DR [Dermatology Reports 2017; 9:7399] [page 55] A retrospective comparative study of the efficacy and safety of two regimens of diphenylcy- clopropenone in the treatment of recalcitrant alopecia areata Tueboon Sriphojanart, Saranya Khunkhet, Poonkiat Suchonwanit Division of Dermatology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Abstract Diphenylcyclopropenone (DPCP) is an effective topical immunotherapy for recal- citrant alopecia areata (AA), which some- times requires prolonged treatment. We developed a new treatment protocol to shorten the duration of therapy. This study aimed to compare the efficacy and safety of the new treatment protocol with the stan- dard treatment protocol in the treatment of recalcitrant AA. We conducted a 6-year ret- rospective comparative study of patients with AA who received one of the DPCP treatment protocols at our institute. Patients’ information was collected and subsequent statistically analyzed. Thirty- nine patients (16 in the new treatment group and 23 in the standard treatment group) were included. There were no statistically significant differences in area of hair regrowth. Mean duration to initial hair regrowth and mean duration to significant hair regrowth in the new treatment group were significantly shorter than in the stan- dard treatment group (P=0.002 and 0.01, respectively). Adverse effects were slightly higher in the new treatment group. The present study reveals the effectiveness and safety of the new treatment protocol, which shortens the duration of DPCP treatment and could represent an alternative regimen. Introduction Alopecia areata (AA) is an autoimmune disease with a genetic basis presenting with non-scarring hair loss both on the scalp and non-scalp areas. The prevalence of AA is approximately 1-2% of the general popula- tion.1 The disease has no sexual and age prevalence. Clinically, patients manifest with well-defined, asymptomatic, non-scar- ring areas of hair loss with characteristic exclamation mark hairs. The prognosis of the disease is quite variable. Spontaneous improvement within 1 year was reported in 34-50% of patients. However, in 14-25%, AA can progress to total scalp hair loss (alopecia totalis; AT) or even entire body hair loss (alopecia universalis; AU), which shows spontaneous improvement in less than 10% of cases.2 Even though AA is not a life threatening disease, it can greatly affect patients’ psychosocial status and quality of life. Compared with the general population, psychological disorders, such as anxiety, depression, and phobias, are more common in patients with AA.3,4 The pathogenesis of AA remains to be determined. Currently, a widely accepted theory is the autoimmune etiology. Specific T-cell lymphocytes, autoantibodies against anagen follicles, and various cytokines such as interferon-γ, interleukins, and tumor necrosis factor-α have been found to play a major role in AA.5 In addition, the immune privilege theory has been recently intro- duced and suggested to play a role in the pathogenesis.6 Therefore, the principle of treatment is to inhibit all the possible etio- logical pathways. There is currently no cur- ative treatment for AA. Treatment modali- ties include corticosteroids, anthralin, cyclosporine, biologic therapy, topical immunotherapy, photochemotherapy, and 308-nm excimer laser, among other.7 However, treatment is still very difficult, particularly in patients with chronic and extensive AA. Currently, topical immunotherapy using diphenylcyclopropenone (DPCP) is one interesting treatment option for recalcitrant case. This treatment was first introduced by Happle et al. in 1983.8 The principle of treatment is induction and repeated elicita- tion of an allergic contact dermatitis by application of contact allergen on affected areas. Its mechanism of action is not fully understood, but antigenic competition from induction of contact dermatitis on the scalp is believed to be a possible mechanism.9 For almost 10 years, we have been per- forming DPCP treatment regularly in patients with AA who do not