Peptides with skin whitening activity and their uses

Abstract

The peptide of the present invention has skin whitening activity by inhibiting melanosome invagination in keratinocytes and promoting melanosome degradation, and can be used as an active ingredient in drugs for treating or preventing hyperpigmentation disorders caused by excessive melanosome deposition or as an active ingredient in skin whitening cosmetics.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the benefit of priority based on Korean Patent Application No. 10 - 2022 - 0155228, filed on November 18, 2022, and all the contents disclosed in the document of the Korean Patent Application are incorporated herein by reference as part of this specification.

[0002] The present invention relates to a peptide having skin - whitening activity and its uses.

Background Art

[0003] Cutaneous melanin exists in the skin, hair follicles, eyes, etc., and has an important function of protecting the skin from ultraviolet rays. However, excessive formation of melanin induces hyperpigmentation diseases such as freckles, chloasma, and dark spots, which in turn induce mental stress and a decline in quality of life. Melanin is mainly synthesized in melanocytes located between the basal layers of the epidermis, under the basal layer, and in hair follicles. The synthesis occurs in melanosomes, which are organelles within melanocytes. These melanosomes move to adjacent keratinocytes via dendritic processes, and as the keratinocytes rise to the outer skin layer, the skin color is shown. Although the mechanism by which melanosomes move from melanocytes to keratinocytes has not been precisely elucidated, it is known that after melanosomes move to the dendritic processes of melanocytes and are released extracellularly, membrane fusion and phagocytosis between melanosomes and keratinocytes are involved.

[0004] The synthesis process of melanin is synthesized from L - tyrosine through DOPA, DOPAquinone, DOPAchrome, DHI (5,6 - dihydroxyindole), and traditionally, the suppression of melanin synthesis has focused on factors that inhibit the activity of tyrosinase, an enzyme that catalyzes the rate - regulating step of the melanin synthesis pathway.

[0005] PCT International Publication Patent WO2020 / 153819 discloses a polypeptide having activity that inhibits the activity of tyrosinase, a key enzyme in the melanin synthesis pathway, and its use in skin whitening. Furthermore, Korean Registered Patent No. 10-1869783 describes a peptide having activity that inhibits melanin production and tyrosinase activity, and its use in skin whitening.

[0006] Up until now, the development of skin whitening agents has focused on inhibiting the activity of tyrosinase, a key enzyme in melanin synthesis. However, from the perspective of achieving synergistic effects on skin whitening through diverse mechanisms of action, it is necessary to develop skin whitening agents with different mechanisms of action and target sites. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2020 / 153819 [Patent Document 2] Korean Registered Patent Publication No. 10-1869783 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The inventors, through research efforts to develop peptides with improved skin whitening activity, experimentally demonstrated that the novel peptide they developed is highly effective in suppressing the invagination of melanosomes into keratinocytes and in degrading melanosomes within keratinocytes, thereby completing the present invention.

[0009] Therefore, the object of the present invention is to provide a novel peptide having skin whitening activity.

[0010] Another object of the present invention is to provide a skin whitening composition containing the aforementioned active peptide as an active ingredient.

[0011] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising the aforementioned peptide as an active ingredient.

[0012] Another object of the present invention is to provide a skin whitening cosmetic composition containing the aforementioned active peptide as an active ingredient. [Means for solving the problem]

[0013] To achieve the aforementioned objective, one aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0014] Another aspect of the present invention is to provide a skin whitening composition containing the peptide as an active ingredient.

[0015] Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising the peptide as an active ingredient.

[0016] Another aspect of the present invention is to provide a skin whitening cosmetic composition containing the peptide as an active ingredient.

[0017] The present invention will be described in detail below.

[0018] 1. Peptides and their activity

[0019] According to one aspect of the present invention, a peptide comprising the amino acid sequence disclosed in Sequence ID No. 1 is provided.

[0020] In this specification, the term "peptide" means a linear molecule formed by the bonding of amino acid residues to each other via peptide bonds.

[0021] The peptide containing the amino acid sequence of SEQ ID NO: 1 of the present invention may be used without modification, but within the range that does not affect the original activity of the peptide, such as skin whitening activity, variants or fragments of amino acids having different sequences due to deletion, insertion, substitution, or combinations thereof of amino acid residues may be used.

