KR101375782B1 - Method of modulating melanogenesis using GDF-15 - Google Patents

Abstract

The present invention relates to a method for regulating the production of melanin, characterized in that it inhibits or increases the expression of GDF-15, and a cosmetic composition for preventing or improving blemishes, and a pharmaceutical composition for preventing or treating vitiligo. Method for controlling melanin production using GDF-15 according to the present invention, by inhibiting the activity of tyrosinase by inhibiting the expression or activity of GDF-15 by inhibiting the production of melanin, or by increasing the expression of GDF-15 Increasing the production of melanin has the effect of effectively improving the skin pigment-related blemishes and vitiligo.

Description

Method of modulating melanogenesis using GDF-15

The present invention relates to a method for controlling the biosynthesis of melanin, characterized in that it inhibits or increases the expression of GDF-15, and a cosmetic composition for preventing or improving blemishes, pharmaceutical composition for preventing or treating vitiligo.

Knowledge of the chemical and enzymatic basis of melanogenesis as a mechanism for regulating the color of the skin is well known. Melanocytes migrate from the embryonic neural crest to the skin to produce melanosomes that produce melanin, the particles they secrete. The key enzyme in melanogenesis is tyrosinase, which leads to a reaction cascade that converts tyrosine to raw polymer melanin. Two tyrosinase-related proteins (TRP's) are known, TRP-1 and TRP-2, which share about 40% homology with tyrosinase and have a catalytic activity as well as a regulatory role in melanogenesis. .

The melanin occurs in an organelle called melanosome (melanosome) in which the enzymes are distributed, and the melanosome containing melanin is later transferred to keratinocytes through melanocyte dendrites. The melanin transferred to the keratinocyte is thus an important factor in determining the skin color.

As the main enzyme involved in melanogenesis is tyrosinase, there are also major constructs for transferring the produced melanosomes to the dendrite of melanocytes. They are called Melanosome Transport Complex (MTC) and are composed of three proteins, Melanoophilin, Rab27a, and Myosin 5a. Rab27a is a protein that binds to the surface of the melanosome and acts as a carrier that recognizes and transports the melanosome. In order to move to the end, Rab27a must meet the myosin 5a protein, a cytoskeleton, and recognize the two proteins in the middle. It's a protein called melanophilin that acts as an adapter to connect and connect. When these three constructs meet to form a complex, the melanosomes can be normally transferred to the cell ends, and ultimately, they can be delivered to keratinocytes.

On the other hand, melanin pigment, which is produced from melanocytes of human body skin, is a phenolic polymer having a complex form of black pigment and protein, and plays an important role in inhibiting skin damage caused by ultraviolet rays irradiated from the sun. Over-synthesis and accumulation of not only causes serious aesthetic problems such as blemishes, freckles, and senile surpluses on the skin, but also promotes skin aging and causes various skin cancers. At this time, melanocytes are slow-growing cells that require mitogen for their growth, including SCF (Grabbe et al., 1994), basal fibroblast growth factor (bFGF) (Halaban et al., 1987), bovine Several factors, including pituitary extract (Wilkins et al., 1985), TPA (Krasagakis et al., 1993), and cAMP stimulating agents are known to proliferate melanocytes in vitro, but are clinically Mitogens that can be used are very rare.

In addition, the migration of melanocytes can cause repimentation of vitiligo. Pigmentation may be a result of melanocyte proliferation or melanogenesis (Lan et al., 2005).

The melanocyte migration or melanogenesis is a very complex process, and growth of normal melanocytes requires various mitogens because different classes of mitogen can properly stimulate melanocyte growth (Abdel -Malek et al., 1992; Eisinger and Marko, 1982; Halaban et al., 1986). Thus, these mitogens can be seen to influence each other on proliferation and / or melanogenesis, whereby bFGF or TPA alone did not induce optimal melanocyte proliferation and binding to a-MSH (Swope et al., 1995) or binding to CT and / or IBMX (Abdel-Malek et al., 1992), respectively, increased their mitogenic effects.

