Peptides having skin whitening activity and uses thereof

Abstract

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

Description

Technical Field

[0001] Cross - reference to related applications

[0002] This application claims priority to Korean Patent Application No. 10-2022-0155229, filed with the Korean Intellectual Property Office on November 18, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to peptides having skin-whitening activity and uses thereof. Background Art

[0004] Skin melanin is present in the skin, hair follicles, eyes, etc., and performs an important function of protecting the skin from ultraviolet rays. However, excessive melanin formation causes hyperpigmentation diseases such as melasma, freckles, senile lentigines, etc., thereby causing mental stress and a decline in quality of life. Melanin is mainly synthesized in melanocytes located between the basal layers of the epidermis or below the basal layer and in hair follicles. It is synthesized in melanosomes, which are organelles within melanocytes. These melanosomes move through dendrites to adjacent keratinocytes, and as the keratinocytes enter the outer layer of the skin, the skin color appears. Although the mechanism by which melanosomes move from melanocytes to keratinocytes has not been elucidated, it is known that melanosomes are released from there to the outside of the cell after moving to the dendrites of melanocytes, and are involved in membrane fusion between melanosomes and keratinocytes and in phagocytosis.

[0005] Regarding the synthesis process of melanin, it is ultimately synthesized from L-tyrosine through dopa (DOPA), dopaquinone, DOPA pigment, and 5,6-dihydroxyindole (DHI). Conventionally, the inhibition of melanin synthesis has focused on factors that inhibit the activity of tyrosinase, which is an enzyme that catalyzes the rate-limiting step of the melanin synthesis pathway.

[0006] PCT International Publication WO 2020 / 153819 discloses polypeptides having the activity of inhibiting tyrosinase activity and uses thereof for skin whitening, where tyrosinase is an important enzyme in the melanin synthesis pathway. In addition, Korean Patent No. 10-1869783 discloses peptides having the activity of inhibiting melanin production and tyrosinase activity and uses thereof for skin whitening.

[0007] To date, the development of skin whitening agents has focused on inhibiting the activity of tyrosinase, which is an important enzyme in melanin synthesis. However, in order to achieve a synergistic effect of skin whitening through various action points, it is necessary to develop skin whitening agents having different mechanisms of action or action points.

[0008] [Prior Art Documents]

[0009] [Patent Document]

[0010] Patent Document 1. WO 2020 / 153819

[0011] Patent Document 2. Korean Patent No. 10-1869783 Summary of the Invention

[0012] Technical problem

[0013] The present inventors have conducted research to develop peptides with improved activity and skin whitening activity. As a result, they have experimentally demonstrated that the novel peptides they have developed have excellent effects of inhibiting the incorporation of melanosomes into keratinocytes and excellent activity of degrading melanosomes, thus completing the present invention.

[0014] Accordingly, an object 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 composition for skin whitening, which comprises the peptide having the above activity as an active ingredient.

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

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

[0018] Technical solution

[0019] To achieve the above objects,

[0020] One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0021] Another object of the present invention is to provide a composition for skin whitening, which comprises the peptide as an active ingredient.

[0022] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hyperpigmentation diseases, which comprises the peptide as an active ingredient.

[0023] Another object of the present invention is to provide a cosmetic composition for skin whitening, which comprises the peptide as an active ingredient.

[0024] The present invention will be described in detail below.

[0025] 1. Peptide and its activity

[0026] According to one aspect of the present invention, there is provided a peptide comprising the amino acid sequence disclosed in SEQ ID NO:1.

[0027] As used herein, the term "peptide" refers to a linear molecule formed by linking amino acid residues together through peptide bonds.

[0028] The peptide of the present invention comprising the amino acid sequence of SEQ ID NO:1 can be used without modification, but within the range that does not affect the original activity of the peptide (such as skin whitening activity), variants or fragments thereof having different amino acid sequences by deletion, insertion, substitution of amino acid residues or a combination thereof can be used.

[0029] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. within the range that its activity remains unchanged.

[0030] The peptides of the present invention include peptides containing amino acid sequences substantially identical to the peptide comprising the amino acid sequence of SEQ ID NO:1 and variants or active fragments thereof. Substantially identical amino acid sequences refer to amino acid sequences having at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95% and at least 97% sequence identity with the amino acid sequence of SEQ ID NO:1, respectively. Additionally, the peptide may further include targeting sequences, tags, labeled residues, and amino acid sequences prepared for specific purposes of increasing the half-life or stability of the peptide.

