Vitamin D supplementation can enhance therapeutic effects of excimer laser in patients with vitiligo

Vitiligo is a disease of acquired depigmentation characterized by the destruction of melanocytes. A theoretical association between low level of 25‐hydroxyvitamin D [25(OH)D] and vitiligo has been previously suggested. The objective of this study was to determine the efficacy of intramuscular injection of cholecalciferol with excimer laser compared with the excimer laser alone for vitiligo treatment.

the detection of anti-melanocyte autoantibodies that activate autoreactive cytotoxic CD8 + T cells. 5The treatment of vitiligo is still challenging.Topical medications (topical corticosteroids or topical calcineurin inhibitors), oral medication (systemic corticosteroids), and phototherapy (psoralen-UVA, narrowband UVB, and excimer laser) are treatment modalities, whereas surgical intervention (epidermal grafting, autologous mini-graft, and dermabrasion) can be considered in patients with refractory vitiligo. 6[9] Previously, there has been a suggested association between vitamin D deficiency and vitiligo. 10Theoretically, vitamin D can influence the pathogenesis of vitiligo.Vitamin D influences melanocyte maturation, differentiation, and melanin synthesis. 11,12In addition, vitamin D has been implicated in the regulation of immune cell proliferation and differentiation.Accumulated data suggest that vitamin D deficiency is associated with a higher risk of autoimmune diseases. 13,14Vitamin D performs physiological functions by interacting on the vitamin D receptor (VDR).VDR genes are located on chromosome 12q12-14 and are known to have more than 25 different polymorphisms. 157][18][19][20] VDR gene polymorphisms (Apal, FokI, TaqI, and BsmI) can influence the risk of vitiligo. 18,20,21searchers have suggested an association between the efficacy of therapeutic modalities for vitiligo and serum 25-hydroxyvitamin D [25(OH)D] levels.Previous studies have demonstrated that narrowband UVB phototherapy increases serum 25(OH)D levels in patients. 22,23After phototherapy, patients with a low initial serum 25(OH)D levels experienced a significant increase in their serum 25(OH)D levels.However, patients with a high serum 25(OH)D level did not show significant changes in their serum 25(OH)D levels. 24e increase in vitamin D levels after phototherapy may improve the therapeutic effects of phototherapy.In addition, studies have shown that topical vitamin D analogs are effective in patients with vitiligo. 25 (3) patients with diseases that can affect the kidneys, such as chronic kidney disease, hyperparathyroidism, or hypoparathyroidism; (4) patients with uncontrolled chronic diseases such as diabetes mellitus, hyperlipidemia, and hypertension; and (5) patients with spot urine calcium levels >30 mg/dL.

| Study design
This prospective, randomized controlled study was approved by the Institutional Review Board of Hallym University Kangnam Sacred Heart Hospital (IRB no.2018-04-017).This study has been registered at ClinicalTrials.gov(NCT05364567).All participants consented to participate in the study and provided written informed consent.All eligible participants were randomly divided into two groups: control and study.Based on previous research, we utilized an online randomization site (http:// www.graph pad.com/ quick calcs/ index.cfm). 26th groups underwent treatment using a 308-nm excimer laser and tacrolimus 0.1% ointment (Protopic, LEO Pharma, Denmark).All the patients received topical tacrolimus ointment twice daily.The study group received an additional vitamin D supplementation via a 200 000 IU intramuscular cholecalciferol injection.Although there may not be a universally standardized dosage for cholecalciferol supplementation, we determined our dosage based on previous studies that demonstrated therapeutic effects. 27,28The total study duration was 6 months, and clinical assessments were conducted monthly.

