Efficacy of isobutylamido thiazolyl resorcinol for prevention of laser‐induced post‐inflammatory hyperpigmentation: A randomized, controlled trial

Q‐switched (QS) Nd: YAG laser is one of the treatment options for solar lentigines (SLs). However, the incidence of post‐inflammatory hyperpigmentation (PIH) is a common complication, especially in dark‐complexioned skin. Isobutylamido thiazolyl resorcinol (ITR) has been reported as a preventive modality for ultraviolet B (UVB)‐induced hyperpigmentation.


| INTRODUC TI ON
Solar lentigines (SLs) are common benign pigmented lesions associated with aging that affect more than 90% of Caucasians older than 50 years of age. 1,2The pathogenesis of SLs is melanocyte hyperproliferation and abnormal melanin retention within keratinocytes, resulting in hyperpigmented macule or patch. 3Numerous treatment modalities have been introduced for the treatment of SLs, which were categorized into topical and physical methods.For topical treatment, various options including hydroquinone (HQ), tretinoin, adapalene, and chemical peeling were commonly used. 2 However, these treatments usually take longer period to achieve a clinical improvement compared to physical therapies such as Q-switched (QS) lasers (e.g., alexandrite, ruby, Nd: YAG), 4,5 picosecond laser, 6,7 intense pulsed light, 8 and cryotherapy. 2r decades, a 532 nm QS Nd: YAG laser has been proved its efficacy in the treatment of SLs.However, one of the most observed complications of QS Nd: YAG lasers is post-inflammatory hyperpigmentation (PIH). 5,7,9,10PIH is caused by the overproduction and abnormal distribution of melanin within the epidermis or dermis that occurred following the cutaneous trauma or inflammation. 11It is characterized by the irregular-shaped macules or patches that can present in a light brown to gray color, depends on the depth of the lesion.PIH occurs in all skin phototype but more frequently affects individuals with dark, complex skin.It can cause a major cosmetic concern and have a negative effect on patients' quality of life. 11though there are several therapeutic options available for PIH, prevention is still the best method.For laser-induced PIH, few options exist regarding the prevention of this condition.
Recently, isobutylamido thiazolyl resorcinol (ITR) has been reported as an effective tool for the prevention of UVB-induced hyperpigmentation. 12erefore, we hypothesized that ITR might be able to prevent PIH due to other modalities as well.The aim of this study was to evaluate the efficacy and safety of ITR for the prevention of laserinduced PIH.

| Participants
A total of 24 healthy participants aged between 42 and 80 years who were clinically diagnosed with SLs by dermatologists were recruited.
Exclusion criteria included active skin infection at the lesions, immunocompromised status, history of skin cancer or keloids, history of laser treatment at the lesions within 3 months prior to the study, history of topical or systemic vitamin A and its derivatives used within 3 months prior to the study, pregnancy, and lactation.

| Treatment
Three SLs clinically similar in size and color to each participant were selected.A computer-generated, blocked-randomized sequence of the treatment groups was concealed in sequentially numbered, opaque, sealed envelopes.According to this random sequence, each participant's SLs were assigned by an investigator to either applied ITR-containing product (0.15% ITR, Eucerin® Ultrawhite spotless spot corrector, Beiersdorf AG, Germany) twice daily (Group A), once daily (Group B), or no application (Group C) for 2 weeks.On Day 14 of the study, all lesions were treated with a single session of 532nm Q-switched Nd: YAG laser (Revlite®, Hoya Conbio, Fremont, CA), with 3-mm spot size and an energy fluence of 0.8-1.5 J/cm 2 at 2 Hz.The clinical endpoint was immediate whitening.Aquaphor® (Beiersdorf AG, Germany) was applied to the treated area 4 times/ day until the crusts peeled off.Participants were advised to avoid sun exposure, apply sunscreen with sun protection factor (SPF) of 50+ and protection grade of UVA (PA) ++++.They were prohibited from concomitant used of other topical bleaching agents throughout the entire study.They were appointed for follow-up for 3 sessions post-laser treatment (i.e., Day 28, Day 42, and Day 70 of the study).

| Outcome evaluation
The primary outcome was the incidence of PIH clinically diagnosed by one blinded dermatologist who was uninvolved in treatment al-

| Characteristic of participants
Twenty-four patients aged 42 to 80 years (mean age: 64.75 ± 11.52 years) completed the study and were analyzed.There were 21 female (87.50%) and 3 male (12.50%) participants.Twelve subjects (50%) had Fitzpatrick (FPT) skin type III, 11 subjects (45.83%) had FPT skin type IV, and 1 subject (4.17%) had FPT skin type V. Three participants (12.50%) had a history of previous treatments with their SLs.All SLs were located on the upper extremities.Baseline mean RMI and mean L* of each group were not statistically significant difference.Twenty-two participants reported that they remembered to apply the medicine as instructed.However, two participants failed to apply it twice a week as required.Patients' characteristics were presented in Table 1.

