SUMMARY INTRODUCTION. Walai-orn Pratchyapurit, MD

Transcription

1 Combined use of two formulations containing diacetyl boldine, TGF-ß1 biomimetic oligopeptide-68 with other hypopigmenting/exfoliating agents and sunscreen provides effective and convenient treatment for facial melasma. Walai-orn Pratchyapurit, MD Department of Medical Services, Institute of Dermatology, Rajathevi, Bangkok, Thailand SUMMARY Background: Treatment of melasma remains a challenge and involves multistep approach. Diacetyl boldine (DAB) stabilizes tyrosinase in its inactive form while TGF-ß1 biomimetic oligopeptide-68 inhibits tyrosinase activity. Aims: To study the efficacy and safety of the combination use of two formulae containing two principal ingredients: DAB and TGF-ß1 biomimetic oligopeptide-68 on facial melasma. Methods: A 12-week comparative study of pre-/posttreatment was conducted in 40 females. Melasma was evaluated by manual MASI score and MASI score with instrumentally graded darkness at baseline, 6 th week, and 12 th week. The author also assessed the safety profile of the two formulae throughout the study. Results: Thirty-eight subjects have completed the study. Their melasma showed improvement at the 6th week and 12th week as compared with baseline (P < 0.05). ne developed severe reaction. Most subjects had temporary, mild skin reaction. About 2.6% of subjects graded themselves markedly improved, 76.3% moderately improved, and 21.1% slightly improved. Conclusion: Combination of DAB serum at night and DAB/TGF-ß1 biomimetic oligopeptide-68/sunscreen cream in the morning and at noon was effective and safe for facial melasma. Key words: melasma, diacetyl boldine, TGF-beta-1 biomimetic oligopeptide-68, MASI score. INTRODUCTION Treatment of melasma remains a challenge, as many therapeutic options often show unsatisfactory results and further complications and relapse are common. In recent years, the mechanism of hyperpigmentation has been increasingly understood. We know that catecholamines, melanocyte membrane receptors and calcium are prerequisite for melanocyte signal transduction 1 in which a complex of ligand/melanocyte surface receptor is initiated in response to extracellular signals, including intrinsic factors such as hormones and extrinsic factors such as UV radiation. 2 After that occurs the multistep biochemical reaction of melanogenesis. The number of α-adrenergic receptors expressing on a melanocyte is related to both keratinocyte-derived catecholamines and intracellular Ca 2+ influx. 3 The phototransduction results in an increase of microphthalmiaassociated transcription factor (MITF). MITF regulates the transcription of the major melanogenic proteins, tyrosinase, tyrosinase-related protein 1 and 2, and protein kinase C-ß (PKC-ß). 4 The transient receptor potential melastatin-1 acting as an active calcium channel in melanocytes is also regulated by MITF. 5 A number of biological compounds, including TGF-ß1, are able to inhibit the function of tyrosinase and related enzymes mainly through down-regulation of MITF. 6,7 When TGF-ß receptor is activated, there is a decrease of melanin synthesis. So, MITF is one of the crucial factors in melanogenesis that can be targeted to induce hypopigmentation in melasma. Protein kinase C-ß expression appears to be restricted to melanocyte, and this explains its well defined role in the biology of melanocyte. 8 PKC-ß needs Ca 2+ to phosphorylate inactive tyrosinase into its active form. 1 In case of MITF expression, one of the intracellular signal transduction pathways is mediated by stimulating PKC-ß via membrane-generated diacylglycerol (DAG) and Ca Thus, interfering in Ca 2+ influx induces inhibition of PKC-ß activity and MITF phosphorylation. In the past, the therapeutic effect of hypopigmenting agents was mainly restricted to inhibitory or cytotoxic effects. Hydroquinone (HQ) has been banned in many countries because of its cytotoxic and mutagenic effects. These concerns have driven research to develop alternative skin-lightening agents with comparable efficacy to HQ but a better safety profile. 9 Advances in the knowledge of melanocyte biology and melanin synthesis have led to new pharmacologic approaches for melasma treatment. Accordingly, a huge number of hypopigmenting agents have been developed and were identified as successfully reducing melanin 1

