Composition for regulating melanin pigment in skin or hair

Abstract

The present invention relates to a composition for regulating melanin pigment in skin or hair, comprising erythropoietin or a derivative thereof. The erythropoietin of the present invention has excellent skin tanning or hair blackening efficacy by promoting melanin production, and can be effectively used for preventing, ameliorating or treating skin pigment diseases or hair pigment diseases.

Description

Composition for controlling melanin pigmentation of skin or hair

[0001] The present invention relates to a composition for controlling melanin pigmentation of skin or hair, comprising erythropoietin or a derivative thereof, and more specifically, to the use of erythropoietin for skin tanning or hair darkening, and for the prevention, treatment and improvement of skin pigmentation diseases or hair pigmentation diseases.

[0002]

[0003] Melanin is synthesized in pigment cells (melanocytes) in the basal layer of the skin by alpha-melanocyte stimulating hormone (α-MSH), cAMP promoter, and ultraviolet rays, and is transferred to surrounding keratinocytes to represent human skin color. Meanwhile, if pigment cells die or become necrotic, vitiligo can develop, causing white patches of various sizes and shapes to appear on the skin. It is a relatively common disease that occurs in about 1% of the population worldwide, with the highest age of onset being 10 to 30 years, and 30% of patients have a family history.

[0004] Current treatments for vitiligo include psoralen ultraviolet light therapy, topical and systemic steroid treatments, and surgical procedures. The most common treatment is topical steroids, but long-term use can lead to side effects, including skin shrinkage.

[0005] Furthermore, with the recent rise in interest in appearance, the use of self-tanning products to maintain a healthy appearance is on the rise. Self-tanning is gaining popularity because it can prevent photoaging caused by overexposure to UV rays, achieve a tanning effect even during seasons without sunlight, and maintain a healthy level of melanin, which plays a crucial role in protecting the body from UV rays. It also offers the advantage of being free from the risk of skin aging and skin cancer caused by excessive melanin synthesis. Recently, self-tanning products bind to keratin in the stratum corneum, temporarily adding pigment to the skin. However, these products often result in uneven skin tone and have poor pigmentation.

[0006] In addition, gray hair, which generally occurs with age, also gives a sense of old age as interest in appearance increases, and it is a cause of stress, especially in young people. Melanin is an important factor that determines hair color. Melanin produced in the pigment cells located at the top of the hair is transferred to the hair cortex cells (especially the keratinocytes surrounding the hair cortex) and moves upward along with hair growth. Gray hair, as a phenomenon of physiological aging, is caused by a decrease in the number of hair pigment cells and a decrease in melanin production due to a decline in the function of the pigment cells. Currently, hair dyeing is used as a solution to gray hair, but dyeing is a temporary method and must be re-dyeed as gray hair grows, and the ingredients contained in hair dyes can be irritants, causing contact dermatitis or skin allergies, which has become a problem.

[0007] Meanwhile, erythropoietin is a secretory protein primarily produced by the kidneys and is known to regulate red blood cell production. For example, when the body's oxygen concentration is low, the kidneys secrete erythropoietin, stimulating red blood cell production in the bone marrow and increasing oxygen supply. Recombinant erythropoietin is primarily used to treat anemia, and is a key component of injections prescribed for patients with anemia, particularly those with chronic renal failure or undergoing cancer treatment.

[0008] Accordingly, the inventor of the present invention conducted research on a substance effective for skin tanning, hair darkening, and pigmentation disorders of the skin or hair, and discovered that erythropoietin can be used to control pigmentation of the skin or hair, thereby completing the present invention.

[0009]

[0010] The technical problem to be achieved by the present invention is to provide a composition for controlling melanin pigmentation of skin or hair.

[0011] In addition, a technical problem to be achieved by the present invention is to provide a cosmetic composition for skin tanning or hair blackening.

[0012] In addition, the technical problem to be achieved by the present invention is to provide a pharmaceutical composition and a cosmetic composition for preventing, treating or improving skin pigmentation disease or hair pigmentation disease.