| Incidence of PIH
The incidence of PIH was highest at 4 weeks post-laser in all groups, which were 5 (20.83%), 7 (29.17%),and 12 (50%) patients in group A, B, and C, respectively.In group A, the incidence of PIH was significantly lower than group C (p = 0.028).However, the incidence of PIH was not statistically significant difference between groups (16.67%, 8.33%, and 4.16%, respectively) at the end of the study.The data is shown in Figures 2, 3, and Table 2.

| Relative melanin index (RMI)
Mean RMI of all groups was statistically significantly lower at Days 28, 42, and 70 of the study when compared to their baseline (p < 0.05).The lowest RMI of each treatment were observed on Day 28 of the study, which were 0.02 ± 0.06, 0.02 ± 0.07, and 0.03 ± 0.06 in group A, B, and C, respectively.However, there was no statistically significant difference of mean RMI among three groups at any visits.The mean RMI of all treatment groups is shown in Table 3.

| Luminance score (L*)
Mean L* was statistically significantly increased in all groups at all follow-ups in comparison to baseline.There was not statistically significant difference between all groups of treatment at all-time points.Table 4 shows mean L* of each group.

| Hyperpigmentation score
Subjects' self-assessment of hyperpigmentation score rated by participants demonstrated an improvement of mean hyperpigmentation score in all groups of treatment.No significant differences in hyperpigmentation score were observed during all follow-ups (Table 5).

| Adverse events
There was no stinging, burning, or contact dermatitis on ITR-treated sites.Crust formation was seen in all laser-treated lesions, which was spontaneously peeled off in 7-14 days following the laser treatment.
Four patients (16.67%) reported mild erythema on laser-treated site after the crust peeled off but resolved without any additional intervention.
b n indicated the number of lesions.

F I G U R E 2
Comparison in the incidence of post-inflammatory hyperpigmentation between ITR twice daily, ITR once daily, and no application at 4 and 8 weeks postlaser.
One patient (4.16%) reported an occurrence of hypopigmentation after crust peeled off, which was spontaneously subsided within 1 month.No participant was required to withdraw early from the study.