respond to other treatments within the first 6 months. The first step of treatment is sensitization of the patient to DPCP. After this is successful- ly completed, the lowest concentration of DPCP is initially applied to the affected area on the patient’s scalp. Then, the con- centration is gradually increased every week until the optimal reaction occurs. We observed that some patients took several weeks or even months to reach the optimal reaction, which could extend the overall treatment duration. We developed a new treatment protocol of topical immunotherapy for recalcitrant AA and have been using it in some patients since 2011. The purpose of developing the new protocol was to minimize the duration of DPCP treatment. After sensitization, multiple concentrations of DPCP were applied to the scalp in individual small areas at the same time. Then, the optimal concentration that could cause mild contact dermatitis was selected to be the first con- centration applied to the patient’s scalp. We believe that our new treatment protocol could reduce the treatment duration and also reduce the cumulative treatment cost compared to the standard protocol. Therefore, to prove our hypothesis, we con- ducted a retrospective comparative study of patients with AA who received DPCP treat- ment at our institute. The objective of the study was to determine whether the new treatment protocol is superior to the stan- dard protocol in the treatment of recalcitrant AA and to compare adverse events between the two treatment protocols. Materials and Methods Study design This is a retrospective comparative study conducted at Ramathibodi Hospital, Mahidol University. Patients’ information from 2011 to 2016 was collected from med- ical records. The study was reviewed and Dermatology Reports 2017; volume 9:7399 Correspondence: Poonkiat Suchonwanit, Division of Dermatology, Ramathibodi Hospital, 270 Rama VI Road, Rajthevi, Bangkok, Thailand 10400. Tel.: +662.201.1141 - Fax: +662.201.1211. E-mail: poonkiat@hotmail.com Key words: Alopecia; alopecia areata; diphen- cyprone; diphenylcyclopropenone; topical immunotherapy. Contributions: TS, SK, PS, study conception and design; TS, SK, acquisition of data; TS, SK, PS, analysis and interpretation of data; TS, PS, drafting of manuscript; PS, critical revision. Conflict of interest: The authors declare no conflict of interest. Received for publication: 7 September 2017. Accepted for publication: 6 November 2017. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). ©Copyright T. Sriphojanart et al., 2017 Licensee PAGEPress, Italy Dermatology Reports 2017; 9:7399 doi:10.4081/dr.2017.7399 No n c om me rci al us e o nly [page 56] [Dermatology Reports 2017; 9:7399] approved by the Mahidol University Institutional Review Board for Ethics in Human Research (protocol number 06-59- 52). Patients Patients with AA who received DPCP treatment using either the standard treat- ment protocol or the new treatment protocol in the outpatient dermatology clinic were included in the study. The diagnosis of AA in each patient was based on clinical and/or histological examination. The inclusion cri- teria were age greater than 18 years, unfa- vorable treatment outcomes with any other topical and systemic treatments for at least 6 months, scalp hair loss greater than 50%, and regular followed up in our clinic at least 6 months after discontinuation of DPCP treatment. The exclusion criteria were incomplete medical records and unsuccess- ful sensitization to DPCP. Demographic data including the age of onset, duration of disease, medical history, family history of AA, type of AA, severity of disease meas- ured using the Severity of Alopecia Tool (SALT) score,10 and nail abnormalities were collected for further analysis. Sensitization to diphenylcyclo- propenone DPCP solution was prepared by dis- solving DPCP powder (Fluka, Sigma- Aldrich Corp, St. Louis, MO, USA) in ace- tone at serial dilutions of 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, and 2.0% and storing the dilution in dark glass vials to prevent