WO2002060404A1

WO2002060404A1 - Composition of miconazole for external application and skin-whitening

Info

Publication number: WO2002060404A1
Application number: PCT/KR2001/000615
Authority: WO
Inventors: Wonhong Woo; Yeunja Mun; Jounghoon Kim
Original assignee: Wonhong Woo; Yeunja Mun; Jounghoon Kim
Priority date: 2001-01-31
Filing date: 2001-04-13
Publication date: 2002-08-08
Also published as: KR20010103057A

Abstract

The present invention relates to a externally using composition for skin-whitening, that contains miconazole. The said composition is stable, free from harmful side effects, and greatly effective on restraining forming melanin pigment and preventing pigmentary deposit of skin, as containing the said miconazole in 0.05 % to 5.00 % of the composition, wherein the said miconazole restrains activity of tyrosinase, main enzyme for forming melanin pigment, and restrains forming melanin pigment.

Description

COMPOSITION OF MICONAZOLE FOR EXTERNAL APPLICATION-AND

SKIN-WHITENING

TECHNICAL FIELD The present invention relates to an externally applied skin-whitening composition containing miconazole, and more particularly, to a composition having an excellent skin whitening effect by containing miconazole as an effective ingredient, wherein the miconazole inhibits tyrosinase activity and melanin production.

BACKGROUND ART

Human skin colors are dependent on content and distribution of melanin pigment in skin cells, according to genetic factors, but it is often found that melanin is accumulated, causing the darkening of skin color. The production of melanin is stimulated by environmental and physical factors such as ultraviolet rays, female hormones, cosmetics, pregnancy, fatigue, and stress. It is known that 60-70% of melanin production is caused by the exposure to ultraviolet rays, taking of female hormone supplements and pregnancy, indicating high possibility of appearance of increased melanin deposits in females. With this reason, pigment-related conditions such as freckles are commonly observed on faces of many women, raising interest in preventing formation of excessive melanin.

Melanin is synthesized in melanocytes, being initiated by tyrosinase enzyme activated by internal and external regulatory factors. Tyrosinase acts on tyrosine to generate dihydroxyphenylalanine (DOPA), and oxidizes DOPA to give dopaquinone. Dopaquinone undergoes a non-enzymatic and spontaneous redox process to yield melanin pigment. Melanin is packaged in organelles called melanosomes, which distributed to neighboring keratinocytes and translocate to epidermal surface through a keratinization process, where melanin pigment protects skin from UN radiation. Also, melanin pigment functions as a free radical scavenger which removes various radicals modifying biological macromolecules such as proteins, lipids, and nucleic acids. However, when melanin is excessively synthesized at some area, or normal physiological functions of the body are impaired owing to aging and skin lesion, synthesized melanin is accumulated, resulting in various pigmentary deposits such as freckles, black spots, and ephelides. Based on the known causes and mechanisms for the darkening of skin, with an aim of reducing melanin formation, there are commonly used depigmenting agents having inhibitory effects on tyrosinase activity or other reactions in the melanin synthesis process in cosmetics and pharmaceutical fields. Examples of the depigmenting agents include tocopherol, vitamin, ascorbic acid, kojic acid, and hydroquinone. However, the inhibitors of prior arts are not suitable for clinical or cosmetic application. That is, ascorbic acid is not useful as a melanin synthesis inhibitor because of its low inhibitory effect versus tyrosinase activity, and its instability. Kojic acid shows high inhibitory activity against tyrosinase, but it is very unstable because it causes a color change when mixed with pharmaceutical compositions and decreases in titer with the lapse of time. Additionally, kojic acid exhibits so high irritation of the skin as to modify melanin pigment or melanocytes.

Also, although hydroquinone shows an excellent inhibitory effect on melanin synthesis, its use is generally limited because of hypersensitivity of skin to it. In particular, the known depigmenting agents have serious problems that freckles remain in some areas even after treatment, harmful side effects occur, and a long time, generally at least 6 months, is required for expected results to be achieved.

Moreover, peeling or laser therapy, which are commonly used for removal of epidermal freckles of a depth of 0.1 mm, also can cause mechanical wounds and aggravate freckles.

