KR20110130282A

KR20110130282A - Composition for treating pigmentary disorder through wif-1 modulation

Info

Publication number: KR20110130282A
Application number: KR1020100049849A
Authority: KR
Inventors: 강희영
Original assignee: 아주대학교산학협력단
Priority date: 2010-05-27
Filing date: 2010-05-27
Publication date: 2011-12-05

Abstract

The present invention relates to a composition for treating or preventing pigment diseases such as vitiligo and blemishes. The present invention also relates to a method for treating or preventing pigment diseases such as vitiligo and blemishes. The present invention uses Wif-1 protein as an active ingredient for treating / preventing vitiligo and uses a substance that inhibits the expression or activity of Wif-1 as an active ingredient for treating / preventing blemishes.

Description

Composition for treating pigmentary disorder through Wif-1 modulation}

The present invention relates to a composition for treating or preventing pigment diseases such as vitiligo and blemishes. The present invention also relates to a method for treating or preventing pigment diseases such as vitiligo and blemishes using the composition.

Vitiligo is the most common disease of acquired pigmentation diseases in which the skin turns white due to the loss or loss of melanin cells, which determine the color of the skin, resulting in a decrease or disappearance of melanin in the skin area. Although vitiligo is not a life-threatening disease, it has a high prevalence of 1 to 2% of the total population and a disease that is not well treated by conventional medical treatments. Particularly when the exposed part is invaded, it is a serious disease that imposes a lot of restrictions on the normal social life.

Topical steroids are applied or injected for the treatment of vitiligo, especially when the lesion area is small, but side effects of steroids are problematic. Another method of treatment is to increase the melanin production by irradiating with ultraviolet rays, but the treatment is often inconvenient to do for a long time. In addition, chemotherapy is difficult to apply to pregnant women and children, and if you have photosensitive hypersensitivity to light, cataracts, etc. can not be treated.

Melasma is an irregular shape, brown spots of various sizes, which occur on the exposed area, especially the face, which is exacerbated by sun exposure, pregnancy, oral contraceptives or some anticonvulsants. Melanin pigments deposit on the cheeks, forehead, and under the eyes mainly symmetrically.

For the treatment of such blemishes, but using a topical coating containing sun block and hydroquinone, retinoids, steroids, azelaic acid, etc., it is not easy to treat.

Therefore, the technical problem to be achieved by the present invention is to provide a composition for treating or preventing such pigmented diseases as vitiligo, blemishes.

Another technical problem to be achieved by the present invention is to provide a method for treating or preventing pigment disease such as vitiligo, blemishes.

In order to achieve the above technical problem, the present invention provides a composition for the treatment or prevention of vitiligo, comprising Wif-1 (Wnt inhibitory factor 1) protein as an active ingredient. This Wif-1 protein has the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a composition for promoting skin melanin production, comprising Wif-1 protein as an active ingredient.

The present invention also provides a method of promoting melanin production or improving vitiligo comprising administering or contacting an effective amount of Wif-1 protein to skin in need of improvement of vitiligo.

The present invention is based on the discovery that when the Wif-1 protein is administered to melanocytes or the like, the melanin content is increased, thereby allowing Wif-1 to be used for the treatment or prevention of vitiligo.

The present invention also provides a method for treating or preventing blemishes by inhibiting the activity of Wif-1 protein in blemishes due to the fact that many Wif-1 proteins are produced in blemish lesions and that Wif-1 is involved in melanogenesis.

Staining lesions Monoclonal or polyclonal antibodies against Wif-1 protein, antisense oligonucleotides complementarily binding to mRNA of Wif-1, siRNA specific to Wif-1 gene to reduce activity of Wif-1 protein Or shRNA, an inactive Wif-1 like protein or fragment thereof, a peptide that binds to the Wif-1 protein, a compound that specifically binds to the mRNA of Wif-1 and inhibits transcription or translation, specific for a protein of Wif-1 Compounds that bind to and inhibit the function of Wif-1 may be used, and such substances may be administered to inhibit or reduce the production of blemishes.

The gene encoding the Wif-1 protein has a nucleotide sequence of SEQ ID NO.

First, the present invention provides a composition for improving blemishes comprising a monoclonal or polyclonal antibody against Wif-1 protein as an active ingredient in order to reduce the activity of the surrounding protein. Antibodies of the invention include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') ₂ and Fv.

