Peptides having skin lightening activity and uses thereof

Abstract

The peptide of the present application has a skin-whitening activity by inhibiting incorporation of melanin into keratinocytes and promoting degradation of melanin. The peptide of the present application can be used as an active ingredient in a medicament for treating or preventing a hyperpigmentation disease caused by excessive deposition of melanin, or as an active ingredient in a cosmetic for skin whitening.

Description

Technical Field

[0001] Cross-references to related applications

[0002] This application claims priority to Korean Patent Application No. 10-2022-0155228, filed with the Korean Intellectual Property Office on November 18, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] This invention relates to peptides with skin-whitening activity and their uses. Background Technology

[0005] Melanin is present in the skin, hair follicles, and eyes, and plays a vital role in protecting the skin from ultraviolet radiation. However, excessive melanin formation leads to hyperpigmentation disorders such as melasma, freckles, and age spots, causing mental stress and a decline in quality of life. Melanin is primarily synthesized in melanocytes located between or below the basal layer of the epidermis and in hair follicles. It is synthesized in melanosomes, organelles within melanocytes. These melanosomes migrate via dendrites to adjacent keratinocytes, and skin color is revealed as keratinocytes migrate into the outer layer of skin. Although the exact mechanism by which melanosomes migrate from melanocytes to keratinocytes is not fully understood, it is known that after migrating to the dendrites of melanocytes, melanosomes are released from there to the outside of the cell and participate in membrane fusion between melanosomes and keratinocytes, as well as phagocytosis.

[0006] Regarding the synthesis of melanin, it ultimately arises from L-tyrosine via dopa (DOPA), dopaquinone, DOPA pigment, and 5,6-dihydroxyindole (DHI). Traditionally, inhibition of melanin synthesis has focused on factors that inhibit the activity of tyrosinases, enzymes that catalyze the rate-controlling steps of the melanin synthesis pathway.

[0007] PCT International Publication WO 2020 / 153819 discloses a polypeptide with activity that inhibits tyrosinase activity and its use for skin whitening, wherein tyrosinase is an important enzyme in the melanin synthesis pathway. Additionally, Korean Patent No. 10-1869783 discloses a peptide with activity that inhibits melanin production and tyrosinase activity and its use for skin whitening.

[0008] To date, the development of skin whitening agents has focused on inhibiting the activity of tyrosinase, a crucial enzyme in melanin synthesis. However, to achieve a synergistic skin whitening effect through various sites of action, it is necessary to develop skin whitening agents with different mechanisms of action or sites of action.

[0009] [Existing Technical Documents]

[0010] [Patent Literature]

[0011] Patent Document 1. WO 2020 / 153819

[0012] Patent Document 2. Korean Patent No. 10-1869783 Summary of the Invention

[0013] Technical issues

[0014] The inventors have conducted research to develop peptides with improved activity and skin-whitening activity. As a result, they have experimentally demonstrated that their newly developed peptides possess excellent effects in inhibiting melanosome incorporation into keratinocytes and exhibit excellent activity in degrading melanosomes, thus completing this invention.

[0015] Therefore, the object of this invention is to provide novel peptides with skin whitening activity.

[0016] Another object of the present invention is to provide a composition for skin whitening, comprising peptides having the above-described activity as active ingredients.

[0017] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation, comprising a peptide having the above-described activity as an active ingredient.

[0018] Another object of the present invention is to provide a cosmetic composition for skin whitening, comprising peptides having the above-described activity as active ingredients.

[0019] Technical solution

[0020] In order to achieve the above objectives,

[0021] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0022] Another object of the present invention is to provide a composition for skin whitening, comprising the peptide as an active ingredient.

[0023] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation, comprising the peptide as an active ingredient.

[0024] Another object of the present invention is to provide a cosmetic composition for skin whitening, comprising the peptide as an active ingredient.

[0025] The present invention will now be described in detail.

[0026] 1. Peptides and their activity

[0027] According to one aspect of the invention, a peptide comprising the amino acid sequence disclosed in SEQ ID NO:1 is provided.

[0028] As used in this article, the term "peptide" refers to a linear molecule formed by linking amino acid residues together through peptide bonds.

[0029] The peptide of the present invention containing the amino acid sequence of SEQ ID NO:1 can be used without modification, but variants or fragments thereof with different amino acid sequences by means of deletion, insertion, substitution of amino acid residues or combinations thereof can be used within the scope of not affecting the original activity of the peptide (e.g., skin whitening activity).

[0030] The peptides of the present invention can be modified by phosphorylation, sulfation, acrylate esterification, glycosylation, methylation, farnesylation, etc., without altering their activity.