On the other hand, vitiligo is an acquired depigmentation disease in which skin with various sizes and forms of melanoma due to the death or necrosis of melanocytes is a relatively common disease in about 1% of the world. There is no difference. The incidence age is most common in 10-30 years, 95% of cases develop before 40 years of age, and 30% of patients have a family history. In the early stage, vitiligo is less pronounced, but it is often clearer with time, and the boundary with normal skin may not be clear, but it may be darker and clearer than normal skin color, and rarely itching. do. White spots grow on areas with hair, especially on the hair and eyebrows. In addition, vitiligo can occur anywhere on the skin, but in particular, bones such as fingers, toes, knees, elbows, etc., protrudes around the mouth, around the nose, around the eyes, under the armpits, the inside of the wrist, and the like. In addition, vitiligo occurs particularly well in areas where wounds occur easily, and is often symmetrical or neurally distributed. In addition, vitiligo may be accompanied by abnormal pigmentation of the iris and retina of the eye, in addition to the appearance of white spots on the skin, diabetes mellitus, pernicious anemia, hypothyroidism or hyperactivity, Down syndrome, biliary cirrhosis, stomach cancer, Adison disease, It can be combined with autoimmune diseases such as alopecia areata and lupus erythematosus.

Therefore, there have been many researches and experiments for treating such vitiligo, and as a result, currently used vitiligo treatment methods include psoralen ultraviolet therapy, local and systemic treatment of steroids, and surgical treatment. Soralene UV therapy is called photochemotherapy, and it is systemic ultraviolet irradiation after taking soralene or partial ultraviolet irradiation after applying soralene. Since it requires long-term treatment, there is a risk of skin aging or skin cancer caused by ultraviolet irradiation. Steroid treatment requires topical application or topical injection with triamcinolone when the lesion area is small, and systemic administration if widespread. This also requires long-term treatment and there is a risk of causing skin and systemic side effects. Surgical treatment is a method of transplanting melanocytes by surgery, which can be divided into skin grafts and cell grafts. For skin grafts, full layer, middle layer and epidermal grafts are used. This method is not always available and can be used only if the lesion has not spread for many years. In the case of treatment with steroid external preparations, which is the most commonly used method, long-term use may cause serious side effects including skin contraction. However, there is an urgent need for a method to solve this problem.

In addition, other major skin diseases, blemishes are caused by hyperpigmentation of the skin and cause a variety of cosmetic problems. Various methods such as chemical peeling, laser treatment, arbutin, Many methods using kojic acid are used, but it is difficult to expect an effect that satisfies the needs of consumers, or the side effects are often severe, causing large problems.

In order to solve the above problems, research is being conducted to treat diseases such as vitiligo and blemishes by directly controlling the biosynthesis of melanin, but there are few reports on breakthrough methods for improving pigmentation.

 If the production of melanin can be controlled, it is possible to treat blemishes caused by skin pigment overdeposition or vitiligo that does not have normal skin color. Therefore, research on a target substance that can control the production of melanin, which can replace conventional treatment, is needed.

While the present inventors have been studying the target of a new pigmentation process that can replace the existing blemishes or vitiligo treatment, the present inventors have confirmed that the production of melanin can be controlled by inhibiting or increasing the expression of GDF-15, thereby completing the present invention. It was.

An object of the present invention to provide a method for controlling the production of melanin by inhibiting or increasing the expression of GDF-15, and to provide a cosmetic composition for preventing or improving blemishes, pharmaceutical composition for preventing or treating vitiligo.

The present invention provides a cosmetic composition for preventing or improving blemishes comprising a growth differentiation factor-15 (GDF-15) expression inhibitor or an activity inhibitor as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating vitiligo comprising GDF-15 as an active ingredient.

In another aspect, the present invention provides a method for regulating melanin production, characterized by inhibiting or increasing the expression of GDF-15.

Method for controlling melanin production using GDF-15 according to the present invention inhibits the activity of tyrosinase by inhibiting the expression or activity of GDF-15 to inhibit the production of melanin, or to increase the expression of GDF-15 melanin By increasing the production of can be effectively improved the skin pigmentation-related opacity and vitiligo.

