Peeling with retinoic acid in microemulsion for treatment of melasma: A double‐blind randomized controlled clinical study

To evaluate the clinical efficacy of peeling with a microemulsion formulation containing 1% retinoic acid.

ted to four peeling sessions, fortnightly on Days 0, 15, 30, and 45, and analyzed at the time intervals of 0, 15, 30, 45, and 60 days.Evaluation was made by using the Melasma Area and Severity Index (MASI) and Melasma Quality of Life (MelasquoL) instrument.
Hemato-biochemical parameters were also evaluated at Days 0 and 60.After obtaining the results, normality was evaluated by means of the Kolmogorov-Smirnov test and afterwards, the following tests were applied: Friedman statistical (to test the effect of the treatments on the MASI index); Wilcoxon, (for comparison between pairs to test the effect of treatments on the MelasQoL index); Kruskal-Wallis, (to test the differences between the groups); and Mann-Whitney, (comparisons between treatments).The level of significance adopted was 5% (α = 0.05).

Results:
The three groups presented a significant reduction in the MASI index, indicating the effect of all the treatments on reducing the melasma (p < 0.001).A significant reduction in the stains was observed with the use of retinoic acid peeling delivered in microemulsion (62%) when compared with the conventional peeling with 1% retinoic acid in a conventional vehicle (26%) and the placebo (12%).There was also a significant reduction in the MelasQoL index (sum of all the aspects) in the three groups, indicating the effect of all the treatments, including the placebo, on the overall quality of life of those with melasma.However, RA 1%M the treatment that promoted the greatest effect on the quality of life of individuals.In percentage terms, the RA 1%M provided a mean reduction of 30% in the MelasQoL index, against 13% of the conventional treatment and only 4% of the placebo.When the hemato-biochemical parameters were compared on Days 0 and 60, there were no significant changes in the results.

| INTRODUC TI ON
Chemical peelings are useful therapeutic tools in the treatment of melasma. 1 Among the chemical peelings, the type with retinoic acid has been widely used, because not only does it remove the pigment deposited on the epidermis, it also has the capacity to inhibit tyrosinase, a key enzyme in melanin production, yet without good effectiveness for dermal melasma. 2 Various drugs may have difficulty in crossing the barrier of the skin and attaining its site of action. 3Nanotechnology has been developed in various fields of medicine, among them dermatology.The physico-chemical characteristics of nanoparticles, such as their size, may contribute to better drug permeation through the skin. 4ny strategies have been proposed for overcoming the barrier function to increase the permeability of substances applied to the skin.Microemulsions are useful approaches capable of delivering drugs through the skin, to a larger extent than simple substances at conventional doses. 1,2In addition to having a good appearance and being thermodynamically stable, microemulsions may increase cutaneous permeation because of their reduced size. 5 this context, the present study proposed to make a clinical evaluation of a microemulsion containing 1% retinoic acid, by means of chemical peeling, with regard to its clinical efficacy in patients with melasma, in addition to testing the hypothesis that peeling in microemulsion would be more effective than the conventional type of procedure.

| MATERIAL S AND ME THODS
An experimental, prospective, double-blind, randomized, controlled clinical study was conducted in patients with melasma, with the aim of evaluating the effects of chemical peeling using a microemulsion containing 1% retinoic acid.Prior to the beginning of the study, a sample calculation of the minimum required sample size was performed, considering the following parameters: test power of 80% (β = 0.20) and error of 5% (α = 0.05) reaching up to 20 individuals per group.
Randomization was performed by a researcher who was not involved in the clinical part of the study, thus guaranteeing allocation concealment.BioEstat 5.0 software (Civil Society Mamiraurá) was used to build a table of random numbers, taking into consideration the sample size for each group, the population size and the treatment used (i.e., treatment or non-treatment).
Before the study began, the research project was sent to the Research Ethics Committee, from which it received a favorable report with No. CAAE: 28825514.8.0000.0055.
Patients with melasma were randomly selected at a private Dermatology clinic, in the city of Vitória da Conquista, Bahia, Brazil.
The sample consisted of 60 patients of the female sex; with melasma of the epidermal, mixed and dermal type; with duration of over 1 year; over the age of 18 years; with Fitzpatrick phototypes from I to IV; and who had the personal wish to participate in the study.All the participants signed the term of free and informed consent that contained an exhaustive explanation about the risks, benefits and possible complications involved.
Excluded from the sample were pregnant women, nursing mothers or those planning to fall pregnant in the following 3 months; patients with other cutaneous diseases in the face; phototypes V and VI; history of recurrent herpes; allergy to retinoic acid or the vehicle; history of Keloids and hypertrophic scars; those making use of topical medications (except sun filters); treatments with lasers, Intense pulsed light (IPL), dermoabrasion or peeling in the last 3 months; use of oral corticotherapy less than a month previously; use of systemic retinoids, cyclosporin, interferon, methotrexate less than 4 months previously; use of photoallergic or photosensitizing drugs less than 1 month previously, and use of hormone replacement therapy.
The entire clinical history of each patient was collected, including personal history (age, sex, marital status, occupation); family history; duration of melasma; relationship with: previous pregnancies; exposure to sun; previous therapies; relationship of melasma with use of contraceptive pills.All the patients were instructed about the use of sun filter.
The microemulsions were prepared in order to obtain an amount equivalent to about 0.5 mg of retinoic acid in chloroform and make up the volume to 100 mL of solution with the same solvent.A standard solution will be prepared at the same concentration, using the same solvent.The amount of a 1 mL syringe distributed over the entire face was applied to each patient.
The absorbances of the resulting solutions were measured at 365 nm using chloroform for zero adjustment.The amount of RA 1%M was calculated from the readings obtained.Alternatively, the