| Phototherapy
The treatments were administered twice a week, not on consecutive days, for 6 months.Phototherapy for treating depigmented areas was performed using a 308-nm xenon chloride excimer laser (E1, Jetema, Seoul, Korea).The initial dose for the excimer laser was decided based on the location of the depigmented lesion with consideration of the previous results of phototherapy 29,30 : eye = 100 mJ/cm 2 , head, face, ear, neck, axilla = 150 mJ/cm 2 ; arm, leg, trunk = 200 mJ/cm 2 ; wrist = 250 mJ/cm 2 ; elbow, knee = 300 mJ/ cm 2 ; hand, foot = 400 mJ/cm 2 ; finger, toe = 600 mJ/cm 2 ; if the erythema disappeared within 24 h, the energy dose was increased by 50 mJ/cm 2 for each treatment.However, if the erythema sustained for 24-48 h, the energy dose was maintained.If the erythema lasted longer than 48 h, the energy dose was reduced by 50-100 mJ/cm 2 in each session.If blistering or pain occurred, the treatment was omitted until symptom resolution was achieved.

| Outcome measures
Clinical evaluation using photographs was performed each month after treatment initiation.The blind evaluation of depigmented lesions was based on the Vitiligo Area Scoring Index (VASI) and physician global assessment (PGA). 31One blinded trained dermatologist evaluated the VASI and PGA using clinical photographs.The VASI is a quantitative clinical tool used to evaluate vitiligo.The VASI score was calculated by multiplying the area and extent of depigmentation.The total body VASI score was calculated by summing the contributions of all body regions.The area and extent of the depigmentation were assessed.The PGA score was calculated to assess improvement after treatment.The score was assigned on a scale from 0 to 4: 0 = 0%, 1 = 1%-25%, 2 = 26%-50%, 3 = 51%-75%, and 4 = 76%-100%.In addition, the presence of adverse events was assessed during each visit.

| Statistical analysis
Considering that the estimated prevalence of vitiligo in Korea is about 0.12%-0.13%,we set a confidence level of 99% with a margin of error of 2.5%. 32Based on this calculation, we set the sample size to 13 for each group.All continuous data are shown as mean ± standard deviation (SD), and categorical variables are expressed as numbers (%).The differences in the VASI scores between the study and control groups were evaluated using the Student's t-test based on the normality test.Comparisons of therapeutic efficacy according to location were evaluated using the Mann-Whitney U-test.To analyze the associations between vitamin D supplementation and improvement, we evaluated the categorical outcomes (VASI50, VASI75, and PGA) using the chi-squared test.Statistical significance was set at p < 0.05.All statistical analyses were performed using IBM SPSS Statistics (version 24.0;IBM Co., Armonk, NY, USA).
The patients were Korean and had Fitzpatrick skin type III or IV.The average duration of vitiligo in patients who completed the study was 19.01 ± 15.17 months and the average serum 25(OH)D level was 13.8 ± 4.0 ng/mL.There were no significant differences in sex ratio, age, duration of vitiligo, or serum 25(OH)D levels between the control and study groups (Table 1).The average cumulative UVB dose was 14.1 J/cm 2 with doses ranging from 3.5 to 45 J/cm 2 , and the corresponding values of the phototherapy with vitamin D supplementation group and phototherapy only group were 17.3 and 11.3, respectively.A total of 126 phototherapies were administered to the treated area.

| Vitiligo Area Scoring Index
We examined the treatment effect by evaluating the change in VASI based on the baseline score.A statistically significant

| The area and the extent of repigmentation
The extent of the depigmented area ranged from 0.3% to 6%.Both groups showed improvement in the extent of repigmentation after treatment (Table 3).All patients in the study group showed at least 10% improvement in the extent of repigmentation after 3 months of treatment, on the contrary, 35.7% of the patients in the control group did not show such improvement (p = 0.021).Regarding the change in area after 3 months of treatment, 66.7% of the study group showed an improvement of more than 25%, while 28.6% of the patients in the control group did not show such improvement (p = 0.05).After 6 months of treatment, 75% of the study group showed more than 50% improvement in the extent of repigmentation; however, only 42.8% of the control group showed improvement in repigmentation, although the difference was not statistically significant (p = 0.098).

