DEPIGMENTATING AGENT IN THE TREATMENT OF MELASMA

TECHNICAL FIELD

This invention relates to a type depigmenting agent for the melanoderma of patients with melasma. This depigmenting agent, containing a combination of corticosteroid and lincosamide, exhibits remarkable depigmenting effect with less side effects.

BACKGROUND ART

Melasma is defined as a facial hypermelanosis of light to dark brown color, occurring commonly on sun-exposed areas that develops slowly and symmetrically. Although no sex or race is spared, melasma appears to be more common in female.

Epidermal hyperpigmentation is charachterized by an increase in the number of active melanocytes and also by increased melanin synthesis. The biosynthesis of melanin is initiated by catalytic oxidation of tyrosine to dihydroxylphenyialanine (DOPA), which is oxidized to dopaquinone and dopachrome. Tyrosinase is a rate-limiting enzyme in melanin biosysnthesis that catalizes the conversion of tyrosine to DOPA, DOPA to DOPAquinone.

The precise cause of melasma is unknown, however, multiple factors have been implicated in the etiopathogenesis of this condition. These included pregnancy, menopause, oral contraceptives, estrogen- progesteron therapies, thyroid dysfunction, cosmetics, malnutrition, mental stress, phototoxic and antiseizure drugs (diphenylhydantoin or mesantoin). But it has been reported that genetic factors and UV radiation are the most important factors.

Melasma can be a cosmetically disfiguring and psychologically

devasting disease. Despite numerous claims that depigmneting agents give effective results in up to 80 percent of users, most clinician continue to express dissatifaction with presently available bleaching and depigmenting agents. Up to now, it has been reported that phenol compounds such as hydroquinone, monobenzyl ether of hydroquinone (MBEH), 4- isopropylcatechol, paratertiarybutyl and amyl phenol, 4-hydroxyanisole have demonstrated potent depigmenting effects in the treatment of melasma and other disoders of skin pigmentation. However, these treatments are not completely satisfactory because high concentrations are required, which results in side effects such as local irritation and irreversibile depigmentation of surrounding normal skin.

Hydroquinone is the most commonly used depigmenting agent for the treatment of melasma. It can be used in combination with 0.1% tretinoin (retinoic acid) with or withtout a corticosteroid. Kligman and

Willis (Arch. Dermatol. 111 :40-48, 1981) have reported that enhanced efficacy of 5% hydroquinon, 0.1% tretinoin, and 0.1% dexamethasone in hyrophilic ointment for the treatment of melasma, ephelides, and postinflammatory hyperpigmentation. Local irritation and allergic contact dermatitis may develop in some patients after the daily and prolonged use of hydroquinon at high concentration (4% to 5%). Other adverse reactions are hypopigmentation, disorders of keratinization, postinflammatory hyperpigmentation and ochronosis. Not all the melasma lesions respond to hydroquinon treatment. Moreover, hydroquinon itself is unstable and may be decomposed.

Topical application of azelaic acid at concnetration of 20% has also been reported to be effective in the treatment of several disoders of hyperpigmwntation including melasma and lentigo maligna. However such formulation revealed adverse reactions including pruritus, mild and

transient erythema, scaling, and burning.

Furthermore, recently tretinoin alone or in combination with hydroxyanisole has been considered as a depigmenting agents in the therapy of of postinflammatory hyperpigmentation and mealsma. Butit it has also been encountered with severe cutaneous side effects such as erthema and/or peeling in the area of application.

Thus there is a need to develop a more stable, more effective, and less irritating depigmenting agent for better management of melasma and other abnormal melanoderma ofthe skin.

DISCLOSURE OF INVENTION

To comply with the shortcomings of conventional drugs, the inventor et al. have conducted intensive studies for the development of a novel formulation demonstrating remarkable depigmenting effects with less side effects.

The object of this invention is to provide a new agent for improved efficacy and reduced toxicity.

The depigmenting agent of this invention contains the combination of corticosteroid and lincosamide.

The corticosteroid contained in the depigmenting agent of this invention may include cortisol, cortisone, cortisterone, deoxycortisterone, aldosterone, prednisolone, triamcinolone, paramethasone, betamethasone, or dexamethasone. It is preferred to select betamethasone valerate or dexamethasone. The content ofthe corticosteroid is in the range of 0.01 to 10%, and preferably in the range of 0.04 to 1%.

The lincosamide contained in the depigmenting agent of this invention may also include lincomycin hydrochloride or clindamycin. The content of the linconsamide is in the range of 0.1 to 50%, and preferably in the range of 1 to 5%.

