Peptides having skin whitening activity and uses thereof

Abstract

The peptides of the present invention exhibit skin whitening activity by inhibiting melanin synthesis in melanocytes and inhibiting melanin body migration. 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 excessive deposition of melanin bodies, or as an active ingredient in a cosmetic for skin whitening.

Description

Cross-reference to related applications

[0001] This application claims priority based on Korean Patent Application No. 10-2023-0138102, filed on October 16, 2023, the entire contents of which are incorporated herein by reference as a part of this specification. Technical Field

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

[0003] Cutaneous melanin, found in the skin, hair follicles, and eyes, plays a crucial role in protecting the skin from UV damage. However, excessive melanin production can lead to hyperpigmentation disorders such as melasma, freckles, and age spots, causing psychological stress and a decreased quality of life. Melanin is primarily synthesized in melanocytes located between or beneath the basal layer of the epidermis and in hair follicles. Melanin is synthesized in melanosomes, organelles within melanocytes. These melanosomes migrate via dendrites to nearby keratinocytes, and as the keratinocytes rise to the outer layer, they contribute to skin color. Melanin synthesis begins with L-tyrosine, which sequentially synthesizes DOPA, DOPAquinone, DOPAchrome, and DHI (5,6-dihydroxyindole), ultimately resulting in melanin. The reactions from L-tyrosine to DOPA and from DOPA to dopaquinone are catalyzed by tyrosinase, the most important enzyme in melanin formation. Additionally, enzymes TRP1 (tyrosinase-associated protein 1) and TRP2 (tyrosinase-associated protein 2) also participate in the synthesis of melanin.

[0004] The mechanism by which melanin-containing melanosomes migrate from melanocytes to keratinocytes is not fully understood. However, it is known that melanosomes migrate to the dendrites of melanocytes and are then released into the extracellular space, followed by membrane fusion and phagocytosis between the melanosomes and keratinocytes. Melanophilin, Rab27a, and myosin 5a proteins are known to be involved in the migration of melanosomes to the dendrites of melanocytes. Rab27a recognizes and binds to melanosomes, while melanophilin links Rab27a to myosin 5a (an intracellular cytoskeletal protein). Therefore, the melanosome forms a complex with Rab27a, melanavitin and myosin 5a proteins and is transported to the terminal part of the dendrite, eventually reaching the keratinocyte (J. Biochem. 2012;151(4):343-351, Lysosome-Related Organelles M Fukuda, Tohoku University, Sendai, Miyagi, Japan 2016 Elsevier Inc).

[0005] PCT International Publication No. WO2020 / 153819 discloses a peptide that inhibits the activity of tyrosinase (a key enzyme in the melanin synthesis pathway) and its use for skin whitening. Furthermore, Korean Patent No. 10-1869783 describes a peptide that inhibits both melanin production and tyrosinase activity and its use for skin whitening. Previous development of skin whitening agents has focused on inhibiting the activity of tyrosinase (a key enzyme in melanin synthesis). However, in order to achieve synergistic skin whitening effects through different sites of action, it is necessary to develop skin whitening agents with different mechanisms and sites of action.

[0006] [Existing technical documents] [Patent Literature] WO2020 / 153819 Korean Patent No. 10-1869783 Summary of the Invention Technical issues The inventors conducted research to develop peptides with improved skin-whitening activity. As a result, the inventors experimentally demonstrated that their newly developed peptides not only inhibit melanin synthesis in melanocytes but also inhibit melanosome migration, and thus can be developed into skin-whitening agents and for the treatment of hyperpigmentation disorders, thereby completing this invention.

[0007] Therefore, one object of the present invention is to provide novel peptides with skin whitening activity.

[0008] Another object of the present invention is to provide a composition for skin whitening, comprising peptides having the above-described activity as active ingredients.

[0009] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising a peptide having the above-described activity as an active ingredient.

[0010] Another object of the present invention is to provide a cosmetic composition for skin whitening, comprising peptides having the above-described activity as active ingredients.

[0011] Technical solution In order to achieve the above objectives, One aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0012] Another aspect of the present invention provides a composition for skin whitening, comprising the peptide as an active ingredient.