Accordingly, as a result of many trials to find a skin whitening substance having no undesirable side effects and having visible depigmenting effects within a short time, the inventors of the present invention discovered that miconazole has an excellent inhibitory effect on tyrosinase activity and melanin formation and is also capable of being used as an effective ingredient of external compositions for skin whitening.

On the other hand, it was reported that miconazole functions as an antifungal agent. For example, Korean Pat. Publication No. 96-03321 discloses that econazole and miconazole, imidazole derivatives, are useful therapeutic agents for skin diseases induced by pathogenic fungi, Trichophyton and Candida species, where skin diseases including tinea ungium, tinea capitis, and tinea corporis result from infection of Trichophyton. Also, miconazole can be applied to a composition for treating and protecting skin against acute inflammation, as described in Korean

Pat. Publicatoin No. 89-00207. However, it is not yet disclosed that miconazole can be applied for inhibition of melanin synthesis, especially as a skin-whitening agent.

DISCLOSURE OF THE INVENTION Accordingly, it is an object of the present invention to provide a new use of miconazole as a skin-whitening agent, having inhibitory effects on melanin formation and on tyrosinase activity.

It is another object of the present invention to provide an externally applied skin-whitening composition containing miconazole as an effective ingredient with a skin whitening effect.

The above objects of the present invention were achieved with the following procedure: a optimal treatment concentration of miconazole was determined by analyzing viability of melanocyte cell line B16, and using the optimal concentration of miconazole, there were investigated effects of miconazole on tyrosinase activity, melanin formation and expression of tyrosinase, where hyperpigmentation was induced by increasing cAMP concetration by the treatment with α-melanocyte stimulating hormone (α-MSH) and forskolin, and miconazole was found to have a skin whitening effect and an inhibitory effect on melanin formation.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Fig. 1 shows cell viability of melanocyte cell line B16 incubated with miconazole at a concentration of 0.1-50.0 μg/ml;

Fig. 2 shows tyrosinase activity in melanocyte cell line B16 incubated with miconazole at a concentration of 0.1 - 10.0 μg/ml;

Fig. 3 shows tyrosinase activity in melanocyte cell line B 16 incubated with miconazole at a concentration of 0.1-10.0 μg/ml as well as hyperpigmentation produced by treatment with α-MSH;

Fig. 4 shows tyrosinase activity in melanocyte cell line B16 incubated with miconazole at a concentration of 0.1-10.0 μg/ml as well as hyperpigmentation produced by the treatment of forskolin;

Fig. 5 shows inhibitory effects of miconazole and hydroquinone on tyrosinase activity;

Fig. 6 shows synthesized melanin amount in melanocyte cell line B16 incubated with miconazole at a concentration of 0.1 - 10.0 μg/ml; and

Fig. 7 is a western blot showing protein expression level of tyrosinase in melanocyte cell line B16 incubated with miconazole at a concentration of 0.1-10.0 μg/ml.

BEST MODES FOR CARRYING OUT THE INVENTION Mouse melanocyte cell line B16, which synthesizes melanin, is used for investigating effects of miconazole on melanogenesis. Miconazole is prepared by being dissolved in dimethyl sulfoxide (DMSO), and its optimal treatment concentration is determined by investigating cell viability of B 16 melanoma cells incubated with miconazole at varying concentrations of 0.1 to 50.0 μg/ml using sulphorhodamine-B (SRB) assay.

According to the present invention, miconazole has an inhibitory effect on the activity of tyrosinase, which initiates melanin synthesis in melanocyte cells. Moreover, miconazole inhibits tyrosinase activity in B16 melanoma cells being stimulated by α-MSH (α-melanocyte stimulating hormone), which induces hyperpigmentation by increasing melanin synthesis through stimulation of associated signal transduction pathways by increasing the intracellular concentration of cAMP. Also, miconazole displays an inhibitory effect on tyrosinase activity in B16 melanoma cells where hyperpigmentation is induced by forskolin. Such an inhibitory effect of miconazole on tyrosinase activity is observed to be very strong in comparison with that of hydroquinone, a conventional depigmenting agent. In addition, the synthesized melanin amount and the expression level of tyrosinase are reduced in B16 melanoma cells that have been treated with miconazole.

