)amino)acetic acid (Bicine), tris(hydroxymethyl)ammonium methane (Tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (Tricine), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]methanesulfonic acid (TES), and piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES).

[0093] The isotonic agent may be used without restriction as long as it is intended for use in the manufacture of hyaluronic acid fillers, and may be included in the buffer solution. Sodium chloride may be used as a preferred isotonic agent.

[0094] In one embodiment, the filler composition of the present invention may further include other substances or combinations of substances that provide beneficial effects when administered to an individual, and may include, but is not limited to, antioxidants, antipruritics, anticellulents, antiscarring agents, anti-inflammatory agents, anesthetics, irritation relievers, vasoconstrictors, vasodilators, antihemorrhagic agents, such as hemostatic agents or anti-fibrinolytic agents, exfoliants, elasticity enhancers, anti-acne agents, colorants, color inhibitors, or moisturizers.

[0095] In one embodiment, the filler composition may 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 it may be used for the treatment of wounds, scars, or stretch marks.

[0096] In one embodiment, the filler composition may be a cosmetic composition, preferably a cosmetic composition for improving wrinkles or tissue volume reduction in soft tissues selected from the group consisting of the neck, chest, buttocks, arms, armpits, hands, legs, and feet.

[0097] In one embodiment, the filler composition may be a pharmaceutical composition, preferably a pharmaceutical composition for the treatment or prevention of wounds, scars, or stretch marks.

[0098] If the filler composition is in the form of a cosmetic composition or a pharmaceutical composition, the technical content and technical features described for cosmetic compositions and pharmaceutical compositions in the above-mentioned composition section apply directly to the filler composition of the present invention. Therefore, in order to avoid excessive complexity in the specification, these will not be described again.

[0099] The filler composition of the present invention may be administered in a dosage form suitable for the above-mentioned use, preferably in the form of an injection.

[0100] The filler composition of the present invention may be subjected to a sterilization process, and may be quantitatively filled into a suitable container, such as a syringe, sealed, and sterilized.

[0101] Applications of peptides In another aspect of the present invention, the present invention provides applications for using a peptide comprising the amino acid sequence of SEQ ID NO: 1 for skin regeneration or improvement of skin condition.

[0102] In another aspect of the present invention, the present invention provides a use for a peptide containing the amino acid sequence of SEQ ID NO: 1 in the manufacture of cosmetics for skin regeneration or skin condition improvement.

[0103] In another aspect of the present invention, the present invention provides applications for the treatment of skin diseases using a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0104] In another aspect of the present invention, the present invention provides an application for use in the manufacture of a pharmaceutical for treating skin diseases using a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0105] In another aspect of the present invention, the present invention provides an application for delaying the hyaluronic acid degradation of a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0106] The technical content and features of the present invention described in relation to the peptides and compositions of the present invention are all applied in the same manner to the uses of the peptides of the present invention. Therefore, in order to avoid excessive complexity in the specification, such technical content and features will not be described redundantly. [Effects of the Invention]

[0107] The peptide of the present invention has the activity to promote skin cell activation and can therefore be used for skin regeneration or improvement of skin condition. Furthermore, the peptide of the present invention has the activity to delay or suppress the degradation of hyaluronic acid by hyaluronidase and reactive oxygen species. When used together with hyaluronic acid fillers during their manufacture, it has the effect of extending the time it takes for hyaluronic acid fillers to be degraded in vivo, thereby improving the performance of the fillers.

[0108] However, the effects of the present invention are not limited to those mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description. [Brief explanation of the drawing]