[0031] The peptides of the present invention comprise peptides containing an amino acid sequence substantially identical to that of the peptide comprising the amino acid sequence of SEQ ID NO:1, and variants or active fragments thereof. A substantially identical amino acid sequence is defined as an amino acid sequence having at least 75%, for example, at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% sequence identity with the amino acid sequence of SEQ ID NO:1, respectively. Additionally, the peptide may further include targeting sequences, tags, labeled residues, and amino acid sequences prepared for a specific purpose of increasing the peptide's half-life or stability.

[0032] The peptides of the present invention may have N-terminal and / or C-terminal modifications that can be induced to select a portion of the amino acid sequence and increase their activity. Through these N-terminal and / or C-terminal modifications, the stability of the peptides of the present invention can be significantly improved, for example, increasing the half-life of the peptide upon in vivo administration. The term "stability" refers not only to in vivo stability, which protects the peptides of the present invention from attack by protein-cleaving enzymes in vivo, but also to storage stability (e.g., storage stability at room temperature).

[0033] N-terminal modification can be the binding of a protective group to the N-terminus of the peptide. The protective group is selected from the group consisting of acetyl groups, fluoreneylmethoxycarbonyl groups, formyl groups, palmitoyl groups, myristyl groups, stearoyl groups, and polyethylene glycol (PEG). C-terminal modification can be the binding of a hydroxyl group (-OH), an amino group (-NH2), a hydrazide group (-NHNH2), etc., to the C-terminus of the peptide, but is not limited to these.

[0034] The peptides of the present invention can be prepared by a variety of methods widely known in the art to which this invention pertains. For example, the peptides of the present invention can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd edition, Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis (US Patent No. 5,516,891).

[0035] The peptides of this invention have skin-whitening activity.

[0036] In one embodiment, the peptide of the present invention has the activity of inhibiting melanosome incorporation into keratinocytes or promoting melanosome degradation.

[0037] Melanosomes produced in melanocytes migrate from melanocytes to keratinocytes. Specifically, the migration of melanosomes to keratinocytes includes the release of melanosomes from melanocytes and the incorporation of melanosomes into keratinocytes. Melanosome incorporation can be expressed differently as either keratinocyte uptake of melanosomes or keratinocyte phagocytosis of melanosomes.

[0038] PAR2 is involved in the process of absorbing melanosomes released from melanocytes through phagocytosis by keratinocytes.

[0039] In one embodiment, the peptide of the present invention has the activity of inhibiting the expression of the protease-activated receptor 2 (PAR2) gene in keratinocytes.

[0040] Receptor 2 for protease activation (PAR2) is a receptor protein located in the cell membrane and is known to regulate inflammatory responses, obesity, metabolism, and cancer, and to act as a protease receptor during infection. PAR2 protein is located in the membrane of keratinocytes and is involved in the uptake (incorporation) of melanosomes by keratinocytes. It is known that inhibition of PAR2 protein activity leads to inhibition of melanosome uptake 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.).

[0041] In one embodiment, the peptide of the present invention has the activity of upregulating the amount of Beclin-1 or LC3-I / II protein or downregulating the amount of p62 protein in keratinocytes. The amounts of Beclin-1, LC3-I / II, and p62 proteins can be the expression levels of these proteins.

[0042] Autophagy is a natural process by which cells remove unnecessary, dysfunctional, or deficient substances (such as denatured proteins). For example, autophagy removes organelles that have degenerated and become dysfunctional by the end of their lifespan. The proteins or organelles removed by autophagy are isolated within the cell in double-membrane vesicles called autophagosomes, and these vesicles then bind to lysosomes to form autophagolysomes, in which they are degraded by lysosomal enzymes.

[0043] It is also known that the degradation of melanosomes is induced by the activation of autophagy flux in keratinocytes (Pigment Cell Melanoma Res. May 2020; 33(3):403-415; Int. J. Mol. Sci 2021, 22,3995).

[0044] Beclin-1 and LC3-I / II proteins are known to be key autophagy marker proteins involved in autophagy, and their levels are known to increase when autophagy is activated.

[0045] p62 is known to be a protein that transports ubiquitinated cargo for autophagic degradation. It is known that p62 expression decreases when autophagy is activated and increases when autophagy is inhibited (Cellular & Molecular Biology Letters (2016) 21:29).

[0046] As described above, the peptides of the present invention can exhibit skin whitening activity and prevent or treat hyperpigmentation by inhibiting the incorporation activity of melanosomes and the activity of degrading melanosomes.

[0047] 2. Compositions for skin whitening and for the prevention, treatment or improvement of hyperpigmentation disorders.

[0048] In another aspect of the invention, a composition for skin whitening is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0049] The peptide of the present invention, comprising the amino acid sequence of SEQ ID NO:1, has skin whitening activity through the mechanism described above.

[0050] In another aspect of the invention, the composition of the invention may be a pharmaceutical composition for the prevention or treatment of hyperpigmentation, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0051] In one embodiment, the pharmaceutical composition comprising a peptide as an active ingredient inhibits the incorporation of melanosomes into keratinocytes or promotes the degradation of melanosomes.

