WO2006088310A1

WO2006088310A1 - A pharmaceutical composition comprising retinoid for prevention and treatment of vitiligo

Info

Publication number: WO2006088310A1
Application number: PCT/KR2006/000524
Authority: WO
Inventors: Won-Young Chey; Ai-Young Lee
Original assignee: Stiefel Laboratories Korea
Priority date: 2005-02-15
Filing date: 2006-02-15
Publication date: 2006-08-24
Also published as: KR100798853B1; KR20060091829A

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of vitiligo comprising retinoid as an effective ingredient, precisely a pharmaceutical composition for the prevention and treatment of vitiligo designed to effectively work for vitiligo caused by apoptosis or necrosis of melanocytes by increasing the cell density by reducing apoptosis of human keratinocytes. The pharmaceutical composition of the invention containing retinoid as an effective ingredient not only prevents skin atrophy caused by corticosteroid, a conventional treatment agent of vitiligo, but also inhibits apoptosis of melanocytes and thus improving pigmentation, so that it can be effectively used for the prevention and treatment of vitiligo.

Description

A PHARMACEUTICAL COMPOSITION COMPRISING RETINOID FOR PREVENTION AND TREATMENT OF VITILIGO

Technical Field The present invention relates to a pharmaceutical composition for the prevention and treatment of vitiligo containing retinoid as an effective ingredient, more precisely, a pharmaceutical composition for the prevention and treatment of vitiligo containing, retinoid wherein the retinoid reduces the destruction of keratinocytes and thus increases the number of cells to 'prevent and treat vitiligo caused by apoptosis or necrosis of melanocytes.

Background Art Vitiligo is an acquired depigmentation syndrome caused by apoptosis or necrosis of melanocytes with showing various sizes and forms of white spots on the skin. This is a very common disease observed in about 1% of total population on earth and shows no specificity to ethnic and regional groups. Vitiligo can start at any age, but it often occurs between the ages of 10 and 30. 95% of patients are attacked before the age of 40 and 30% of patients has family history.

The patterns of vitiligo are various sizes of circular or irregular white spots. In early stage of vitiligo, depigmentation is not so severe but as time goes by the spots become clearer, darker and more obvious than normal skin color even though the boundary between the spots and normal skin might be unclear. In some cases, it itches. When the white spots appear in areas with hair such as head or eyebrows, white hair might be growing. Vitiligo can appear anywhere on the skin, but in particular, it appears frequently on bone protrusion areas such as a finger, a toe, a knee and an elbow or around the mouth, around the nose, around the eyes, the armpit and the inner wrist. Rarely, it appears on mucosa including lips or genitals, and such parts that can be easily wounded are major target places. Vitiligo is distributed according to bilateral symmetry or nervous system. In addition to simple white spots on the skin, vitiligo causes dyschromatosis in the iris and the retina of the eye. Vitiligo has been associated with certain diseases such as diabetes mellitus, pernicious anemia, hypothyroidism, hyperthyroidism, Down's syndrome, biliary cirrhosis, stomach cancer, Addison's disease, alopecia areata, and autoimmune diseases like lupus erythematosus, etc.

A concrete and singular cause leading up to this event has not been established, however there are various theories such as autoimmune theory, neurogenic theory, self-destruct theory, etc, which may account for vitiligo and they have some overlapping aspects worthy of consideration. According to the autoimmune theory, autoantibody is expressed against melanocytes, causing the destruction or malfunction of melanocytes, or lymphokine is secreted by cytotoxic lymphocytes or activated lymphocytes, causing the destruction of melanocytes. According to the neurogenic theory, abnormality of catecholamine biosynthesis and increase of monoamine oxidase produce hydrogen peroxide in relation to stress, resulting in the destruction of melanocytes. Based on the neurogenic theory, vitiligo occurs by the routes of ganglions after neuronal damage or stress. According to the self-destruct theory, the accumulation of phenol complex, which is the final or intermediate metabolite of melanin synthesis, causes the destruction of melanocytes. Besides, inherent cellular defect, heredity, apoptosis and abnormal calcium metabolism are considered to find out a reason for vitiligo.