[0109] [Figure 1] These experimental results demonstrate that the peptide of the present invention has the effect of promoting the proliferation of NIH3T3 cells (mouse fibroblasts) and HaCaT cells (human keratinocyte cell line). [Figure 2] These experimental results demonstrate that the peptide of the present invention promotes the expression of collagen and hyaluronic acid (HA) in NIH3T3 cells (mouse fibroblasts) and HaCaT cells (human keratinocyte cell line). [Figure 3] This experimental result shows that the peptide of the present invention has the effect of delaying the degradation of hyaluronic acid by hyaluronic acid-degrading enzymes in a hyaluronic acid solution. [Figure 4] This experimental result shows that the peptide of the present invention has the effect of delaying the degradation of hyaluronic acid by hyaluronic acid-degrading enzymes in BDDE-crosslinked hyaluronic acid fillers. [Figure 5] This experimental result shows that the peptide of the present invention has the effect of delaying the decomposition of hyaluronic acid by reactive oxygen species (hydrogen peroxide) in a hyaluronic acid solution. [Figure 6] This experimental result shows that the peptide of the present invention has the effect of delaying the degradation of hyaluronic acid by reactive oxygen species (hydrogen peroxide) in BDDE-crosslinked hyaluronic acid fillers. [Figure 7a] These experimental results demonstrate that the peptide of the present invention remains stable even at a high temperature of 50°C. [Figure 7b] These experimental results demonstrate that the peptide of the present invention remains stable even in the pH range of 4 to 10. [Figure 8] This is a schematic diagram illustrating the effect of the peptide of the present invention on delaying the degradation of hyaluronic acid by hyaluronic acid-degrading enzymes. [Modes for carrying out the invention]

[0110] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0111] Manufacturing Example 1: Production of Peptides and Peptide Complexes Peptides having the amino acid sequence of SEQ ID NO: 1, as shown in Table 1 below, were synthesized using an automated peptide synthesizer (CEM Liberty, CEM Corporation, USA). These synthesized peptides were then separated into pure molecules using C18 reversed-phase high-performance liquid chromatography (HPLC) (Thermo Fisher Scientific, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm x 100 mm, 1.7 μm, Waters Co, USA).

[0112] [Table 1]

[0113] The efficacy of the peptide of Sequence ID No. 1, which was manufactured as described above, was evaluated through the following experiment.

[0114] Experimental Example 1: Analysis of Cell Proliferation The effect of the peptide of Sequence ID No. 1, produced in Production Example 1, on cell proliferation was evaluated.

[0115] NIH3T3 cells (mouse fibroblasts) and HaCaT cells (human keratinocyte cell line) were compared in a 1x10⁻¹⁶ sample. 4 Cells were seeded into a 96-well plate at a density of cells / well and cultured for 24 hours. The cells were washed once with serum-free DMEM medium. Peptide medium solutions at concentrations of 1.6 μM, 8 μM, 40 μM, 200 μM, and 1000 μM were prepared by adding different amounts of peptide to 200 μL of serum-free DMEM medium, and the washed cells were treated with the prepared peptide medium solutions. Next, the treated cells were cultured for 72 hours in a 37°C CO2 incubator, washed twice with PBS, and then MTT (Sigma, Cat. No.: M2003, USA) solution was dispensed into each well to a final concentration of 0.5 mg / ml. Next, after being shielded from light, the cells were cultured for 4 hours in a 37°C CO2 incubator. Absorbance was measured at 540 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA).

[0116] As shown in Figure 1, the experimental results clearly demonstrated a cell proliferation-promoting effect in all two skin cell lines, NIH3T3 cells and HaCaT cells, after peptide treatment.

[0117] Experimental Example 2: Analysis of Collagen and Hyaluronic Acid (HA) Expression Enhancement The effect of the peptide of Sequence ID No. 1, produced in Production Example 1, on the expression of collagen and hyaluronic acid (HA) was confirmed.

[0118] NIH3T3 cells (mouse fibroblasts) and HaCaT cells (human keratinocyte cell line) were compared in a 1x10⁻¹⁶ sample. 4Cells were seeded into 24-well plates at a density of cells / well and cultured for 24 hours. The cells were washed once with serum-free DMEM medium. Peptide medium solutions were prepared by adding different amounts of peptide to 500 μL of serum-free DMEM medium to concentrations of 50 μM, 100 μM, and 200 μM, and the washed cells were treated with the prepared peptide medium solutions. In this process, 100 nM TGF-β1 was used for NIH3 T3 cells and 100 nM IGF-1 was used for HaCaT cells as the positive control group. Next, the treated cells were cultured for 72 hours in a CO2 incubator at 37°C. The cell culture medium was collected, centrifuged, and the supernatant was transferred to a tube. The obtained supernatants were subjected to ELISA analysis using a Collagen ELISA kit (abcam, Cat No.: ab210579, UK) and an HA ELISA kit (ECHELON biosciences, Cat No.: K-1200, USA), respectively.

