Depigmentation of black guinea pig skin by topical application of cysteaminylphenol, cysteinylphenol, and related compounds

Phenol and catechol were combined with sulfur to develop new melanocytotoxic agents. Among these synthetic compounds, 4-S-cysteaminylphenol (4-S-CAP) and 4-S-cysteinylphenol (4-S-CP), which showed an in vivo antimelanoma effect, were evaluated for cytotoxicity to normal epidermal melanocytes using hydroquinone (HQ) as the control. Topical application of 4-S-CAP on the skin of black guinea pigs revealed a marked depigmentation of black skin. 4-S-Cysteinylphenol also showed some depigmenting potency. 2-S-Cysteinylhydroquinone, which was made by combining cystine with HQ, on the other hand, did not show any depigmenting effect. Depigmentation of black skin by 4-S-CAP appeared to derive from: a decrease in the number of functioning melanocytes; a decrease in the number of melanosomes synthesized within the melanocytes and transferred to keratinocytes; and destruction of the membranous organelles of the melanocytes. None of these degenerative changes was observed in the keratinocytes, indicating the selective effect of 4-S-CAP on melanocytes.     

Selective cytotoxicity of N-acetyl-4-S-cysteaminylphenol on follicular melanocytes of black mice

Previous in vivo studies have shown that 4-S-cysteaminylphenol (4-S-CAP) and N-acetyl-4-S-cysteaminylphenol (N-Ac-4-S-CAP) have antimelanoma effects and that N-Ac-4-S-CAP produced a 98% depigmentation of hair follicles of black mice. This study investigated the process of selective melanocytotoxicity by N-Ac-4-S-CAP through light and electron microscopy studies of hair follicles obtained from newborn black mice treated with N-Ac-4-S-CAP. Visible changes in follicular melanocytes were found 4 h after intraperitoneal (i.p.) administration. Clumps of melanin granules and areas of melanocytic nuclear condensation were seen in the hair follicles. On electron microscopy there was progressive destruction of melanocytes with swelling of membranous organelles, nuclear condensation, and vacuolation of the cytoplasm, culminating in completely necrotic cells. None of these changes were demonstrated in the surrounding keratinocytes. N-Ac-4-S-CAP appears to have specific, cytotoxic effects on melanocytes actively producing eumelanin. The drug may not affect precursor or dormant melanocytes which retain the ability to become active, melanin-producing cells.     

N-acetyl-4-S-cysteaminylphenol as a new type of depigmenting agent for the melanoderma of patients with melasma

BACKGROUND AND DESIGN.--Melasma is a difficult medical problem to treat. Hydroquinone is administered to many patients, but it is unstable and local irritation and dermatitis may develop after a prolonged use at a high concentration. This study introduces a new depigmenting agent, N-acetyl-4-S-cysteaminylphenol, for better management of melanoderma in patients with melasma. RESULTS.--Our study, based on a retrospective observation of 12 patients using 4% N-acetyl-4-S-cysteaminylphenol in oil-in-water emulsion, showed a complete loss (8%), a marked improvement (66%), or a moderate improvement (25%) of melasma lesions. Visible changes of melanoderma can be seen in 2 to 4 weeks after daily topical application. This depigmentation was associated with a decrease in the number of functioning melanocytes and in the number of melanosomes transferred to keratinocytes. N-acetyl-4-S-cysteaminylphenol is the tyrosinase substrate, and, on exposure to tyrosinase, it formed a melanin-like pigment. CONCLUSIONS.--A phenolic thioether, N-acetyl-4-S-cysteaminylphenol, is a new type of depigmenting agent for the better management of melasma. It is much more stable and less irritating to the skin than hydroquinone, and it is specific to melanin-synthesizing cells.     

Retinoic acid synergistically enhances the melanocytotoxic and depigmenting effects of monobenzylether of hydroquinone in black guinea pig skin

