Peptides with skin whitening activity and uses thereof

Abstract

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

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0155229, filed on November 18, 2022, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a peptide having skin whitening activity and uses thereof. [Background technology]

[0003] Cutaneous melanin is present in the skin, hair follicles, and eyes and plays an important role in protecting the skin from ultraviolet rays. However, excessive melanin production can lead to hyperpigmentation disorders such as age spots, freckles, and dark spots, as well as psychological stress and a decline in quality of life. Melanin is primarily synthesized in melanocytes, located between and below the basal layer of the epidermis and in hair follicles. It is synthesized in melanosomes, organelles within melanocytes. These melanosomes then travel via dendrites to nearby keratinocytes, where they emerge from the epidermis and contribute to the development of skin color. The exact mechanism by which melanosomes move from melanocytes to keratinocytes has not been elucidated, but it is known that after melanosomes move to the dendrites of melanocytes and are released outside the cells, membrane fusion between melanosomes and keratinocytes and phagocytosis are involved.

[0004] Melanin is synthesized through the process of L-tyrosine, DOPA, DOPAquinone, DOPAchrome, and finally 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-regulating step in the melanin synthesis pathway.

[0005] PCT International 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. Korean Patent Registration No. 10-1869783 also discloses a peptide that inhibits melanin production and tyrosinase activity, and its use for skin whitening.

[0006] Until now, the development of skin whitening agents has focused on inhibiting the activity of tyrosinase, a key enzyme in melanin synthesis. However, in order to achieve a synergistic effect through various sites of action in skin whitening, it is necessary to develop skin whitening agents with different mechanisms and sites of action. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2020 / 153819 [Patent Document 2] Korean Patent Registration No. 10-1869783 Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have made research efforts to develop peptides with improved skin whitening activity, and as a result have experimentally proven that the novel peptides they have developed inhibit the invagination of melanosomes into keratinocytes and have excellent activity in degrading melanosomes within keratinocytes, thereby completing the present invention.

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

[0010] Another object of the present invention is to provide a skin whitening composition containing, as an active ingredient, a peptide having the above-mentioned activity.

[0011] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating hyperpigmentation diseases, which comprises a peptide having the above-mentioned activity as an active ingredient.

[0012] A further object of the present invention is to provide a cosmetic composition for skin whitening, which contains as an active ingredient a peptide having the above-mentioned activity. [Means for solving the problem]

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

[0014] Another aspect of the present invention provides a composition for skin whitening, comprising the peptide as an active ingredient.

[0015] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating hyperpigmentation disorders, comprising the peptide as an active ingredient.

[0016] Yet another aspect of the present invention provides a cosmetic composition for skin whitening, comprising the peptide as an active ingredient.

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

[0018] 1. Peptides and their activities

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

[0020] As used herein, the term "peptide" refers to a linear molecule formed by amino acid residues joined together by peptide bonds.

[0021] The peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 1 may be used without modification, but amino acid variants or fragments having different sequences due to deletion, insertion, substitution, or a combination thereof of amino acid residues may also be used, as long as the original activity of the peptide, for example, skin whitening activity, is not affected.

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

[0023] The peptides of the present invention include peptides comprising an amino acid sequence substantially identical to that of a peptide comprising the amino acid sequence of SEQ ID NO: 1, as well as variants thereof or active fragments thereof. The term "substantially identical amino acid sequence" refers to an amino acid sequence having 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. The peptides may further comprise a targeting sequence, a tag, a labeled residue, or an amino acid sequence specifically engineered to increase half-life or peptide stability.

[0024] The peptides of the present invention may be modified at the N- and / or C-terminus by selecting a portion of the amino acid sequence to increase their activity. Such N- and / or C-terminal modifications can significantly improve the stability of the peptides of the present invention, for example, increasing the half-life of the peptides when administered in vivo. The term "stability" refers not only to in vivo stability, which protects the peptides of the present invention from attack by in vivo protease enzymes, but also to storage stability (e.g., storage stability at room temperature).

[0025] The N-terminal modification may be a protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl 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 C-terminal modification may be a protecting group such as a hydroxyl group (-OH), an amino group (-NH), or an azide (-NHNH) attached to the C-terminus of the peptide, but is not limited thereto.