2 pigment. For instance, the TGF-ß1 biomimetic oligopeptide-68 (ß-White TM ) is able to reduce melanocytes dendriticity and melanin production with higher activity than vitamin C and arbutin in an in vitro test. 10 Likewise, diacetyl boldine (DAB) inhibits tyrosinase activation by exerting dual effect as an α-adrenergic antagonist and interfering with Ca 2+ influx. 1 DAB also displays antioxidant activity. 11 Its activity on tyrosinase is dose-dependent, and more active than kojic acid s one. 1 A clinical study in a small group of Caucasians and Asians has demonstrated the improvement of solar lentigines and facial melasma after treatment with topical 4% DAB and 5% ß-White. 10 In sum, TGF-ß1 biomimetic oligopeptide-68 diminishes the tyrosinase production and DAB deactivates the remaining tyrosinase. The purpose of this study is to evaluate the efficacy and tolerance of the combined use of DAB serum and DAB/TGF-ß1 biomimetic oligopeptide-68 /sunscreen cream on facial melasma. MATERIALS AND METHODS OBJECTIVES This 12-week, comparative pre-/posttreatment clinical trial was conducted in 40 subjects with mild-to-severe facial melasma between vember 2012 May Ethical pattern This study was approved by the IRB and IE Committee of the Institute of Dermatology, Bangkok, Thailand and conducted accordingly to the ethical standards of the Declaration of Helsinki, principles of Good Clinical Practice (GCP/ICH), and local regulations. The patients were fully informed about the purpose of the study. After providing written consent, they were screened with the below inclusion and exclusion criteria. SELECTION CRITERIA FOR THE STUDY POPULATION Inclusion criteria Subjects with facial melasma at least on both cheeks aged between 25 and 65 willing to adhere to all the protocol requirements. Exclusion criteria Subjects who had received bleaching, keratolytic or peeling agents, like topical steroid, vitamin A derivative, α- or ß-hydroxy acids, laser or intense pulsed light within 2 months preceding the screening visit; those with side effects from previous treatments and those with other facial conditions that would distort the results and concept of this study were excluded. Patients allergic to parabens, sodium metabisulfite/sulfite, and to constituents included in the primary test products, that is, glycolic acid, arbutin, ascorbic acid, salicylic acid, licorice, extracts from boldo tree, and TGF-ß1 biomimetic oligopeptide-68 were not recruited in the study. Those with the following conditions were also excluded: pregnant/ lactating female subjects, those having active medical problems/ infection/skin cancer on the face, subjects under oral isotretinoin within 6 months prior to screening visit, or currently using any antiaging product. EVALUATION Test products Two hypopigmenting formulae were tested in this study: Neotone and Neotone Radiance (ISISPHARMA, Lyon, France). The former combines 4% DAB with five other active ingredients, namely licorice extract, ascorbic acid, glycolic acid, salicylic acid, and alpha-arbutin, which respectively prevent melanin and free radical production, absorb UV radiation, exert anti-inflammatory action and accelerate desquamation. The latter incorporates 4% DAB and 0.05% TGF-ß1 biomimetic oligopeptide-68 (as 5% ß-White TM ) together with ascorbic 2 acid and broad spectrum UVA and UVB filters with SPF50+. The former formula is mentioned as DAB serum and the latter formula as DAB/TGF-ß1/sunscreen cream. Study protocol The subjects were interviewed, submitted to physical examination, photographs were taken, and instrumental measures performed on screening day (D0), at the 6 th week and the end of the study (12 th week). The following data were recorded: skin phototype, history regarding age of onset and duration of melasma, outdoor activity, hours of sun exposure, and precipitating factors of melasma such as pregnancy or oral contraceptive. The subjects also filled a subject s static global assessment for melasma and expressed their satisfaction toward the test products. Application of test products The applications of the two products took place at night, in the morning and at noon: The DAB serum, containing exfoliating agents, on a clean skin at bedtime in order to reduce the risk of irritation if exposed to sunlight. The product was gently applied on each affected area, and then spread evenly all over the face; in total 0.6 to 0.8 g was needed for the entire face. After DAB serum dried up, the subjects applied a moisturizer (Sensylia 24 h, ISISPHARMA). The DAB/TGF-ß1/ sunscreen cream was applied twice daily in the morning and at noon in the same manner as for a sunscreen. The subjects were not allowed to use any other commercial sunscreen or moisturizer. They could carry on daily activities as usual, but strictly avoiding excessive sunlight. Safety monitoring Adverse events were monitored throughout the study. The patients could contact the investigator around the clock. Serious adverse events (if any) had to be reported to the Thai FDA. Subject s satisfaction On