Peptide having activity of skin whitening and uses thereof

Abstract

The peptide of the present invention has skin-whitening activity by inhibiting the uptake of melanosomes in keratinocytes and promoting the degradation of melanosomes. The peptide of the present invention may be used as an active ingredient in drugs for the treatment or prevention of hyperpigmentation diseases caused by the excessive deposition of melanosomes, or as an active ingredient in skin-whitening cosmetics.

Description

Peptides having skin whitening activity and their uses

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2025-0032913 filed on March 13, 2025, and all contents disclosed in said Korean Patent Application are incorporated herein as part of this specification.

[0003] [Technology Field]

[0004] The present invention relates to a peptide having skin whitening activity and the use thereof.

[0005]

[0006] Cutaneous melanin is present in the skin, hair follicles, and eyes, performing the important function of protecting the skin from ultraviolet rays. However, the excessive formation of melanin causes hyperpigmentation disorders such as melasma, freckles, and age spots, leading to 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. Synthesis takes place in melanosomes, which are organelles within melanocytes. These melanosomes then move through dendrites to adjacent keratinocytes, and as the keratinocytes rise to the surface of the skin, skin color is produced. Although the mechanism by which melanosomes move from melanocytes to keratinocytes has not been precisely elucidated, it is known that melanosomes move to the dendrites of melanocytes, are released extracellularly from them, and subsequently, membrane fusion and phagocytosis between the melanosomes and keratinocytes are involved. It is known that receptors such as PAR-2 ​​(protease-activated receptor 2), KGFR (keratinocyte growth factor receptor), and TLRs (Toll-like receptors) are involved in the regulation of phagocytosis by melanosomes by keratinocytes.

[0007] The synthesis of melanin is ultimately achieved through the progression from L-tyrosine to DOPA, DOPAquinone, DOPAchrome, and DHI (5,6-dihydroxyindole); traditionally, the inhibition 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.

[0008] PCT International Patent Publication WO2020 / 153819 discloses a polypeptide having activity that inhibits the activity of tyrosinase, a key enzyme in the melanin synthesis pathway, and the use of the same for skin whitening. Additionally, Korean Registered Patent No. 10-1869783 describes a peptide having activity that inhibits melanin production and the activity of tyrosinase, and the use of the same for skin whitening.

[0009] The development of skin whitening agents to date has focused on inhibiting the activity of tyrosinase, a key enzyme in melanin synthesis. However, in order to achieve synergistic effects through various points of action, there is a need to develop skin whitening agents with different mechanisms of action or sites of action.

[0010] [Prior Art Literature]

[0011] [Patent Literature]

[0012] WO 2020 / 153819

[0013] Republic of Korea Registered Patent No. 10-1869783

[0014]

[0015] As a result of research efforts to develop a peptide with improved activity having skin whitening activity, the inventors experimentally proved that the novel peptide developed by the inventors has excellent inhibitory activity on the uptake of melanosomes into keratinocytes and excellent activity promoting the degradation of melanosomes in keratinocytes, and confirmed that the peptide of the present invention can be used as an active ingredient in a skin whitening agent, thereby completing the present invention.

[0016] Therefore, the objective of the present invention is to provide a novel peptide having skin whitening activity.

[0017] Another objective of the present invention is to provide a skin whitening composition comprising a peptide having the activity described above as an active ingredient.

[0018] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders comprising a peptide having the activity described above as an active ingredient.

[0019] Another objective of the present invention is to provide a cosmetic composition for skin whitening comprising a peptide having the activity described above as an active ingredient.

[0020]

[0021] In order to achieve the above purpose,

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

[0023] Another aspect of the present invention provides a skin whitening composition comprising the peptide as an active ingredient.

[0024] 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.

[0025] Another aspect of the present invention provides a cosmetic composition for skin whitening comprising the peptide as an active ingredient.

[0026]

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

[0028]

[0029] 1. Peptides and their activity

[0030] According to one aspect of the present invention, a peptide comprising the amino acid sequence disclosed in SEQ ID NO. 1 is provided.

[0031] [Amino acid sequence of Sequence No. 1]

[0032] KLGTWTYDGSV (Lys-Leu-Gly-Thr-Trp-Thr-Tyr-Asp-Gly-Ser-Val)

[0033] The term "peptide" in this specification refers to a linear molecule formed by amino acid residues being joined together by peptide bonds.

[0034] The peptide containing the amino acid sequence of SEQ ID NO. 1 of the present invention may be used without modification, but a variant or fragment of an amino acid having a different sequence may be used by deletion, insertion, substitution, or a combination thereof of amino acid residues within a range that does not affect the original activity of the peptide, such as skin whitening activity.