[0026] The peptides of the present invention can be prepared by various methods well known in the art, including chemical synthesis techniques well 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 (U.S. Patent No. 5,516,891).

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

[0028] In one embodiment, the peptide of the present invention has the activity of inhibiting melanosome invagination or promoting melanosome degradation in keratinocytes.

[0029] Melanosomes produced in melanocytes are transferred from melanocytes to keratinocytes, and the transfer of melanosomes to keratinocytes involves the process of melanosome release from melanocytes and the process of melanosome invagination into keratinocytes. The process of melanosome invagination can be expressed differently as melanosome uptake by keratinocytes or phagocytosis of melanosomes by keratinocytes.

[0030] The process of phagocytic absorption of melanosomes released from melanocytes by keratinocytes involves PAR2 (protease activated receptor 2), TLR3 (Toll-like receptors 3), and KGFR (keratinocyte growth factor receptors).

[0031] In one embodiment, the peptide of the present invention inhibits the expression of one or more genes selected from the group consisting of PAR2 (protease activated receptor 2) gene, TLR3 (Toll-like receptor 3) gene, and KGFR (keratinocyte growth factor receptors) gene in keratinocytes.

[0032] 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. PAR2 protein is present in the membrane of keratinocytes and is involved in the keratinocyte's absorption (invagination) of melanosomes. Suppressing the activity of PAR2 protein is known to inhibit the absorption of melanosomes by keratinocytes (Exp Cell Res. 2000 Jan 10;254(1):25-32; Pigment Cell Res. 2001 Aug;14(4):236-42; Exp Dermatol. 2003;12 Suppl 2:5-12).

[0033] The TLR3 protein is known to be a receptor protein that recognizes pathogens and plays an important role in activating innate immunity. It is known that stimulation of the TRR3 protein in keratinocytes induces improved melanosome absorption (J Dermatol Sci. 2019 Dec;96(3):168-177), and that the TRR3 protein in melanocytes promotes melanosome transfer to keratinocytes (Int. J. Mol. Sci. 2020, 21, 9769).

[0034] KGFR is a tyrosine kinase receptor expressed in many types of epithelial cells, activated by four known ligands, FGF-1, FGF-3, FGF-7, and FGF-10, and known to be involved in epithelial cell proliferation, differentiation, wound healing, etc. KGFR is also known to promote melanosome migration to keratinocytes (J Invest Dermatol. 2005 Dec;125(6):1190-9., Journal of Investigative Dermatology Vol. 128, Issue 3, March 2008, pp.558-567).

[0035] As described above, the peptide of the present invention can exhibit skin whitening activity and preventive or therapeutic effects on hyperpigmentation disorders due to its melanosome invagination inhibitory activity and melanosome degrading activity.

[0036] 2. Compositions for skin whitening and prevention, treatment or improvement of hyperpigmentation disorders

[0037] In another aspect of the present invention, there is provided a composition for skin whitening, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0038] The peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 1 has skin whitening activity via the mechanism described above.

[0039] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating hyperpigmentation diseases, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0040] In one embodiment, a pharmaceutical composition containing the peptide as an active ingredient inhibits melanosome invagination or promotes melanosome degradation in keratinocytes.

[0041] In one embodiment, a pharmaceutical composition containing the peptide as an active ingredient suppresses the expression of one or more genes selected from the group consisting of PAR2 (protease activated receptor 2) gene, TLR3 (Toll-like receptor 3) gene, and KGFR (keratinocyte growth factor receptors) gene in keratinocytes.

[0042] As used herein, the term "hyperpigmentation disorder" refers to a disorder caused by an excessive increase in the amount of melanin in the skin. Thus, an excessive increase in the amount of melanin in the skin results in darker skin. Hyperpigmentation disorders may be caused by, but are not limited to, sun exposure, skin inflammation such as acne (post-inflammatory hyperpigmentation), trauma, hormonal imbalance, or medication.

[0043] In one embodiment, the hyperpigmentation disorder may be age spots, freckles, age spots, solar lentigo, or hyperpigmentation that occurs after skin inflammation, such as hyperpigmentation that occurs after trauma, ultraviolet rays, or inflammatory skin disorders such as acne.

