Peptides with skin-whitening activity and their uses

Abstract

The peptides of the present invention exhibit skin-whitening activity by inhibiting the absorption of melanosomes in keratinocytes. Therefore, the peptides of the present invention can be used as active ingredients in medicaments for treating or preventing hyperpigmentation caused by excessive melanosome deposition, or as active ingredients in cosmetics for skin whitening.

Description

Cross-references to related applications

[0001] This application claims priority based on Korean Patent Application No. 10-2023-0138104, filed on October 16, 2023, the entire contents of which are incorporated herein by reference as a part of this specification. Technical Field

[0002] This invention relates to peptides with skin-whitening activity and their uses. Background Technology

[0003] Cutaneous melanin, found in the skin, hair follicles, and eyes, plays a crucial role in protecting the skin from UV damage. However, excessive melanin production can lead to hyperpigmentation disorders such as melasma, freckles, and age spots, causing psychological stress and a decreased quality of life. Melanin is primarily synthesized in melanocytes located between or beneath the basal layer of the epidermis and in hair follicles. Melanin is synthesized in melanosomes, organelles within melanocytes. These melanosomes migrate via dendrites to nearby keratinocytes, and as the keratinocytes rise to the outer layer, they contribute to skin color. The exact mechanism by which melanosomes migrate from melanocytes to keratinocytes is not fully understood, but it is known that melanosomes migrate to the dendrites of melanocytes and are then released into the extracellular space, followed by membrane fusion and phagocytosis between the melanosomes and keratinocytes. Receptors such as PAR-2 ​​(protease-activated receptor 2), KGFR (keratinocyte growth factor receptor), and TLR (Toll-like receptors) are known to be involved in the regulation of phagocytosis of melanosomes by keratinocytes.

[0004] Melanin is synthesized from L-tyrosine through the processes of DOPA, DOPAquinone, DOPAchrome, and DHI (5,6-dihydroxyindole). Traditionally, inhibition of melanin synthesis has focused on factors that suppress tyrosinase activity, the rate-controlling enzyme in the melanin synthesis pathway.

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

[0006] Previous development of skin whitening agents has focused on inhibiting the activity of tyrosinase (a key enzyme in melanin synthesis). However, in order to achieve synergistic skin whitening effects through different sites of action, it is necessary to develop skin whitening agents with different mechanisms and sites of action.

[0007] [Existing Technical Documents] [Patent Literature] WO2020 / 153819 Korean Patent No. 10-1869783 Summary of the Invention Technical issues The inventors conducted research to develop peptides with improved skin-whitening activity. As a result, the inventors experimentally demonstrated that their novel peptides exhibit excellent inhibitory activity against the uptake of melanosomes into keratinocytes, and confirmed that the peptides of this invention can be used as active ingredients in skin-whitening agents, thus completing this invention.

[0008] Therefore, one object of the present invention is to provide novel peptides with skin whitening activity.

[0009] Another object of the present invention is to provide a composition for skin whitening, comprising peptides having the above-described activity as active ingredients.

[0010] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising a peptide having the above-described activity as an active ingredient.

[0011] 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.

[0012] Technical solution In order to achieve the above objectives, One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0013] Another aspect of the present invention provides a composition for skin whitening, comprising the peptide as an active ingredient.

[0014] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising the peptide as an active ingredient.

[0015] Another aspect of the present invention provides a cosmetic composition for skin whitening, comprising the peptide as an active ingredient.

[0016] The present invention will now be described in detail.

[0017] 1. Peptides and their activities According to one aspect of the invention, a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 is provided.

[0018] [Amino acid sequence of SEQ ID NO:1] KLKKDRMQRS(Lys-Leu-Lys-Lys-Asp-Arg-Met-Gln-Arg-Ser) As used in this article, the term "peptide" refers to a linear molecule formed by amino acid residues linked together by peptide bonds.

[0019] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention can be used without modification, but variants or fragments of amino acids having different sequences by means of deletion, insertion, substitution or combination thereof may also be used without affecting the original activity of the peptide (e.g., skin whitening activity).

[0020] The peptides of the present invention can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc., without changing their activity.

[0021] The peptides of the present invention comprise: peptides comprising an amino acid sequence substantially identical to that of the peptide comprising the amino acid sequence of SEQ ID NO: 1, and variants thereof or active fragments thereof. A substantially identical amino acid sequence means an amino acid sequence having at least 75%, for example, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 1. Furthermore, the peptide may additionally comprise a targeting sequence, a tag, labeled residues, or an amino acid sequence prepared for a specific purpose of increasing half-life or peptide stability.