[0022] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. within the range that does not change its activity.

[0023] 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, variants thereof, 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 further include a targeting sequence, a tag, a labeled residue, a half-life, or an amino acid sequence produced for a specific purpose to increase peptide stability.

[0024] The peptide of the present invention may be one in which a partial site of the amino acid sequence is selected and N-terminal and / or C-terminal modifications are induced to increase its activity. Such N-terminal and / or C-terminal modifications can significantly improve the stability of the peptide of the present invention, for example, increase the half-life during in vivo administration of the peptide. The term "stability" includes not only in vivo stability that protects the peptide of the present invention from the attack of proteolytic enzymes in the body, but also storage stability (for example, room temperature storage stability).

[0025] The N-terminal modification may involve attaching a protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl 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 attaching a hydroxyl group (-OH), an amino group (-NH2), to the C-terminus of the peptide. hydrazino group ( Hydazino Group It may be a combination of , -NHNH2, etc., but is not limited to this.

[0026] 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).

[0027] The peptide of the present invention has skin whitening activity.

[0028] In one embodiment, the peptide of the present invention has the activity to suppress the invagination of melanosomes in keratinocytes or to promote the degradation of melanosomes.

[0029] Melanosomes produced by melanocytes migrate from melanocytes to keratinocytes. This migration of melanosomes to keratinocytes includes the release of melanosomes from melanocytes and the invagination of melanosomes into keratinocytes. The invagination of melanosomes can be described differently from uptake of melanosomes by keratinocytes or phagocytosis of melanosomes by keratinocytes.

[0030] PAR2 is involved in the process by which melanosomes released from melanocytes are absorbed by keratinocytes through phagocytosis.

[0031] In one embodiment, the peptide of the present invention has the activity to suppress the expression of the PAR2 (protease activated receptor 2) gene in keratinocytes.

[0032] The aforementioned PAR2 (protease-activated receptor 2) is a receptor protein present in the cell membrane and is known to regulate inflammatory responses, obesity, metabolism, and cancer, and to act as a sensor for proteolytic enzymes during infection. The PAR2 protein is present in the membrane of keratinocytes and is involved in the absorption (invagination) of melanosomes by keratinocytes. It is known that inhibiting the activity of the PAR2 protein suppresses the absorption of melanosomes by keratinocytes (Exp Cell Res. 2000 Jan 10;254(1):25-32; Pigment Cell Res. 2001 Aug;14(4):236-42; Exp Dermatol. 2003;12 Suppl 2:5-12).

[0033] In one embodiment, the peptide of the present invention has the activity to upwardly regulate the amount of Beclin-1 protein or LC3-I / II protein, or downwardly regulate the amount of p62 protein, in keratinocytes. The amounts of Beclin-1, LC3-I / II, and p62 proteins may be the expression levels of the proteins.

[0034] Autophagy is a natural process within cells that removes unnecessary, dysfunctional, or degenerated substances (e.g., denatured proteins) that have reached the end of their lifespan. For example, organelles that have reached the end of their lifespan, become degenerated, and have reduced function are removed by autophagy. The proteins or organelles targeted for removal by autophagy are sequestered within the cell into vesicles called autophagosomes, which have a double-membrane structure. These vesicles then recombine with lysosomes to form autophagolysosomes, where degradation occurs by lysosomal enzymes.

[0035] It is also known that the activation of autophagy flux in keratinocytes induces the degradation of melanosomes (Pigment Cell Melanoma Res. 2020 May; 33(3): 403-415; Int. J. Mol. Sci. 2021, 22, 3995).

[0036] The aforementioned Beclin-1 protein and LC3-I / II protein are known autophagy marker proteins that are major factors involved in autophagy, and it is known that when autophagy is activated, the levels of Beclin-1 protein and LC3-I / II protein increase.

[0037] The aforementioned p62 protein is known to be a protein that carries ubiquitinated cargoes for autophagy breakdown, and it is known that when autophagy is activated, the expression of p62 protein decreases, and conversely, when autophagy is suppressed, the expression of p62 protein increases (Cellular & Molecular Biology Letters (2016) 21:29).

