Efficacy and tolerability of a depigmenting gel serum comprising tranexamic acid, niacinamide, 4‐butylresorcinol, phytic acid, and a mixture of hydroxy acids that targets the biological processes regulating skin melanogenesis

The diverse causes of hyperpigmentation and complex nature of melanogenesis make it a challenge to manage. Current approaches either fail to deliver effective pigmentation control or have undesirable safety profiles that preclude their long‐term use.


| INTRODUC TI ON
Disorders of hyperpigmentation, including melasma, postinflammatory hyperpigmentation, solar lentigines, ephelides, and age spots, have a significant impact on an individual's quality of life and are a common reason for seeking treatment. 1Since the 1960s, hydroquinone (HQ) has been the gold standard treatment for hyperpigmentation. 2 Although highly effective, HQ can produce irritation, contact dermatitis, and exogenous ochronosis, especially during its prolonged use. 2 Several safety concerns have also been raised around long-term HQ exposure.Safer but equally effective alternatives are therefore sought.
The excess melanin synthesis that precedes hyperpigmentation starts with an extrinsic stimulus, such as ultraviolet radiation, inflammation, or hormonal changes, that induces keratinocytes and fibroblasts to release paracrine factors that stimulate the production of melanin pigment within melanosomes. 3Following melanin production, the melanin-loaded melanosomes migrate to the dendrites of melanocytes, where, upon maturation, they are transferred to keratinocytes. 4 As these pigmented keratinocytes terminally differentiate, the melanosomes are degraded before the mature keratinocytes are finally removed from the skin by desquamation. 5The complex biological processes regulating skin melanogenesis make it exquisitely amenable to targeting with substances that can interrupt or modulate it, and as a result, numerous ingredients that act on one or more of these steps have been identified.
Combining ingredients that target one or more steps in the melanogenesis pathway is an appealing approach for pigmentation control for the potential synergies it offers.Taking this as a paradigm we have developed a cosmetic gel serum containing multiple ingredients that have been demonstrated to act on each of the biological processes regulating the production of melanin and its distribution in the skin (Figure 1).Firstly, tranexamic acid has been demonstrated to block the induction of melanogenesis in human melanocytes. 6,7Secondly, 4-butylresorcinol, a powerful tyrosinase inhibitor and tyrosinase-related protein-1 inhibitor, inhibits the production of new melanin in melanocytes. 8[12] D112, 1 month after termination of treatment.The product also demonstrated very good skin tolerability.

Conclusion:
A gel serum comprising tranexamic acid, niacinamide, 4-butylresorcinol, and hydroxy acids, designed to target the biological processes regulating skin melanogenesis, demonstrates rapid, robust, and sustained pigmentation control in this cohort.

| Test product
The investigational product (Melaclear Advanced™; MADV; ISDIN, Barcelona, Spain) is a gel serum whose principal ingredient is tranexamic acid at a concentration of 3%.It also contains a combination of other ingredients known to interfere with melanin biosynthesis and melanin distribution (Table 1).

| In vitro depigmenting potential of MADV
The capacity of MADV to reduce pigmentation was first determined in vitro using a reconstructed epidermis (RHEm) comprising melanocytes from an Asian donor (MelanoDerm™ MEL-300-A; MatTek Corporation, Ashland, MA).MADV (10 mg/cm 2 ) was topically applied to the surface of the RHEm every 2 days for up to 21 days.Untreated RHEm was used as a control.Five RHEm were used for each treatment group.
Melanin was extracted from the RHEm by incubating it in a solution of 1% sodium bicarbonate for 30 min.The RHEm was then placed in an aqueous-based solubilizer (Solvable™; PerkinElmer Inc., Waltham, MA) and incubated overnight at 95°C.After vortexing and centrifugation, the optical density (OD) was measured at 490 nm, and melanin quantity was determined according to a standard curve.
Results were expressed as percent reduction of melanin content versus untreated control tissue.

| Evaluation of RHEm viability
The effect of MADV on the viability of the RHEm was determined Participants were instructed to apply MADV to the face and neck twice daily (1-2 pumps = mean 0.46 g ± 0.14 g/application; AM and PM) for 3 months (84 days).A SPF50 facial sunscreen was also provided, and subjects were instructed to apply it in the morning and throughout the day for the duration of the study (until D112).
Efficacy was assessed through a combination of instrumental measurement and clinical evaluation: • The number and surface area of brown spots in the face were determined by VISIA®-CR (Canfield Scientific, Parsippany, NJ). Brown spots were detected using RBX® (red/brown/X) Technology (Canfield Scientific, Parsippany, NJ). 13 • Skin color (melanin index) of one selected spot was evaluated using a Mexameter MX18® (Courage + Khazaka Electronic GmbH, Köln, Germany) connected to a 5 mm probe.
• Skin luminosity in the face was determined by a dermatologist using a 5-point clinical score, where: 0 = not luminous/dull; 1 = slightly luminous; 2 = medium luminosity; 3 = moderate luminosity; and 4 = very luminous and bright.
• Hyperpigmentation in the face was evaluated by a dermatologist using the mMASI (modified Melasma Area Severity Index) scoring method.According to the mMASI, the whole face is divided into four areas: 30% the forehead (F), 30% right malar TA B L E 1 Anti-pigmentation ingredients in MADV and their putative targets of pigmentation.