[0022] The peptides of the present invention may be modified at the N-terminus and / or C-terminus to select a portion of the amino acid sequence and enhance their activity. Such N-terminal and / or C-terminal modifications can significantly enhance the stability of the peptides of the present invention and, for example, increase the half-life of the peptides when administered in vivo. The term "stability" above is intended to cover not only in vivo stability (which protects the peptides of the present invention from attack by in vivo protein-cleaving enzymes) but also storage stability (e.g., room temperature storage stability).

[0023] N-terminal modification can be a modification in which a protecting group selected from the group consisting of acetyl, fluoreonylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, and polyethylene glycol (PEG) is attached to the N-terminus of the peptide. The aforementioned C-terminal modification can be, but is not limited to, a hydroxyl (-OH), amino (-NH2), or azide (-NHNH2) group attached to the C-terminus of the peptide.

[0024] 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. ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis (US Patent No. 5,516,891).

[0025] The peptides of this invention have skin whitening activity.

[0026] In one embodiment, the peptide of the present invention has the activity of inhibiting the absorption (uptake) of melanosomes in keratinocytes.

[0027] Melanosomes produced in melanocytes migrate from melanocytes to keratinocytes. This migration of melanosomes to keratinocytes involves the release of melanosomes from melanocytes and their incorporation into keratinocytes. Alternatively, this incorporation of melanosomes can be represented as the absorption (uptake) of melanosomes by keratinocytes or the phagocytosis of melanosomes by keratinocytes.

[0028] PAR2 (protease-activated receptor 2), TLR3 (Toll-like receptor 3), and KGFR (keratinocyte growth factor receptor) are involved in the uptake of melanosomes released from melanocytes via phagocytosis by keratinocytes.

[0029] In one embodiment, the peptide of the present invention has the activity of inhibiting the expression of the TLR3 (Toll-like receptor 3) gene or the KGFR (keratinocyte growth factor receptor) gene in keratinocytes.

[0030] TLR3 protein is known to be a receptor protein that recognizes pathogens and plays a crucial role in activating innate immunity. Stimulation of TLR3 protein in keratinocytes is known to induce enhanced melanosome uptake (J Dermatol Sci. 2019 Dec;96(3):168-177), and TLR3 protein in melanocytes is also known to promote the migration of melanosomes to keratinocytes (Int. J. Mol. Sci. 2020, 21, 9769).

[0031] KGFR is a tyrosine kinase receptor expressed in many types of epithelial cells and activated by four known ligands, FGF-1, FGF-3, FGF-7, and FGF-10. It is known to participate in epithelial cell proliferation, differentiation, and wound healing. In addition, KGFR is known to promote the migration of melanosomes to keratinocytes (J Invest Dermatol. 2005 Dec;125(6):1190-9., Journal of Investigative Dermatology Vol 128, Issue 3, March2008, p. 558-567).

[0032] As described above, the peptides of the present invention can exhibit skin whitening activity and preventive or therapeutic effects on hyperpigmentation diseases through their inhibitory activity on melanosome absorption.

[0033] 2. Compositions for skin whitening and compositions for the prevention, treatment or improvement of hyperpigmentation disorders. In another aspect of the invention, a composition for skin whitening is provided, the composition comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0034] Through the above mechanism, the peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention has skin whitening activity.

[0035] In another aspect of the invention, the invention provides a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0036] In one embodiment, a pharmaceutical composition comprising a peptide as an active ingredient inhibits the absorption of melanosomes in keratinocytes.

[0037] In one embodiment, a pharmaceutical composition comprising a peptide as an active ingredient inhibits the expression of the TLR3 (Toll-like receptor 3) gene or the KGFR (keratinocyte growth factor receptor) gene in keratinocytes.

[0038] In this specification, the term "hyperpigmentation disorder" refers to a condition caused by an excessive increase in the amount of melanin in the skin. Therefore, the skin color darkens due to the excessive increase in melanin levels. Causes of hyperpigmentation disorders include, but are not limited to, sun exposure, skin inflammation such as acne, trauma, hormonal imbalances, and drug-induced hyperpigmentation (post-inflammatory hyperpigmentation).

[0039] In one implementation, hyperpigmentation disorders can be melasma, freckles, age-related hyperpigmentation, solar lentigo, or post-inflammatory hyperpigmentation of the skin.

[0040] The pharmaceutical compositions of the present invention may include a therapeutically effective amount of the peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 1.

