Peptide having activity of skin whitening and uses thereof

Abstract

A peptide of the present invention exhibits the activity of skin whitening by inhibiting melanosome absorption in keratinocytes. The peptide of the present invention can be used as an active ingredient in drugs for the treatment or prevention of hyperpigmentation disorders caused by excessive deposition of melanosomes, or as an active ingredient in cosmetics for skin whitening.

Description

Peptides with skin whitening activity and their uses

[0001] [Cross-citation with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0138104, filed October 16, 2023, the entire contents of which are incorporated herein by reference.

[0003] [Technical Field]

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

[0005] Cutaneous melanin exists in the skin, hair follicles, and eyes, and plays an important role in protecting the skin from ultraviolet rays. However, excessive melanin formation can cause hyperpigmentation diseases such as melasma, freckles, and liver spots, which can lead to mental stress and a decline in quality of life. Melanin is mainly synthesized in melanocytes located between or below the basal layer of the epidermis and in hair follicles. Melanin is synthesized in melanosomes, which are organelles within melanocytes. These melanosomes then travel to nearby keratinocytes via dendrites, and when the keratinocytes rise to the epidermis, they reveal skin color. The exact mechanism by which melanosomes move from melanocytes to keratinocytes is not fully understood, but it is known that melanosomes move to the dendrites of melanocytes, are released from them, and subsequently undergo membrane fusion and phagocytosis between melanosomes and keratinocytes. 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 of melanosomes by keratinocytes.

[0006] Melanin synthesis is ultimately synthesized from L-tyrosine through DOPA, DOPAquinone, DOPAchrome, and DHI (5,6-dihydroxyindole). Traditionally, inhibition of melanin synthesis has focused on factors that inhibit the activity of tyrosinase, an enzyme that catalyzes the rate-controlling step of the melanin synthesis pathway.

[0007] PCT International Patent Publication No. WO2020 / 153819 discloses a polypeptide 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.

[0008] Until now, the development of skin whitening agents has focused on inhibiting the activity of tyrosinase, a key enzyme in melanin synthesis. However, to achieve synergistic skin whitening effects through multiple targets, the development of skin whitening agents with different mechanisms of action and targets is necessary.

[0009] [Prior Art Literature]

[0010] [Patent Document]

[0011] WO 2020 / 153819

[0012] Republic of Korea Patent No. 10-1869783

[0013] The present inventors have conducted research efforts to develop a more improved peptide having skin whitening activity, and as a result, have experimentally proven that the novel peptide developed by the present inventors has an excellent inhibitory activity on the uptake of melanosomes into keratinocytes, and have confirmed that the peptide of the present invention can be used as an active ingredient of a skin whitening agent, thereby completing the present invention.

[0014] Accordingly, the purpose of the present invention is to provide a novel peptide having skin whitening activity.

[0015] Another object of the present invention is to provide a skin whitening composition comprising a peptide having the above-described activity as an active ingredient.

[0016] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hyperpigmentation disease, which comprises a peptide having the above-described activity as an active ingredient.

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

[0018] To achieve the above purpose,

[0019] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0020] Another aspect of the present invention provides a skin whitening composition comprising the peptide as an active ingredient.

[0021] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating hyperpigmentation disease, comprising the peptide as an active ingredient.

[0022] Another aspect of the present invention provides a cosmetic composition for skin whitening comprising the peptide as an active ingredient.

[0023] The present invention is described in detail below.

[0024] 1. Peptides and their activities

[0025] According to one aspect of the present invention, a peptide comprising an amino acid sequence disclosed in SEQ ID NO: 1 is provided.

[0026] [Amino acid sequence of sequence number 1]

[0027] KLKKDRMQRS (Lys-Leu-Lys-Lys-Asp-Arg-Met-Gln-Arg-Ser)

[0028] The term "peptide" in this specification means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.

[0029] The peptide comprising 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 by deletion, insertion, substitution, or a combination thereof of amino acid residues may be used within a range that does not affect the original activity of the peptide, such as skin whitening activity.

[0030] 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.

[0031] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to a peptide comprising an amino acid sequence of SEQ ID NO: 1, and a variant or active fragment thereof. The substantially identical amino acid sequence refers to an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more, with the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0032] The peptide 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 increase its activity. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptide of the present invention, for example, increasing the half-life of the peptide when administered in vivo. The term "stability" encompasses not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0033] The above N-terminal modification may be a modification in which 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) is bonded to the N-terminus of the peptide. The above C-terminal modification may be a modification in which a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), or the like is bonded to the C-terminus of the peptide, but is not limited thereto.

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

[0035] The peptide of the present invention has skin whitening activity.

