Assessing effectiveness of adding niosomal atorvastatin 1% ointment to topical calcineurin inhibitor treatment in non‐segmental vitiligo

Treatment of vitiligo is still a big challenge for dermatologists. The efficacy of statins in the treatment of vitiligo is controversial.


| INTRODUC TI ON
Vitiligo is a chronic dermatosis with an incidence rate of 0.5-1%.
Vitiligo can have detrimental effects of health-related quality of life (QoL).Aetiologic factors include genetics, autoimmunity, and biochemical, neurochemical, and environmental factors.[3][4][5][6] Autoreactive melanocyte-specific lymphocytes, CD8+ lymphocytes secreting interferon gamma (IFN-ƴ), tumor necrosis factor α (TNFα), and other T helper-1(TH-1) related cytokines along with increased interleukin (IL)-17 have a central role in pathogenesis.[5][6][7][8] Although there are various therapeutic methods for vitiligo, treatment is still a big challenge for dermatologists.One major problem is acral depigmentation for which treatment can be more challenging. 2,3,9,10Common topical options are topical corticosteroids (TCs) and topical calcineurin inhibitors (TCIs). 11Long-term usage of these drugs might be expensive and also have complications.Also, these drugs are not equally effective in different parts of the body; for instance, the best treatment response with TCIs have been observed for face and neck lesions. 12Patients under treatment with other methods such as phototherapy, laser therapy, and systemic immunosuppressants usually do not show proper long-term response. 2,3,9,10atins are competitive inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG CoA) reductase and have prophylactic and therapeutic use in cardiovascular disorders. 13There are increasing evidences about their anti-inflammatory and immunomodulatory roles.
5][16][17] Moreover, it has been reported that statins, especially atorvastatin, simvastatin, and pravastatin are effective in faster healing of ulcers. 18,191][22] In this pilot randomized placebo-controlled trial (RCT), we aim to investigate any possible therapeutic effect using topical 1% niosomal atorvastatin combined with topical 0.1% tacrolimus compared to placebo combined with 0.1% tacrolimus in treatment of nonsegmental vitiligo.

| Materials
Polyoxyethylene (2) cetyl ether (Brij® 52), cholesterol, and atorvastatin calcium were purchased from Sigma-Aldrich (USA).All other chemicals and solvents were of analytical grade and obtained from Merck (Germany).We used 0.1% tacrolimus cream (Tacril®, Tehran Chemie, Iran) for comparison in both intervention and control groups.
Atorvastatin, cholesterol, and Brij 52 were dissolved in 5 mL of chloroform, and the organic solvent was evaporated by a rotary evaporator (Heidolf, Germany) at 65°C.Normal saline was used as the hydration medium and mixed with the above thin lipid film at 65°C for 45 min.Niosomal suspension was observed by optical microscope (Olympus, Japan) as round and multilamellar vesicles (MLVs) without any aggregation.The prepared niosomes were mixed with Eucerin to form hydrophilic ointment of lipid vesicular atorvastatin.The patients were randomly allocated to two different groups based upon the random number table.The intervention group received topical 0.1% tacrolimus cream and topical 1% niosomal atorvastatin ointment, twice a day.Patients were requested to apply the two formulations with at least 1 h interval.The patients in control group were prescribed the 0.1% tacrolimus cream and placebo ointment (which looked exactly the same as niosomal atorvastatin ointment).The investigators, the patients, and the person who gave the patients the medications did not know which one was placebo.

| Study design
Drugs were stored at room temperature.The data were analyzed using the SPSS® software, version 18 (SPSS Inc, Chicago, Il, USA).The statistical significance level was a p value <0.05.For comparison, we used mean values between the groups and due to the fact that our data had a normal distribution, we used Student's t-test for our analyses.