| DISCUSS ION
The pathogenesis of SLs is related to chronic intermittent ultraviolet exposure, which results in increased melanin production and abnormal pigment retention by keratinocytes. 2,3The laser-based therapies are frequently used for the treatment of SLs.Of all the lasers modalities, QS 532-nm Nd: YAG laser is well-established method with excellent outcome. 4,5Despite its efficacy, there is a high a incidence of adverse effects such as PIH.Lasers-induced PIH is more frequently occur in people with darker skin complexion including Asian population.Its incidence has been reported as 10-47% in previous studies, which can cause a major cosmetic concern to patients. 9,10rious modalities have been introduced to prevent laserinduced PIH (e.g., topical corticosteroids, sunscreen, and tranexamic acid). 13,14However, the efficacy is still debated since treatment outcomes are generally unpredictable.Recently, ITR has  gained attention as an alternative treatment modality for hyperpigmentation since it was proclaimed as the most potent human tyrosinase inhibitor. 15,16According to previous studies, ITR represents an effective and safe ingredient against hyperpigmented lesions such as melasma and SLs. 17,18Moreover, it has been shown that ITR has an inhibitory effect against UVB-induced hyperpigmentation in human study. 12 our randomized controlled trial, the incidence of PIH was significantly lower in group A (i.e., ITR twice daily group) when compared to group C (i.e., no application) at 4 weeks post-laser (i.e., Day 42 of the study) with the incidence of 20.83% versus 50% (p = 0.028).However, the incidence of PIH was not significantly different between group B and group C (29.17% vs. 50%, p = 0.103).At 8 weeks post-laser (i.e., Day 70 of the study), the incidence of PIH was 16.67%, 8.33%, and 4.16% in groups A, B, and C, respectively.A delay onset of PIH in both group A and B is probably due to the inhibitory effect of ITR on human tyrosinase.However, after the application was discontinued, the tyrosinase inhibitory effect was declined.
In addition, ITR did not provide post-laser anti-inflammatory effect.
Therefore, anti-inflammatory agent should be co-administered as a part of post-laser care to prevent PIH. 13 In terms of RMI and L*, both were statistically significantly different when compared to baseline.However, no significant differences were observed between three groups of treatment at all visits.This could be due to a shorter duration of ITR application.In contrast to a previous study by Vachiramon et al., 12 a 3-week application of ITR twice daily showed a preventive effect of UVB-induced hyperpigmentation.In addition, a difference in the pathogenesis of laser-induced PIH and UVBinduced hyperpigmentation might contribute to this finding.
The pathogenesis of laser-induced PIH has been proposed to the principle of "sublethal laser irradiation".Initially, QS Nd: YAG produces high-energy radiation that transform into the rapid rising in temperature and destroy melanosomes in the pigmented area through the process called "selective photothermolysis". 19As a laser passed through pigmented tissue, non-pigmented surrounding dermal fibroblast was also co-incidental exposed to lower-energy level of laser irradiation and was biostimulated to release melanogenic cytokine and paracrine.It was confirmed by an in vitro study by Burd et al.,20 that this sublethal radiation from QS Nd: YAG laser stimulated fibroblasts and significantly upregulated the expression of some particular paracrine factors that have been known to play a pivotal role in the melanogenesis, such as stem cell factor (SCF), basic fibroblast growth factor (b-FGF), and hepatocyte growth factor (HGF), and a progressive increase in melanin content was also observed in cultured melanoma cell line in that study. 20,21Moreover, the inflammatory process caused by the thermal during laser treatment brings about the generation of cytokines, chemokines, and other inflammatory mediators.The presence of these molecules results in melanogenesis by increase tyrosinase activity, finally resulting in PIH as a result of laser irradiation. 22ereas the pathogenesis of UVB-induced PIH was explained by the upregulation of α-melanocyte-stimulating hormone receptor expression and activity, leaded to an elevated level of intracellular cAMP and then induced the expression of tyrosinase and its related protein. 23,24Therefore, PIH from laser irradiation was regulated by more processes involving with fibroblasts, keratinocytes, and melanocytes compared to PIH caused from UVB.Additionally, laser treatment may cause direct damage to the deeper part of the skin, resulting in PIH that is more challenging to treat.
Although a 2-week of twice-daily ITR application before laser treatment showed some positive effects for laser-induced PIH.
However, tyrosinase inhibitory effect is probably declined after discontinuation.6 to apply 0.33% brimonidine gel 1 h before performing QS Nd: YAG and picosecond laser could prevent PIH development.
Regarding TXA, the intralesional TXA injection provide the benefit for PIH prevention of SLs treated with 532-nm QS Nd: YAG laser. 27However, oral TXA can be used, but the results depend on the dosage.According to Kato et al., 28 oral TXA 750 mg/day did not prevent PIH from 694.5-nm QS Ruby laser.However, oral TXA 1500 mg/day showed a reduction in dermatoscopic pigment granules when compared to placebo group. 14r CO 2 laser resurfacing and fractional CO 2 laser, the use of sunscreen and petrolatum on the first day after fractional ablative CO 2 laser showed a positive effect in terms of PIH prevention when compared to petrolatum alone. 29However, the application of combined 10% glycolic acid, 4% HQ, and 0.025% tretinoin 4 weeks before ablative CO2 laser resurfacing did not provide the benefit in terms of PIH prevention. 30ilizing medical applications before laser surgery can significantly decrease the occurrence of PIH following the laser treatments.
However, the disadvantage is that they can be time-consuming.
Therefore, it is advisable to recommend this technique for individuals at risk of developing PIH from laser treatments, such as those with dark complexioned skin or those unable to avoid sun exposure after laser treatments.Previous studies demonstrated the efficacy of using ITR in preventing UV-induced hyperpigmentation. 12 terms of side effects, there were four patients (16.67%) developed some degree of erythema, and one patient (4.16%) developed hypopigmentation on QS Nd: YAG laser-treated site.These side effects were minor and tolerable.Two weeks application of ITR in our study did not produce any side effects resulting in the good compliance of patients.This emphasized the advantage of ITR over HQ, which can usually cause skin irritation and ochronosis.
There are some limitations in our research.Firstly, the small sample size that could have decreased the power in detecting the statistically significant difference among three groups of treatment.
Secondly, our study was conducted in patients who were treated with 532-nm Q-switched Nd: YAG laser only.The result may not be applicable to PIH associated with other types of laser or energybased devices.
In conclusion, our study demonstrated that two-week applica- This prospective, randomized, controlled, evaluator-blinded study was conducted at a university-based hospital (Ramathibodi Hospital, Mahidol University, Bangkok, Thailand).We performed our study in compliance with the Declaration of Helsinki and was approved by the Institutional Review Board of Human Rights Related to Research Involving Human Subjects, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Protocol number COA.MURA2021/701) and Thai Clinical Trials Registry (Identification number TCTR20230119003).All participants signed a written informed consent prior to study initiation.
location.PIH was defined by worsening of the pigmented lesion on digital photographs at Day 42 and Day 70 of the study (i.e., 4 and 8 weeks post-laser treatment) compared to the photograph at Day 28 of the study (i.e., 2 weeks post-laser treatment).Secondary outcome included the changes in color of SLs, determined by melanin index (MI), luminance score (L*), and patients' self-grading hyperpigmentation score evaluated at baseline and all follow-ups.MI was measured by a three-dimensional skin imaging (Antera 3D®, Miravex Limited, Dublin, Ireland).The difference between MI of the SLs and the surrounding normal skin was referred to relative melanin index (RMI), which indicated the intensity of pigmentation relative to normal skin around lesions.The lower RMI indicated that the color of the measured lesion was close to the normal skin.RMI was calculated as this following formula: RMI = (MI of SLs) -(MI of surrounding normal skin).Colorimeter (DSM II Colormeter®, Cortex Technology, Denmark) was utilized to perform objective assessment of the luminance score (L*), which represented skin lightness on a scale ranging from 0 (total black) to 100 (total white).We measured L* repeatedly three times and calculated the mean luminance score (mean L*).All subjects were requested to rate a self-grading hyperpigmentation score based on a 10-cm visual analogue scale (VAS) since Day 28 (2 weeks after laser).The score ranges from 0 to 10 points, where 0 is comparable to surrounding normal skin and 10 is for the darkest lesion.The protocol flow chart is presented in Figure 1.

