Thiamidol containing treatment regimens in facial hyperpigmentation: An international multi‐centre approach consisting of a double‐blind, controlled, split‐face study and of an open‐label, real‐world study

Abstract Objective Tyrosinase is the rate‐limiting enzyme in melanogenesis. Thiamidol is the most potent inhibitor of human tyrosinase out of 50 000 tested compounds. In clinical studies, it was shown to improve facial hyperpigmentation, post‐inflammatory hyperpigmentation and age spots significantly. To identify the optimal number of daily Thiamidol applications, we conducted a split‐face study comparing the efficacy and tolerability of four‐times with two‐times daily application. Subsequently, we evaluated the efficacy and tolerability of a typical face care regimen containing Thiamidol in a real‐world study. Methods The split‐face study was double‐blind, randomized, controlled, including two Thiamidol containing products (serum and day care SPF 30). The serum was applied twice daily on one half of the face and the day care SPF30 twice‐daily on the whole face. The real‐world study was open‐label, observational, including three Thiamidol containing products (day care SPF 30 in the morning, serum and night care in the evening). In both studies, subjects with mild‐to‐moderate facial hyperpigmentation applied the products over 12 weeks. Assessments included clinical and subjective grading of hyperpigmentation, skin condition, hemi‐/modified MASI, chromameter and clinical photography. Results In the split‐face study (n = 34), hyperpigmentation, skin roughness and hMASI improved all significantly (P < 0.001) versus baseline, with first visible results after two weeks of twice‐daily application. The four‐times daily application led to significant improvement versus the two‐times daily application. In the real‐world study (n = 83), all evaluated parameters, including skin condition and chromametry (n = 30), improved significantly (P < 0.001) in comparison with baseline and the corresponding preceding visits. The subjects judged the cosmetic properties of the products positively. In both studies, the products were well tolerated. Conclusion Four‐times daily Thiamidol improves facial hyperpigmentation significantly more than two‐times daily and is well tolerated by the subjects. The real‐world study with a typical face care regimen containing Thiamidol shows improvement of facial hyperpigmentation and confirms tolerability. Furthermore, the data provide evidence for the suitability of this three‐product Thiamidol regimen for day‐to‐day life.

CONCLUSION: Four-times daily Thiamidol improves facial hyperpigmentation significantly more than two-times daily and is well tolerated by the subjects. The real-world study with a typical face care regimen containing Thiamidol shows improvement of facial hyperpigmentation and confirms tolerability. Furthermore, the data provide evidence for the suitability of this three-product Thiamidol regimen for day-to-day life. , l'hyperpigmentation, la rugosit e de la peau et l'hMASI se sont tous am elior ees de mani ere significative (P < 0.001) par rapport a la ligne de base, avec des effets devenus visibles apr es deux semaines d'application deux fois par jour. L'application quatre fois par jour a apport e une am elioration significative par rapport a l'application deux fois par jour. Dans l' etude en situation r eelle (n = 83), tous les param etres evalu es, y compris l' etat de la peau et la chromam etrie (n = 30) se sont am elior es significativement (P < 0.001) par rapport a la ligne de base et aux visites pr ec edentes correspondantes. Les sujets ont jug e positivement les propri et es cosm etiques des produits. Dans les deux etudes, les produits ont et e bien tol er es. CONCLUSION: L'application de Thiamidol quatre fois par jour, am eliore l'hyperpigmentation du visage de mani ere plus significative que deux fois par jour et est bien tol er e par les sujets. L' etude en situation r eelle avec un r egime de soins du visage typique contenant du Thiamidol montre une am elioration de l'hyperpigmentation faciale et confirme la tol erance. En outre, les donn ees fournissent des preuves de l'ad equation de ce r egime de trois produits a base de Thiamidol pour le soin quotidien.