First, polyclonal antibodies can be prepared by various procedures well known in the art. For example, human Wif-1 proteins or fragments thereof can include a variety of hosts, including but not limited to rabbits, mice, mice, and horses, to induce the production of serum containing polyclonal antibodies specific for human antigens. May be administered to the animal. Various adjuvants may be used to increase the immune response, depending on the host species, and the adjuvant may be Freund's adjuvant (complete or incomplete), mineral gels such as aluminum hydroxide, or surface active substances such as lysolecithin. , Such as pluronic polyol, polyanion, peptide, oil emulsion, keyhole limpet hemocyanin, dinitrophenol, and BCG (bacille Calmette-Guerin) and Coryhebacterium parvum Potentially useful human adjuvants include, but are not limited to. Such adjuvants are well known in the art.

Monoclonal antibodies of the present invention are known in the art to which the present invention pertains to the synthesis of antibodies, for example, hybridoma technology (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), phage display Technology (Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991), prepared by chemical synthesis or preferably by recombinant expression techniques. Can be. More specifically, monoclonal antibodies are described in Harlow et al., Antibodies: A Laboratory Manual, 2 ^nd ed ., Cold Spring Harbor Laboratory Press (1988); Or Hammering et al., Monoclonal Antibodies and T-Cell Hybridomas , Elsevier (1981), 563-681. The documents are hereby incorporated by reference in their entirety.

Methods of preparing and screening specific antibodies using hybridoma technology are generally well known in the art. Briefly, in one embodiment, mice can be immunized with Wif-1 or fragments thereof and once an immune response is detected, for example, an antibody specific for an antigen is detected in mouse serum, a mouse spleen is obtained and splenocytes. ) Are separated. Splenocytes are then fused to any suitable myeloma cells, such as those of the cell line SP2 / 0-Agl4 available in ATCC (Accession Number CRL-1581) by well known techniques. Hybridomas are selected and cloned by limiting dilution. Hybridoma clones are then analyzed by methods well known in the art to cells that secrete antibodies capable of binding to cell-related Wif-1 or fragments thereof. In general, ascites fluid containing high levels of antibodies can be produced by injecting positive hybridoma clones into mice.

For some reason, it is desirable to use chimeric, humanized, or fully human antibodies, particularly in the in vivo use and in vivo detection assays of antibodies in humans. Chimeric antibodies are molecules in which different parts of the antibody are derived from immunoglobulin molecules from different species. For example, without limitation, chimeric antibodies may have light and / or heavy chain variable regions derived from mouse antibodies and light and / or heavy chain constant regions derived from human immunoglobulins. Methods for generating chimeric antibodies are known in the art (eg, Morrison, Science. 229 (4719): 1202-1207 (1985); Oi et al., Bio Techniques. 4 (3): 214-221 (1986); Gillies et al., J. Enmunol.Methods. 125 (1-2): 191-202 (1989); and US Pat. Nos. 4,816,397, 4,816,567, and 5,807,715, which are incorporated by reference. The entirety of which is incorporated herein).

The present invention also provides a composition for improving blemishes comprising siRNA or shRNA specific to the Wif-1 gene as an active ingredient, using RNA interference (RNAi) technology to reduce the activity of the surrounding bleeding protein.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in which degradation of the corresponding mRNA occurs by introducing two stranded chain RNAs (dsRNAs) corresponding to the Wif-1 coding gene into the cell or organism. . RNAi is a very powerful way to create the desired knockout or 'knockdown' at the RNA level since multiple cell divisions continue before gene expression is restored by the RNAi effect. RNAi has been found to be successful in human cells, including human embryonic kidney and HeLa cells (Elbashir et al. Nature, 411,494-498, 2001).

In other words, siRNA refers to double stranded RNA of about 20 nucleotides in length capable of mediating RNA interference or gene silencing, and shRNA refers to five to nine bases in the sense and antisense sequences of the siRNA target sequence. It refers to RNA of a short hairpin structure positioned between the loop (loop). siRNAs or shRNAs are known to specifically bind to target mRNAs having complementary sequences to induce RNA interference through cleavage of mRNAs of target genes. In one embodiment of the invention, siRNA or shRNA that inhibits the expression of the Wif-1 protein may have a sequence complementary to the mRNA of the gene encoding Wif-1.