[0013]

[0014] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0015]

[0016] In order to achieve the above technical task, one embodiment of the present invention provides a composition for controlling melanin pigmentation of skin or hair, comprising erythropoietin or a derivative thereof.

[0017] In an embodiment of the present invention, the erythropoietin may be derived from a mammal or cell line.

[0018] In an embodiment of the present invention, the derivative of erythropoietin may include any one of the amino acid sequences of SEQ ID NOs: 1 to 4.

[0019] In order to achieve the above technical task, another embodiment of the present invention provides a cosmetic composition for skin tanning or hair blackening comprising erythropoietin or a derivative thereof.

[0020] In order to achieve the above technical task, another embodiment of the present invention provides a pharmaceutical composition for preventing or treating a skin pigmentation disease or a hair pigmentation disease, comprising erythropoietin or a derivative thereof.

[0021] In an embodiment of the present invention, the skin pigmentation disease may be vitiligo.

[0022] In an embodiment of the present invention, the hair pigment disease may be white hair.

[0023] In order to achieve the above technical task, another embodiment of the present invention provides a cosmetic composition for preventing or improving skin pigmentation disease or hair pigmentation disease, comprising erythropoietin or a derivative thereof.

[0024]

[0025] The present invention relates to a composition for controlling melanin pigmentation of skin or hair, comprising erythropoietin or a derivative thereof. The erythropoietin of the present invention promotes melanin production, thereby exhibiting excellent skin tanning or hair blackening effects, and can be usefully utilized for preventing, improving, or treating skin pigmentation diseases or hair pigmentation diseases.

[0026]

[0027] The effects of the present invention are not limited to the above-described effects, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0028]

[0029] Figure 1 shows the results of examining changes in skin color by treating mouse skin with recombinant human erythropoietin (rhEPO) protein to determine the skin pigmentation-enhancing ability of erythropoietin protein according to one embodiment of the present invention.

[0030] Figures 2 and 3 show the results of pixel analysis based on photographs of skin tissue treated with rhEPO protein.

[0031] Figure 4 shows the results of quantifying melanin pigment extracted from skin tissue treated with rhEPO protein by absorbance.

[0032] Figure 5 shows the results confirming that erythropoietin receptor (EPOR) is expressed in keratinocytes and melanocytes, which are major cells of the stratum corneum of the skin.

[0033] Figure 6 shows the results of confirming whether rhEPO protein is toxic to melanocytes.

[0034] Figures 7 to 13 illustrate the results of homology-based structural modeling and molecular dynamics experiments for the selection of peptide fragment sequences derived from full-length erythropoietin.

[0035] Figures 14 and 15 show the results of a blackening experiment on a human skin tissue model using a peptide derived from full-length erythropoietin according to one embodiment of the present invention.

[0036] Figure 16 shows the results of a western blot on human-derived artificial skin tissue treated with a peptide derived from full-length erythropoietin.

[0037] Figure 17 shows the western blot results for mouse skin tissue treated with rhEPO protein.

[0038] Figures 18 to 20 show the results of confirming the cytotoxicity of peptides derived from full-length EPO against major skin cell lines.

[0039]

[0040] Hereinafter, the present invention will be described in detail.

[0041]

[0042] The present invention relates to a composition for controlling melanin pigmentation of skin or hair.

[0043] The composition of the present invention comprises erythropoietin or a derivative thereof.

[0044] The above erythropoietin may include erythropoietin derived from any mammal, including humans. The above erythropoietin may include not only the naturally occurring, i.e. wild-type form, but also derivatives, analogs, variants, mutes, or mutants thereof, as long as they exhibit the biological effects of the wild-type form.

