WO2009020267A1 - Competitors of microphthalmia transcription factor and the cosmetic composition comprising thereof - Google Patents

Abstract

The present invention relates to MITF (microphthalmia transcription factor) competitors, precisely MITF competitors selected by using the protein chip for screening MITF competitors being able to suppress the expression of pigmentation related enzymes by inhibiting the binding of MITF with promoter of the pigmentation related enzyme gene and a cosmetic composition for whitening comprising the same. The composition of the present invention has skin whitening activity but no cytotoxicity, so that it can be effectively used for cosmetics for skin whitening or as a whitening agent.

Description

[DESCRIPTION]

[invention Title]

COMPETITORS OF MICROPHTHALMIA TRANSCRIPTION FACTOR AND THE COSMETIC COMPOSITION COMPRISING THEREOF

[Technical Field]

The present invention relates to a competitor of MITF (microphthalmia transcription factor) and a cosmetic composition for whitening comprising the same.

[Background Art]

MITF (microphthalmia transcription factor) is a protein specifically expressed in melanocytes and an important regulation factor involved in melanin synthesis. Skin hyperpigmentation is caused by the generation of black eumelanin and yellow or red pheomelanin by oxidation of tyrosine of melanocytes in Golgi mediated by such pigment related enzymes as tyrosinase, Tyrpl (tyrosinase related protein 1), Tyrp2 (tyrosinase related protein 2), DCT (dopachrom tautomerase) , DHICA (dihydroxyindole carboxylic acid) oxidase, etc. These enzymes are expressed specifically in melanocytes. MITF is bound to E-box

(CATGTG) existing in the promoter of the pigmentation related enzyme gene such as tyrosinase, so that it accelerates the binding of N-terminal transcription activation domain (TAD) and C-tecminal TAD domain to the

E-box, resulting in the expressions of the pigment related enzymes to induce melanin biosynthesis. So, the binding of

MITF with E-box is the key factor in regulation of the expressions of major enzymes involved in pigment generation.

To disclosure the mechanism of whitening, studies on

ET-I (endothelin-1) that is the key factor in the growth and differentiation of melanocytes, PAR-2 that is the important factor for transduction of melanosome, and MITF inducing melanin synthesis are actively undergoing. It has been reported that the MITF/E-box binding inhibitor, MITF- DN and PIAS3 (protein inhibitor of activated STAT3) suppress the expression of related enzymes. So, the MITF/E-box specific inhibitor is expected to be effectively used as a therapeutic agent for skin hyperpigmentation and as a whitening material for functional cosmetics. However, the development of a whitening material targeting MITF is still slow and limited in intracellular screening. Therefore, it is required to establish a novel extracellular screening system.

The present inventors previously reported the protein chip for screening the MITF (microphthalmia transcription factor) and E-box binding specific inhibitor and a method for screening the MITF competitor using the same (Korean Patent Publication No. 10-2005-0031365) . The protein chip and the screening method facilitated multiple, simultaneous experiments and screening of a target material having low concentration and low molecular weight via HTS (high throughput screening) . So, they can be effectively used for the production of an advanced material for cosmetics and a therapeutic agent for skin diseases by screening target molecules related with whitening.

The present inventors synthesized 27 candidates prepared by copying the binding site of MITF and E-box for the inhibitor of MITF expression working by binding to the promoters of MITF and tyrosinase gene, and then confirmed the MITF inhibiting effect after applying the candidates in the protein chip and demonstrated their potentials as the composition for functional cosmetics for whitening from melanin synthesis inhibition test and cytotoxicity test. Three candidates among the above candidates that demonstrated the best effect were selected and proceeded to EMSA (Electrophoretic Mobility Shift Assay) . As a result, the present inventors completed this invention by confirming that the inhibition effect of the above candidates was resulted from the inhibition of the binding of MITF to tyrosinase gene promoter.

[Disclosure] [Technical Problem] It is an object of the present invention to provide a

MITF competitor that can be used as an advanced material for cosmetics for whitening or a therapeutic agent for skin diseases and a cosmetic composition for whitening comprising the same.

[Technical Solution]

To achieve the above object, the present invention provides a cosmetic composition for whitening comprising the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box as an active ingredient.

The present invention also provides a melanin synthesis inhibitor containing the binding inhibitor as an active ingredient.

The present invention further provides a pharmaceutical composition for skin whitening comprising the binding inhibitor as an active ingredient.

The present invention also provides a health food for skin whitening comprising the binding inhibitor as an active ingredient.

The present invention also provides a method for inhibiting melanin synthesis comprising the step of administering the binding inhibitor to a subject. The present invention also provides a method for skin whitening comprising the step of applying the binding inhibitor on melanin pigmented skin.

The present invention also provides a use of the binding inhibitor for the production of a cosmetic composition for skin whitening.

In addition, the present invention also provides a use of the binding inhibitor for the production of a pharmaceutical composition for skin whitening.

[Advantageous Effect]

The composition of the present invention has the effect of skin whitening but no cytotoxicity, so that it can be effectively used as cosmetics for whitening or as a whitening agent.

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Fig.l is a set of a graph and a diagram illustrating the optimum concentration of MITF in the protein chip: a: binding image of MITF with oligo-DNA in the protein chip; and, b: a diagram representing the signal values obtained from the image of Fig. Ia.

Fig. 2 is a set of a graph and a diagram illustrating the optimum concentration of oligo-DNA: a: binding image of MITF with oligo-DNA in the protein chip; and, b: a diagram representing the signals obtained from the image of Fig. 2a. From the calculation by Langmuir Isotherm, Ki constant was determined as 17.23.

Fig. 3 is a diagram illustrating the inhibiting effect of the 27 MITF competitor candidates of the invention on MITF and oligo-DNA binding confirmed in the protein chip (left: 0.025 mg/m#, right: 0.25 mg/m#) .

Fig. 4 is a graph representing the signal values obtained from the image of Fig. 3. Fig. 5 is a graph illustrating the melanin synthesis inhibiting effect and cytotoxicity of the MITF competitors of the invention confirmed by animal tests.

Fig. 6 is a photograph illustrating the binding between MITF and E-box confirmed by EMSA: Molecular marker (M) , negative control (C) , α-MSH untreated nuclear extract + biotin-labeled oligo-DNA (1- a) , 5x diluted α-MSH untreated nuclear extract + biotin- labeled oligo-DNA (1-b), α-MSH treated nuclear extract + biotin-labeled oligo-DNA (2-a) , 5x diluted α-MSH treated nuclear extract + biotin-labeled oligo-DNA (2-b) , 5x diluted α-MSH treated nuclear extract + non-labeled oligo-DNA (2-c) .

Fig. 7 is a photograph illustrating the inhibiting effect of the competitor candidates on MITF/E-box binding confirmed by EMSA:

Molecular marker (M) , negative control 1 (C) , negative control 2 (N. C), α-MSH treated nuclear extract + 8x diluted #18 candidate + biotin-labeled oligo-DNA (1), α-MSH treated nuclear extract + 4x diluted #18 candidate + biotin-labeled oligo-DNA (2), α-MSH treated nuclear extract + 2x diluted #18 candidate + biotin-labeled oligo- DNA (3), α-MSH treated nuclear extract + Ix diluted #18 candidate + biotin-labeled oligo-DNA (4) .

[Best Mode]

Hereinafter, the present invention is described in detail .

The present invention provides a cosmetic composition for whitening comprising the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box as an active ingredient.

The binding inhibitor is bound to the oligo-DNA containing E-box, changing the binding site between MITF and E-box. Therefore, this inhibitor interrupts MITF not to be functioning as a transcription factor, so that it can be a candidate for the MITF competitor. The MITF competitor candidates are preferably those compounds represented by formula 1 - formula 27 at table 1, more preferably N ' - [1- (4-Chloro-phenyl ) -meth- (E) -ylidene] -N-

(4, 6-di-piperidin-l-yl- [1,3,5] triazin-2-yl) -N-methyl- hydrazine, 3-{ [ 4- (4-Fluoro-phenylamino) -β-piperidin-1-yl-

