WO2020226201A1 - Pharmaceutical composition for preventing or treating skin hypochromatism, health functional food, and cosmetic composition - Google Patents

Abstract

A pharmaceutical composition comprising nilotinib or a pharmaceutically acceptable salt thereof according to the present invention can be used for the prevention or treatment of skin hypochromatism. Furthermore, a health functional food and a cosmetic composition, comprising the nilotinib or a salt thereof can be used for the prevention or amelioration of skin hypochromatism.

Description

Pharmaceutical composition for preventing or treating skin hypopigmentation, health functional food and cosmetic composition

The present invention relates to a pharmaceutical composition, a health functional food, and a cosmetic composition for preventing or treating skin hypopigmentation.

Melanin produced by melanocytes present in the base layer of the epidermis of human skin refers to a phenolic polymer material having a complex form of black pigment and protein. The melanin determines the skin color of a person according to its concentration and distribution, and also serves to protect the human skin from ultraviolet rays.

Excessive production of melanin causes pigmentation such as spots and freckles. On the contrary, if the production of melanin is excessively suppressed, skin lesions of vitiligo may appear, the number and function of melanocytes in the hair root may decrease, resulting in white hair, and hypopigmentation may occur, which is a risk factor for skin cancer. Therefore, studies that promote melanin production and induce blackening are very important as well as whitening-related studies that inhibit melanin production.

These vitiligo, white hair, and hypopigmentation are not only unsightly in appearance, but also cause many difficulties in social activities. Thus, in the case of vitiligo and hypopigmentation, there are many cases where they are treated using surgical procedures or temporarily concealed by using external skin preparations such as cosmetics, and in the case of white hair, many people try to temporarily hide gray hair by dyeing it. However, since these methods are not fundamental prevention, improvement, or treatment methods, studies on melanin production promoters are necessary for a more fundamental solution.

In this regard, Korean Patent Registration No. 10-1669359 discloses a composition for promoting melanin synthesis comprising a mixture of azalea extract, salicornia extract, and oyster oak extract, but each extract is used by mixing several extracts. There is a problem that the preparation of raw materials and the manufacturing process are complicated.

On the other hand, nilotinib is 4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-N-[5-(4-methyl-1H-imidazol-1-yl )-3-(trifluoromethyl)phenyl]benzamide (Figure 1A). A particularly useful salt of nilotinib is nilotinib hydrochloride monohydrate. These therapeutic compounds have utility as inhibitors of the protein tyrosine kinase (TK) activity of Bcr-Abl. Examples of diseases that can be treated with the therapeutic compound include chronic myelogenous leukemia and gastrointestinal stromal tumors.

Accordingly, the inventors of the present invention were researching a substance capable of effectively promoting melanin production and less irritation to the human body in order to fundamentally solve vitiligo, white skin disease, bleaching nevus and hypopigmentation in addition to the above problems, nilotinib The present invention was completed by confirming that the production of melanin was promoted to induce blackening, and that there was no skin irritation and cytotoxicity, and thus the stability of the human body was excellent.

In addition, an object of the present invention is to provide a pharmaceutical composition for preventing or treating skin hypopigmentation comprising nilotinib or a pharmaceutically acceptable salt thereof.

In addition, it is an object of the present invention to provide a health functional food for preventing or improving skin hypopigmentation, including nilotinib or a food physiologically acceptable salt thereof.

In addition, it is an object of the present invention to provide a cosmetic composition for preventing or improving skin hypopigmentation comprising nilotinib or a cosmetically acceptable salt thereof.

1. Nilotinib (Nilotinib) or a pharmaceutical composition for the prevention or treatment of skin hypopigmentation comprising a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to the above 1, wherein the skin hypopigmentation is one selected from the group consisting of vitiligo, white skin disease, and bleaching nevus.

3. Nilotinib (Nilotinib) or a health functional food for preventing or improving skin hypopigmentation containing a food acceptable salt thereof.

4. In the above 3, wherein the skin hypopigmentation is one selected from the group consisting of vitiligo, white skin disease, and bleaching nevus, health functional food.

5. Nilotinib (Nilotinib) or a cosmetic composition for preventing or improving skin hypopigmentation containing a cosmetically acceptable salt thereof.