Most studies on vitiligo have been focused on melanocytes. However, melanocytes of the skin form a functional structural unit with neighboring keratinocytes, indicating that studies on keratinocytes are required in relation to the study on vitiligo. As a matter of fact, keratinocytes of a vitiligo patient show a functional defect reducing the activities of catalase and glutathione reductase (Challreuter K. U. et al . , J Invest Dermatol 97: 1081-1085, 1991) and the absorption of calcium (Schallreuter K. U. et al., Arch Dermatol Res 280: 137-139, 1988) and a structural defect causing vacuolization and extracellular evasion of a granule out of a cell (Moellmann G. et al, J. Invest Dermatol 79: 321-330, 1982; Bhawan J. et al., J Cutan Pathol 10: 207-212, 1983). Vacuolization in keratinocytes is known to be induced by the damage by hydrogen peroxide (Maresca V. et al . , J. Invest Dermatol 109; 310-313, 1997) . In fact, hydrogen peroxide is one of the most powerful stimulator inducing apoptosis of keratinocytes (Kucchle M. K. et al . , Cell Death Differentiation 7; 566-573, 2000). The present inventors earlier confirmed that apoptosis of keratinocytes is increased in the region of vitiligo (Lee A. Y. et al., Br J Dermatol 151: 995-1003, 2004), compared with a normal skin of the same patient, and thereby the level of stem cell factor (SCF) , which is produced in keratinocytes, is reduced in the region of vitiligo, suggesting the potential for apoptosis of melanocytes (Lee A. Y et al., J Invest Dermatol 124: 976-983, 2005).

The conventional treatments for vitiligo are psoralen UV treatment, local and systemic treatments of steroid and surgical treatment, etc. Psoralen UV treatment is also called as photochemotherapy, which is that a systemic UV irradiation after psoralen is orally administered or a local UV irradiation after psoralen is applied to the skin. Psoralen UV treatment is a long-term treatment, having a potential skin aging or skin cancer. To treat with steroid, a local application or a local injection using triamcinolone is performed when a lesion is small and a systemic application is required when a lesion is large and found all over the body. The treatment with steroid is also a long-term care, so that it might cause side effects affecting the skin and/or the entire body. Surgical treatment is a way to transplant melanocytes, which is classified into skin graft and cell transplantation. For the skin graft, full-thickness graft, split-thickness graft and epidermal graft are performed, which are though not always carried out and only applicable when a lesion is not wider for years .

Among those treatments, topical steroid preparation is widely used. However, the long-term treatment with steroid may cause side effects including skin atrophy, which have to be solved for the successful treatment of vitiligo.

Retinoic acid inhibits comedones formation and reduces ductal cornification, so it has been used as an anti-acne drug. In addition, retinoic acid reduces migration or adhesion of melanocytes, inhibits proliferation of melanocytes (Fligiel S. E. et al . , J. Cutan Pathol 19; 27-33, 1992) and reduces dendrites of melanocytes, resulting in lightening of pigment, so that it used to be used for the treatment of melasma (Ortonne J. P., Br J Dermatol 127 suppl 41: 43-47, 1992). According to a recent report that retinoic acid can reduce skin atrophy caused by corticosteroid, it is expected for retinoic acid to inhibit side effects carried by corticosteroid.

Therefore, the present inventors prepared a pharmaceutical composition containing retinoid as an effective ingredient and then completed the invention by confirming that the composition of the invention not only prevents skin atrophy by corticosteroid but also proliferates human keratinocytes and inhibits apoptosis to up-regulate SCF and other melanocyte growth factors, so that it is effectively used for the treatment of vitiligo by increasing melanocytes with restoring pigment.

Disclosure Technical Problem

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of vitiligo that can not only prevent side effects of corticosteroid inducing skin atrophy but also induce melanocytes proliferation and increase coloring effect of the cells.