[0044] The pharmaceutical composition of the present invention may comprise a therapeutically effective amount of a peptide comprising the amino acid of SEQ ID NO:1 of the present invention.

[0045] The term "therapeutically effective amount" means an amount sufficient for the peptide, which is the active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, for example, an amount sufficient for achieving efficacy in treating or preventing hyperpigmentation disorders.

[0046] As used herein, the term "prevention" means reducing the risk of contracting a disease or disorder, and refers to 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.

[0047] As used herein, the term "treatment" means alleviating a disease or disorder and includes any action that prevents or reduces the progression of a disease or one or more of its clinical symptoms, thereby improving or modifying the symptoms of the disease.

[0048] In the present invention, the prevention or treatment of hyperpigmentation diseases 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 or promoting the degradation of melanosomes.

[0049] The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.

[0050] The pharmaceutically acceptable carriers are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0051] The pharmaceutical composition of the present invention may further contain, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like, but is not limited to these.

[0052] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, (19th ed., 1995, Williams & Wilkins).

[0053] The pharmaceutical composition of the present invention may be administered by any suitable route for treating hyperpigmentation disorders, for example, orally or parenterally, and in the case of parenteral administration, it 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 preventive or therapeutic activity for hyperpigmentation disorders of the skin, it is preferably administered topically, such as by application to the skin.

[0054] 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 variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, diet of the patient, administration time, administration route, excretion rate, and reaction sensitivity.

[0055] The pharmaceutical composition of the present invention may be prepared in a unit dose form or in a multi-dose container by formulating it with pharmaceutically acceptable carriers and / or excipients by a method easily performed by a person skilled in the art to which the invention pertains. In this case, the dosage form may be a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granules, tablets, or capsules, and may further contain a dispersing agent or stabilizer.

[0056] The pharmaceutical composition of the present invention may be a topical skin preparation. The topical skin preparation is a preparation that can be applied externally to 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 delivery patch, drug-containing bandage, lotion, or a combination thereof. The topical skin preparation may be appropriately blended with ingredients commonly used in topical skin preparations such as cosmetics and 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, as needed. The topical skin preparation may also contain, as appropriate, sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; drugs such as caffeine, tannin, licorice extract, glabridin, various herbal medicines, tocopherol acetate, glycyrrhizin, tranexamic acid, and derivatives or salts thereof; vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, and sugars such as glucose, fructose, and trehalose.

[0057] In another aspect, the present 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.

[0058] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient inhibits melanosome invagination or promotes melanosome degradation in keratinocytes.

[0059] In one embodiment, a cosmetic composition containing the peptide of the present invention as an active ingredient suppresses the expression of one or more genes selected from the group consisting of PAR2 (protease activated receptor 2) gene, TLR3 (Toll-like receptor 3) gene, and KGFR (keratinocyte growth factor receptors) gene in keratinocytes.

[0060] The cosmetic composition may be prepared in any form commonly used in the technical field to which the present invention pertains, and may be a topical skin preparation, such as, but not limited to, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, spray, etc.

[0061] The cosmetic composition may be prepared in various forms such as a solution, sol-gel, emulsion, oil, wax, aerosol, etc., including, but not limited to, a softening lotion, a nourishing lotion, 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.

[0062] The cosmetic composition of the present invention may contain other additives such as excipients and carriers, and it is possible to apply and blend in the required amounts of common ingredients blended in general skin cosmetics.

[0063] When the cosmetic composition is in the form of a paste, cream, or gel, the carrier component may be animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or the like.

[0064] 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, and particularly when the cosmetic composition is in the form of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be further included, but is not limited thereto.

[0065] When the cosmetic composition is in the form of a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, and examples of such solvents include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, and sorbitan fatty acid esters.

[0066] When the 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, or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth may be used as a carrier component.

[0067] When the cosmetic composition is in the form of a surfactant-containing cleanser, the carrier component may be a fatty alcohol sulfate, a fatty alcohol ether sulfate, a sulfosuccinic acid monoester, isethionic acid, an imidazolinium derivative, methyl taurate, sarcosinate, a fatty acid amide ether sulfate, an alkylamidobetaine, a fatty alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a lanolin derivative, or an ethoxylated glycerol fatty acid ester.