[0045] The above derivatives are functional equivalents or derivatives of erythropoietin which retain the basic erythropoietin structure and are obtained by substitution of one or more atoms or molecular groups or radicals, in particular substitution of sugar chains such as ethylene glycol, or which differ in their amino acid sequences from those of naturally occurring human or animal erythropoietin proteins at least in one position but which have essentially high homology at the amino acid level and similar biological activity. The homology refers to the degree of relationship between two or more polypeptide molecules and means a sequence identity of at least 80% or more, preferably at least 90% or more. Differences between erythropoietin derivatives and native erythropoietin can occur, for example, through mutations such as deletions, substitutions, insertions, additions, base exchanges or recombination of the nucleotide sequence encoding the erythropoietin amino acid sequence. Therefore, the derivative comprises a mutant erythropoietin molecule, i.e., an erythropoietin mutant protein.

[0046] The above erythropoietin derivative may, for example, comprise any one of the amino acid sequences of SEQ ID NOs: 1 to 4.

[0047] The above analogs include compounds that do not have an identical amino acid sequence to erythropoietin, but whose three-dimensional structure closely resembles erythropoietin and thus possesses corresponding biological activity. For example, they may be compounds that contain amino acid residues responsible for binding of erythropoietin to its receptor in an appropriate three-dimensional configuration and thus can stimulate essential surface properties of the erythropoietin binding site.

[0048] The above erythropoietin can be produced in various ways, for example, by isolating it from human urine or from the urine or plasma (including serum) of patients with aplastic anemia. Human erythropoietin can also be obtained from tissue cultures of human renal cancer cells, from lymphoma cells capable of producing human erythropoietin, and from fusion cell cultures obtained by fusion of human cell lines.

[0049] Additionally, the erythropoietin may be derived from a cell line that produces it. Specifically, the erythropoietin may be produced by genetic engineering, which recombinantly produces the desired protein using DNA or RNA encoding the appropriate amino acid sequence, for example, in bacteria, yeast, plant cell lines, or animal cell lines.

[0050] The above melanin pigment regulation means promoting melanin production in the skin or hair, and may include use for darkening the skin or hair.

[0051]

[0052] The present invention relates to a cosmetic composition for skin tanning or hair blackening.

[0053] The cosmetic composition of the present invention comprises erythropoietin or a derivative thereof.

[0054] Erythropoietin and its derivatives are as described above.

[0055] The above skin tanning may refer to imparting color to the skin using artificial means, preferably chemical means. The cosmetic composition for skin tanning of the present invention may include a composition that produces an artificial tan similar to that produced by long-term exposure to sunlight, as well as a composition that imparts a slight pigmentation to the skin, which is not easily recognized as an artificial tan but rather creates a subtle color on the skin that makes the skin appear healthier.

[0056] The above hair blackening refers to an increase in the color pigment of the hair, and in particular, it can refer to a phenomenon in which the amount of melanin pigment increases, causing the hair to appear dark.

[0057] The above cosmetic composition may be formulated as a skin, lotion, toner, cosmetic soap, body wash, serum, cleansing lotion, essence, nourishing cream, pack, massage cream, etc., and may also be formulated as a softening toner, astringent toner, nourishing toner, eye cream, eye essence, cleansing foam, cleansing water, powder, body lotion, body cream, body oil, body essence, makeup base, foundation, shampoo, or rinse, but is not limited thereto.

[0058] In addition, when used as a cosmetic composition, additional substances may be added according to the formulation of the external skin agent or cosmetic. For example, but not limited to, when the formulation is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component, and when the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included. In addition, when the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and preferably, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used, but is not limited thereto.When the formulation is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components, and when the formulation is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used as carrier components, but are not limited thereto.

[0059]

[0060] The present invention relates to a pharmaceutical composition and a cosmetic composition for preventing, treating or improving skin pigmentation diseases or hair pigmentation diseases.

[0061] The pharmaceutical composition and cosmetic composition of the present invention comprise erythropoietin or a derivative thereof.

[0062] Erythropoietin and its derivatives are as described above.

[0063] The above skin or hair pigment disease may refer to a disease caused by deposition or lightening of pigment in the skin or hair.

[0064] The skin pigmentation disorder may be, for example, vitiligo. The hair pigmentation disorder may be, for example, albinism.