screening visit, the subjects completed a questionnaire regarding how they felt about their melasma with regard to its darkness and severity as follows: 1 = no evidence of hyperpigmentation, 2 = only minimal evidence of hyperpigmentation, and 3 = significant hyperpigmentation. After treatment at the 6 th week and 12 th week, they graded the melasma as follows: 0 = clear of pigmentation, 1 = markedly improved (75%), 2 = moderately improved (50%), 3 = slightly improved (25%), 4 = no improvement, and 5 = worse. They also rated product efficacy, expressed their satisfaction, and informed about adverse reaction. Efficacy assessments Instrumental measurement A narrowband spectrophotometer (Mexameter MX16, Courage + Khazaka Electronic GMBH, Köln, Germany) was used for quantifying changes in melanin and erythema levels. A decrease in Mexameter readings indicated improvement of melasma lesion. On the clean face, a technician performed three successive measurements of melanin index (MI) on each designated area on the face, and the mean value was used for statistical analysis. Clinical measurement Melasma was categorized by a Wood light (366 nm) into epidermal, dermal, and mixed type at screening visit and documented under the same condition at baseline and at each visit by Visioface1000D Camera. In the process of photograph taking, three photographs at fixed front, right, and left lateral views were taken. Afterward, all photographs were analysed in flashlight mode and UV mode to enhance contrast and saved as jpeg files in their original resolution, then sent to MASI scoring raters. MASI scoring The efficacy of the combination treatment was evaluated by three experienced dermatologists with the manual MASI scoring 12 and by the MASI scoring with instrumentally graded darkness. Manual MASI scoring Further to darkness (D), homogeneity (H) and area of involvement (A) of the melasma were clinically rated according to the reference set of clinical photographs, while the H and A parameters were reassessed

3 Table 1. The MASI grading scale showed matching pairs of the manual (original) darkness scale and instrumentally graded darkness scale Grade Darkness (D) 0 Absent 1 Slight 2 Mild 3 Marked 4 Severe Darkness (D) by Mexameter (a.u.) rmal skin: Minimal pigment visibly detected by UV light: 0.5 > 100 to 150 Slight: 1 > > Mild: 2 > > Marked: 3 > > Maximum: 4 > 450 through highest value Homogeneity (H) rmal skin Area (A) of involvement Slight: specks of involvement <10% Mild: small patchy areas of involvement 1.5 cm diameter Marked: patches of involvement > 1.5 cm diameter Maximum: uniform skin involvement without any clear areas 10 29% 30 49% 50 69% % % and confirmed by digital photographs. An average value for D, H and A parameters from three raters was used to obtain the MASI score: 0.3 (D F + H F )A F (D MR + H MR )A MR (D ML + H ML )A ML (D C + H C )A C giving a maximum of 48 and minimum of F: forehead; MR: right malar region; ML: left malar region; C: chin. MASI score with instrumentally graded darkness In fact, an individual s constitutive skin color and extrapigmentation from hypermelanogenic process collectively contribute to ultimate visualized color of melasma. The MI of normal skin in people with dark skin tone may be higher than that of melasma in people with light skin tone. It is the difference in color between melasma and surrounding normal skin or in the other word a relative MI value, that reflects the genuine severity of melasma, not the darkness of melasma (or absolute MI value) alone. The MI numeric values collected from the subjects were converted into MI darkness score and then calculated to obtain sum MASI score in the same manner as the manual MASI score. The clinical scale of darkness from the manual MASI score matched with new instrumental scale of darkness to obtain the socalled MASI score with instrumentally graded darkness. Statistical analysis Statistical analysis has been carried out using Microsoft Excel 2007, and Megastat free software (Informer Technologies Inc., CA, USA), Epi Info TM version 7. All statistics were applied at 5% level of significance with two-sided test. multiple corrections were performed to adjust the level of significance for efficacy analysis. RESULTS Demographic characteristics All enrolled subjects (n = 40) and their baseline characteristics are shown in Table 2; 38 of 40 subjects (95%) completed the trial, assessed the clinical response, and reported compliance (Table 3). Most subjects were middle-aged and 97.4% aged 30 years or over. All of them had only facial melasma which mostly affected the malar region. Twenty-eight subjects (70%) had at least one-time experience of pregnancy in the past (time back