[0038] As described above, the peptide of the present invention can exert skin whitening activity and preventive or therapeutic effects against hyperpigmentation disorders through its melanosome invagination inhibitory activity and melanosome degradation activity.

[0039] 2. Composition for skin whitening, prevention, treatment, or improvement of hyperpigmentation disorders

[0040] In another aspect of the present invention, a skin whitening composition is provided that contains a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0041] The peptide containing the amino acid sequence of Sequence ID No. 1 of the present invention has skin whitening activity through the mechanism described above.

[0042] In another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders comprising a peptide containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0043] In one embodiment, a pharmaceutical composition containing the peptide as an active ingredient inhibits the invagination of melanosomes in keratinocytes or promotes the degradation of melanosomes.

[0044] In one embodiment, a pharmaceutical composition containing the peptide as an active ingredient suppresses the expression of the PAR2 (protease activated receptor 2) gene in keratinocytes, or increases the amount of Beclin-1 protein or LC3-I / II protein, or decreases the amount of p62 protein. The amounts of Beclin-1, LC3-I / II, and p62 proteins may be the expression levels of the respective proteins.

[0045] In this specification, the term "hyperpigmentation disorder" refers to a disorder resulting from an excessive increase in the amount of melanin in the skin. Thus, an excessive increase in the amount of melanin in the skin causes the skin to darken. Causes of hyperpigmentation disorders may include, but are not limited to, sun exposure, skin inflammation such as acne (post-inflammatory hyperpigmentation), trauma, hormonal imbalances, or drug use.

[0046] In one embodiment, the hyperpigmentation disorder may be a freckle, age spot, solar lentigo, or hyperpigmentation that occurs after inflammation of the skin. The hyperpigmentation that occurs after inflammation of the skin may be a hyperpigmentation that occurs after trauma, ultraviolet radiation, or an inflammatory skin disease such as acne.

[0047] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of the peptide containing the amino acid of SEQ ID NO: 1 of the present invention.

[0048] 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 hyperpigmentation disorders.

[0049] 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.

[0050] 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.

[0051] In the present invention, prevention or treatment of hyperpigmentation disorders may involve removing the cause of hyperpigmentation in the skin or suppressing the progression to hyperpigmentation, and specifically, this may involve suppressing the invagination of melanosomes in keratinocytes or promoting the degradation of melanosomes.

[0052] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.

[0053] 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.

[0054] 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.

[0055] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy (19th ed., 1995, Williams & Wilkins).

[0056] The pharmaceutical composition of the present invention may be administered via any suitable route for treating hyperpigmentation disorders, 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 hyperpigmentation disorders of the skin, it is preferable to apply it by topical administration, such as by applying it to the skin.

[0057] The dosage of the pharmaceutical composition may be 0.0001 ug to 100 mg, 0.001 ug to 100 mg, 0.01 ug to 100 mg, 0.1 ug to 100 mg, or 1.0 ug to 1000 mg per day, but is not limited thereto, and can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity.

[0058] The pharmaceutical compositions of the present invention may be manufactured in unit volume form or contained in multi-dose containers 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.

[0059] 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 hyperpigmentation 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.

[0060] In another aspect of the present invention, the present invention provides a skin whitening cosmetic composition comprising a peptide containing the amino acid sequence of Sequence ID No. 1 as an active ingredient.

[0061] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient suppresses the invagination of melanosomes in keratinocytes or promotes the degradation of melanosomes.

[0062] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient suppresses the expression of the PAR2 (protease activated receptor 2) gene in keratinocytes, or adjusts the amount of Beclin-1 protein or LC3-I / II protein upward, or adjusts the amount of p62 protein downward. The amounts of Beclin-1, LC3-I / II, and p62 proteins may be the expression levels of the respective proteins.

[0063] 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.

[0064] 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.

[0065] The cosmetic composition of the present invention may contain excipients, carriers, and other additives, and it is possible to apply and incorporate ordinary ingredients commonly used in general skin cosmetics in the necessary amounts.

[0066] 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.