[0031] The peptides of the present invention may have N-terminal and / or C-terminal modifications that select a part of the amino acid sequence and increase its activity. Through these N-terminal and / or C-terminal modifications, the stability of the peptides of the present invention can be significantly improved. For example, the half-life of the peptide can be increased when administered in vivo. The term "stability" refers to not only in vivo stability, which protects the peptides of the present invention from attack by in vivo proteolytic enzymes, but also storage stability (such as storage stability at room temperature).

[0032] The N-terminal modification can be the binding of a protective group to the N-terminal of the peptide, and the protective group is selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristoyl group, a stearoyl group, and polyethylene glycol (PEG). The C-terminal modification can be the binding of a hydroxyl group (-OH), an amino group (-NH 2 ), an azide group (-NHNH 2 ) etc. to the C-terminal of the peptide, but is not limited thereto.

[0033] The peptides of the present invention can be prepared by various methods widely known in the technical field to which the present invention pertains. For example, the peptides of the present invention can be prepared using 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, Ill. (1984)) or liquid-phase synthesis techniques (U.S. Patent No. 5,516,891).

[0034] The peptides of the present invention have the activity of whitening the skin.

[0035] In one embodiment, the peptides of the present invention have the activity of inhibiting the incorporation of melanosomes in keratinocytes or promoting the degradation of melanosomes.

[0036] Melanosomes produced in melanocytes move from melanocytes to keratinocytes. In particular, the movement of melanosomes to keratinocytes includes the process of releasing melanosomes from melanocytes and the process of incorporating melanosomes into keratinocytes. The process of incorporating melanosomes can be alternatively expressed as the uptake of melanosomes by keratinocytes or the phagocytosis of melanosomes by keratinocytes.

[0037] Protease-activated receptor (PAR2), Toll-like receptor 3 (TLR3), and keratinocyte growth factor receptor (KGFR) are involved in the absorption process of melanosomes released from melanocytes through keratinocyte phagocytosis.

[0038] In one embodiment, the peptides of the present invention inhibit the expression of one or more genes selected from the group consisting of protease-activated receptor 2 (PAR2) gene, Toll-like receptor 3 (TLR3) gene, and keratinocyte growth factor receptor (KGFR) gene in keratinocytes.

[0039] PAR2 is a receptor protein present in the cell membrane and is known to regulate inflammatory responses, obesity, metabolism, and cancer, and acts as a sensor for proteases during infection. The PAR2 protein is present in the membrane of keratinocytes and is involved in the absorption (incorporation) of melanosomes by keratinocytes. Inhibition of PAR2 protein activity is known to result in inhibition of 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.).

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

[0041] KGFR, which is a tyrosine kinase receptor, is known to be expressed in many types of epithelial cells, is activated by four known ligands (i.e., FGF-1, FGF-3, FGF-7, and FGF-10), and is involved in the proliferation and differentiation of epithelial cells and wound healing. In addition, KGFR is also known to promote the migration of melanosomes into keratinocytes (J Invest Dermatol. 2005 Dec;125(6):1190-9., Journal of Investigative Dermatology Volume 128, Number 3, March 2008, p. 558-567).

[0042] As described above, the peptide of the present invention can exhibit skin whitening activity and the effect of preventing or treating hyperpigmentation diseases by inhibiting the activity of incorporating melanosomes and the activity of degrading melanosomes.

[0043] 2. Composition for skin whitening and for preventing, treating or improving hyperpigmentation diseases

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

[0045] The peptide of the present invention having the amino acid sequence of SEQ ID NO:1 has skin whitening activity through the mechanism described above.

[0046] In another aspect of the present invention, the composition of the present invention can be a pharmaceutical composition for preventing or treating hyperpigmentation diseases, which comprises a peptide having the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0047] In one embodiment, the pharmaceutical composition comprising the peptide as an active ingredient inhibits the incorporation of melanosomes into keratinocytes or promotes the degradation of melanosomes.

[0048] In one embodiment, a pharmaceutical composition comprising a peptide inhibits the expression of one or more genes selected from the group consisting of protease-activated receptor 2 (PAR2) gene, Toll-like receptor 3 (TLR3) gene, and keratinocyte growth factor receptor (KGFR) gene in keratinocytes.

[0049] As used herein, the term "hyperpigmentation disease" refers to a disease caused by an excessive increase in the amount of melanin in the skin. Thus, due to the excessive increase in the amount of melanin in the skin, the skin color becomes darker. The causes of hyperpigmentation diseases can be, for example, exposure to sunlight, skin inflammation such as acne (post-inflammatory hyperpigmentation), injury, hormonal imbalance, or drugs, but are not limited thereto.