[0052] In one embodiment, a pharmaceutical composition comprising a peptide as an active ingredient inhibits the expression of the protease-activated receptor 2 (PAR2) gene in keratinocytes, upregulates the amount of Beclin-1 or LC3-I / II protein, or downregulates the amount of p62 protein. The amounts of Beclin-1, LC3-I / II, and p62 proteins can be the expression levels of these proteins.

[0053] As used in this article, the term "hyperpigmentation disorder" refers to a condition caused by an excessive increase in the amount of melanin in the skin. As a result, the skin becomes darker due to the excessive amount of melanin. Causes of hyperpigmentation disorder can include, but are not limited to, sun exposure, skin inflammation such as acne (post-inflammatory hyperpigmentation), injury, hormonal imbalances, or medications.

[0054] In one implementation, hyperpigmentation disorders can be melasma, freckles, age spots, sunspots, or excessive pigmentation in post-inflammatory skin. Post-inflammatory hyperpigmentation can occur after injury, ultraviolet radiation, or inflammatory skin conditions such as acne.

[0055] The pharmaceutical compositions of the present invention may include a therapeutically effective amount of the peptide of the present invention comprising the amino acid of SEQ ID NO:1.

[0056] The term "therapeutic effective amount" refers to an amount sufficient to enable a peptide to exert its activity or efficacy, such as an amount sufficient to treat or prevent hyperpigmentation, wherein the peptide is the active ingredient in the pharmaceutical composition of the present invention.

[0057] As used in this article, the term “prevention” refers to reducing the risk of developing a disease or condition, and refers to all actions taken to suppress or delay the onset of a disease by preventing the progression of the disease or one or more of its clinical symptoms.

[0058] As used herein, the term “treatment” means to alleviate a disease or symptom, and includes all actions that improve or beneficially alter the symptoms of a disease by stopping or reducing the progression of the disease or one or more of its clinical symptoms.

[0059] In this invention, prevention or treatment of hyperpigmentation can be achieved by removing the cause of hyperpigmentation in the skin or inhibiting the development of hyperpigmentation, and specifically, by inhibiting the incorporation of melanosomes into keratinocytes or promoting the degradation of melanosomes.

[0060] The pharmaceutical compositions of the present invention may include pharmaceutically acceptable carriers.

[0061] Pharmaceutically acceptable carriers commonly used in formulations include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc., but are not limited to these.

[0062] In addition to the above-mentioned components, the pharmaceutical composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but the components are not limited thereto.

[0063] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy (19th edition, 1995, Williams & Wilkins).

[0064] The pharmaceutical compositions of the present invention can be administered via any route suitable for treating hyperpigmentation disorders, such as oral or parenteral administration. In the case of parenteral administration, the compositions can be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical application, transdermal application, etc. Since the pharmaceutical compositions of the present invention have activity in preventing or treating hyperpigmentation disorders, topical application of the compositions is preferable, for example, by applying them to the skin.

[0065] The dosage of the pharmaceutical composition may be from 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 daily, but is not limited thereto. The pharmaceutical composition may be prescribed in various ways depending on factors such as preparation method, method of administration, patient's age, weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and responsiveness.

[0066] The pharmaceutical compositions of the present invention can be formulated in unit dosage forms using pharmaceutically acceptable carriers and / or excipients, according to methods readily practiced by those skilled in the art, or the pharmaceutical compositions can be prepared by placing them in multi-dose containers. In particular, the formulations can be in the form of solutions, suspensions, or emulsions in oil or aqueous media, or can be in the form of extracts, powders, granules, tablets, or capsules, and may additionally include dispersants or stabilizers.

[0067] The pharmaceutical compositions of the present invention can be topical skin preparations. Topical skin preparations are preparations that can be used by applying them to the external skin. When the pharmaceutical compositions of the present invention are used as topical skin preparations, they can be applied to areas of skin with excessive pigmentation. The topical skin preparations can be creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal delivery patches, bandages containing the medicine, lotions, or combinations thereof. The topical skin preparations can be appropriately mixed as needed with ingredients commonly used in topical skin preparations (e.g., cosmetics and pharmaceuticals), such ingredients as aqueous components, oil-based components, powder components, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. The topical skin preparation may be appropriately mixed with the following: metal chelating agents (e.g., disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc.), caffeine, tannins, licorice extract, glycyrrhizin, various herbs, tocopheryl acetate, glycyrrhizic acid, drugs (e.g., tranexamic acid and its derivatives or salts), vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, and sugars (e.g., glucose, fructose, trehalose, etc.).

[0068] In another aspect of the invention, the compositions of the invention provide a cosmetic composition for skin whitening, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0069] In one embodiment, the cosmetic composition comprising the peptide of the present invention as an active ingredient inhibits the incorporation of melanosomes into keratinocytes or promotes the degradation of melanosomes.