Figure 1 is treated with 0.1, 1, 10, 20 μM each of histamine in SK-MEL-2 human melanoma cells, melanogenesis-related proteins TRP-1, TRP-2, tyrosinase (tyrosinase) And shows the results of confirming the expression of GDF-15 by Western blot.
Figure 2 is a diagram showing the result of inhibiting the gene of GDF-15 with a gene-specific siRNA, and whether the GDF-15 gene is effectively inhibited (a), through which the production of melanin is inhibited (b).
3 is a diagram showing the results confirmed by Western blot whether the expression (b) of tyrosinase (a), TRP-1, TRP-2 is reduced by inhibiting GDF-15 gene expression.
4 is a diagram showing the results of confirming the mobility change of SK-MEL-2 human melanoma cells transfected with GDF-15 specific siRNA.
5 is a diagram showing the results of confirming the increase in melanin biosynthesis (a) and the increase in mobility of SK-MEL-2 human melanoma cells (b) in SK-MEL-2 human melanoma cells induced overexpression of GDF-15. .

The present invention provides a method for regulating melanin production, characterized by inhibiting or increasing the expression of growth differentiation factor-15 (GDF-15).

The GDF-15, a member of the transforming growth factor-β cytokine superfamily, was first identified as a macrophage-inhibiting cytokine-1 (MIC-1) and later also referred to as placental transforming growth factor-β. (Bootcov 1997, Proc Natl Acad Sci 94: 11514-11519; Tan 2000, Proc Natl Acad Sci 97: 109-114). In addition, GDF-15 has been reported to be overexpressed in melanoma and higher in biopsies of metastatic melanoma tissue than normal melanocytes.

Inhibiting or increasing the expression of GDF-15 may use a method of inducing overexpression by using a GDF gene specific expression or activity inhibitor or by introducing a GDF-15 gene into melanocytes and controls the expression of a gene or protein. Methods known in the art can be used without limitation.

The present invention provides a cosmetic composition for preventing or improving blemishes comprising a growth differentiation factor-15 (GDF-15) expression inhibitor or an activity inhibitor as an active ingredient.

The cosmetic composition for preventing or improving blemishes of the present invention may inhibit or inhibit the activity of tyrosinase by inhibiting the expression or activity of GDF-15, thereby inhibiting the production of melanin and thus preventing or improving blemishes caused by hyperpigmentation.

The GDF-15 expression inhibitor is characterized in that at least one selected from the group consisting of antisense nucleotides, siRNA (short interfering RNA) and shRNA (short hairpin RNA) complementary to the mRNA of the GDF-15 gene.

The GDF-15 activity inhibitor is characterized in that at least one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers and antibodies that complementarily bind to GDF-15 protein.

The GDF-15 gene specific siRNA preferably has a nucleotide sequence of SEQ ID NO: 1 (5'-ACAUGCACGCGCAGAUCAATT-3) or SEQ ID NO: 2 (5'-UUGAUCUGCGCGUGCAUGUTT-3).

The blemishes refer to the development of light, hyperpigmented lesions of light brown, dark brown or even black color on the central part of the face such as cheeks, forehead, nose and chin.

In addition, in the present invention, the cosmetic composition, in addition to the GDF-15 expression inhibitors or activity inhibitors and other components that can give a synergistic effect to the main effect, preferably within the range that does not impair the main effect of the present invention. It may contain.

The cosmetic composition includes a GDF-15 expression inhibitor or an active inhibitor according to the present invention as an active ingredient, and various creams, cleansing foams, soaps, sunscreens and skins, lotions, essences, nutrition creams, massage creams and cleansing creams, and the like. Basic cosmetics such as packs and color cosmetics including powders, foundations and makeup bases, and the like. In the cosmetic composition of each formulation, other ingredients in addition to the GDF-15 expression inhibitor or activity inhibitor may be appropriately selected and blended by those skilled in the art according to the formulation or purpose of use of other cosmetics. To this end, it may also comprise various excipients which are acceptable for cosmetic compositions, which act as diluents, dispersants or carriers for the active ingredients in the composition. Excipients other than water or excipients containing water include emollients, solvents, wetting agents, fats and oils, emulsifiers, surfactants, organic and inorganic pigments, ultraviolet absorbers, preservatives, fungicides, antioxidants, pH adjusting agents in liquid or solid form. , Fragrances, thickeners or powders. However, the present invention is not limited thereto.

In the cosmetic composition of the present invention, GDF-15 expression inhibitor or activity inhibitor is preferably contained in 0.01 to 5% by weight based on the total weight of the cosmetic composition.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating vitiligo comprising growth differentiation factor-15 (GDF-15) as an active ingredient.