[0036] In one embodiment, the peptide of the present invention has the activity of inhibiting the uptake of melanosomes in keratinocytes.

[0037] Melanosomes produced in melanocytes move from melanocytes to keratinocytes. This movement of melanosomes to keratinocytes involves the release of melanosomes from melanocytes and the incorporation of melanosomes into keratinocytes. This incorporation of melanosomes can be expressed in different ways: as the uptake of melanosomes by keratinocytes or as the phagocytosis of melanosomes by keratinocytes.

[0038] PAR2 (protease activated receptor 2), TLR3 (Toll-like receptor 3), and KGFR (keratinocyte growth factor receptor) are involved in the process of uptake of melanosomes released from melanocytes by phagocytosis of keratinocytes.

[0039] 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.

[0040] The TLR3 protein is known to be a receptor protein that recognizes pathogens and plays a crucial role in activating innate immunity. It is known that stimulation of the TLR3 protein in keratinocytes induces enhanced melanosome uptake (J Dermatol Sci. 2019 Dec;96(3):168-177), and it is also known that the TRR3 protein in melanocytes promotes the movement of melanosomes into keratinocytes (Int. J. Mol. Sci. 2020, 21, 9769).

[0041] 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).

[0042] As described above, the peptide of the present invention can exhibit skin whitening activity and preventive or therapeutic effects on hyperpigmentation diseases through melanosome absorption inhibition activity.

[0043] 2. Composition for skin whitening, composition for preventing, treating or improving hyperpigmentation disease

[0044] In another aspect of the present invention, a skin whitening composition is provided, which comprises a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0045] The peptide comprising the amino acid sequence of sequence number 1 of the present invention has skin whitening activity through the mechanism described above.

[0046] In another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating hyperpigmentation disease, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0047] In one embodiment, a pharmaceutical composition comprising the peptide as an active ingredient inhibits the uptake of melanosomes in keratinocytes.

[0048] In one embodiment, a pharmaceutical composition comprising the peptide as an active ingredient inhibits the expression of a TLR3 (Toll-like receptor 3) gene in keratinocytes, or inhibits the expression of a KGFR (keratinocyte growth factor receptor) gene.

[0049] As used herein, the term "hyperpigmentation disorder" refers to a condition caused by an excessive increase in melanin in the skin. This excessive increase in melanin darkens the skin's color. Causes of hyperpigmentation disorders include, but are not limited to, exposure to sunlight, skin inflammation (post-inflammatory hyperpigmentation) such as acne, trauma, hormonal imbalance, or medication.

[0050] In one embodiment, the hyperpigmentation disorder may be melasma, freckles, age-related pigmentation, solar lentigo, or post-inflammatory hyperpigmentation of the skin.

[0051] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of a peptide comprising the amino acid of sequence number 1 of the present invention.

[0052] 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 efficacy in treating or preventing hyperpigmentation disease.

[0053] The term "prevention" as used herein means reducing the risk of developing a disease or disorder, and means any action that inhibits or delays the onset of a disease by preventing the disease or one or more of its clinical symptoms from progressing.

[0054] The term "treatment" as used herein means alleviating a disease or disorder, and includes any action that improves or beneficially alters the symptoms of a disease by arresting or reducing the progression of the disease or one or more of its clinical symptoms.

[0055] In the present invention, prevention or treatment of hyperpigmentation disease may be by removing the cause of hyperpigmentation in the skin or inhibiting the progression to hyperpigmentation, and specifically, by inhibiting the incorporation of melanosomes into keratinocytes.

[0056] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0057] The pharmaceutically acceptable carriers mentioned above 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.

[0058] The pharmaceutical composition of the present invention may additionally include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., but is not limited thereto.

[0059] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, (19th ed., 1995, Williams & Wilkins).

[0060] The pharmaceutical composition of the present invention may be administered by any suitable route for treating hyperpigmentation diseases, for example, orally or parenterally, and in the case of parenteral administration, may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, etc. Since the pharmaceutical composition of the present invention has an activity for preventing or treating hyperpigmentation diseases of the skin, it is preferably applied by topical administration such as application to the skin.

[0061] 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 formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

[0062] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the method can be performed. 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, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0063] The pharmaceutical composition of the present invention may be a topical skin preparation. The topical skin preparation is a preparation that can be applied to the outside of the skin and used. When the pharmaceutical composition of the present invention is used as a topical skin preparation, it may be applied to the skin, specifically, to an area of ​​the skin where hyperpigmentation has occurred. The topical skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The topical skin preparation may be appropriately mixed 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 a combination thereof, as needed. The above skin external preparation may also appropriately contain metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glabridin, hot water extract of the fruit of Kalin; various herbal medicines; tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0064] In another aspect of the present invention, the present invention provides a cosmetic composition for skin whitening comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0065] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient inhibits the uptake of melanosomes in keratinocytes.