Technical Solution To achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of vitiligo containing retinoid as an effective ingredient.

The present invention also provides a treatment method for a vitiligo patient comprising the administering retinoid.

The present invention further provides an inhibitor of vitiliginous epidermal cell apoptosis containing retinoid as an effective ingredient. The present invention also provides an accelerant of vitiliginous keratinocyte proliferation containing retinoid as an effective ingredient.

Hereinafter, the present invention is described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of vitiligo containing retinoid as an effective ingredient.

The retinoid of the present invention can be selected from a group consisting of retinol, retinal, retinoic acid, tazarotene and adapalene, and among these, retinoic acid is preferred. Herein, the retinoic acid includes tretinoin, isotretinoin and etretinate, and among them tretinoin is used in a preferred embodiment of the invention. To confirm whether a pharmaceutical composition of the invention containing retinoid can improve a lesion of a vitiligo patient, tretinoin, a kind of retinoid, has been administered for 12 months to vitiligo patients. As a result, 43.5% of patients showed improvement, and in particular, 30.6% of patients showed improvement within 6 months (Fig. 1) .

The present inventors investigated the effect of a composition of the invention containing retinoid on the regulation of SCF (stem cell factor) of epidermal cells of keratinocytes . For the investigation, normal epidermis and depigmented epidermis samples were treated with tretinoin and vehicle, and then the expression of SCF therein was measured by using anti-SCF antibody. As a result, the level of SCF in depigmented epidermis each treated with tretinoin and vehicle was lower than that in normal epidermis. From the comparison between depigmented epidermis groups, it was confirmed that the depigmented epidermis treated with tretinoin showed higher SCF level than the other depigmented epidermis treated with vehicle, in improved groups, and similar result was observed in non- reacted groups (Fig. 3 and Fig. 4) .

The present invention also provides a treatment method for a vitiligo patient comprising the following steps :

1) Administering the pharmaceutical composition of claim 1 to a vitiligo patient; and

2) Terminating the administering or administering the composition additionally after checking the lesion of the patient treated with the composition in step 1) .

As shown in Fig. 1, a composition of the invention containing retinoid was locally applied to the lesion of a vitiligo patient and after a while, skin color was observed to examine if the epidermal cells of the lesion were recovered to normal cells, based on which the additional application of the composition was determined. The application frequency and the application amount can be determined according to the conditions of a patient.

The present invention also provides an inhibitor of epidermal cell apoptosis containing retinoid as an effective ingredient.

The present invention further examined whether retinoid of the invention was involved in apoptosis and the expressions of apoptosis related proteins, active caspase-3, 8 and 9. Normal epidermis and depigmented epidermis treated respectively with tretinoin and vehicle were sectioned, followed by TUNEL assay to examine apoptosis. The expression levels of the active caspases were examined by the same manner as used for SCF. As a result, the number of TUNEL positive cells in the depigmented epidermis sections was increased or similar to that in normal epidermis sections. From the comparison between the depigmented epidermis groups, it was confirmed that in improved groups, the number of TUNEL positive cells in the tretinoin treated epidermis was less than that in vehicle treated epidermis. In the meantime, in non-reacted groups, the number of TUNEL positive cells in the tretinoin treated epidermis was similar to that in the vehicle treated epidermis (Fig. 5). The expressions of active caspase-3, 8 and 9 in the depigmented epidermis were increased or similar to those in normal epidermis. From the comparison between depigmented epidermis groups, it was confirmed that the expression levels of the active caspases in the tretinoin treated groups were lower than those in the vehicle treated groups, among improved groups, but the expression levels in non-reacted groups were all similar (Fig. 6) . The present invention also provides an accelerant of keratinocyte proliferation containing retinoid as an effective ingredient.