| Skin tolerability
Skin tolerability (i.e., appearance of erythema, oedema, vesicle, bulla, papule, scab, dryness, coloration, and soap effect) was evaluated by a dermatologist at each visit on D14, D28, D56, D84, and D112.Intensity of erythema and oedema was assessed using an ordinal scale (slight, moderate, or severe).The number of vesicles and papules was assessed according to an ordinal scale (1-2 vesicles or papules; more than 2 vesicles or papules).Dryness and coloration were assessed using an ordinal scale (slight, moderate, or severe).
Participants were also asked to assess sensations of discomfort (i.e., heating, stinging, and pruritus) when using the product throughout the duration of the study.

| Effect of MADV on melanin production in vitro
With respect to D0, melanin production in untreated control RHEm was increased by 275.7% on D14 (p < 0.001) and by 441% on D21 (p < 0.001).Over the same period, melanin production in MADVtreated RHEm was only increased by 37.8% on D14 and by 45.8% on D21, representing a relative reduction of total melanin content in the RHEm of 50.0% on D14 (p < 0.001) and 67.0% on D21 (p < 0.001) (Table 2; Figure 2).Notably, melanin levels were not significantly different on D14 than they were on D21.MADV had no effect on viability at either time point (data not shown).

| Clinical depigmenting efficacy
Thirty-five subjects were enrolled in the study (Table 3).

| Skin tolerability
No skin reactions or sensations of discomfort were detected by the dermatologist or reported by the subjects during the study.

| DISCUSS ION
MADV was designed to target skin melanogenesis using a combination of ingredients that, in isolation, have been clinically demonstrated to reduce skin pigmentation.As both a monotherapy, 14 and in combination with other depigmenting agents, 15 3% tranexamic acid has proven to be comparable to 3%-4% HQ for the treatment of melasma and to reduce skin pigmentation levels.Similarly, 4% niacinamide demonstrated equivalent efficacy as 4% HQ for the treatment of melasma in a double-blinded randomized split-face study. 16butylresorcinol (0.3%) has also demonstrated superiority over other tyrosinase inhibitors for the treatment of age spots, with a faster onset of improvement and maintenance of pigmentation control after treatment termination. 17 vitro, MADV strongly inhibited melanin production without cytotoxicity, suggesting that it acts by inhibiting melanin biosynthesis rather than through any cytotoxic effect on melanocytes like HQ. 18 In clinical use, twice-daily application of MADV in combination with an SPF50 sunscreen led to significant reductions in instrumental (brown spots count and area, melanin index) and clinical (IGA, skin luminosity, mMASI) measures of hyperpigmentation.Notably, these effects were seen rapidly, with significant reductions in brown spots and melanin index observed within 14 days.A similarly rapid effect has been reported for the combination of 2 or 3% tranexamic acid with niacinamide and a tyrosinase inhibitor (alpha-arbutin).15 Interestingly, statistically significant improvements to clinical measures of efficacy were only observed from D28 onwards, possibly reflecting the lower sensitivity of such measurements.The greatest rate of change in most parameters was observed between D28 and D56, highlighting the benefit of prolonged use of MADV for pigmentation control. Th effects of MADV were also maintained for at least 4 weeks following cessation of treatment, and notably some parameters, such as the melanin index, were significantly improved without further treatment.Furthermore, no relapse of clinical parameters was observed after cessation of treatment at D84.This is important for efficacy and consumer satisfaction, especially considering the highly recurrent nature of hyperpigmentation.19 A longerterm follow-up period, however, would provide valuable insight on the sustained efficacy of MADV.
A limitation of this study is the lack of a comparator such as HQ or another known depigmenting agent or a placebo control group in the in vitro and clinical testing that would permit an objective and more robust assessment of the efficacy of MADV.
Nevertheless, as a proof-of-concept study and in combination with the in vitro data, the depigmenting potential of MADV is compelling.Additionally, because this study was performed in subjects with majority Fitzpatrick skin types II to III (91.4%), follow-up studies in subjects with darker skin types would also be valuable, especially considering that such individuals are particularly susceptible to hyperpigmentation and recalcitrant to treatment. 20Including a wider range of skin types would also provide a more comprehensive understanding of the efficacy and safety of MADV across different populations.In this study, a sunscreen was provided to limit further stimulation of melanin production for its duration.Although not explicitly tested, this sunscreen contains no ingredients at concentrations likely to have a depigmenting action themselves, so the effects observed are likely to be attributable to MADV alone.Nevertheless, a comparison of MADV versus this sunscreen alone would help exclude any possibility that the decreases in hyperpigmentation observed are unrelated to treatment with MADV.Further in vitro studies examining the effects of MADV would also be valuable to define its mechanism of action and confirm that it is not melanocidal.

| CON CLUS IONS
Through a combination of in vitro and clinical studies, we have demonstrated that MADV, a gel serum comprising tranexamic acid, niacinamide, 4-butylresorcinol, phytic acid, and a mixture of hydroxy acids, is effective at reducing hyperpigmentation, with quantifiable changes in skin pigmentation detected as early as week 2, which continue through week 12 and persist without relapse for 1 month after treatment termination.This new targeted depigmenting serum was demonstrated to be an effective and well-tolerated treatment for hyperpigmentation in this cohort.