[0065] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, which is commonly used in the preparation of pharmaceuticals, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0066] The pharmaceutical composition of the present invention may further include, in addition to the above components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, body weight, sex, degree of disease symptoms, food, administration time, administration route, excretion rate, and reaction sensitivity, and a generally skilled physician can easily determine and prescribe an effective dosage for the desired treatment. Meanwhile, the dosage of the pharmaceutical composition of the present invention is not limited thereto and may be 0.01-2000 mg / kg (body weight) per day.

[0067] The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or skin application. It is preferable that the route of administration of the pharmaceutical composition of the present invention be determined depending on the type of disease to which it is applied.

[0068] The pharmaceutical composition of the present invention can be manufactured in a unit dosage form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains, and the method can be performed. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0069] The cosmetic composition of the present invention can be formulated as a skin, lotion, toner, cosmetic soap, body wash, serum, cleansing lotion, essence, nourishing cream, pack, massage cream, etc. for the purpose of preventing or improving skin pigmentation disease or hair pigmentation disease, and can also be manufactured and processed in the form of an emollient toner, an astringent toner, a nourishing toner, an eye cream, an eye essence, a cleansing foam, a cleansing water, a powder, a body lotion, a body cream, a body oil, a body essence, a makeup base, a foundation, a scalp lotion, a scalp cream, a scalp oil, a scalp essence, a shampoo, a rinse, etc.

[0070] The cosmetic composition of the present invention refers to a skin product manufactured and processed using raw materials or ingredients having functionality useful to the human body, and is meant to be applied or used for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological action.

[0071] In addition, when used as a cosmetic composition, additional substances may be added according to the formulation of the external skin agent or cosmetic. For example, but not limited to, when the formulation is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component, and when the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included. In addition, when the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and preferably, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used, but is not limited thereto.When the formulation is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components, and when the formulation is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used as carrier components, but are not limited thereto.

[0072]

[0073] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0074]

[0075] Experimental methods and results

[0076] 1. Obtaining erythropoietin protein

[0077] Recombinant human erythropoietin protein (rhEPO) was purchased from Thermo Fisher Scientific as Human EPO (Erythropoietin) Recombinant Protein, PeproTech®, Gibco, produced for research use.

[0078]

[0079] 2. Confirmation of EPO's ability to improve skin pigmentation

[0080] To investigate the skin pigmentation-enhancing ability of EPO protein, mouse skin was excised and cultured for 9 days. The cells were divided into two groups: one exposed to UV light for 5 minutes daily (UV+) and the other not (UV-). Under identical culture conditions, the experimental group treated with recombinant human erythropoietin (rhEPO) protein and the control group treated with only the solvent were compared.

[0081] As a result, under UV-conditions without UV irradiation, the experimental group treated with 0.4 nM rhEPO showed an overall darker skin color compared to the control group treated with only solvent.

[0082] Under UV+ conditions with UV irradiation, the skin color of the control group not treated with rhEPO also turned dark due to the effect of UV irradiation, but the experimental group treated with 0.4 nM rhEPO turned darker than the control group.

[0083] The rhEPO-treated experimental group showed overall darkening of the skin, with a particularly strong darkening centered around the hair follicles. This suggests the potential for hair darkening (Fig. 1).

[0084] In addition, as a result of performing pixel analysis on the photographs of the cultured skin tissues of Fig. 1, a histogram by brightness (darkness) was obtained as shown in Fig. 2 (white 255 points / black 0 points).

[0085] As a result, it can be seen that in the skin not irradiated with UV rays (UV-), more pixels with dark colors were found in the experimental group treated with rhEPO, and relatively fewer pixels with light colors were found (Box 1 on the left).

[0086] In the UV-irradiated skin (UV+), more pixels with darker colors were found in the rhEPO-treated experimental group (box 2 on the right).