degradation from ultraviolet light. At the first visit, the patient was sensitized by application of 2% DPCP at the inner aspect of the upper arm over a 4×4 cm2 area. DPCP was left on the sensitized area for 24 hours then washed off. After 72 h, patient was evaluated for an eczematous reaction to detect whether sensitization to DPCP had occurred. Treatment with standard regimen Two weeks after sensitization, DPCP treatment starting with the 0.0001% con- centration was applied at the affected scalp area every week. First, DPCP was left on the scalp for 24 h and then washed off with gentle shampoo. Each week, concentration of DPCP was titrated upward to reach a tol- erable eczematous reaction, which was mild pruritus and mild erythema lasting not more than 48 h. The concentration that produced optimal eczematous reaction was main- tained at subsequent treatments. If the con- centration of DPCP used on the scalp revealed negative or below the optimal reaction, then the concentration of DPCP would be titrated upward. The concentra- tion may be titrated upward or downward to maintain the optimal eczematous reaction. Treatment with new treatment regimen Two weeks after sensitization, the scalp was mapped for applying DPCP. Then, six consecutive concentrations of DPCP (0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, and 0.5%) were applied on the scalp over 3×3 cm2 areas separated by a distance of 4 cm. DPCP was left on the scalp for 24 h and then washed off with gentle shampoo. One week later, the concentration that created a mild eczematous reaction was chosen to be the first applied. The starting concentration was applied at the affected scalp area and left for 24 h and then washed off. The con- centration of DPCP was adjusted every week to maintain a tolerable eczematous reaction. Clinical assessment The patients were followed up weekly for evaluation of clinical responses and side effects. Treatment was withdrawn if there were no signs of hair regrowth at 6 months. If there was any clinical response, treatment was continued until complete hair regrowth. For patients with incomplete hair regrowth at 1 year after treatment, percentage of hair regrowth at 1 year was recorded as their treatment outcome. All patients were fol- lowed up monthly for 6 months after dis- continuation of treatment. The clinical assessment was divided into efficacy and safety. Efficacy assess- ment included the treatment response reported as percentages of area of hair regrowth, change in SALT score from base- line, and duration of clinical responses (ini- tial response and significant response). The initial response was defined as appearance of any new regrowth hair within treated sites and significant response was defined as greater than 75% hair regrowth. Failure of treatment was considered when hair regrowth was not observed after the first 6 months of treatment. Relapse was defined as more than 25% hair loss after complete hair regrowth. Safety information was col- lected from the adverse effects recorded. Statistical analysis Statistical analysis was performed using the SPSS statistics version 18.0 (SPSS Inc., Chicago, IL, USA). Chi-square test, Fisher exact test, t-test, and Wilcoxon rank-sum test were performed to compare demo- graphic data, efficacy, and adverse effects. The results were considered statistically significant at P<0.05. Results From a total of 65 patients with AA who received DPCP treatment during the last 5 years, 39 patients were eligible and were included in the study. Sixteen patients were treated with the new treatment regimen and 23 patients were treated with the standard regimen. Epidemiological data and baseline clinical characteristics of all patients are compared and summarized in Table 1. The groups were comparable in terms of age, gender, duration of disease, underlying dis- ease, scalp area involvement, and nail involvement. Table 2 demonstrates the comparison of treatment outcomes between the 2 treatment groups. Eight patients in the new treatment group and 12 patients in the standard