AUTH O R CO NTR I B UTI O N S
MF, AB, and CG designed the studies.AB wrote the paper.All authors revised the manuscript.

ACK N OWLED G M ENTS
The authors thank all subjects for their participation in the study.
All studies and publication costs were wholly funded by ISDIN, the manufacturer of the product under study.

CO N FLI C T O F I NTER E S T S TATEM ENT
MF, AB, JB, AFdH, CT, and EJ are employees of ISDIN, the manufacturer of the product under study.CG was employed by ISDIN at the time of the studies and now acts as a consultant to ISDIN.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.

E TH I C S S TATEM ENT
The study protocol (PT.06.01 V05) was approved on January

K
E Y W O R D S hyperpigmentation, melanogenesis, melasma, pigmentation, tranexamic acid F I G U R E 1 Schematic representation of the events in melanogenesis pathway targeted by MADV.(1) Induction of melanogenesis is inhibited by tranexamic acid.(2) The production of new melanin is inhibited by 4-butylresorcinol.(3) The transfer of melanin from melanocytes to keratinocytes is inhibited by niacinamide.(4) The removal of melanin-containing keratinocytes from the skin is enhanced by the action of phytic acid, mandelic acid, and gluconolactone.In this study, we evaluated the efficacy of this gel serum by first measuring its capacity to inhibit melanin production in vitro using a melanocyte-containing, reconstructed human epidermis model.Next, its clinical efficacy and safety were addressed in an openlabel, non-comparative study in subjects with slight to moderate facial hyperpigmentation.

2 . 4 |
by measuring MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reduction at D14 and D21.Optical density of tissue extracts, proportional to the number of living cells and their metabolic activity, was measured at 540 nm with a microplate reader (VersaMax, Molecular Devices, CA).Viability of MADV-treated tissues was compared to untreated tissues at equivalent time points.Clinical depigmenting efficacy of MADV Depigmenting efficacy of MADV was assessed in a single-center non-comparative study involving 35 women aged 35-70 years with skin phototypes II-IV and slight to moderate hyperpigmented spots on the face.The study protocol (PT.06.01 V05) was approved on January 4, 2021, by the Independent Ethics Committee of Clinica Dr. Carlos Ramos, Lousado, Portugal.Prior written informed consent was obtained from all subjects, and all studies were performed in full respect of the Declaration of Helsinki (1964) and its subsequent amendments and following COLIPA guidelines for the Evaluation of the Efficacy of Cosmetic Products.Good clinical practice was maintained throughout.Exclusion criteria included: (i) use of topical retinoids during the 3 months prior to the onset of the study; (ii) having undertaken antiaging or aesthetic treatment within 6 months of entry into the study; (iii) initiation or change of hormonal treatment during the study or in the 3 months preceding it; (iv) pregnancy and nursing; and (v) intensive sun or UVA exposure within 2 weeks preceding the study and throughout its duration.Concomitant use of products that contain vitamin C, AHAs/BHAs, retinoids, and exfoliating treatments was prohibited throughout the study.

F I G U R E 2
Four subjects did not complete the study due to personal reasons unrelated to the use of MADV.Two subjects withdrew at D14, and one each on D28 and D56.There were no additional withdrawals after this time point.Efficacy, skin acceptability, and tolerability of MADV were TA B L E 2 Effect of MADV on melanin synthesis in RHEm on D14 and D21.(±1.27)*** 21.14 (±2.16)***Note: Values represent the mean melanin concentration (±SEM) of 5 independent experiments per treatment group at each time point.Statistical comparisons are calculated versus untreated RHEm at the equivalent time points.***p < 0.001.Representative images of melanin content (Warthin-Starry staining) in RHEm at (A) D0, (B) untreated RHEm at D9, and (C) MADV-treated RHEm at D9. thus assessed on 33 subjects until D14, on 32 subjects until D28, and on 31 subjects on D56, D84, and D112.

F I G U R E 4
Evolution of brown spots from baseline (D0) to D112 in RBX Brown images captured by VISIA®-CR (Subject #29; female, age 41, Fitzpatrick Skin Phototype III).
4, 2021, by the Independent Ethics Committee of Clinica Dr. Carlos Ramos, Lousado, Portugal.Prior written informed consent was obtained from all subjects, and all studies were performed in full respect of the Declaration of Helsinki (1964) and its subsequent amendments and following COLIPA guidelines for the Evaluation of the Efficacy of Cosmetic Products.Good clinical practice was maintained throughout.