[0068] When the cosmetic composition is in the form of a hair shampoo, the peptide of the present invention may be mixed with base ingredients for forming a shampoo, such as a thickener, surfactant, viscosity modifier, moisturizer, pH adjuster, preservative, essential oil, etc. The thickener may be CDE, the surfactant may be an anionic surfactant LES or an amphoteric surfactant cocobetaine, the viscosity modifier may be polyquaternary, the moisturizer may be glycerin, and the pH adjuster may be citric acid or sodium hydroxide. The preservative may be grapefruit extract, and in addition, essential oils such as cedarwood, peppermint, and rosemary, silk amino acids, pentaol, or vitamin E may be added.

[0069] The components contained in the cosmetic composition may further include, in addition to the peptide of the present invention as an active ingredient and a carrier component, components commonly used in cosmetic compositions, such as, for example, common adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, but are not limited thereto.

[0070] The peptide of the present invention may be contained in the above-mentioned composition, pharmaceutical composition or cosmetic composition at a concentration of 0.01 uM to 1000 uM, and specifically, the peptide of the present invention may be contained in the above-mentioned composition, pharmaceutical composition or cosmetic composition at a concentration of 0.01 uM to 1000 uM; 0.05 uM to 800 uM, 0.05 uM to 700 uM, 0.05 uM to 600 uM, 0.05 uM to 500 uM, 0.05 uM to 300 uM, 0.05 uM to 200 uM; 0.1 uM to 800 uM, 0.1 uM to 700 uM, 0.1 The concentration of the hydroxybenzoate may be, but is not limited to, 0.1 uM to 600 uM, 0.1 uM to 500 uM, 0.1 uM to 300 uM, 0.1 uM to 200 uM; 1 uM to 800 uM, 1 uM to 700 uM, 1 uM to 600 uM, 1 uM to 500 uM, 1 uM to 300 uM, 1 uM to 200 uM; 5 uM to 800 uM, 5 uM to 700 uM, 5 uM to 600 uM, 5 uM to 500 uM, 5 uM to 300 uM, or 5 uM to 200 uM.

[0071] 3. Uses of the peptides of the present invention

[0072] In another aspect of the present invention, there is provided the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for skin lightening or for the prevention, treatment or amelioration of hyperpigmentation disorders.

[0073] In another aspect of the present invention, there is provided a method for skin whitening, comprising administering a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising said peptide to a subject in need of skin whitening.

[0074] In another aspect of the present invention, there is provided a method for preventing or treating a hyperpigmentation disease, comprising administering a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide to a subject in need of such prevention or treatment.

[0075] In another aspect of the present invention there is provided the 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 disorders.

[0076] In another aspect of the present invention, there is provided the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of skin lightening cosmetics. [Effects of the Invention]

[0077] The peptide of the present invention exhibits skin whitening activity by inhibiting melanosome invagination in keratinocytes and promoting melanosome degradation, and therefore can be used as an active ingredient in drugs for treating or preventing hyperpigmentation disorders caused by excessive melanosome deposition or as an active ingredient in skin whitening cosmetics.

[0078] However, the effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. [Brief explanation of the drawings]

[0079] [Figure 1] 1 shows the results of a melanin content analysis showing that the peptide of the present invention inhibits melanosome invagination in HaCaT cells. [Figure 2a] 1 shows the results of PCR analysis showing that the peptide of the present invention concentration-dependently reduces the mRNA level of PAR2, a receptor involved in melanosome invagination in HaCaT cells. [Figure 2b] The graph shows the measured band density values ​​of the electrophoresis in Figure 2a. [Figure 3a] 1 shows the results of PCR analysis demonstrating that the peptide of the present invention reduces the mRNA level of TLR3, a receptor involved in melanosome invagination, in HaCaT cells. [Figure 3b] The band density measured in the electrophoresis of Figure 3a is shown in a graph. [Figure 4a] 1 shows the results of PCR analysis showing that the peptide of the present invention reduces the mRNA level of KGFR, a receptor involved in melanosome invagination in HaCaT cells. [Figure 4b] The band density measured in the electrophoresis of Figure 4a is shown in a graph. [Figure 5] 1 shows the results of a melanosome content analysis demonstrating that the peptides of the present invention induce melanosome degradation in HaCaT cells. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention will be described in more detail below with reference to examples. However, the following examples are provided to specifically illustrate the present invention, and the scope of the present invention is not limited to the following examples.