[0050] In one embodiment, the hyperpigmentation disease can be melasma, freckles, senile lentigines, solar lentigines, or post-inflammatory hyperpigmentation of the skin. Post-inflammatory hyperpigmentation can be hyperpigmentation that occurs after injury, ultraviolet rays, or skin inflammatory diseases such as acne.

[0051] The pharmaceutical composition of the present invention may comprise a therapeutically effective amount of the peptide of the present invention comprising the amino acids of SEQ ID NO:1.

[0052] The term "therapeutically effective amount" refers to an amount sufficient for the peptide to achieve its activity or efficacy, for example, an amount sufficient to achieve the efficacy of treating or preventing hyperpigmentation diseases, and the peptide is the active ingredient of the pharmaceutical composition of the present invention.

[0053] As used herein, the term "prevention" refers to reducing the risk of developing a disease or disorder, and refers to all actions that inhibit or delay the onset of a disease by preventing the progression of the disease or one or more of its clinical symptoms.

[0054] As used herein, the term "treatment" refers to alleviating a disease or disorder, and includes all actions 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 symptoms.

[0055] In the present invention, preventing or treating hyperpigmentation diseases can be removing the cause of hyperpigmentation in the skin or inhibiting the development of hyperpigmentation, and specifically, it can inhibit the incorporation of melanosomes into keratinocytes or promote the degradation of melanosomes.

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

[0057] Pharmaceutically acceptable carriers commonly used in formulations include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc., but the carriers are not limited thereto.

[0058] In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but the components are not limited thereto.

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

[0060] The pharmaceutical composition of the present invention can be administered by any route suitable for treating hyperpigmentation diseases, such as oral or parenteral administration. In the case of parenteral administration, the composition can 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 the activity of preventing or treating hyperpigmentation diseases, topical application of the composition is desirable, for example, by applying it to the skin.

[0061] The dose of the pharmaceutical composition can 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. The pharmaceutical composition can be prescribed in various ways according to factors such as the formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient.

[0062] The pharmaceutical composition of the present invention can be prepared in unit dosage form by using pharmaceutically acceptable carriers and / or excipients and according to methods that can be easily implemented by those skilled in the art, or the pharmaceutical composition can be prepared by placing it in a multi-dose container. In particular, the formulation can be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or can be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or a stabilizer.

[0063] The pharmaceutical composition of the present invention may be a topical skin preparation. A topical skin preparation is a preparation that can be used by applying it to the outside of the skin. When the pharmaceutical composition of the present invention is used as a topical skin preparation, it can be applied to the skin area where hyperpigmentation occurs. The topical skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspending agent, transdermal delivery patch, bandage containing a drug, lotion, or a combination thereof. The topical skin preparation may be appropriately mixed with components commonly used in topical skin preparations (such as cosmetics and drugs) as needed, such components as aqueous components, oil-based components, powder components, alcohols, humectants, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or a combination thereof. The topical skin preparation may be appropriately mixed with the following: metal chelating agents (such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc.), caffeine, tannins, licorice extract, glabridin, various herbs, tocopheryl acetate, glycyrrhizic acid, drugs (such as tranexamic acid and its derivatives or salts, etc.), vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, and saccharides (such as glucose, fructose, trehalose, etc.).

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

[0065] In one embodiment, the cosmetic composition comprising the peptide of the present invention as an active ingredient inhibits the incorporation of melanosomes into keratinocytes or promotes the degradation of melanosomes.

[0066] In one embodiment, the cosmetic composition comprising the peptide of the present invention as an active ingredient inhibits the expression of one or more genes selected from the group consisting of protease-activated receptor 2 (PAR2) gene, Toll-like receptor 3 (TLR3) gene, and keratinocyte growth factor receptor (KGFR) gene.

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

[0068] The cosmetic composition can be prepared in various forms, such as solutions (e.g., softening lotions, nourishing lotions, nourishing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, packs, sprays, powders, hair tonics, hair creams, hair lotions, hair shampoos, hair rinses, hair conditioners, hair sprays, hair aerosols, pomades, gels, etc.), sol-gels, emulsions, oils, waxes, aerosols, etc., but not limited thereto.

[0069] The cosmetic composition of the present invention may include other additives, such as excipients and carriers, and may incorporate and mix common ingredients typically mixed in general skin cosmetics as needed.

[0070] When the formulation of the cosmetic composition is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc, zinc oxide, etc. can be used as carrier components.