[0087] Therefore, rhEPO increased skin pigmentation even in skin that was not irradiated with UV rays, and the increase in skin pigmentation was further increased in skin that was irradiated with UV rays.

[0088] If the above tendency is expressed as pixel-by-pixel contrast, it can be seen that skin darkening due to rhEPO becomes more evident (Fig. 3).

[0089]

[0090] 3. Confirmation of increased melanin pigmentation

[0091] The results of extracting melanin pigment from the cultured skin tissue of Fig. 1 and quantifying it by absorbance are as shown in Fig. 4.

[0092] As with the image pixel analysis, the amount of melanin in the rhEPO-treated group increased in the group not treated with UV light, and in the group treated with UV light, the amount of melanin in the rhEPO-treated group increased.

[0093] In particular, the amount of melanin extracted from skin treated with UV rays was similar to that of skin treated only with rhEPO without UV rays treatment.

[0094] This suggests that simply applying rhEPO to the skin can produce effects similar to tanning from UV rays.

[0095]

[0096] 4. Confirmation of erythropoietin receptor (EPOR) expression

[0097] To determine the types of skin cells expressing erythropoietin receptors, mouse-derived pigment cell line Clone M3 (melanocyte), skin keratinocyte cell line 308 (keratinocyte), undifferentiated adipocyte cell line 3T3-L1 (fibroblast), human-derived skin keratinocyte cell line HaCaT (keratinocyte), and skin dermal cell line CCD (fibroblast) were cultured, and the cells were lysed with RIPA buffer to obtain homogenized cell line protein solutions. These protein solutions were used for protein electrophoresis (SDS-PAGE). Western blot experiments to confirm protein expression were performed using anti-tyrosinase antibody (1:1000), anti-erythropoietin receptor antibody (1:1000, R&D Systems), and a monoclonal antibody to β-actin (1:1000) as a control. In Western blot experiments, secondary antibody reactions were performed with goat anti-mouse IgG (H+L) antibody conjugated with HRP (1:2000, Invitrogen, 31430) and goat anti-rabbit IgG antibody conjugated with HRP (1:2000, EMD Millipore, AP132P). Protein electrophoresis was performed with a Hoefer apparatus, and protein transfer from PAGE gel to nitrocellulose membrane (0.45 μm pore, Bio-rad) was performed using a Bio-rad mini-Trans-Blot Cell. The antibody-bound blotting membrane was washed with ECL solution (Thermo Scientific™, SuperSignal). TM The luminescence reaction was induced with West Pico PLUS Chemiluminescent Substrate, and imaged using an imaging system for chemiluminescence measurement (WSE-6200 LuminoGraph ll, ATTO Korea).

[0098] As a result, we confirmed that the erythropoietin receptor (EPOR) is expressed in melanocytes and keratinocytes, which are highly associated with skin pigmentation (Fig. 5). Therefore, the pigment-enhancing effect of rhEPO protein appears to be mediated by the EPOR signaling pathway.

[0099]

[0100] 5. Confirmation of EPO cytotoxicity

[0101] To determine whether rhEPO protein is toxic to melanocytes, various toxicity tests (CCK-8 assay, MTT assay) were performed on three types of pigmented cell lines (Clone M3, B16 / F1, and B16 / F10). Regardless of cell line type, no significant cytotoxicity was observed in any rhEPO-treated experimental group compared to the control group (Fig. 6).

[0102]

[0103] 6. Design of peptide fragment sequences derived from full-length EPO

[0104] Based on the circular sequence (ML1, LQLHVDKAVSGLRSLTTLLRALG (SEQ ID NO: 5)) centered on the EPOR binding helix of the full-length EPO protein, 31 modified EPO proteins were designed and manufactured by changing the physicochemical properties of the helix structure surface by increasing the proportion of hydrophobic amino acids or increasing the proportion of polar (charged) amino acids.

[0105] Among them, the four peptides primarily used in in vitro experiments were MLP, MLP-C, MLP-H, and ML1-h3 (SEQ ID NOs: 1 to 4) (Table 1). These were selected by selecting sequences with relatively high energy values ​​for the receptor binding model during the in silico experiment, and among them, four peptides with relatively short peptide sequences and thus relatively low production costs were selected.