group had >75% area of hair regrowth. Seven patients in each group had complete hair regrowth. Failure of treatment was reported in 2 and 3 patients in the new and standard treatment group, respectively. Regarding area of hair regrowth, there were no statisti- cally significant differences between the two groups. For treatment duration, mean duration to initial hair regrowth in the new treatment group was significantly shorter than that in the standard treatment group (10.5±2.6 weeks and 14.2±3.9 weeks, respectively; P=0.002). Moreover, mean duration to >75% hair regrowth was 24.3±4.2 weeks in the new treatment group and 29.2±6.5 weeks in the standard treat- ment group, representing a statistically sig- nificant difference (P=0.01). Regarding relapse of AA, there was no significant dif- ference in both number of patients and median duration to relapse between the 2 groups (P=0.81 and 0.62, respectively). Adverse effects were slightly higher in the new treatment group, without statistical sig- nificance. Discussion Topical immunotherapy using DPCP has been proven effective in the treatment of patients with recalcitrant AA. This method is recommended as first-line thera- py in patients with AA who have more than 50% scalp area involvement.11 Its efficacy, in terms of acceptable regrowth, has been reported in previous studies with variable response. A systematic review reported that the average response rate of DPCP treat- ment in 26 studies was 53.75%.12 Our study reported a significant response rate of 50% in the new treatment group and 52.1% in the standard treatment group, which are compa- rable with the average response rate. Article No n c om me rci al us e o nly Previous studies reported response rates of DPCP ranging from 5 to 85%.8,13-20 The variability in clinical response rate between studies may be attributed to different study designs, treatment protocols, baseline dis- ease severity, therapeutic response grading systems, and follow-up periods. To mini- mize this problem in future studies, clinical trials using a standardized method in clini- cal assessment is recommended. Regarding percentage of hair regrowth, the present study found similar clinical response rates between the 2 groups. The data confirms that the efficacy of the new treatment protocol was not inferior in com- parison with the standard treatment proto- col. In addition, this retrospective study reveals the advantage of the multiple DPCP treatment protocol in shortening the dura- tion of treatment. Our study showed that mean duration to initial response and mean duration to >75% hair regrowth in the new treatment group were significantly shorter than in the standard treatment group, which was approximately 5 weeks. Our results are comparable with a previous study using multiple concentrations of DPCP in patients with AT or AU, which revealed a benefit of shortened treatment duration.21 When comparing the treatment duration using data from the standard treatment group, the average duration to significant hair regrowth was comparable with previ- ous studies. Chiang et al.22 and El Khoury et al.23 reported durations to significant hair regrowth of 28 and 31.74 weeks, respec- tively. Therefore, the new treatment proto- col could shorten the response time, which normally takes several months to reach a significant clinical response, and could be considered an alternative regimen to short- en the therapeutic period. Application of multiple concentrations of DPCP at the first treatment assisted the physician in identify- ing the optimal concentration in a shorter period. Reduction of the overall treatment duration could reduce the cost of treatment and other expenses such as transportation, accommodation, and income loss while undergoing DPCP treatment. The relapse rates in each treatment group in the present study were less than that reported in previous studies, which range between 45.1 and 70%.19,20,24-26 The difference in the relapse rate may be due to different definitions of relapse and the dura- tion of follow-up. Although the