[0070] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient inhibits the expression of the protease-activated receptor 2 (PAR2) gene in keratinocytes, upregulates the amount of Beclin-1 or LC3-I / II protein, or downregulates the amount of p62 protein. The amounts of Beclin-1, LC3-I / II, and p62 proteins can be the expression levels of these proteins.

[0071] The cosmetic composition can be prepared in any formulation commonly prepared in the technical field to which this invention pertains, and can be a topical skin preparation. For example, the cosmetic composition can be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto.

[0072] The cosmetic composition can be prepared in various forms, such as solutions (e.g., softening lotions, nourishing lotions, nourishing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, packs, sprays, powders, hair activators, hair creams, hair lotions, hair shampoo, hair rinses, hair conditioners, hair sprays, hair aerosols, pomades, gels, etc.), sol-gels, emulsions, oils, waxes, aerosols, etc., but not limited to these.

[0073] The cosmetic compositions of the present invention may include other additives, such as excipients and carriers, and may be applied and mixed as needed with commonly used ingredients mixed in general skin cosmetics.

[0074] When the formulation of a cosmetic composition is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin wax, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc., can be used as carrier ingredients.

[0075] When the formulation of a cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, when the cosmetic composition is a spray, it may also include a propellant, such as chlorofluorocarbon, propane / butane, or dimethyl ether, but is not limited thereto.

[0076] When a cosmetic composition is in the form of a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol aliphatic esters, polyethylene glycol, sorbitan fatty acid esters, etc.

[0077] When the formulation of a cosmetic composition is a suspension, liquid phase diluents (such as water, ethanol, and propylene glycol), suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester, polyoxyethylene dehydrated sorbitol ester, etc.), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth gum, etc. can be used as carrier components.

[0078] When the formulation of a cosmetic composition is a surfactant-containing detergent, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurate, sarcosine esters, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, etc., can be used as carrier components.

[0079] When the cosmetic composition is formulated as a shampoo, the peptides of the present invention can be mixed with base ingredients used to form the shampoo, such as thickeners, surfactants, viscosity modifiers, humectants, pH adjusters, preservatives, and essential oils. CDE can be used as a thickener; LES (anionic surfactant) and coco betaine (amphotericidal surfactant) can be used as surfactants; polyquartz salts can be used as viscosity modifiers; glycerin can be used as a humectant; and citric acid and sodium hydroxide can be used as pH adjusters. Grapefruit extract and the like can be used as preservatives; additionally, essential oils of cedarwood, peppermint, and rosemary, as well as silk amino acid, pentanol, or vitamin E can be added.

[0080] In addition to the peptides and carrier components of the present invention, the active ingredients included in the cosmetic composition may include, but are not limited to, auxiliary ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances.

[0081] The peptides of the present invention can be included in the above-described compositions, pharmaceutical compositions, or cosmetic compositions at concentrations from 0.01 μM to 1000 μM, and specifically, the peptides of the present invention can be in concentrations of 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, or 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, etc. Concentrations of 0.1 μM to 500 μM, 0.1 μM to 300 μM, or 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, or 1 μM to 200 μM; or 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM are included in, but not limited to, the above-described compositions, pharmaceutical compositions, or cosmetic compositions.

[0082] 3. Uses of the peptides of the present invention

[0083] In another aspect of the invention, a peptide comprising the amino acid sequence of SEQ ID NO:1 is provided for skin whitening or for the prevention, treatment or improvement of hyperpigmentation.

[0084] In another aspect of the invention, a skin whitening method is provided, comprising administering to a subject who requires skin whitening a peptide comprising the amino acid sequence of SEQ ID NO:1 or a composition comprising said peptide.

[0085] In another aspect of the invention, a method for preventing or treating hyperpigmentation disorders is provided, comprising administering to a subject who requires prevention or treatment of hyperpigmentation disorders a peptide comprising the amino acid sequence of SEQ ID NO:1 or a composition comprising said peptide.

[0086] In another aspect of the invention, the use of a peptide comprising the amino acid sequence of SEQ ID NO:1 is provided in the preparation of a medicament for the prevention or treatment of hyperpigmentation disorders.

[0087] In another aspect of the invention, the use of a peptide comprising the amino acid sequence of SEQ ID NO:1 is provided in the preparation of cosmetics for skin whitening.

[0088] Beneficial effects

[0089] The peptides of this invention exhibit skin-whitening activity by inhibiting the incorporation of melanosomes into keratinocytes and promoting the degradation of melanosomes. Therefore, the peptides of this invention can be used as active ingredients in pharmaceuticals for treating or preventing hyperpigmentation caused by excessive melanosome deposition, or as active ingredients in cosmetics for skin whitening.