[0106] SEQ ID NO NAME Sequence 1MLPLHVDKAVSGLRSLTTLRA2MLP-CRHVKKRVKGLKSLTTLLRA3MLP-HLHVLKAVSGLLTLTMIRRA4ML1-h3LQLHVLKAVAGLRTLTMIRRALA

[0107]

[0108] 7. Homology Modeling with Molecular Dynamics

[0109] Using the bound structure of full-length EPO protein and EPOR, of which the structure has already been elucidated (PDB: 1EER), as a template, the binding structure of the previously designed full-length EPO-derived peptide fragment sequence and EPOR was modeled (Homology Modeling). Using Modeller, the most widely used analysis tool for homology-based structure prediction, 500 models were generated for each sequence, and the final binding structure model was derived through molecular dynamics modeling. The top 10% (50 models) of the generated models were selected based on the Discrete Optimized Protein Energy (DOPE) Score, which evaluates the stability of the structural model, and then the biopython code was used to analyze the tendency of the single EPOR-dual EPOR binding mode within the top models based on the number of interactions between the peptide fragment sequence and EPOR. At this time, when the number of interactions between the second subunit of the two identical subunits constituting the EPOR and the peptide fragment sequence was 0, it was judged as a single EPOR binding mode, and when it was 1 or more, it was judged as a dual EPOR binding mode.

[0110] The results are shown in Figures 7 to 13. In all peptide sequences, the energy values ​​of the single binding mode and the double binding mode showed similar patterns, and when simulating the same peptide sequence, the DOPE score value of the double binding mode in all sequences showed a smaller energy value than the DOPE score of the single binding mode. However, it was found that three peptides, including ML1-L2, ML1-K2, and ML1-R2, still showed high energy score values ​​(high instability).

[0111] Accordingly, based on the minimum value of the DOPE score indicating structural stability, four types of peptides, including MLP, MLP-C, MLP-H, and ML1-h3, were finally selected among 31 design peptides.

[0112]

[0113] 8. Darkening Experiments on Human Skin Tissue Models

[0114] KeraSkin (Biosolution), an artificial skin model utilizing human-derived keratinocytes (skin epidermal cells) and melanocytes, was treated with 0.4 nM of full-length EPO (rhEPO; recombinant human Erythropoietin), which is an active concentration in the literature, or the EPO-derived peptides of the present invention (SEQ ID NOs: 1 to 4), and then cultured for 10 days. Peptide treatment was performed under UV-shielded conditions, and artificial skin treated with UVB for 1 minute without peptide treatment was used as a positive control. All experiments were repeated three times to confirm reproducibility (n=3).

[0115] When observed with the naked eye immediately after culture, the artificial skin tissue treated with EPO peptide was darker overall and had many black spots compared to the control group (Cont) (Fig. 14).

[0116] Additionally, to quantitatively measure the degree of color change, all artificial skin tissues were photographed at the same camera height under the same illumination, and pixel analysis was performed using ImageJ. Each pixel in the photograph was measured as a value range from white (255) to black (0), and this was converted into a histogram format and graphically depicted as a violin plot in Figure 15.

[0117] As a result of the experiment, the violin plot of the artificial skin surface (top of Fig. 15) confirmed that all peptide-treated groups were blackened (close to a value of 0). In the negative control group (Control), the most pixels had values ​​around 150 (red dotted line), but in the positive control group (UV+Control), the most pixel values ​​changed from 150 to 130. Compared to the negative and positive control groups, all peptide-treated groups had values ​​less than 150 (bottom of the red dotted line).

[0118] In particular, the MLC peptide showed the largest black shift, and ML1-h3 also showed a comparable black shift.

[0119] In the violin plot at the bottom of the artificial skin (bottom of Fig. 15), black shift was observed in all peptide treatment groups, and similarly, the black shift of the largest number of pixels was observed in ML-C.