Introduction
Hyperpigmentation is a frequent cosmetic problem characterized by hyperpigmentation on sun-exposed areas because of increased melanogenesis [1,2]. Although the natural sun-induced production of melanin is desirable for its photoprotective function and the tanning effect, accumulation of abnormal amounts in specific parts of the skin resulting in darker patches is undesirable and poses an aesthetic problem [3][4][5].
The first rate-limiting step of melanogenesis is the oxidation of tyrosine to dopaquinone by tyrosinase [6,7]. Therefore, tyrosinase inhibitors are in the focus of the research for the treatment of hyperpigmentation [7][8][9][10]. Acting immediately and reversibly, they are considered safer than, for example hydroquinone [11] which is not allowed in the EU for cosmetic use [12]. So far, most tyrosinase inhibitors lack clinical efficacy [13] because they were identified by testing on mushroom and not on human tyrosinase [8,14,15]. The purification of soluble variants of human tyrosinase [16] allowed the identification of Thiamidol (isobutylamido thiazolyl resorcinol) as its most potent inhibitor [11,17,18] out of 50 000 screened compounds.
In melanocyte cultures, Thiamidol was superior to widely used tyrosinase inhibitors such as kojic acid, arbutin, or hydroquinone showing potent and reversible inhibition of melanin production [17]. In addition, the resorcinol derivative being a strictly competitive tyrosinase inhibitor and not a substrate is not converted to a toxic and potentially leukoderma-inducing quinone [17]. In vivo Thiamidol twice-daily reduced visibly and significantly age spots [17], the modified melasma area and severity index (mMASI) [19] in comparison with control (sunscreen only; P ≤ 0.001) [20], and post-inflammatory acne hyperpigmentation [21] and was always very well tolerated.
So far, its efficacy and tolerability were shown in twice-daily applications. To identify the optimal number of daily applications, we did a split-face study in a clinical setting, comparing the efficacy and tolerability of four-times with two-times daily application in women with mild-to-moderate facial hyperpigmentation. In a second study in the same target group, we investigated the efficacy and tolerability in a day-to-day life setting (real-world study) using a typical three-product face care regimen [22] (serum, day care with SPF 30 and night care cream) containing Thiamidol.

Study design
This was a single-centre, double-blind, randomized, controlled, split-face clinical study conducted in a centre in the United States (Texas Research Center, Texas 75081).
After approval by the institutional review board, the study was conducted following the Declaration of Helsinki and the ICH GCP guidelines as applicable to cosmetic products. Before study inclusion, the subjects received the patient information and provided written, informed consent conforming to Title 21 Code of Federal Regulations (CFR) 50.25, including consent for photography.

Study population and treatment
Eligible for the study were healthy subjects (25-65 years, Fitzpatrick skin type I-IV) with mild-to-moderate hyperpigmentation (3-6 according to a modified Griffith's 10-point scale, where 0 = none and 9 = severe [23]) on both sides of the face and willing to use the two test products. Pregnancy, allergies to skin care products, hormone replacement therapy or oral contraception within the previous 3 months, topical hyperpigmentation treatment within the previous 4 months, any topical or systemic medication known to affect skin ageing or dyschromia within the previous 8 weeks, any facial treatments, or photosensitizing or immunosuppressive drug application within the previous 6 months, high energy treatments, plastic surgery, or ablative laser resurfacing within the previous 12 months, extensive UV or other irradiation during the study, suntan, scars, birthmarks, skin cancer, chronic diseases (e.g. rosacea, psoriasis and acne) or strong hair growth on the face, poorly adjusted hypo-or hyperthyreosis, health condition or pre-existing or dormant dermatologic disease on the face that could interfere with the outcome of the study, or concomitant participation to another clinical study were all exclusion criteria.
The subjects were allowed to follow their usual make-up and cleansing routine and applied the treatment regimen for 12 weeks as follows: Twice-daily serum on the assigned left or right side of the face and day care with SPF30 on the whole face, that means, one side of the face received four Thiamidol applications and the other two applications (Table 1); the subjects were not allowed to use any face care or make-up product in the mornings of the study visits. The INCI compositions of the study products are described in Table 2.

Study design
This was a single-arm, open-label, prospective, observational, realworld study conducted in one centre in Germany (HAUTZENTRUM K€ oln) and two centres in Argentina (CLAIM, Buenos Aires; Laser Therapy Clinic Dr. Agustina Vila Echag€ ue, Buenos Aires). In Germany, the study received approval by the local Ethics Committee and in Argentina by the corresponding Institutional Review Board. The study was conducted according to the Declaration of Helsinki and the ICH GCP guidelines as applicable to cosmetic products. All subjects received the patient information and provided written, informed consent, including photography.