The present inventors also investigated the effect of retinoid of the invention on cell proliferation. Particularly, normal epidermis and depigmented epidermis treated respectively with tretinoin and vehicle were sectioned, followed by Kiβ7 screening assay for growing cells. As a result, the number of Ki67 positive cells in the vehicle treated depigmented epidermis was less than that in normal epidermis. But the number of Ki67 positive cells in the tretinoin treated epidermis was more than or similar to that in normal epidermis. From the comparison between the depigmented groups, the number of Ki67 positive cells in the tretinoin treated group was similar to or a little higher than that in the vehicle treated group. The above results were consistent with both of improved groups and of non-reacted groups (Fig. 5).

The present inventors investigated the effect of retinoid of the invention on cell proliferation using cultured normal keratinocytes and melanocytes. Particularly, MTT assay kit examining cell proliferation by a spectroscopic method was used and optical densities of keratinocytes and melanocytes were measured with increasing the concentration of tretinoin. As a result, optical density of keratinocytes was increased with the increase of tretinoin concentration (Fig. 7), while optical density of melanocytes was not increased (Fig. 8) . That is, the number of keratinocytes was increased with the increase of tretinoin concentration but the number of melanocytes was not increased with the increase of tretinoin concentration.

The present inventors further investigated the effect of retinoid of the invention on the expression of SCF of normal keratinocytes. The expression level was measured by using anti-SCF antibody. As a result, the level of SCF was increased with the increase of tretinoin concentration (Fig. 9) .

Therefore, a composition of the invention can be effectively used for the prevention and treatment of vitiligo by inhibiting apoptosis ^' and promoting the proliferation of human keratinocytes to up-regulate SCF and melanocyte growth factor, resulting in the proliferation and coloring of melanocytes .

A pharmaceutical composition for the prevention and treatment of vitiligo of the invention can additionally contain, in addition to retinoid, one or more effective ingredients having the same or similar functions to the retinoid. The composition of the present invention can also include, in addition to the above-mentioned effective ingredients, one or more pharmaceutically acceptable carriers for the administration. A pharmaceutically acceptable carrier can be selected or be prepared by mixing more than one ingredients selected from a group consisting of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrose solution, glycerol and ethanol. Other general additives such as anti- oxidative agent, buffer solution, bacteriostatic agent, etc, can be added. In order to prepare injectable solutions such as aqueous solution, suspension and emulsion, diluents, dispersing agents, surfactants, binders and lubricants can be additionally added. The composition of the present invention can further be prepared in suitable forms for each disease or according to ingredients by following a method represented in Remington's Pharmaceutical Science (the newest edition) , Mack Publishing Company, Easton PA.

The composition of the invention can be administered preferably parenterally (for example, intravenous, hypodermic, intracavaty injections, local application or visual pathway application) , and local application is more preferred. The local application is to cure the skin and mucous membrane, for which the composition can be formulated in the form of paste ointment, cream, milk, powder, invasive pad, solution, gel, spray, lotion or suspension. Other formulations such as microgranules or ultra microgranules, lipotropic or polymeric follicles or polymeric additives capable of controlled release and hydrogel can be prepared. A composition for the local application can be formulated in the form of hydrophobic or hydrophilic according to the clinical purpose. The effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The pharmaceutical composition of the invention contains retinoid at the level of 0.001 - 1% by weight and administration frequency is preferably once or several times a day.

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

Fig. 1 shows a set of photographs (upper part) illustrating the result of placebo control test in which the right hand of a patient, as an experimental group, was treated with a pharmaceutical composition of the invention containing retinoid, and the left hand of the patient, as a control, was treated with a placebo vehicle (V) , and a line graph (lower part) illustrating the number of patients with the improvement by the composition of the present invention, according to the period of time,

Fig. 2 is a graph showing the degrees of improvement of the depigmented areas respectively treated with retinoid and vehicle (pigmented area after the treatment/pigmented area before the treatment) ,

ATRA : Depigmented epidermis treated with retinoid, V : Depigmented epidermis treated with vehicle

Fig. 3 is a set of photographs showing the results of

Western blotting comparing the expression of SCF (stem cell factor) and the expression of β-actin in the improved group treated with retinoid (upper part) and in the non-reacted group,

Normal : Normal epidermis of a patient, Vehicle: Depigmented epidermis of a patient treated with vehicle,

ATRA : Depigmented epidermis of a patient treated with retinoid,

Actin : A protein used as a standard for the comparison of the expressions

Fig. 4 is a bar graph showing the relative ratio of SCF expression in depigmented epidermis treated with vehicle and retinoid to SCF expression in normal epidermis among improved groups and non-reacted groups each treated with vehicle (V) and retinoid (A) .