| The comparison of therapeutic efficacy according to the location
Since the therapeutic efficacy of excimer laser can differ depending on the location or site of vitiligo, we compared the efficacy according to the site of vitiligo between the two groups (Figure 3).For this analysis, we classified patients into those with only the head or neck and those with other sites.At Month 1, the study group with head or neck involvement showed significantly greater reduction in VASI scores compared with the control group with head or neck (59.4% vs. 12.4%; p = 0.044, Figure 3A).After 6 months of treatment, the VASI score showed an 84.5% improvement in the initial score in the study group with only the head or neck and 55.6% in the control group with only the head or neck, although this was not significant.
At Months 2 and 6, the study group with other sites showed significantly greater improvement in mean percentage change in VASI scores compared with the control group involving other sites (58.2% vs. 29.3% and 83.4% vs. 53.5%;p = 0.031 and p = 0.042, Figure 3B).

| Physician global assessment
The number of patients in each group classified using the PGA scale was analyzed (Figure 4).When comparing the percentage of patients reaching PGA ≥3, the study group showed a higher ratio than the control group after 3 and 6 months of treatment (p = 0.02 and p = 0.009).

| Adverse events
During the study period, seven patients (26.9%) experienced long-lasting erythema or erythema with a burning sensation after phototherapy.However, all patients completed the treatment safely without a discontinuation owing to adverse effects.

| DISCUSS ION
Low serum vitamin D levels have been suggested to increase the risk of autoimmune diseases, such as multiple sclerosis, type 1 diabetes mellitus, and systemic lupus erythematosus. 16In recent meta-analyses, serum 25(OH)D deficiency also showed an association with the risk of vitiligo, featuring an autoimmune nature. 18,19,33However, studies on the use of topical or systemic vitamin D for the treatment of vitiligo are limited.Patients with pediatric vitiligo who received calcipotriol, a topical vitamin D analog, in combination with phototherapy, showed favorable repigmentation. 34,35In addition, previous study has shown that phototherapy combined with topical calcipotriol has more favorable efficacy in adult patients than phototherapy only. 25Little research have shown the efficacy of oral vitamin D supplementation in treating vitiligo. 36,37  receptors and vitamin D can be directly involved in regulating melanin synthesis. 11In an in vitro study, melanocytes cultured with vitamin D increased the size of dendrites, tyrosinase levels, and number of dendrites. 12It is probably due to the ultraviolet irradiation that causes the skin pigmentation by photochemically converting pro-vitamin D to vitamin D. Fourth, the low serum calcium level, which can be induced by low serum vitamin D level, has a role in the pathogenesis of vitiligo.Previous study showed that vitiligo patients had the decreased serum calcium levels compared with controls. 44rthermore, it is speculated that decreased intracellular calcium inhibits tyrosinase activity in melanocytes, resulting in decreased melanin synthesis via perturbed redox status. 45 this study, intramuscular vitamin D supplementation with phototherapy showed more favorable results in vitiligo patients with low levels of 25(OH)D than in those who received phototherapy alone.
Phototherapy has been widely recognized as the primary treatment modality for vitiligo.However, phototherapy has limitations, such as long treatment durations of several months to years, which sometimes result in unsatisfactory outcomes.Our study showed a significantly superior therapeutic effect of combined therapy with vitamin D and phototherapy compared with phototherapy only 1 month after initiating the treatment, and this effect was sustained until 6 months of treatment.
The effects of excimer laser vary depending on the location of vitiligo. 46Therefore, this study classified patients with lesioned sites and analyzed the therapeutic efficacy according to the location.We found that vitamin D exerted a synergistic effect, regardless of location.At Month Change in the area of repigmentation the injection of cholecalciferol that we employed in this study. 27,28 conclusion, vitamin D supplementation produced synergistic effects with phototherapy in the treatment of vitiligo with vitamin D deficiency.At the first visit to the clinic, we suggest that patients with