In addition, shRNAs are used to overcome the high cost of synthesis of siRNAs, short duration of RNA interference effects due to low transfection efficiency, and can be used as adenovirus, lentivirus, and plasmid expression vector systems from promoters of RNA polymerase III. It can be introduced into cells and expressed using. shRNAs are known to be converted into siRNAs with the correct structure by siRNA processing enzymes (Dicer or RNase III) present in the cell to induce silencing of target genes.

Methods for preparing such siRNAs include chemical synthesis of oligonucleotides directly (Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC, Shi Y. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.Proc Natl Acad Sci USA 99: 5515-20), (Brummelkamp TR, Bernards R, Agami R. (2002) A system for stable expression of short interfering RNAs in mammalian Science 296: 550-3), Method for cleaving long double-stranded RNA synthesized by in vitro transcription using enzymes such as RNase III family enzymes (Paul CP, Good PD, Winer I, Engelke DR. (2002) Effective expression of small interfering RNA in human cells.Nature Biotechnology 20: 505-8.), Expression by intracellular delivery of siRNA expression plasmids or viral vectors (Lee NS, Dohjima T, Bauer G, Li H, Li MJ, Ehsani A, Salvaterra P, Rossi J. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.Nature Biotechnology 20: 500-5) and expression by intracellular delivery of polymerase chain reaction (PCR) -induced siRNA expression cassettes (Castanotto D, Li H, Rossi JJ. (2002) Functional siRNA expression from transfected PCR products. RNA8: 1454-60), but is not limited thereto.

The present invention also provides a blemish-improving composition comprising an antisense oligonucleotide that complementarily binds to the mRNA of Wif-1 as an active ingredient in order to reduce the activity of the surrounding bleeding protein.

Antisense oligonucleotides refer to oligomers that hybridize with target sequences in RNA by Watson-Crick base pairing to form oligomeric heteroduplexes between mRNA and RNA in the target sequence. Antisense oligonucleotides have complete or approximate sequence complementarity to the target sequence and can inhibit the expression of the target gene by altering the processing of mRNA that blocks or inhibits translation of the mRNA and produces splicing variants.

In addition, the present invention, in addition to the antibody, siRNA, etc. in addition to the activity of the Wif-1 protein around the blemishes or reduced activity of the Wif-1 like protein or fragment thereof, a peptide that binds to and inhibits the activity, Wif-1 protein, Wif- Provides a composition for improving blemishes containing a compound that specifically binds to mRNA of 1 to inhibit transcription or translation, a compound that specifically binds to a protein of Wif-1 and inhibits the function of Wif-1 as an active ingredient Such compositions can be applied to the skin to inhibit or reduce the production of blemishes.

The present invention also provides a method for improving blemishes by applying an effective amount of a substance that reduces the activity of the Wif-1 protein mentioned above to skin in need of blemishes.

The composition for treating or preventing pigment diseases of the present invention may be a solid preparation, a semisolid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, and the like. Solid form preparations may include, but are not limited to, excipients, flavors, binders, preservatives, disintegrants, lubricants, fillers and the like. Semi-solid preparations include, but are not limited to, creams, lotions, emulsifiers, lining agents, and the like, and may be prepared by adding suitable colorants, flavoring agents, stabilizers, viscosity agents, surfactants, and the like. Liquid formulations include, but are not limited to, solutions such as water, alcohols, propylene glycol solutions, suspensions, emulsions, and the like, and may be prepared by adding suitable colorants, flavors, stabilizers, viscosity agents, and the like.

However, the composition of the present invention is preferably a semi-solid formulation that can be applied to the skin in view of the nature to be applied to the skin. Such semi-solid formulations can be prepared by the following exemplary method.

First, vitiligo or blemish improving lotion may be prepared as follows. 0.1% by weight of the active ingredient of the present invention, 5% by weight of glycerin, 3% by weight of dipropylene glycol, 0.5% by weight of hyaluronic acid, 0.1% by weight of polyoxyethylene hydrogenated castor oil, 0.1% by weight of polyethylene oleyl ether, 5% by weight of ethanol, A skin lotion may be prepared by mixing 0.15% by weight of a preservative, an appropriate amount of perfume and pigment, and a residual amount of purified water.