[0071] When the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as carrier components, and in particular, when the cosmetic composition is a spray, a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether may also be included, but not limited thereto.

[0072] When the cosmetic composition is in the form of a solution or emulsion, solvents, solubilizers or emulsifiers can be used as carrier components, such as water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butanediol, oils, glycerol fatty esters, polyethylene glycol, sorbitan fatty acid esters, etc.

[0073] When the formulation of the cosmetic composition is a suspension, liquid diluents (such as water, ethanol and propylene glycol), suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethylated sorbitan esters, polyoxyethylene sorbitan esters, etc.), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc. can be used as carrier components.

[0074] When the formulation of the cosmetic composition is a surfactant-containing cleanser, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurine, sarcosine esters, fatty acid amide ether sulfates, alkyl amide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acids, etc. can be used as carrier components.

[0075] When the formulation of the cosmetic composition is shampoo, the peptides of the present invention can be mixed with the base components for forming the shampoo, such as thickeners, surfactants, viscosity regulators, humectants, pH regulators, preservatives, and essential oils. CDE can be used as a thickener; LES (anionic surfactant) and coco betaine (amphoteric surfactant) can be used as surfactants; polyquarter can be used as a viscosity regulator; glycerol can be used as a humectant; and citric acid and sodium hydroxide can be used as pH regulators. Grapefruit extract, etc. can be used as preservatives; in addition, essential oils of cedar, mint, and rosemary, as well as silk amino acid, amyl alcohol, or vitamin E can be added.

[0076] In addition to the peptides and carrier components of the present invention, the components included as active ingredients in the cosmetic composition can include auxiliary components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, but are not limited thereto.

[0077] The peptides of the present invention can be included in the above composition, pharmaceutical composition, or cosmetic composition at a concentration of 0.01 μM to 1000 μM, and specifically, the peptides of the present invention can be at 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, or 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, or 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, or 1 μM to 200 μM; or 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM in the above composition, pharmaceutical composition, or cosmetic composition, but are not limited thereto.

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

[0079] In another aspect of the present invention, there is provided a peptide comprising the amino acid sequence of SEQ ID NO:1, for skin whitening or for preventing, treating or improving hyperpigmentation diseases.

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

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

[0082] 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 in the preparation of a medicament for preventing or treating hyperpigmentation diseases.

[0083] 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 in the preparation of a cosmetic for skin whitening.

[0084] Advantageous effects

[0085] The peptide of the present invention exhibits skin whitening activity by inhibiting the incorporation of melanosomes into keratinocytes and promoting the degradation of melanosomes. Therefore, the peptide of the present invention can be used as an active ingredient in a medicament for treating or preventing hyperpigmentation diseases caused by the deposition of excessive melanosomes, or as an effective ingredient in a cosmetic for skin whitening.

[0086] However, the effects of the present invention are not limited to the above effects, and those skilled in the art will clearly understand other effects not mentioned from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The results of melanin content analysis are shown, illustrating that the peptide of the present invention inhibits the incorporation of melanosomes into HaCaT cells.

[0088] Figure 2a The results of PCR analysis are shown, illustrating that the peptide of the present invention reduces the mRNA level of PAR2, a receptor related to melanosome incorporation, in a concentration-dependent manner in HaCaT cells.

[0089] Figure 2b A graph showing Figure 2a the measured values of the band densities of the electrophoresis shown.

[0090] Figure 3a The results of PCR analysis are shown, demonstrating that the peptide of the present invention reduces the mRNA level of TLR3 in HaCaT cells, and TLR3 is a receptor associated with melanosome incorporation.

[0091] Figure 3b Shows Figure 3a a graph of the measured band densities of the electrophoresis described.

[0092] Figure 4a The results of PCR analysis are shown, demonstrating that the peptide of the present invention reduces the mRNA level of KGFR in HaCaT cells, and KGFR is a receptor associated with melanin incorporation.

[0093] Figure 4b Shows Figure 4a a graph of the measured band densities of the electrophoresis described.

[0094] Figure 5 The results of melanosome content analysis are shown, demonstrating that the peptide of the present invention induces the degradation of melanosomes in HaCaT cells. Detailed Description of the Invention

[0095] Hereinafter, the present invention will be described in detail. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0096] Preparation Example 1: Preparation of Peptide

[0097] 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 then these synthesized peptides were purified using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA).

[0098] [Table 1]

[0099]

[0100] The efficacy of the prepared peptide of SEQ ID NO: 1 was evaluated by the following experiments.