[0090] However, the effects of the present invention are not limited to those described above, and those skilled in the art will clearly understand from the following description other effects not mentioned. Attached Figure Description

[0091] Figure 1 The results of melanin content analysis are shown, illustrating that the peptide of the present invention inhibits the incorporation of melanosomes into HaCaT cells.

[0092] Figure 2a The results of PCR analysis show that the peptide of the present invention reduces the mRNA level of PAR2 in HaCaT cells in a concentration-dependent manner. PAR2 is a receptor associated with melanosome incorporation.

[0093] Figure 2b Explanation is shown Figure 2a A graph showing the measured values ​​of band density from electrophoresis.

[0094] Figure 3 The results of melanin content analysis are shown, illustrating the peptide-induced degradation of melanosomes in HaCaT cells according to the present invention.

[0095] Figure 4a The results of Western blot analysis show that, compared with the negative control (CON), in HaCaT cells, treatment with the peptides of the present invention increased the levels of Beclin-1 and LC3-I / II proteins (i.e., autophagy-related marker proteins) while decreasing the level of p62 protein.

[0096] Figures 4b to 4d It shows Figure 4a A graph showing the measured values ​​of band density in the PAGE gel. Detailed Implementation

[0097] The present invention will be described in detail below. However, the following embodiments specifically illustrate the present invention, and the content of the present invention is not limited to the following embodiments.

[0098] Preparation Example 1: Preparation of Peptides

[0099] Peptides with the amino acid sequence of SEQ ID NO:1 shown in Table 1 were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and then purified using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was an ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, WatersCo, USA).

[0100] [Table 1]

[0101]

[0102] The efficacy of the peptide prepared by SEQ ID NO:1 was evaluated through the following experiments.

[0103] Experimental Example 1: Incorporation Analysis via Melanosome Phagocytosis

[0104] To confirm whether the peptide of SEQ ID NO:1 prepared in Preparation Example 1 inhibits the incorporation of melanosomes through phagocytosis in keratinocytes.

[0105] Human keratinocyte cell line (HaCaT cells) were used at 3×10 5Cells were seeded at a density of 6-well cell culture plates and cultured for 24 hours in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. The cells were then washed once with serum-free DMEM. A solution of the peptide of SEQ ID NO:1 from Example 1 was added to 3 mL of serum-free DMEM to prepare peptide solutions at concentrations of 50 μM, 100 μM, and 200 μM, and these solutions were added to the cultured cells respectively. Specifically, the negative control group (CON, (+)) was the untreated group, and the positive control group was treated with 500 nM of 6-amino-1-[4-(3-methyl-1-oxobutyl)-1-piperazinyl]-1-hexanone hydrochloride (ENMD-1068, Sigma-Aldrich). Then, after culturing them in a CO2 incubator at 37°C for 24 hours, 10 μg / mL of melanosomes isolated from human epidermal melanocyte-darkly pigmented donors (HEM-DP) were additionally added to the cultured cells excluding the negative control (CON) group. Subsequently, the cells were cultured in a CO2 incubator at 37°C for 48 hours, washed three times with PBS, treated with 1X TE for 10 minutes, and HaCaT cells were collected. HaCaT cells were recovered using a centrifuge, dissolved in 1M NaOH, and aliquoted into 96-well plates. Melanin content was measured by absorbance at 490 nm using an ELISA reader.

[0106] As a result of the experiment, such as from Figure 1 The results of the melanin content analysis shown in the figure confirm that the incorporation of melanosomes in HaCaT cells is inhibited by the peptide of SEQ ID NO:1.

[0107] Experimental Example 2: Analysis of PAR2 gene expression related to melanosome phagocytosis

[0108] An experiment was conducted to investigate the effect of the peptide of SEQ ID NO:1 prepared in Example 1 on the expression of protease-activated receptor 2 (PAR2), a gene related to melanosome phagocytosis.

[0109] HaCaT cells were used at a rate of 3 × 10 5Cells were seeded at a density of 6-well plates and cultured for 24 hours in DMEM medium containing 10% FBS. The cells were then washed once with serum-free DMEM medium, and a solution of the peptide from SEQ ID NO. 1 of 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, respectively, which were then dispensed into the cells. Specifically, the negative control group (CON, (+)) was the untreated group, and the positive control group was treated with 0.02X and 0.05X protease inhibitors (PIs). TM Cells were treated with a protease inhibitor cocktail (Roche, CH). After culturing at 37°C in a CO2 incubator for 24 hours, 4 units of trypsin were added to the cultured cells excluding the negative control (CON) group. Subsequently, cells were cultured at 37°C in a CO2 incubator for 16 hours, washed three times with PBS, and RNA was isolated using easy blue (iNtRON, catalog number: 17061, Korea). After quantifying the amount of isolated RNA, 2000 ng of RNA was dispensed into each tube, and cDNA was synthesized using a cDNA synthesis kit (Enzynomics, catalog number: RT200, Korea). PCR was performed using primers targeting the PAR2 gene shown in Table 2 and a PCR kit (Enzynomics, catalog number: P581T, Korea). The PCR products were then electrophoresed on a 1.2% agarose gel, and the bands were detected and analyzed using the Bio-Rad gel imaging system.