[0035] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., within a range that does not change its activity.

[0036] The peptide of the present invention comprises a peptide having a substantially identical amino acid sequence to the peptide having the amino acid sequence of SEQ ID NO. 1, a variant thereof, or an active fragment thereof. The substantially identical amino acid sequence means an amino acid sequence having at least 75%, e.g., 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. Additionally, the peptide may further include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

[0037] The peptide of the present invention may have N-terminal and / or C-terminal modifications induced to select a portion of the amino acid sequence and increase its activity. Through such N-terminal and / or C-terminal modifications, the stability of the peptide of the present invention can be significantly improved, for example, by increasing the half-life of the peptide upon in vivo administration. The term "stability" includes not only stability in vivo, which protects the peptide of the present invention from attack by protein-cleaving enzymes in vivo, but also storage stability (e.g., room temperature storage stability).

[0038] The above N-terminal modification may be 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) attached to the N-terminus of the peptide. The above C-terminal modification may be a protecting group such as a hydroxyl group (-OH), an amino group (-NH2), or a hydrazino group (-NHNH2) attached to the C-terminus of the peptide, but is not limited thereto.

[0039] The peptide of the present invention can be prepared by various methods widely known in the art to which the present invention belongs. For example, the peptide of the present invention can be prepared according to chemical synthesis methods known in the art, in particular 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).

[0040] The peptide of the present invention has skin whitening activity.

[0041] In one embodiment, the peptide of the present invention has activity that inhibits the uptake of melanosomes in keratinocytes.

[0042] Melanosomes produced in melanocytes migrate from melanocytes to keratinocytes, and this migration of melanosomes to keratinocytes includes the process of releasing melanosomes from melanocytes and the process of invagination of melanosomes into keratinocytes. The invagination process of melanosomes may also be described as uptake by keratinocytes or phagocytosis by keratinocytes.

[0043] PAR2 (protease activated receptor 2), KGFR (keratinocyte growth factor receptor), and TLR3 (Toll-like receptor 3) are involved in the process of absorption of melanosomes released from melanocytes by phagocytosis of keratinocytes.

[0044] In one embodiment, the peptide of the present invention has activity that inhibits the expression of the PAR2 (protease activated receptor 2) gene, KGFR (keratinocyte growth factor receptor) gene, or TLR3 (Toll-like receptor 3) gene in keratinocytes.

[0045] The aforementioned PAR2 is a receptor protein present in the cell membrane that 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 process of keratinocytes absorbing (invaginating) melanosomes; it is known that inhibiting the activity of the PAR2 protein inhibits 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.).

[0046] The above KGFR is a tyrosine kinase receptor that is expressed in many types of epithelial cells and is activated by four known ligands, FGF-1, FGF-3, FGF-7, and FGF-10, and is known to be involved in the proliferation, differentiation, and wound healing of epithelial cells. In addition, KGFR is also 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, March 2008, p. 558-567).

[0047] The above TLR3 is a receptor protein present on the cell membrane and is a type of innate immune system; it is known to be expressed in skin epithelial cells, dendritic cells, etc. TLR3 protein activates the immune response, and it is known that stimulating TLR3 increases the uptake of melanosomes into keratinocytes (Journal of Dermatological Science Vol 96, Issue 3, Dec 2019, p. 168-177., Pigment Cell & Melanoma Research Vol 31, Issue 5, Sep 2018, p. 570-584).

[0048] In one embodiment, the peptide of the present invention has activity that promotes the degradation of melanosomes.

[0049] Melanosomes absorbed (invaginated) into keratinocytes can be broken down by autophagy.

[0050] Autophagolysosomes, formed by the fusion of autophagosomes and lysosomes in keratinocytes, can degrade melanosomes absorbed into keratinocytes.

[0051] The above autophagy activation can be confirmed by an increase in the expression of Beclin-1 protein, which is involved in the formation of autophagosomes, and the ratio of LC3-II to LC3-I (LC3-II / LC3-I).

[0052] In addition, p62 is known to be degraded within the lysosome during autophagy, so a decrease in p62 may indicate that autophagy is activated (Methods Enzymol. 2009:452:181-97. Monitoring autophagic degradation of p62 / SQSTM1).

[0053] As described above, the peptide of the present invention can exhibit skin whitening activity and preventive or therapeutic efficacy against hyperpigmentation disorders through the activity of inhibiting absorption and promoting degradation of melanosomes.