[0067] 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 further included, but are not limited thereto.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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. CDE may be used as the thickener, LES, an anionic surfactant, and cocobetaine, an amphoteric surfactant, as the surfactant, polyquartz may be used as the viscosity modifier, glycerin may be used as the humectant, and citric acid and sodium hydroxide may be used as the pH adjuster. Grapefruit extract may be used as the preservative, and essential oils such as cedarwood, peppermint, and rosemary, as well as silk amino acids, pentaol, or vitamin E may also be added.

[0072] 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.

[0073] The peptide of the present invention may be included in the aforementioned compositions, pharmaceutical compositions, or cosmetic compositions at concentrations 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, and 0.1 μM to 700 μM. It may be present in concentrations of uM~600uM, 0.1uM~500uM, 0.1uM~300uM, 0.1uM~200uM; 1uM~800uM, 1uM~700uM, 1uM~600uM, 1uM~500uM, 1uM~300uM, 1uM~200uM; 5uM~800uM, 5uM~700uM, 5uM~600uM, 5uM~500uM, 5uM~300uM, or 5uM~200uM, but is not limited to these concentrations.

[0074] 3. Uses of the peptide of the present invention

[0075] In another aspect of the present invention, the present invention provides applications for using a peptide containing the amino acid sequence of SEQ ID NO: 1 for skin whitening purposes, or for preventing, treating, or improving hyperpigmentation disorders.

[0076] In another aspect of the present invention, a method for skin whitening is provided, comprising the step of administering a peptide containing the amino acid sequence of SEQ ID NO: 1, or a composition containing the peptide, to a subject (subject) that requires skin whitening.

[0077] In another aspect of the present invention, the present invention provides a method for preventing or treating hyperpigmentation disorder, comprising the step of administering a peptide containing the amino acid sequence of SEQ ID NO: 1, or a composition containing the peptide, to a subject (subject) in need of prevention or treatment of hyperpigmentation disorder.

[0078] In another aspect of the present invention, the present invention provides a use for a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for the prevention or treatment of hyperpigmentation disorders.

[0079] In another aspect of the present invention, the invention provides applications for peptides comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of skin whitening cosmetics. [Effects of the Invention]

[0080] The peptide of the present invention exhibits skin whitening activity by inhibiting the invagination of melanosomes in keratinocytes and promoting the degradation of melanosomes. Therefore, the peptide of the present invention can be used as an active ingredient in drugs for the treatment or prevention of hyperpigmentation disorders caused by excessive melanosome deposition, or as an active ingredient in skin whitening cosmetics.

[0081] 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]

[0082] [Figure 1] This melanin content analysis shows that the peptide of the present invention suppresses melanosome invagination in HaCaT cells. [Figure 2a] This PCR analysis shows that the peptide of the present invention reduces the mRNA level of PAR2, a receptor involved in melanosome invagination in HaCaT cells, in a concentration-dependent manner. [Figure 2b] Figure 2a shows a graph of the measured band densities in the electrophoresis. [Figure 3] This is a melanosome content analysis result showing that the peptide of the present invention induces the degradation of melanosomes in HaCaT cells. [Figure 4a] This Western blot analysis shows that peptide treatment according to the present invention increases the levels of the autophagy marker proteins Beclin-1 and LC3-I / II, and decreases the level of p62 protein in HaCaT cells compared to the negative control group (CON). [Figure 4b] The graph shows the measured band density values ​​for the PAGE gel shown in Figure 4a. [Figure 4c] The graph shows the measured band density values ​​for the PAGE gel shown in Figure 4a. [Figure 4d] The graph shows the measured band density values ​​for the PAGE gel shown in Figure 4a. [Modes for carrying out the invention]

[0083] 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.

[0084] Manufacturing Example 1: Peptide Production

[0085] Peptides having the amino acid sequence of SEQ ID NO: 1, as shown in Table 1 below, were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA). These synthesized peptides were then separated into pure molecules using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm x 100 mm, 1.7 μm, Waters Co, USA).

[0086] [Table 1]

[0087] The efficacy of the peptide of Sequence ID No. 1, which was manufactured as described above, was evaluated through the following experiment.