to last pregnancy 1 36 years, mean 15.4 ± 9.7 years) and 10 subjects (26.3%) were menopaused (menopause duration 1 15 years, mean 5.4 ± 4.8 years). The subjects who denied family history of melasma (22/40, 55%) slightly outnumbered the subjects who reported melasma (18/40, 45%) in mother and sister (18/18), and father and brother (2/18). Nineteen subjects (47.5%) underwent medical and/or surgical treatments for melasma before the trial and were treated with HQ, corticosteroids, and tretinoin at different occasion or together at a time. At baseline, some subjects reported symptoms of sensitive-prone skin in the past two months (see details in Table 2). Only 8 of 40 (20%) subjects had a previous history of asthma, hypertension, dyslipidemia, or chronic eczema. Melasma severity was reported as minimal (score 2) in 7 of 40 (17.5%) and significant (score 3) in 33 of 40 (82.5%) subjects. 3 Table 2. Subjects and baseline characteristics (n = 40) Patients characteristics N or Mean (± SD) Range, % Age (year, mean ± SD) 44.9 (± 9.3) 27 to 63 Work type* Indoor Outdoor Table 3. Comparison of study parameters at baseline, the 6 th week and 12 th week (n = 38) An asterisk (*) depicted significant difference in mean value between baseline and 12th week (P < 0.001) % 13.2% Duration of melasma (year) 7.0 (± 5.0) 1 20 Skin type Type 3 Type % 60.0% Daily sun exposure (minutes, hours) 2.4 h (± 2.0) <30 min to 10 h Melasma type Epidermal Mixed (epidermal and dermal type) Family history of melasma History of oral pill use History of previous treatment History of bleaching agents use Previous experience of skin sensitivity Dryness Pruritus Burning & stinging sensation Scaling Erythema Urticaria Parameters MASI score By manual By instrumentally graded darkness Baseline (mean ± SD) 19.4 ± ± th week (mean ± SD) 16.2 ± ± % 62.5% 45.0% 55% 30.0% 70.0% 47.5% 52.5% 47.5% 52.5% 32.5% 20% 10% 7.5% 2.5% 2.5% 12 th week (mean ± SD) 10.2 ± 6.5* 12.3 ± 6.9* Melanin pigment (a.u.): Facial melasma ± ± ± 61.9*

4 Efficacy results evaluated by subject s satisfaction Two cases left the study (5% dropout rate) because they moved to new domicile far away from the research base. Thirty-eight subjects have completed the combination regimen and filled a subject s static global assessment form. The lightening effect of the combination use of DAB serum and DAB/TGF-ß1/ sunscreen cream was clinically noticed by the subjects as early as the 2 nd week of treatment. The skin tone was improved in most cases and melasma lesion gradually faded over time (Fig. 1). Only one subject complained that her right cheek became darker at the 6 th week but turned better at the 12 th week, and her complaint was confirmed true by the spectrophotometer. At the end of study, 1 of 38 subject (2.6%) graded her face as markedly improved, 29 of 38 subjects (76.3%) moderately improved, and 8 of 38 subjects (21.1%) slightly improved. In one case a patch of melasma completely cleared at the 6 th week but reappeared afterward because of unprotected sun exposure. Twenty-three subjects (60.5%) were pleased with the combination regimen. As regards patient satisfaction, the overall satisfaction was significantly in favour of DAB/TGF-ß1/sunscreen cream over DAB serum because the former promptly made the face look lighter. Five subjects (13.2%) with dark skin tone were pleased with the result of treatment, but they did not like the all-white look after applying the DAB/TGF-ß1/sunscreen cream. Figure 1. Comparison of melasma at baseline and at 12 th week. Baseline Efficacy results evaluated by MASI score The average score for D parameter obviously changed, but H and A parameters showed slight change along the course of treatment. There was low correlation (r 2 = 0.36, P 0.01) between average score of D parameter by raters and by instrumental rating. teworthy, the mean value of MASI score with both MASI scoring systems showed similar trend of significant improvement of melasma at P value < (Fig. 2a). The manual MASI score dropped 16.5% at 6 th week, additional 37.7% at 12 th week, and in total 47.4% when compared the baseline with 12-week score. While the MASI score with instrumentally graded darkness dropped 18.7% at 6 th week, additional 29.3% at 12 th week, and in total 42.5% when compared the baseline to the 12-week score. In brief, the percentage of melasma improvement rated by manual MASI score was higher than by MASI score with instrumentally graded darkness. The author found that this combined clinic/instrumental grading of darkness was very useful for melasma rating, especially when clinical feature of melasma fell between two clinical grades and was difficult to judge by bare eye. Efficacy results evaluated by