Monobenzylether of hydroquinone (MBEH) has long been utilized for the depigmentation therapy of patients with extensive vitiligo. In this approach, the normally pigmented areas surrounding vitiligo lesions are depigmented to achieve a uniform skin tone. One of the important disadvantages of MBEH therapy, however, is the resistance of a considerable number of vitiligo patients against the depigmenting effect of this agent. We have previously proposed that the glutathione-dependent cytoprotection of melanocytes can be impaired through the inhibition of the enzyme glutathione S-transferase by retinoic acid (RA). The combination of RA with melanocytotoxic agents could thus lead to increased susceptibility of melanocytes to such compounds. In this study we have shown, for the first time, that the melanocytotoxic and depigmenting effects of MBEH are synergistically enhanced when it is combined with RA. The treatment of black guinea pig skin with RA (0.025%) alone induced no significant changes in the number of epidermal melanocytes and no skin depigmentation. On the other hand, MBEH (10%) produced mild to moderate skin depigmentation and reduced the average number of melanocytes from 76 (+/-5)/field (magnification: x 40) in control sites, to 42 (+/-6)/field in the depigmented skin. The RA (0.025%)-MBEH (10%) combination, however, produced a complete degree of depigmentation in the majority of treated sites after 10 days of application and reduced the average number of melanocytes to only 6 (+/-6)/field. RA-MBEH combination serves as a very potent skin depigmenting formula and now awaits future assessments of its potential use for the treatment of extensive vitiligo.     

The treatment of hypermelanosis with 4-isopropylcatechol

Over the past 6 years sixty-eight patients have been treated with I% or 3% 4-isopropylcatechol (4-IPC). Fifty-four had melasma, the others a variety of disorders of pigmentation. Two-thirds of the patients were significantly improved. Twenty patients had skin irritation due to 4-IPC and four developed an allergic contact dermatitis. One patient developed confetti-like areas of depigmentation in the 4-IPC-treated areas. Light and electron microscopic studies showed that in the 4-IPC-treated areas there was a loss and damage of the melanocytes, but the keratinocytes and Langerhans cells were unaffected. Melanosomal complexes containing many melanosomes were frequently found in the keratinocytes of the 4-IPC-treated negro skin. 4-Isopropylcatechol is a potent depigmenting agent and is of use in the topical therapy of selected patients with hypermelanosis. However, like other substituted phenols and hydroquinone it is irritant to the skin and should be used with caution.     

Retinoid therapy of pigmentary disorders

Topical retinoids such as all-trans-retinoic acid (RA), 13-cis-retinoic acid (isotretinoin), retinol, retinaldehyde, tazarotene, and adapalene have been shown to improve dyspigmentation of photodamaged skin including mottling and actinic lentigines. RA monotherapy has also been demonstrated to improve melasma and postinflammatory hypermelanosis. Furthermore, RA in combination with hydroquinone or 4-hydroxyanisole, or azelaic acid increases the potency of depigmenting agents for the treatment of melasma, actinic lentigines, and postinflammatory hypermelanosis. The basic mechanisms underlying these effects are not completely identified. Topical retinoids stimulate the cell turn-over of epidermal keratinocytes and promote a decrease in melanosome transfer and a rapid loss of melanins via epidermopoiesis. Topical retinoids are also involved in the control of cell differentiation. Retinoid-induced changes in the stratum corneum and the permeability barrier may also facilitate the penetration of depigmenting agents in the epidermis and increase their bioavailability, leading to increased depigmentation. In addition, several in vitro studies demonstrate that cis and trans-retinoic acid inhibit UV-B stimulated melanogenesis in term of tyrosinase activity and melanin synthesis. It is likely that topical retinoids modulate epidermal melanin count via a direct action on melanocytes and epidermal keratinocytes.     

Synthesis of cysteinylphenol,cysteaminylphenol, and related compounds,and in vivo evaluation of antimelanoma effect

Summary Phenolic and catecholic compounds were synthesized, by combination with cysteine or cysteamine through thioether bond, and their antimelanoma and melanocytotoxic effects were evaluated. Among nine compounds tested, 4-S-cysteaminylphenol (CAP) resulted in an increase in the life span (% ILS) of melanoma-bearing mice and in the growth inhibition (% GI) of melanoma tissue. 4-S-Cysteinylphenol (CP) and its methyl ester form also showed some increase in % GI. The 2-S-isomers of CP and CAP and diphenolic derivatives of CP did not show any significant antimelanoma effect. In addition, the s.c. injection of 4-S-CAP and 4-S-CP, in particular 4-S-CAP, caused the depigmentation of black hair which was manifested by loss of functioning melanocytes, as seen under light microscopy. The 4-S-CAP appears to provide a basis for development of a new class of antimelanoma and melanocytotoxic agents that are more stable than catecholic compounds, which have been most widely utilized as a source of rational chemotherapy for malignant melanoma.     