[0088] Experimental Example 1: Analysis of invagination by melanosome phagocytosis

[0089] We confirmed whether the peptide of Sequence ID No. 1, produced in Production Example 1, suppresses phagocytosis-induced invagination of melanosomes in keratinocytes.

[0090] HaCaT cells (Human keratinocyte cell line) 3 x 10 5 Cells were seeded into 6-well cell culture plates at a density of cells / well and cultured for 24 hours in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% FBS. Next, the cells were washed once with serum-free DMEM medium. Peptide SEQ ID NO: 1 from Preparation Example 1 was added to 3 mL of serum-free DMEM medium to prepare peptide solutions at concentrations of 50 μM, 100 μM, and 200 μM, and these were added to the cultured cells. In this procedure, the negative control group (CON, (+)) was the untreated group, and 500 nM ENMD-1068 (6-Amino-1-[4-(3-methyl-1-oxobutyl)-1-piperazinyl]-1-hexanone hydrochloride, Sigma-Aldrich) was used as the positive control group. Next, after culturing for 24 hours at 37°C in a CO2 incubator, 10 ug / ml melanosomes isolated from HEM-DP (Human Epidermal Melanocyte, darkly pigmented donor) were added to the cultured cells, excluding the negative control group (CON). Then, the cells were cultured for 48 hours at 37°C in a CO2 incubator, washed three times with PBS, and treated with 1X TE for 10 minutes to collect HaCaT cells. After recovering the HaCaT cells using a centrifuge, they were lysed with 1M NaOH and dispensed into 96-well plates. Melanin content was measured by absorbance at 490 nm using an ELISA reader.

[0091] As can be seen from the experimental results and the melanin content analysis results in Figure 1, we confirmed that the peptide of Sequence ID No. 1 suppresses melanosome invagination in HaCaT cells.

[0092] Experimental Example 2: Expression Analysis of Melanosome Phagocytosis-Related PAR2 Genes

[0093] We conducted experiments to investigate the effect of the peptide of Sequence ID No. 1, produced in Production Example 1, on the expression of the melanosome phagocytosis-related gene PAR2 (protease activated receptor 2).

[0094] HaCaT cells 3 x 10 5 Cells were inoculated into 6-well plates at a density of cells / well and cultured for 24 hours in DMEM medium containing 10% FBS. Next, the cells were washed once with serum-free DMEM medium, and then peptide SEQ ID NO: 1 of Preparation Example 1 was added to 3 mL of serum-free DMEM medium to prepare peptide solutions at concentrations of 10 μM, 50 μM, and 100 μM, which were then dispensed into the cells. The negative control group (CON, (+)) was treated as the untreated group, while the positive control group was treated with 0.02X and 0.05X protease inhibitors (PI) (cOmplete® Protease Inhibitor Cocktail, Roche, CH). Next, the cells were cultured for 24 hours at 37°C in a CO2 incubator, after which 4 units of trypsin were added to the cultured cells, excluding the negative control group (CON). Next, the cells were cultured for 16 hours at 37°C in a CO2 incubator, washed three times with PBS, and then RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). After quantifying the amount of isolated RNA, 2000 ng of RNA was dispensed per tube and cDNA was synthesized using a cDNA synthesis kit (enzynomics, Cat. No.: RT200, Korea). PCR was carried out using primers targeting the PAR2 gene and a PCR kit (enzynomics, Cat. No.: P581T, Korea) as shown in Table 2. Next, the PCR products were subjected to electrophoresis on a 1.2% agarose gel, and the bands were detected and analyzed using the Bio-Rad gel image system.

[0095] [Table 2]

[0096] The experimental results, as shown in Figures 2a and 2b, confirmed that the peptide of Sequence ID No. 1 decreased the mRNA level of the PAR2 gene, a melanosome invagination-related receptor, in a concentration-dependent manner in HaCaT cells. This result confirmed that the peptide of Sequence ID No. 1 suppresses melanosome invagination.

[0097] Experiment Example 3: Melanosome Degradation Analysis

[0098] We analyzed whether the peptide of Sequence ID No. 1, produced in the above-mentioned Production Example 1, degrades melanosomes.