[0041] The term "therapeutic effective amount" refers to an amount sufficient to enable the peptide (the active ingredient of the pharmaceutical composition of the present invention) to achieve its activity or efficacy, such as an amount sufficient to achieve the efficacy of treating or preventing hyperpigmentation disorders.

[0042] As used herein, the term “prevention” means reducing the risk of the development of a disease or disorder, and refers to any action that inhibits or delays the onset of a disease by preventing the progression of the disease or one or more of its clinical symptoms.

[0043] As used herein, the term “treatment” means to alleviate a disease or disorder and includes any action that improves or beneficially alters the symptoms of a disease by halting or reducing the progression of the disease or one or more of its clinical symptoms.

[0044] In this invention, prevention or treatment of hyperpigmentation can be achieved by eliminating the cause of hyperpigmentation in the skin or inhibiting the progression of hyperpigmentation, and specifically by inhibiting the incorporation of melanosomes into keratinocytes.

[0045] The pharmaceutical compositions of the present invention may include pharmaceutically acceptable carriers.

[0046] Pharmaceutically acceptable carriers are those commonly used in formulations, including but not limited to 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, and mineral oil.

[0047] In addition to the above-mentioned components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but is not limited thereto.

[0048] Suitable pharmaceutically acceptable carriers and formulations are described in detail in "Remington: The Science and Practice of Pharmacy" (19th ed., 1995, Williams & Wilkins).

[0049] The pharmaceutical compositions of the present invention can be administered via any suitable route 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 of the skin, they are preferably applied topically, such as by applying them to the skin.

[0050] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg per day, and may be prescribed in various ways depending on factors such as the method of preparation, method of administration, patient's age, weight, sex, pathological condition, food intake, time of administration, route of administration, excretion rate and sensitivity to response.

[0051] The pharmaceutical compositions of the present invention can be formulated into unit dosage forms using pharmaceutically acceptable carriers and / or excipients, or by means readily practicable by those skilled in the art, or by inserting them into multi-dose containers. In this case, the dosage form can be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granules, tablet, or capsule, and may additionally include dispersants or stabilizers.

[0052] The pharmaceutical compositions of the present invention can be topical skin preparations. Topical skin preparations are preparations that can be applied externally to the skin. When the pharmaceutical compositions of the present invention are used as topical skin preparations, they can be applied to the skin, specifically to areas of skin that have already developed hyperpigmentation. Topical skin preparations can be creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, medicated bandages, lotions, or combinations thereof. Topical skin preparations can be appropriately mixed as needed with ingredients commonly used in topical skin preparations (such as cosmetics or pharmaceuticals), such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. The aforementioned topical skin preparations may also contain, as appropriate, metal chelating agents (such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid); caffeine, tannins, verapamil, licorice extract, glycyrrhizin, etc. pawpaw ( Pseudocydonia sinensis Hot water extracts of the fruit; various herbs; tocopheryl acetate, glycyrrhizic acid, tranexamic acid and their derivatives or salts thereof; and vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid and sugars (such as glucose, fructose and trehalose).

[0053] In another aspect of the invention, the invention provides a cosmetic composition for skin whitening, comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0054] In one embodiment, a cosmetic composition including the peptide of the present invention as an active ingredient inhibits the absorption of melanosomes in keratinocytes.

[0055] In one embodiment, a cosmetic composition including the peptide of the present invention as an active ingredient inhibits the expression of the TLR3 (Toll-like receptor 3) gene or the KGFR (keratinocyte growth factor receptor) gene in keratinocytes.

[0056] Cosmetic compositions can be formulated into any dosage form commonly prepared in the technical field to which this invention pertains, and can be topical skin preparations. For example, cosmetic compositions can be formulated into solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, and sprays, but are not limited thereto.

[0057] Cosmetic compositions can be formulated in various forms, such as solutions, sol-gels, emulsions, oils, waxes, and aerosols, including but not limited to: flexible toners, nourishing toners, moisturizing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, face masks (packs), sprays, loose powders, hair tonics, hair creams, hair lotions, hair shampoo, hair rinses, hair conditioners, hair sprays, hair aerosols, hair waxes, and gels.

[0058] The cosmetic compositions of the present invention may include other additives, such as excipients and carriers, and can be applied and mixed with commonly used ingredients in general skin cosmetics as needed.

[0059] When the dosage form of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, astragalus gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0060] When the cosmetic composition is in the form of a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, a propellant such as chlorofluorocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto.