L : Depigmented epidermis N : Normal epidermis

Fig. 5 is a set of photographs of fluorescent microscope. Normal epidermis and depigmented epidermis respectively treated with retinoid and vehicle were double stained with TUNEL (green) and Ki67 (red) to examine apoptosis and cell proliferation. The upper part shows the epidermis of improved groups and the lower part presents the epidermis of non-reacted groups,

Arrow : TUNEL (green) Arrowhead : Kiβ7 (red)

Fig. 6 is a set of photographs showing the expressions of apoptosis related proteins, active caspase 8, 9 and 3, in epidermis of both improved groups and non- reacted groups by using an antibody capable of detecting the protein. The right three lanes illustrates the detection in patients of improved groups and the left three lanes illustrates the detection in patients of non-reacted groups,

N : Normal epidermis,

V : Depigmented epidermis treated with vehicle, A : Depigmented epidermis treated with retinoid Fig. 7 is a bar graph showing the result of MTT assay, in which the optical density of melanocytes is not increased with the increase of retinoid concentration to 0, 1 and 100 nM,

Fig. 8 is a bar graph showing the result of MTT assay, in which the optical density of keratinocytes is increased with the increase of retinoid concentration,

Fig. 9 is a set of photographs showing that the expression of SCF protein was increased in keratinocytes with the increase of retinoid concentration.

Best Mode

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.

Experimental Example 1: Placebo controlled study on the tretinoin treatment to a vitiligo patient A control group treated with a placebo (vehicle) and an experimental group treated with tretinoin were paired up for placebo-controlled paired comparison left-right study. One side of hands, arms, legs, bodies, abdomens and faces of 62 vitiligo patients were treated with tretinoin and the other sides of those of the same patients were treated with vehicle produced by the same manufacturer (Stiefel Labs. Ltd., Singapore) . After the application of tretinoin or vehicle, local corticoid was applied once or twice a day. The formation of pigmentation around hair follicle and the diffusion of repigmentation were observed in depigmented areas treated tretinoin and vehicle. Photographs were taken every two weeks for 3 - 6 months for comparison, and two dermatologists examined the results and at the same time, the same photographs taken before and after the treatment were given to researchers not informed about those patients for objective analysis using a microscope equipped with Leika Qwin V₃. Once one of experimental groups was judged to be improved more, the experiment was stopped.

As a result, in 27 patients among 62 patients, the area treated with tretinoin showed better repigmentation than the area treated with vehicle (Fig. 1, upper part) . Those groups improved by tretinoin were named improved groups and those not improved by tretinoin were named non- reacted groups. In improved groups, 4 patients (6.5%) showed improvement within one month from the treatment of tretinoin, and so did 9 patients (14.5%) within 3 months, 13 patients (21%) within 4 months, 17 patients (27.4%) within 5 months and 19 patients (30.6%) within 6 months (monthly total). Although 3 patients (4.8%) showed improvement about a year later, most of the patients showed fast improvement (Fig. 1, lower part). Analysis using Leika Qwin V₃ was performed with 13 patients of improved groups and 9 of non-reacted groups. As a result, the depigmented area treated with tretinoin was improved significantly (P = 0.01), compared with the depigmented area treated with vehicle (improvement degree = pigmented area after treatment/pigmented area before treatment) (Fig. 2) .