spectrophotometer As shown in Figure 2b, the combination treatment on the face significantly induced pigment reduction steadily down toward the 12 th week of treatment (P value<0.05). ticeably, the combination use could exert its action even in cases with moderate to severe facial melasma who failed or developed side effects from other previous bleaching agents. Figure 2. (a) The improvement of facial melasma evaluated by manual MASI score and MASI score with instrumentally graded darkness, at baseline, 6 th week, 12 th week (n = 38). The asterisk depicted significant difference in mean value of MASI score between baseline and 12th week (P < 0.001). (b) Melanin index on the face at baseline and after treatment with DAB serum and DAB/TGF-b oligopeptide-68/sunscreen cream at 6 th week, 12 th week (n = 38). The asterisk depicted significant difference in mean value of Melanin index between baseline and 12 th week (P < 0.05). (a) 25 Manual MASI score MASI score with instrumentally-graded darkness 12 th week MASI score * 12.3 * 10.2 P< Baseline 6 th week 12 th week (b) 350 Combination treatment (face) Melanin index (a.u.) * Baseline 6 th week 12 th week 4

5 Safety profile patient dropped out of the study because of treatment-related adverse events. 8 (21.1%) from 40 subjects reported adverse events from the combination use; that is, erythema (1 40, 2.5%), pruritus (8 40, 20.0%), skin exfoliation (3 40, 7.5%), and dryness (1 40 subjects, 2.5%). The side effects were acceptable by all subjects as they were very mild in intensity and disappeared by the first month. There was no case of telangiectasia or skin atrophy reported after the 12-week treatment. Luckily, the author had a chance to follow-up several subjects who continued using the combination treatment after this project had been closed, so far after 6 months all subjects showed no skin side effects which usually occurred on long-termed use of HQ or triple combination of HQ, steroid, and tretinoin. Discussion The harmonious combination scale of clinic/instrumentally graded darkness used in this study more or less reduced human prejudice and brought forth a better level of clinical measurement of MASI scoring. In case of clinical dilemma or inflamed melasma, a researcher s rating can be confident on the MI grading scale. Pandya et al. 13 found that the H parameter of the MASI scoring was the most difficult component to reliably assess and removal of this parameter did not alter reliability or validity measures of the MASI score. This study found that the H and A parameters showed little impact on a comparative result of time related MASI score. Perhaps, just a validated clinic/instrumental grading of melasma darkness which specifically developed for three major groups of ethnic skin type (i.e., African, Asian and Hispanic, Caucasian) was sufficient and practical for assessing and following-up melasma severity. Most subjects responded well to the combination treatment, and only one exceptional case had darkening of melasma. Besides the fact that inflammation from treatment may induce postinflammatory hyperpigmentation, it was presumed that abnormal melanin might partake in variable success rate. 14 However, the exact mechanism of this phenomenon remains unclear and needs further investigation in the future. It is no doubt that licorice extract, ascorbic acid, glycolic acid, salicylic acid, alpha-arbutin, which are incorporated in the DAB serum, and that sunscreen as a part of the combined formula of DAB/TGF-ß1/sunscreen cream also take part in the end result of skin colour. In general, when compared to 4% HQ cream, these cosmetic ingredients at a safe concentration provide weak lightening effect. This study showed that together with DAB and TGF-ß1 biomimetic oligopeptide-68, they can induce more pigment reduction than exerting their action alone. Once people stop treatment for melasma, they will have melasma back. Thus, there is high tendency for them to keep seeking for treatments, as we know that the adverse reaction of hypopigmenting agents is dose and duration related. Most of patients went through those treatments repeatedly or persistently for months leading to a number of adverse effects including an irreversible consequence. Fortunately, the adverse reaction from this combination treatment was very mild and transient. It seemed that one patient could continue the treatment for several months without serious side effect, and the clinical benefits could be assumed to be more noticeable over time. Interestingly, the cases allergic to HQ in this study could bear DAB serum and DAB/TGF-ß1/sunscreen cream very well. So, based on this result, the combination regimen could be recommended as