[1, 3, 5] triazin-2-yl] -hydrazonomethyl } -phenol, 4- [1- (2, 4- Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] -2- (3-fluoro- phenyl) -4H-oxazol-5-one, { 4- [3-Isobutyl-2- [(Z) -4-methoxy- phenylimino] -4-oxo-thiazolidin- (5Z) -ylidenemethyl] -2- methoxy-phenoxy } -acetic acid, N-tert-Butyl-2- ( (2-chloro- benzyl) -{2- [5- (3, 4-dimethoxy-phenyl) -tetrazol-2-yl] - acetyl } -amino) -2-thiophen-2-yl -acetamide, N, N-Diphenyl-2- [ 3-pyridin-4-yl-5- (thiazol-2-ylcarbamoylmethylsulfanyl) - [1, 2, 4] triazol-1-yl] -acetamide, (E) -3- [4- (3-Chloro- benzoylamino) -2, 5-diethoxy-phenylcarbamoyl] -acrylic acid, N- [ (4-Methoxy-benzylcarbamoyl) -methyl] -N- (3-methoxy- phenyl) -N' -thiazol-2-yl-succinamide, N- [3- (4-Ethoxy- phenoxy) -5-m-tolyloxy-phenyl] -2- (3-nitro- [1,2,4] triazol-1- yl) -acetamide, { 1- [2- (4-Chloro-phenoxy) -ethyl] -IH- benzoimidazol-2-ylsulfanyl}-acetic acid, 3-{ [ (2-Methoxy-5- methyl-phenyl) -methyl-carbamoyl] -methyl } -1-methyl-lH- indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l- octyl-2-oxo-3-m-tolylimidazolidin-4-yl) -3-m-tolylurea, 1- (4-Ethoxy-phenyl) -5-methoxy-2-methyl-lH-indole-3- carboxylic acid, N- (3-Chloro-4-methyl-phenyl) -N ' - (4 , 6- dimethyl-pyrimidin-2-yl ) -N ' ' - (3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro-benzoyl) -4-hydroxy-5-oxo-2-phenyl-2 , 5- dihydro-pyrrol-1-yl] -butyric acid, and most preferably 3- { [4- (4-Fluoro-phenylamino) -β-piperidin-1-yl- [1,3,5] triazin-2-yl] -hydrazonomethy1 } -phenol, 4- [1- (2, 4- Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] -2- (3-fluoro- phenyl) -4H-oxazol-5-one and { 1- [2- (4-Chloro-phenoxy ) - ethyl] -lH-benzoimidazol-2-ylsulfanyl } -acetic acid. Among these compounds, N ' - [1- (4-Chloro-phenyl) -meth- (E) - ylidene] -N- (4, 6-di-piperidin-l-yl- [1,3,5] triazin-2-yl) -N- methyl-hydrazine, 3-{ [4- (4-Fluoro-phenylamino) -6- piperidin-1-yl- [ 1, 3, 5] triazin-2-yl ] -hydrazonomethyl } - phenol, 4- [1- (2, 4-Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] - 2- (3-fluoro-phenyl) -4H-oxazol-5-one, { 4- [3-Isobutyl-2- [(Z) -4-methoxy-phenylimino] -4-oxo-thiazolidin- (5Z) - ylidenemethyl] -2-methoxy-phenoxy } -acetic acid, N-tert- Butyl-2- ( (2-chloro-benzyl) -{2- [5- (3, 4-dimethoxy-phenyl) - tetrazol-2-yl] -acetyl} -amino) -2-thiophen-2-yl-acetamide, N,N-Diphenyl-2- [3-pyridin-4-yl-5- (thiazol-2- ylcarbamoylmethylsulfanyl )-[l,2,4]triazol-l-yl] -acetamide, (E) -3- [4- (3-Chloro-benzoylamino) -2, 5-diethoxy- phenylcarbamoyl] -acrylic acid, N- [ (4-Methoxy- benzylcarbamoyl) -methyl] -N- (3-methoxy-phenyl) -N¹ -thiazol- 2-yl-succinamide, N- [3- (4-Ethoxy-phenoxy) -5-m-tolyloxy- phenyl] -2- (3-nitro- [1, 2, 4] triazol-1-yl) -acetamide, {1- [2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl } - acetic acid, 3- { [ (2-Methoxy~5-methyl-phenyl) -methyl- carbamoyl] -methyl } -l-methyl-lH-indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l-octyl-2-oxo-3-m- tolylimidazolidin-4-yl) -3-m-tolylurea, 1- (4-Ethoxy- phenyl) -5-methoxy-2-methyl-lH-indole-3-carboxylic acid, N- (3-Chloro-4-methyl-phenyl) -N ' - (4 , 6-dimethyl-pyrimidin-2- yl) -N ' ' - (3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro- benzoyl) ~4-hydroxy-5-oxo-2-phenyl-2, 5-dihydro-pyrrol-l- yl] -butyric acid can inhibit the binding of MITF with oligo-DNA containing E-box on the protein chip. And particularly, 3-{ [4- (4-Fluoro-phenylamino) -6-piperidin-l- yl-[l,3,5]triazin-2-yl] -hydrazonomethy1 } -phenol, 4- [1-

(2, 4-Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] -2- (3-fluoro- phenyl) -4H-oxazol-5-one and ( 1- [2- ( 4-Chloro-phenoxy) - ethyl] -lH-benzoimidazol-2-ylsulfanyl } -acetic acid surpassed other compounds in melanin synthesis inhibiting effect without causing cytotoxicity in animal tests, making them be selected as excellent candidates.

In a preferred embodiment of the present invention, concentrations of MITF and oligo-DNA containing E-box on the protein chip for screening the MITF competitors were investigated to judge the optimum METF/E-box binding. As a result, the optimum concentrations were respectively 125 βg /mi and 2 μg/mi (see Figs. 1 and 2) .

The present inventors synthesized 27 candidates capable of inhibiting MITF not to be functioning as a transcription factor by modifying the binding site between MITF and E-box, which is prepared by copying the binding site between MITF and E-box. And the inventors further investigated if bhe competitor candidates could inhibit the binding of MITF (microphthalmia transcription factor) to oligo-DNA containing E-box on the protein chip. As shown in Table 2, 15 MITF competitor candidates were selected (see Figs. 3 and 4) . The present inventors also investigated the inhibiting effect of MITF competitor candidates on inhibition of melanin synthesis in animal cells and performed cytotoxicity test as well to judge whether the MITF competitor candidates could be used as a cosmetic composition for whitening. The experimental group treated with the selected MITF competitor candidates was compared with PTU treated group. And as a result, MITF competitor candidates #3, #5 and #18, demonstrating excellent melanin synthesis inhibiting effect with less toxicity, were selected (see Fig. 5) .

In a preferred embodiment of the present invention, it was investigated whether the selected candidates could be active to inhibit the binding of MITF to E-box. To do so, EMSA (Electrophoretic Mobility Shift Assay) was employed in order to examine protein-DNA binding interaction detected by chemiluminescence. Particularly, non-labeled oligo-DNA was added to compete with biotin- labeled oligo-DNA for MITF. As a result, it was confirmed that MITF bound specifically to E-box (see Fig. 6 lane 2-c) . As the concentration of the candidate #18 increased, band moved to upper part of the gel by MITF/oligo-DNA binding became thinner (see Fig. 7 lanes 1 - 4) . So, the candidate #18 was confirmed to have an excellent inhibiting effect on MITF/E-box binding as a MITF competitor. In addition, it was also confirmed that HTS (High-throughput screening) using a protein chip designed to include MITF can be a useful method for the selection of a whitening agent.

The binding competitor that inhibits the binding of MITF (microphthalmia transcription factor) to oligo-DNA containing E-box on the protein chip (referred as MITF competitor hereinafter) prevents excessive melanin synthesis, so that it can be used for a cosmetic composition for whitening.

The preferable content of the MITF competitor in the total weight of the composition of the invention is 0.005 - 50 weight%, but it can be lower or higher than that according to the purpose of use as long as the whitening effect is maintained and toxicity is not caused. The composition of the invention can additionally include, in addition to the MITF competitor, one or more active ingredients having similar or same functions.

The appropriate formulations for the cosmetics prepared with the cosmetic composition for whitening comprising the MITF competitor of the present invention as an active ingredient are exemplified by solution, gel, solid or dough anhydride, emulsion where oil phase is dispersed in aqueous phase, suspension, micro emulsion, micro capsule, micro granule or ionic (liposome) , non-ionic vesicle dispersing agents, cream, toner, lotion, powder, ointment, spray or conceal stick and aerosol containing a foam type or pressed propel lant. The preferable formulations of the cosmetic composition for whitening of the present invention are exemplified by skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, essence, nutrient essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, milk lotion, press powder, loose powder, eye shadow, etc. The cosmetic composition for whitening of the present invention can additionally include, in addition to the MlTF competitor, fat material, organic solvent, dissolving agent, thickener and gelation agent, softener, antioxidant, suspending agent, stabilizer, foaming agent, aromatic, surfactant, water, ionic or non-ionic emulsifying agent, filler, metal ion blocker and chelating agent, preserving agent, vitamin, blocker, moisturing agent, essential oil, pigment, dye, hydrophilic or lipophilic activator, lipid vesicle or other supplements generally used for cosmetics. And, the content of each ingredient mentioned above is the amount generally used in the field of dermatology.

In addition to the form of cosmetics containing the MITF competitor of the invention, the competitor can be formulated as a locally or systemically applicable supplement generally used in the field of dermatology by containing a dermatologically acceptable medium or substrate .

The cosmetics containing the MITF competitors of the present invention can be delivered to customers as packed in proper containers, considering viscosity. For example, lotion can be packed in a bottle, a roll-ball applicator, a capsule or other containers equipped with propeller-driving aerosol device or manual pump. Cream can be packed in an unchangeable bottle, a squeezing container such as tube or a jar with lid. The present invention also provides a melanin synthesis inhibitor comprising the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box as an active ingredient. In a preferred embodiment of the present invention, the binding inhibitor of the invention inhibited melanin synthesis 10 - 20% greater than PTU, known as the conventional melanin synthesis inhibitor (see Fig. 5) , so the binding inhibitor of the present invention can be provided as a melanin synthesis inhibitor.

The melanin synthesis inhibitor of the preset invention can additionally include general excipients, disintegrating agents, sweeteners, lubricants and flavors, and can be formulated as tablets, capsules, powders, granules, suspensions, emulsions, syrups and other liquid preparations by the conventional method. Particularly, the melanin synthesis inhibitor can be prepared in the forms for oral-administration such as tablets, troches, lozenges, water-soluble or fat-soluble suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. To provide the composition as the forms of tablet and capsule, binders such as lactose, sacarose, sorbitol, mannitol, starch, amilopectin, cellulose or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch or sweet potato starch; and lubricants such as magnesium stearate, calcium stearate, sodium stearylfumarate or polyethyleneglycol wax can be included. In the: case of capsules, liquid carriers such as fatty oil can be additionally included. The melanin synthesis inhibitor can be aidministered orally or parenterally. The parenteral administration is exemplified by hypodermic injection, intravenous injection, intramuscular injection and intrathoracic injection. To prepare formulations for parenteral administration, the melanin synthesis inhibitor of the present invention is mixed with stabiiizer or buffer in water to give suspension, which is prepared as units of ampoules or vials. The effective dosage of the active ingredient of the present invention can be determined by in vivo absorption rate of the active ingredient, inactivation rate, excretion rate, age, gender and health condition of a patient, severity of a disease, etc. In general, the dosage of the MITF competitor for oral administration to an adult is 0.1 ~ 0.2 g/kg per day and administration frequency is once a day or preferably a few times a day, and preferably 0.1 ~ 10 fflg/kg per day.

The present invention further provides a pharmaceutical composition for skin whitening comprising the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box as an active ingredient.

The pharmaceutical composition of the present invention can be prepared as various formulations for oral- administration, for example tablets, powders, capsules, troches, liquids, suspensions or in other formulations such as ointments, etc. The pharmaceutical formulations prepared by using the conventional carriers can be administered orally or parenterally, for example local application on skin. The effective dosage of the active ingredient for administration depends on age and conditions of a patient, but generally 10 ~ 500 mg per day (for adult), and preferably 50 ~ 300 mg per day. The administration frequency can be determined by a doctor or a pharmacist, which would be a couple of times a day and preferably 1 - 6 times a day.

The present invention also provides a health food for skin whitening comprising the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box as an active ingredient.