[0081] Production Example 1: Production of peptides

[0082] 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 the synthesized peptides were purified and separated using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was an ACQUITY UPLC BEH300 C18 (2.1 mm x 100 mm, 1.7 μm, Waters Co., USA).

[0083] [Table 1]

[0084] The efficacy of the prepared peptide of SEQ ID NO: 1 was evaluated through the following experiment.

[0085] Experimental Example 1: Analysis of melanosome invagination by phagocytosis

[0086] It was confirmed whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 inhibits the invagination of melanosomes in keratinocytes by phagocytosis.

[0087] HaCaT cells (human keratinocyte cell line) 3x10 5Cells were seeded into 6-well cell culture plates at a density of 100 cells / well and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS for 24 hours. After washing the cells once with serum-free DMEM, the peptide of SEQ ID NO: 1 from Preparation Example 1 was added to 3 mL of serum-free DMEM to prepare peptide solutions at concentrations of 50 μM, 100 μM, and 200 μM. These solutions were then added to the cultured cells. A negative control (CON, (+)) was used as an untreated group, and 500 nM ENMD-1068 (6-Amino-1-[4-(3-methyl-1-oxobutyl)-1-piperazinyl]-1-hexanone hydrochloride, Sigma-Aldrich) was used as a positive control. After 24 hours of incubation in a CO2 incubator at 37°C, melanosomes (10 μg / ml) isolated from HEM-DP (Human Epidermal Melanocyte, darkly pigmented donor) were added to the cultured cells, except for the negative control (CON) group. After 48 hours of incubation in a CO2 incubator at 37°C, the cells were washed three times with PBS and then treated with 1X TE for 10 minutes to harvest the HaCaT cells. The HaCaT cells were collected using a centrifuge, dissolved in 1M NaOH, and dispensed into a 96-well plate. The melanin content was measured by absorbance at 490 nm using an ELISA reader.

[0088] As a result of the experiment, as can be seen from the melanin content analysis results in FIG. 1, it was confirmed that the peptide of SEQ ID NO: 1 inhibited melanosome invagination in HaCaT cells.

[0089] Experimental Example 2: Expression analysis of melanosome phagocytosis-related PAR2 gene

[0090] The effect of the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 on the expression of melanosome phagocytosis-related gene PAR2 (protease activated receptor 2) in keratinocytes was examined.

[0091] 3x10 HaCaT cells 5 Cells were seeded into 6-well plates at a density of 1000 cells / well and cultured for 24 hours in DMEM medium containing 10% FBS. After washing once with serum-free DMEM, the peptide of SEQ ID NO: 1 from Preparation Example 1 was added to 3 mL of serum-free DMEM to prepare peptide solutions at concentrations of 10 μM, 50 μM, and 100 μM. These solutions were then dispensed onto the cells. The negative control group (CON, (+)) was untreated, and the positive control group was treated with 0.02X or 0.05X protease inhibitor (PI) (cOmplete™ Protease Inhibitor Cocktail, Roche, CH). After culturing for 24 hours in a CO2 incubator at 37°C, 4 units of trypsin were added to the cultured cells, except for the negative control group (CON). The cells were then cultured for 16 hours at 37°C in a CO2 incubator, washed twice with PBS, and RNA was isolated using Easy Blue (IntRON, Cat. No. 17061, Korea). After quantifying the amount of isolated RNA, 2000 ng of RNA was dispensed per tube and cDNA was synthesized using a cDNA synthesis kit (Enzynomics, Cat. No. RT200, Korea). PCR was performed using primers targeting the PAR2 gene (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.

[0092] [Table 2]

[0093] As a result of the experiment, as shown in Figure 2a and Figure 2b, it was confirmed that the peptide of SEQ ID NO: 1 concentration-dependently reduced the mRNA level of the PAR2 gene, a receptor related to melanosome phagocytosis (invagination), in HaCaT cells. This result confirmed that the peptide of SEQ ID NO: 1 inhibits melanosome invagination.