According to the present invention, a new use of miconazole as a skin- whitening agent is provided on the basis of its strong inhibitory effects on tyrosinase activity and melanin formation. Thus, a composition containing miconazole can be applied to external uses including cosmetics and pharmaceutical compositions with a skin whitening effect. According to the present invention, provided is an externally applied skin- whitening composition containing miconazole as an effective ingredient, and the composition contains miconazole at an amount of 0.05-5.0 % by weight, and preferably, at an amount of 0.1-2.0%) by weight. If the composition of the present invention contains miconazole at less than 0.05%, skin-whitening effect may be poor. In contrast, if the amount of miconazole exceeds 5.0%, skin irritation may occur. Accordingly, it is more preferable that the composition of the present invention contains miconazole at an amount of 0.1-2.0%) by weight, and most preferably, at an amount of 2.0% by weight. The externally applied skin- whitening composition of the present invention can also contain other substances used in conventional cosmetics and medical products for treatment, such as other whitening agents, moisturizers, antioxidants, oils, UV absorbents, surfactants, thickeners, alcohols, powders, dyes, perfumes, hydrophilic materials, water, and nutrient creams. For external application, the skin- whitening composition of the present invention can also be formulated to various forms, such as cream, fluid cream, ointment, lotion, or pack, but the formulation is not limited to these.

The present invention will be explained in more detail with reference to the following examples in conjunction with the accompanying drawings. However, the following examples are provided only to illustrate the present invention, and the present invention is not limited to them.

REFERENCE EXAMPLE 1 : Preparation of miconazole

Miconazole (Sigma, USA) was dissolved in dimethyl sulfoxide (DMSO) and diluted in suitable concentrations for use in the below Examples. REFERENCE EXAMPLE 2 : Cultivation of mouse B 16 melanoma cells

Mouse melanoma cell line B16 was purchased from Korean Cell Line Bank. 1X10⁵ of B16 melanoma cells per culture flask were incubated in 10% fetal bovine serum (FBS)-containing DMEM medium for 24 hours at 37°C under 5% CO₂, and miconazole was then added at various concentrations of 0.1-10 μg/ml, and incubation was proceeded for 3 more days. The cultured cells were used in the following Examples to investigate effects of miconazole on sulphorhodamine- B production, tyrosinase activity, melanin production, protein expression level, etc.

REFERENCE EXAMPLE 3 : Sulphorhodamine-B (SRB) assay Sulphorhodamine-B (Sigma, USA) assay was performed to determine viable cell number when Blόmelanoma cells were exposed to miconazole. B16 melanoma cells was seeded to a 96- well plate at a density of lxlO⁴ cells per well and incubated for 24 hours, and after addition of miconazole in various amounts, further incubated for 48 hours. Each of test and control groups incubated respectively with or without minaconazole was composed of three wells. The cultivated cells were then fixed with a solution of 50% trichloroacetate (TCA) at 4°C for 1 hour, stained with a mixture of 0.4% sulphorhodamine-B (SRB) and 1% acetic acid, and after four washes with 1% acetic acid, dried. The dried cells were dissolved with protein stain in 10 mM Tris base to measure absorbance at 540 nm.

REFERENCE EXAMPLE 4 : Inhibitory effect on tyrosinase activity

An inhibitory effect of miconazole on tyrosinase activity was investigated as follows: B16 melanoma cells incubated with or without miconazole, which is a test or a control group, were harvested and lysed with 100 μl of lysis buffer (1% Triton X-100, 10 mM sodium phosphate, pH 7.0, 0.1 mM PMSF at 4°C with shaking once every 30 min, and then centrifuged. The obtained supernatant was used for the tyrosinase activity assay, in which 50 μl supernatant was supplemented with 100 μl of 100 mM sodium phosphate buffer (pH 7.0), and after incubation for 5 min at 30°C and then addition of 50 μl of 100 mM L-DOPA, change in optical density was measured at 405 nm over 1 hour. Inhibition level for tyrosinase activity by miconazole was calculated according to the below formula.