In the composition of this depigmenting agent of this invention, the molar concentration ratio between corticosteroid and lincosamide is in the range of 1 : 1 to 1 : 1000, preferably in the range of 1 : 10 to 1 : 100.

Besides the depigmenting agent of this invention may be used as a skin cream where hydrophilic ointment base is added to a combination of corticosteroid and lincosamide, other formulation may include lotion, solution, suspension, ointment, aerosol (spray), foam, paste, gel, or patch.

The depigmenting agent of this invention has some advantages in that a) it may inhibit the melanin synthesis with no effect on tyrosinase activity in melanoma cells, and b) at the same time, it may inhibit the proliferation of melanoma cells, thus demonstrating better depigmenting effects with less side effects.

While melanoma cells are treated with corticosteroid alone, the proliferation of the cells was inhibited, however, tyrosinase activity and melanin synthesis were increased. Whereas treatment with linosamide alone had no effect on tyrosinase activity and inhibited melanin synthesis although the proliferation ofthe cells are also inhibited. According to this invention, this depigmenting agent containing a combination of corticosteroid and lincosamide may have synergic or additional effects on melanogenesis, compared with a single-drug regimen.

Since the depigmenting agent of this invention has no effect on

tyrosinase activity in melanoma cells, it is beneficial since the rebound phenomenon of melanin overproduction is not expected to occur after discontinuation, and this is very important in maintaining homeostasis. In addition,therapeutic effects of topical corticosteroid may mediated immunological suppression of hypermelanogesis, and simultaneous treatment of lincosamide may counteract corticosteroid-induced melanogenesis. Thus the combination of these drugs may turn out to be ideal in the better depigmenting effects.

The depigmenting agent of this invention is a novel which has not been used as a depigmenting agent in the past, and any experimental findings of this agent have not been published yet.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 show the comparison of clinical improvement of a patient between before treatment (A) and after treatment (B), There is a marked improvement of mealsma after topical application of the skin cream(Example 2) for 6 weeks.

Fig. 2 show the comparison of clinical improvement of a patient between before treatment (A) and after treatment (B), There is a complete loss of melasma after topical application of the skin cream (Example 2) for 6 weeks.

Fig. 3 show the comparison of clinical improvement of a patient before treatment (A) and one year after drug discontinuation (B), There is a marked improvement and no recurrence after drug discontinuation.

BEST MODE FOR CARRYING OUT THE INVENTION

This invention is detailed in more detail by the following examples and experimental examples, but the claims are not limited to these examples and experimental examples.

Example 1

A skin cream of this invention was prepared by the combination of 0.05% dexamethasone and 2% lincomycin hydrochloride.

Example 2 A skin cream was prepared by mixing a skin-hydrophilic ointment base with 0.05% betamethasone valerate and 2% lincomycin hydrochloride and topically applied on the melanoderma of the melasma patients by a double blind method.

Experimental example 1: Inhibitory effects on the growth of melanoma cells

Melanoma cells were treated with dexamethasone, lincomycin hydrochloride, or dexamethasone/lincomycin hydrochloride (1 : 1) in various concentration ranges of 10 ^"9 M to 10 ^"4 to investigate the inhibitory effects of these drugs on the growth of melanoma cells. Also, so as to determine the optimum concentration ratio between dexamethasone and lincomycin hydrochloride, cells treated with fixed dose of 10 ^"6 M lincomycin hydrochloride in conjuntin with various dose of dexamethasone, in the range of 10 ^"9 M to lO^M.

B16 murine melanoma cells (Fiedler IJ, Nature, 242:148-149, 1973),were routinely cultured in a minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS). Cells were seeded in 24-well plates at 2 X IO ³ cells/well, treated with drugs on next day and cultured at 37 ^° C for 4 days in air containing 5% CO2. Every 2 days, the media was freshly changed. The growth rate of melanoma cells was

determined by MTT assay 4 days after drug treatment, and expressed as a percentage of untreated cells.

MTT assay was performed as follows: MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was dissolved in phosphate-buffered saline (PBS) and 50μi of this solution was added to each well of 24-well plate. After 4 hrs, 200μi dimethylsulfoxide (DMSO) and 50μ£ glycin buffer (pH 10.5) were added to the well and agitated for 10 min. Aliquots of 200μi from each well of agitated plates were transferred to 96-well plate and the absorbance was read at 540 nm using a Microwell System Reader 210 (Organon, Tekinika, Austria) and repeated it with 4 wells of each treated group three times. The data were statistically analyzed using Student's t test and ANOVA test. The results were demonstrated in the following table 1.