[0094] Experimental Example 3: Expression analysis of melanosome phagocytosis-related TLR3 gene

[0095] The effect of the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 on the expression of melanosome phagocytosis-related gene TLR3 (Toll-like receptors 3) was examined.

[0096] 3x10 HaCaT cells 5 The cells were seeded into 6-well plates at a density of 1000 cells / well and cultured for 24 hours in DMEM medium containing 10% FBS. The cells were then washed once with serum-free DMEM medium, and 2 mL of serum-free DMEM medium was added to the cells. The cells were then cultured for 24 hours in a CO2 incubator at 37°C. The peptide of SEQ ID NO: 1 from Preparation Example 1 was added to 3 mL of DMEM medium containing 2% serum to prepare peptide solutions with concentrations of 10 μM, 50 μM, 100 μM, and 200 μM, and this solution was added to the cells. After 30 minutes, 30 μg / mL of poly(I:C) was added. After 24 hours of culture in a CO2 incubator at 37°C, the cells were washed twice with PBS, and RNA was isolated using Easy Blue (iNtRON, Cat. No.: 17061, Korea). After quantifying the amount of isolated RNA, 2000 ng of RNA was dispensed per tube and cDNA was synthesized using a cDNA synthesis kit (enzynomics, Cat. No.: RT200, Korea). PCR was performed using primers targeting the TLR3 gene listed in Table 3 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.

[0097] [Table 3]

[0098] As a result of the experiment, as shown in Figure 3a and Figure 3b, it was confirmed that the peptide of SEQ ID NO: 1 reduced the mRNA level of the TLR3 gene, a receptor related to melanosome phagocytosis (invagination), in HaCaT cells compared to the control group. This result confirmed that the peptide of SEQ ID NO: 1 inhibits melanosome invagination.

[0099] Experimental Example 4: Expression analysis of melanosome phagocytosis-related KGFR gene

[0100] The effect of the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 on the expression of melanosome phagocytosis-related gene KGFR (keratinocyte growth factor receptors) was examined.

[0101] 3x10 HaCaT cells 5Cells were seeded into 6-well plates at a density of 1000 cells / well and cultured in DMEM medium containing 10% FBS for 24 hours. After washing once with serum-free DMEM medium, 2 mL of serum-free DMEM medium was added to the cells and cultured in a CO2 incubator at 37°C for 24 hours. Next, peptide solutions of 10 μM, 50 μM, 100 μM, and 200 μM were prepared by adding the peptide of SEQ ID NO: 1 from Preparation Example 1 to 3 mL of DMEM medium containing 2% serum. These solutions were added to the cells and 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). The amount of isolated RNA was quantified, and 2000 ng of RNA was dispensed per tube, and cDNA was synthesized using a cDNA synthesis kit (enzynomics, Cat. No. RT200, Korea). PCR was performed using primers targeting the KGFR gene in Table 4 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.

[0102] [Table 4]

[0103] As shown in Figures 4a and 4b, the experimental results confirmed that the peptide of SEQ ID NO: 1 reduced the mRNA level of the KGFR gene, a melanosome phagocytosis-related receptor, in HaCaT cells compared to the control group. This result confirmed that the peptide of SEQ ID NO: 1 inhibits melanosome invagination.

[0104] Experimental Example 5: Melanosome degradation analysis

[0105] The peptide of SEQ ID NO: 1 prepared in Preparation Example 1 was analyzed to determine whether it degrades melanosomes.

[0106] 3x10 HaCaT cells5 Cells were seeded into 6-well plates at a density of 100 cells / well and cultured in DMEM medium containing 10% FBS for 24 hours. Next, melanosomes isolated from HEM-DP (Human Epidermal Melanocyte, darkly pigmented donor) were added to 2 mL of DMEM medium containing 2% serum to prepare a melanosome solution at a concentration of 10 μg / mL. This solution was then dispensed onto the cells and cultured for 48 hours at 37°C in a CO2 incubator.