Depigmentation therapies for normal skin in vitiligo universalis

If vitiligo involves most of the body, it might be easier to depigment the normal remaining skin rather than to attempt repigmentation. We reviewed the literature to date regarding available therapies for depigmenting the normal skin in vitiligo universalis. Our review revealed that the threshold regarding what percentage of body surface area qualifies as depigmentation is variable among practitioners. Monobenzyl ether of hydroquinone (MBEH) is the most widely used depigmenting agent and has few side‐effects. Tretinoin in combination with MBEH is able to speed depigmentation of the skin. Monomethylether of hydroquinone has also been used successfully for depigmentation. Eighty‐eight per cent phenol is also effective in depigmenting the skin but its application on large areas is toxic for liver and kidney. Different types of lasers are also available to destruct the melanocytes selectively, but this technique can be painful and expensive. Cryotherapy is a cheap depigmenting therapy but, because of scarring risk, it should only be used by experienced dermatologists. No trials have compared the efficacy of the above‐mentioned well‐established depigmentation agents/techniques. Certain drugs such as imatinib, imiquimod and diphencyprone, which are used to treat other diseases, caused depigmentation as a side‐effect. Some depigmentation agents used for branding cattle can also serve as topical depigmentation agents. In conclusion, comparative clinical trials are needed to compare the efficacy of various depigmentation agents/techniques. In particular, topical imatinib, imiquimod and diphencyprone may be considered as potential depigmenting agents, which require further investigation. This review revealed that MBEH is safe and effective depigmenting agent.     

Melanocytotoxicity and the mechanism of activation of gamma-L-glutaminyl-4-hydroxybenzene

gamma-L-Glutaminyl-4-hydroxybenzene is converted by the tyrosinase of the common mushroom, Agaricus bisporus, to the toxic, dormancy-inducing metabolite 2-hydroxy-4-imino-2,5-cyclohexadiene-1-one. Hydroxylation of gamma-L-glutaminyl-4-hydroxybenzene by mammalian tyrosinase was monitored by determining tritium water release from gamma-L-glutaminyl-[3,5-(3)H[4-hydroxybenzene and occurred at only 25% of the rate found with tyrosine. The dihydroxy product of the hydroxylation reaction, gamma-L-glutaminyl-3,4-dihydroxybenzene, was not oxidized by the mammalian enzyme. Therefore, oxidation of gamma-L-glutaminyl-4-hydroxybenzene to sulfhydryl-reactive quinones by mammalian tyrosinase is an unlikely explanation for the hair depigmentation and inhibition of melanocarcinoma growth observed following administration of this compound. Cleavage of gamma-L-glutaminyl-4-hydroxybenzene by gamma-glutamyl transpeptidase releasing p-aminophenol was demonstrated. p-Aminophenol was an active depigmenting and melanocytotoxic compound. N2-Methyl-gamma-L-glutaminyl-4-hydroxybenzene was synthesized, differing from gamma-L-glutaminyl-4-hydroxybenzene only by the presence of a methylated amide linkage. This chemical modification resulted in a compound resistant to cleavage by gamma-glutamyl transpeptidase and lacking in melanocytotoxic activity. gamma-Glutamyl transpeptidase cleavage is proposed as the route for transformation of gamma-L-glutaminyl-4-hydroxybenzene into an active inhibitor of melanocytes.     

Langerhans' cells in hair follicles of the depigmenting C57Bl/Ler-vit.vit mouse. A model for human vitiligo

The C57Bl/Ler-vit.vit mouse grows a black pelage after birth. During successive hair molts, the fur loses its pigmentation. By 6 months of age, most of the fur of the animal is white. The epidermis of the ears and tail also loses its pigmentation. Histologic studies confirm that in the epidermis and hair follicles there is an absence of pigment cells identifiable by various histochemical or electron microscopic techniques. This mouse may be an excellent model in which to study the role of Langerhans' cells and the immune response in the pathogenesis of vitiligo, a study not easily done in humans. From results of prior studies, we postulated that if Langerhans' cells were involved in the destruction of melanocytes, they would be abnormal (either more or less numerous) in number during the active phase of depigmentation and normal in number after depigmentation was complete. To determine whether the Langerhans cell (Ia+/adenosine triphosphatase dendritic epidermal cell) might be involved in destruction of pigment cells, we quantified the number of Ia+ and adenosine triphosphatase dendritic cells in the hair follicles in skin from the ear, abdomen, back, and tail from male C57Bl/Ler-vit.vit mice while the fur and skin were depigmenting and after depigmentation was almost completed. We found that Langerhans' cells were normal in number during depigmentation and were most numerous after depigmentation. Previous studies indicate that Langerhans' cells in these mice are functionally defective and respond poorly to some contact allergens. From these morphologic and functional data, we conclude that Langerhans' cells probably are uninvolved in causing depigmentation in these mice. We also observed that the epithelium of hair follicles has a significantly higher (up to 1600/mm2) population density of Langerhans' cells than interfollicular skin.     