The MITF competitor of the present invention can be used as food additive. In that case, the MITF competitor can be added as it is or as mixed with other food or other food components according to the conventional method. The mixing ratio of active ingredients can be regulated according to the purpose of use (prevention, health enhancement or hygiene) .

The forms or kinds of the health food herein are not limited. The forms of the health food that can contain the MITF competitor are exemplified by tablets, capsules, powders, granules, liquids and pills, etc, and the kinds of the health food are exemplified by dairy products including yogurt, cheese, butter and ice cream, breads, chocolates, candies, snacks, cookies, pizza, ramyuns, other noodles, gums, soups, beverages, tea, drinks, alcohol drinks and vitamin complex, etc, and in fact every kinds of general food applicable for the health food can be included.

The health food of the present invention can additionally include various flavors or natural carbohydrates, etc, like other conventional health foods. The natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xilytole, sorbitol and erythritol. Besides, natural sweetening agents such as thaumatin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be included as a sweetening agent. The content of the natural carbohydrate is preferably 0.01-0.04 g and more preferably 0.02-0.03 g in 100 mi of the health food of the present invention.

In addition to the ingredients mentioned above, the health food of the present invention can include in variety of nutrients, vitamins, minerals, flavors, coloring agents, pectic acid and its salts, algini c acid and its salts, organic acid, protective colloidal viscosifiers, pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 001-0.1 weight part per 100 weight part of the health food of the present invention.

The present invention also provides a method for inhibiting melanin synthesis comprising the step of administering the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box to a subject.

The subject herein can be vertebrates and preferably mammals and more preferably animals on experiments such as rats, rabbits, guinea pigs, hamsters, dogs and cats and most preferably apes such as chimpanzees and gorillas. The present invention also provides a method for skin whitening comprising the step of applying the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box on the melanin pigmented skin.

The binding inhibitor of the present invention is capable of regulating the pigment synthesis of melanocytes. Thus, the binding inhibitor of the invention can be applied on a target area of the skin of the mammals to regulate or prevent the production of pigment thereon. Examples of such application are as follows:

1. Vanity products, making skin brighter, in particular targeting dark skin;

2. Reducing pigmented spots of skin such as freckles or senile plaques;

3. Reducing traces of uneven pigmentation and irregular skin color;

4. Treating hyperpigmentation-related medical symptoms such as liver spots, ochronosis and lentigines; 5. Making hair pigmentation lighter when applied on the skin containing pigmented hair follicles; β. Reducing postinflammatory hyperpigmentation caused by trauma, acne, invasive surgery, face lifting, laser treatment or plastic surgery; and, 7. Reducing skin pigmentation on normal skin area near the vitiligo area by reducing the difference between normal skin tone and vitiligo skin tone.

The binding inhibitor used for whitening of the present invention is the product for local application on human skin, so that it can be formulated as cream, gel, hydro cream or milk lotion or oil. The binding inhibitor can also include excipients appropriate for the application on face and neck. At this time, the appropriate excipients are required to have stability and adhesivity as well as resistance and high affinity to skin for enhancing pleasure and easiness. It is another example of the application of the composition of the present invention that small amount of the composition (approximately 1 mi - 5 m£) is applied on a target skin area by being pushed out from a container or an applicator and then spread or rubbed by hand, finger or other proper devices.

The present invention also provides a use of the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box for the production of a cosmetic composition for whitening.

In addition, the present invention also provides a use of the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box for the production of a pharmaceutical composition for skin whitening.

[Mode for Invention] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Preparation of MBPek-MITE'

<1-1> Construction of MBPek-MITF expression vector The expression vector for the fusion protein comprising MBP (maltose binding protein) and MITF (microphthalmia transcription factor) containing enterokinase cleavage site was constructed.

PCR was performed using glutathione-S-transferase (GST)-MITF vector provided by Dr. Kitamura (Department of Dermatology, University of Yamanashi, Faculty of Medicine, Yamanashi, Japan) , as a template with the primer set comprising sequences each represented by SEQ. ID. NO: 1 and NO: 2 by the following conditions, to obtain MITF gene nucleotide. Another PCR was performed using pMAL-c2X (New England Biolabs, England) vector as a template with the primer set comprising sequences each represented by SEQ. ID. NO: 3 and NO: 4 by the following conditions to obtain MBPek gene nucleotide. The MBPek gene nucleotide was amplified to contain enterokinase cleavage site. PCR was performed using DNA polymerase (Unipfu, Takara, Japan) as follows; predenaturation at 94°C for 5 minutes, denaturation at 94 ^°C for 1 minute, annealing at 55°C for 1 minute, polymerization at 72 "C for 1 minute, 30 cycles from denaturation to polymerization, and final extension at

72 ^°C for 10 minutes.

The MITF and MBPek gene nucleotides obtained by the PCRs were treated with restriction enzymes EcoR I and Sal I , and inserted into pET vector (Novagen, USA) . As a result the pETMBPek-MITF expression vector was constructed.

<l-2> Expression and purification of MBPek-MITF recombinant fusion protein

MBPek-MITF protein was mass-expressed from the pETMBpek-MITF vector and purified.

Escherichia coli BL21 (DE3) was transformed with the pETMBPek-MITF vector constructed by the method of Example

<1-1>. The transformed DE3 was pre-cultured for 12 hours, followed by main-culture until ODβoo reached 0.6. IPTG (isopropyl-β-D-thiogalactopyranoside) was treated thereto at the final concentration of 1 mM, followed by culture for 4 hours to induce the expression of MBPek-MITF protein. The cell culture solution was obtained, followed by centrifugation at 6,000 rpm for 20 minutes to collect cells. The cells were suspended in buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0) . After floating, the cells were lysed by sonication for 1 and half hours with the cycle of 6 minute- lysis-5 minute rest. The cell lysate was centrifuged at 12,000 rpm for 30 minutes to separate supernatant and precipitate. The precipitate was suspended in 8M urea, which was stored in deep freezer. The supernatant was filtered with 0.2 μm filter, which proceeded to 1.1 cm x 30 cm MBP affinity chromatography column (Millipore, USA) , leading to the separation of MBPek-MITF by treating elution buffer [binding buffer (20 mM Tris-HCl, 0.2 M NaCl, 1 mM EDTA, pH 7.4) containing 10 mM maltose] at the speed of 4 m^/min. The urea suspension stored in deep freezer proceeded to SDS-PAGE together with washing fraction and eluting fraction obtained from the MBPek-MITF column separation process.

Example 2: Preparation of protein chip <2-l> Substrate modification

Substrate modification was performed for the fixation of the MBPek-MITF protein obtained in Example 1 thereon. β-Cyclodextrin (Sigma, USA) having similar chemical structure to maltose capable of binding with MBP (Maltose Binding Protein) was dissolved in 0.1 N NaOH solution at the concentration of 70 mg/mt, in which glass substrate (CEL Associates, USA) coated with epoxy functional group thereon was soaked, followed by reaction at 40^°C for 20 hours to coat the glass substrate with β-cyclodextrin. The substrate was washed with TDW and then soaked in 1% BSA

(Qbiogene, USA) for 10-20 minutes, resulting in blocking of the remaining epoxy functional group. The substrate was washed again with TDW, and moisture thereon was eliminated.

The resultant substrate was stored at 4^°C. β-cyclodextrin reacts specifically with MBP, so that it plays a role in fixing MBP conjugated protein specifically on the glass substrate. The substrate modified by the above process was preferably used within a week.

<2-2> Dropping of MBPek-MITF protein The MBPek-MITF protein obtained in Example 1 was dropped on the modified substrate prepared in Example <2-l> to construct protein chip for the screening of MlTF competitors .

Tetraethylene glycol (final cone. : 25%, Sigma, USA) was added to the buffer containing 0 (1% BSA), 31.2, 62.5, 125, 250 or 500 βg/mi of MBPek-MITF. 19 μl (per 11 mm x 10 mm substrate) of the solution was dropped on the substrate modified in Example <2-l>. The substrate was covered to spread the protein evenly on the surface, followed by reaction at room temperature for one hour with maintaining the humidity as 50-60% to induce MBPek-MITF binding. As a result, the protein chip for screening MITF competitors was prepared. Upon completion of the reaction, the protein chip was washed and then used for the following experiments.

Example 3: Confirmation of the binding between MITF and E- box (CATGTG)

<3-l> Construction of Cy3-labeled oligo-DNA

Cy3-labeled oligo-DNA containing E-box (CATGTG; SEQ. ID. NO: 7) that was able to bind with MITF was constructed.

The oligo-DNA was obtained by PCR using the gene

(Entrez GeneID 22173) containing tyrosinase promoter (Tyr-

P) E-box (CATGTG) as a template with Tyr-P forward primer represented by SEQ. ID. NO: 5 and Tyr-P reverse primer represented by SEQ. ID. NO: 6 according to the above conditions. To detect fluorescent signal presenting the binding of MITF with E-box on the substrate, 5' region of the forward primer was labeled with fluorescent material Cy3 (Bioneer, Korea) (J. M. Jung et al., Anal Biochem. , 330, 251, 2004; M. L. Bulyk et al., Proc. Natl. Acad. Sci., 98 , 7158 , 2001 ) .

<3-2> Binding of MITF with E-box (CATGTG)

<3-2-l> Proper concentration of M]TF Proper concentration of MITF in the protein chip for screening MITF competitors for the binding of MITF with E- box was determined.

The protein chip constructed in Example 2 was washed with PBST-I (0.05% Tween 20 in PBS) (sigma, USA), PBST-2 (0.01% Triton X-100 in PBS) (sigma, USA), PBS (sigma, USA) and TDW stepwise, and moisture was eliminated to be ready for oligo-DNA binding. The Cy3-labeled oligo-DNA constructed in Example <3-l> was dissolved in TE buffer {10 mM Tris-Cl, 0.1 raM EDTA, pH = 8.0) at the concentration of 100 μM, followed by denaturation in 100^°C distilled water for 5 minutes. Then, the solution was cooled down at room temperature slowly to be double stranded.