Study population and treatment
Healthy subjects (25-60 years, Fitzpatrick skin type I-IV) with mild-to-moderate facial hyperpigmentation on both sides of the face and willing to use the three-product treatment regimen were included. Pregnancy, or hormone replacement therapy within the previous 3 months, topical hyperpigmentation treatment within the previous 2 months, any facial treatments, or photosensitizing or immunosuppressive drug application within the previous 6 months, extensive UV or other irradiation during the study, suntan, scars, birthmarks, skin cancer, chronic diseases (e.g. rosacea, psoriasis and acne) or strong hair growth on the face, poorly adjusted hypo-or hyperthyreosis, known allergic reactions to any of the ingredients of the test regimen, or concomitant participation to another clinical study were all exclusion criteria.
The subjects were allowed to follow their usual make-up, cleansing, and sunscreen routine and applied the treatment regimen for 12 weeks as follows (Table 1): • In the morning, day care cream with SPF 30; • In the evening, serum and night care cream.
The subjects were not allowed to use any face care product in the mornings of the study visits. The INCI compositions of the study products are described in Table 2.

Statistics
We analysed the data descriptively (number, percentage, mean, standard deviation [SD], minimum and maximum) and used the Wilcoxon matched-pairs signed-ranks test to compare visits and face sides. In the case of multiple comparisons, we used the Bonferroni-Holm correction of the level of significance. Differences were considered statistically significant at a P < 0.005 level.

Split-face study
Demographic characteristics Between April and August 2017, a total of 34 females aged 49.5 AE 8.5 years (range 25-64) were enrolled (Table 3).

Clinical photography
Clinical photography demonstrated a visible improvement of the hyperpigmentation from baseline to Week 12 after treatment with the combination of serum and day care SPF30 (Fig. 1).

Self-grading of skin condition
The self-assessment also demonstrated at all-time points statistically significant improvement of the mean hyperpigmentation scores of    Fig. 4). The hMASI decreased from 6.12 at baseline to 3.69 at week 12 at the face sides treated with the combination and from 6.42 at baseline to 5.15 at week 12 at the sides treated with the day SPF30 cream only.

Tolerability
The analysis of the tolerability assessments showed no statistically significant changes from baseline for any parameter (erythema, oedema, dryness, burning, stinging or itching) at any time point for both sides of the face.

Demographic characteristics
Between August 2018 and April 2019, a total of 83 subjects (82 females) with a mean age of 44.7 AE 9.2 years (range 27-71) were enrolled. Demographics are summarized in Table 3.

Clinical photography
The improvement of hyperpigmentation after 12 weeks of treatment with the treatment regimen (day care SPF30, serum and night care) was documented by clinical photography (Figs 5 and 6).

Clinical grading of skin condition
In the course of the study, the means of all the skin condition parameters as assessed by the investigators significantly improved in comparison with baseline and with the previous visits (P < 0.001; Fig. 7). At week 12, mean evenness improved by 93.8% (from 1.6 AE 0.9 to 3.1 AE 0.5), radiance by 94.1% (from 1.7 AE 0.7 to 3.3 AE 0.6) and smoothness by 50.0% (from 2.2 AE 1.0 to 3.3 AE 0.6) versus baseline. From baseline to week  12, the evenness improved in 94% (n = 78) of the subjects and remained unchanged in 6.0% (n = 5), the radiance improved in 92.8% (n = 77) and remained unchanged in 7.2% (n = 6), and the smoothness improved in 78.3% (n = 65), remained unchanged in 18.1% (n = 15) and worsened in 3.6% (n = 3) of the subjects.

mMASI
The mMASI improved significantly from 8.5 AE 3.9 at baseline to 3.6 AE 2.6 at week 12 (P < 0.001). The improvement was statistically significant also when comparing visits 2-4 to the corresponding preceding visits (P ≤ 0.001; Fig. 9).

Chromameter
The mean luminosity value (L* parameter) increased significantly (P < 0.001) from 58.4 AE 3.5 at baseline to 59.6 AE 3.2 at week 4, to 60.5 AE 2.9 at week 8 and to 60.7 AE 2.8 at week 12, corresponding to an average change of 4.02%. The differences were significant between all possible pairs of L* means (P < 0.001) (Fig. 10).  Subjective assessment of product performance Over 90% of the subjects rated all subjective performance questions positively, answering the respective questions with 'yes'. The corresponding results are listed in Table 4.