6. The cosmetic composition of the above 5, wherein the skin hypopigmentation is one selected from the group consisting of vitiligo, white skin disease, and bleaching nevus.

In addition, the pharmaceutical composition of the present invention can prevent or treat skin hypopigmentation, particularly vitiligo, bleaching nevus, white skin disease, and the like.

In addition, the health functional food and cosmetic composition of the present invention can prevent or improve skin hypopigmentation, particularly vitiligo, bleaching nevus, white skin disease, and the like.

1A is a diagram showing the structure of nilotinib, and B is a diagram showing the cytotoxicity of nilotinib in HM3KO melanoma cells.

2 shows the effect of nilotinib on pigment formation in HM3KO melanoma cells, where A shows the effect according to the nilotinib dose, and B shows the effect over time.

3 shows the effect of nilotinib on the expression of a pigment-forming gene in HM3KO melanoma cells, where A shows the mRNA level by RT-PCR, and B shows the protein level by Western blot.

4 shows the effect of nilotinib on intracellular signaling, where A shows the protein level by Western blot, and B and C shows the results of the treatment of H89 and nilotinib.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating skin hypopigmentation comprising nilotinib or a pharmaceutically acceptable salt thereof.

Nilotinib of the present specification is a compound of Formula 1 below, and the IUPAC Name is 4-methyl- N -[3-(4-methyl-1 H -imidazol-1-yl)-5-(trifluoromethyl)phenyl]-3 -[(4-pyridin-3-ylpyrimidin-2-yl)amino]benzamide, molecular formula is C ₂₈ H ₂₂ F ₃ N ₇ O, molecular weight is a compound corresponding to 529.5245 g/mol:

[Formula 1]

The nilotinib may be derived from nature or may be synthesized using a known organic synthesis method.

The nilotinib may be a non-protein compound, a peptide, an extract of a plant-derived tissue or cell, or a product obtained by culturing a microorganism (eg, bacteria or fungi, and particularly yeast).

The pharmaceutical composition according to the present invention may be provided as a pharmaceutical composition containing an active ingredient alone or one or more pharmaceutically acceptable carriers, excipients, or diluents.

In the present invention, "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition.

The term "pharmaceutically acceptable salt" of the present invention means a salt prepared using a specific compound according to the present invention and a relatively non-toxic acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a sufficient amount of a base with the neutral form of such a compound in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include salts or similar salts of sodium, potassium, calcium, ammonium, organic amine, or magnesium. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a sufficient amount of acid with the neutral form of such compounds in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable acid addition salts are salts of inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate ion, phosphoric acid, monohydrogen phosphate ion, dihydrogen phosphate ion, sulfuric acid, hydrogen sulfate ion, hydroiodic acid or phosphorous acid, And salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. And salts of amino acids (eg, arginine) and salts of organic acids such as glucuronic acid.

Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from the parent compound containing an acidic or basic moiety. In general, these salts are prepared by reacting the form of the free acid or base of these compounds with a stoichiometrically appropriate amount of a base or acid in water or in an organic solvent or in a mixture of the two. In general, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.

Furthermore, the pharmaceutical composition of the present invention may be provided by mixing with a conventionally known composition for preventing or treating skin hypopigmentation. That is, the pharmaceutical composition of the present invention can be administered in parallel with a known compound having a preventive or therapeutic effect on skin hypopigmentation.

In the present invention, "administration" means introducing a predetermined substance to an individual by an appropriate method, and "individual" means all organisms such as rats, mice, livestock, including humans capable of possessing skin hypopigmentation. . As a specific example, it may be a mammal including a human.

If necessary, the pharmaceutical composition of the present invention may additionally include a composition having a known effect of preventing or treating skin hypopigmentation.

Compositions for preventing or treating such skin hypopigmentation include thistle extract, extract of Artemisia capillaris Thunberg, or isoproxidine 7- O- (6'- O - p -coumaroyl)-β- Glucopyranoside (isofraxidin 7- O -(6'- O - p -coumaroyl)-β-glucopyranoside), lemur extract, red bean extract, casino A, casino B, casino E, casino H, etc. However, it is not limited thereto.