Table 1. Growth inhibition of melanoma cells treated with dexamethaxone, lincomycin hydrochloride, and dexamethaxone/lincomycin hydrochlide at various concentration

^* mean ± SE : percentage(%) of a control n=6

When melanoma cells were treated with lincomycin hydrochloride alone, the cells growth was inhibited in a dose dependent manner and the maximum growth inhibition (84%) was at 10 ^"8 M or 10 ^"7 M.

When melanoma cells were treated with dexamethasone alone, the cells growth was also inhibited in a dose dependent manner. The maximum growth inhibition was observed at 10 ^'8 M and 10 ^'7 M. A single regimen of dexamethasone showed more inhibitory effects than lincomycin hydrochloride alone.

When melanoma cells were treated with the equimolar combination

of dexamethasone and lincomycin hydrochloride, the cells growth was inhibited in a dose dependent manner and the maximum growth inhibition of 41% was observed at 10 ^"8 M or 10 ^"7 M. This combination demonstrated more inhibitory effects than a single treatment of dexamethasone or lincomycin hydrochloride.

When melanoma cells were treated with the combination of dexamethasone/ 10 ^"6 M lincomycin hydrochloride, the maximum growth inhibition of 55% was observed at 10 ^"8 M and 10 ^"7 M of dexamethasone concentration. The molar ratio of the two drugs (dexamethasone: lincomycin hydrochloride) for a maximum inhibitory effects of melanoma cells was between 1 : 10 to 1 : 100. Experimental example 2: Inhibitory effects of melanin synthesis in melanoma cells

Melanoma cells were treated with dexamethasone, lincomycin hydrochloride, or dexamethasone/lincomycin hydrochloride ( 1 : 1 ) in each concentration range of 10 ^"9 M to 10 ^'4 M to investigate the inhibitory effects of these drugs on the melanin synthesis in melanoma cells. Also, so as to determine the optimum concentration ratio between dexamethasone and lincomycin hydrochloride, the concentration of lincomycin hydrochloride was fixed to 10 ^'6 M and that of dexamethasone was in the range of 10 ^"9 M to 10 ^"4 M for this experiment. Hence, the melanin content was quantified according to the procedure described by Hosoi et al. (Cancer Res, 45; 1474-1478, 1985).

B16 murine melanoma cells were seeded in petri dish at 2 X I O ⁴ cells/ml. The cells were treated with above drugs and cultured for 4 days.

Melanoma cells were harvested and washed with PBS twice, agitated sufficiently by addition of 0.4 ml of PBS containing 1% Nonidet P-40 and centrifuged at 13,000 X g for 10 min. The pellet of cells was

solubilized at 80 ^° C for 2 hrs after addition of IN sodium hydroxide and 10% DMSO. The absorbance was read at 475 nm using a spectrophotometer. According to a standard curve using a synthetic melanin, melanin content was assayed. Protein estimation was performed as reported by Lowry et al. (J. Biol. Chem., 193: 265-271, 1951 ).

The melanin content was calculated and expressed in μg /mg protein.

Stastistical comparison group data was performed using Student's t test and ANOVA test. The test results appear in the following tables 2 and 3.

Table 2 : Inhibition of melanin synthesis in melanoma cells treated with dexamehtasone, lincomycin hydrochloride, or dexamethasone/lincomycin hydrochloride at various concentration

^* Mean +- SE : Melanin( μ g)/total protein(mg) n=6

Table 3: Inhibition of melanin synthesis in melanoma cells treated with dexamehtasone/10 ^'6 lincomycin hydrochloride.

^* Mean +- SE : Melanin( μ g)/total protein(mg)

When melanoma cells were treated with dexamethasone alone, melanin synthesis in melanoma cells was increased in a dose dependent manner.

However, when melanoma cells were treated with lincomycin hydrochloride alone, melanin synthesis in melanoma cells was inhibited in a dose dependent manner. The melanin synthesis in melanoma cells was most significantly inhibited at high concentrations.

When melanoma cells were treated with equimolar combination of dexamethasone and lincomycin hydrochloride (1 : 1 ), melanin synthesis in melanoma cells was inhibited in a dose response curve and melanin synthesis was most significantly inhibited at 10 ^'7 M or 1 O^M.

When melanoma cells were treated with a combination of dexamethasone/ 10 ^"6 M lincomycin hydrochloride, the maximum growth inhibition of melanin synthesis in melanoma cells was observed at less than 10 ^"7 M of dexamethasone concentration. The molar ratio of the two drugs (dexamethasone : lincomycin hydrochloride) for a maximum inhibitory effects of melanoma cells was less than 1 : 10.