[0101] Experimental Example 1: Analysis of Incorporation by Phagocytosis of Melanosomes

[0102] Confirm whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 inhibits the incorporation of melanosomes by phagocytosis in keratinocytes.

[0103] The human keratinocyte cell line (HaCaT cells) was seeded at a density of 3×10 5 cells / well in a 6-well cell culture plate and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS for 24 hours. Then, the cells were washed once with serum-free DMEM. A solution of the peptide of SEQ ID NO:1 prepared in 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, and each of the solutions was added to the cultured cells. In particular, the negative control group (CON, (+)) was the untreated group, and the positive control group was treated with 500 nM of 6-amino-1-[4-(3-methyl-1-oxobutyl)-1-piperazinyl]-1-hexanone hydrochloride (ENMD-1068, Sigma-Aldrich). Then, after culturing them in a CO 2 incubator at 37°C for 24 hours, melanosomes (10 μg / mL) isolated from human epidermal melanocyte-darkly pigmented donors (HEM-DP) were additionally added to the cultured cells excluding the negative control (CON) group. Thereafter, the cells were cultured in a CO 2 incubator at 37°C for 48 hours, washed three times with PBS, treated with 1X TE for 10 minutes, and HaCaT cells were collected. HaCaT cells were recovered using a centrifuge, dissolved in 1 M NaOH, and dispensed into a 96-well plate. The melanin content was measured by absorbance at 490 nm using an ELISA reader.

[0104] As a result of the experiment, as can be seen from the results of the melanin content analysis shown in Figure 1 , it was confirmed that the incorporation of melanosomes into HaCaT cells was inhibited by the peptide of SEQ ID NO:1.

[0105] Experimental Example 2: Analysis of the expression of PAR2 gene related to melanosome phagocytosis

[0106] An experiment was conducted on the effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of protease-activated receptor 2 (PAR2) in keratinocytes, which is a gene related to melanosome phagocytosis.

[0107] HaCaT cells were seeded at 3×10 5Cells were seeded at a density of cells / well in a 6-well plate and cultured in DMEM medium containing 10% FBS for 24 hours. Then, the cells were washed once with serum-free DMEM medium, and a solution of the peptide of SEQ ID NO.1 prepared in Preparation Example 1 was added to 3 mL of serum-free DMEM medium to prepare peptide solutions with concentrations of 10 μM, 50 μM, and 100 μM, respectively, and the solutions were each dispensed into the cells. In particular, the negative control group (CON, (+)) was the untreated group, and the positive control group was treated with 0.02X and 0.05X protease inhibitor (PI) (cOmplete TM Protease Inhibitor Cocktail, Roche, CH). Then, after culturing in a CO 2 incubator at 37 °C for 24 hours, 4 units of trypsin were added to the cultured cells excluding the negative control (CON) group. Thereafter, the cells were cultured in a CO 2 incubator at 37 °C for 16 hours, washed twice with PBS, and RNA was isolated using easy blue (iNtRON, product number: 17061, Korea). After quantifying the amount of the isolated RNA, 2000 ng of RNA was dispensed into each tube, and cDNA was synthesized using a cDNA synthesis kit (Enzynomics, product number: RT200, Korea). PCR was performed using primers targeting the PAR2 gene shown in Table 2 and a PCR kit (Enzynomics, product number: P581T, Korea). Next, the PCR products were electrophoresed on a 1.2% agarose gel, and the bands were detected and analyzed using a Bio-Rad gel imaging system.

[0108] [Table 2]

[0109]

[0110] As a result of the experiment, as Figure 2a and 2b shown, it was confirmed that the peptide of SEQ ID NO:1 decreased the mRNA level of the PAR2 gene, which is a receptor related to melanosome phagocytosis (incorporation), in a concentration-dependent manner in HaCaT cells. These results confirmed that the peptide of SEQ ID NO:1 inhibits the incorporation of melanosomes.

[0111] Experimental Example 3: Expression analysis of the TLR3 gene related to melanosome phagocytosis

[0112] An experiment was conducted on the effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of Toll-like receptor 3 (TLR3), which is a gene related to melanosome phagocytosis.