In the present invention, the route of administration of the pharmaceutical composition is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, This includes topical, sublingual or rectal.

The composition of the present invention may be administered orally or parenterally, and when administered parenterally, it is preferable to select an injection method for external use of the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. , But is not limited thereto.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the composition is preferably administered at 0.01 to 1000 mg/kg/day, preferably 0.1 to 500 mg/kg/day, but is not limited thereto. The administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient in the extract, such as starch, calcium carbonate, and sucrose. Or it is prepared by mixing lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycero gelatin, and the like may be used.

The skin hypopigmentation may be, for example, vitiligo, white skin disease, bleaching nevus, etc., but is not limited thereto.

In the case of nilotinib, it may exhibit a preventive or therapeutic effect of skin hypopigmentation by increasing pigment formation in melanoma cells, and specifically, Tyro, a rate-limiting enzyme in melanin formation as well as melanin formation. Synase activity may be increased, and more specifically, expression of MITF and TRP1, which are pigment formation-related genes, may be increased, but is not limited thereto.

In addition, nilotinib can reduce the phosphorylation of AKT related to AKT signaling, which negatively regulates the pigment formation process, and the PKA inhibitor H89, which is the backbone of the pigment formation process, related to PKA signaling, is a cAMP reaction element binding protein ( cAMP response element-binding protein, CREB) phosphorylation can be inhibited, and nilotinib can activate it, but is not limited thereto.

In addition, the present invention relates to a health functional food for preventing or improving skin hypopigmentation, including nilotinib or a food physiologically acceptable salt thereof.

The skin hypopigmentation may be, for example, vitiligo, white skin disease, bleaching nevus, and the like, but is not limited thereto, and the effect and mechanism of action of nilotinib may be within the above-described range.

In the present invention, "food-wise acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition.

In the present invention, the term "food-friendly salt" refers to a salt prepared using a specific compound according to the present invention and a relatively non-toxic acid or base, and may be within the above-described range for "salt".

The health functional food of the present invention may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including at least one of carriers, diluents, excipients and additives. Foods to which the extract of the present invention can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

Additives that may be further included in the present invention include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), Pactic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents and one or more components selected from the group consisting of pulp can be used. .

Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and heavy ingredients (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid. And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, the composition according to the present invention may contain pulp for the manufacture of natural fruit juice and vegetable beverages. These components may be used independently or in combination.

Specific examples of the carrier, excipient, diluent and additive are, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate And it is preferable that at least one selected from the group consisting of mineral oil is used.

When formulating the health functional food of the present invention, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. that are commonly used.

In addition, the present invention relates to a cosmetic composition for preventing or improving skin hypopigmentation, comprising nilotinib or a cosmetically acceptable salt thereof.

The skin hypopigmentation may be, for example, vitiligo, white skin disease, bleaching nevus, and the like, but is not limited thereto, and the effect and mechanism of action of nilotinib may be within the above-described range.

In the present invention, "cosmetically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition.

In the present invention, "cosmetically acceptable salt" means a salt prepared using a specific compound according to the present invention and a relatively non-toxic acid or base, and may be within the above-described range for "salt".

In the present invention, the cosmetic composition may additionally include components conventionally added to the cosmetic composition, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and conventional adjuvants and carriers such as fragrances.

The cosmetic composition may be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil , Powder foundation, emulsion foundation, wax foundation, and spray may be formulated, but are not limited thereto. More specifically, lotions such as flexible lotions or nutritious lotions, emulsions such as facial lotions and body lotions, creams such as nutritional creams, moisture creams, eye creams, essences, cosmetic ointments, sprays, gels, packs, sunscreens, and makeup It can be prepared in a formulation such as a foundation, such as a base, a liquid type, a solid type, or a spray type, a powder, a cleansing cream, a cleansing lotion, a makeup remover such as a cleansing oil, a cleansing foam, a soap, a body wash, and the like. In this case, the cosmetic composition is a surfactant, an emulsifier, a soap acid, a solvent, a colorant, a preservative, an antioxidant, an antifoaming agent, an antibacterial agent, an anti-redeposition agent, an enzyme, a plant or mineral oil, a fat, a fluorescent substance, a fungicide, a hydrophobic inducing substance, It may contain excipients including moisturizers, fragrances, fragrance carriers, preservatives, proteins, silicones, solubilizers, sugar derivatives, sunscreen agents, vitamins, plant extracts, waxes, and the like.