Essence for improving vitiligo or blemishes may be prepared as follows. 3% by weight of active ingredient of the present invention, 1% by weight of cetostearyl alcohol, 1% by weight of monostearic acid, 0.5% by weight of beeswax, 5% by weight of squalane, 3% by weight of isocetyloctanoate, 0.3% by weight of dimethylsiloxane, monostearic acid Sorbitan 0.5 wt%, monostearate polyethylene glycol 8 wt%, glycerin 4 wt%, propylene glycol 0.2 wt%, carboxypolymer 0.22 wt%, triethanolamine 0.25 wt% Essence can be prepared.

Lotion for improving vitiligo or blemishes may be prepared as follows. 1% by weight of active ingredient of the present invention, 0.8% by weight of cetostearyl alcohol, 1% by weight of monostearic acid, 0.5% by weight of beeswax, 0.5% by weight of stearic acid, 7% by weight of liquid paraffin, 5% by weight of squalane, 3% by weight of macadamia oil %, 2% by weight of isocetyloctanoate, 0.3% by weight of dimethylsiloxane, 0.5% by weight of sorbitan monostearate, 1.2% by weight of polyethylene glycol monostearate, 4% by weight of glycerin, 4% by weight of propylene glycol, 4% by weight of betaine, A lotion may be prepared by mixing 0.12% by weight of carboxypolymo, 0.15% by weight of triethanolamine, 0.25% by weight of a preservative, an appropriate amount of perfume and colorant, and a residual amount of purified water.

Cream for improving vitiligo or blemishes may be prepared as follows. 5% by weight of the active ingredient of the present invention, 3% by weight of cetostearyl alcohol, 2% by weight of monostearic acid, 0.5% by weight of beeswax, 8% by weight of liquid paraffin, 7% by weight of squalane, 4% by weight of isocetyloctanoate, tablet jojoba Oil 4%, dimethylsiloxane 0.3%, monostearate sorbitan 1%, monostearic acid polyethylene glycol 1.2%, glycerin 6%, propylene glycol 4%, betaine 4%, xanthan gum 0.06%, A cream may be prepared by mixing 0.10% by weight of triethanolamine, 0.25% by weight of a preservative, an appropriate amount of perfume and pigment, and a residual amount of purified water.

In order to achieve the object of the present invention, treatment or prevention of pigment diseases, the composition of the present invention may be administered from about 0.01 mg / kg to about 10 g / kg daily, from about 0.1 mg / kg to about 1 g / kg Daily doses are preferred. However, the dosage may vary depending on the route and formulation of administration, the condition of the patient (age, gender, weight, etc.), the severity of the condition being treated and the like. If desired, the total daily dose may be divided for convenience and divided several times throughout the day.

The present invention provides a composition useful for improving pigment diseases such as vitiligo and blemishes. The present invention also provides a method useful for improving pigment diseases such as vitiligo and blemishes.

1 is a result of evaluating the effect of Wif-1 protein treatment on the proliferation of melanocytes.
2 is a result of evaluating the effect of Wif-1 protein treatment on melanocyte production of melanocytes.
3 is a result of evaluating the effect of Wif-1 protein treatment on the expression of tyrosinase and beta-catenin.
4 is a result of evaluating the effect of Wif-1 protein treatment on melanocyte production of melanocytes in melanocyte and fibroblast co-culture.
5 is a result showing that the Wif-1 protein is more expressed in blemishes lesions.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

The effects of Wif-1 on the proliferation and melanogenesis of melanocytes were evaluated as follows.

Evaluation of proliferation of melanocytes

Melanocytes were obtained and cultured as follows. The fat was first removed from the human foreskin and then cut into 4 mm x 4 mm size. After 4ml 0.25% trypsin was incubated for 4 hours at 37 ℃. Thereafter, only the epidermis (epidermis) was placed in 7 ml of F-12 medium, vortexed for 1 minute, and then placed in a 100 ml dish and incubated in a 37 ° C. incubator. Test cells were selected from one cell in three passages. Thereafter, the cells were seeded (seedig), and the cells were divided into 130,000 cells in 6 cm plates and incubated again at 37 ° C.

When the cells were grown in a 6 cm dish, the control group, Wif-1 (0.5 µg / ml) and Wif-1 (5 µg / ml) were mixed with melanocyte drug treatment medium MCDB153. Then incubated for 5 days at 37 ℃. After 5 days washed with PBS and 200μl EDTA / trypsin was put in 37 ℃ incubator for 1 minute. 2 ml of F-12 medium was added thereto, and 200 µl was used for counting the cells, and the remaining cells were used for the following melanin content measurement. Cell number was measured by Coulter counter machine, the results are shown in FIG.