[0113] HaCaT cells were seeded in 6-well plates at a density of 3×10 5 cells / well and cultured in DMEM medium containing 10% FBS for 24 hours. Then, the cells were washed once with serum-free DMEM medium, and then 2 mL of serum-free DMEM medium was added to the cells. Then the cells were cultured in a CO 2 incubator at 37 °C for 24 hours. The peptide of SEQ ID NO:1 prepared in 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, and each of these solutions was added to the cells. After 30 minutes, polyinosinic:polycytidylic acid (poly(I:C)) at 30 μg / mL was additionally added. Subsequently, the cells were cultured in a CO 2 incubator at 37 °C for 24 hours, washed twice with PBS, and RNA was isolated using easy blue (iNtRON, product number: 17061, Korea). After quantifying the amount of the isolated RNA, 2000 ng of RNA was dispensed into each tube, and cDNA was synthesized using a cDNA synthesis kit (Enzynomics, product number: RT200, Korea). PCR was performed using the primers targeting the TLR3 gene in Table 3 and a PCR kit (Enzynomics, product number: P581T, Korea). Then, the PCR products were electrophoresed on a 1.2% agarose gel, and the bands were detected and analyzed using a Bio-Rad gel imaging system.

[0114] [Table 3]

[0115]

[0116] As a result of the experiment, as Figure 3a and 3b shown, it was confirmed that, compared with the control group, the peptide of SEQ ID NO:1 reduced the mRNA level of the TLR3 gene in HaCaT cells, and TLR3 is a receptor related to melanosome phagocytosis (incorporation). These results confirmed that the peptide of SEQ ID NO:1 inhibits the incorporation of melanosomes.

[0117] Experimental Example 4: Expression analysis of the KGFR gene related to melanosome phagocytosis

[0118] An experiment was conducted on the effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of keratinocyte growth factor receptor (KGFR), which is a gene related to melanosome phagocytosis.

[0119] HaCaT cells were seeded at 3×10 5The cells were seeded at a density of cells / well in a 6-well plate and cultured in DMEM medium containing 10% FBS for 24 hours. Then, the cells were washed once with serum-free DMEM medium, and then 2 mL of serum-free DMEM medium was added to the cells. Then the cells were cultured in a CO 2 incubator at 37 °C for 24 hours. The peptide of SEQ ID NO:1 of Preparation Example 1 was added to 3 mL of DMEM medium containing 2% serum to prepare peptide solutions at concentrations of 10 μM, 50 μM, 100 μM, and 200 μM, and each of these solutions was added to the cells and the cells were cultured in a CO 2 incubator at 37 °C for 6 hours. Subsequently, after washing the cells twice with PBS, RNA was isolated using easy blue (iNtRON, product number: 17061, Korea). After quantifying the amount of isolated RNA, the RNA was aliquoted at 2000 ng per tube, and cDNA was synthesized using a cDNA synthesis kit (Enzynomics, product number: RT200, Korea). PCR was performed using the primers targeting the KGFR gene in Table 4 and a PCR kit (Enzynomics, product number: P581T, Korea). Then, the PCR products were electrophoresed on a 1.2% agarose gel, and the bands were detected and analyzed using a Bio-Rad gel imaging system.

[0120] [Table 4]

[0121]

[0122] As a result of the experiment, as Figure 4a and 4b shown, it was confirmed that, compared with the control group, the peptide of SEQ ID NO:1 decreased the mRNA level of the KGFR gene in HaCaT cells, and the KGFR gene is a receptor related to melanosome phagocytosis. These results confirmed that the peptide of SEQ ID NO:1 inhibits the incorporation of melanosomes.

[0123] Experimental Example 5: Melanosome degradation analysis

[0124] An analysis was performed on whether the peptide of SEQ ID NO:1 prepared in Preparation Example 1 degrades melanosomes.

[0125] HaCaT cells were seeded at 3×10 5The cells were seeded at a density of cells / well in a 6-well plate and cultured in DMEM medium containing 10% FBS for 24 hours. Then, melanosomes isolated from human epidermal melanocytes - dark pigment donor (HEM-DP) were added to 2 mL of DMEM medium containing 2% serum to prepare a melanosome solution at a concentration of 10 μg / mL, which was distributed to the cells, and the cells were incubated in a CO 2 incubator at 37 °C for 48 hours.

[0126] Subsequently, 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 of 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 the peptide solutions were additionally distributed to the cells. Specifically, the negative control group (CON) was the untreated group, where the negative control group (+) was treated only with the melanosome solution, while the positive control groups included the group treated with the melanosome solution and 100 nM and 200 nM rapamycin (Rapa), and the group treated with the melanosome solution and 200 μM and 500 μM resveratrol (RSV). Then, the cells were cultured in a CO 2 incubator at 37 °C for 72 hours, washed three times with PBS, then treated with 1X TE for 10 minutes, and HaCaT cells were collected. The HaCaT cells were recovered using a centrifuge, dissolved in 1 M NaOH, and distributed into a 96-well plate. The content of melanin was measured by absorbance at 490 nm using an ELISA reader.