When the formulation of the cosmetic composition is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide are used as carrier components. Can be used.

When the formulation of the cosmetic composition is a powder or spray, toss, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, Propellants such as propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, and the like may be used.

When the formulation of the cosmetic composition is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid Amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters, and the like may be used.

Hereinafter, examples will be described in detail to illustrate the present invention in detail.

Example

1. Experimental method and materials

(1) cell culture

The human melanoma cell line HM3KO was maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Life Technologies Corporation, Grand Island, NY). The compound nilotinib was obtained from Enzo Life Sciences Inc. (Farmingdale, NY) was purchased, dissolved in dimethyl sulfoxide (DMSO), and then diluted in culture medium (the final concentration of DMSO is 0.1%). The PKA inhibitor H89 was purchased from Sigma-Aldrich (St. Louis, MO).

(2) Cytotoxicity test

HM3KO melanoma cells were treated with nilotinib for 24 hours, after which the medium was 0.5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) It was replaced with a fresh medium containing the solution. Cells were incubated for an additional 4 hours, after which formazan crystals were dissolved in DMSO. Cell viability was determined by measuring the optical density at 570 nm using an ELISA reader.

(3) melanin content and tyrosinase activity

For the determination of pigment formation, cells were collected and pelleted by centrifugation. The melanin pigment was dissolved in 1 N NaOH at 100° C. for 30 minutes, and was quantified by measuring the optical density at 405 nm. To determine tyrosinase activity, cells were lysed in Pro-Prep protein extraction solution (Intron, Daejeon, Korea), and then the lysate was purified by centrifugation. After quantification, 250 μg of total protein in 100 μl lysis buffer was transferred to a 96-well plate, and 100 μl 1 mM _L- DOPA was added. After incubation at 37° C. for 30 minutes, the absorbance was measured at 405 nm.

(4) Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated using an Easy-blue RNA extraction kit (Intron, Daejeon, Korea). 2 mg of total protein was reverse transcribed with the moloney-murine leukaemia virus (M-MLV) reverse transcriptase (RTase) (Elpis Biotech, Daejeon, Korea). Aliquots of the RT mixture were subjected to PCR cycles with an appropriate primer set. The sequences of the primers are as follows: MITF, 5'-ACCTTCTCTTTGCCAGTCCA-3' (SEQ ID NO: 1) and 5'-CGGATATAGTCCACGGATCG-3' (SEQ ID NO: 2); Tyrosinase, 5'-AGGCAGAGGTTCCTGTCAGA-3' (SEQ ID NO: 3) and 5'-CTATGCCAAGGCAGAAAAGC-3' (SEQ ID NO: 4); TRP1, 5'-CTCCTGCACACCTTCACAGA-3' (SEQ ID NO: 5) and 5'-TCAGTGAGGAGAGGCTGGTT-3' (SEQ ID NO: 6); GAPDH, 5'-CGACCACTTTGTCAAGCTCA-3' (SEQ ID NO: 7) and 5'-AGGGGTCTACATGGCAACTG-3' (SEQ ID NO: 8).

(5) Western blot analysis

Cells were harvested by centrifugation, and then lysed in Pro-Prep protein extraction solution (Intron, Daejeon, Korea). After vigorous pipetting, the extract was centrifuged at 15,000 rpm for 15 minutes. Total protein was measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL). Samples (20-30 μg protein per lane) were run on SDS-polyacrylamide gels, transferred onto a nitrocellulose membrane, and incubated with the appropriate antibody overnight at 4° C. with gentle agitation. Thereafter, the blot was incubated with a peroxidase-conjugated secondary antibody for 30 minutes at room temperature, and visualized by enhanced chemiluminescence (Intron, Daejeon, Korea). The following primary antibodies were used in this experiment: MITF, tyrosinase, TRP1 (Santa Cruz Biotechnologies, Santa Cruz, CA); Actin (Sigma-Aldrich, St. Louis, MO); phospho-AKT, AKT, phospho-CREB, CREB (Cell Signaling Technology, Danvers, MA).