[0054]

[0055] 2. Composition for skin whitening, composition for the prevention, treatment, or improvement of hyperpigmentation disorders

[0056] In another aspect of the present invention, a skin whitening composition is provided that comprises a peptide having the amino acid sequence of SEQ ID NO. 1 as an active ingredient.

[0057] The peptide containing the amino acid sequence of SEQ ID NO. 1 of the present invention has skin whitening activity through a mechanism as described above.

[0058] In another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of hyperpigmentation disease comprising a peptide having the amino acid sequence of SEQ ID NO. 1 as an active ingredient.

[0059] In one embodiment, a pharmaceutical composition containing the peptide as an active ingredient inhibits the absorption of melanosomes in keratinocytes.

[0060] In one embodiment, a pharmaceutical composition comprising the peptide as an active ingredient inhibits the expression of the PAR2 (protease activated receptor 2) gene in keratinocytes, inhibits the expression of the KGFR (keratinocyte growth factor receptor) gene, or inhibits the expression of the TLR3 (Toll-like receptor 3) gene.

[0061] In this specification, the term "hyperpigmentation disorder" refers to a disorder caused by an excessive increase in the amount of melanin in the skin. As such, the skin color becomes darker due to an excessive increase in the amount of melanin in the skin. The causes of hyperpigmentation disorder may be, for example, exposure to sunlight, skin inflammation such as acne (post-inflammatory pigmentation), trauma, hormonal imbalance, or the use of drugs, but are not limited thereto.

[0062] In one embodiment, the hyperpigmentation disorder may be melasma, freckles, senile pigmentation spots, solar lentigo, or hyperpigmentation occurring after inflammation of the skin.

[0063] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of a peptide comprising the amino acid of SEQ ID NO. 1 of the present invention.

[0064] The above term "therapeutically effective amount" means an amount sufficient for the peptide, which is an active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, for example, an amount sufficient to achieve the efficacy of treating or preventing hyperpigmentation disorders.

[0065] In this specification, the term "prevention" means reducing the risk of contracting a disease or disorder, and refers to any act of suppressing or delaying the onset of a disease by preventing the progression of the disease or one or more clinical signs thereof.

[0066] In this specification, the term "treatment" means alleviating a disease or disability and includes all acts that improve or beneficially alter the symptoms of a disease by preventing or reducing the progression of the disease or one or more of its clinical signs.

[0067] In the present invention, the prevention or treatment of hyperpigmentation disease may involve removing the cause of hyperpigmentation in the skin or inhibiting the progression to hyperpigmentation, and specifically, may involve inhibiting the invagination of melanosomes in keratinocytes.

[0068] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0069] The above pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0070] The pharmaceutical composition of the present invention may be a composition comprising a therapeutically effective amount of a peptide containing the amino acid of SEQ ID NO. 1; and a pharmaceutically acceptable carrier.

[0071] The pharmaceutical composition of the present invention may additionally include, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., but is not limited thereto.

[0072] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, (19th ed., 1995, Williams & Wilkins).

[0073] The pharmaceutical composition of the present invention may be administered by any suitable route for treating hyperpigmentation disorders, for example, orally or parenterally. In the case of parenteral administration, it may be administered via intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, topical administration, transdermal administration, etc. Since the pharmaceutical composition of the present invention has preventive or therapeutic activity for hyperpigmentation disorders of the skin, it is preferable to apply it via topical administration, such as by application to the skin.

[0074] The dosage of the above pharmaceutical composition may be 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg per day, but is not limited thereto, and may be prescribed in various ways depending on factors such as the method of formulation, mode of administration, age, body weight, sex, pathological condition of the patient, food, time of administration, route of administration, excretion rate and response responsiveness.

[0075] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation 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, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0076] The pharmaceutical composition of the present invention may be a topical skin preparation. The topical skin preparation is a formulation in a form that can be applied to the outside of the skin. 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. The topical skin preparation may be appropriately formulated as needed with ingredients commonly used in topical skin preparations such as cosmetics or pharmaceuticals, for example, 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 above topical skin preparation may also appropriately incorporate metal chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, licorice extract, glabridin, various herbal medicines, preparations such as tocopherol acetate, glycyrrhizin, tranexamic acid and its derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0077] In another aspect of the present invention, the present invention provides a cosmetic composition for skin whitening comprising a peptide having the amino acid sequence of SEQ ID NO. 1 as an active ingredient.

[0078] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient inhibits the absorption of melanosomes in keratinocytes.