The buffer solution comprising 0 (1% BSA), 31.2, 62.5,

125, 250 and 500 /zg/m£ of MBPek-MITF was dropped on the protein chip using microarrayer CM-1000 (Proteogen, Korea) and 320 μm pin (SMPlO, Telechem, USA), respectively. The

Oligo-DNA was mixed with DNA reaction mixture [poly dldC

(0.25 mg/m£; Amersham Pharmacia Biotech, USA), binding buffer (10 mM HEPES pH 7.9, 50 mM KCl, 2.5 mM DTT, 0.1 mM EDTA, 0.05% NP-40, 10% Glycerol, 5% BSA; used after at least 14 -16 hours of storage at 4 "C), tetraethylene glycol

(final cone: 25%)] . At this time, the mixing ratio of the oligo-DNA to the DNA reaction mixture was 2:8. The mixture was dropped on the protein chip above at regular intervals (1 mm) by 0.125, 0.25, 0.5, 1, 2, 4, 8 and 10 μg/vd, making three lines of dropping. Then, the glass substrate was left at room temperature for 1 hour with maintaining high humidity as at least 60% to induce the binding of MITF with oligo-DNA. The substrate was washed with PBST-I, PBST--2, PBS and TDW in that order and moisture was eliminated.

To detect fluorescent signal by Cy3 indicating the binding of MITF with oligo-DNA, GenePix 4100A scanner (Axon, USA) was used to image the fluorescent signal (Fig. Ia) and GenePix 4.1 program (Axon, USA) was used to evaluate the binding signals as numbers.

As shown in Fig. Ia, signals were developed as images and then evaluated as numbers which were displayed as a graph as shown in Fig. Ib. As a result, the optimum M] TF concentration exhibiting the maximum signal at minimum concentration was proved to be 125 βg/mi.

<3-2-2> Concentration of oligo-DNA containing E-box

Proper concentration of oligo-DNA for the binding of MITF with E-box was determined. The double-stranded oligo-DNA obtained in Example <3-2-l> was dropped on the protein chip, pre-loaded with the buffer containing MBPek-MITF by 125

at regular intervals (1 ram) by 0, 0.125, 0.5, 0.75, 1, 1.25, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 βg/mt, making four lines of dropping (Fig. 2a) .

To detect fluorescent signal by Cy3 indicating the binding of MITF with oligo-DNA, GenePix 4100A scanner was used to image the fluorescent signal (Fig. 2a) and GenePix 4.1 program was used to evaluate the binding signals as numbers.

As shown in Fig. 2a, signals were developed as images and then evaluated as numbers which were displayed as a graph with SigmaPlot 2001 as shown in Fig. 2b. As a result, kinetics of DNA binding with MITF showed Langmuir isotherm and its kinetic constant K₁ was determined as 17.23. And the maximum signal value was 1.04 ^χlθ⁵ (Mathematical Formula 1) . It was also confirmed that the optimum concentration of oligo-DNA on the protein chip loaded with the buffer containing MBPek-MITF by 125 μg/mi was 2 βg/M,.

[Mathematical Formula l]

Therefore, the binding of MITF with oligo-DNA containing E-box on the protein chip for screening MITF competitors was confirmed and bhe optimum concentrations of

MITF and oligo-DNA for the binding of MITF with E-box were

Example 4: MITF competitor candidates inhibiting the binding of MITF with E-box <4-l> MITF compebitor candidates MITF competitor candidates capable of competing with MITF for binding with E-box were selected.

Synthetic compounds able to interrupt MITF not to play a role as a transcription factor by changing the binding site of MITF with E-box after binding with MITF were selected by molecular modeling. Particularly, compounds of the commercial data base were virtually screened by using the predicted site for MITF/E-box binding. First, binding mode and energy value obtained from ^ΛDOCK4' program (//dock. compbio . ucsf . edu) were analyzed and then 4 score functions [Ludi (hydrogen bond, ionic bond, hydrophobic molecular surface and rotating bond considered function) / LigScore (vDW, polar surface area in between target and ligand considered function) / PLPl-2 (Pairwise Linear Potentials; hydrogen bond, repulsive force and contacting force considered function) / PMF (Potential of Mean Force; every atom in between target and ligand considered enercjy function) ] calculated from ^λLigandFit' program (Accelrys Inc., USA) were evaluated to reselect the compounds from the virtually screened compounds. The reselected compounds were purchased from each corresponding production company (Table 1) .

As a result, as shown in Table 1, total 27 MITF competitor candidates were selected and each candidate was dissolved in DMSO at the concentration of 0.5 mg/ml for further experiments.

[Table l] MITF competitor candidates

No. Name

6- [3- (4-Fluoro-benzoyl) -A- hydroxy-2- (4-isopropyl- phenyl) -5-oxo-2, 5-dihydro- pyrrol-1-yl] -hexanoic acid

N ' - [ 1- ( 4-Chloro-phenyl ) -meth-

(E) -ylidene] -N- ( 4 , 6-di- piperidin-1-yl- [1, 3, 5] triazin-

2-yl) -N-methyl-hydrazine

3-{ [4- (4-Fluoro-phenylamino! β-piperidin-1-yl-

3

[1,3,5] triazin-2-yl] - hydrazonomethyl } -phenol

2-Diphenylacetylamino-5, 6- dihydro-4H- cyclopenta [b] thiophene-3- carboxylic acid

4- [1- (2, 4-Bis-benzyloxy- phenyl) -meth- (Z) -ylidene] -2-

(3-fluoro-phenyl ) -4H-oxazol-5-

N- [3- (4-Ethoxy-phenoxy) -5-m- ASINEX, tolyloxy-phenyl] -2- (3-nitro- C₂₅H₂₃N₅O₆ Russia

[1,2,4] triazol-1-yl) -acetamide

N, N '-Bis- (4-ethoxy-phenyl) -6-

(5-methyl- [1, 3, 4] thiadiazol-2- ASINEX,

C₂₂H₂₃N₇O₂S₂ ylsulfanyl) - [1, 3, 5] triazine- Russia

2 , 4-diamine

{ 1- [2- (4-Chloro-phenoxy) -

ASINEX, ethyl] -lH-benzoimidazol-2- :₁₇H₁₅C1N₂O₃S Russia ylsulfanyl} -acetic acid

[4- (3, 4-Dimethyl-phenyl) -2- (4- ASINEX, methoxy-phenylamino) -thiazol- C₂₀H₂₀N₂O₃S Russia

5-yl] -acetic acid

3-{ [ (2-Methoxy-5-methyl- phenyl) -methyl-carbamoyl] - C_{2 I}H₂₂N₂O₄ CHEMDIV, USA methyl } -l-methyl-lH-indole-2- carboxylic acid

2- (Benzylcarbamoyl- methylsulfanyl) -4-oxo-3- [2- (4- sulfamoyl-phenyl) -ethyl] -3, 4- C₂₇H₂₆N₄O₆S₂ CHEMDIV, USA dihydro-quinazoline-7- carboxylic acid methyl ester

1-hydroxy-l- (5, 5-dimethyl-l- octyl-2-oxo-3-m- IBSCREEN,

C₂₈H₄₀N₄O₃ tolylimidazolidin-4-yl) -3-m- Russia tolylurea

1- (4-Ethoxy-phenyl) -5-methoxy- IBSCREEN,

2-methyl-lH-indole-3- C₁₉H₁₉NO₄ Russia carboxylic acid

N- (3-Chloro-4-methyl-phenyl) -

N ' - (4 , 6-dimethyl-pyrimidin-2- IBSCREEN, yl) -N¹ ' - (3-oxo-butyryl) - C₁₈H₂₀ClN₅O₂ Russia guanidine

4- [3- (4-Chloro-benzoyl) -4- hydroxy-5-oxo-2-phenyl-2, 5- CHEMBRIDGE,

C₂₁H₁₈ClNO₅ dihydro-pyrrol-1-yl] -butyric USA acid

5- [5- (4-Fluoro-phenyl) -3- thiophen-2-yl-4 , 5-dihydro- IBSCREEN,

Ci₈Hi₇FN₂O₃S pyrazol-1-yl] -5-oxo-pentanoic Russia acid

3- ( 3 , 4-Dichloro-benzyloxy) - KEYORGANICS,

C₁₅H₉NO₃SCl₂ thieno [ 2 , 3-b] pyridine-2- England carboxylic acid ^J

<4-2> Protein chip for screening MITF competitor candidates

The protein chip on which MITF was dropped was prepared to make the MITF competitor candidates compete with MITF for binding with oligo-DNA containing E-box.

Considering that the signals on the protein chip might be weakened by competition, the concentration of MITF to be loaded on the protein chip was raised from the expected optimum concentration of^" 125 βg/mi to 500 βg/mi. Particularly, tetraethylene glycol (final cone: 25%, Sigma, USA) was mixed with the buffer containing MBPek-MITF by 500 βg/xΑl, which was dropped on the modified substrate prepared in Example <2-l> by 80 μi (per 25 mm x 50 mm substrate) . The substrate was covered to spread the protein evenly on the surface, followed by reaction at room temperature for one hour with maintaining the humidity as 50-60% to induce MBPek-MITF binding. As a result, the protein chip for screening MITF competitors was prepared. Upon completion of the reaction, the protein chip was washed and then used for the following experiments.

<4-3> MITF-E-box binding inhibitory effect of MITF competitor candidates The MITF-E-box binding inhibitory effect of the MITF competitor candidates obtained in Example <4-l> was investigated.

The protein chip prepared in Example <4-2> was washed with PBST-I, PBST-2, PBS and TDW stepwise and moisture was eliminated, to be ready for the binding of the MITF competitor candidates with oligo-DNA. The fluorescent material-labeled oligo-DNA constructed in Example <3-l> was dissolved in the DNA reaction mixture used in Example <3-2- 1> at the concentration of 4 βg/M,, followed by denaturation in 100 ^°C distilled water for 5 minutes. Then, the solution was cooled down at room temperature slowly to be double stranded. The 27 MITF competitor candidates obtained in Example <4-l> were added to the DNA reaction mixture at the concentration of 0.25 and 0.025 nig/ml.