[0127] As a result of the experiment, it can be seen from the change in the melanosome content in Figure 3 that it was confirmed that treatment with the peptide of SEQ ID NO:1 induced the degradation of melanosomes in HaCaT cells.

[0128] Preparation Example 2: Preparation of a pharmaceutical composition

[0129] 2-1. Preparation of an ointment

[0130] 5 g of the peptide of the present invention

[0131] 20 g of cetyl palmitate

[0132] 40 g of cetyl alcohol

[0133] 40 g of stearyl alcohol

[0134] 80 g of isopropyl myristate

[0135] Polysorbate 60 g

[0136] Propyl paraoxybenzoate 1 g

[0137] Methyl paraoxybenzoate 1 g

[0138] Phosphoric acid and purified water in sufficient amounts

[0139] Prepare an ointment by incorporating the above components in the specified amounts according to a conventional ointment preparation method.

[0140] 2-2. Preparation of powder

[0141] The peptide of the present invention 2 g

[0142] Lactose 1 g

[0143] Prepare a powder by mixing the above components and then filling the mixture into an airtight bag.

[0144] 2-3. Tablet preparation

[0145] The peptide of the present invention 100 mg

[0146] Corn starch 100 mg

[0147] Lactose 100 mg

[0148] Magnesium stearate 2 mg

[0149] Prepare tablets by mixing the above components and then compressing the mixture according to a conventional tablet preparation method.

[0150] 2-4. Capsule preparation

[0151] The peptide of the present invention 100 mg

[0152] Corn starch 100 mg

[0153] Lactose 100 mg

[0154] Magnesium stearate 2 mg

[0155] Prepare capsules by mixing the above components and then filling the mixture into gelatin capsules according to a conventional capsule preparation method.

[0156] 2-5. Pill preparation

[0157] The peptide of the present invention 1 g

[0158] Lactose 1.5 g

[0159] Glycerol 1 g

[0160] Xylitol 0.5 g

[0161] After mixing the above ingredients, pills are prepared in an amount of 4 g per pill according to a conventional method.