Chronic actinic dermatitis with vitiligo-like depigmentation

This report describes two patients suffering from severe chronic actinic dermatitis. Unusual widespread vitiligo-like depigmentation occurred during the course of the disease. The progression of these lesions was triggered by the chronic actinic dermatitis. Loss of pigment and complete absence of tyrosinase positive melanocytes were found in depigmented skin of both cases. Immunohistological investigation of the inflammatory infiltrate in case 2 revealed a predominance of CD-8 positive cytotoxic/-suppressor lymphocytes. Analysing the adjacent pigmented epidermis of progressive depigmenting lesions a dense exocytosis of CD-8 T-cells was notable. This distribution suggests cytotoxic destruction of melanocytes as the cause for the vitiligo-like depigmentation.     

The Yucatan miniature swine as an in vivo model for screening skin depigmentation.

The usefulness of the Yucatan miniature pig as a screen for skin dipigmenting activity by topical application was evaluated with standard compounds. This is a naturally occurring breed of swine with light brown to dark brown skin that is relatively hairless. The skin morphology, including the pattern of pigment distribution, in this breed of swine closely resembles the human skin. Test compounds examined in this study included the three standard compounds with known clinical depigmenting activity, hydroquinone (HQ), 4-hydroxyanisole (4HA) and tert-butyl catechol (TBC), each at a 5% concentration. Test materials in 25 microliters of propylene glycol/ethanol (50:50) were applied topically twice daily, 7 days a week for 90 days to test sites on each side of the dorsal mid-line. Test sites were graded weekly for variation in pigmentation and local irritation. After 90 days of test material application, skin biopsies of the test sites were taken for histological evaluation. Topical application of HQ, 4HA and TBC promoted marked skin depigmentation which was substantiated by reductions of pigment and melanocytes observed on microscopic examination. While both HQ and TBC produced marked local irritation, 4HA was only mildly irritating. These results suggest that the Yucatan pig, could be a potentially useful model for screening compounds with skin depigmenting activity.     

The depigmenting effect of RALGA in C57BL/6 mice

BACKGROUND: It has been known for a long time that the topical use of retinoic acid (RA) produces mild depigmentation of human skin. However, RA has two major disadvantages for its utilisation as a topical depigmenting compound. First, RA can act as an irritant and can produce considerable erythema and exfoliation of skin. Second, RA has a relatively weak depigmenting ability compared to other known depigmenting chemicals. OBJECTIVE: In this study, we show that RALGA, a combination of the less irritant retinoid retinaldehyde (RAL; 0.1%) and glycolic acid (6.4%), has a higher skin-depigmenting potential than RA 0.05% in the tail skin of C57BL/6 mice. This effect was observed in reducing the number of functioning melanocytes and/or in inhibiting their ability to synthesise melanin. In addition, the visually recognisable depigmenting effect of RALGA was evident earlier than that of RA, i.e. only after 1 week of application. RALGA may therefore serve as a depigmenting product for the treatment of skin hyperpigmentary disorders. Postacne hyperpigmented lesions represent a very common pigmentary problem among acne patients. RALGA may thus act as an anti-acne product, due to the presence of RAL--an RA precursor--which could simultaneously remove the postacne hyperpigmented lesions in such patients.     

An alternative approach to depigmentation by soybean extracts via inhibition of the PAR-2 pathway

The protease-activated receptor 2, expressed on keratinocytes but not on melanocytes, has been ascribed functional importance in the regulation of pigmentation by phagocytosis of melanosomes. Inhibition of protease-activated receptor 2 activation by synthetic serine protease inhibitors requires keratinocyte-melanocyte contact and results in depigmentation of the dark skinned Yucatan swine, suggesting a new class of depigmenting mechanism and agents. We therefore examined natural agents that could exert their effect via the protease-activated receptor 2 pathway. Here we show that soymilk and the soybean-derived serine protease inhibitors soybean trypsin inhibitor and Bowman-Birk inhibitor inhibit protease-activated receptor 2 cleavage, affect cytoskeletal and cell surface organization, and reduce keratinocyte phagocytosis. The depigmenting activity of these agents and their capability to prevent ultraviolet-induced pigmentation are demonstrated both in vitro and in vivo. These results imply that inhibition of the protease-activated receptor 2 pathway by soymilk may be used as a natural alternative to skin lightening.     