2 | ME THODS 2 . 1 |
phototherapy in treating vitiligo patients with low serum 25(OH)D levels compared with those treated with phototherapy alone.
Vitamin D deficiency was evaluated based on serum 25(OH)D levels.Serum 25(OH)D levels were measured using chemiluminescent immunoassay (Atellica Solution; Siemens Healthineers, Erlangen, Germany).Participants with serum 25(OH) vitamin D levels below 20 ng/mL were defined as having a vitamin D deficiency.The study group with vitamin D deficiency was administered an intramuscular injection of 200 000 IU cholecalciferol (Kwangdong Pharmaceutical Co., Seoul, Korea) once at baseline.

TA B L E 1
Figure 2.

TA B L E 2 F I G U R E 2
phototherapy and phototherapy with vitamin D supplementation groups.Since there have been reports demonstrating no significant difference in improving vitamin D levels between oral and injection forms, this study chose the injection form considering patient compliance.27,38,39After 3 and 6 months of treatment, the proportion of patients who reached VASI75 was significantly higher in the study group than in the control group.In our study, the VASI score showed an 83.6% improvement in the initial score in the study group and a 54.7% improvement in the control group after 6 months of treatment.Although the mechanism of action of vitamin D remains unclear, several mechanisms underlying its role in vitiligo pathogenesis have been suggested.First, low levels of serum vitamin D are associated with Th1 mediated autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.The serum levels of 25(OH)D can not only affect the risk of autoimmune disease but also affect disease activity. 40Second, vitamin D plays a role in regulating the proliferation and differentiation of immune cells, resulting in the modulation of immune activity.Because immune cells, including T cells, B cells, dendritic cells, and macrophages, express the vitamin D receptor, vitamin D can regulate immune responses. 41-43Third, melanocytes expressing 1,25(OH) 2 D

3
Treatment evaluation criterion based upon the change in the extent and area of repigmentation.involvement attained a significantly greater reduction in mean percentage change in VASI scores compared with the control group with other sites involvement.After 6 months of treatment, the study group achieved improvements of 84.5% (head or neck) and 83.5% (other sites), while the control group showed improvements of 55.6% (head or neck) and 53.5% (other sites).Our study showed that vitamin D supplementation can enhance the therapeutic effect of phototherapy in patients with vitiligo and vitamin D deficiency.Furthermore, according to a previous systematic review paper, the effectiveness of topical vitamin D analogs can vary depending on their combination with specific types of phototherapy.47This study demonstrated that applying topical vitamin D analogs was effective with narrowband UVB but not with excimer laser.Since our study confirmed the beneficial effects of intramuscular vitamin D supplementation in combination with excimer laser therapy, our method of vitamin D supplementation differs from that discussed in the systemic review on the therapeutic outcomes of topical vitamin D and phototherapy.In the future, it is important to assess the therapeutic benefits of the combined treatment approach with narrowband UVB and intramuscular vitamin D administration.This study had some limitations.First, this study had a small sample size.Further large-scale studies are necessary to validate the therapeutic effects of vitamin D supplementation in patients with vitiligo.Second, we could not measure changes in serum 25(OH)D levels after vitamin D supplementation.However, previous studies have shown a significant improvement in serum vitamin D levels with

F I G U R E 3
Changes in mean VASI (Vitiligo Area Scoring Index) in percentage according to the involved area.(A) At Month 1, the study group with head or neck involvement showed significantly greater reduction in VASI scores compared with the control group with head or neck involvement (p = 0.044).(B) At Months 2 and 6, the study group with other sites involvement showed significantly greater improvement in mean percentage change in VASI scores compared with the control group with other sites involvement (p = 0.031 and p = 0.042).*p < 0.05, compared with the control group.vitiligo should check their serum 25(OH)D levels and be recommended adequate vitamin D supplementation.Intramuscular injection of cholecalciferol can be a safe supplementary option to phototherapy for treating patients with vitiligo and vitamin D deficiency.