[0107] Next, melanosomes isolated from HEM-DP were added to 3 mL of DMEM medium containing 2% serum to prepare a melanosome solution at a concentration of 10 μg / mL, and the peptide of SEQ ID NO: 1 from Preparation Example 1 was added to 3 mL of DMEM medium containing 2% serum to prepare peptide solutions at concentrations of 10 μM, 50 μM, 100 μM, and 200 μM. The melanosome solution and peptide solution were then added to the cells. In this case, the negative control group (CON) was an untreated group, the negative control group (+) was a group treated with only the melanosome solution, and the positive control groups were a group treated with the melanosome solution and 100 nM or 200 nM rapamycin (Rapa) and a group treated with the melanosome solution and 200 μM or 500 μM resveratrol (RSV). The cells were then cultured for 72 hours at 37°C in a CO2 incubator, washed three times with PBS, and then treated with 1XTE for 10 minutes to collect HaCaT cells. HaCaT cells were collected using a centrifuge, dissolved in 1M NaOH, and dispensed into a 96-well plate. The amount of melanin was measured by absorbance at 490 nm using an ELISA reader.

[0108] As a result of the experiment, as can be seen from the change in melanosome content in FIG. 3, it was confirmed that treatment with the peptide of SEQ ID NO: 1 induced the degradation of melanosomes in HaCaT cells.

[0109] Preparation Example 2: Preparation of pharmaceutical composition

[0110] 2-1. Ointment manufacturing 5g of the peptide of the present invention Cetyl palmitate 20g Cetanol 40g 40g stearyl alcohol Myristan Isopropyl 80g Polysorbate 60g Propyl parahydroxybenzoate 1g Methyl parahydroxybenzoate 1g Phosphoric acid and purified water (appropriate amount)

[0111] According to a conventional method for manufacturing an ointment, an ointment was prepared containing the above ingredients in the amounts indicated.

[0112] 2-2. Powder manufacturing 2g of the peptide of the present invention Lactose 1g

[0113] The ingredients were mixed and filled into an airtight bag to prepare a powder.

[0114] 2-3. Tablet manufacturing 100mg of the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0115] The above ingredients were mixed and then compressed into tablets according to a conventional tablet manufacturing method.

[0116] 2-4. Capsule manufacturing 100mg of the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0117] The above ingredients were mixed and then filled into gelatin capsules according to a conventional capsule manufacturing method to prepare capsules.

[0118] 2-5.Making of rounds Peptide of the present invention 1g Lactose 1.5g 1g glycerin Xylitol 0.5g

[0119] The ingredients were mixed and then prepared in a conventional manner so that each pill weighed 4 g.

[0120] Production Example 3: Production of cosmetic composition

[0121] 3-1. Cream production Peptide of the present invention 4.6 parts by weight Cetostearyl alcohol 2.8 parts by weight Beeswax 2.6 parts by weight Stearic acid 1.4 parts by weight Lipophilic glycerin monostearate 2 parts by weight PEG-100 stearate 1 part by weight Sorbital sesquioleate 1.4 parts by weight Jojoba oil 4 parts by weight Squalene 3.8 parts by weight Polysorbate 60 1.1 parts by weight Macadamia oil 2 parts by weight Tocopherol acetate 0.2 parts by weight Methylpolysiloxane 0.4 parts by weight Ethylparaben 0.1 parts by weight Propylparaben 0.1 parts by weight Euxyl K-400 0.1 parts by weight 1,3-butylene glycol 7 parts by weight Methylparaben 0.05 parts by weight Glycerin 6 parts by weight d-Panthenol 0.2 parts by weight Triethanolamine 0.2 parts by weight pt41891 0.2 parts by weight p-H2O 46.05 parts by weight