[0079] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient inhibits the expression of the PAR2 (protease activated receptor 2) gene in keratinocytes, inhibits the expression of the KGFR (keratinocyte growth factor receptor) gene, or inhibits the expression of the TLR3 (Toll-like receptor 3) gene.

[0080] The above cosmetic composition may be prepared in any formulation conventionally manufactured in the technical field to which the present invention belongs, and may be an external application for the skin. For example, it may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto.

[0081] The above cosmetic composition may be manufactured in various forms such as softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, pomade, gel, etc., but is not limited thereto.

[0082] The cosmetic composition of the present invention may include excipients, carriers, and other additives, and it is possible to incorporate ordinary ingredients used in general skin cosmetics in the necessary amounts.

[0083] In the case where the formulation of the above cosmetic composition is in the form of a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0084] In the case where the formulation of the above cosmetic composition is in the form of 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, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto.

[0085] In the case where the formulation of the above cosmetic composition is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a 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.

[0086] In the case where the formulation of the above 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 sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used as carrier components.

[0087] In the case where the formulation of the above cosmetic composition is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0088] In the case where the formulation of the above cosmetic composition is a hair shampoo, base components for composing the shampoo, such as a thickener, a surfactant, a viscosity modifier, a moisturizer, a pH adjuster, a preservative, and an essential oil, may be mixed with the peptide of the present invention. CDE may be used as the thickener, LES which is an anionic surfactant and coco betaine which is an amphoteric surfactant may be used as the surfactant, polyquaternium may be used as the viscosity modifier, glycerin may be used as the moisturizer, and citric acid or sodium hydroxide may be used as the pH adjuster. Grapefruit extract, etc., may be used as the preservative, and in addition, essential oils such as cedarwood, peppermint, and rosemary, silk amino acids, pentaol, or vitamin E may be added.

[0089] The ingredients included in the above cosmetic composition may further include, in addition to the peptide and carrier components of the present invention as active ingredients, ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, but are not limited thereto.

[0090] The peptide of the present invention may be included in the above-described composition, pharmaceutical composition, or cosmetic composition at a concentration of 0.01 μM to 1000 μM, specifically, the peptide of the present invention is 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; It may be included at a concentration of 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, but is not limited thereto.

[0091]

[0092] 3. Uses of the peptide of the present invention

[0093] In another aspect of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO. 1 is provided for use in skin whitening, or for the prevention, treatment, or improvement of hyperpigmentation disorders.

[0094] In another aspect of the present invention, a skin whitening method is provided comprising the step of administering a peptide having the amino acid sequence of SEQ ID NO. 1 or a composition having said peptide to a subject requiring skin whitening.

[0095] In another aspect of the present invention, a method for preventing, treating, or improving a hyperpigmentation disease is provided, comprising the step of administering a peptide containing the amino acid sequence of SEQ ID NO. 1 or a composition containing said peptide to a subject who requires prevention, treatment, or improvement of a hyperpigmentation disease.

[0096] The above-mentioned object may be a human or an animal other than a human.

[0097] In another aspect of the present invention, a use of a peptide comprising the amino acid sequence of SEQ ID NO. 1 is provided for the manufacture of a medicament for the prevention or treatment of hyperpigmentation disorders.

[0098] In another aspect of the present invention, a use of a peptide comprising the amino acid sequence of SEQ ID NO. 1 is provided for the manufacture of cosmetics for skin whitening.

[0099] In the above-mentioned methods for preventing, treating, or improving hyperpigmentation disorders, uses for preventing, treating, or improving hyperpigmentation disorders, uses for manufacturing compositions for preventing, treating, or improving hyperpigmentation disorders, or uses for manufacturing cosmetics, other technical details including peptides and compositions are identical to those described in the skin whitening composition, pharmaceutical composition for treating or preventing hyperpigmentation disorders, or cosmetic composition, which are aspects of the present invention; therefore, they are referred to by reference and are not described redundantly.

[0100]

[0101] The peptide of the present invention inhibits the absorption of melanosomes in keratinocytes and promotes their degradation. Therefore, the peptide of the present invention can be used as an active ingredient in a drug for the treatment or prevention of hyperpigmentation caused by excessive deposition of melanosomes, or as an active ingredient in a cosmetic for skin whitening.

[0102] However, the effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the following description.

[0103]

[0104] Figure 1 shows the results of measuring the absorption rate of melanosomes isolated after treating HaCaT cells with the peptide of the present invention or the positive control ENMD-1068.

[0105] Figure 2 shows the results of measuring the mRNA level of PAR2 after treating HaCaT cells with the peptide or protease inhibitor of the present invention and then treating them with trypsin. The band values ​​represent the relative expression level with the control set to 1, calculated by comparing the band density measurement result of the PAR2 gene with the densitometry result of the GAPDH band, which is the loading control.