(6) statistical analysis

All data were derived from at least 3 independent experiments. Results were expressed as a percentage controlling the ± standard deviation (SD). The data were statistically evaluated by the student's t-test using one-way ANOVA or SPSS software v 22.0 (IBM, Seoul, Korea). Statistical significance was set at p <0.01.

2. Experiment result

Nilotinib was originally developed as a small molecule inhibitor for BCR-ABL tyrosine kinase (Fig. 1A). We first confirmed the cytotoxic effect of nilotinib on HM3KO melanoma cells. As a result, nilotinib did not show significant cytotoxicity up to a dose of 1.0 μM (FIG. 1B).

When HM3KO melanoma cells were treated with nilotinib, the pigmentation of melanoma cells was greatly increased, which was confirmed by the color of the cell pellet. Consistent with these results, quantitative analysis indicated that the melanin content was significantly increased by the dose-dependent manner of nilotinib. Since tyrosinase is a rate-limiting enzyme in melanogenesis, we measured tyrosinase activity using cell lysates. As a result, treatment with nilotinib remarkably increased tyrosinase activity (Fig. 2A). The possibility of nilotinib inducing pigmentation was confirmed again in a time course study. As shown in FIG. 2B, nilotinib increased melanin content and tyrosinase activity in a time-dependent manner.

Nilotinib increased tyrosinase activity in HM3KO melanoma cells, and we tested whether nilotinib influenced gene expression for pigment formation. RT-PCR showed that nilotinib treatment significantly increased the expression of pigmentation-related genes such as MITF, tyrosinase and TRP1 (Fig. 3A). Consistent with these results, protein levels for MITF, tyrosinase, and TRP1 were also significantly increased by nilotinib (Fig. 3B).

Pigment formation is a process in which many signaling events are cooperatively involved. To describe the mechanism of action of nilotinib, we investigated whether nilotinib influences intracellular signaling pathways. We first evaluated the effect of nilotinib on AKT signaling, which negatively regulates the pigment formation process. As a result, phosphorylation of AKT was significantly reduced by nilotinib in HM3KO melanoma cells. Next, we evaluated the phosphorylation of cAMP response element-binding protein (CREB), a well-established downstream of cAMP/PKA signaling, and thus nilotinib for cAMP/PKA signaling. The effect of was tested. In contrast to AKT signaling, nilotinib significantly increased the phosphorylation of CREB (Fig. 4A). Since PKA signaling is the backbone of the pigment formation process, we further investigated the effect of nilotinib on PKA signaling. We pretreated HM3KO melanoma cells with the PKA inhibitor H89, and then treated with nilotinib. The pretreatment of H89 showed the result of remarkable inhibition of nilotinib-induced phosphorylation of CREB, confirming the effective blocking of PKA signaling (FIG. 4B). As expected, pretreatment of H89 markedly inhibited nilotinib-induced pigment formation in melanoma cells along with inhibition of tyrosinase activity (FIG. 4C). These results indicate that activation of PKA signaling by nilotinib is one of the important mechanisms of action for inducing pigment formation.

Claims (6)

Nilotinib (Nilotinib) or a pharmaceutical composition for the prevention or treatment of skin hypopigmentation comprising a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 1, wherein the skin hypopigmentation is one selected from the group consisting of vitiligo, white skin disease, and bleaching nevus. Nilotinib (Nilotinib) or a health functional food for preventing or improving skin hypopigmentation, including a food acceptable salt thereof. The health functional food according to claim 3, wherein the skin hypopigmentation is one selected from the group consisting of vitiligo, white skin disease, and bleaching nevus. Nilotinib (Nilotinib) or a cosmetic composition for preventing or improving skin hypopigmentation comprising a cosmetically acceptable salt thereof. The cosmetic composition of claim 5, wherein the skin hypopigmentation is one selected from the group consisting of vitiligo, white skin disease, and bleaching nevus.

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