As shown in Figure 1, melanocytes were more proliferated when the Wif-1 protein of the present invention.

Melanin content evaluation

The cultured cells were centrifuged (RPM 11000, 5 minutes) and the supernatant was aspirated to separate only pellets. Then, 100 μl of 1N NAOH was added to the pellet and incubated at 37 ° C. for 1.5 hours. During the incubation was vortexed every 30 minutes. Then, the melanin content was measured using a spectrophotometer, and the result is shown in FIG.

As shown in Figure 2, the melanin content produced in the same number of melanocytes increased when the Wif-1 protein treatment of the present invention.

For western blot analysis, the melanocytes were lysed after 5 μg / ml Wif-1 protein for 5 days. 10 ug of protein per lane was electrophoresed using sodium dodecyl sulphate-polyacrylamide gel and blotted onto nitrocellulose paper. Blots were incubated with rabbit polyclonal antibodies against tyrosinase, diluted 1: 2000 and then treated with horseradish peroxidase-binding secondary antibody. Bound antibodies were analyzed using the Enhanced Chemiluminescence Plus Kit (Amersham Int., UK).

As shown in FIG. 3, Wif-1 of the present invention increased the expression of tyrosinase involved in the production of melanin pigment in the skin in melanocytes and also increased the expression of beta-catenin. Wif-1 of the invention is thought to be via Wnt signaling.

The effect of Wif-1 on the proliferation and melanogenesis of melanocyte-fibroblasts was evaluated as follows.

Melanocytes -Proliferation evaluation of fibroblasts

First, after removing the fat from the human foreskin (foreskin), cut into 4mm x 4mm size, 4ml 0.25% trypsin and incubated for 4 hours at 37 ℃. Then, epidermis (epidermis) in 7ml of F-12 medium, dermis in 7ml RPMI1640 medium (dermis) was pulled out, vortexed for 1 minute and put in a 100ml dish and incubated in a 37 ℃ incubator. The test cells were selected from one cell in passage 3. Afterwards, the fibroblasts were seeded with 10 ⁶ , then the pellets of cells of 2 ml of collagen matrix and fibroblasts 10 ⁶ were mixed and spread on the edge of 100 ml dish, followed by F Washed 5 times with -12 medium, and 7 ml of 10% FBS / DMEM was added thereto and incubated in a 37 ° C. incubator for 24 hours. After 24 hours, 6 × 10 ⁵ melanocytes were added, and the medium was mixed with 5 ml of F-12 medium and 5 ml of 10% FBS / DMEM medium, and cultured at 37 ° C.

One control group, Wif-1 (5µg / ml) was selected and mixed with melanocyte drug treatment medium MCDB153 and incubated at 37 ° C. for 5 days. After 5 days, 500 µl of EDTA / trypsin after PBS washing was placed in a 37 ° C incubator for 2 minutes, and 5 ml of F-12 medium was added thereto. 500 μl of cells were placed in the tube to be counted, and the remaining cells were placed in a 15 ml tube. 500 μl of cells in the tube to be counted and 1 ml of icestone were mixed and counted in a Coulter counter machine.

Melanin content evaluation

The cultured cells were centrifuged (RPM 11000, 5 minutes) and the supernatant was aspirated to separate only pellets. Then, 100 μl of 1N NAOH was added to the pellet and incubated at 37 ° C. for 1.5 hours. During the incubation was vortexed every 30 minutes. Thereafter, the melanin content was measured using a spectrophotometer, and the results are shown in FIG. 4.

As shown in Figure 4, the treatment of Wif-1 according to the present invention significantly increased melanin production.

Lesions and adjacent normal skin of bleeding patients were subjected to a 2 mm punch biopsy. Samples were fixed in formalin and embedded in paraffin to make blocks, and sections were made to observe expression changes by immunohistochemical staining using antibodies against Wif-1 (R & D, 1:10). A streptavidin horseradish peroxidase attached with a streptavidin capable of binding to biotin attached to a secondary antibody was used, and 3-amino-9-ethylcarbazole (AEC) was used. The results are shown in Fig.

As shown in Figure 6, it was confirmed that more Wif-1 protein is expressed in the blemish lesion.