Inhibition efficiency versus tyrosinase activity (%) = 100-(absorbance of test sample / absorbance of control sample)χl00

EXAMPLE 1 : Effect of miconazole on viability of B16 melanoma cells Viability of B16 melanoma cells was investigated in order to determine a treatment concentration of miconazole.

The miconazole as prepared above was added to B16 melanoma cells at various concentrations of 0.1 to 50 μg/ml, and cell viability was measured using Sulphorhodamine-B (SRB) assay, which shows proliferation of cells by measuring cellular protein concentration. As a result, there were no differences in proliferation rates between melanoma cells treated with miconazole concentrations up to 20 μg/ml and a control group incubated without miconazole. In contrast, proliferation of B16 melanoma cells was inhibited at miconazole concentratons over 20 μg/ml, while proliferation of melanoma treated with miconazole at 50 μg/ml was reduced to 45% of that of a control group. The results are shown in

Fig. 1.

EXAMPLE 2 : Inhibitory effect of miconazole on tyrosinase activity

Step 1 : Inhibitory effect of miconazole on tyrosinase activity To analyze an effect of miconazole on melanin production, activity of tyrosinase, which is the most important enzyme in the melanin synthesis pathway, was investigated. As test groups, B16 melanoma cells were treated with miconazole of from 0.1 to 10 μg/ml, and a control group was incubated without miconazole. After incubation for 48 hours, tyrosinase activity was examined according to the procedure described in Reference Example 4. Test groups incubated with miconazole at 0.1, 1, 5, and 10 μg/ml showed reduced tyrosinase activities of 86, 75, 67, and 4.6 %, respectively, compared to the control group. Accordingly, tyrosinase activity was significantly inhibited in proportion to increase in miconazole concentration (refer to Fig. 2).

Step 2 : Inhibitory effect of miconazole on tyrosinase activity when hyperpigmentation is induced by α-MSH

To investigate an inhibitory effect of miconazole on hyperpigmentation in melanocytes stimulated by UV and other environmental factors, an effect of miconazole was observed when hyperpigmentation was induced by α-melanocyte stimulating hormone (α-MSH), which stimulates melanin synthesis by increasing intracellular cAMP concentration via its receptor and through associated signal transduction pathways.

B16 melanoma cell were treated with or without miconazole of from 0.1 to 10 μg/ml, and after 30 min, α-MSH was added to 10 nM and incubation was continued for 48 hours. The melanoma cells treated with only α-MSH showed the increased tyrosinase activity of about 2.3 times higher than a control group.

In contrast, in the melanoma cells treated with both α-MSH and miconazole at a concentration of 1, 5, or 10 μg/ml, tyrosinase activities were reduced to 64, 21, or 1.1 %, respectively, compared to the cells treated with only α-MSH. Accordingly, it was found that miconazole significantly inhibits tyrosinase activity upon hyperpigmentation being induced by α-MSH (refer to Fig. 3).

Step 3 : Inhibitory effect of miconazole on tyrosinase activity when hyperpigmentation is induced by forskolin The effect of miconazole on tyrosinase activity was investigated when B16 melanoma cells were stimulated with forskolin, which mimics the action of α- MSH by increasing intracellular cAMP concentration through activation of adenylate cyclase enzyme.

B16 melanoma cells were treated with or without miconazole of from 0.1 to 10 μg/ml, and after 30 min, forskolin was added to 20 nM and incubation was continued for 48 hours. The cells with only forskolin exhibited increased tyrosinae activity of about 6 times higher than a control group, whereas the cells treated with both forskolin and miconazole of 0.1, 1, 5, or 10 μg/ml displayed reduced tyrosinase activities of 88.6, 64.8, 15.9, or 1.5 %, respectively, in comparison with the cells treated with only forskolin. Accordingly, it was confirmed that miconazole significantly inhibited tyrosinase activity in cells exhibiting hyperpigmentation induced by forskolin (refer to Fig. 4).

Step 4 : Comparison of inhibitory effects of miconazole and hydroquinone on tyrosinase activity In cultivated B 16 melanoma cells using the same method as in Reference

Example 2, tyrosinase activities were analyzed accordingly to various concentrations of miconazole and hydroquinone, and the results are given in Fig. 5. As demonstrated in Fig. 5, miconazole strongly inhibited tyrosinase activity in the mouse melanoma cells. Especially, in comparison with hydroquinone, a known whitening agent, miconazole showed very strong inhibition of tyrosinase activity at the cellular level.