The pharmaceutical composition for preventing or treating vitiligo of the present invention can effectively improve vitiligo, a pigment-related disease of the skin, by increasing the production of melanin by overexpressing GDF-15.

The vitiligo is a disease in which the skin color turns white due to the death of melanocytes, which is a relatively common disease that occurs in about 1-2% of the world's population regardless of race and gender. Although the age at which it develops is not fixed, more than half of patients appear before the age of 20 and are not covered by clothing, such as the face or hands, or areas where the skin is folded, such as the groin or armpits, or protruding bones such as elbows, knees, and hand joints. It looks good around the body's holes, such as the skin on the stomach, mouth, or nose. It usually appears symmetrically, but can only be found on one or both sides along the ganglion, sometimes only in one or several areas, and in rare cases, almost all of the skin becomes white.

In the present invention, the pharmaceutical composition may contain, in addition to GDF-15, other ingredients that can give a synergistic effect to the main effect, preferably within a range that does not impair the main effect of the present invention.

In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient and diluent in addition to the active ingredient described above for administration.

For example, carriers, excipients and diluents include lactose, textose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil.

The pharmaceutical composition of the present invention may be prepared in various parenteral or oral administration forms according to known methods. As representative of formulations for parenteral administration, isotonic aqueous solutions or suspensions are preferred for injectable formulations. The injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The effective dosage of the pharmaceutical composition of the present invention may vary depending on the age and gender of the patient, but may be administered at 0.001 to 100 mg / kg.

Hereinafter, the present invention will be described in detail with reference to Production Examples and Examples. However, the following Preparation Examples and Examples are illustrative of the present invention, and the content of the present invention is not limited by the following Production Examples and Examples.

Example  By histamine GDF Increased expression of --15 and tyrosinase

SK-MEL-2 human melanoma cells were distributed and used by the Korean Cell Line Bank. Hereinafter, DMEM (dulbecco's modified eagle's medium) containing 10% fetal bovine serum (FBS) and 100 units / penicillin-streptomycin (100 units / antibacterial antimycotic) was used for cell culture. .

SK-MEL-2 human melanoma cells were cultured in 6-well plates and histamine was added by 0.1, 1, 10, 20 μM by concentration. After 24 hours, the whole cell extract was prepared and analyzed by 4-12% SDS-PAGE. Expression of melanogenesis-related proteins TRP-1, TRP-2, tyrosinase and GDF-15 were analyzed by Western blot.

 The results are shown in Fig.

As shown in FIG. 1, as a result of treating histamine with SK-MEL-2 human melanoma cells, expression of TRP-1, TRP-2, tyrosinase, and GDF-15 associated with melanin biosynthesis was increased. there was.

Example  2. GDF -15 specific siRNA  Confirmation of melanogenesis inhibition during treatment

Since overexpression of GDF-15 is induced by histamine and melanin production is promoted, it was confirmed whether melanin production was inhibited when the gene of overexpressed GDF-15 was inhibited with a gene-specific siRNA. GDF-15 gene specific siRNA used the sequence of Table 1 below.

GDF-15 specific siRNA sequence SEQ ID NO: order Characteristic One 5'-ACAUGCACGCGCAGAUCAATT-3 siRNA for GDF-15 2 5'-UUGAUCUGCGCGUGCAUGUTT-3 siRNA for GDF-15

SEQ ID NOS: 1 and 2 were introduced into SK-MEL-2 human melanoma cells using lipofectamine RNAiMAX (invitrogen). After 48 hours, melanin production was measured in the total cell extract. In addition, the siRNA treatment of the melanocytes was confirmed by optical microscopy whether the formation of dendritic cells (dendrocytes) representing the melanin formation process was reduced.

The results are shown in Fig.

As shown in FIG. 2, the treatment of GDF-15-specific siRNA effectively inhibited the expression of GDF-15 (a) and decreased the production of melanin that was increased by histamine treatment (b). Therefore, it can be seen that by controlling the expression of GDF-15 can control the biosynthesis of melanin.