Conclusion:
The chemical peeling performed with RA 1%M was effective for the treatment of melasma, and was shown to be superior to the peeling performed with retinoic acid in a conventional vehicle, in reducing the stains and improving the quality of life of patients.All the patients were instructed to make daily use of sun filter (SPF30).
The groups were submitted to peeling that consisted of four fortnightly applications on Day 0, 15, 30, and 45, and analyses were performed on Day 0, 15, 30, 45, and 60.
The procedure was performed in the following manner: initially the skin was cleaned with gauze dampened with 3% acetone in alcohol to remove oiliness and impurities, and afterwards the product was homogeneously applied on the entire face by using a swab.The product remained in contact with the skin for a period of 6 h, and was then removed by washing with soap and water.All products used (conventional, microemulsion and placebo) were in identical packaging with only letters identifying what it was about.The product applicator did not know what he was applying.
Clinical exams and digital photographs (Canon Rebel 60 D) were taken before and after each procedure; that is on Day 0, 15, 30, 45, and 60.Photographs were taken from the front view (measured at 90°), right and left views (measured at 45°), and the measurements were made with the aid of a goniometer.Afterwards, the photographs were analyzed by two dematologists, in which they followed the criteria of the MASI index (melasma area and severity index).
Data were obtained from these two indices (MASI and Melas-QoL), and were subsequently submitted to statistical analysis.
To evaluate the possible systemic effects of the treatment, the patients were submitted to hemato biochemical analyses by means

| RE SULTS
By means of the intragroup comparisons the authors observed that at each evaluation, there was a reduction in the MASI index in the three groups.However, by means of the intergroup comparisons, they observed that only the group treated by peeling with RA 1%M differed significantly from the placebo group and the conventional method (RA 1%) in the evaluations after baseline, demonstrated that RA 1%M was more efficient than the conventional method and the placebo treatment of melasma.In percentage terms, the authors observed that after 60 days, RA 1%M provided a mean reduction of 62% in the MelasQoL index, against 26% of the conventional treatment and only 12% of the placebo.(Figure 2).
The treatment with RA 1%M was the only one capable of improving almost all aspect of the quality of life of the melasma patients.The only aspect conventional treatment did not improve.was the aspect of depression, and the placebo promoted improvement in only four aspects (appearance, frustration, embarrassment, and demonstration of affection).Moreover, the treatment with RA 1%M was more efficient than the conventional type in three aspects (frustration r and relationships); and than the placebo, in eight aspects (appearance, frustrations, embarrassment, depressive feeling, relationships, attraction, feeling of importance, and sense of freedom).Whereas conventional treatment was more efficient than the placebo in only two aspects (feeling of importance and sense of freedom).
The authors observed a reduction in the MelasQoL index (sum of all aspects) in the three groups (Figure 3).However, RA 1%M was the treatment that promoted the greatest effect on the quality of life of individuals, followed by conventional treatment with retinoic acid.In percentage terms, the authors observed that after treatment, RA 1%M provided a mean reduction of 30% in the MelasQoL index, against 13% of the conventional treatment and only 4% of the placebo.
Figure 4 shows the results of the clinical aspect of the patients of this study.
With regard to laboratory exams, there were no significant changes between Day 0 and after treatment.

