Patients fulfilling the inclusion criteria will be recruited after their informed written consent (Appendix 1 and 2). A detailed clinical history and examination will be done, and findings recorded in a pre-designed proforma (Appendix 3). The vitiligo patch selected for NCES will be clinically photographed using a smart phone or a hand-held camera in a well-lit room, in a uniform setting without magnification with standard setting of contrast and brightness. The size of the vitiligo patch will be estimated using the graph paper method. Dermoscopic examination of the selected vitiligo patch will also be done and photograph taken, and findings recorded in a pre-designed proforma (Appendix 3). A 5 ml venous blood sample will be collected under aseptic precautions for complete blood count and serum biochemistry, and a Mantoux test will be performed.

Randomization, allocation concealment and blinding

Patients will be randomized to receive either tofacitinib (Group A) or placebo (Group B) using block randomization method with variable block size. Numbers will be prepared by computer generated random tables. Envelopes will be opaque and sealed with each containing details of the drug. These envelopes will be made by a person not directly or indirectly involved in the study. One envelope will be taken per patient and opened by one of the investigators (who is not involved in performing NCES or evaluation of any outcome measures) and the medicine (tofacitinib or placebo) will be prescribed based on the group to which the patient has been randomized. The patient, dermatologist performing the NCES and the investigator evaluating the treatment response will be blinded. 

4 mm skin punch biopsies will be performed from the vitiligo patch selected for NCES and subjected to RT-PCR and ELISA analysis.

Treatment protocol

Group A: Oral tofacitinib 5mg BD x 6 months, OD x 3 months

Group B: Oral placebo BD x 6 months, OD x 3 months

Source of drugs for the study

Tofacitinib will be provided to the patients free of cost for the duration of study by INTAS PHARMACEUTICALS. They will not provide any financial support, and will have no role in data collection, interpretation of results, statistical analysis, manuscript preparation or submission to journal for publication.

Patients in both the treatment groups will undergo NCES after 2 months of receiving tofacitinib or placebo. 4 mm skin punch biopsy will be repeated from the vitiligo patch selected for NCES before the procedure, and subjected to immunofluorescence and flowcytometric analysis and RT-PCR.

Drug adverse effects will be evaluated by clinical evaluation and monitoring of complete blood count and serum biochemistry at monthly intervals for the first 3 months and then at 6 months.

Transplantation of the non-cultured epidermal cell suspension

Harvesting the graft

About one-fourth to one-tenth the size of the recipient area will be selected as the donor site, usually on non-cosmetically important site, like the thighs, buttocks or waist. Under aseptic precautions and EMLA (lignocaine+ prilocaine) topical anesthesia or local infiltration of 1% lignocaine with adrenaline, a split thickness skin graft (STSG) will be taken from donor site. The recipient site will be covered with a paraffin gauze, gauze piece and surgical pad and the dressing will be secured using adhesive tape.

Preparation of NCES

The STSG will be transported to the laboratory in PBS in a sealed sterile centrifuge tube after proper labelling. There, the skin graft will be transferred to Trypsin-EDTA solution (0.25% trypsin and 0.05% EDTA, (Gibco BRL) in a Petri dish and incubated at 37°C for 90 minutes to separate the epidermis from the dermis. After 45 minutes, the trypsin– EDTA solution will be pipetted out and phosphate buffered saline (PBS) will be added and the tissue teased with sterile forceps so as to separate the cells from the tissue. The solid waste of tissue will be removed and the suspension will be centrifuged at 1000 rpm for 10 minutes. The supernatant will then be discarded and the pellet, containing cells from the stratum basale and lower half of the stratum spinosum that are rich in melanocytes and basal keratinocytes will be taken.

Transplanting the NCES

Under strict asepsis, the recipient vitiliginous areas will be anaesthetized by lignocaine 1% or topical EMLA. It will be dermabraded using diamond burr until uniform pinpoint bleeding is noted. After that, NCES suspended in PBS will be applied and uniformly spread. It will then be covered with dry collagen sheet, gauze piece and then sterile surgical pad. The area will be immobilized with dynaplast, and patient will be advised to restrict movement at the site. Patients will be started on 1 week course of antibiotics and anti-inflammatory agents. Dressings will be removed after one week and patients will be asked to expose the area to sunlight 10 minutes every day from 10th day onwards.

Sample collection

1.      A 5 ml of venous blood sample will be collected using the standard aseptic precautions for complete blood count, liver and renal function test and fasting lipid profile, at visits 1, 3, 4, 5, 12 and 24 (six visits). Testing for HBsAg, anti-HCV and HIV-1 and -2, and latent TB (Mantoux test) will be done only at visit 1.

2.      A clean catch mid-stream urine sample will be collected for routine microscopy at visit 1 only.  

3.      Skin punch biopsies from the vitiligo patch selected for NCES will be taken from 10 patients of tofacitinib group at the baseline (visit 1) in and 3 months (visit 5) later (post-tofacitinib treatment) for analysis of mRNA expression of vitiligo activity biomarkers (Cytokines/chemokines and receptors such as IFN-gamma, IL15, IL-10, CXCL9, CXCL10, CCR3 etc; transcription factors such as FOXP3, EOMES, RUNX1, GATA3, JAKs, STATs etc) by customized PCR array (supplied by Qiagen) (exploratory study).

4.      Two 4 mm skin punch biopsies from the vitiligo patch selected for NCES will be taken at the baseline (visit 1) in another 20 consecutive consenting patients from each treatment group (20 x 2 = 40) and at 3 months after receiving tofacitinib or placebo (visit 5) for analysis of mRNA expression (of target genes)  and protein levels of vitiligo activity (based on the results of PCR array) and melanogenesis (BMP, TYR, TRP1, MITF etc) biomarkers by RT-PCR and/or ELISA to validate the results of PCR array analysis (validation study).

In addition, biopsies from vitiligo (n=10) and non-vitiligo controls (n=10) will also be taken for RT-PCR and ELISA analysis for comparison with cases.

Outcome parameters

Primary outcome

1.      Proportion of patients with >80% repigmentation in the treated vitiligo patch at 6 months and 1 year, in both groups

2.      Mean percentage repigmentation in the treated vitiligo patch at 6 months and 1 year, in both groups

Secondary outcome

1.      Proportion of patients with residual ‘achromic fissure’ in the treated vitiligo patch at 6 months, in both groups

2.      Proportion of patients with a Vitiligo noticeability scale score of >4 in both groups

3.      Change in the lesional skin tissue biomarkers at baseline vs post-tofacitinib priming prior to NCES (n=20 in each group)

4.      Change in the lesional skin tissue biomarkers at baseline vs post-placebo priming prior to NCES (n=20 in each group)

5.      Comparison of the lesional skin tissue biomarkers post-tofacitinib priming prior to NCES vs vitiligo patches >1 year of clinical disease stability (n=10 in each group)

6.      Comparison of the lesional skin tissue biomarkers post-tofacitinib priming prior to NCES vs normal skin of healthy controls (n=10 in each group)

7.      Comparison of baseline clinical and dermoscopic features in vitiligo patches with >80% repigmentation vs <80% repigmentation

8.      Comparison of sequential dermoscopic findings in vitiligo patches that showed clinical signs of disease reactivation at any time post NCES vs that did not