relapse rate of topical immunotherapy with DPCP is quite high, it is considered to be the most successful treatment for severe types of AA.27 Common adverse effects of DPCP treat- ment described in the literature are severe eczematous reaction, wide spread eczema, urticarial reaction, severe dermographism, and cervical lymphadenopathy.28-30 The present study shows that adverse effects were slightly higher in the new treatment protocol group, without statistical signifi- cance. Using a higher concentration during the early treatment period could increase the possibility of severe reactions. Fortunately, the adverse effects were tolera- ble and rapidly disappeared within a few days after providing symptomatic treat- ment. None of the patient dropped out of the treatment. Limitations of this study are the retro- spective design and small number of patients. Therefore, we were unable to assess patients’ history and clinical evalua- tion completely. The small sample size may account for the lack of statistically signifi- cant results. Prospective clinical trials with a large number of patients could overcome this limitation. Conclusions Topical immunotherapy is an effective treatment option for patients with chronic Article Table 1. Demographics and clinical characteristics of the patients in the 2 treatment groups. Variables Standard regimen New regimen P value Number 23 16 N/A Mean age (year) 35.5±5.2 32.9±4.6 0.11 Sex (male:female) 9:14 6:10 0.91 Mean age at onset (year) 26.9±5.6 23.5±5.8 0.07 Mean duration of disease (week) 56.9±10.2 61.9±12.6 0.17 Underlying diseases, n (%) Atopic disease 3 (13) 1 (6.2) 0.49 Autoimmune thyroid 1 (4.3) 1 (6.2) 0.79 Family history of AA, n (%) 2 (8.6) 3 (18.8) 0.35 Nail involvement, n (%) 8 (34.8) 9 (56.2) 0.18 Type of alopecia areata, n (%) Multiple patches 9 (39.2) 6 (37.4) 0.81 Alopecia totalis 8 (34.8) 7 (43.7) Alopecia universalis 6 (26) 3 (18.9) Baseline area involvement, n (%) 51-75% 5 (21.7) 3 (18.9) 0.95 76-99% 5 (21.7) 4 (25) 100% 13 (56.6) 9 (56.1) AA, alopecia arata. Table 2. Response to treatments and adverse effects observed in patients in the 2 treat- ment groups. Variables Standard regimen New regimen P value Area of hair regrowth, n (%) no hair regrowth 3 (13) 2 (12.5) 0.99 1-25% 2 (8.6) 2 (12.5) 26-50% 2 (8.6) 1 (6.2) 51-75% 4 (17.7) 3 (18.9) 76-99% 5 (21.7) 1 (6.2) 100% 7 (30.4) 7 (43.7) Mean duration to initial response (week) 14.2±3.9 10.5±2.6 0.002* Mean duration to >75% hair regrowth (week)# 29.2±6.5 24.3±4.2 0.01* Relapse, n (%) 5 (21.7) 4 (25) 0.81 Median duration to relapse (week) 14 (8-23) 11 (6-18) 0.62 Adverse effects, n (%) Blister formation 1 (4.3) 1 (6.2) 0.9 Widespread eczema 3 (13) 6 (37.4) Dyspigmentation 1 (4.3) 2 (12.5) Lymphadenopathy 7 (30) 8 (50) *Statistically significant; #data collected from patients with >75% hair regrowth. [Dermatology Reports 2017; 9:7399] [page 57] No n c om me rci al us e o nly [page 58] [Dermatology Reports 2017; 9:7399] and extensive AA. The present study shows that the new treatment protocol is effective and safe, and could shorten the duration of DPCP treatment. Our new treatment proto- col could be an alternative regimen for the treatment of AA. References 1. Willemsen R, Vanderlinden J, Roseeuw D, Haentjens P. Increased history of childhood and lifetime traumatic events among adults with alopecia areata. J Am Acad Dermatol 2009;60:388-93. 2. Mcdonagh AJ, Messenger AG. The pathogenesis of alopecia areata. Dermatol Clin 1996;14:661-70. 3. Rafique R, Hunt N. Experiences and coping behaviours of adolescents in Pakistan with alopecia areata: an inter- pretative phenomenological analysis. Int J Qual Stud Health Well-being 2015;10:26039. 4. Hunt N, Mchale S. The psychological impact of alopecia. BMJ 2005;331:951- 3. 5. Gregoriou S, Papafragkaki D, Kontochristopoulos G, et al. Cytokines and other mediators in alopecia areata. Mediators Inflamm 2010;2010:928030. 6. Kang H, Wu WY, Lo BK, et al. Hair fol- licles from alopecia areata patients exhibit alterations in immune privilege- associated gene expression in advance of hair loss. J Invest Dermatol 2010;130:2677-80. 