[0110] [Table 2]

[0111]

[0112] As a result of the experiment, such as Figure 2a and 2b As shown, the peptide of SEQ ID NO:1 was confirmed to reduce the mRNA level of the PAR2 gene, a receptor involved in melanosome incorporation, in HaCaT cells in a concentration-dependent manner. These results confirm that the peptide of SEQ ID NO:1 inhibits melanosome incorporation.

[0113] Experimental Example 3: Melanosome Degradation Analysis

[0114] An analysis was conducted to determine whether the peptide of SEQ ID NO:1 prepared in Preparation Example 1 degraded melanosomes.

[0115] HaCaT cells were used at a rate of 3 × 10 5Cells were seeded at a density of 10% FBS in 6-well plates and cultured for 24 hours. Melanosomes isolated from human epidermal melanocyte-deep pigment donors (HEM-DP) were then added to 2 mL of DMEM medium containing 2% serum to prepare a concentration of 10 μg / mL, which was dispensed into the cells, and the cells were incubated in a CO2 incubator at 37°C for 48 hours.

[0116] Subsequently, melanosomes isolated from HEM-DP were added to 3 mL of DMEM medium containing 2% serum to prepare a melanosome solution with a concentration of 10 μg / mL, and the peptide of SEQ ID NO:1 of Preparation Example 1 was added to 3 mL of DMEM medium containing 2% serum to prepare peptide solutions with concentrations of 10 μM, 50 μM, 100 μM, and 200 μM, and the melanosome solution and peptide solution were separately aliquoted into the cells. Specifically, the negative control group (CON) was the untreated group, wherein the negative control group (+) was treated with melanosome solution only, while the positive control group included groups treated with melanosome solution and 100 nM and 200 nM rapamycin (Rapa), and groups treated with melanosome solution and 200 μM and 500 μM resveratrol (RSV). The cells were then cultured in a CO2 incubator at 37°C for 72 hours, washed three times with PBS, treated with 1XTE for 10 minutes, and HaCaT cells were collected. HaCaT cells were recovered using a centrifuge, dissolved in 1M NaOH, and aliquoted into 96-well plates. Melanin content was measured by absorbance at 490 nm using an ELISA reader.

[0117] As a result of the experiment, from Figure 3 The changes in melanosome content confirm that treatment with the peptide of SEQ ID NO:1 induces melanosome degradation in HaCaT cells.

[0118] Experimental Example 4: Activation Analysis of Autophagy Flux

[0119] Western blot analysis confirmed the effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the activation of autophagy flux in human keratinocytes in terms of autophagy markers (i.e., Beclin-1, LC3-I, LC3-II and p62 proteins).

[0120] HaCaT cells were used at a rate of 3 × 10 5Cells were seeded at a density of 1 / well in 6-well cell culture plates and cultured for 24 hours in DMEM medium containing 10% FBS. Cells were then washed once with serum-free DMEM medium, and the peptide of SEQ ID NO:1 from 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. Specifically, the negative control group (CON) was the untreated group, while the positive control group consisted of groups treated with 100 nM and 200 nM rapamycin (Rapa) and groups treated with 50 μM and 100 μM resveratrol (RSV). Cells were then cultured in a CO2 incubator at 37°C for 3 hours, washed twice with PBS, and lysed by adding cell lysis buffer (Whole Cell Extraction Kit, Millipore, USA). Samples were prepared by treatment with 5X sample buffer and SDS-PAGE was performed using an 8% SDS-PAGE gel. Proteins separated by SDS-PAGE were transferred to a PVDF membrane. Blocking was performed for 30 minutes at room temperature using 5% skim milk. The following primary antibodies were diluted 1:1000 in 5% BSA and reacted with the membrane for 2 hours: anti-Beclin-1 antibody (Cell signaling, catalog number: #3495, USA), anti-LC3-I / II antibody (Cell signaling, catalog number: #4108, USA), and anti-p62 antibody (Cell signaling, catalog number: #5114, USA). Subsequently, the antibodies were washed three times with 0.1% PBS-T (PBS of 0.1% Tween-20) for 10 minutes each time, and goat anti-rabbit IgG (Jackson Immune Research, catalog number: 111-035-033, USA) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 1 hour. Subsequently, the product was washed three times with 0.1% PBS-T (PBS of 0.1% Tween-20) for 10 minutes each time, and detected on the membrane using ECL solution (GE Healthcare, catalog number: RPN2232, USA).