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Experimental example 3: Effects on tyrosinase activity in melanoma cells

Melanoma cells were treated with dexamethasone, lincomycin hydrochloride,or dexamethasone/lincomycin hydrochloride (1 : 1) in various concentration ranges of 10 ^'8 M to I O ^"4 M to investigate the effects of these drugs on tyrosinase activity in melanoma cells. Also, so as to determine the optimum concentration ratio between dexamethasone and lincomycin hydrochloride, the eel treated with the fixed dose of 10 ^{" 6} M lincomycin hydrochloride in conjunction with various dose of dexamethasone in the range of 10 ^"9 M to 10 ^'4 M. Tyrosinase activity of melanoma cells was measured by a method of Bouchard et al. (J. Exp. Med., 169; 2029-2042, 1989).

B16 murine melanoma melanoma cells were seeded in 100 mm of petri dish at 2 X IO ⁴ cells/ml. The cells were treated with above drugs at indicated concentrations and cultured for 4 days.

Melanoma cells were washed with PBS twice and harvested after treatment with trypsin/EDTA. The pellet of the cells was washed with PBS twice and agitated sufficiently by addition of 0.4 ml of PBS containing 1% Nonidet P-40. The mixture was centrifuged at 13,000 X g for 10 mins to transfer the supernatant. A reacting solution of 20 μ Ci/ml [3H] L-tyrosine, 2% Nonidet P-40, 0.2 mM L-tyrosine and 2 mM L- DOPA was added to aliquots of 0.2 ml supernatant. The mixture was incubated at 37 ^° C for 1 hr and the reaction was terminated by addtion of 0.2 ml of charcoal solution ( 100 mg/ml in 0.1 M citric acid).

The reaction mixture was left on ice for 30 mins and centrifuged at

2,000 X g for 5 mins. After obtaining the supernatant, 2 ml of scintillation

fluid (Aquazol II) was added to aliquots of 0.2 ml supernatant and tyrosinase activity was repeatedly measured from 6 dishes, at least two times.

<Tyrosinase activity =

Free ³ H_0 release by test cell lysate x l00 >

³ H_0 release of control reaction mixutre

The data were statistically analyzed using Student's t-test and

ANOVA test. The results were demonstrated in the following tables 4 and

5.

Table 4: Tyrosine activity in melanoma cells treated with dexamethasone, lincomycin hydrochloride, or dexamethasone/lincomycin hydrochlorie at various concentration

^* Mean ± SE : Percentage(%) of control n=6

Table 5 : Tyrosinase activity in melanoma cells treated with dexamethasone/10 ⁶ M lincomycin hydrochloride.

^* Mean +- SE : Percentage (%) of control When melanoma cells were treated with dexamethasone alone, the tyrosinase activity was increased in a dose dependent manner. When melanoma cells were treated with lincomycin hydrochloride alone, the tyrosinase activity in the cells was slightly increased in a dose dependent manner.

When melanoma cells were treated with a equimolar combination of dexamethasone and lincomycin hydrochloride, or the combination of various dose of dexamethasone and 10 ^"6 M lincomycin hydrochloride, the tyrosinase activity in the cells was slightly increased in a dose dependent manner and there was no significant difference compared with a single regimen of lincomycin hydrochloride.

Thus, the combination treatment of dexamethasone and lincomycin hydrochloride did not significantly chnage tyrosinase activity.

Experimental example 4: Effects on the tyrosinase mRNA level in melanoma cells

Melanoma cells were treated with dexamethasone, lincomycin hydrochloride, or dexamethasone/lincomycin hydrochloride (1 : 1) in

various concentration ranges of 10 ^"8 M to 10 ^"4 to investigate the effects of these drugs on the tyrosinase mRNA level in melanoma cells by Nothern blot analysis.

According to a method of Kim et al. (Mol. Cell. Biol., 12;8, 3636-

3643, 1992), mRNA in melanoma cells were extracted from oligo (dT) cellulose column chromatography, fractionated on 1% formaldehyde gel via electrophoresis, and transferred to a Gene Screen Plus membrane. After UV cross-linking and prehydbridization in Church buffer, the blots were hybridized with a radiolabeled probe which were isolated from the clone MTY81 IC . Next day, the filters were washed twice at 65 ^° C in 2 X SSC/0.1% SDS for 20 min each, then washed twice at 65 ^° C in 2 X SSC/0.1% SDS each. The band intensity of the autoradiogram was measured using a personal densitometer SI (Molecular Dynamics, Sunnyvale, CA, USA). The tyrosinase mRNA transcript level was expressed as a percentage of untreated control.