| DISCUSS ION
The hypothesis previously formulated was confirmed, since the results showed evidence that chemical RA 1%M was effective for the treatment of melasma, and shown to be superior to RA 1% in a conventional vehicle for the treatment of this disease.F I G U R E 4 Clinical aspect of the face of patients after application of peelings and placebo; 1%-1% retinoic acid Peeling (RA1%); 2%-1% Retinoic acid in microemulsion peeling (RA1%M) and 3-Placebo.
promoted a reduction to the order of 42% in the melasma, showing evidence of greater reduction in the disease when compared with peeling performed with 1.0% retinoic acid in a conventional vehicle that promoted a 26.1% improvement, suggesting the influence of concentration on the effectiveness of the product.Nevertheless, peeling performed at 1% in a microemulsioned vehicle presented a reduction in the melasma with more significant results, with a mean reduction of 62% in the MASI index showing evidence of the clinical importance of this study.This is because, significant efficacy could be obtained with a low concentration of this drug in clearing the stains on the skin.Furthermore, by using a low concentration of this drug, it was possible to avoid side effects such as erythema, itching and desquamation, among others.
In addition, the group treated with placebo showed improvement during treatment.This may be attributed to the better care patients were taking of their skin; that is, they began to use sun filter regularly in their routines.
In addition to the better final result, after the second application of peeling; that is, on the 30th day of evaluation, the authors could observe a significant reduction in MASI, when compared with RA 1% in the conventional formulation and the placebo.This suggests that RA 1%M obtained a good result in a lower number of applications.
These results suggest that peeling in microemulsion was able to cross the skin barrier more efficiently when compared with standard peeling.Thereby, better results could be obtained by using a lower concentration of the active substance.
From the point of view of penetration, the skin acts as a nanoporous mechanical barrier, perforated by a large number of almost semicircular canals or pathways.The majority of publications have estimated that these hydrophilic "pores" have a mean diameter ranging from 0.4 to 36.0 nm.As the majority of molecules capable of permeating, cross the skin through these intercellular microcanals, diverse techniques have been proposed to improve this trajectory and transpose the molecular architecture represented by the corneocytes and multiple layers of intercellular lipids.It is in this point that nanometric carriers participate actively.Therefore, this system of releasing active principles for topical application aim to: (1) facilitate the vehiculation of products, increasing the efficacy of the formulation, and improving the esthetics of the end product; (2)   maximize the time the composite remains on the skin, minimizing its transdermal absorption; and (3) release the products in specific areas. 6,7rthermore, significant improvement was verified in the quality of life of the patients, measured by MelasQoL.The results of the present study corroborate the findings of 4 who tested lactic acid peeling and concluded that it was effective and safe as monotherapy for the treatment of melasma.However, the authors pointed out limitations in the study due to the fact that it was not a controlled study.
The authors of the present study took care to conduct a controlled, randomized, double-blind clinical study.
Various studies have used standardized questionnaires to evaluate the quality of life. 8,9These questionnaires may elucidate questions that patients do not expose, recognizing aspects that must be worked on with greater emphasis.Quality of Life questionnaires may including the placebo, on the overall quality of life of patients with melasma.These results may be justified by the fact that the patients were taking better care of themselves and had received information about how to take better care of the skin and protect themselves against exposure to ultravoilet rays.The quality of life index was higher in the patients of the Group that received the application of RA 1%M, suggestive of resulting from the reduction in skin stains.
There may be hypertriglyceridemia, hypercholesterolemia, and elevation of hepatic enzymes as side effects of the use of retinoids, mainly with systemic use. 7In this study, the authors proved that these side effects-common with the use of retinoids-did not occur.
In order to avoid any changes in the evaluation of the patients' blood, the patients were asked to fill in a diary with information about their diet and the use of medications or drugs.No patient was excluded from the sample as no abnormality was presented by them.These results indicated that in spite of peeling being microemulsioned, and having the capacity to penetrate into the viable epidermis, it is not extensively permeated through the skin, and therefore, fewer systemic side effects are perceived.This is important, because it concerns a safe medication-one that involves few risks to the health of individuals.Thus, with the results obtained, the authors of this study suggest that the product containing retinoic acid in microemulsion for peeling may be safely used.However, it is worth mentioning a limitation of the study, which was the lack of an objective evaluation, since the evaluations carried out were subjective.
It is important to emphasize the need for studies that evaluate the penetration of these products into the skin, since the conclusions of the present study concern only the improvement in melasma.This is a suggestion for future studies.

