This was a comparative, non-inferiority, randomized, investigator-blind, parallel-group investigation conducted in a single center in Brazil. The planned duration of the study was a 3-month treatment period, which included four visits.

The protocol of this study was approved by an independent ethics committee from the Pró-Cardíaco Hospital, Rio De Janeiro, Brazil. The investigation was conducted in accordance with the principles of Resolution 466/2012 of the Conselho Nacional de Saúde do Brazil, ethical principles stipulated in the Declaration of Helsinki of 1964 (and its subsequent amendments), and Good Clinical Practice defined by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. The patients provided consent for the publication of patient images. ClinicalTrials.gov identifier NCT06787846.

The criteria for inclusion in this study were as follows: female patients with skin phototypes I–IV; patient age > 18 years; and diagnosis of mild-to-severe epidermal or mixed facial melasma.

All patients received verbal and written information concerning the study in accordance with the applicable local regulations and guidelines. This information explained the nature, purpose, and risks of the study. In addition, it emphasized that participation is voluntary and that the patient may withdraw from the study at any time and for any reason. The patients provided written informed consent for their participation in the study.

MB3 is a new facial depigmenting serum containing 0.5% 2-MNG, 10% niacinamide, Cystoseira tamariscifolia extract, lipohydroxy acid, carnosine, retinyl palmitate, and dipotassium glycyrrhizate.

The patients were randomized to receive treatment at home with MB3 or hydroquinone 4% (registered as a cosmetic standard of care in Brazil) for 3 months. The treatment involved facial application of MB3 twice daily (morning and evening) or hydroquinone 4% cream once daily (evening).

In both groups, patients also applied a tinted sun protection factor (SPF) 50+ with high ultraviolet A-protection factor (UVA-PF) sunscreen with good visible light protection (Anthelios UVMUNE 400 tinted cream; La Roche-Posay Laboratoire Dermatologique) to the area of treatment twice daily (morning and midday with a minimum of a 10-min interval between applications).

Evaluations of the application area were conducted at day (D) 0, D28, D56, and D84 of treatment by a dermatologist (clinical scores) or by the patients (self-assessment).

The primary endpoint was the non-inferiority analysis at D84 based on the mean change from baseline of Modified Melasma Area and Severity Index (mMASI). The mMASI is a modified version of the MASI (the most common outcome measure used for studies on melasma) after eliminating homogeneity measurement [22]. This measure was employed to assess the area and darkness of the melasma on three areas of the face (i.e., forehead, right and left cheeks, and chin) (score range 0–24). The mMASI score has previously demonstrated excellent reliability and good validity for assessing the severity of melasma [23]. Thus, it is commonly used for the evaluation of efficacy in clinical trials [24]. In the absence of a published minimal clinically important difference (MCID) for mMASI, we decided to set 1.3 as the non-inferiority margin, representing approximately 50% of the difference versus placebo at D84 in this study [25].

The secondary efficacy endpoints included mMASI, Melasma Quality of Life questionnaire (MELASQoL), and Patient Unique Stigmatization Holistic tool in Dermatology (PUSH-D) scores at each visit. The MELASQoL is a dermatology-specific instrument used to assess the impact of melasma on health-related QoL [26]. The instrument includes 10 questions, which are evaluated by the patients using a scale ranging from 1 (not bothered at all) to 7 (bothered all the time). The total MELASQoL score ranges from 7 to 70, with a higher score signifying worse QoL. This instrument has been validated in clinical trials and translated in numerous languages [10]. PUSH-D is a 17-item questionnaire offering a comprehensive view of the degree of stigmatization in visible skin disorders, as well as the comparability of stigmatization levels across various skin conditions [27]. It measures two pertinent dimensions, namely felt stigma (eight items; range 0–32) and enacted stigma (nine items; range 0–36). The stigmatization total score ranges from 0 (no stigma) to 68 (high stigma).

Safety was evaluated by the investigator and the patients.

All statistical analyses were performed with SAS® version 9.4 or higher (SAS institute, Cary, NC, USA) at the 5% significance level using a two-sided test. Normality was evaluated by the Kolmogorov–Smirnov test at the 1% threshold.

Quantitative efficacy variables were analyzed by an analysis of covariance (ANCOVA). Non-inferiority was achieved if the upper bound of the two-sided 95% confidence interval (CI) was < 1.3. For the mMASI analyses, missing values were imputed using the last observation carried forward (LOCF) method. All quantitative efficacy parameters were analyzed using an ANCOVA or by the Mann–Whitney test if normality was rejected. Non-quantitative parameters were analyzed using the chi-squared test.