| RE SULTS
From our 20 patients, 13 were female (65%), nine of which were in the intervention group (90%).The mean age of total participants was 34.9 ± 12.36.Mean age for intervention and control groups was 37.5 ± 13.7 and 32.3 ± 10.9, respectively.Duration of vitiligo in intervention was 2.05 ± 1.86 years, and for the control group, it was 1.68 ± 1.06 years.Minimum and maximum durations in the intervention group were 0.4 and 6 years, respectively.Minimum and maximum durations in the control group were 0.3 and 3 years, respectively.
At the final visit, we re-assessed the VASI score.The minimum, maximum, and mean of VASI scores before and after treatment are shown in Table 1.
We found statistically significant difference in VASI score before and after treatment in both intervention and control groups with p values of 0.01 and 0.03, respectively (Figures 1 and 2).However, comparison of the VASI score change between groups was not statistically significant (p = 0.62).
In terms of adverse reactions at the end of the study, no patient reported pain or redness.Two patients in the control group had pruritus.Two patients in the intervention group and one patient in the control group reported burning sensation.There was no significant difference between the groups in terms of pruritus and burning and VASI score (p values 0.9 and 0.2 for pruritus and burning, respectively).
The mean patient satisfaction in the intervention group was 5 ± 1.4, and for control group, it was 3.5 ± 1.9; the difference between groups was not statistically significant (p = 0.9).
There was no significant difference between gender and VASI score between groups (p = 0.16 for intervention group and p = 0.19 for control group).
There was no significant correlation between VASI score and vitiligo type in each group; neither was a significant correlation between intervention and control groups (p = 0.39 for intervention group and p = 0.35 for control group; p = 0.9 for between groups analysis).Also, we could not find any statistically significant correlation between VASI score and disease duration (p = 0.54 for intervention group and p = 0.74 for control group).It was the same for age (p = 0.60 for intervention group and p = 0.52 for control group).
At the end, by assessing the laboratory results, we could not find any significant correlation in each group and between groups regarding any of the measure laboratory values (data will be accessible on request).

| DISCUSS ION
In our study, VASI score changed significantly before and after treatment in each group; however, there was no significant difference between intervention group and control group regarding VASI score.
We also found no significant difference between VASI score and gender, age, disease duration, laboratory data, and patients' satisfaction.We also did not have any significant adverse effect in each group.
Niezgoda et al in 2019 investigated repigmentation of vitiligo lesions after using atorvastatin and/or simvastatin ointments in 24 patients with active vitiligo in extremities for 3 months.They did not find any significant improvement in vitiligo lesions based on VASI scores. 9Their findings were in line with our analyses because we also did not find a statistically significant change in VASI score when atorvastatin was compared to placebo.However, the major difference between our study and the abovementioned study is the fact that they did not have a control group, and they only reported the observations in each patient before and after treatment.however, comparison between the control group and the intervention group showed no significant difference. 26The difference between their study and ours is the route of statin administration and also the topical treatment used as an adjunct.They also had a much larger sample size which provides their study with more power.