| CON CLUS ION
By conducting this study, the authors could conclude that: RA 1%M was shown to be more effective in the treatment of melasma, in addition to having promoted improvement in the quality of life of pa- second application of RA 1%M, suggesting that with a lower number of applications, a beneficial effect of this treatment could already be visualized; RA 1%M was shown to be more efficient, even with the use of lower concentrations of retinoic acid, when compared with peelings performed with conventional vehicles and there were no changes in the hemato-biochemical parameters of the volunteers between Day 0 and 60, suggesting that in addition to being effective, the RA 1%M is also safe for use in humans.

3 ( 1 (
mentation and homogeneity are evaluated on a scale ranging from 0 to 4 (0 = absent; 1 = light; 2 = mild; 3 = marked; 4 = maximum).The MASI score is calculated by adding the sum of the pigmentation and homogeneity scores, multiplied by the area of involvement in each of the four domains on the face.The formula used is MASI = 0.PM + HM) AM.The MASI score ranges from 0 to 48.The patients also answered the MelasQoL questionnaire (melasma quality of life) on Day 0 and 60.

F I G U R E 1 2 . 1 |
Flowchart of the procedures performed. of blood collection, in which the hepatic transaminases (TGO, TGO), alkaline phosphatase, gama-gt, hemogram, total cholesterol, LDL, HDL, VLDL, and Triglycerides were dosed on Day 0 and 60. 3 After this the data were analyzed by the Student's-t-test.Statistical procedure for analyzing the clinical study Initially the presuppositions were verified for the use of the repeated measures analysis of variance (homoscedasticity, normality, and sphericity).After normality had been rejected by means of the Kolmogorov-Smirnov test, the authors proceeded with nonparametric statistics.The Friedman test was used to test the effect of treatments on the MASI index, with the comparisons throughout the evaluations (0, 15, 30, 45, and 60) being made by means of the Wilcoxon test.The Wilcoxon test was also used to test the effect of the treatments on the MelasQoL index.The Kruskal-Wallis test was used to test the differences between the Groups (RA1%, RA1%M and placebo) in each evaluation, with the comparisons between treatments being made by means of the Mann-Whitney test.The level of significance adopted was 5% (α = 0.05).The data were tabulated and analyzed in the software program IBM SPSS Statistics for Windows (IBM SPSS.21.0, 2012: IBM Corp.).
Magalhães et al. (2011) 4 conducted a studying which they evaluated the effect of peeling of retinoic acid in 30 melasma patients, who were submitted to the application of 5% or 10% retinoic acid peelings, and subsequent evaluation of the MASI index and quality of life evaluation by means of MelasQoL.The results found by the cited authors showed that the 5% and 10% retinoic acid peeling F I G U R E 3 Effect of treatments on melasma, according to evaluation periods.MASI, melasma area and severity index; AR1%, 1% retinoic acid; AR1%M, 1% retinoic acid in microemulsion.*p-value < 0.001 (Friedman test) for intragroup comparison **p-value < 0.05 (Kruskal-Wallis Test) for intergroup comparison.† p-value < 0.05 (Wilcoxon test) for all treatments compared with the previous assessment.‡ pvalue < 0.05 (Mann-Whitney test): AR1% = AR1%M and placebo; AR1%M ≠ placebo; #p-value < 0.05 (Mann-Whitney test): AR1% = placebo; AR1%M ≠ AR1% and placebo.
be used to enable a more objective evaluation of this combination of subjective factors.The use of questionnaires in clinical practice allows identification of the aspects most influenced by a certain health condition and evaluate the effectiveness of the intervention strategy used in the treatment of patients.5,10In the present study, associated with the MASI index, the authors used MelasQoL with the purpose of evaluating the quality of life before and after application of the peelings.Cestari et al. (2006), 6 proposed a study whose objective was to validate the melasma quality of life questionnaire for the Brazilian Portuguese language.This study demonstrated that this questionnaire was a valid instrument and could be used to evaluate the quality of life in response to the treatment of melasma in Brazilian patients.Based on the findings of the cited authors, the authors of the present study decided to use this instrument.Despite this evaluation, the results revealed a significant reduction in the MelasQoL index in the three groups, indicating the effect of all the treatments,