[0013] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising the peptide as an active ingredient.

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

[0015] The present invention will now be described in detail.

[0016] 1. Peptides and their activities According to one aspect of the invention, a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1 is provided.

[0017] [Amino acid sequence of SEQ ID NO:1] THTISR(Thr-His-Thr-Ile-Ser-Arg) As used in this article, the term "peptide" refers to a linear molecule formed by amino acid residues linked together by peptide bonds.

[0018] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention can be used without modification, but variants or fragments of amino acids having different sequences by means of deletion, insertion, substitution or combination thereof may also be used without affecting the original activity of the peptide (e.g., skin whitening activity).

[0019] The peptides of the present invention can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, etc., without changing their activity.

[0020] The peptides of the present invention comprise: peptides comprising an amino acid sequence substantially identical to that of the peptide comprising the amino acid sequence of SEQ ID NO: 1, and variants thereof or active fragments thereof. A substantially identical amino acid sequence means an amino acid sequence having at least 75%, for example, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 1. Furthermore, the peptide may additionally comprise a targeting sequence, a tag, labeled residues, or an amino acid sequence prepared for a specific purpose of increasing half-life or peptide stability.

[0021] The peptides of the present invention may be modified at the N-terminus and / or C-terminus to select a portion of the amino acid sequence and enhance their activity. Such N-terminal and / or C-terminal modifications can significantly enhance the stability of the peptides of the present invention and, for example, increase the half-life of the peptides when administered in vivo. The term "stability" above is intended to cover not only in vivo stability (which protects the peptides of the present invention from attack by in vivo protein-cleaving enzymes) but also storage stability (e.g., room temperature storage stability).

[0022] N-terminal modification can be a modification in which a protecting group selected from the group consisting of acetyl, fluoreonylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, and polyethylene glycol (PEG) is attached to the N-terminus of the peptide. C-terminal modification can be, but is not limited to, a hydroxyl (-OH), amino (-NH2), or azide (-NHNH2) group attached to the C-terminus of the peptide.

[0023] The peptides of the present invention can be prepared by a variety of methods widely known in the art to which this invention pertains. For example, the peptides of the present invention can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis (US Patent No. 5,516,891).

[0024] The peptides of this invention have skin whitening activity, as well as preventive or therapeutic activity against hyperpigmentation disorders.

[0025] The peptides of this invention have the activity of inhibiting melanin synthesis in melanocytes.

[0026] In the signaling pathway of melanin synthesis enhanced by α-MSH in melanocytes, MITF (microphthalmia-associated transcription factor) is used as a key positive transcription factor for melanin synthesis, and CREB (cAMP response element binding protein) is a known transcription factor that promotes MITF expression (EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 173-185, 2020).

[0027] In one embodiment, under conditions that induce (promote) melanin synthesis, the peptide of the present invention inhibits the increase in CREB phosphorylation levels in melanocytes.

[0028] Under conditions that induce (promote) melanin synthesis, the peptides of the present invention have the activity of inhibiting melanosome migration in melanocytes.

[0029] In one implementation, melanin synthesis can be induced by α-MSH.

[0030] Melanosomes produced in melanocytes migrate to the ends of melanocyte dendrites, are released from the melanocytes, and are incorporated into adjacent keratinocytes, eventually being delivered to the keratinocytes. It is known that factors such as Rab27a, MYO5A (myosin VA), and MLPH (melavisin) are involved in the melanosome migration process in melanocytes (J. Biochem. 2012;151(4):343-351, Lysosome-Related Organelles M Fukuda, Tohoku University, Sendai, Miyagi, Japan 2016 Elsevier Inc).

[0031] In one embodiment, under conditions that promote melanin synthesis, the peptide of the present invention has the activity of inhibiting the upregulation of expression levels of melanosome migration-related factors Rab27a, MYO5A (myosin VA), and MLPH (melanophilin) ​​in melanocytes.

[0032] As described above, the peptides of the present invention can exhibit skin whitening activity and preventive or therapeutic effects on hyperpigmentation diseases by inhibiting melanin synthesis in melanocytes and inhibiting melanosome migration.