[0121] As a result of the experiment, such as in Figures 4a to 4d As can be seen, treatment with the peptide of SEQ ID NO:1 confirmed an increase in the levels of Beclin-1 and LC3-I / II proteins and a decrease in the level of p62 protein in HaCaT cells compared to the negative control (CON). These results indicate that the peptide of SEQ ID NO:1 promotes melanosome degradation by activating autophagy flux in HaCaT cells.

[0122] Preparation Example 2: Preparation of Pharmaceutical Composition

[0123] 2-1. Preparation of Ointment

[0124] The peptide of the present invention, 5g

[0125] 20g palmitate hexadecyl palmitate

[0126] 40g of hexadecane alcohol

[0127] 40g of octadecyl alcohol

[0128] Myristane isopropyl 80g

[0129] Polysorbate 60g

[0130] propyl paraoxybenzoate 1g

[0131] methyl paraoxybenzoate 1g

[0132] Sufficient phosphoric acid and purified water

[0133] According to conventional ointment preparation methods, the ointment is prepared by incorporating the above-mentioned ingredients in a specified amount.

[0134] 2-2. Preparation of Powder

[0135] The peptide 2g of the present invention

[0136] 1g lactose

[0137] The powder is prepared by mixing the above ingredients and then filling the mixture into an airtight bag.

[0138] 2-3. Tablet Preparation

[0139] The peptide of this invention, 100mg

[0140] 100mg of corn starch

[0141] 100mg lactose

[0142] Magnesium stearate 2mg

[0143] Tablets are prepared by mixing the above ingredients and then compressing the mixture according to conventional tablet preparation methods.

[0144] 2-4. Capsule Preparation

[0145] The peptide of this invention, 100mg

[0146] 100mg of corn starch

[0147] 100mg lactose

[0148] Magnesium stearate 2mg

[0149] Capsules are prepared by mixing the above ingredients and then filling the mixture into gelatin capsules according to conventional capsule preparation methods.

[0150] 2-5. Preparation of pills

[0151] The peptide 1g of the present invention

[0152] 1.5g lactose

[0153] 1g of glycerin

[0154] xylitol 0.5g

[0155] After mixing the above ingredients, pills are prepared in a quantity of 4g per pill according to conventional methods.

[0156] Preparation Example 3: Preparation of Cosmetic Composition

[0157] 3-1. Preparation of Cream

[0158] 4.6 parts by weight of the peptide of the present invention

[0159] Cetosterol 2.8 parts by weight

[0160] 2.6 parts by weight of beeswax

[0161] 1.4 parts by weight of stearic acid

[0162] 2 parts by weight of lipophilic glyceryl monostearate

[0163] 1 part by weight of PEG-100 stearate

[0164] Sorbitol sesquioleate 1.4 parts by weight

[0165] 4 parts by weight of jojoba oil

[0166] 3.8 parts by weight of squalane

[0167] Polysorbate 60 1.1 parts by weight

[0168] Macadamia oil (2 parts by weight)

[0169] Tocopherol acetate 0.2 parts by weight

[0170] 0.4 parts by weight of methyl polysiloxane

[0171] 0.1 parts by weight of ethyl p-hydroxybenzoate

[0172] 0.1 parts by weight of propylparaben

[0173] Euxyl K-400 0.1 parts by weight

[0174] 1,3-Butenediol 7 parts by weight

[0175] 0.05 parts by weight of methylparaben

[0176] 6 parts by weight of glycerin

[0177] 0.2 parts by weight of D-panthenol

[0178] 0.2 parts by weight of triethanolamine

[0179] pt 41891 0.2 parts by weight

[0180] p-H2O 46.05 parts by weight

[0181] 3-2. Preparation of lotion

[0182] 3.5 parts by weight of the peptide of the present invention

[0183] Cetosterol 1.6 parts by weight

[0184] 1.4 parts by weight of stearic acid

[0185] 1.8 parts by weight of lipophilic glyceryl monostearate

[0186] 2.6 parts by weight of PEG-100 stearate

[0187] 0.6 parts by weight of sorbital sesquioleate

[0188] 4.8 parts by weight of squalene

[0189] Macadamia oil (2 parts by weight)

[0190] 2 parts by weight of jojoba oil

[0191] Tocopherol acetate 0.4 parts by weight

[0192] 0.2 parts by weight of methyl polysiloxane

[0193] 0.1 parts by weight of ethyl p-hydroxybenzoate

[0194] 0.1 parts by weight of propylparaben

[0195] 1,3-Butenediol 4 parts by weight

[0196] 0.1 parts by weight of methylparaben

[0197] 0.1 parts by weight of xanthan gum

[0198] 4 parts by weight of glycerin

[0199] 0.15 parts by weight of D-panthenol

[0200] 0.1 parts by weight of allantoin

[0201] Calcium carbonate (2% aqueous solution) 4 parts by weight

[0202] 0.15 parts by weight of triethanolamine

[0203] 3 parts by weight of ethanol

[0204] pt 41891 0.1 parts by weight

[0205] p-H2O 48.3 parts by weight

[0206] 3-3. Preparation of softening detergent

[0207] The peptide of this invention is 0.2 wt%.