[0099] HaCaT cells 3 x 10 5 Cells were inoculated into 6-well plates at a density of cells / well and cultured for 24 hours in DMEM medium containing 10% FBS. Next, melanosomes isolated from HEM-DP (Human Epidermal Melanocyte, darkly pigmented donor) were added to 2 mL of DMEM medium containing 2% serum to prepare a concentration of 10 ug / ml, which was then dispensed into cells and cultured for 48 hours at 37°C in a CO2 incubator.

[0100] Next, a 10 ug / ml melanosome solution was prepared by adding melanosomes isolated from HEM-DP to 3 mL of DMEM medium containing 2% serum. Then, peptide solutions of 10 uM, 50 uM, 100 uM, and 200 uM concentrations were prepared by adding the peptide of SEQ ID NO: 1 from Preparation Example 1 to 3 mL of DMEM medium containing 2% serum. The melanosome solution and peptide solution were then added to the cells by aliquoting. The negative control group (CON) was the untreated group, the negative control group (+) was the melanosome solution treatment group, and the positive control group was treated with melanosome solution and 100 nM or 200 nM rapamycin (Rapa), or with melanosome solution and 200 uM or 500 uM resveratrol (RSV). Next, the cells were cultured for 72 hours at 37°C in a CO2 incubator, washed three times with PBS, and then treated with 1XTE for 10 minutes to extract the HaCaT cells. After recovering the HaCaT cells using a centrifuge, they were dissolved in 1M NaOH and dispensed into 96-well plates. Melanin levels were measured by absorbance at 490 nm using an ELISA reader.

[0101] The experimental results, as can be seen from the changes in melanosome content in Figure 3, confirmed that treatment with the peptide of Sequence ID No. 1 induces melanosome degradation in HaCaT cells.

[0102] Experimental example 4: Autophagic flow activation analysis

[0103] The effect of the peptide of Sequence ID No. 1, produced in Production Example 1, on the activation of autophagy flux in human keratinocytes was confirmed by Western blotting analysis against autophagy markers Beclin-1, LC3-I, LC3-II, and p62 proteins.

[0104] HaCaT cells 3 x 10 5Cells were inoculated into 6-well cell culture plates at a density of cells / well and cultured for 24 hours in DMEM medium containing 10% FBS. Next, the cells were washed once with serum-free DMEM medium, and peptide SEQ ID NO: 1 of Preparation Example 1 was added to 1 mL of serum-free DMEM medium to prepare peptide solutions at concentrations of 50 μM, 100 μM, and 200 μM, which were then dispensed into the cells. The negative control group (CON) was the untreated group, and the positive control groups were rapamycin-treated groups (Rapa) with 100 nM and 200 nM, and resveratrol-treated groups (RSV) with 50 μM and 100 μM. Next, the cells were cultured for 3 hours at 37°C in a CO2 incubator, washed twice with PBS, and lysed by adding cell lysis buffer (Whole Cell Extraction Kit, Millipore, US). Samples were prepared by processing with 5X sample buffer, and SDS-PAGE was performed using an 8% SDS-PAGE gel. Proteins separated via SDS-PAGE were transferred to a PVDF membrane. Blocking was performed at room temperature for 30 minutes using 5% skim milk. The following primary antibodies were diluted 1:1000 in 5% BSA and reacted with the membrane for 2 hours. Primary antibodies: anti-Beclin-1 antibody (cell signaling, Cat. No.: #3495, USA), xuan-LC3-I / II antibody (cell signaling, Cat. No.: #4108, USA), xuan-p62 antibody (cell signaling, Cat. No.: #5114, USA). Next, the membrane was washed three times for 10 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), and Goat anti-rabbit IgG (Jackson Immune Research, Cat. No.: 111-035-033, USA) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 1 hour. Then, the membrane was washed three times for 10 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS), and detection was performed on film using ECL solution (GE Healthcare, Cat. No.: RPN2232, USA).

[0105] As can be seen from the experimental results in Figures 4a to 4d, treatment with the peptide of Sequence ID No. 1 increased the levels of Beclin-1 protein and LC3-I / II protein, and decreased the level of p62 protein in HaCaT cells compared to the negative control group (CON). This result suggests that the peptide of Sequence ID No. 1 activates autophagy flux in HaCaT cells and promotes the degradation of melanosomes.