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

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

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

[0036] In one embodiment, a pharmaceutical composition comprising the peptide of the present invention as an active ingredient inhibits melanin synthesis in melanocytes or inhibits melanosome migration.

[0037] In one embodiment, a pharmaceutical composition comprising the peptide of the present invention as an active ingredient, under conditions that promote melanin synthesis, inhibits in melanocytes (i) the increase in phosphorylation of CREB (cAMP response element binding protein), (ii) the increase in expression levels of MITF (microphthalmia-associated transcription factor), TYR (tyrosinase), or TYRP1 (tyrosinase-associated protein 1), or (iii) the increase in expression levels of Rab27a, MYO5A, or MLPH (melanavitin).

[0038] In this specification, the term "hyperpigmentation disorder" refers to a disease caused by an excessive increase in the amount of melanin in the skin.

[0039] Therefore, the skin color darkens due to an excessive increase in melanin levels. Causes of hyperpigmentation include, but are not limited to, sun exposure, skin inflammation such as acne, trauma, hormonal imbalances, and drug-induced hyperpigmentation (post-inflammatory hyperpigmentation).

[0040] In one implementation, hyperpigmentation disorders can be melasma, freckles, age-related hyperpigmentation, solar lentigo, or post-inflammatory hyperpigmentation of the skin. Post-inflammatory hyperpigmentation of the skin can be caused by trauma, ultraviolet radiation, or inflammatory skin conditions such as acne.

[0041] The pharmaceutical compositions of the present invention may include a therapeutically effective amount of the peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 1.

[0042] The term "therapeutic effective amount" refers to an amount sufficient to enable the peptide (the active ingredient of the pharmaceutical composition of the present invention) to achieve its activity or efficacy, such as an amount sufficient to achieve the efficacy of treating or preventing hyperpigmentation disorders.

[0043] As used herein, the term “prevention” means reducing the risk of the development of a disease or disorder, and refers to any action that inhibits or delays the onset of a disease by preventing the progression of the disease or one or more of its clinical symptoms.

[0044] As used herein, the term “treatment” means to alleviate a disease or disorder and includes any action that improves or beneficially alters the symptoms of a disease by halting or reducing the progression of the disease or one or more of its clinical symptoms.

[0045] In this invention, the prevention or treatment of hyperpigmentation can be to eliminate the cause of hyperpigmentation in the skin or inhibit the progression of hyperpigmentation, and specifically to inhibit the incorporation of melanosomes into keratinocytes or promote the decomposition of melanosomes.

[0046] The pharmaceutical compositions of the present invention may include pharmaceutically acceptable carriers.

[0047] Pharmaceutically acceptable carriers are those commonly used in formulations, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0048] In addition to the above-mentioned components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but is not limited thereto.

[0049] Suitable pharmaceutically acceptable carriers and formulations are described in detail in "Remington: The Science and Practice of Pharmacy" (19th ed., 1995, Williams & Wilkins).

[0050] The pharmaceutical compositions of the present invention can be administered via any suitable route for treating hyperpigmentation disorders, such as oral or parenteral administration. In the case of parenteral administration, the compositions can be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical application, transdermal application, etc. Since the pharmaceutical compositions of the present invention have activity in preventing or treating hyperpigmentation disorders of the skin, they are preferably applied topically, such as by applying them to the skin.

[0051] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg per day, and may be prescribed in various ways depending on factors such as the method of preparation, method of administration, patient's age, weight, sex, pathological condition, food intake, time of administration, route of administration, excretion rate and sensitivity to response.

[0052] The pharmaceutical compositions of the present invention can be formulated into unit dosage forms using pharmaceutically acceptable carriers and / or excipients, or by means readily practicable by those skilled in the art, or by inserting them into multi-dose containers. In this case, the dosage form can be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granules, tablet, or capsule, and may additionally include dispersants or stabilizers.