Experimental Example 2 : Western blotting of SCF

Normal pigmented epidermis and depigmented epidermis respectively treated with tretinoin and vehicle were frozen in liquid nitrogen and then pulverized into powders. The epidermis samples were homogenized in a buffer cooled with ice containing 50 mM Tris-base (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.1% Tween-20 and protease inhibitor (0.1 mM phenylmethylsulfonyl-fluoride, 5

aprotinin, and 5 βg/mi leupeptin) . Centrifugation was performed at 12,000 g at 4°C for 30 minutes to obtain a supernatant. The protein concentration in the supernatant was measured by using DC protein analysis kit (Bio-Red Laboratories, Inc., Herculus,

CA) . 20 βg of protein was separated from each by 7% SDS- PAGE, which was transferred onto a nitrocellulose membrane.

The membrane was dipped in a blocking solution prepared by adding non-fat dry milk (NFDM) at the level of 5% (W/V) to

TTBS (Tris-buffered saline) supplemented with 10 mM Tris

(pH 7.6), 150 mM NaCl and 0.1% Tween-20, followed by standing at room temperature for one hour. The membrane was put in an anti-Human SCF antibody (mouse monoclonal antibody; Santa Cruz, CA) solution diluted in the blocking solution at the ration of 1:1,000, followed by reaction at 4^°C for overnight. Then, the membrane was washed with TTBS and dipped in anti-Mouse antibody (Jackson Laboratories, West Grove, PA) or anti-Rabbit containing horseradish peroxidase diluted in the blocking solution at the ratio of 1/1,000, which was standing at room temperature for one hour. After washing with TTBS for 30 minutes, the membrane was treated with chemiluminescent solution (ECL Kit; Amersham Life Science, Buckinghamshire, England) for one minute, and then exposed on X-ray film (Hyperfilm; Amersham Life Science, Buckinghamshire, England) . To quantify the protein therein, an antibody capable of detecting β-actin was used. First, a stripping buffer (0.1 M Glycine, pH 2.5) was used to eliminate antibodies attached on the protein of the membrane surface. Then, the membrane was put in anti-actin antibody (mouse monoclonal antibody; Sigma, St. Louis, MO) solution diluted in the blocking solution at the ratio of 1/5,000, which was standing for one hour at room temperature. Next, secondary antibody was treated by the same manner as described above. The film was developed right after and then the density of a protein band was investigated. As a result, the level of SCF was lower in depigmented epidermis than in normal epidermis (Fig. 3). In 7 patients of the improved group, among 13 patients expressing SCF, those treated with tretinoin showed significantly higher SCF levels than those treated with vehicle (Fig. 3, upper part and Fig. 4), while SCF levels were not much different in 6 patients of the non-reacted group (Fig. 3, lower part and Fig. 4) .

Experimental example 3: Apoptosis and cell proliferation 1) TUNEL assay

Epidermis samples were fixed in 4% paraformaldehyde and then dehydrated, which were put in paraffin wax. The samples were cut into 3 μm thick sections, followed by deparaffination by using xylene. The sections were put in alcohol solutions serially, resulting in rehydration. Apoptosis in normal epidermis and depigmented epidermis treated with tretinoin and vehicle respectively was examined by TUNEL assay (Terminal deoxynucleotide transferase mediated dUTP Nick End Labeling assay; In situ cell death detection kit, Roche, Mannheim, Germany) . Sections were washed with PBS, followed by pre-treatment with proteinase K (Invitrogen, Carlsbad, CA) for 15 - 30 minutes. The sections were washed again with PBS, followed by reaction in TUNEL reaction mixture included in the kit for one hour at 37 ^°C . After washing with distilled water, nuclei were counter-stained with Hoechst 33258 (Sigma, St. Louis, MO) , followed by observation under a fluorescent microscope (Nikon Eclipse TE300, Japan) . For the quantitative analysis of apoptosis, 4 different regions were randomly selected from normal and depigmented epidermis treated respectively with tretinoin and vehicle, and pictures of them were taken. The numbers of apoptotic cells and total cells were counted. The number of apoptotic cells was calculated by % proportionally to the number of total cells (apoptotic cells/total cells) .