[0106] Figure 3 shows the results of measuring the mRNA level of KFGR after treating HaCaT cells with the peptide of the present invention and rhKGF protein. The band values ​​represent the relative expression level with the control set to 1, calculated by comparing the band density measurement result of the KFGR gene with the densitometry result of the GAPDH band, which is the loading control.

[0107] Figure 4 shows the results of measuring the mRNA level of TLR3 after treating HaCaT cells with the peptide of the present invention and Poly(I:C). The band values ​​represent the relative expression level with the control set to 1, calculated by comparing the band density measurement result of the TLR3 gene with the densitometry result of the GAPDH band, which is the loading control.

[0108] Figure 5 shows the results of measuring the amount of residual melanosomes after treating HaCaT cells with isolated melanosomes and treating them with the peptide of the present invention, resveratrol, or rapamycin. The values ​​in the graph represent the relative amount of melanosomes with the control set to 100, measured by measuring the absorbance at a wavelength of 490 nm.

[0109] Figure 6 shows the results of treating HaCaT cells with the peptide of the present invention, resveratrol, or rapamycin, and measuring the degree of autophagy activation for each. The values ​​for Beclin-1 and p62 represent the relative expression levels with the control set to 1, calculated by determining the actin band density measurement results of the loading control. The values ​​for LC3-I and LC3-II represent the relative results with the control set to 1, calculated by determining LC3-II / LC3-I using the LC3-I and LC3-II band density measurement results of each treatment group.

[0110]

[0111] The present invention will be described in detail below by way of examples. However, the following examples are intended to specifically illustrate the present invention, and the scope of the present invention is not limited by the following examples.

[0112]

[0113] Preparation Example 1: Preparation of a peptide and a peptide complex

[0114] Peptides having the amino acid sequence of SEQ ID NO. 1 listed in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and these synthesized peptides were purified using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). An ACQUITY UPLC BEH300 C18 column (2.1 mm x 100 mm, 1.7 µm, Waters Co, USA) was used.

[0115] Sequence No. Peptide Amino Acid Sequence 1KLGTWTYDGSV (Lys-Leu-Gly-Thr-Trp-Thr-Tyr-Asp-Gly-Ser-Val)

[0116] The efficacy of the peptide of sequence number 1 prepared above was evaluated through the following experiment.

[0117]

[0118] Experimental Example 1: Analysis of absorption by melanosomal phagocytosis

[0119] It was confirmed whether the peptide prepared in Preparation Example 1 above inhibits uptake by phagocytosis of melanosomes in keratinocytes.

[0120] HaCaT cells (Human keratinocyte cell line) 3 x 10 5Cells were seeded into 6-well cell culture plates at a cell / well density and cultured for 24 hours in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS. Subsequently, the cells were replaced with serum-free DMEM medium, and the peptide of Preparation Example 1 was added to the serum-free DMEM medium to prepare peptide solutions at concentrations of 100 μM and 200 μM, which were then added to the cultured cells. The negative control was the peptide-untreated group, and 500 μM of ENMD-1068 (6-Amino-1-[4-(3-methyl-1-oxobutyl)-1-piperazinyl]-1-hexanone hydrochloride, Sigma-Aldrich) was used as the positive control. Subsequently, after incubating in a 37°C, CO2 incubator for 24 hours, 10 μg / ml of melanosomes isolated from B16F10 cells (mouse melanoma cell line) were added to the cultured cells, excluding the control group. Then, after incubating in a 37°C, CO2 incubator for 48 hours and washing three times with PBS, the HaCaT cells were collected. The HaCaT cell pellet was recovered using a centrifuge, lysed with 1M NaOH, and dispensed into a 96-well plate. To measure the melanin content, the absorbance at a wavelength of 490 nm was measured using a spectrophotometer.

[0121] As shown in the experimental results and the melanin content analysis results in Figure 1, it was confirmed that the uptake of melanosomes in HaCaT cells was inhibited by the peptide of Preparation Example 1.

[0122]

[0123] Experimental Example 2: Analysis of PAR2 Expression, a Gene Related to Melanosomal Phagocytosis

[0124] Keratinocytes uptake melanosomes from nearby melanocytes through phagocytosis, and receptors that regulate this melanosome phagocytosis by keratinocytes include PAR2, KGFR, and TLRs.

[0125] Experiments were conducted to determine the effect of the peptide prepared in Preparation Example 1 above on the expression of the PAR2 (protease activated receptor 2) gene, which is a gene related to melanosomal phagocytosis.