EXAMPLE 3 : Inhibitory effect of miconazole on melanin synthesis

A quantitative examination was made of the inhibitory effect of miconazole on melanin synthesis.

Melanoma B16 cells cultivated in Reference Example 2 were washed twice with PBS and harvested by centrifugation, and the obtained cell pellet was then resuspended in 1 ml of distilled water, sonicated, and centrifuged to give a pellet. With an aim to obtain acid-insoluble materials, the pellet was dissolved by incubation for 1 hour at 80°C with 300 μl of IN sodium hydroxide containing two pellet volume of DMSO. Optical density was then measured at 475 run, and melanin concentration was determined using a standard curve, prepared using commercially available synthetic melanin (Sigma, USA). An inhibitory effect of miconazole on melanin synthesis was calculated according to the below formula.

Inhibitory effect on melanin synthesis (%) = 100 - (Absorbance of each sample / Absorbance of control) x 100

Melanin amount in each group was analyzed after treatment with or without miconazole of from 0.1 to 10 μg/ml and incubating for 72 hours. The test groups treated with miconazole of 0.1, 1, 5, or 10 μg/ml displayed reduced melanin amounts of 93, 90, 77, or 51 %, respectively, compared to the control group. Accordingly, it was found that miconazole inhibits the synthesis of melanin pigment in melanocytes (refer to Fig. 6).

EXAMPLE 4 : Inhibitory effect of miconazole on expression of tyrosinase Expression level of tyrosinase was investigated by Western blotting using anti-tyrosinase antibody.

5 l0⁶ cells of test groups incubated with miconazole of 1, 5, and 10 μg/ml and a control group without miconazole were washed with PBS and lysed with cell lysis buffer for 30 min at 4°C, and then centrifuged for 30 min at 20,000χg. The obtained supernatant, which contains cellular proteins, was concentrated by using an Amicon system and protein concentration was analyzed with the use of

Bradford assay. For Western blotting, 50 μg of protein was electrophoresed in a

10 % SDS-polyacrylamide gel, and then transferred to a nitrocellulose (NC) membrane. The NC membrane was incubated with a blocking buffer (5% skim milk) for 2 hours at room temperature (RT), and then with anti-tyrosinase antibody

(Santacruz, USA) at 1 :500 dilution for 1 hour, and after washing three times, with anti-mouse horseradish peroxidase conjugate at 1 :1,000 dilution. Protein expression level of tyrosinase was detected using the ECL system, and the results are shown in Fig. 7.

It was found that test groups treated with miconazole showed lower protein expression levels of tyrosinase than the control group, and the expression level of tyrosinase was more significantly reduced at higher miconazole concentrations, indicating that miconazole significantly inhibits the expression of tyrosinase as well as enzyme activity of tyrosinase.

Upon consideration of the overall results of the above Examples, it is demonstrated that miconazole can prevent hyperpigmentation thanks to its excellent inhibitory effects on tyrosinase activity and melanin formation.

According to the present invention, the externally applied skin-whitening compositions containing miconazole as an effective ingredient were prepared as creams in Preparation Example 1 and Comparative Preparation Example 1, below, and also, a cream without miconazole was prepared as Comparative Preparation Example 1 described below, followed by tests for stability and inhibitory effect on hyperpigmentation.

PREPARATION EXAMPLE 1 : Preparation of creams containing miconazole

The externally applied skin-whitening composition containing miconazole was formulated as creams according to Table 1, below.

TABLE 1 Compositions for externally applied skin-whitening cream formulation

EXAMPLE 5 : Test for color stability of the externally applied skin- whitening composition

Color stability was tested for the externally applied skin-whitening composition. A cream of Preparation Example 1 was prepared according to Table 1 , and a composition for a cream of Comparative Preparation Example 1 was the same as that of Preparation Example 1 with the exception of sodium hydrogen sulfite. The obtained creams were then separately put into a transparent glass vessel, covered, and maintained at room temperature to observe changes in color, where the observation was carried out on the first day, and the second, the fourth and the sixth weeks. The results are shown in Table 2, below.