[0066] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient inhibits the expression of the TLR3 (Toll-like receptor 3) gene in keratinocytes, or inhibits the expression of the KGFR (keratinocyte growth factor receptor) gene.

[0067] The above cosmetic composition may be prepared in any formulation commonly manufactured in the technical field to which the present invention pertains, and may be a topical skin preparation. For example, it may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto.

[0068] The above cosmetic composition can be manufactured in various forms such as a solution, sol-gel, emulsion, oil, wax, aerosol, etc., such as a flexible toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, a powder, a hair tonic, a hair cream, a hair lotion, a hair shampoo, a hair rinse, a hair conditioner, a hair spray, a hair aerosol, a pomade, a gel, etc., but is not limited thereto.

[0069] The cosmetic composition of the present invention may include other additives such as excipients and carriers, and it is possible to apply and mix as much as necessary the usual ingredients mixed in general skin cosmetics.

[0070] When the formulation of the above cosmetic composition is in the form of a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0071] When the formulation of the above 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, 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.

[0072] When the formulation of the above cosmetic composition is in the form of a solution or emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier component, and examples thereof include 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.

[0073] When the formulation of the above cosmetic composition is in the form of a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0074] When the formulation of the above cosmetic composition is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0075] When the formulation of the above cosmetic composition is a hair shampoo, base ingredients for forming a shampoo, such as a thickener, a surfactant, a viscosity modifier, a moisturizer, a pH modifier, a preservative, and essential oils, 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, polyquat 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 modifier. Grapefruit extract or the like may be used as the preservative, and in addition, essential oils such as cedarwood, peppermint, and rosemary, as well as silk amino acids, pentaol, or vitamin E may be added.

[0076] The ingredients included in the above cosmetic composition may further include, in addition to the peptide of the present invention and the carrier component as active ingredients, ingredients commonly used in cosmetic compositions, such as conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, but are not limited thereto.

[0077] The peptide of the present invention can 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 can be included in a concentration of 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 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, but is not limited to, 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.

[0078] 3. Uses of the peptide of the present invention

[0079] 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 preventing, treating or improving hyperpigmentation diseases.

[0080] In another aspect of the present invention, a skin whitening method is provided, comprising administering to a subject in need of skin whitening a peptide comprising an amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide.

[0081] In another aspect of the present invention, a method for preventing or treating hyperpigmentation disease is provided, comprising administering to a subject in need of prevention or treatment of hyperpigmentation disease a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide.

[0082] In another aspect of the present invention, there is provided a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for the prevention or treatment of hyperpigmentation diseases.

[0083] In another aspect of the present invention, the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of cosmetics for skin whitening is provided.

[0084] The peptide of the present invention inhibits the uptake of melanosomes in keratinocytes. Therefore, the peptide of the present invention can be used as an active ingredient in a drug for treating or preventing hyperpigmentation caused by excessive melanosome deposition, or as an effective ingredient in a skin whitening cosmetic.

[0085] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0086] Figure 1 shows the results of measuring the absorption rate of melanosomes isolated from HaCaT cells and treated with the peptide of the present invention or the positive control ENMD-1068.

[0087] Figure 2 shows the results of measuring the mRNA level of the TLR3 gene after treating HaCaT cells with Poly(I:C), a synthetic TLR3 agonist, and then treating them with the peptide of the present invention. The band values ​​are calculated by comparing the densitometry results of the band densitometry of the TLR3 gene with the densitometry results of the GAPDH band, which is a loading control, and then indicating the relative expression level with the control as 1.

[0088] Figure 3 shows the results of measuring the mRNA level of KFGR after treating HaCaT cells with the peptide of the present invention. The band values ​​are calculated by comparing the densitometry results of the KFGR gene bands with the densitometry results of the GAPDH bands, which are the loading control, and then represent the relative expression level, with the control group being set at 1.

[0089] Figure 4a is a confocal microscopic photograph showing the fluorescence of microspheres introduced into the cells after treating HaCaT cells with microspheres and treating them with Poly(I:C) and the peptide of the present invention.

[0090] Figure 4b is a graph showing the fluorescence values ​​of microspheres introduced into cells in each control and experimental group.

[0091] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0092] Manufacturing Example 1: Manufacturing of peptides and peptide complexes

[0093] A peptide having the amino acid sequence of SEQ ID NO: 1 shown in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was purified using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm Х 100 mm, 1.7 μm, Waters Co, USA).