[0126] HaCaT cells 3 x 10 5Cells were inoculated into 6-well plates at a cell-to-well density and cultured for 24 hours in DMEM medium containing 10% FBS. After replacing the cells with serum-free DMEM medium, the peptide of 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, which were then dispensed into the cells. The negative control was the peptide-untreated group, and the positive control was treated with 0.02X and 0.05X protease inhibitor cocktail (Roche, Switzerland). Subsequently, after incubating for 24 hours in a CO2 incubator at 37°C, 4 units of trypsin were added to the cultured cells, excluding the control group. Subsequently, the samples were incubated in a CO2 incubator at 37°C for 16 hours and washed twice with PBS, after which RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). After quantifying the amount of isolated RNA, cDNA was synthesized using an RNA and cDNA synthesis kit (enzynomics, Cat. No.: RT200, Korea). PCR was performed using primers targeting the PAR2 gene shown in Table 2 below and a PCR kit (enzynomics, Cat. No.: P581T, Korea). Subsequently, the PCR products were subjected to electrophoresis on a 1.2% agarose gel, and the bands were detected and analyzed using a Bio-Rad gel image system.

[0127] As a result of the experiment, as shown in Figure 2, it was confirmed that the expression of PAR2 was increased by treatment with trypsin (serine protease), an activator of PAR2, in HaCaT cells, and that the increased expression level of PAR2 was reduced again by treatment with the peptide of Preparation Example 1.

[0128]

[0129] Experimental Example 3: Analysis of KGFR Expression, a Gene Related to Melanosomal Phagocytosis

[0130] Experiments were conducted to determine the effect of the peptide of SEQ ID NO. 1 prepared in Preparation Example 1 above on the expression of the KGFR (Keratinocyte growth factor receptor) gene, which is related to melanosomal phagocytosis.

[0131] HaCaT cells 3 x 10 5 Cells were seeded into 6-well plates at a cell-to-well density and cultured in DMEM medium containing 10% FBS for 24 hours in a 37°C CO2 incubator. After replacing the medium with serum-free DMEM, the cells were cultured for an additional 24 hours in a 37°C CO2 incubator. The serum-free DMEM medium was pretreated for 30 minutes with a solution containing the peptide of Preparation Example 1 at concentrations of 50 μM, 100 μM, and 200 μM, with the negative control being the peptide-untreated group. 50 ng / mL of rhKGF protein was added to the cultured cells, excluding the control group, and cultured for an additional 6 hours in a 37°C CO2 incubator. After washing twice with PBS, RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). After quantifying the amount of isolated RNA, cDNA was synthesized using an RNA and cDNA synthesis kit (enzynomics, Cat. No.: RT200, Korea). PCR was performed using primers targeting the KGFR gene shown in Table 2 below and a PCR kit (enzynomics, Cat. No.: P581T, Korea). Subsequently, the PCR products were subjected to electrophoresis on a 1.2% agarose gel, and the bands were detected and analyzed using a Bio-Rad gel image system.

[0132] As a result of the experiment, as shown in Figure 3, it was confirmed that the expression of the KGFR gene was reduced in a concentration-dependent manner by treatment with the peptide of Preparation Example 1 in HaCaT cells.

[0133]

[0134] Experimental Example 4: Analysis of TLR3 Expression, a Gene Related to Melanosomal Phagocytosis

[0135] Experiments were conducted to determine the effect of the peptide of Sequence No. 1 prepared in Preparation Example 1 above on the expression of the TLR3 (Toll-like receptor 3) gene, which is related to melanosomal phagocytosis.

[0136] HaCaT cells 3 x 10 5Cells were seeded into 6-well plates at a cell / well density and cultured in DMEM medium containing 10% FBS for 24 hours in a 37°C CO2 incubator. After replacing the cells with serum-free DMEM medium, they were cultured for an additional 24 hours in a 37°C CO2 incubator. After replacing the serum-free DMEM medium with 2% serum DMEM medium, the cells were pretreated for 30 minutes with a solution containing the peptide of Preparation Example 1 at concentrations of 50 μM, 100 μM, and 200 μM, with the negative control being the peptide-untreated group. Poly(I:C) at a concentration of 30 μg / mL was applied to the cultured cells, excluding the control group, and cultured for an additional 24 hours in a 37°C CO2 incubator. After washing twice with PBS, RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). After quantifying the amount of isolated RNA, cDNA was synthesized using an RNA and cDNA synthesis kit (enzynomics, Cat. No.: RT200, Korea). PCR was performed using primers targeting the KGFR gene shown in Table 2 below and a PCR kit (enzynomics, Cat. No.: P581T, Korea). Subsequently, the PCR products were subjected to electrophoresis on a 1.2% agarose gel, and the bands were detected and analyzed using a Bio-Rad gel image system.