As a result, TUNEL positive cells were observed in the lower and upper granular layers of epidermis and the numbers of the cells were significantly different among groups. Among improved groups, the depigmented epidermis treated with tretinoin had lower number of TUNEL positive cells than that treated with vehicle (Fig. 5, upper part) . In the meantime, in non-reacted groups, the depigmented epidermis treated with tretinoin had similar TUNEL positive cell number to that treated with vehicle (Fig. 5, lower part) .

2) Immunohistochemical staining of Ki67 and counter- staining with TUNEL

For immunofluorescent staining of Ki67, normal epidermis and depigmented epidermis respectively treated with tretinoin and vehicle were fixed in paraffin, which were then cut into 3 μm thick sections. After deparaffination and rehydration, the sections were dipped in a citrate solution (100 mM citrate, pH 6.0) and boiled for one minute, followed by washing with Tris buffer. The sections were put in a 10% FBS (fetal bovine serum) solution, which stood at room temperature for two hours. The fractions were put in a 1:200 diluted anti-Kiβ87 antibody (mouse monoclonal antibody; Zymed, South San Francisco, CA), followed by reaction for overnight. After washing with PBS, the sections were reacted with biotin- labeled anti-Mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for two hours. Upon completion of the reaction, the sections were washed with PBS and reacted with Alex Fluor 594 conjugate (Molecular Probes, Eugene, OR) linked streptavidin. Counter-staining of the sections was performed by TUNEL method by the same manner as described above, followed by observation under a fluorescent microscope (Nikon Eclipse TE300, Japan) . As a result, Ki67 positive cells were mainly spread in the lower part of the epidermis, and the number of Ki67 positive cells was more or less increased in the tretinoin treated groups, compared with the vehicle treated groups. The increase was not apparent between the improved group and the non-reacted group. After counter-staining with TUNEL, it was confirmed that in improved groups, the depigmented epidermis treated with tretinoin had less TUNEL positive cells but more Ki67 positive cells than the depigmented epidermis treated with vehicle (Fig. 5, upper part) . On the other hand, in non-reacted groups, the number of TUNEL positive cells in the depigmented epidermis treated with tretinoin was similar to that in the depigmented epidermis treated with vehicle and the number of Ki67 positive cells was also similar or a little higher than that treated with vehicle (Fig. 5, lower part) . The above results indicate that in tretinoin treated groups, tretinoin reduces apoptosis significantly but increases cell proliferation, increasing the total cell number.

Experimental example 4 : Western blotting of active caspase Western blotting was performed by the same manner as used for SCF examination, except different primary antibodies were used for active caspase 3 (rabbit polyclonal antibody; Pharmingen, San Diego, CA) , 8 (rabbit polyclonal antibody; Pharmingen, San Diego, CA) and 9 (rabbit polyclonal antibody; Pharmingen, San Diego, CA) .

As a result, compared with normal epidermis, the tretinoin treated improved group showed lower expression levels of active caspase 3, 8 and 9 than the vehicle treated improved group. In the meantime, the expression levels of active caspase 3, 8 and 9 were similar between non-reacted groups (Fig. 6) .

Experimental example 5: MTT assay <5-l> Culture of normal melanocytes

A normal skin sample was treated with dispase (#295 825, Roche, Mannheim, Germany) for one hour to separate epidermis from dermis. The epidermis was treated with trypsin for 10 minutes to prepare single epidermal cell suspension. After centrifugation, the cells were cultured in MCDB153 medium containing vitamin E, 12-0- tetradecanoylphorbol 13-acetate, bovine pituitary extract, insulin, transferrin, selenium and basic fibroblast growth factor. When cells were attached onto the bottom of the culture flask, SCF was added. After proliferation, cells were separated from the flask and inoculated into a new culture flask. Before separating melanocytes, XB-2 cells were prepared as follows. XB-2 cells were cultured for 4 hours together with 10 βg of mitomycin C without supplying 3T3 and then treated with trypsin to prepare each cell suspension. Then, cells were inoculated into a new culture flask (5 x 10⁴ cells/m^) . Melanocytes 3-10 times sub- cultured were used for experiments.