[0106] Manufacturing Example 2: Manufacturing of Pharmaceutical Compositions

[0107] 2-1. Manufacturing of ointments 5g of the peptide of the present invention Cetyl palmitate 20g Cetanol 40g Stearyl alcohol 40g Myristan isopropyl 80g Polysorbate 60g Propyl parahydroxybenzoate 1g Methyl parahydroxybenzoate 1g Appropriate amounts of phosphoric acid and purified water

[0108] An ointment was manufactured by incorporating the aforementioned components in the indicated concentrations according to a standard method for manufacturing ointments.

[0109] 2-2. Manufacturing of powdered medicines 2g of the peptide of the present invention Lactose 1g

[0110] The aforementioned components were mixed and filled into airtight pouches to produce a powder.

[0111] 2-3. Manufacturing of Tablets 100 mg of the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0112] After mixing the aforementioned components, tablets were manufactured by compressing them according to a standard tablet manufacturing method.

[0113] 2-4. Manufacturing of Capsules 100 mg of the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0114] After mixing the aforementioned components, the mixture was filled into gelatin capsules according to a standard capsule manufacturing method to produce capsules.

[0115] 2-5. Manufacturing of the ball 1g of the peptide of the present invention Lactose 1.5g Glycerin 1g Xylitol 0.5g

[0116] After mixing the aforementioned ingredients, the product was manufactured according to a standard method to produce 4g per pill.

[0117] Manufacturing Example 3: Manufacturing of Cosmetic Compositions

[0118] 3-1. Cream Manufacturing 4.6 parts by weight of the peptide of the present invention Cetostearyl alcohol 2.8 parts by weight Beeswax 2.6 parts by weight 1.4 parts by weight of stearic acid Lipophilic glyceryl monostearate 2 parts by weight PEG-100 stearate 1 part by weight 1.4 parts by weight of sorbital sesquioleate Jojoba oil 4 parts by weight Squalene 3.8 parts by weight Polysorbate 60 1.1 parts by weight Macadamia oil 2 parts by weight Tocopherol acetate 0.2 parts by weight Methylpolysiloxane 0.4 parts by weight Ethylparaben 0.1 parts by weight Propylparaben 0.1 parts by weight Euxyl K-400 0.1 parts by weight 1,3-Butylene glycol 7 parts by weight Methylparaben 0.05 parts by weight Glycerin 6 parts by weight d-Panthenol 0.2 parts by weight Triethanolamine 0.2 parts by weight pt41891 0.2 parts by weight p-H2O 46.05 parts by weight

[0119] 3-2. Manufacturing of lotion 3.5 parts by weight of the peptide of the present invention Cetostearyl alcohol 1.6 parts by weight 1.4 parts by weight of stearic acid Lipophilic glyceryl monostearate 1.8 parts by weight PEG-100 stearate 2.6 parts by weight 0.6 parts by weight of sorbital sesquioleate Squalene 4.8 parts by weight Macadamia oil 2 parts by weight Jojoba oil 2 parts by weight Tocopherol acetate 0.4 parts by weight Methylpolysiloxane 0.2 parts by weight Ethylparaben 0.1 parts by weight Propylparaben 0.1 parts by weight 1,3-Butylene glycol 4 parts by weight Methylparaben 0.1 parts by weight Xanthan gum 0.1 parts by weight Glycerin 4 parts by weight d-Panthenol 0.15 parts by weight Allantoin 0.1 parts by weight Calcium carbonate (2% aq. Sol) 4 parts by weight Triethanolamine 0.15 parts by weight 3 parts by weight of ethanol pt41891 0.1 parts by weight p-H2O 48.3 parts by weight

[0120] 3-3. Manufacturing of softening lotion 0.2% by weight of the peptide of the present invention Ethanol 10.0% by weight Polyoxyethylene sorbitan polylaurate 1.0% by weight Methyl parahydroxybenzoate 0.2% by weight Glycerin 5.0% by weight 1,3-Butyl glycol 6.0% by weight Incense (appropriate amount) Dye appropriate amount Purified water (appropriate amount) Total 100