<110> Ajou University Industry Cooperation Foundation <120> Composition for treating pigmentary disorder through Wif-1 modulation <130> P10-121 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 379 <212> PRT <213> Wif-1 protein <400> 1 Met Ala Arg Arg Ser Ala Phe Pro Ala Ala Ala Leu Trp Leu Trp Ser 1 5 10 15 Ile Leu Leu Cys Leu Leu Ala Leu Arg Ala Glu Ala Gly Pro Pro Gln 20 25 30 Glu Glu Ser Leu Tyr Leu Trp Ile Asp Ala His Gln Ala Arg Val Leu 35 40 45 Ile Gly Phe Glu Glu Asp Ile Leu Ile Val Ser Glu Gly Lys Met Ala 50 55 60 Pro Phe Thr His Asp Phe Arg Lys Ala Gln Gln Arg Met Pro Ala Ile 65 70 75 80 Pro Val Asn Ile His Ser Met Asn Phe Thr Trp Gln Ala Ala Gly Gln 85 90 95 Ala Glu Tyr Phe Tyr Glu Phe Leu Ser Leu Arg Ser Leu Asp Lys Gly 100 105 110 Ile Met Ala Asp Pro Thr Val Asn Val Pro Leu Leu Gly Thr Val Pro 115 120 125 His Lys Ala Ser Val Val Gln Val Gly Phe Pro Cys Leu Gly Lys Gln 130 135 140 Asp Gly Val Ala Ala Phe Glu Val Asp Val Ile Val Met Asn Ser Glu 145 150 155 160 Gly Asn Thr Ile Leu Gln Thr Pro Gln Asn Ala Ile Phe Phe Lys Thr 165 170 175 Cys Gln Gln Ala Glu Cys Pro Gly Gly Cys Arg Asn Gly Gly Phe Cys 180 185 190 Asn Glu Arg Arg Ile Cys Glu Cys Pro Asp Gly Phe His Gly Pro His 195 200 205 Cys Glu Lys Ala Leu Cys Thr Pro Arg Cys Met Asn Gly Gly Leu Cys 210 215 220 Val Thr Pro Gly Phe Cys Ile Cys Pro Pro Gly Phe Tyr Gly Val Asn 225 230 235 240 Cys Asp Lys Ala Asn Cys Ser Thr Thr Cys Phe Asn Gly Gly Thr Cys 245 250 255 Phe Tyr Pro Gly Lys Cys Ile Cys Pro Pro Gly Leu Glu Gly Glu Gln 260 265 270 Cys Glu Ile Ser Lys Cys Pro Gln Pro Cys Arg Asn Gly Gly Lys Cys 275 280 285 Ile Gly Lys Ser Lys Cys Lys Cys Ser Lys Gly Tyr Gln Gly Asp Leu 290 295 300 Cys Ser Lys Pro Val Cys Glu Pro Gly Cys Gly Ala His Gly Thr Cys 305 310 315 320 His Glu Pro Asn Lys Cys Gln Cys Gln Glu Gly Trp His Gly Arg His 325 330 335 Cys Asn Lys Arg Tyr Glu Ala Ser Leu Ile His Ala Leu Arg Pro Ala 340 345 350 Gly Ala Gln Leu Arg Gln His Thr Pro Ser Leu Lys Lys Ala Glu Glu 355 360 365 Arg Arg Asp Pro Pro Glu Ser Asn Tyr Ile Trp 370 375 <210> 2 <211> 1994 <212> DNA <213> Gene encoding Wif-1 protein <400> 2 gtctaaacgg gaacagccct ggctgaggga gctgcagcgc agcagagtat ctgacggcgc 60 caggttgcgt aggtgcggca cgaggagttt tcccggcagc gaggaggtcc tgagcagcat 120 ggcccggagg agcgccttcc ctgccgccgc gctctggctc tggagcatcc tcctgtgcct 180 gctggcactg cgggcggagg ccgggccgcc gcaggaggag agcctgtacc tatggatcga 240 tgctcaccag gcaagagtac tcataggatt tgaagaagat atcctgattg tttcggaggg 300 gaaaatggca ccttttacac atgatttcag aaaagcgcaa cagagaatgc cagctattcc 360 tgtcaatatc cattccatga attttacctg gcaagctgca gggcaggcag aatacttcta 420 tgaattcctg tccttgcgct ccctggataa aggcatcatg gcagatccaa ccgtcaatgt 480 ccctctgctg ggaacagtgc ctcacaaggc atcagttgtt caagttggtt tcccatgtct 540 tggaaaacag gatggggtgg cagcatttga agtggatgtg attgttatga attctgaagg 600 caacaccatt ctcaaaacac ctcaaaatgc tatcttcttt aaaacatgtc aacaagctga 660 gtgcccaggc gggtgccgaa atggaggctt ttgtaatgaa agacgcatct gcgagtgtcc 720 tgatgggttc cacggacctc actgtgagaa agccctttgt accccacgat gtatgaatgg 780 tggactttgt gtgactcctg gtttctgcat ctgcccacct ggattctatg gagtgaactg 840 tgacaaagca aactgctcaa ccacctgctt taatggaggg acctgtttct accctggaaa 900 atgtatttgc cctccaggac tagagggaga gcagtgtgaa atcagcaaat gcccacaacc 960 ctgtcgaaat ggaggtaaat gcattggtaa aagcaaatgt aagtgttcca aaggttacca 1020 gggagacctc tgttcaaagc ctgtctgcga gcctggctgt ggtgcacatg gaacctgcca 1080 tgaacccaac aaatgccaat gtcaagaagg ttggcatgga agacactgca ataaaaggta 1140 cgaagccagc ctcatacatg ccctgaggcc agcaggcgcc cagctcaggc agcacacgcc 1200 ttcacttaaa aaggccgagg agcggcggga tccacctgaa tccaattaca tctggtgaac 1260 tccgacatct gaaacgtttt aagttacacc aagttcatag cctttgttaa cctttcatgt 1320 gttgaatgtt caaataatgt tcattacact taagaatact ggcctgaatt ttattagctt 1380 cattataaat cactgagctg atatttactc ttccttttaa gttttctaag tacgtctgta 1440 gcatgatggt atagattttc ttgtttcagt gctttgggac agattttata ttatgtcaat 1500 tgatcaggtt aaaattttca gtgtgtagtt ggcagatatt ttcaaaatta caatgcattt 1560 atggtgtctg ggggcagggg aacatcagaa aggttaaatt gggcaaaaat gcgtaagtca 1620 caagaatttg gatggtgcag ttaatgttga agttacagca tttcagattt tattgtcaga 1680 tatttagatg tttgttacat ttttaaaaat tgctcttaat ttttaaactc tcaatacaat 1740 atattttgac cttaccatta ttccagagat tcagtattaa aaaaaaaaaa attacactgt 1800 ggtagtggca tttaaacaat ataatatatt ctaaacacaa tgaaataggg aatataatgt 1860 atgaactttt tgcattggct tgaagcaata taatatattg taaacaaaac acagctctta 1920 cctaataaac attttatact gtttgtatgt ataaaaaaaa aaaaaaaaaa aaaaaaaata 1980 aaaaaaaaaa aaaa 1994