7. Delamere FM, Sladden MM, Dobbins HM, Leonardi-Bee J. Interventions for alopecia areata. Cochrane Database Syst Rev 2008:Cd004413. 8. Happle R, Hausen BM, Wiesner- Menzel L. Diphencyprone in the treat- ment of alopecia areata. Acta Derm Venereol 1983;63:49-52. 9. Sutherland L, Laschinger M, Syed ZU, Gaspari A. Treatment of alopecia areata with topical sensitizers. Dermatitis 2015;26:26-31. 10. Olsen EA, Hordinsky MK, Price VH, et al. Alopecia areata investigational assessment guidelines—Part II. National Alopecia Areata Foundation. J Am Acad Dermatol 2004;51:440-7. 11. Alkhalifah A, Alsantali A, Wang E, et al. Alopecia areata update: part II. Treatment. J Am Acad Dermatol 2010;62:191-202. 12. Jang YH, Jung HJ, Moon SY, et al. Systematic review and quality analysis of studies on the efficacy of topical diphenylcyclopropenone treatment for alopecia areata. J Am Acad Dermatol 2017;77:170-2. 13. Hull SM, Cunliffe WJ. Successful treat- ment of alopecia areata using the con- tact allergen diphencyprone. Br J Dermatol 1991;124:212-3. 14. Van Der Steen PH, Van Baar HM, Perret CM, Happle R. Treatment of alopecia areata with diphenylcyclopropenone. J Am Acad Dermatol 1991;24:253-7. 15. Van Der Steen PH, Boezeman JB, Happle R. Topical immunotherapy for alopecia areata: re-evaluation of 139 cases after an additional follow-up peri- od of 19 months. Dermatology 1992;184:198-201. 16. Cotellessa C, Peris K, Caracciolo E, et al. The use of topical diphenylcyclo- propenone for the treatment of exten- sive alopecia areata. J Am Acad Dermatol 2001;44:73-6. 17. Aghaei S. Topical immunotherapy of severe alopecia areata with diphenylcy- clopropenone (DPCP): experience in an Iranian population. BMC Dermatol 2005;5:6. 18. Firooz A, Bouzari N, Mojtahed F, et al. Topical immunotherapy with diphen- cyprone in the treatment of extensive and/or long-lasting alopecia areata. J Eur Acad Dermatol Venereol 2005;19:393-4. 19. Avgerinou G, Gregoriou S, Rigopoulos D, et al. Alopecia areata: topical immunotherapy treatment with diphen- cyprone. J Eur Acad Dermatol Venereol 2008;22:320-3. 20. Ohlmeier MC, Traupe H, Luger TA, Bohm M. Topical immunotherapy with diphenylcyclopropenone of patients with alopecia areata—a large retrospec- tive study on 142 patients with a self- controlled design. J Eur Acad Dermatol Venereol 2012;26:503-7. 21. Thuangtong R, Varothai S, Triwongwaranat D, Rujitharanawong C. Multi-concentration level patch test guided diphenyl cyclopropenone (DPCP) treatment in alopecia totalis or alopecia universalis, J Med Assoc Thai 2017;100:86-92. 22. Chiang KS, Mesinkovska NA, Piliang MP, Bergfeld WF. Clinical Efficacy of Diphenylcyclopropenone in alopecia areata: retrospective data analysis of 50 patients. J Investig Dermatol Symp Proc 2015;17:50-5. 23. El Khoury J, Abd-El-Baki J, Succariah F, et al. Topical immunomodulation with diphenylcyclopropenone for alopecia areata: the Lebanese experi- ence. Int J Dermatol 2013;52:1551-6. 24. Bolduc C, Shapiro J. DPCP for the treatment of alopecia areata. Skin Therapy Lett 2000;5:3-4. 25. Wiseman MC, Shapiro J, Macdonald N, Lui H. Predictive model for immunotherapy of alopecia areata with diphencyprone. Arch Dermatol 2001;137:1063-8. 26. Luk NM, Chiu LS, Lee KC, et al. Efficacy and safety of diphenylcyclo- propenone among Chinese patients with steroid resistant and extensive alopecia areata. J Eur Acad Dermatol Venereol 2013;27:e400-5. 27. Hoffmann R, Happle R. Topical immunotherapy in alopecia areata. What, how, and why? Dermatol Clin 1996;14:739-44. 28. Hull SM, Cunliffe WJ. Treatment of alopecia areata with diphenylcyclo- propenone. J Am Acad Dermatol 1992;26:276-7. 29. Alam M, Gross EA, Savin RC. Severe urticarial reaction to diphenylcyclo- propenone therapy for alopecia areata. J Am Acad Dermatol 1999;40:110-2. 30. Skrebova N, Nameda Y, Takiwaki H, Arase S. Severe dermographism after topical therapy with diphenylcyclo- propenone for alopecia universalis. Contact Dermatitis 2000;42:212-5. Article No n c om me rci al us e o nly