[0122] 3-2. Lotion manufacturing Peptide of the present invention 3.5 parts by weight Cetostearyl alcohol 1.6 parts by weight Stearic acid 1.4 parts by weight Lipophilic glycerin monostearate 1.8 parts by weight PEG-100 stearate 2.6 parts by weight Sorbital sesquioleate 0.6 parts by weight Squalene 4.8 parts by weight Macadamia oil 2 parts by weight Jojoba oil 2 parts by weight Tocopherol acetate 0.4 parts by weight Methylpolysiloxane 0.2 parts by weight Ethylparaben 0.1 parts by weight Propylparaben 0.1 parts by weight 1,3-butylene glycol 4 parts by weight Methylparaben 0.1 parts by weight Xanthan gum 0.1 parts by weight Glycerin 4 parts by weight d-Panthenol 0.15 parts by weight Allantoin 0.1 parts by weight Calcium carbonate (2% aq. Sol) 4 parts by weight Triethanolamine 0.15 parts by weight Ethanol 3 parts by weight pt41891 0.1 parts by weight p-H2O 48.3 parts by weight

[0123] 3-3. Manufacturing softening lotion Peptide of the present invention 0.2% by weight Ethanol 10.0% by weight Polyoxyethylene sorbitan polylaurate 1.0% by weight Methyl parahydroxybenzoate 0.2% by weight Glycerin 5.0% by weight 1,3-butyl glycol 6.0% by weight Incense (appropriate amount) Dye appropriate amount Purified water (appropriate amount) Total 100

[0124] 3-4. Manufacturing of nutritional lotion Peptide of the present invention 0.1% by weight Vaseline 2.0% by weight Sorbitan sesquioleate 0.8% by weight Polyoxyethylene oleyl ethyl 1.2% by weight Methyl parahydroxybenzoate (appropriate amount) Propylene glycol 5.0% by weight Ethanol 3.2% by weight Carboxyvinyl polymer 18.0% by weight Dye appropriate amount Incense (appropriate amount) Purified water (appropriate amount) Total 100

[0125] 3-5. Essence production Peptide of the present invention 5.0% by weight Propylene glycol 10.0% by weight Glycerin 10.0% by weight Sodium hyaluronate aqueous solution (1%) 5.0% by weight Ethanol 3.2% by weight Polyoxyethylene hydrogenated castor oil 1.0% by weight Methyl parahydroxybenzoate 0.1% by weight Incense (appropriate amount) Purified water (appropriate amount) Total 100

[0126] 3-6. Pack manufacturing Peptide of the present invention 0.5% by weight Glycerin 5.0% by weight Propylene glycol 4.0% by weight Polyvinyl alcohol 15.0% by weight Ethanol 8.0% by weight Polyoxyethylene oleyl ethyl 1.0% by weight Methyl parahydroxybenzoate 0.2% by weight Incense (appropriate amount) Dye appropriate amount Purified water (appropriate amount) Total 100

[0127] The composition ratio is a mixture of suitable ingredients in a preferred embodiment, but the ingredients or the blending ratio may be arbitrarily modified depending on the demand class, demand country, use, regional and ethnic preferences, etc.

[0128] The above describes exemplary embodiments of the present application, but the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the field can make appropriate modifications within the scope of the claims of the present application.

Claims

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

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

4. The pharmaceutical composition for preventing or treating hyperpigmentation diseases according to claim 3, wherein the peptide (i) inhibits melanosome invagination or (ii) promotes melanosome degradation in keratinocytes.

5. 5. The pharmaceutical composition for preventing or treating hyperpigmentation diseases according to claim 4, wherein the peptide inhibits the expression of one or more genes selected from the group consisting of PAR2 (protease activated receptor 2) gene, TLR3 (Toll-like receptor 3) gene, and KGFR (keratinocyte growth factor receptor) gene in keratinocytes.

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

7. 4. The pharmaceutical composition for preventing or treating hyperpigmentation diseases according to claim 3, wherein the hyperpigmentation disease is age spots, freckles, age spots, solar lentigo, or hyperpigmentation that occurs after skin inflammation.

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

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

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

11. 11. The skin whitening cosmetic composition according to claim 10, wherein the peptide inhibits the expression of one or more genes selected from the group consisting of PAR2 (protease activated receptor 2) gene, TLR3 (Toll-like receptor 3) gene, and KGFR (keratinocyte growth factor receptor) gene in keratinocytes.

12. The cosmetic composition for skin whitening according to claim 9, wherein the cosmetic composition is for external application to the skin.

13. 13. The cosmetic composition for skin whitening according to claim 12, wherein the cosmetic composition is in one or more dosage forms 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 rinsing agent, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray.

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