[0119] As shown in Figure 2, experimental results revealed significant increases in collagen expression and hyaluronic acid (HA) expression in all NIH3T3 and HaCaT cells at peptide concentrations ranging from 50 μM to 200 μM. These results suggest that the peptide of the present invention stimulates skin cells and promotes the production of the extracellular matrix (ECM).

[0120] Experimental Example 3: Analysis of the delayed effect of hyaluronic acid (HA) degradation by hyaluronic acid-degrading enzymes. We evaluated whether the peptide of Sequence ID No. 1 produced in Production Example 1 has the effect of delaying the degradation of hyaluronic acid by hyaluronic acid-degrading enzymes.

[0121] 1. Evaluation of delayed hyaluronic acid degradation in hyaluronic acid solution Hyaluronic acid was dissolved in PBS to a concentration of 1.5% by weight and hydrated. 10 g of this solution was then dispensed into a conical tube. To the dispensed hyaluronic acid solution, peptide solutions were added to achieve final peptide concentrations of 0 ppm (no peptide added), 100 ppm, 1000 ppm, and 5000 ppm, respectively. 2 mL of hyaluronidase from bovine testes (Hadase, Sigma) was added to each solution. Samples were taken at 0 and 30 minutes, and viscosity was measured using a rheometer (single shear stress) to compare viscosity values ​​based on peptide concentration. As described above, 1.5% by weight hyaluronic acid solutions with peptides added at each concentration were treated with hyaluronidase, and the enzymatic reaction was maintained for 30 minutes to decompose the hyaluronic acid. After the decomposition reaction, the viscosity of the hyaluronic acid solution was measured to confirm the effect of delaying decomposition. The experimental results, as shown in Figure 3, confirmed that the degradation of hyaluronic acid was delayed as the peptide concentration increased. Since the difference in degradation delay efficacy between 1000 ppm and 5000 ppm was not significant, the experiment proceeded at a concentration of 1000 ppm thereafter.

[0122] 2. Evaluation of delayed hyaluronic acid degradation with cross-linked fillers BDDE (1,4-Butanediol diglycidyl ether) crosslinked hyaluronic acid fillers containing or not containing the peptide from Production Example 1 were prepared, and Congo Red and hyaluronidase (HAdase) were prepared as absorbent dyes. 20 μl of 1 mM Congo Red was placed in a 1.5 mL Eppendorf tube, and 100 μl each of the peptide-free filler (control group filler) and the peptide-containing filler were placed on top of the absorbent dye. 350 μl of 0.15 mg / ml (10⁸ units) of hyaluronidase was then added to each. The samples were placed in a 37°C incubator, and 100 μl of the supernatant was taken at 6 hours and 24 hours and transferred to 96 wells. Absorbance was measured at 500 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA). As described above, BDDE-crosslinked hyaluronic acid filler samples containing and without peptides were prepared, and the degree of degradation of the hyaluronic acid filler by hyaluronic acid-degrading enzymes was compared depending 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 the diffusion of the dye. That is, the principle was used in which the fluidity of the sample increases and the diffusion of the dye increases when hyaluronic acid is degraded by the degrading enzyme. As shown in Figure 4, the experimental results confirmed that the degradation delay was improved by more than 15% after 24 hours due to the peptide contained in the sample after treatment with the hyaluronic acid-degrading enzyme.

[0123] Experimental Example 4: Analysis of the effect of delaying the degradation of hyaluronic acid (HA) by reactive oxygen species. We evaluated whether the peptide of Sequence ID No. 1 produced in Production Example 1 has the effect of delaying the degradation of hyaluronic acid by reactive oxygen species.

[0124] 1. Evaluation of delayed hyaluronic acid degradation in hyaluronic acid solution Hyaluronic acid was dissolved in PBS to a concentration of 1.5% by weight and hydrated. 10 g of this solution was then dispensed into a conical tube. Peptide solutions were added to the dispensed hyaluronic acid solutions to achieve final peptide concentrations of 0 ppm (no peptide added), 100 ppm, 1000 ppm, and 5000 ppm, respectively. 2 mL of 500 mM hydrogen peroxide (H2O2) was added to each solution. Sampling was performed at 0, 30, 60, and 180 minutes, and viscosity was measured using a rheometer (single shear stress) to compare viscosity values ​​based on peptide concentration. As described above, 1.5% by weight hyaluronic acid solutions with peptides added at each concentration were treated with hydrogen peroxide to maintain the decomposition reaction by reactive oxygen species for 60 and 180 minutes, thereby decomposing the hyaluronic acid. After the decomposition reaction, the viscosity of the hyaluronic acid solution was measured to confirm the effect of delaying decomposition. The experimental results, as shown in Figure 5, confirmed that the degradation of hyaluronic acid was delayed as the peptide concentration increased.