[0061] When the dosage form of a cosmetic composition is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, and examples of such components include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glyceryl fatty acid esters, polyethylene glycol, or fatty acid esters of sorbitol.

[0062] When the dosage form of a cosmetic composition is a suspension, liquid diluents (such as water, ethanol, or propylene glycol), suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester, and polyoxyethylene dehydrated sorbitol ester), microcrystalline cellulose, aluminum hydroxide, bentonite, agar, or astragalus gum can be used as carrier components.

[0063] When the dosage form of the above cosmetic composition is a surfactant-containing cleanser, fatty alcohol sulfates / esters, fatty alcohol ether sulfates / esters, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurate / esters, sarcosinates / esters, fatty acid amide ether sulfates / esters, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters can be used as carrier components.

[0064] When the cosmetic composition is in the form of a shampoo, it 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 (an anionic surfactant) and cocamidopropyl betaine (an amphoteric surfactant) can be used as surfactants, polyquaternium salts can be used as viscosity modifiers, glycerin can be used as a humectant, and citric acid or sodium hydroxide can be used as a pH adjuster. Grapefruit extract and similar ingredients can be used as preservatives; additionally, essential oils such as cedarwood, peppermint, and rosemary, as well as amino acids, pentaol, or vitamin E can be added.

[0065] In addition to the peptides of the present invention as active ingredients and carrier ingredients, the cosmetic composition may also include ingredients commonly used in cosmetic compositions, such as conventional adjuvants, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, but is not limited thereto.

[0066] 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. Specifically, the peptides of the present invention can be in concentrations of 0.01 μM to 1000 μM; 0.05 μM to 800 μM; 0.05 μM to 700 μM; 0.05 μM to 600 μM; 0.05 μM to 500 μM; 0.05 μM to 300 μM; 0.05 μM to 200 μM; 0.1 μM to 800 μM; 0.1 μM to 700 μM; 0. Concentrations from 1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1 μM to 200 μM; 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM are included, but not limited to.

[0067] 3. Uses of the peptides of the present invention 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 disorders.

[0068] In another aspect of the invention, a skin whitening method is provided, comprising administering to a subject requiring skin whitening a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising said peptide.

[0069] In another aspect of the invention, a method for preventing or treating hyperpigmentation disorders is provided, the method comprising administering to a subject requiring prevention or treatment of hyperpigmentation disorders a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising said peptide.

[0070] 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.

[0071] 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 manufacture of cosmetics for skin whitening.

[0072] Beneficial effects The peptides of this invention inhibit the absorption of melanosomes in keratinocytes. Therefore, the peptides of this invention can be used as active ingredients in medicines for treating or preventing hyperpigmentation caused by excessive melanosome deposition, or as effective ingredients in skin-whitening cosmetics.

[0073] 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

[0074] Figure 1 The results show the absorption rate of melanosomes isolated from HaCaT cells, melanosomes treated with the peptide of the present invention or the positive control ENMD-1068.

[0075] Figure 2 The results show the measurement of TLR3 gene mRNA levels after HaCaT cells were treated with poly(I:C) (a synthetic TLR3 agonist) and then with the peptide of the present invention. Band values ​​were calculated by comparing the densitometry results of the TLR3 gene band with the densitometry results of the GAPDH band (loading control), and then expressing the relative expression level of the control as 1.

[0076] Figure 3The results of measuring KFGR mRNA levels after treating HaCaT cells with the peptides of the present invention are shown. Band values ​​were calculated by comparing the optical density of the KFGR gene band with that of the GAPDH band (loading control), and then expressing the relative expression level of the control as 1.

[0077] Figure 4a This is a confocal micrograph showing the fluorescence of the microspheres introduced into the cells after HaCaT cells were treated with microspheres and then treated with poly(I:C) and the peptide of the present invention.

[0078] Figure 4b This is a graph showing the fluorescence values ​​of the microspheres introduced into the cells in each control and experimental group. Detailed Implementation

[0079] The present invention will now be described in detail through embodiments. However, the following embodiments are for illustrative purposes only, and the scope of the present invention is not limited to these embodiments.

[0080] Preparation Example 1: Preparation of Peptides and Peptide Complexes Peptides with the amino acid sequence SEQ ID NO: 1 shown in Table 1 were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were purified using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The chromatographic column used was an ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA).

[0081] [Table 1]

[0082] The efficacy of the peptide prepared in SEQ ID NO: 1 was evaluated through the following experiments.