Detection of environmental depigmenting substances

We systematically screened the depigmenting capacity of several phenols, catechols and organic antioxidants. Clear-cut depigmentation was achieved with monomethyl ether of hydroquinone (MMH) and tertiary butyl catechol (TBC) using black guinea pigs and black mice as animal models. A goal was to establish a reliable in vivo method to demonstrate or to predict the depigmenting action of chemicals on mammalian melanocytes. There was no universal solvent or optimal body site, although all tested areas could be depigmented. Irritation induced by some vehicles and test materials produced false positive responses. False negative responses with known depigmenting chemicals were observed. Utilizing these observations, we propose a model for screening medicinal and industrial chemicals for depigmenting capacity.     

Depigmentation therapy in vitiligo universalis with cryotherapy and 4‐hydroxyanisole

Vitiligo is a disease characterized by the loss of melanocytes, resulting in progressive depigmentation of skin, and areas of normally pigmented skin can be of cosmetic concern. Several options have been tried to remove the pigment and make the skin a more even colour. We present an easy and effective therapeutic procedure based on single‐session cryotherapy followed by topical 4‐hydroxyanisole (4‐HA).     

Current clinical use of depigmenting agents

A variety of topical depigmenting agents have been used clinically, with varying degrees of success. To date, the most effective topical treatment is a triple-combination agent containing hydroquinone (HQ), tretinoin, and fluocinolone acetonide. However, its use is associated with relatively high frequencies of adverse reactions, and therefore there is a necessity to produce effective topical depigmenting agents with fewer adverse effects. Several processes can be targeted for the treatment of hyperpigmentation; specifically, regulation of melanogenesis by inhibiting tyrosinase activity, a key enzyme in melanin synthesis, represents a major therapeutic target. Another option is regulation of melanosomes by manipulation of their formation or transfer. In addition, depigmenting agents can act through antioxidant or anti-inflammatory activities. We compared the tyrosinase inhibitory activity and cytotoxicity of HQ with those of other cosmetic ingredients. The results showed that although HQ was a strong tyrosinase inhibitor, it was cytotoxic at high concentrations. By contrast, 4-n-butylresorcinol effectively controlled tyrosinase activity without showing toxicity at high concentrations. These findings indicated that 4-n-butylresorcinol had the potential to act as an effective depigmenting agent, while producing less irritation than the currently available agents.     

Ebselen is a new skin depigmenting agent that inhibits melanin biosynthesis and melanosomal transfer

We assessed the ability of ebselen, a glutathione peroxidase mimic, to reduce pigmentation in various models. In murine B16 melanocytes, 25 μm ebselen inhibited melanogenesis and induced a depolymerisation of actin filaments. In co-cultures of B16 melanocytes with BDVII keratinocytes, a pretreatment of melanocytes with ebselen resulted in a strong inhibition of melanosome transfer to keratinocytes, as shown under optical and electron microscopy. In reconstructed epidermis, topical 0.5% ebselen led to a twofold decrease of melanin without affecting the density of active melanocytes. A similar result was obtained with topical 0.5% ebselen in black guinea pig ears. Ebselen induced a decrease of epidermal melanin parallel to a localisation of melanin and melanosomes in the basal layer. Ebselen appears as a new depigmenting compound that inhibits melanin synthesis and melanosome transfer to keratinocytes.     

A Simple Assay Method for Melanosome Transfer

Pigmentation is induced by production of melanin in specialized organelles termed melanosomes and by transfer of these organelles from melanocytes to surrounding keratinocytes. The chemical basis of melanogenesis is relatively well known but the mechanism of melanosome transfer is not well studied. Various pigmentary disorders and cosmetic applications require the use of depigmenting agents. Currently available topical agents used for the reduction of pigmentation mainly include tyrosinase inhibitors and/or melanocyte-cytotoxic agents. Recently, several agents have been introduced to inhibit melanosome transfer from melanocytes to keratinocytes. However, an experimental model for melanosome transfer is not well established. In this study, a simple assay method using flow cytometry is described.     

Depigmenting action of corticosteroids. Experimental study on guinea pigs.

The depigmenting action of corticosteroids was studied on the black skin of the guinea-pig by cutaneous applications followed by repeated histological sections. These experiments demonstrated the depigmenting action of triamcinolone acetonide and betamethasone 17-valerate. The results of this study show the influence of concentration and chemical formula of the corticosteroids on the process of depigmentation. The histological study did not reveal any particular morphological aspect of melanocytes capable of suggesting a mechanism of action by these chemical compounds at the cellular level.