[0053] The pharmaceutical compositions of the present invention can be topical skin preparations. Topical skin preparations are preparations that can be applied externally to the skin. When the pharmaceutical compositions of the present invention are used as topical skin preparations, they can be applied to the skin, specifically to areas of skin that have already developed hyperpigmentation. Topical skin preparations can be creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, medicated bandages, lotions, or combinations thereof. Topical skin preparations can be appropriately mixed as needed with ingredients commonly used in topical skin preparations (such as cosmetics or pharmaceuticals), such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. Topical preparations may also contain, as appropriate, metal chelators (such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid); caffeine, tannins, verapamil, licorice extract, glycyrrhizin, etc. pawpaw ( Pseudocydonia sinensis Hot water extracts of the fruit; various herbs; tocopheryl acetate, glycyrrhizic acid, tranexamic acid and their derivatives or salts thereof; and vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid and sugars (such as glucose, fructose and trehalose).

[0054] In another aspect of the invention, the invention provides a cosmetic composition for skin whitening, comprising: a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0055] In one embodiment, a cosmetic composition including the peptide of the present invention as an active ingredient inhibits melanin synthesis in melanocytes or inhibits melanosome migration.

[0056] In one embodiment, a cosmetic composition comprising the peptide of the present invention as an active ingredient, under conditions that induce melanin synthesis, inhibits in melanocytes (i) the increase in phosphorylation of CREB (cAMP response element binding protein), (ii) the increase in expression levels of MITF (microphthalmia-associated transcription factor), TYR (tyrosinase), or TYRP1 (tyrosinase-associated protein 1), or (iii) the increase in expression levels of Rab27a, MYO5A, or MLPH (melanavitin).

[0057] Cosmetic compositions can be formulated into any dosage form commonly prepared in the technical field to which this invention pertains, and can be topical skin preparations. For example, cosmetic compositions can be formulated into solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, and sprays, but are not limited thereto.

[0058] Cosmetic compositions can be formulated in various forms, such as solutions, sol-gels, emulsions, oils, waxes, and aerosols, including but not limited to: flexible toners, nourishing toners, moisturizing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, face masks (packs), sprays, loose powders, hair tonics, hair creams, hair lotions, hair shampoo, hair rinses, hair conditioners, hair sprays, hair aerosols, hair waxes, and gels.

[0059] The cosmetic compositions of the present invention may include other additives, such as excipients and carriers, and can be applied and mixed with commonly used ingredients in general skin cosmetics as needed.

[0060] When the dosage form of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, astragalus gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0061] When the cosmetic composition is in the form of a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, a propellant such as chlorofluorocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto.

[0062] When the dosage form of a cosmetic composition is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, and examples of such components include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glyceryl fatty acid esters, polyethylene glycol, or fatty acid esters of sorbitol.

[0063] When the dosage form of a cosmetic composition is a suspension, liquid diluents (such as water, ethanol, or propylene glycol), suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethyl sorbitol ester, and polyoxyethylene dehydrated sorbitol ester), microcrystalline cellulose, aluminum hydroxide, bentonite, agar, or astragalus gum can be used as carrier components.

[0064] When the dosage form of the cosmetic composition is a surfactant-containing cleanser, fatty alcohol sulfates / esters, fatty alcohol ether sulfates / esters, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurate / esters, sarcosinates / esters, fatty acid amide ether sulfates / esters, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters can be used as carrier components.

[0065] When the cosmetic composition is in the form of a shampoo, it can be mixed with base ingredients used to form the shampoo, such as thickeners, surfactants, viscosity modifiers, humectants, pH adjusters, preservatives, and essential oils. CDE can be used as a thickener, LES (an anionic surfactant) and cocamidopropyl betaine (an amphoteric surfactant) can be used as surfactants, polyquaternium salts can be used as viscosity modifiers, glycerin can be used as a humectant, and citric acid or sodium hydroxide can be used as a pH adjuster. Grapefruit extract and similar ingredients can be used as preservatives; additionally, essential oils such as cedarwood, peppermint, and rosemary, as well as amino acids, pentaol, or vitamin E can be added.

[0066] In addition to the peptides of the present invention as active ingredients and carrier ingredients, the cosmetic composition may also include ingredients commonly used in cosmetic compositions, such as conventional adjuvants, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, but is not limited thereto.