<5-2> Culture of normal keratinocytes

Single epidermal cell suspension was prepared by the same manner as described above for the culture of melanocytes. After centrifugation, the cells were cultured in KGM medium (#CC-3101; BioWhittaker, Walkersville, MD) containing bovine pituitary extract, human epidermal cell growth factor, insulin and hydrocortisone. After proliferation, the cells were separated from the flask and inoculated into a new culture flask.

<5-3> MTT assay

To investigate the effect of tretinoin on the proliferation of melanocytes, MTT assay kit (#4890-25-01, R & D, Minneapolis, MN) was used. The concentrations of tretinoin used for the assay were 0, 1 and 100 nM. To investigate the effect of tretinoin on the proliferation of keratinocytes, MTT assay kit was also used. The concentrations of tretinoin used for the assay were 0, 1 and 100 nM.

As a result, melanocytes were neither proliferated nor decreased with the concentration of 0 - 100 nM of tretinoin (Fig. I)₁ while keratinocytes were proliferated

(Fig. 8) . And 0 - 100 nM of tretinoin increased the level of SCF produced in keratinocytes (Fig. 9) .

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

Manufacturing Example: Preparation of formulations for local application 1. Ointment

Retinoic acid 0.020 g

Isopropyl myristate 81.700 g

Mineral oil fluid 9.1 g

Silica 9.180 g

2. Cream

Retinoic acid 0.100 g

Lanolin emulsion alcohol, wax 39.900 g

Methyl para-hydroxybenzoate 0.075 g Propyl para-hydroxybenzoate 0.075 g Sterilized deionized water 59.85 g

Total 100 g

3. Lotion Retinoic acid 0.100 g

Polyethylene glycol (PEG 400) 69.900 g

95% ethanol 30.000 g

Industrial Applicability As explained hereinbefore, retinoid of the invention not only prevents skin atrophy by local corticosteroid but also proliferates human keratinocytes and inhibits apoptosis, that is retinoid of the invention increases the amounts of SCF and various melanocyte growth factors, so that it can be effectively used for the prevention and treatment of vitiligo by proliferating and pigmentating melanocytes .

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

What is claimed is

1. A pharmaceutical composition for the prevention and treatment of vitiligo containing retinoid as an effective ingredient .

2. The pharmaceutical composition for the prevention and treatment of vitiligo as set forth in claim 1, wherein the retinoid is selected from a group consisting of retinol, retinal, retinoic acid, tazarotene and adapalene.

3. The pharmaceutical composition for the prevention and treatment of vitiligo as set forth in claim 2, wherein the retinoic acid is selected from a group consisting of tretinoin, isotretinoin and etretinate.

4. The pharmaceutical composition for the prevention and treatment of vitiligo as set forth in claim 1, wherein the composition is formulated in an appropriate form for local application such as paste ointment, cream, milk, powder, invasive pad, solution, gel, spray, lotion or suspension.

5. A treatment method for a vitiligo patient comprising the following steps: 1) Administering the pharmaceutical composition of claim 1 to a vitiligo patient; and

2) Terminating the administering or additionally administering the composition after checking the lesion of the patient treated with the composition in step 1) .

6. An inhibitor of epidermal cell apoptosis containing retinoid as an effective ingredient.

7. The inhibitor of epidermal cell apoptosis as set forth in claim 6, wherein the inhibitor characteristically reduces the expression of an active caspase.

8. The inhibitor of epidermal cell apoptosis as set forth in claim 7, wherein the active caspase is one of caspase 3, caspase 8 and caspase 9.

9. An accelerant of keratinocyte proliferation containing retinoid as an effective ingredient.

10. The accelerant of keratinocyte proliferation as set forth in claim 9, wherein the accelerant characteristically increases the level of SCF (stem cell factor) .