[0162] Preparation Example 3: Preparation of a cosmetic composition

[0163] 3-1. Preparation of a cream

[0164] 4.6 parts by weight of the peptide of the present invention

[0165] 2.8 parts by weight of cetostearyl alcohol

[0166] 2.6 parts by weight of beeswax

[0167] 1.4 parts by weight of stearic acid

[0168] 2 parts by weight of lipophilic glycerol monostearate

[0169] 1 part by weight of PEG-100 stearate

[0170] 1.4 parts by weight of sorbital sesquioleate

[0171] 4 parts by weight of jojoba oil

[0172] 3.8 parts by weight of squalane

[0173] 1.1 parts by weight of polysorbate 60

[0174] 2 parts by weight of macadamia oil

[0175] 0.2 parts by weight of tocopherol acetate

[0176] 0.4 parts by weight of methylpolysiloxane

[0177] 0.1 parts by weight of ethyl p-hydroxybenzoate

[0178] 0.1 parts by weight of propyl p-hydroxybenzoate

[0179] 0.1 parts by weight of Euxyl K-400

[0180] 7 parts by weight of 1,3-butylene glycol

[0181] 0.05 parts by weight of methyl p-hydroxybenzoate

[0182] 6 parts by weight of glycerol

[0183] 0.2 parts by weight of D-panthenol

[0184] 0.2 parts by weight of triethanolamine

[0185] 0.2 parts by weight of pt 41891

[0186] p-H 2 46.05 parts by weight of O

[0187] 3-2. Preparation of lotion

[0188] 3.5 parts by weight of the peptide of the present invention

[0189] 1.6 parts by weight of cetostearyl alcohol

[0190] 1.4 parts by weight of stearic acid

[0191] 1.8 parts by weight of lipophilic glycerol monostearate

[0192] 2.6 parts by weight of PEG-100 stearate

[0193] 0.6 parts by weight of sorbital sesquioleate

[0194] 4.8 parts by weight of squalene

[0195] 2 parts by weight of macadamia oil

[0196] 2 parts by weight of jojoba oil

[0197] 0.4 parts by weight of tocopherol acetate

[0198] 0.2 parts by weight of methylpolysiloxane

[0199] 0.1 parts by weight of ethyl p-hydroxybenzoate

[0200] 0.1 parts by weight of propyl p-hydroxybenzoate

[0201] 4 parts by weight of 1,3-butylene glycol

[0202] 0.1 parts by weight of methyl p-hydroxybenzoate

[0203] 0.1 parts by weight of xanthan gum

[0204] 4 parts by weight of glycerol

[0205] 0.15 parts by weight of D-panthenol

[0206] Allantoin 0.1 part by weight

[0207] Calcium carbonate (2% aqueous solution) 4 parts by weight

[0208] Triethanolamine 0.15 part by weight

[0209] Ethanol 3 parts by weight

[0210] pt 41891 0.1 part by weight

[0211] p-H 2 O 48.3 parts by weight

[0212] 3-3. Preparation of softening lotion

[0213] The peptide of the present invention 0.2 wt%

[0214] Ethanol 10.0 wt%

[0215] Polyoxyethylene sorbitan polyolaurate 1.0 wt%

[0216] Methyl paraoxybenzoate 0.2 wt%

[0217] Glycerol 5.0 wt%

[0218] 1,3-Butanediol 6.0 wt%

[0219] Fragrance Sufficient

[0220] Dye Sufficient

[0221] Purified water Sufficient

[0222] Total 100

[0223] 3-4. Preparation of nourishing lotion

[0224] The peptide of the present invention 0.1 wt%

[0225] Vaseline 2.0 wt%

[0226] Sorbitan sesquioleate 0.8 wt%

[0227] Polyoxyethylene oleyl ethyl 1.2 wt%

[0228] Methyl paraoxybenzoate Sufficient

[0229] Propylene glycol 5.0 wt%

[0230] Ethanol 3.2 wt%

[0231] Carboxyvinyl polymer 18.0 wt%

[0232] Dye sufficient amount

[0233] Fragrance sufficient amount

[0234] Purified water sufficient amount

[0235] Total 100

[0236] 3 - 5. Preparation of essence

[0237] Peptide of the present invention 5.0 wt%

[0238] Propylene glycol 10.0 wt%

[0239] Glycerol 10.0 wt%

[0240] Sodium hyaluronate (1%) aqueous solution 5.0 wt%

[0241] Ethanol 3.2 wt%

[0242] Polyoxyethylene hydrogenated castor oil 1.0 wt%

[0243] Methyl p - hydroxybenzoate 0.1 wt%

[0244] Fragrance sufficient amount

[0245] Purified water sufficient amount

[0246] Total 100

[0247] 3 - 6. Preparation of membrane (pack)

[0248] Peptide of the present invention 0.5 wt%

[0249] Glycerol 5.0 wt%

[0250] Propylene glycol 4.0 wt%

[0251] Polyvinyl alcohol 15.0 wt%

[0252] Ethanol 8.0 wt%

[0253] Polyoxyethylene oleyl ethyl 1.0 wt%

[0254] Methyl paraoxybenzoate 0.2 wt%

[0255] Fragrance sufficient amount

[0256] Dye sufficient amount

[0257] Purified water in sufficient amount

[0258] Total 100

[0259] The above composition ratio is a mixture of suitable components in a preferred embodiment, but its components or mixing ratio can be arbitrarily modified according to regional or ethnic preferences (such as demand segmentation, countries of demand, and intended use).

[0260] In the foregoing, representative embodiments of the present application have been described by way of exemplary embodiments, but the scope of the present application is not limited to the above 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 SEQ ID NO:

1.

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

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

4. The pharmaceutical composition according to claim 3, wherein in keratinocytes, the peptide: (i) inhibits the incorporation of melanosomes; or (ii) promotes the degradation of melanosomes.

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

6. The pharmaceutical composition according to claim 3, wherein the hyperpigmentation disease is a disease that occurs when the amount of melanin in the skin increases excessively.

7. The pharmaceutical composition according to claim 3, wherein the hyperpigmentation disease is melasma, freckles, senile lentigines, solar lentigines, or post-inflammatory hyperpigmentation of the skin.

8. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is a skin preparation for external use.

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

10. The cosmetic composition according to claim 9, wherein in keratinocytes, the peptide: (i) inhibits the incorporation of melanosomes; or (ii) promotes the degradation of melanosomes.

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

12. The cosmetic composition according to claim 9, wherein the cosmetic composition is a skin preparation for external use.

13. The cosmetic composition according to claim 12, wherein the cosmetic composition is at least one formulation selected from the group consisting of solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, foundation, emulsion foundation, wax foundation, and spray.

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