[0067] The peptides of the present invention can be included in the above-described compositions, pharmaceutical compositions, or cosmetic compositions at concentrations from 0.01 μM to 1000 μM. Specifically, the peptides of the present invention can be in concentrations of 0.01 μM to 1000 μM; 0.05 μM to 800 μM; 0.05 μM to 700 μM; 0.05 μM to 600 μM; 0.05 μM to 500 μM; 0.05 μM to 300 μM; 0.05 μM to 200 μM; 0.1 μM to 800 μM; 0.1 μM to 700 μM; 0. Concentrations from 1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1 μM to 200 μM; 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM are included, but not limited to.

[0068] 3. Uses of the peptides of the present invention In another aspect of the invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1 is provided for skin whitening, or for the prevention, treatment or improvement of hyperpigmentation disorders.

[0069] In another aspect of the invention, a skin whitening method is provided, comprising administering to a subject requiring skin whitening a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising said peptide.

[0070] In another aspect of the invention, a method for preventing or treating hyperpigmentation disorders is provided, the method comprising administering to a subject requiring prevention or treatment of hyperpigmentation disorders a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising said peptide.

[0071] In another aspect of the invention, the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 is provided in the preparation of a medicament for the prevention or treatment of hyperpigmentation disorders.

[0072] In another aspect of the invention, the use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 is provided in the preparation of cosmetics for skin whitening.

[0073] Beneficial effects The peptides of this invention exhibit skin-whitening activity by inhibiting melanin synthesis and melanosome migration in melanocytes. Therefore, the peptides of this invention can be used as active ingredients in medicaments for treating or preventing hyperpigmentation caused by excessive melanosome deposition, or as effective ingredients in skin-whitening cosmetics.

[0074] However, the effects of the present invention are not limited to those described above, and those skilled in the art will clearly understand from the following description other effects not mentioned. Attached Figure Description

[0075] Figure 1 The results of measuring melanin content after treating B16F10 cells with α-MSH, the peptide of Preparation Example 1, and arbutin are shown.

[0076] Figure 2 The results show the determination of CREB phosphorylation levels after treating B16F10 cells with α-MSH, the peptide from Preparation Example 1, and arbutin. Band values ​​were calculated by normalizing the densitometric intensity of the p-CREB band relative to the densitometric intensity of the actin band (loading control) and expressed as relative expression levels compared to the control set to 1.

[0077] Figure 3 The results show the expression levels of genes MITF, TYR, TYRP1, MYO5A, and MLPH, as determined by RT-PCR, after treating B16F10 cells with α-MSH, the peptide from Preparation Example 1, and arbutin. MITF, TYR, and TYRP1 are factors associated with melanin production, while MYO5A and MLPH are factors associated with melanosome migration. Band values ​​were calculated by normalizing the optical density intensity of each marker band relative to the optical density intensity of the GAPDH band (the loading control) and expressed as relative expression levels compared to the control set to 1.

[0078] Figure 4 The following Western blot analysis results are presented after treating B16F10 cells with α-MSH, the peptide from Preparation Example 1, and arbutin, showing the expression levels of MITF, tyrosinase, and TYRP1, as well as Rab27a and melanavitin. MITF, tyrosinase, and TYRP1 are factors associated with melanin production, while Rab27a and melanavitin are factors associated with melanosome migration. Band values ​​were calculated by normalizing the optical density intensity of each marker band relative to the optical density intensity of the actin band (the loading control) and expressed as relative expression levels compared to the control set to 1. Detailed Implementation

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

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

[0081] Table 1

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

[0083] Experimental Example 1: Analysis of Melanin Content To confirm whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 above inhibits melanin synthesis in melanocytes.

[0084] B16F10 mouse melanoma cells were used at a rate of 7 × 10⁻⁶ 4 Cells were seeded at a density of 1 / 2 well in 6-well cell culture plates and cultured for 24 hours. Subsequently, the cells were replaced with medium containing 2% FBS and treated with 200 ng / mL α-MSH (α-melanocyte-stimulating hormone) and the peptide prepared in Preparation Example 1, followed by incubation for 72 hours. At this time, the positive control was treated with 200 ng / mL α-MSH and 500 μM arbutin. After harvesting the cultured cells, cell lysates were prepared by treatment with 1N NaOH. The absorbance at 450 nm was measured using a spectrophotometer.