[0137] As shown in Figure 4, experimental results confirmed that the expression of TLR3 in HaCaT cells was increased by treatment with poly(I:C), an activator of TLR3, and that the increased expression level of TLR3 was reduced again by treatment with the peptide of Preparation Example 1.

[0138]

[0139] Experimental Example 5: Quantitative Analysis of Melanosome Degradation

[0140] It was confirmed whether the peptide of SEQ ID NO. 1 prepared in the above Preparation Example 1 promotes the degradation of melanosomes absorbed by keratinocytes.

[0141] HaCaT cells 3 x 10 5 Cells were seeded into 6-well cell culture plates at a cell / well density and cultured for 24 hours in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS. After replacing the DMEM medium containing 10% FBS with 2% serum DMEM medium, melanosomes isolated from B16F10 cells were treated to the cultured cells (excluding the control group) at a concentration of 10 μg / mL, and cultured in a 37°C CO2 incubator for 48 hours. Subsequently, the medium was removed and replaced with fresh 2% serum DMEM medium. Peptide solutions at concentrations of 50 μM, 100 μM, and 200 μM were prepared by adding the peptide of Preparation Example 1, and these were added to the replaced 2% serum DMEM medium and cultured in a 37°C CO2 incubator for 72 hours. In this case, the negative control was the peptide-untreated group, and 200 μM resveratrol or 100 nM rapamycin was used as the positive control. After washing three times with PBS, HaCaT cells were collected and lysed with 1 M NaOH, and the absorbance at a wavelength of 490 nm was measured using a spectrophotometer.

[0142] As shown in the experimental results and the melanin content analysis results in Figure 5, it was confirmed that the degradation of melanosomes in HaCaT cells was promoted by the peptide of Preparation Example 1.

[0143]

[0144] Experimental Example 6: Analysis of Autophagy Activation Related to Melanosome Degradation

[0145] Keratinocytes are known to absorb melanosomes from nearby melanocytes through phagocytosis and degrade these absorbed melanosomes through the activation of autophagy.

[0146] It was confirmed whether the peptide of SEQ ID NO. 1 prepared in Preparation Example 1 above promotes the autophagy activation of melanosomes.

[0147] HaCaT cells 3 x 10 5Cells were seeded into 6-well cell culture plates at a cell-to-well density and cultured in a 37°C CO2 incubator for 24 hours. After replacing the culture medium with serum-free medium, the peptide of Preparation Example 1 was added to the serum-free medium to prepare peptide solutions at concentrations of 50 μM, 100 μM, and 200 μM, which were then added to the cultured cells. For the positive control group, 50 μM or 100 μM of resveratrol or 100 nM or 200 nM of rapamycin were used. Each sample was washed with PBS, treated with lysis buffer to prepare HaCaT cell lysates, and BCA was quantified to prepare equal amounts of protein samples from each sample. The prepared protein samples were subjected to electrophoresis using a 10% SDS-PAGE gel. Subsequently, proteins separated by SDS-PAGE were transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 30 minutes. Primary antibodies against the markers (Beclin-1, LC3-I, LC3-II, and p62) and actin listed in Table 3 below were each diluted in 3% BSA (Bovine Serum Albumin) at a ratio of 1:1000, reacted with the membrane at 4°C for 16 hours, and washed a total of three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS). The secondary antibody listed in Table 3 below was diluted in 5% skim milk powder at a ratio of 1:2000, reacted with the secondary antibody at room temperature for 1 hour, and then washed a total of 3 times with 0.1% PBS-T for 15 minutes each.After treatment with ECL solution (ECL solution, GE Healthcare, USA), the degree of protein expression was analyzed using an imaging system (Cytiva, ImageQuant 800) and is shown in Fig. 6. The values ​​indicated for the Beclin-1 and p62 bands in Fig. 6 represent the relative expression levels calculated by comparing the Beclin-1 and p62 band density values ​​to the loading control (Actin) band density value, with the control set to 1. The values ​​indicated for the LC3-I and LC-II bands in Fig. 6 represent the relative results calculated by comparing the ratio of LC3-II to LC3-I of each treatment group (LC3-II / LC3-I), with the control set to 1.