Tolerability
The investigators rated the tolerability of the product as very good in 73.5% (n = 61), as good in 22.9% (n = 19) and as satisfactory in 3.6% (n = 3) of the subjects.

Discussion
The split-face study demonstrated in the clinical setting and already after two weeks that four-times daily Thiamidol was visibly and significantly more effective than twice-daily in all assessments and corresponding time points. The release of the active from the two products (serum and day care) is expected to be comparable. Therefore, the results from the split-face study are indicating the benefit of a higher application frequency over a lower. The significant improvement continued until the end of the study in both sides of the face. The second study showed the efficacy, day-to-day acceptability and excellent tolerability, of a typical face care regimen containing the human tyrosinase inhibitor in a real-world setting. These results underline the real-life suitability of the products and their compatibility with the usual make-up and cleansing routines. The reduction of the MASI Score was in both studies significant, considerable, and in-line with earlier made observations [20]. The value of hMASI in the split-face study was smaller because, per definition, it represents the area of half the face. Duplication of the hMASI corresponds to mMASI in the real-world study. We chose the modified MASI because it was shown to be a reliable, valid and responsive to change method for the assessment of melasma severity. Besides, mMASI was demonstrated to be more convenient to use than MASI [25].
In addition to hyperpigmentation also skin roughness, evenness, radiance and smoothness improved significantly. Investigators and subjects confirmed the visible improvements, which were also documented by clinical photography and chromametry. The two latter standardized methods are objective, excluding interobserver variability and difficulty of remembering the baseline condition at the subsequent visit [24]. However, subjective assessments of efficacy are also essential to estimate compliance, especially for conditions such as hyperpigmentation, the treatment of which is challenging and frustrating for patients and physicians [5,10].
In the real-world study, the overall skin improvements and tolerability of the three-product regimen led to good overall acceptance. Over 90% of the subjects agreed that the products have a pleasant texture and are skin-compatible. Results, which are supporting adherence to the treatment. Therefore, we conclude the suitability of this three-product regimen for day-to-day life.
The studies were conducted on both sides of the hemisphere, covered most seasons except summer and various sun intensities to reflect real-world conditions. Based on an earlier study [20], in which half of the patients applied the treatment during the low ambient-UV time of the year and the other half during the high ambient-UV period, we expected no seasonal effects. Nevertheless, as UV-radiation can promote or worsen hyperpigmentation [28,29], sunscreen use is mandatory [30,31]. Therefore, SPF 30 was added in the day care product, which was used by all subjects, excluding thus possible confounding by sun exposure. Because  subjects were not allowed to use other face care products for the duration of the studies, we can assume that no other substances influenced the results. Furthermore, a safety assessment including photosensitivity tests demonstrated that Thiamidol does not induce UV sensitivity (data on file). As hyperpigmentation may have intrinsic or extrinsic triggers [32] affecting all ages, we included adult subjects of 25-65 years in the two studies. The studies' populations were too small for agespecific sub-group analyses, something to consider for the future. However, the lightening effect of Thiamidol on age spots was shown earlier [17]. Similarly, previous studies (and yet unpublished results) showed that the molecule is efficacious in all ethnicities [20]. Thus, in the two studies reported here, we did not focus on ethnic groups. Nevertheless, as hyperpigmentation has a higher prevalence in darker skin types [33], a future study could focus on those. Furthermore, because of the higher prevalence, we focused here on women. Only one man was included in the real-world study, not allowing sex-specific conclusions.
The results reported here supplement and confirm the so far with Thiamidol conducted investigations. Unfortunately, it was not possible to determine all parameters in every centre, affecting the comparability of the results. However, the overall design, duration, visit frequency and methods chosen here are typical for this kind of studies [34][35][36].
In conclusion, Thiamidol four-times daily is well tolerated and provides significant visible improvement of hyperpigmentation versus baseline and twice-daily application. The regimen approach with the three products (serum, day care SPF30 and night care) is also well tolerated, effective and suitable for the day-to-day life of people suffering from facial hyperpigmentation.