[0094] Amino acid sequence of the peptide sequence 1KLKKDRMQRS (Lys-Leu-Lys-Lys-Asp-Arg-Met-Gln-Arg-Ser)

[0095] The efficacy of the peptide of sequence number 1 manufactured above was evaluated through the following experiment.

[0096] Experimental Example 1: Analysis of absorption by melanosome phagocytosis

[0097] It was confirmed whether the peptide manufactured in the above manufacturing example 1 inhibits the uptake of melanosomes by phagocytosis in keratinocytes.

[0098] HaCaT cells (human keratinocyte cell line) were cultured at 3 x 10 5The cells were seeded in a 6-well cell culture plate at a density of 10 cells / well and cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS for 24 hours. 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 50 μM, 100 μM, and 200 μM, which were then added to the cultured cells. The negative control group was a group not treated with peptide, and the positive control group used 500 μM ENMD-1068 (6-Amino-1-[4-(3-methyl-1-oxobutyl)-1-piperazinyl]-1-hexanone hydrochloride, Sigma-Aldrich). Subsequently, after culturing for 24 hours in a CO2 incubator at 37°C, 10 μg / ml melanosomes isolated from B16F10 cells (mouse melanoma cell line) were additionally added to the cultured cells, excluding the negative control (CON) group. Subsequently, after culturing for 48 hours in a CO2 incubator at 37°C, HaCaT cells were harvested after washing three times with PBS. The HaCaT cell pellet was collected using a centrifuge, dissolved in 1 M 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.

[0099] As can be seen from the melanin content analysis results in Fig. 1, it was confirmed that the uptake of melanosomes in HaCaT cells was inhibited by the peptide of Manufacturing Example 1.

[0100] Experimental Example 2: Analysis of TLR3 expression, a gene related to melanosome phagocytosis.

[0101] The effect of the peptide manufactured in the above Manufacturing Example 1 on the expression of the TLR3 (Toll-like receptor 3) gene, a gene related to melanosome phagocytosis, was tested.

[0102] HaCaT cells were cultured at 3 x 10 5 The cells were seeded in 6-well plates at a density of 10 cells / well and cultured in a CO2 incubator at 37°C for 24 hours, replaced with DMEM medium containing 2% FBS. The peptide of Preparation Example 1 was added to the DMEM medium containing 2% FBS to prepare peptide solutions at concentrations of 10 μM, 50 μM, 100 μM, and 200 μM, and these solutions were added to the cells and treated for 30 minutes. Subsequently, 30 μg / mL of poly(I:C) (Polyinosinic-Polycytidylic acid), a TLR3 agonist, was additionally added, and cultured in a CO2 incubator at 37°C for 24 hours. After washing twice with PBS, RNA was isolated using easy blue (iNtRON, Cat. No.: 17061, Korea). After quantifying the amount of isolated RNA, 2,000 ng of RNA was aliquoted per tube and cDNA was synthesized using a cDNA synthesis kit (enzynomics, Cat. No.: RT200, Korea). PCR was performed using primers targeting the TLR3 gene in Table 2 and a PCR kit (enzynomics, Cat. No.: P581T, Korea). The PCR products were then electrophoresed on a 1.2% agarose gel, and bands were detected and analyzed using a Bio-Rad gel image system.

[0103] As a result of the experiment, as shown in Fig. 2, when HaCaT cells were treated with Poly (I:C), an agonist of TLR3, the expression of the TLR3 gene increased, and it was confirmed that this increased expression level of the TLR3 gene was suppressed by treating with the peptide of Preparation Example 1.

[0104] Experimental Example 3: Analysis of KGFR expression, a gene related to melanosome phagocytosis.

[0105] The effect of the peptide of sequence number 1 manufactured in the above manufacturing example 1 on the expression of the melanosome phagocytosis-related gene KGFR (Keratinocyte growth factor receptor) gene was tested.

[0106] HaCaT cells were cultured at 3 x 10 5 Cells were seeded into 6-well plates at a density of 10 cells / well and cultured in DMEM medium containing 10% FBS for 24 hours. The cells were replaced with serum-free DMEM medium and cultured in a CO2 incubator at 37°C for 24 hours. The peptide of Preparation Example 1 was added to serum-free DMEM medium to prepare peptide solutions at concentrations of 10 μM, 50 μM, 100 μM, and 200 μM, which were dispensed onto the cells and further cultured in a CO2 incubator at 37°C for 6 hours. 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 in Table 2 below and a PCR kit (enzynomics, Cat. No.: P581T, Korea). The PCR products were then electrophoresed on a 1.2% agarose gel, and bands were detected and analyzed using a Bio-Rad gel image system.