Example  3. GDF -15 specific siRNA  Inhibition of tyrosinase activity and TRP-1, by treatment TRP -2 reduced expression

By inhibition of GDF-15 gene expression, it was confirmed by Western blot whether tyrosinase, TRP-1 and TRP-2 expression, which are involved in melanin biosynthesis, were also reduced. SK-MEL-2 human melanoma cells were transfected with GDF-15 specific si-RNA for 48 hours. SK-MEL-2 human melanoma cells were treated with histamine at 10 mM and incubated for 24 hours to induce melanin biosynthesis. After 24 hours of histamine treatment, total cell extracts were obtained, which were analyzed by SDS-PAGE at 4-12%.

The results are shown in Fig.

As shown in Figure 3, by inhibiting the expression of GDF-15 it was confirmed that the tyrosinase (a) involved in melanin biosynthesis and the expression of the related proteins TRP-1, TRP-2 is reduced (b)

Example  4. GDF -15 specific siRNA  By treatment, Melanoma  Reduced cell mobility

SK-MEL-2 human melanoma cells were transfected with GDF-15 specific siRNA for 48 hours. Mobility of transfected SK-MEL-2 cells was examined using the Boyden Chamber Kit. The transwell insert of the lower chamber was coated with fibronectin 10 mg / ml and treated with SKMEL-2 human melanoma cells ⁵ × 10 ⁴ per well and treated with histamine 10 mM. After culturing SK-MEL-2 cells, the cells adhered to the bottom of the insert were fixed with Diff-quick and cells were randomly counted at four or more sites.

The results are shown in Fig.

As shown in FIG. 4, the increased mobility of SK-MEL-2 human melanoma cells induced by histamine treatment was inhibited to a similar level as before histamine treatment by treatment with GDF-15 specific siRNA.

Example  5. GDF -15 increased melanin biosynthesis by induction of overexpression and Melanoma  Confirmation of Increased Cell Mobility

SK-MEL-2 human melanoma cells (5 x 10 ^4/6-well) were transfected the plasmids with the GDF-15 gene. After the histamine (10 μM) was added to the transfected SK-MEL-2 human melanoma cells, melanin production was measured and the mobility of SK-MEL-2 cells was confirmed in the same manner as in Example 4.

The results are shown in Fig.

As shown in FIG. 5, significant increase in melanogenesis was confirmed in histamine-treated cells as well as SK-MEL-2 cells transfected with GDF-15 (a). In addition, it was confirmed that significant increase in the mobility of SK-MEL-2 cells in SK-MEL-2 cells transfected with GDF-15 (b). Thus, it can be seen that overexpression of GDF-15 in melanoma cells can increase melanogenesis and increase the mobility of melanoma cells.

Formulation Example 1 . Cosmetics  Formulation

1.1 Softener (Skin Lotion) (Unit: wt%)

GDF-15 expression inhibitor or activity inhibitor 0.1 to 0.2

Butylene Glycol 2.0

Propylene Glycol 2.0

Carboxy Vinyl Polymer 0.1

Fiji-12 nonylphenyl ether 0.2

Polysorbate 80 0.4

Ethanol 10.0

Triethanolamine 0.1

Preservatives, colorings, flavors

1.2 Nutrients Milk lotion ) (Unit: wt%)

GDF-15 expression inhibitor or activity inhibitor 0.1 to 0.2

Beeswax 4.0

Polysorbate 60 1.5

Solbitan Sesquioleate 1.5

Liquid Paraffin 0.5

Caprylic / Capric Triglycerides 5.0

Glycerin 3.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Carboxy Vinyl Polymer 0.1

Triethanolamine 0.2

Preservatives, colorings, flavors

Formulation example  2. Pharmaceutical Formulations

2.1 Sanje  Produce

GDF-15: 2 g

Lactose: 1 g

The above components were mixed and packed in airtight bags to prepare powders.

2.2 Preparation of Tablets

GDF-15: 100 mg

Corn starch: 100 mg

Lactose: 100 mg

Magnesium Stearate: 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

2.3 Preparation of Capsules

GDF-15: 100 mg

Corn starch: 100 mg

Lactose: 100 mg

Magnesium Stearate: 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Attach an electronic file to a sequence list

Claims (7)

A cosmetic composition for preventing or improving blemishes, comprising as an active ingredient siRNA (short interfering RNA) that complementarily binds to mRNA of a growth differentiation factor-15 (GDF-15) gene.
delete delete According to claim 1, wherein the siRNA is characterized in that it has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, cosmetic composition for preventing or improving blemishes.
delete delete delete

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