2. 5 |
Statistical analysisCategorial data (e.g., gender, underlying disease, Fitzpatrick skin type) were expressed as numbers and percentages.Continuous data (e.g., mean L*, RMI) were presented with mean and standard deviation (SD).Mixed-effects linear regression model was used to test continuous variables, and mixed-effects logistic regression was used to test proportions between ITR-treated side and control side on each visit.Two-sided p-values less than 0.05 were considered statistically significant.All statistical tests were analyzed using STATA version 14.0 (StataCorp LLC, College Station, TX).

F I G U R E 3
Clinical photographs of 3 solar lentigines of a 63-year-old female at baseline, assigned to applied ITR twice daily at baseline, 2-weeks, 4-weeks, and 8-weeks post-laser (A-D); ITR once daily at baseline, 2-weeks, 4-weeks, and 8-weeks post-laser (E-H); and no application at baseline, 2-weeks, 4-weeks, and 8-weeks post-laser (I-L).ITR, isobutylamido thiazolyl resorcinol.TA B L E 2 Incidence of PIH between ITR twice daily (Group A), ITR once daily (Group B) and no application (Group C), assessed by a blinded dermatologist at Day 28, 42, and 70 (i.e., 2, 4, and 8 weeks post-laser).
Patients with FPT skin IV to VI needed 12 weeks to meet statistically significant difference in terms of PIH improvement, whereas a significant improvement at 2 weeks was observed in patients with FPT skin II to III.This study reflected that people with darker skin complexions may need a longer duration of ITR application to achieve clinical improvement.Based on the aforementioned pathophysiology of PIH, which described about inflammatory process that can increase melanocytic activity, a previous study from Cheyasak et al.13 revealed that 2 days application of topical corticosteroids immediately after laser treatment has shown significant reduction of incidence of PIH.Therefore, we suggested that the combination of ITR and topical corticosteroids may produce an additional synergistic effect to optimize the benefit in prevention of PIH, as both agents target on different pathogenesis of laser-induced PIH.Apart from topical corticosteroids, topical brimonidine, intralesional tranexamic acid (TXA) injection, oral TXA, and sunscreen application also provide some positive effects on the prevention of laser-induced PIH.According toLee and Hong   et al., tion of ITR prior to QS Nd: YAG 532-nm laser treatment may provide some benefit for the prevention of laser-induced PIH.We recommend the ITR application should be continued after laser treatment to further inhibition of tyrosinase.Longer duration of application including continuous application after the laser treatment, and coadministration with other anti-inflammatory agents may resulted in more effective prevention of laser-indued PIH.Further study with larger sample size, longer duration of ITR administration, and with different types of laser and energy-based devices is warranted to evaluate the efficacy of ITR for the prevention of PIH.
TA B L E 1

Day Incidence of PIH Group A, n (%) Group B, n (%) Group C, n (%) p-Value (A vs. C) p-Value (B vs. C) p-Value (A vs. B)
*p-Values <0.05 are considered statistically significant.