[0107] As a result of the experiment, as shown in Fig. 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.

[0108] Experimental Example 4: Poly(I:C)-induced phagocytosis assay - microsphere uptake assay

[0109] The effect of the peptide manufactured in the above Manufacturing Example 1 on Poly(I:C)-induced melanosome phagocytosis was confirmed through a microsphere uptake experiment.

[0110] HaCaT cells were cultured at 3 x 10 4 The cells were seeded in a 6-well plate at a density of 10 cells / well and cultured in a CO2 incubator at 37°C for 24 hours using DMEM medium containing 10% FBS. After replacing the medium 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 pretreated to the cells for 1 hour. Poly (I:C) (Polyinosinic-Polycytidylic acid) at 30 μg / mL and microspheres (FluoSpheres) TM , diameter: 1 μm) and cultured for 24 hours in a 37℃ CO2 incubator. The negative control group was treated only with Poly(I:C) and microspheres without peptide treatment. Cell fixation was performed by treating with 4% formaldehyde for 30 minutes. After treating with Phalloidin iFluor 488 reagent (Abcam, UK), the cells were reacted for 1 hour. After treating with mounting medium containing DAPI (Invitrogen, USA), confocal microscopy (400x magnification) was used for observation. The fluorescence value of microspheres introduced into the cells was analyzed using the Image J program.

[0111] As shown in Figs. 4a and 4b, the experimental results showed that the absorption of microspheres increased by the Poly (I:C) treatment, and that the increased absorption of microspheres was reduced by the peptide treatment of Preparation Example 1.

[0112] PCR primer sequence list (Table 2)

[0113] Primer name sequence (5'->3') Sequence number TLR3 Forward (5') GCC GTC TAT TTG CCA CAC AC (3') 2TLR3 Reverse (5') GCA GTC AGC AAC TTC ATG GC (3') 3KGFR Forward (5') TGA CCA AAC GTA TCC CCC TG (3') 4KGFR Reverse (5') GGT GTC TGC CGT TGA AGA GA (3') 5GAPDH Forward (5') GGA GCC AAA AGG GTC ATC AT (3') 6GAPDH Reverse (5') GTG ATG GCA TGG ACT GTG GT (3') 7

[0114] Although representative embodiments of the present application have been described above as examples, the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the relevant field will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A peptide comprising the amino acid sequence of sequence number 1.

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

3. A pharmaceutical composition for preventing or treating hyperpigmentation disease, comprising the peptide of claim 1 as an active ingredient.

4. In claim 3, A pharmaceutical composition for preventing or treating hyperpigmentation disease, wherein the above peptide inhibits the uptake of melanosomes in keratinocytes.

5. In claim 4, The above peptide is expressed in keratinocytes. (i) inhibiting the expression of the TLR3 (Toll-like receptor 3) gene, or (ii) A pharmaceutical composition for preventing or treating hyperpigmentation disease, which suppresses the expression of the KGFR (keratinocyte growth factor receptor) gene.

6. In claim 3, A pharmaceutical composition for preventing or treating hyperpigmentation disease, wherein the hyperpigmentation disease is a disease caused by an excessive increase in the amount of melanin in the skin.

7. In claim 3, A pharmaceutical composition for preventing or treating hyperpigmentation disease, wherein the hyperpigmentation disease is a melasma, freckle, senile pigmentation, solar lentigo, or hyperpigmentation occurring after inflammation of the skin.

8. In claim 3, The pharmaceutical composition above is a pharmaceutical composition for the prevention or treatment of hyperpigmentation disease, which is an external skin agent.

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

10. In claim 9, A cosmetic composition for skin whitening, wherein the above peptide inhibits the absorption of melanosomes in keratinocytes.

11. In claim 10, The above peptide is expressed in keratinocytes. (i) (i) inhibiting the expression of the TLR3 (Toll-like receptor 3) gene, or (ii) A cosmetic composition for skin whitening that inhibits the expression of the KGFR (keratinocyte growth factor receptor) gene.

12. In claim 9, The above cosmetic composition is a skin whitening cosmetic composition that is an external skin agent.

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

14. A method for preventing or treating hyperpigmentation disease, comprising administering the peptide of claim 1 or a composition comprising the peptide to a subject in need of prevention or treatment of hyperpigmentation disease.

15. In claim 14, A method for preventing or treating hyperpigmentation disease, wherein the hyperpigmentation disease is a melasma, freckle, age-related pigmentation, solar lentigo, or hyperpigmentation occurring after inflammation of the skin.

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