[0083] Experimental Example 1: Analysis of Absorption via Melanosome Phagocytosis To confirm whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 inhibits the absorption of melanosomes by phagocytosis in keratinocytes.

[0084] HaCaT cells (human keratinocyte cell line) were used at a rate of 3 × 10⁻⁶. 5Cells were seeded at a density of 10% FBS in 6-well cell culture plates and cultured for 24 hours in DMEM (Dubai modified Eagle medium). Subsequently, the cells were replaced with serum-free DMEM medium, and the peptide from Preparation Example 1 was added to the serum-free DMEM medium to prepare peptide solutions at concentrations of 50 μM, 100 μM, and 200 μM. These peptide solutions were then added to the cultured cells. The negative control group was the group not treated with the peptide, and the positive control group was treated with 500 μM ENMD-1068 (6-amino-1-[4-(3-methyl-1-oxobutyl)-1-piperazinyl]-1-hexanone hydrochloride, Sigma-Aldrich). The cells were then cultured in a CO2 incubator at 37°C for 24 hours, and then 10 μg / ml of melanosomes isolated from B16F10 cells (mouse melanoma cell line) were added to the cultured cells, except for the control group. Subsequently, the cells were cultured in a CO2 incubator at 37°C for 48 hours, then washed three times with PBS to harvest HaCaT cells. The HaCaT cell pellet was collected using a centrifuge, dissolved in 1M NaOH, and aliquoted into 96-well plates. To measure melanin content, absorbance at 490 nm was measured using a spectrophotometer.

[0085] As a result of the experiment, from Figure 1 The analysis of melanin content in the samples confirmed that the peptide in Preparation Example 1 inhibited the absorption (uptake) of melanosomes in HaCaT cells.

[0086] Example 2: Expression analysis of TLR3 (a gene involved in melanosome phagocytosis) The effect of the peptide prepared in Preparation Example 1 on the expression of the TLR3 (Toll-like receptor 3) gene was evaluated. TLR3 is a gene involved in melanosome phagocytosis.

[0087] HaCaT cells were used at a rate of 3 × 10 5Cells were seeded at a density of 10 μM / well in 6-well cell culture plates, replaced with DMEM medium containing 2% FBS, and cultured in a CO2 incubator at 37°C for 24 hours. The peptides from Preparation Example 1 were added to DMEM medium containing 2% FBS to prepare peptide solutions at concentrations of 10 μM, 50 μM, 100 μM, and 200 μM. These peptide solutions were then added to the cells and treated for 30 minutes. Subsequently, 30 μg / mL of poly(I:C) (polyinosine-polycytidylic acid, a TLR3 agonist) was added, and the cells were cultured in a CO2 incubator at 37°C for 24 hours. Cells were washed twice with PBS, and RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). The amount of isolated RNA was quantified, and 2000 ng of RNA was aliquoted into each tube, and cDNA was synthesized using a cDNA synthesis kit (enzynomics, Cat. No.: RT200, Korea). PCR was performed using primers targeting the TLR3 gene and a PCR kit (enzynomics, Cat. No.: P581T, Korea) as shown in Table 2. 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.

[0088] As a result of the experiment, such as Figure 2 As shown, the expression level of the TLR3 gene was confirmed to increase when HaCaT cells were treated with poly(I:C) (which is a TLR3 agonist), and the increased expression level of the TLR3 gene was inhibited by treatment with the peptide of Preparation Example 1.

[0089] Example 3: Expression analysis of KGFR (a gene involved in melanosome phagocytosis) The effect of the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 on the expression of the KGFR (keratinocyte growth factor receptor) gene, which is related to melanosome phagocytosis, was evaluated.

[0090] HaCaT cells were used at a rate of 3 × 10 5Cells were seeded at a density in 6-well cell culture plates and cultured for 24 hours in DMEM medium containing 10% FBS. Cells were then replaced with serum-free DMEM medium and cultured in a CO2 incubator at 37°C for 24 hours. Peptide solutions of 10 μM, 50 μM, 100 μM, and 200 μM were prepared by adding the peptide from Preparation Example 1 to serum-free DMEM medium and dispensed onto cells, which were then further cultured in a CO2 incubator at 37°C for 6 hours. Cells were washed twice with PBS and RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). The amount of isolated RNA was quantified, and cDNA was synthesized using an RNA and cDNA synthesis kit (enzynomics, Cat. No.: RT200, Korea). PCR was performed using primers and a PCR kit targeting the KGFR gene as shown in Table 2 (enzynomics, Cat. No.: 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.