[0208] 10.0 wt% ethanol

[0209] Polyoxyethylene dehydrated sorbitol polylaurate 1.0 wt%

[0210] Methyl paraoxybenzoate 0.2 wt%

[0211] Glycerin 5.0 wt%

[0212] 1,3-Butanediol 6.0wt%

[0213] Sufficient amount of fragrance

[0214] Sufficient pigment

[0215] Sufficient purified water

[0216] Total 100

[0217] 3-4. Preparation of Nourishing Wash

[0218] The peptide of this invention is 0.1 wt%.

[0219] Vaseline 2.0 wt%

[0220] 0.8 wt% of dehydrated sorbitan sesquioleate

[0221] Polyoxyethylene oleyl ethyl 1.2 wt%

[0222] Sufficient amount of methyl paraben

[0223] Propylene glycol 5.0 wt%

[0224] 3.2 wt% ethanol

[0225] Carboxyvinyl polymer 18.0 wt%

[0226] Sufficient pigment

[0227] Sufficient amount of fragrance

[0228] Sufficient purified water

[0229] Total 100

[0230] 3-5. Preparation of the serum

[0231] The peptide of this invention is 5.0 wt%.

[0232] Propylene glycol 10.0 wt%

[0233] Glycerin 10.0 wt%

[0234] 5.0 wt% sodium hyaluronate (1%) aqueous solution

[0235] 3.2 wt% ethanol

[0236] Polyoxyethylene hydrogenated castor oil 1.0 wt%

[0237] Methylparaben 0.1 wt%

[0238] Sufficient amount of fragrance

[0239] Sufficient purified water

[0240] Total 100

[0241] 3-6. Preparation of membranes (packs)

[0242] The peptide of this invention is 0.5 wt%.

[0243] Glycerin 5.0 wt%

[0244] Propylene glycol 4.0 wt%

[0245] Polyvinyl alcohol 15.0 wt%

[0246] 8.0 wt% ethanol

[0247] Polyoxyethylene oleyl ethyl 1.0 wt%

[0248] Methyl paraoxybenzoate 0.2 wt%

[0249] Sufficient amount of fragrance

[0250] Sufficient pigment

[0251] Sufficient purified water

[0252] Total 100

[0253] The above composition ratio is a mixture of suitable ingredients in the preferred embodiment, but its composition or mixing ratio can be arbitrarily modified according to regional or ethnic preferences (e.g., demand segmentation, demand country and intended use).

[0254] In the foregoing, representative embodiments of this application have been described by way of exemplary implementation, but the scope of this application is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate modifications within the scope described in the claims of this application.

Claims

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

1.

2. A composition for skin whitening, comprising the peptide of claim 1 as an active ingredient.

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

4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is a skin preparation for external use.

5. Use of the peptide of claim 1 in the preparation of a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders.

6. The use according to claim 5, wherein in keratinocytes, the peptide: (i) Inhibit the incorporation of melanosomes; or (ii) Promotes the degradation of melanosomes.

7. The use according to claim 5, wherein in keratinocytes, the peptide: (i) Inhibit the expression of the protease-activated receptor 2 (PAR2) gene, or (ii) Upregulate the amount of Beclin-1 or LC3-I / II protein, or downregulate the amount of p62 protein.

8. The use as claimed in claim 5, wherein the hyperpigmentation disorder is a disorder that occurs when the amount of melanin in the skin is excessively increased.

9. The use according to claim 5, wherein the hyperpigmentation disorder is melasma, freckles, age spots, sunspots, or hyperpigmentation of post-inflammatory skin.

10. A cosmetic composition for skin whitening, comprising the peptide of claim 1 as an active ingredient.

11. The cosmetic composition of claim 10, wherein the cosmetic composition is a skin preparation for external use.

12. The cosmetic composition of claim 10, wherein the cosmetic composition is at least one formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, powders, surfactant-containing detergents, oils, and sprays.

13. The cosmetic composition of claim 12, wherein the surfactant-containing cleanser is a lotion or soap.

14. The cosmetic composition of claim 11, wherein the cosmetic composition is a formulation selected from the group consisting of powder foundation, lotion foundation, and wax foundation.

15. Use of the peptide of claim 1 in the preparation of a cosmetic composition for skin whitening.

16. The use according to claim 15, wherein in keratinocytes, the peptide: (i) Inhibit the incorporation of melanosomes; or (ii) Promotes the degradation of melanosomes.

17. The use according to claim 15, wherein in keratinocytes, the peptide: (i) Inhibit the expression of the protease-activated receptor 2 (PAR2) gene, or (ii) Upregulate the amount of Beclin-1 or LC3-I / II protein, or downregulate the amount of p62 protein.

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