On the protein chip constructed in Example <4-2>, the mixed solution comprising the oligo-DNA and MITF competitor candidates obtained above was dropped by using microarrayer CM-1000 (Proteogen, Korea) and 320 μm pin (SMPlO, Telechem, USA) in two lines at regular intervals (1 mm), for which the time that took for the pin to touch the slide was set as 0.1 second and the size of the designated clone was determined as 300 μm. As shown in Fig. 3, the fist line, which was control, was loaded with the oligo-DNA solution alone prepared in Example <3-2-l> to induce normal MITF-E- box binding, while the second line was loaded with the DMSO which was used to dissolve the candidates of the present invention in order to investigate the effect of DMSO on MITF-E-box binding. At this time, the time that took for the pin to touch the slide was also set as 0.1 second and the size of the designated clone was determined as 300 μm. The third and the forth lines were loaded with the mixed solution comprising the oligo-DNA and MITF competitor candidates. Then, the glass substrate was left at room temperature for 1 hour with maintaining high humidity as at least 60% to induce the binding of MITF with oligo-DNA. The substrate was washed with PBST-I, PBST-2, PBS and TDW in that order and moisture was eliminated.

To detect fluorescent signal by Cy3 indicating the binding of MITF with oligo-DNA, GenePix 4100A scanner was used to image the fluorescent signal (Fig. 3) and GenePix 4.1 program was used to evaluate the binding signals as numbers, which were displayed as a graph (Fig. 4). Considering the DMSO treated group as a standard, those candidates demonstrating better effect than DMSO were selected.

As a result, as shown in Table 2, 15 MITF competitor candidates were selected.

[Table 2] 15 MITF competitor candidates selected by protein chip experiment

Example 5: Melanin synthesis inhibition and cytotoxicity of MITF competitor candidates <5-l> Cell culture

B16F10 murine melanoma cell line (hereinafter, B16F10; ATCC (American Type Culture Collection), USA) was cultured in DMEM (Dulbecco's modified Eagle's medium; GIBCO, USA) supplemented with 10% (v/v) FBS (fetal bovine serum; Sigma, USA), 100 units/m^ of penicillin (Invitrogen, USA) and 100 βg/mt of streptomycin (Invitrogen, USA) in a 37 ^°C, 5% CO₂ incubator. The cells were sub-cultured every three days until the maximal passage number of 30.

<5-2> Melanin test

The present inventors investigated the inhibitory effect of the MITF competitor candidates selected in Example 4 on melanin synthesis in Bl 6F10 cells. The method for measuring melanin level was based on the method of Hosoi (Hosoi et al . , CANCER RESEARCH 45:1474-1478, 1985) .

The B16F10 cells were cultured in β-well plate at the concentration of β^χlθ⁴ cells/well using the same culture medium and conditions as those of Example <5-l> for 24 hours. Then, 40 βg/ni of each candidate selected in Example 4 was treated thereto. DMSO was treated to the negative control and 15 /zg/m£ of PTU (N-Phenylthiourea; Sigma, USA) , known as a melanin synthesis inhibitor, was treated to the positive control. The B16F10 cells treated with the candidates, PTU and DMSO, were cultured for 48 hours. Then, the cells were collected by treating with trypsin containing EDTA. The cells were washed twice with PBS and suspended in 200 mi of IN NaOH containing 10% DMSO, followed by heating in a 80 ^"C bath for one hour. The cells were cooled down to release melanin and then OD₄₀₅ was measured by using ELISA (enzyme-linked immunosorbent assay) reader [MDS (Molecular Device) , USA] . Melanin production was calculated by the following Mathematical Formula 2. The non-treated group OD in the Mathematical Formula 2 indicates the negative control group OD.

[Mathematical Formula 2]

Melanin Production (%) = (treated group OD/non- treated group OD) x 100

<5-3> Cytotoxicity test

Cytotoxicity test was performed to investigate whether the MITF competitor candidates selected in Example 4 could be used as a cosmetic composition for whitening. The B16F10 cells were cultured in β-well plate at the concentration of 2.5^χlO³ cells/well using the same culture medium and conditions as those of Example <5-l> for 24 hours. Then, 40 βg/mt of each candidate selected in Example 4 was treated thereto. DMSO was treated to the negative control and 15 βg/ml of PTU, known as a melanin synthesis inhibitor, was treated to the positive control. The B16F10 cells treated with the candidates, PTU and DMSO, were cultured for 48 hours. Then, the cells were treated with 100 jtøfl/well MTT (3- (4 , 5-dimethyl-2-thiazolyl) -2, 5- diphenyl-2H-tetrazolium bromide; Sigma, USA) dissolved in PBS (5 mg/m^) . After 4 hours of culture, the medium was removed. Formazan was dissolved by adding DMSO (100 μJL /well) , and OD₅₄₀ was measured by using ELISA microplate reader [MDS (Molecular Device) , USA] . Cell survival rate was calculated according to the following Mathematical Formula 3. The non-treated group OD in the Mathematical Formula 3 indicates the negative control group OD.

[Mathematical Formula 3] Cell Survival Rate (%) = (treated group OD/non- treated group OD) x 100

As shown in Fig. 5, the experimental groups treated with the MITF competitor candidates selected in Example 4 were compared with the group treated with PTU. As a result, the candidates #3, #5 and #18, demonstrating less toxicity but higher melanin inhibition effect, were selected.

Example 6: EMSA To confirm whether the MITF competitor candidates selected in Example 5 had the activity of inhibiting

MITF/E-box binding, EMSA (Electrophoretic Mobility Shift

Assay) which is able to investigate the binding of protein- DNA by chemiluminescence, was performed.

<6-l> Nuclear extract

The MITF protein used for EMSA was nuclear extract isolated from BlβFlO cells. The BlβFlO cells were inoculated on lOOπ Petri dish at the concentration of 10⁶ cells/m^, followed by culture for 24 hours until mono layer was formed. Then, α-MSH, the melanin synthesis inducer, was treated thereto at the concentration of 100 nM, followed by culture for 6 more hours and then the cells were recovered. The recovered cells were washed with cold PBS twice and resuspended in 400 μJL of buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, and 0.5 mM DTT plus protease inhibitors; Roche, Swiss] . The cells were reacted in ice for 10 minutes. After mixing for 10 seconds, centrifugation was performed at 14,000 rpm for 10 seconds. Buffer C [20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 25% (v/v) glycerol plus protease inhibitors; Roche, Swiss] was added thereto by double the amount of pellet, and the cells were suspended and reacted in ice for 30 minutes. After centrifugation for 2 minutes, the supernatant was obtained, measured and stored at -70^°C. Nuclear extract was also isolated from fibroblasts by the same manner as described above to prepare the negative control .

<6-2> Preparation of biotin-labeled oligo-DNA

Biotin-labeled oligo-DNA containing E-box (CATGTG) capable of binding with MITF was constructed.

The oligo-DNA was synthesized by the same manner as described in Example <3-l>, except, that biotin was used to label the 5'of the forward primer instead of the fluorescent material Cy3 to detect, the binging of MITF with E-box on nylon membrane (Bioneer, Korea) (J. M. Jung et al., Anal Biochem. , 330, 251, 2004; M. L. Bulyk et al . , Proc. Natl. Acad. Sci._r 98, 7158, 2001) . In the meantime, the oligo-DNA used for the control group was synthesized without being labeled with biotin.

<6-3> Confirmation of MITF/E-box binding by EMSA EMSA was performed to confirm the binding of nuclear extract containing MITF with oligo-DNA.

EMSA was performed using LightShif Chemiluminesecent

EMSA Kit (PIERCE, USA) according to the manufacturer's instruction. Particularly, native polyacrylamide gel (8 x 8 x 0.1 cm (WHD)), the pre-run gel, was prepared using 0.5x TBE buffer. 20 fd of the binding buffer (1O binding buffer, 50% glycerol, 100 inM MgCl₂, 1 βg/≠ poly (dldC) , 1% NP-40; Fluka, Japan) comprising each nuclear extract obtained in Example <6~1> at the concentrations of 0, 2.5 or 0.5 βg/ml and 2 μϋ of 20 f mol oligo-DNA labeled with biotin prepared in Example <β-2> was reacted at room temperature for 20 minutes. 5 βi of 5^χ loading dye was added thereto and mixed softly, resulting in the loading sample. The loading sample containing biotin-labeled oligo-DNA alone was used as a molecular marker, and the loading sample wherein the nuclear extract of BlGE¹IO was substituted with the nuclear extract of fibroblasts was used as a negative control .

As shown in Fig. 6, the loading samples were injected in the native polyacrylamide gel stepwise by 200 βi each in the order of the molecular marker (M) , the negative control

(C), the α-MSH non-treated nuclear extract group (1-a), 5x diluted α-MSH non-treated nuclear extract group (1-b) , α-

MSH treated nuclear extract group (2-a) , 5x diluted α-MSH treated nuclear extract group (2-b) and 5x diluted α-MSH treated nuclear extract group + non-labeled oligo-DNA group (2-c) . 100 V of voltage was applied on the gel of 8 x 8 x 0.1 cm (WHD), followed by electrophoresis until the loading dye went down to 2/3 of the gel. Electrophoresis was stopped and a nylon membrane having (-) charge was put on the gel, followed by electrophoresis again to move the oligo-DNA of the present invention having (+) charge separated on the gel to the nylon membrane. Electrophoresis was performed with 380 mA for 30 minutes using 0.5^χ TBE buffer as tank buffer, and at this time temperature was maintained up to 10 ^°C. UV was irradiated on the nylon membrane for 1 minute, after completing electrophoresis, to crosslink the oligo-DNA to the nylon membrane. To investigate the effect of the candidates of the present invention on MITF/E-box binding by measuring the level of oligo-DNA, 66.7 μi of streptavidin-conjugat ed HRPC (horseradish peroxidase C) was mixed with 20 ift£ of blocking buffer. The oligo-DNA cross-linked nylon membrane was soaked in the above mixed solution for 15 minutes, to which 6 m£ of luminol and 6 vd of stable peroxide solution were added, followed by luminescent reaction at room temperature for 10 minutes. The luminescence was detected by chemiluminescence meter (FLA-5000; Fuji Film, Japan).

As a result, as shown in Fig. 6, the oligo-DNA not bound to MITF of the nuclear extract was located in the band in lane M. The moving speed of the protein bound DNA on the gel became slower, compared with the protein not- bound DNA, indicating that the protein bound DNA was located upper part. It was confirmed that oligo-DNA did not bind with the protein of the nuclear extract isolated from fibroblasts, the negative control, in lane C. In lanes 1-a and 1-b, the MITF/E-box binding was confirmed even though MITF expression was not induced by α-MSH. In lanes 2-a and 2-b, the MITF/E-box binding was confirmed and the MITF expression was induced by α-MSH. In lane 2-c, the non-labeled oligo-DNA competed with biotin-labeled oligo- DNA for MITF, confirming that the MITF/E-box binding was specific.