The level of mRNA for beta-actin was determined in the same manner as described above. The results were demonstrated in the following table 6.

Table 6 : Tyrosinase mRNA transcript level in melanoma cells treated with dexamethasone, lincomycin hydrochloride or dexamethasone/lincomycin hydrochloride at various concentration

^* Mean SE : Percentage(%) of untreated control n=3

When melanoma cells were treated with dexamethasone alone, tyrosinase mRNA transcript level in melanoma cells was increased in a dose dependent manner. However, when melanoma cells were treated with lincomycin hydrochloride alone, there was no increase in tyrosinase mRNA transcript level in melanoma cells.

When melanoma cells were treated with a combination of dexamethasone and lincomycin hydrochloride, there was no increase in tyrosinase mRNA transcript level in melanoma cells. This means that the increase of tyrosinase mRNA transcript level induced by dexamethasone was obliterated by lincomycin hydrochloride.

Therefore, the results revealed that the combination of dexamethasone and lincomycin hydrochloride had no effects on tyrosinase mRNA transcript level,sequentially tyrosinase activity did not change.

From the above results, a combination of dexamethasone and lincomycin hydrochloride has several advantages in that a) it may inhibit the melanin biosynthesis wihtout change of tyrosinase activity in melanoma cells, and b) at the same time, it may inhibit the growth of melanoma cells. Thus this combination seems to be better than any other previous agent in depigmenting effects and side effects.

Experimental example 5: Double blind control trials using a skin cream containing dexamethasone and lincomycin hydrochloride.

Fourty four patients (41 feamle and 3 males) suffering from abnormal pigmentation of melasma were enrolled for this study, divided into two groups, a control group was topically applied with vehicle on the melanoderma and the other group was applied with a skin cream containing betamethasone valerate and lincomycin hydrochloride of Example 2.

Accordingly to the selection criteria of subjects, we excluded the ones who have a history of administration or have been administering with topical tretinoin, systemic steroid, hydroquinone, tathion, or vitamin C within six months. The underlying diseases causing melasma were assessed by physical examination and history taking. A clinical type of melasma by pattern was assigned to each patient as centrofacial, mandibular, or malar. Wood's light was used to determined melasma type as epiderma, dermal, or mixed. Mixed and indeterminant types were considered equivalent for purpose of this study.

the subjects were applied with a skin cream after washing face before sleep once daily. A total of 19 patients were dropped from the study because of not using the medication regularly or frquent abscence to follow-up visit.

The clinical effects were observed at every 2 weeks for 6 weeks for final assessment, grouped into the following categories as described by Jimbow K, et al. (Arch Dermatol, 127: 528-1534, 1991).

- N (no effect) no visible change of pigmentation

- G (good) a moderate improvement, the decrease of visible pigmentation, but still showing a clear visible border of abnormal pigmentation.

- F (fair) a marked improvement, a marked decrease of visible pigmentation ,but there is still some visible border between melasma lesion and normal- looking skin.

- E (excellent) a complete loss, no visible border between non-treated normal skin and treated normal skin.

Changes in clinical parameters of melasma between this study group and vehicle- treated groups were compared using a two-sample t test and X ² test.

The results were summarized in the following table 7.

Table 7 : Depigmenting effects of Example 2

Clinical improvement : N ; No effect

G ; Good F ; Fair E ; Excellent

^* P= 0.0001

In vehicle-control group, 10 patients from the enrolled 19 patients completed a six-week follow-up, 9 patients (90%) showed no clinical improvement of melasma except 1 patient (10%).

In study group applied with a skin cream containing betamethasone valerate and lincomycin hydrochloride, 15 patients from the enrolled 25 patients completed a six-week follw-up, 14 patients (94%) showed clinical improvement of melasma except 1 patient (7%). The clinical

details of 14 patients were as follows: 6 patients were "good", 7 patients were 'fair'(Fig.1 and Fig. 3) and 1 patient was "excellent'(Fig. 2).

To assess tolerability, the degree burning, itching, erythema, and peeling, capillary dilatation were assessed at each visit based on scale 0 through 4 (0, abscent; 1, mild; 2, moderately severe;3, severe;4)

The results were summarized in the following table 8.

Table 8 : Side effects of Example 2

^* P= 0.1

As shown in the above table 8, some side effects were observed but the severity was mild and there was no significant difference between two groups (p=0.1).

It is suggested that a skin cream containing betamethasone valerate and lincomycin hydrochloride must be safe and have remarkable depigmenting effects with less side effects.