[0120]

[0121] 9. Western Blot on Human Skin Tissue Model

[0122] A portion of the above skin tissue was rapidly frozen in liquid nitrogen, disrupted using a Bead Mill Homogenizer, and dissolved in RIPA buffer. The protein concentration of all samples was adjusted to a constant 65 ng / 10 μl, and SDS-PAGE electrophoresis and Western blotting were performed.

[0123] The antibodies used are as follows.

[0124] pJAK2: cell signaling #3776

[0125] pSTAT5: cell signaling #9351

[0126] pAkt: cell signaling #9271

[0127] pErk: cell signaling #9101

[0128] JAK2: cell signaling #3230 (125kDa)

[0129] STAT5: cell signaling #25656 (90kDa)

[0130] Akt: cell signaling #9272 (60kDa)

[0131] Erk: cell signaling #9102 (42 / 44kDa)

[0132] GAPDH: Merck #MAB374

[0133] As a result, compared to the control group, pJAK2 and pSTAT5 increased in all peptide groups, whereas pAkt increased in MLP, MLC, and ML1-h3, and pErk showed a large increase in MLH, indicating differences in signaling. This shows that there are differences in the activation of EPOR downstream signals depending on the peptide (Fig. 16).

[0134]

[0135] 10. Western Blot on Mouse Skin Tissue

[0136] Mouse skin tissue was collected using a 6-mm skin punch and cultured for 10 days through explant culture. During culture, tissue was divided into experimental and control groups: EPO-treated, non-EPO-treated, and UVB-treated. The cultured tissue was homogenized using a Bead Mill Homogenizer, dissolved in RIPA buffer, and 20-30 μg per well was subjected to SDS-PAGE, followed by Western blotting.

[0137] The antibodies used are as follows.

[0138] pJAK2: cell signaling #3776

[0139] pSTAT5: cell signaling #9351

[0140] pAkt: cell signaling #9271

[0141] pErk: cell signaling #9101

[0142] JAK2: cell signaling #3230 (125kDa)

[0143] STAT5: cell signaling #25656 (90kDa)

[0144] Akt: cell signaling #9272 (60kDa)

[0145] Erk: cell signaling #9102 (42 / 44kDa)

[0146] GAPDH: Merck #MAB374

[0147] As a result, melanization induced by full-length EPO appeared to increase overall phosphorylation of pJAK2, pAkt, pSTAT5, and pERK. This appears to be due to EPOR signaling activation, as previously reported. In melanization-induced tissues treated simply with UVB without peptide treatment, unlike EPOR activation, no increase in phosphorylation of pJAK2, pAkt, pSTAT5, and pErk was observed (Fig. 17).

[0148]

[0149] 11. Cytotoxicity test of EPO-derived peptides

[0150] The toxicity of each peptide was tested against B16 / F1 (Fig. 18), HaCaT (Fig. 19), and 3T3-L1 (Fig. 20) cell lines, which are major cell types of the skin: keratinocytes, melanocytes, and fibroblasts. Toxicity was less than 5% for all cell lines.

Claims

1. A composition for controlling melanin pigmentation of skin or hair, comprising erythropoietin or a derivative thereof.

2. A composition according to claim 1, wherein the erythropoietin is derived from a mammal or cell line.

3. A composition according to claim 1, wherein the derivative of erythropoietin comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 4.

4. A cosmetic composition for skin tanning or hair blackening containing erythropoietin or a derivative thereof.

5. A pharmaceutical composition for preventing or treating skin pigmentation disease or hair pigmentation disease containing erythropoietin or a derivative thereof.

6. A pharmaceutical composition according to claim 5, wherein the skin pigment disease is vitiligo.

7. A pharmaceutical composition according to claim 5, wherein the hair pigment disease is gray hair.

8. A cosmetic composition for preventing or improving skin pigmentation disease or hair pigmentation disease containing erythropoietin or a derivative thereof.

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