[0091] As a result of the experiment, such as Figure 3 As shown, this confirms that in HaCaT cells, treatment with the peptide prepared in Example 1 reduced the expression level of the KGFR gene in a concentration-dependent manner.

[0092] Experimental Example 4: Analysis of Poly(I:C)-induced Phagocytosis—Analysis of Microsphere Absorption The effect of the peptide prepared in Preparation Example 1 on poly(I:C)-induced phagocytosis of melanocytes was evaluated by microsphere uptake (microsphere uptake) experiments.

[0093] HaCaT cells were used at a rate of 3 × 10 4 Cells were seeded at a density of 10% FBS in 6-well cell culture plates and cultured in a CO2 incubator at 37°C for 24 hours. The medium was then replaced with serum-free DMEM, and the peptides from Preparation Example 1 were added to serum-free DMEM to prepare peptide solutions at concentrations of 50 μM, 100 μM, and 200 μM. Cellular peptides were pretreated with these solutions for 1 hour. Cells were then treated with 30 μg / mL of poly(I:C) (polyinosine-polycytidylic acid) and microspheres (FluoSpheres). TMCells were treated with poly(I:C) microspheres (1 μm in diameter) and then cultured in a CO2 incubator at 37°C for 24 hours. A negative control was treated with poly(I:C) microspheres without peptide treatment. Cells were fixed by treatment with 4% formaldehyde for 30 minutes. Cells were treated with iFluor 488 reagent (Abcam, UK) and allowed to react for 1 hour. Cells were then observed using a DAPI-containing mounting medium (Invitrogen, USA) and observed using a confocal microscope (400× magnification). The fluorescence values ​​of the microspheres internalized into the cells were analyzed using ImageJ.

[0094] As a result of the experiment, such as Figure 4a and Figure 4b As shown, it was confirmed that the absorption of microspheres was increased by poly(I:C) treatment, while the increased absorption of microspheres was reduced by treatment with the peptide of Preparation Example 1.

[0095] List of PCR primer sequences (Table 2) [Table 2]

[0096] Although representative embodiments of this application have been described above by way of examples, the scope of this application is not limited to the specific embodiments described above, and those skilled in the art can make appropriate changes within the scope described in the claims of this application.

Claims

1. A peptide comprising 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 disorders, comprising the peptide of claim 1 as an active ingredient.

4. The pharmaceutical composition for preventing or treating hyperpigmentation disorders according to claim 3, wherein, The peptide inhibits the absorption of melanosomes in keratinocytes.

5. The pharmaceutical composition for preventing or treating hyperpigmentation disorders according to claim 4, wherein, The peptide inhibits the expression of (i) TLR3 (Toll-like receptor 3) gene or (ii) KGFR (keratinocyte growth factor receptor) gene in keratinocytes.

6. The pharmaceutical composition for preventing or treating hyperpigmentation disorders according to claim 3, wherein, Hyperpigmentation is a condition that occurs when there is an excessive increase in the amount of melanin in the skin.

7. The pharmaceutical composition for preventing or treating hyperpigmentation disorders according to claim 3, wherein, The hyperpigmentation disorders mentioned are melasma, freckles, age-related hyperpigmentation, solar lentigines, or post-inflammatory hyperpigmentation.

8. The pharmaceutical composition for preventing or treating hyperpigmentation disorders according to claim 3, wherein, The pharmaceutical composition is a topical skin preparation.

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

10. The cosmetic composition for skin whitening according to claim 9, wherein, The peptide inhibits the absorption of melanosomes in keratinocytes.

11. The cosmetic composition for skin whitening according to claim 10, wherein, The peptide inhibits the expression of (i) TLR3 (Toll-like receptor 3) gene or (ii) KGFR (keratinocyte growth factor receptor) gene in keratinocytes.

12. The cosmetic composition for skin whitening according to claim 9, wherein, The cosmetic composition is a topical skin preparation.

13. The cosmetic composition for skin whitening according to claim 12, wherein, The cosmetic composition is at least one dosage form selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing detergents, oils, powder foundations, emulsion foundations, wax foundations, and sprays.

14. A method for preventing or treating hyperpigmentation disorders, comprising administering to a subject requiring prevention or treatment of hyperpigmentation disorders the peptide of claim 1 or a composition comprising said peptide.

15. The method for preventing or treating hyperpigmentation disorders according to claim 14, wherein, The hyperpigmentation disorders mentioned are melasma, freckles, age-related hyperpigmentation, solar lentigines, or post-inflammatory hyperpigmentation.

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