Variables
Vanderweil et al in 2017 studied the effects of oral simvastatin (40 mg daily for the first month and 80 mg for the remaining 5 months) in 15 vitiligo patients.They reported a 26% increase in the mean VASI score after 6 months of treatment in the treatment group (95% confidence interval −45:97%) and 0% change for the control group (95% confidence interval −5:5%) but the differences between groups were not significant (p = 0.094); the worsening in the treatment group was a result of nine patients in the inflammatory phase of the disease. 22ese results are contradictory to our study and the other studies.
There was worsening of disease during the time of study.
In F I G U R E 2 Right, before treatment; left, 50% residual pigmentation at the end of treatment in placebo group.
This is a triple blind, pilot, placebo-controlled clinical trial performed in patients with non-segmental vitiligo diagnosed by a dermatologist at dermatology clinic in 2020-2021.The inclusion criteria are non-pregnant, non-breastfeeding individuals more than 18 years of age, with no other systemic or cutaneous diseases.The exclusion criteria include patients with more than 20% involvement in body surface area, any hypersensitivity reaction to statins, inflammatory skin disorders, taking oral statins or any immunosuppressive treatment in the previous 8 weeks, phototherapy in the previous 4 weeks, alcohol or illicit drug use and skin cancers in the previous 5 years.The patients who fulfilled the inclusion criteria and did not have any exclusion criteria were chosen; given the appropriate information about the study and after their agreement, they signed the informed consent form.Patients were informed that they should not use systemic statins, or phototherapy throughout the study period and sun-tanning was prohibited during the study period.If they were required to start any new medication, they should inform the investigators.
At the first session, the investigators recorded the VASI score,23 took photographs of lesions, and ordered the required laboratory values including complete blood count (CBC) with differential, blood urea nitrogen (BUN), creatinine, liver function tests (LFTs), thyroid function tests (TFTs), creatine phosphokinase (CPK), c-reactive protein (CRP), erythrocyte sedimentation rate (ESR), lipid profile, and beta human chorionic gonadotropin (β-HCG) for women in reproductive age.VASI measures the affected body surface and has a range of 0% (lowest score) to 100% (highest score).It is determined by multiplication of hand units (HU) in residual pigmentation (RP).Body surface is divided into head and neck, upper extremities (including axillae) except hands, trunk, lower extremities (including buttocks and inguinal areas) except feet, hands, and feet.Each HU is equal to 1% surface involvement.RP has a range of 0%-100% and is determined as follows: RP 100% means full depigmentation, RP 75% means there is more depigmentation than pigmentation, RP 50% means pigmentation and depigmentation areas are roughly equal, RP 25% means pigmentation is more that depigmentation, and RP 0% means there is no area of depigmentation.At the second session 1 month later, we measure the amount of ointment they had used and if required, we prescribed more of the drugs and assessed and recorded any adverse reactions.At the last session at 3 months after the start of the trial, we re-calculated the VASI score, patients satisfaction [based on VAS (verbal analog scale, one being the least satisfaction and 10 being the most satisfaction)], took photographs of the lesions at the same room, same level light, and the same camera.We also reordered the laboratory data at the end of the trial.
In a RCT performed by Nguyen et al in 2017, the investigators compared topical simvastatin combined with narrowband UVB (NB-UVB) phototherapy with NB-UVB phototherapy alone.They reported the results of 29 patients and used VASI score for assessment 6 months after the start of the trial.VASI score for control group was 2.95 ± 1.39 (p = 0.04) and for intervention group was 4.38 ± 1.44 (p = 0.005); however, they found no significant correlation in VASI score change between groups.They also used VETF (vitiligo European Task Force) spreading score for their assessment and using this tool, they did not find any significant VETF score change either in each group or between groups.24Although they had a different regimen for combination therapy, their main finding was in accordance to what we have found.Zhang et al in their 2020 study on five vitiligo patients used oral simvastatin with different dosages (mostly 20 mg/day, with some patients receiving 40 mg/day) combined with topical simvastatin.They used VETF tool for assessment at 4 and 8 weeks after treatment.Their results show a significant improvement in three patients and no change in the other two.None of the participants reported an adverse effect and based upon their results the investigators stated that oral simvastatin was safe with potential effectiveness.25 Their regimen for treatment was the same between our study and Zhang study; however, the routes of administration were different.The other differences between this study and ours are their smaller sample size and having no control.In Iraji et al study in 2017, oral simvastatin 80 mg/day combined with topical betamethasone ointment was compared with betamethasone ointment alone on 88 patients with more than 20% body surface area involvement for 12 weeks.They used VASI score for assessment at 4, 8, and 12 weeks after treatment.They found a significant VASI score change in both control and intervention groups; 2015, Agarwal et al investigated the effects of intraperitoneal injection of simvastatin in repigmentationor prevention the induction of vitiligo lesions in mice models.They reported both prevention and reversal of depigmentation in mice and hypothesized that this might F I G U R E 1 Right, before treatment; left, less than 25% residual pigmentation at the end of treatment in niosomal atorvastatin group.CLI N I C A L TR I A L R EG I S TR ATI O N N U M B E RIRCT20200714048099N1.E TH I C S S TATEM ENTThis study was approved by the local ethical committee of Kerman University of medical sciences with ethical code IR.KMU.AH.REC.1398.132.andregistered in the Iranian registry of clinical trial with IRCT20200714048099N1. O RCI D Mahin Aflatoonian https://orcid.org/0000-0002-9373-8682R E FE R E N C E S 1. Farajzadeh S, Aflatoonian M, Mohammadi S, Vares B, Amiri R. Epidemiological aspects and disease association of childhood vitiligo.J Pakistan Assoc Dermatol.2015;25(2):105-110.