Claims

Wif-1 (Wnt inhibitory factor 1) composition for the treatment or prevention of vitiligo, characterized in that it comprises a protein as an active ingredient.

A composition for promoting skin melanin production, comprising Wif-1 (Wnt inhibitory factor 1) protein as an active ingredient.

A method of promoting melanin production comprising administering or contacting an effective amount of a Wnt inhibitory factor 1 (Wifi-1) protein.

A method of treating vitiligo comprising administering or contacting an effective amount of a Wnt inhibitory factor 1 (Wifi-1) protein to skin in need of improvement.

Wif-1 (Wnt inhibitory factor 1) A composition for treating or preventing blemishes, characterized in that it comprises a substance that inhibits the expression or activity of the protein as an active ingredient.

The method of claim 5, wherein the substance is a composition for the treatment or prevention of blemishes, characterized in that the antibody to the Wif-1 protein.

The method of claim 5, wherein the substance is a siRNA or shRNA specific to the Wif-1 protein coding gene, characterized in that the composition for the treatment or prevention of blemishes.

The method of claim 5, wherein the substance is an antisense oligonucleotide that complementarily binds to the mRNA of Wif-1 composition for the treatment or prevention of blemishes.

The method according to claim 5, wherein the substance specifically binds to and inactivates a Wif-1 like protein or fragment thereof, a peptide that inhibits the activity of Wif-1 that binds to the Wif-1 protein, and the mRNA of Wif-1. Or a compound that inhibits translation, or a compound that specifically binds to a protein of Wif-1 to inhibit the function of Wif-1.

A method of treating or preventing blemishes, which inhibits the activity of the Wnt-1 (Wnt inhibitory factor 1) protein.