[0085] As a result of the experiment, from Figure 1 The analysis of melanin content in the cells confirmed that the melanin content in B16F10 cells increased after α-MSH treatment, and the increased melanin content decreased in a concentration-dependent manner after treatment with the peptide of Preparation Example 1.

[0086] Experiment Example 2: Analysis of the expression regulation of MITF, a transcription factor involved in melanin production By measuring the phosphorylation level of CREB (cAMP response element binding protein), the effect of the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 above on the expression regulation of MITF (microphthalmia-associated transcription factor), a transcription factor for melanin synthesis in melanocytes, was confirmed under conditions that promote melanin synthesis. In the α-MSH-induced melanin synthesis signaling pathway in melanocytes, CREB is known to act as a transcription factor for MITF and participate in regulating MITF expression (EXPERIMENTAL AND THERAPEUTIC MEDICINE 20:173-185, 2020).

[0087] B16F10 mouse melanoma cells were used at a rate of 3 × 10⁻⁶ 5 Cells were seeded at a density of 1:1000 in 6-well cell culture plates and cultured for 24 hours. The medium was then replaced with serum-free medium and incubated for another 24 hours. Cells were treated with 200 ng / mL α-MSH and the peptide from Preparation Example 1 and incubated for 10 minutes. At this time, the positive control group was treated with 200 ng / mL α-MSH and 500 μM arbutin. After washing with PBS, cell lysates were prepared by treatment with lysis buffer, and BCA was quantified to prepare an equal volume of protein samples. The prepared cell lysates and protein samples were electrophoresed using a 10% SDS-PAGE gel. The proteins separated by SDS-PAGE were transferred to a PVDF membrane and blocked with 5% skim milk for 30 minutes at room temperature. Primary antibodies against p-CREB (Cell Signaling, USA) and actin (Santa Cruz, USA) were diluted 1:1000 in 3% BSA and reacted with the PVDF membrane at 4°C for 16 hours. The membrane was washed three times for 15 minutes each time with 0.1% PBS-T (0.1% Tween-20 in PBS). Then, the secondary antibody (Jackson ImmunoResearch, USA) was diluted 1:2000 in 5% skim milk and reacted at room temperature for 1 hour. Cells were then washed three times for 15 minutes each time with 0.1% PBS-T (0.1% Tween-20 in PBS). After treatment with ECL solution (GE Healthcare, USA), expression levels were analyzed using an ImageQuant 800 (Cytiva, USA).

[0088] As a result of the experiment, from Figure 2 The analysis results of the phosphorylation level of CREB confirmed that the phosphorylation level of CREB increased by α-MSH treatment, and the increased phosphorylation level of CREB was reduced in a concentration-dependent manner by treatment with the peptide of Preparation Example 1.

[0089] Experiment Example 3: Expression Analysis of Factors Involved in Melanin Formation and Melanoma Body Migration—mRNA Expression Analysis Using RT-PCR The effects of the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 on the expression of melanin synthesis-related factors MITF (microphthalmia-associated transcription factor), TYR (tyrosinase), TYRP1 (tyrosinase-associated protein 1), and MYO5A (myosin VA) and MLPH (melanocyte avidin) in melanocytes were confirmed by measuring mRNA expression levels using RT-PCR.

[0090] B16F10 mouse melanoma cells were used at a rate of 5 × 10⁻⁶ 4 Cells were seeded at a density of 200 ng / well in 6-well cell culture plates and cultured for 24 hours. The medium was then replaced with 2% FBS and treated with 200 ng / mL α-MSH and the peptide from Preparation Example 1, followed by incubation for 72 hours. The positive control group was treated with 200 ng / mL α-MSH and 500 μM arbutin. After washing with PBS, RNA was isolated by treatment with 300 μl of Easy Blue (intron, Korea). The isolated RNA was quantified, and cDNA synthesis was performed using a cDNA synthesis kit (enzynomics, Korea). PCR was performed using the primers and PCR premix (enzynomics, Korea) listed in Table 2. PCR products were electrophoresed on a 1.5% agarose gel. Bands were measured using a Bio-Rad gel imaging system.