an alternative for those who are intolerant to HQ, retinoids, and steroids. Besides effective treatment, elimination of possible causative factors, the successful treatment of melasma requires highly reliable sun protection as well as sufficient duration of treatment time. Photoprotective measures like avoidance of sun exposure and regular use of a broad spectrum UVA and UVB sunscreens (SPF 30) are always advised. This study revealed that the subjects applied much lower amount of sunscreen than the recommended dose. Therefore, every single melasma patient needs more education on sunscreen use such as sunscreen is just a complement to shade, clothing, and broad-brimmed hats, 15 and wearing sunscreen does not mean a person can spend longer periods of time in the sun. Patients must learn how to properly apply a sunscreen, and how to achieve adequate dose and uniform distribution of sunscreen (see details in ref.16). So, a very high SPF value may not be as important as sufficient amount of sunscreen, frequency, and right moment of sunscreen application. 15 Certainly, DAB serum and DAB/TGF-ß1 /sunscreen cream are formulated for normalizing skin colour. Meanwhile, DAB/TGF-ß1/sunscreen cream can be used as a sunscreen on a routine schedule (in the morning and at noon), but an extra sunscreen is recommended at any moment of the day when necessary. If so, we can assume the better and quicker improvement of melasma. CONCLUSION The combination use of DAB serum at night and DAB/TGF-ß1 biomimetic oligopeptide-68/sunscreen cream in the morning and at noon generated statistical improvement of melasma with MASI evaluation and spectrophotometer measurement. This combination worked well on new cases with all staged severity of melasma and even those who failed from previous treatments. Their combination provided a better choice in term of pigment reduction, particularly for patients who are allergic to hydroquinone. As we learned from this study, they caused no serious side effects. The incorporation of hypopigmenting agents with sunscreen simplifies the daily facial care for melasma patients while obtaining lightening effect and routine photoprotection at a time. 5

6 BIBLIOGRAPHICAL REFERENCES 1. Mas-Chamberlin C, Peschard O, Leroux R et al. Di-acetyl-nor-aporphines: novel molecules and novel mechanism to inhibit melanogenesis. S OFW- Journal 2004; 130: Barankin B, Silver SG, Carruthers A. The skin in pregnancy. J Cutan Med Surg 2002; 6: Schallreuter KU. Epidermal adrenergic signal transduction as part of the neuronal network in the human epidermis. J Investig Dermatol Symp Proc 1997; 2: Park HY, Yaar M. Disorders of melanocytes: biology of melanocytes. In: L Goldsmith, S Katz, B Gilchrest et al., eds. Fitzpatrick s Dermatology in General Medicine, 8th edn., Vol. 1. New York: McGraw-Hill Professional, 2012: p Miller AJ, Mihm MC Jr. Melanoma. N Engl J Med 2006; 355: Brenner M, Hearing VJ. Modifying skin pigmentation approaches through intrinsic biochemistry and exogenous agents. Drug Discov Today Dis Mech 2008; 5: e Kim DS, Park SH, Park KC. Transforming growth factor beta1 decreases melanin synthesis via delayed extracellular signal-regulated kinase activation. Int J Biochem Cell Biol 2004; 36: Park HY, Wu H, Killoran CE et al. The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-beta on melanosomes. J Cell Sci 2004; 117: Draelos ZD, Yatskayer M, Bhushan P et al. Evaluation of a kojic acid, emblica extract, and glycolic acid formulation compared with hydroquinone 4% for skin lightening. Cutis 2010; 86: Lucas Meyer Cosmetics (formerly Unipex Innovations). ß-WhiteTM Technical file. [online] n.d., [cited 2015 March 13]: [33 screens] Available from URL: White.pdf 11. Zhu W, Gao J. The use of botanical extracts as topical skin-lightening agents for the improvement of skin pigmentation disorders. J Investig Dermatol Symp Proc 2008; 13: Kimbrough-Green CK, Griffiths CE, Finkel LJ et al. Topical retinoic acid (tretinoin) for melasma in black patients. A vehicle controlled clinical trial. Arch Dermatol 1994; 130: Pandya A, Berneburg M, Ortonne JP et al. Guidelines for clinical trials in melasma. Pigmentation Disorders Academy. Br J Dermatol 2006; 156: Moncada B, Sahagun-Sanchez LK, Torres-Alvarez B et al. Molecular structure and concentration of melanin in the stratum corneum of patients with melasma. Photodermatol Photoimmunol Photomed 2009; 25: Jungman E, Maibach HI. Enhancing sunscreen efficacy in the real world? J Dermatol Treat 2010; 21: Jeanmougin M, Bouloc A, Schmutz JL. A new sunscreen application technique to protect more efficiently from ultraviolet radiation. Photodermatol Photoimmunol Photomed 2014; 30: ISISPHARMA, SPECIALIST IN SKIN HOMEOSTASIS