<6-4> Confirmation of inhibitory effect of MITF competitor candidates on MITF/E-box binding by EMSA

EMSA was performed to investigate the inhibitory effect of the MITF competitor candidates selected in Example 5 on the binding of nuclear extract containing MITF with oligo-DNA.

1 /it of the candidate #18 prepared in Example <4-l> at different concentrations prepared by diluting 8, 4, 2, and 1 fold, which was selected among three candidate compounds selected in Example 5, was added to the binding solution comprising each nuclear extract obtained in Example <6-l> at the concentration of 0 or 0.5 μg/mi and 2 /it of 2 f mol biotin-labeled oligo-DNA obtained in Example <6-2>. The total volume of the mixture was made to 20 μϋ, followed by reaction at room temperature for 20 minutes. 5 μi of 5^χ loading dye was added thereto and mixed softly, resulting in the loading sample. EMSA was performed by the same manner as described in Example <6-3>. At this time, the loading sample containing biotin-labeled oligo-DNA alone was used as a molecular marker, and the loading sample wherein the nuclear extract of B16F10 was substituted with the nuclear extract of fibroblasts was used as a negative control 1. And the loading sample containing DMSO instead of the candidate #18 was used as a negative control 2.

As shown in Fig. 7, the loading samples were injected in the order of the molecular marker (M) , the negative control 1 (C) , the negative control 2 (N, C) , the α-MSH treated nuclear extract + 8x diluted candidate #18 group (1), the α-MSH treated nuclear extract + 4x diluted candidate #18 group (2), the α-MSH treated nuclear extract + 2x diluted candidate #18 group (3), the α-MSH treated nuclear extract + Ix diluted candidate #18 group (4), and electrophoresed by the same manner as described in Example <6-3>. The protein transportation to nylon membrane and chemiluminescence were measured by the same manner as described in Example <β-3>.

As a result, as shown in Fig. 7, in lanes 1 -4, as the concentration of each candidate material increased, the thickness of the band migrated to the upper part of the gel, resulted from the binding of MITF with oligo-DNA, was thinner. This result indicates that the candidate #18, as the MITF competitor, has excellent, direct inhibitory effect on the MITF/E-box binding.

It was also confirmed that HTS (high-throughput screening) using the protein chip designed to have MITF can be a very useful method to select a whitening agent.

The Manufacturing Examples for the composition of the present invention are described hereinafter.

Manufacturing Example 1 : Preparation of cosmetics for whitening, which comprises MITF competitor as an active ingredient

<!-!> Preparation of skin softener

Toner containing MITF competitor as an active ingredient was prepared according to the composition of

Table 3.

[Table 3]

part;

10.00

1.00

0.05

0.10

0.05

0.01

0.02

L.00

2.00

1.00 Ethanol 30.00

Preserving agent Small amount

Coloring acjent Small amount

Flavor Small amount

Purified water Proper amount

<l-2> Preparation of nutrition cream

Nutrition cream containing MITF competitor as an active ingredient was prepared according to the composition of Table 4.

[Table 4]

Preserving agent Proper amount

Flavor Proper amount

Purified water Proper amount

Manufacturing Example 2: Preparation of melanin synthesis inhibitor containing MITF competitor as an active ingredient

<2-l> Preparation of syrups

Syrups containing MITF competitor as an active ingredient were prepared according to the composition of Table 5.

[Table 5]

0.8

25.4

0.04

0.4

60

<2-2> Preparation of tablets

Tablets containing MITF competitor as an active ingredient were prepared according to the composition of Table 6.

[Table β]

Component Content (weight part;

250

175.9

180

32

160

50

Manufacturing Example 3: Preparation of pharmaceutical formulations containing MITF competitor as an active ingredient

<3-l> Preparation of powders

MITF competitor 2 g

Lactose 1 g

Powders were prepared by mixing all the above components, which were filled in airtight packs according to the conventional method for preparing powders.

<3-2> preparation of tablets MITF competitor 100 nig Corn starch 100 mg Lactose 100 rag Magnesium stearate 2 rag

Tablets were prepared by mixing all the above components by the conventional method for preparing tablets,

<3-3> Preparation of capsules MITF competitor 100 rag

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg Capsules were prepared by mixing all the above components, which were filled in gelatin capsules according to the conventional method for preparing capsules.

Manufacturing Example 4: Preparation of health food containing MITF competitor as an active ingredient

<4-l> Preparation of dairy products

5 - 10 weight part of the MITF competitor of the present invention was added to mi Lk. Dairy products for skin whitening such as butter and ice cream were prepared with the milk mixture according to the conventional method.

<4-2> Preparation of flour food

0.5 ~ 5.0 weight part of the MITF competitor of the present invention was added to the flour. Health foods for skin whitening such as bread, cake, cookies, crackers and noodles were prepared with the flour mixture according to the conventional method.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the; art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

[CLAIMS]

[Claim l]

A cosmetic composition for whitening comprising the binding inhibitor of MITF (microphthalmia transcription factor) with oligo-DNA containing E-box as an active ingredient .

[Claim 2]

The cosmetic composition for whitening according to claim 1, wherein the binding inhibitor is selected from the group consisting of N ' - [1- (4-Chloro-phenyl) -meth- (E) - ylidene] -N- (4, β-di-piperidin-1-yl- [1, 3, 5] triazin-2-yl) -N- methyl-hydrazine, 3-{ [4- (4-Fluoro-phenylamino) -6- piperidin-1-yl- [ 1, 3, 5] triazin-2-yl] -hydrazonomethyl } - phenol, 4- [1- (2 , 4-Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] - 2- (3-fluoro-phenyl) -4H-oxazol-5-one, { 4- [3-Isobutyl-2- [(Z) -4-methoxy-phenylimino] -4-oxo-thiazolidin- (5Z) - ylidenemethyl] -2-methoxy-phenoxy } -acetic acid, N-tert- Butyl-2- ( (2-chloro-benzyl) -{2- [5- (3, 4-dimethoxy-phenyl) - tetrazol-2-yl] -acetyl } -amino) -2-thiophen-2-yl-acetamide, N,N-Diphenyl-2- [3-pyridin-4-yl-5- (thiazol-2- ylcarbamoylmethylsulfanyl) -[1,2,4] triazol-1-yl] -acetamide, (E) -3- [4- (3-Chloro-benzoylamino) -2, 5-diethoxy- phenylcarbamoyl] -acrylic acid, N- [ (4-Methoxy- benzylcarbamoyl ) -methyl ] -N- ( 3-methoxy-phenyl ) -N ' -thiazol- 2-yl-succinamide, N- [3- (4-Ethoxy-phenoxy) -5-m-tolyloxy- phenyl] -2- (3-nitro- [l,2,4]triazol-l-yl) -acetamide, { 1- [2-

(4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl }- acetic acid, 3-{ [ (2-Methoxy-b-methyl-phenyl) -methyl- carbamoyl] -methyl } -l-methyl-lH-indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l-octyl-2-oxo-3-m- tolylimidazolidin-4-yl) -3-m-tolylurea, 1- (4-Ethoxy- phenyl) -5-methoxy-2-methyl-lH-indole-3-carboxylic acid, N-

(3-Chloro-4-methyl-phenyl ) -N ' - ( 4 , 6-dimethyl-pyrimidin-2- yl) -N¹ ' - (3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro- benzoyl) -4-hydroxy-5-oxo-2-phenyl-2 , 5-dihydro-pyrrol-l- yl] -butyric acid.

[Claim 3] The cosmetic composition for whitening according to claim 1, wherein the binding inhibitor is selected from the group consisting of 3-{ [4- (4-Fluoro-phenylamino) -6- piperidin-1-yl- [1,3,5] triazin-2-yl] -hydrazonomethyl } - phenol, 4- [1- (2, 4-Bis~benzyloxy-phenyl) -meth- (Z) -ylidene] - 2- (3-fluoro-phenyl) -4H-oxazol-5-one and { 1- [2- (4-Chloro- phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl } -acetic acid.

[Claim 4]

A melanin synthesis inhibitor comprising the binding inhibitor of MITF with oligo-DNA containing E-box as an active ingredient.

[Claim 5]

The melanin synthesis inhibitor according to claim 4, wherein the binding inhibitor is selected from the group consisting of N' - [1- (4-Chloro-phenyl) -meth- (E) -ylidene] -N- (4, β-di-piperidin-1-yl- [1, 3, 5] triazin-2-yl) -N-methyl- hydrazine, 3-{ 1_.4- (4-Fluoro-pheny Lamino) -6-piperidin-l-yl- [1,3,5] triazin-2-yl] -hydrazonomethy1 } -phenol, 4- [1- (2, 4- Bis-benzyloxy-phenyl) -meth- (Z) -yli dene] -2- (3-fluoro- phenyl) -4H-oxazol-5-one, {4- [3-Isobutyl-2- [(Z) -4-methoxy- phenylimino] -4-oxo-thiazolidin- (5Z) -ylidenemethyl] -2- methoxy-phenoxy} -acetic acid, N-tert-Butyl-2- ( (2-chloro- benzyl) - { 2- [5- (3, 4-dimethoxy-phenyl ) -tetrazol-2-yl] - acetyl } -amino) -2-thiophen-2-yl-acetamide, N, N-Diphenyl-2- [3-pyridin-4-yl-5- (thiazol-2-ylcarbamoylmethylsulfanyl) - [1,2,4] triazol-l-yl]-acetamide, (E) -3- [4- (3-Chloro- benzoylamino) -2, 5-diethoxy-phenylcarbamoyl] -acrylic acid, N- [ (4-Methoxy-benzylcarbamoyl) -methyl] -N- (3-methoxy- phenyl) -N' -thiazol-2-yl-succinamide, N- [3- (4-Ethoxy- phenoxy) -5-m-tolyloxy-phenyl] -2- (3-nitro- [1,2,4] triazol-1- yl) -acetamide, {1- [2- (4-Chloro-phenoxy) -ethyl] -]H- benzoimidazol-2-ylsulfanyl} -acetic acid, 3-{ [ (2-Methoxy-5- methyl-phenyl) -methyl-carbamoyl] -methyl }-l-methyl-lH- indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l- octyl-2-oxo-3-m-tolylimidazolidin-4-yl) -3-m-tolylurea, 1- (4-Ethoxy-phenyl) -5-methoxy-2-methyl-lH-indole-3- carboxylic acid, N- (3-Chloro-4-methyl-phenyl ) -N ' - (4 , 6- dimethyl-pyrimidin-2-yl ) -N ' ' - ( 3-oxo-butyryl ) -guanidine and 4- [3- (4-Chloro-benzoyl) -4-hydroxy-5-oxo-2-phenyl-2, 5- dihydro-pyrrol-1-yl] -butyric acid.