[0148] As shown in the experimental results and the results in Figure 6, the peptide of Preparation Example 1 increased Beclin-1 expression and the ratio of LC3-II to LC3-I (LC3-II / LC3-I) in HaCaT cells, indicating that the formation of autophagosomes increased. In addition, it was confirmed that the peptide of Preparation Example 1 promoted the degradation of melanosomes through a decrease in the expression of p62, which is known to be degraded within autophagolysosomes.

[0149] List of PCR primer sequences (Table 2)

[0150] Primer Name Sequence (5'->3') Sequence Number PAR2 Forward(5') TGC TAG CAG CCT CTC TCT CC (3') 2PAR2 Reverse(5') CTT CAA GGG GAA CCA GAT GA (3') 3KGFR Forward(5') TGA CCA AAC GTA TCC CCC TG (3') 4KGFR Reverse(5') GGT GTC TGC CGT TGA AGA GA (3') 5TLR3 Forward(5') GCC GTC TAT TTG CCA CAC AC (3') 6TLR3 Reverse(5') GCA GTC AGC AAC TTC ATG GC (3') 7GAPDH Forward(5') GGA GCC AAA AGG GTC ATC AT (3') 8GAPDH Reverse(5') GTG ATG GCA TGG ACT GTG GT (3') 9

[0151] Information on markers and primary and secondary antibodies against actin (Table 3)

[0152] Primary Antibody Manufacturer Cat. No. Secondary Antibody Manufacturer Cat. No.Beclin-1 binding antibodyCell Signaling(Danvers, MA, USA)3495Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L)Jackson ImmunoResearch(West Grove, PA, USA)115-035-003LC3-Ⅰ,LC3-Ⅱ binding antibodyCell Signaling(Danvers, MA, USA)4108Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L)Jackson ImmunoResearch(West Grove, PA, USA)115-035-003SQSTM1 / p62 binding antibodyCell Signaling (Danvers, MA, USA)5114Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L)Jackson ImmunoResearch(West Grove, PA, USA)115-035-003Actin binding antibodySanta cruz (Dallas, TX, USA)sc-47778Peroxidase-AffiniPure Rabbit Anti-Goat IgG (H+L)Jackson ImmunoResearch(West Grove, PA, USA)305-035-003

[0153]

[0154] Although representative embodiments of the present application have been described above by way of example, the scope of the present application is not limited to such specific embodiments, and those skilled in the art will be able to make appropriate modifications within the scope described in the claims of the present application.

Claims

1. A peptide comprising the amino acid sequence of Sequence No.

1.

2. A skin whitening composition 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. In Claim 3, A pharmaceutical composition for the prevention or treatment of hyperpigmentation, wherein the above-mentioned peptide inhibits the absorption of melanosomes in keratinocytes.

5. In Claim 4, The above peptide is in keratinocytes (i) inhibiting the expression of the PAR2 (protease activated receptor 2) gene, or (ii) suppressing the expression of the KGFR (keratinocyte growth factor receptor) gene, or (iii) A pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, which inhibits the expression of the TLR3 (Toll-like receptor 3) gene.

6. In Claim 3, A pharmaceutical composition for the prevention or treatment of hyperpigmentation, wherein the above peptide promotes the degradation of melanosomes in keratinocytes.

7. In Claim 3, A pharmaceutical composition for the prevention or treatment of hyperpigmentation disease, wherein the above-mentioned hyperpigmentation disease is a disease caused by an excessive increase in the amount of melanin in the skin.

8. In Claim 3, A pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, wherein the hyperpigmentation disorder is melasma, freckles, senile pigment spots, solar lentigo, or hyperpigmentation occurring after inflammation of the skin.

9. In Claim 3, The above pharmaceutical composition is a topical application for the skin, a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders.

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

11. In Claim 10, A cosmetic composition for skin whitening in which the above-mentioned peptide inhibits the absorption of melanosomes in keratinocytes.

12. In Claim 11, The above peptide is in keratinocytes (i) inhibiting the expression of the PAR2 (protease activated receptor 2) gene, or (ii) suppressing the expression of the KGFR (keratinocyte growth factor receptor) gene, or (iii) A cosmetic composition for skin whitening that inhibits the expression of the TLR3 (Toll-like receptor 3) gene.

13. In Claim 10, A cosmetic composition for skin whitening, wherein the above-mentioned peptide promotes the degradation of melanosomes in keratinocytes.

14. In Claim 10, The above cosmetic composition is a skin whitening cosmetic composition that is an external application to the skin.

15. In Claim 14, The above cosmetic composition is one or more formulations selected from the group consisting of solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, for skin whitening.

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