TABLE 2 Color stability of an externally applied skin whitening composition

As apparent in Table 2, even after storage for 6 weeks at room temperature, severe color changes were not observed in the cream containing miconazole.

EXAMPLE 6 : Inhibitory effect of an externally applied skin- whitening composition on pigment deposition

To both forearms of 20 healthy volunteers were applied aluminum foils having two rows of six holes of 7 mm diameter, and the foil areas were then irradiated with an UV ray using ORIEL solar simulator 1 ,000 W at a level of 60 m/cm² at a distance of 10 cm. Before UV irradiation, the portions to be irradiated were disinfected with a solution of 70 % ethanol. From 3 days before UN irradiation to three weeks after UN irradiation, to the lower parts of arm were individually applied the creams prepared in Preparation Example 1 and Comparative Preparation Example 2 in a straight row twice a day. Pigmentation level was observed with naked eyes and evaluated as no effect, some effect, and significant effect. The results are shown in Table 3, below.

TABLE 3 Inhibitory effect of an externally applied skin whitening composition on formation of pigment deposition

As shown in Table 3, the externally applied skin- whitening composition containing miconazole prepared in Preparation Example 1 displayed a whitening effect in 17 of 20 volunteers, especially an excellent whitening effect in comparison with the cream-type composition prepared in Comparative Preparation Example 2, which contains ascorbic acid as a known whitening agent, and exhibited no side effects to skin, demonstrating that minaconazole is an excellent whitening agent having stability while reducing hyperpigmentation, such as freckles and ephelides.

Based on the above Examples, following are various examples of formulations containing miconazole for the externally applied skin-whitening compositions. However, the following examples are provided only to illustrate the present invention, and the present invention is not limited to them.

PREPARATION EXAMPLE 2 : Ointment formulation of an externally applied skin-whitening composition TABLE 4 Composition of skin- whitening ointment

EXAMPLE 7 : Whitening effect of the skin- whitening ointment on freckles

To the portions having freckles of 30 volunteers with freckles, regardless of their sex, was applied the skin-whitening ointment prepared in Preparation Example 2 twice a day for 60 days, and state of the freckles was observed once every 10 days. The results are shown in Table 5.

TABLE 5 Effect of the skin- whitening ointment on lightening of freckles

As shown in the above Table 5, the usefulness represents a sum of values of very useful, useful and a little useful as a percentage, and it was found that the skin- whitening ointment of the present invention was effective for removal of freckles, confirming that the skin-whitening ointment has an excellent skin whitening effect. PREPARATION EXAMPLE 3 : Solution formulation of an externally applied skin-whitening composition

TABLE 6 Composition of skin- whitening solution

PREPARATION EXAMPLE 4 : Milky lotion formulation of an externally applied skin-whitening composition

TABLE 7 Composition of skin- whitening milk lotion

PREPARATION EXAMPLE 5 : Pack formulation of an externally applied skin- whitening composition

TABLE 8 Composition of skin- whitening pack

INDUSTRIAL APPLICABILITY

As described in the above Examples and Preparation Examples, a new use of miconazole as a skin-whitening agent, which is widely known as antifungal agent, was demonstrated by confirming its inhibitory effects on tyrosinase activity, melanin formation and expression of tyrosinase enzyme. Moreover, in cosmetic application, miconazole showed high stability, no side effects and rapid whitening effect. Accordingly, the externally applied skin-whitening composition containing miconazole of the present invention is very useful for cosmetic and pharmaceutical applications.

Claims

1. An externally applied skin-whitening composition comprising of miconazole as an effective ingredient at an amount of 0.05 to 5.0 % by weight.

2. The externally applied skin-whitening composition as set forth in claim 1, wherein the miconazole is comprised at an amount of 0.01 to 2.0 % by weight.

3. An externally applied skin- whitening agent, containing the composition of claim 1 as an effective ingredient.

4. The externally applied skin-whitening agent as set forth in claim 3, wherein the agent is in the formulation form of cream, ointment, solution, lotion, or pack.