[0125] 2. Evaluation of delayed hyaluronic acid degradation with cross-linked fillers BDDE (1,4-Butanediol diglycidyl ether) crosslinked hyaluronic acid fillers containing or not containing the peptide from Production Example 1 were prepared, and Congo Red and hyaluronidase (HAdase) were prepared as absorbent dyes. 20 μl of 1 mM Congo Red was placed in a 1.5 mL Eppendorf tube, and 100 μl each of the peptide-free filler (control group filler) and the peptide-containing filler were placed on top of the absorbent dye. 350 μl of 500 mM hydrogen peroxide (H2O2) was then added to each. The samples were placed in a 37°C incubator, and 100 μl of the supernatant was taken at 1 hour, 3 hours, 6 hours, 24 hours, 48 ​​hours, and 72 hours and transferred to 96 wells. Absorbance was measured at 500 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA). As described above, BDDE-crosslinked hyaluronic acid filler samples containing and without peptides were prepared, and the degree of degradation of the hyaluronic acid filler by reactive oxygen species was compared depending 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 the diffusion of the dye. That is, the principle was used in which the fluidity of the sample increases and the diffusion of the dye increases when hyaluronic acid is degraded by reactive oxygen species. Experimental results, as shown in Figure 6, confirmed that the degradation delay was improved by more than 50% after 24 hours of treatment with hydrogen peroxide (reactive oxygen species) due to the peptide contained in the sample. In the 48-hour treated sample, most of the hyaluronic acid was degraded in the peptide-free filler, while an 85% degradation delay effect was confirmed when peptides were included.

[0126] Experimental Example 5: Evaluation of pH and thermal stability of peptides The pH stability and thermal stability of the peptide of Sequence ID No. 1 produced in Production Example 1 were evaluated.

[0127] pH stability was evaluated as follows: A 1000 ppm diluted solution of the peptide from Production Example 1 was prepared, and the initial pH of this solution was measured (pH = 3.21). 0.5 M NaOH was added, and the solution was titrated to pH 4, pH 7, and pH 10, respectively. Each sample was filtered through a 0.22 μm filter. The area values ​​were compared using HPLC with pH 7 as the reference.

[0128] The thermal stability was evaluated as follows: A 1000 ppm diluted solution of the peptide from Production Example 1 was prepared, dispensed 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. Area values ​​were compared by HPLC, with pH 7 as the baseline and 25°C as the baseline.

[0129] The experimental results, as shown in Figures 7a and 7b, confirmed that the peptide from Production Example 1 remained stable even at a high temperature of 50°C and in the pH range of 4 to 10.

[0130] While the above describes representative embodiments of this application, the scope of this application is not limited to the specific embodiments described above, and any person with ordinary skill in the art can appropriately modify the claims of this application.

Claims

1. A peptide containing 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 promoting the expression of collagen or hyaluronic acid.

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

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

6. The cosmetic composition for improving skin condition according to claim 5, wherein the improvement of skin condition is the enhancement of skin elasticity, the improvement of skin wrinkles, the enhancement of skin moisture, the suppression of skin aging, the recovery of skin damage caused by ultraviolet rays, the protection of skin from ultraviolet rays, or the improvement of skin wounds.

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

8. The pharmaceutical composition for the prevention or treatment of a skin disease according to claim 7, wherein the skin disease is a skin wound or a skin scar.

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

10. The composition according to claim 9, wherein the decomposition of the hyaluronic acid is by decomposition by hyaluronic acid-degrading enzymes (hyaluronidase) or by reactive oxygen species.

11. A filler composition comprising the peptide and hyaluronic acid described in claim 1.

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

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

14. The filler composition according to claim 11, wherein the filler composition is 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 is used for the treatment of wounds, scars, or stretch marks.

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