[0091] [Table 2]

[0092] As a result of the experiment, from Figure 3 The results showed that the expression of MITF, TYR, and TYRP1 genes, which are factors related to melanin production, and MYO5A and MLPH genes, which are factors related to melanosome migration, was increased by α-MSH treatment, and the increased gene expression levels were reduced again by treatment with the peptide of Preparation Example 1.

[0093] Example 4: Expression Analysis of Factors Involved in Melanin Production and Melanoma Body Migration—Protein Expression Analysis Using Western Blotting The effects of the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 on the expression of factors related to melanin synthesis in melanocytes, such as MITF (microphthalmia-associated transcription factor), tyrosinase (TYR), TYRP1 (tyrosinase-associated protein 1), and factors related to melanosome migration, such as Rab27a and melanavitin, were confirmed by measuring protein expression levels using Western blotting.

[0094] B16F10 mouse melanoma cells were used at a rate of 7 × 10⁻⁶ 4 Cells were seeded at a density of 2% per well in 6-well cell culture plates and cultured for 24 hours. The medium was then replaced with medium containing 2% FBS, followed by treatment with 200 ng / mL α-MSH and the peptide from Preparation Example 1, and incubated for 72 hours. At this time, the positive control group was treated with 200 ng / mL α-MSH and 500 μM arbutin. After washing with PBS, cell lysates were prepared by treatment with lysis buffer, and BCA was quantified to prepare equal volumes of protein samples. The prepared cell lysates and protein samples were electrophoresed using a 10% SDS-PAGE gel. The proteins separated by SDS-PAGE were transferred to a PVDF membrane and blocked with 5% skim milk for 30 minutes at room temperature. Primary antibodies against MITF, TYR, TYRP1, Rab27a, and melanavitin (Cell signaling, USA) and against actin (Santa Cruz, USA) were diluted 1:1000 in 3% BSA and reacted with PVDF membranes at 4°C for 16 hours. The membranes were washed three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS). Secondary antibody (Jackson Immuno Research, USA) was diluted 1:2000 in 5% skim milk and reacted at room temperature for 1 hour. The membranes were washed three times for 15 minutes each with 0.1% PBS-T (0.1% Tween-20 in PBS). The membranes were treated with ECL solution (GE Healthcare, USA) and expression levels were analyzed using an ImageQuant 800 (Cytiva, USA) instrument.

[0095] As a result of the experiment, from Figure 4 The results showed that the expression of MITF, TYR, and TYRP1 proteins, which are factors related to melanin production, as well as Rab27a and melanavitin proteins, which are factors related to melanosome migration, was increased by α-MSH treatment, and the expression levels of the above-mentioned increased proteins were reduced again by treatment with the peptide of Preparation Example 1.

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

Claims

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

1.

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

3. A pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders, comprising the peptide of claim 1 as an active ingredient.

4. The pharmaceutical composition for preventing or treating hyperpigmentation disorders according to claim 3, wherein, The peptide inhibits (i) melanin synthesis or (ii) melanosome migration in melanocytes.

5. The pharmaceutical composition for preventing or treating hyperpigmentation disorders according to claim 4, wherein, The peptide has the following activities: (i) Inhibit the increase in phosphorylation levels of CREB (cAMP response element binding protein); (ii) Inhibit the increase in expression levels of MITF (microphthalmia-associated transcription factor), TYR (tyrosinase), or TYRP1 (tyrosinase-associated protein 1); or (iii) Inhibit the increase in expression levels of Rab27a, MYO5A, or MLPH (melanavitin) in melanocytes induced during melanin synthesis.

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

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

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

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

10. The cosmetic composition for skin whitening according to claim 9, wherein, The peptide inhibits (i) melanin synthesis or (ii) melanosome migration in melanocytes.

11. The cosmetic composition for skin whitening according to claim 10, wherein, The peptide has the following activities: (i) Inhibit the increase in phosphorylation levels of CREB (cAMP response element binding protein); (ii) Inhibit the increase in expression levels of MITF (microphthalmia-associated transcription factor), TYR (tyrosinase), or TYRP1 (tyrosinase-associated protein 1); or (iii) Inhibit the increase in expression levels of Rab27a, MYO5A, or MLPH (melanavitin) in melanocytes induced during melanin synthesis.

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

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

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

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

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