[Claim 6]

The melanin synthesis inhibitor according to claim 4, wherein the binding inhibitor is selected from the group consisting of 3- { [4- (4-Fluoro-phenylamino) -6-piperidin-l- yl-[l,3,5]triazin-2-yl] -hydrazonomethy1 } -phenol, 4- [ 1- (2, 4-Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] -2- (3-fluoro- phenyl) -4H-oxazol-5-one and { 1- [2- (4-Chloro-phenoxy ) - ethyl] -lH-benzoimidazol-2-ylsulfariyl } -acetic acid.

[Claim 7]

A pharmaceutical composition for skin whitening, which comprises the binding inhibitor of MITF with oligo- DNA containing E-box as an active ingredient.

[Claim 8]

The pharmaceutical composition for skin whitening according to claim 7, wherein the binding inhibitor is selected from the group consisting of N¹ - [1- (4-Chloro- phenyl) -meth- (E) -ylidene] -N- (4, 6-di-piperidin-l-yl-

[1,3, 5] triazin-2-yl) -N-methyl-hydrazine, 3-{ [4- (4-Fluoro- phenylamino) -β-piperidin-1-yl- [ 1, 3, 5] triazin-2-yl] - hydrazonomethyl } -phenol, 4- [1- (2 , 4-Bis-benzyloxy-phenyl) - meth- (Z) -ylidene] -2- (3-fluoro-phenyl) -4H-oxazol-5-one, {4-

[3-Isobutyl-2- [ (Z) -4-methoxy-phenylimino] -4-oxo- thiazolidin- (5Z) -ylidenemethyl] -2-methoxy-phenoxy} -acetic acid, N-tert-Butyl-2- ( (2-chloro-benzyl) -{2- [5- (3, 4- dimethoxy-phenyl) -tetrazol-2-yl] -acetyl } -amino) -2- thiophen-2-yl-acetamide, N, N-Dipheriyl-2- [3-pyridin-4-yl~5-

(thiazol-2-ylcarbamoylmethylsulfanyl) -[1,2,4] triazol-1- yl] -acetamide, (E) -3- [4- (3-Chloro-benzoylamino) -2, 5- diethoxy-phenylcarbamoyl] -acrylic acid, N- [ ( 4-Methoxy- benzylcarbamoyl) -methyl] -N- (3-methoxy-phenyl ) -N ' -thiazol- 2-yl-succinamide, N- [3- (4-Ethoxy-phenoxy) -5-m-tolyloxy- phenyl] -2- (3-nitro- [l,2,4]triazol-l-yl) -acetamide, { 1- [2-

(4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl } - acetic acid, 3- { [ (2-Methoxy-5-methyl-phenyl ) -methyl- carbamoyl] -methyl } -l-methyl-lH-indole-2-carboxylic aci d, 1-hydroxy-l- (5, 5-dimethyl-l-octyl-2-oxo-3-m- tolylimidazolidin-4-yl) -3-m-tolylurea, 1- (4-Ethoxy- phenyl) -5-methoxy-2-methyl-lH-indole-3-carboxylic acid, N-

(3-ChIoro-4-methyl-phenyl) -N ' - (4 , 6-dimethyl-pyrimidin-2- yl) -N' ' - (3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro- benzoyl) -4-hydroxy-5-oxo-2-phenyl-2, 5-dihydro-pyrrol-l- yl ] -butyric acid .

[Claim 9]

The pharmaceutical composition for skin whitening according to claim 7, wherein the binding inhibitor is selected from the group consisting of 3-{ [4- (4-Fluoro- phenylamino) -6-piperidin-l-yl- [1,3, 5] triazin-2-yl] - hydrazonomethyl } -phenol, 4- [ 1- (2 , 4-Bis-benzyloxy-phenyl ) - meth- (Z) -ylidene] -2- (3-fluoro-phenyl) -4H-oxazol-5-one and { 1- [2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2- ylsulfanyl} -acetic acid.

[Claim lθ]

A health food for skin whitening comprising the binding inhibitor of MITF with oligo-DNA containing E-box as an active ingredient.

[Claim 11]

The health food for skin whitening according to claim 10, wherein the binding inhibitor is selected from the group consisting of N ' - [1- (4-Chloro-phenyl) -meth- (E) - ylidene] -N- (4, 6-di-piperidin-l-yl- [1, 3, 5] triazin-2-yl) -N- methyl-hydrazine, 3-{ [4- (4-Fluoro-phenylamino) -6- piperidin-1-yl- [1,3, 5] triazin-2-yl] -hydrazonomethyl } - phenol, 4- [1- (2, 4-Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] - 2- (3-fluoro-phenyl) -4H-oxazol-5-one, { 4- [3-Isobutyl-2- [(Z) -4-methoxy-phenylimino] -4-oxo-thiazolidin- (5Z) - ylidenemethyl] -2-methoxy-phenoxy } -acetic acid, N-tert- Butyl-2- ( (2-chloro-benzyl) -{2- [5- (3, 4-dimethoxy-phenyl ) - tetrazol-2-yl] -acetyl } -amino) -2-thiophen-2-yl-acetamide, N,N-Diphenyl-2- [3-pyridin-4-yl-5- (thiazol-2- ylcarbamoylmethylsulfanyl) -[l,2,4]triazol-l-yl] -acetamide, (E) -3- [4- (3-Chloro-benzoylamino) -2, 5-diethoxy- phenylcarbamoyl] -acrylic acid, N- [ ( 4-Methoxy- benzylcarbamoyl ) -methyl ] -N- ( 3-methoxy-phenyl ) -N ' -thiazol- 2-yl-succinamide, N- [3- (4-Ethoxy-phenoxy) -5-m-tolyloxy- phenyl] -2- (3-nitro- [1, 2, 4 ] triazol-1-yl) -acetamide, {1- [2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl } - acetic acid, 3- { [ (2-Methoxy-5-methyl-phenyl ) -methyl- carbamoyl] -methyl } -l-methyl-lH-indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l-octyl~2-oxo-3-m- tolylimidazolidin-4-yl) -3-m-tolylurea, 1- (4-Ethoxy- phenyl) -5-methoxy-2-methyl-lH-indole-3-carboxylic acid, N- (3-ChIoro-4 -methyl-phenyl ) -N ' - ( 4 , β-dimethyl-pyrimidin-2- yl) -N¹ '-(3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro- benzoyl) -4-hydroxy-5-oxo-2-phenyl-2 , 5-dihydro-pyrrol-l- yl] -butyric acid.

[Claim 12] The health food for skin whitening according to claim 10, wherein the binding inhibitor is selected from the group consisting of 3- { [4- (4-Fluoro-phenylamino) - 6- piperidin-1-yl- [1,3,5] triazin-2-yl ] -hydrazonomethyl } - phenol, 4- [1- (2, 4-Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] - 2- (3-fluoro-phenyl) -4H-oxazol-5-one and { 1- [2- (4-Chlor o- phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl}-acetic acid.

[Claim 13]

A method for inhibiting melanin synthesis comprising the step of administering the binding inhibitor of MITF with oligo-DNA containing E-box to a subject.

[Claim 14]

The method for inhibiting melanin synthesis according to claim 13, wherein the binding inhibitor is selected from the group consisting of N ' - [1- ( 4-Chloro- phenyl) -meth- (E) -ylidene] -N- (4, 6-di-piperidin-l-yl- [1, 3, 5] triazin-2-yl) -N-methyl-hydrazine, 3-{ [4- (4-Fluoro- phenylamino) -6-piperidin-l-yl- [1,3, 5] triazin-2-yl] - hydrazonomethyl} -phenol, 4- [1- (2 , 4-Bis-benzyloxy-phenyl )- meth- (Z) -ylidene] -2- (3-fluoro-phenyl) -4H-oxazol-5-one, { 4- [3-Isobutyl-2- [ (Z) -4-methoxy-phenylimino] -4-oxo- thiazolidin- (5Z) -ylidenemethyl] -2-methoxy-phenoxy} -acetic acid, N-tert-Butyl-2- ( (2-chloro-benzyl) -{2- [5- (3, 4- dimethoxy-phenyl ) -tetrazol-2-yl] -acetyl } -amino) -2- thiophen-2-yl-acetamide, N, N-Diphenyl-2- [3-pyridin-4-yl-5- (thiazol-2-ylcarbamoylmethylsulfanyl) -[l,2,4]triazol-l- yl] -acetamide, (E) -3- [4- (3-Chloro-benzoylamino) -2, 5- diethoxy-phenylcarbamoyl] -acrylic acid, N- [ ( 4-Methoxy- benzylcarbamoyl) -methyl] -N- (3-methoxy-phenyl) -N' -thiazol- 2-yl-succinamide, N- [3- (4-Ethoxy-phenoxy) -5-m-tolyloxy- phenyl] -2- (3-nitro- [1,2, 4 ] triazol-1-yl) -acetamide, {1- [2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl } - acetic acid, 3-{ [ (2-Methoxy-5-methyl-phenyl) -methyl- carbamoyl] -methyl } -l-methyl-lH-indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l-octyl~2-oxo-3-m- tolylimidazolidin-4-yl) -3-m-tolylurea, 1- (4-Ethoxy- phenyl ) -5-methoxy-2-methyl-lH-indole-3-carboxylic acid, N- (3-ChIoro-4 -methyl-phenyl ) -N ' - (4 , 6-dimethyl-pyrimidin-2- yl) -N ' ' - (3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro- benzoyl) -4-hydroxy-5-oxo-2-phenyl-2, 5-dihydro-pyrrol-l- yl] -butyric acid.