[0121] 3-4. Manufacturing of nourishing lotion 0.1% by weight of the peptide of the present invention Vaseline 2.0% by weight Sorbitan sesquioleate 0.8% by weight Polyoxyethylene oleylethyl 1.2% by weight Methyl parahydroxybenzoate (appropriate amount) Propylene glycol 5.0% by weight Ethanol 3.2% by weight Carboxyvinyl polymer 18.0% by weight Dye appropriate amount Incense (appropriate amount) Purified water (appropriate amount) Total 100

[0122] 3-5. Essence Manufacturing 5.0% by weight of the peptide of the present invention Propylene glycol 10.0% by weight Glycerin 10.0% by weight Sodium hyaluronate aqueous solution (1%) 5.0% by weight Ethanol 3.2% by weight Polyoxyethylene hydrogenated castor oil 1.0% by weight Methyl parahydroxybenzoate 0.1% by weight Incense (appropriate amount) Purified water (appropriate amount) Total 100

[0123] 3-6. Pack manufacturing 0.5% by weight of the peptide of the present invention Glycerin 5.0% by weight Propylene glycol 4.0% by weight Polyvinyl alcohol 15.0% by weight Ethanol 8.0% by weight Polyoxyethylene oleylethyl 1.0% by weight Methyl parahydroxybenzoate 0.2% by weight Incense (appropriate amount) Dye appropriate amount Purified water (appropriate amount) Total 100

[0124] The aforementioned composition ratio is a mixture of preferred components as in the preferred example, but the components or blending ratio may be arbitrarily modified depending on regional and ethnic preferences such as the demand segment, the country of demand, and the intended use.

[0125] Although typical embodiments of this application have been described above, the scope of this application is not limited to such specific embodiments, and any person with ordinary skill in the art may modify the claims of this application as appropriate.

Claims

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

1.

2. A skin whitening composition comprising the peptide described in claim 1 as an active ingredient.

3. A pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising the peptide described in claim 1 as an active ingredient.

4. The pharmaceutically acceptable composition for the prevention or treatment of hyperpigmentation disorders according to claim 3, wherein the peptide (i) inhibits the invagination of melanosomes or (ii) promotes the degradation of melanosomes in keratinocytes.

5. The peptide is present in keratinocytes, (i) Suppress the expression of the PAR2 (protein activated receptor 2) gene, or (ii) The pharmaceutical composition for the prevention or treatment of hyperpigmentation disorder according to claim 4, which adjusts the amount of Beclin-1 protein or LC3-I / II protein upward or the amount of p62 protein downward.

6. The pharmaceutical composition for the prevention or treatment of hyperpigmentation disorder according to claim 3, wherein the hyperpigmentation disorder is a disorder caused by an excessive increase in the amount of melanin in the skin.

7. The pharmaceutical composition for the prevention or treatment of hyperpigmentation disorder according to claim 3, wherein the hyperpigmentation disorder is a freckle, age spot, solar lentigo, or hyperpigmentation that occurs after inflammation of the skin.

8. The pharmaceutical composition for the prevention or treatment of hyperpigmentation disorder according to claim 3, wherein the pharmaceutical composition is a topical skin preparation.

9. A cosmetic composition for skin whitening containing the peptide described in claim 1 as an active ingredient.

10. The skin whitening cosmetic composition according to claim 9, wherein the peptide (i) inhibits the invagination of melanosomes or (ii) promotes the degradation of melanosomes in keratinocytes.

11. The peptide is present in keratinocytes, (i) Suppress the expression of the PAR2 (protein activated receptor 2) gene, or (ii) The skin whitening cosmetic composition according to claim 10, wherein the amount of Beclin-1 protein or LC3-I / II protein is adjusted upward, or the amount of p62 protein is adjusted downward.

12. The cosmetic composition described above is a topical skin preparation, as described in claim 9, for skin whitening.

13. The cosmetic composition for skin whitening according to claim 12, wherein the cosmetic composition is one or more dosage forms selected from the group consisting of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, and spray.

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