[Claim 15] The method for inhibiting melanin synthesis according to claim 13, wherein the binding inhibitor is selected from the group consisting of 3-{ [4- (4-Fluoro- phenylamino) -6-piperidin-l-yl- [1,3, 5] triazin-2-yl] - hydrazonomethyl } -phenol, 4- [1- (2 , 4-Bis-benzyloxy-phenyl) - meth- (Z) -ylidene] -2- (3-fluoro-phenyl) -4H-oxazol-5-one and { 1- [2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2- ylsulfanyl } -acetic acid.

[Claim lβl A method for skin whitening comprising the step of applying the binding inhibitor of MITF with oligo-DNA containing E-box on the melanin pigmented skin.

[Claim 17] The method for skin whitening according to claim 16, wherein the binding inhibitor is selected from the group consisting of N' - [1- (4-Chloro-phenyl) -meth- (E) -ylidene] -N- (4 , β-di-piperidin-1-yl- [l,3,5]triazin-2-yl) -N-methyl- hydrazine, 3-{ [4- (4-Fluoro-phenylamino) -6-piperidin-l-yl- [1, 3, 5] triazin-2-yl] -hydrazonomethyl } -phenol, 4-[l-(2,4- Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] -2- (3-fluoro- phenyl) -4H-oxazol-5-one, { 4- [3-Isobutyl-2- [(Z) -4-methoxy- phenylimino] -4-oxo-thiazolidin- (5Z) -ylidenemethyl] -2- methoxy-phenoxy } -acetic acid, N-tert-Butyl-2- ( (2-chloro- benzyl) -{2- [5- (3, 4-dimethoxy-phenyl ) -tetrazol-2-yl] - acetyl } -amino) -2-thiophen-2-yl-acetamide, N, N-Diphenyl-2- [3-pyridin-4-yl-5- (thiazol-2-ylcarbamoylmethylsulfanyl) - [1,2, 4] triazol-1-yl] -acetamide, (E) -3- [4- (3-Chloro- benzoylamino) -2, 5-diethoxy-phenylcarbamoyl] -acrylic acid, N- [ (4-Methoxy-benzylcarbamoyl) -methyl] -N- (3-methoxy- phenyl) -N ' -thiazol-2-yl-succinamide, N- [3- (4-Ethoxy- phenoxy) -5-m-tolyloxy-phenyl] -2- (3-nitro- [1,2,4] triazol-1- yl) -acetamide, {l-[2- (4-Chloro-phenoxy) -ethyl] -IH- benzoimidazol-2-ylsulfanyl } -acetic acid, 3- { [ (2-Methoxy-5- methyl-phenyl ) -methyl-carbamoyl ] -methyl } -1-methyl-lH- indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l- octyl-2-oxo-3-m-tolylimidazolidin-4-yl) -3-m-tolylurea, 1- ( 4-Ethoxy-phenyl ) -δ-methoxy-Σ-methyl-lH-indole-S- carboxylic acid, N- (3-Chloro-4~methyl-phenyl ) -N ' - (4 , 6- dimethyl-pyrimidin-2-yl) -N¹ ' - (3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro-benzoyl) -4-hydroxy-5-oxo-2-phenyl-2, 5- dihydro-pyrrol-1-yl] -butyric acid.

[Claim 18] The method for skin whitening according to claim ] 6, wherein the binding inhibitor is selected from the group consisting of 3- { [4- (4-Fluoro-phenylamino) -6-piperidin-l- yl- [1,3,5] triazin-2-yl] -hydrazonomethyl } -phenol, 4- [ 1-

(2, 4-Bis-benzyloxy-phenyl) -meth- (Z) -ylidene] -2- (3-fluoro- phenyl) -4H-oxazol-5-one and { 1- [2- (4-Chloro-phenoxy) - ethyl] -lH-benzoimidazol-2-ylsulfanyl} -acetic acid.

[Claim 19]

A use of the binding inhibitor of MITF with oligo-DNA containing E-box for the preparation of the cosmetic composition for whitening.

[Claim 20]

The use according to claim 19, wherein the binding inhibitor is selected from the group consisting of N ' — | 1— (4-Chloro-phenyl ) -meth- (E) -ylidene] -N- (4, β-di-piperidin-1- yl- [1, 3, 5] triazin-2-yl) -N-methyl-hydrazine, 3-{ [4- ( 4 - Fluoro-phenylamino) -6-piperidin-l-yl- [1, 3, 5] triazin-2-yl] - hydrazonomethyl} -phenol, 4- [1- (2, 4-Bis-benzyloxy-phenyl ) - meth- (Z) -ylidene] -2- (3-fluoro-phenyl) -4H-oxazol-5-one, {4- [3-Isobutyl-2- [(Z) -4-methoxy-phenyl imino] -4-oxo- thiazolidin- (5Z) -ylidenemethyl] -2-methoxy-phenoxy } -acetic acid, N-tert-Butyl-2- ( (2-chioro-benzyl) -{2- [5- (3, 4- dimethoxy-phenyl ) -tetrazol-2-yl] -acetyl } -amino) -2- thiophen-2-yl-acetamide, N, N-Diphenyl-2- [3-pyridin-4-yl-5- (thiazol-2-ylcarbamoylmethylsulfanyl) - [1, 2, 4] triazol-1- yl] -acetamide, (E) -3- [4- (3-Chloro-benzoylamino) -2, 5- diethoxy-phenylcarbamoyl] -acrylic acid, N- [ (4-Methoxy- benzylcarbamoyl) -methyl] -N- (3-methoxy-phenyl) -N' -thiazol- 2-yl-succinamide, N- [3- (4-Ethoxy-phenoxy) -5-m-tolyloxy- phenyl] -2- (3-nitro- [1, 2 , 4 ] triazol-1-yl) -acetamide, { 1- [ 2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl } - acetic acid, 3- { [ (2-Methoxy-5-methyl-phenyl) -methyl- carbamoyl] -methyl } -l-methyl-lII-indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l-octyl-2-oxo-3-m- tolylimidazolidin-4-yl) -3-m-tolylurea, 1- (4-Ethoxy- phenyl) -5-methoxy-2-methyl-lH-indole-3-carboxylic acid, N- (3-Chloro-4-metbyl-phenyl) -N' - (4, β-dimethyl-pyrimidin-2- yl) -N ' ' - (3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro- benzoyl) -4-hydroxy-5-oxo-2-phenyl~2, 5-dihydro-pyrrol-l- yl] -butyric acid.

[Claim 21]

The use according to claim 19, wherein the binding inhibitor is selected from the group consisting of 3— { 14— (4-Fluoro-phenylamino) -6-piperidin-l-yl- [1, 3, 5] triazin-2- yl] -hydrazonomethyl} -phenol, 4- [1- (2, 4-Bis-benzyloxy- phenyl) -meth- (Z) -ylidene] -2- (3-fluoro-phenyl ) -4H-oxazol-5- one and { 1- [2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol- 2-ylsulfanyl}-acetic acid.

[Claim 22]

A use of the binding inhibitor of MITF with oligo-DNA containing E-box for the preparation of the pharmaceutical composition for skin whitening.

[Claim 23]

The use according to claim 22, wherein the binding inhibitor is selected from the group consisting of N'-[l- (4-Chloro-phenyl) -meth- (E) -ylidene] -N- (4, 6-di-piperidin-l- yl- [1, 3, 5] triazin-2-yl) -N-methyl-hydrazine, 3-{ [4- (4- Fluoro-phenylamino) -β-piperidin-1-yl- [1,3,5] triazin-2-yl] - hydrazonomethyl } -phenol, 4- [1- (2 , 4-Bis-benzyloxy-phenyl ) - meth- (Z) -ylidene] -2- (3-fluoro-phenyl) -4H-oxazol-5-one, { 4- [3-Isobutyl-2- [ (Z) -4-methoxy-phenylimino] -4-oxo- thiazolidin- (5Z) -ylidenemethyl] -2-methoxy-phenoxy } -acetic acid, N-tert-Butyl-2- ( (2-chloro-benzyl) -{2- [5- (3, 4- dimethoxy-phenyl ) -tetrazol-2-yl] -acetyl } -amino) -2- thiophen-2-yl-acetamide, N, N-Diphenyl-2- [3-pyridin-4-yl-5- (thiazol-2-ylcarbamoylmethylsulfanyl) -[1,2,4] triazol-1- yl] -acetamide, (E) -3- [4- (3-Chloro-benzoylamino) -2, 5- diethoxy-phenylcarbamoyl] -acrylic acid, N- [ ( 4-Methoxy- benzylcarbamoyl ) -methyl ] -N- ( 3-methoxy-phenyl ) -N ' -thiazol- 2-yl-succinamide, N- [3- (4-Ethoxy-phenoxy) -5-m-tolyloxy- phenyl] -2- (3-nitro- [1,2, 4] triazol-1-yl) -acetamide, {l-[2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol-2-ylsulfanyl } - acetic acid, 3- { [ (2-Methoxy-5-methyl-phenyl) -methyl- carbamoyl] -methyl }-l-methyl-lH-indole-2-carboxylic acid, 1-hydroxy-l- (5, 5-dimethyl-l-octyl-2-oxo-3-m- tolylimidazolidin-4-yl) -3-m-tolylurea, l-(4-Ethoxy- phenyl) -5-methoxy-2-methyl-lH-indole-3-carboxylic acid, N- ( 3-Chloro-4 -methyl-phenyl) -N ' - ( 4 , 6-dimethyl-pyrimidin-2 - yl) -N¹ ' - (3-oxo-butyryl) -guanidine and 4- [3- (4-Chloro- benzoyl) -4-hydroxy-5-oxo-2-phenyl-2, 5-dihydro-pyrrol-l- yl] -butyric acid. [Claim 24]

The use according to claim 22, wherein the binding inhibitor is selected from the group consisting of 3-{[4- (4-Fluoro-phenylamino) -6-piperidiri-l-yl- [1, 3, 5] triazin-2- yl] -hydrazonomethyl} -phenol, 4- [1- (2, 4-Bis-benzyloxy- phenyl) -meth- (Z) -ylidene] -2- (3-fluoro-phenyl) -4H-oxazol-5- one and { 1- [2- (4-Chloro-phenoxy) -ethyl] -lH-benzoimidazol- 2-ylsulfanyl } -acetic acid.