Clinics in Dermatology (2015) 33, 300–304
Treatment of alopecia areata with squaric acid dibutylester Nikki D. Hill, MD a,⁎, Kristin Bunata, BBA b , Adelaide A. Hebert, MD a a
Department of Dermatology University of Texas at Houston Health Science Center, 6655 Travis Street #600, Houston, TX 77030 University of Texas at Houston Medical School, 6431 Fannin Street, Houston, TX 77030
b
Abstract Squaric acid dibutylester is a topical sensitizing agent utilized for the treatment of alopecia areata. The mechanism of action is not fully understood, but is believed to redirect the inflammatory response by invoking allergic contact dermatitis. Several studies have compared the efficacy of squaric acid dibutylester to other treatments vs placebo with favorable results. This contribution reviews the history of the topical sensitizing agent squaric acid dibutylester and discusses the mechanism of action, the use in alopecia areata, and the efficacy and safety of this therapeutic agent. © 2015 Elsevier Inc. All rights reserved.
Introduction The treatments for alopecia areata (AA) are numerous, and many of them are quite ineffective.1 The lack of randomized double-blind controlled trials and the incidence of spontaneous hair regrowth render validating a therapy difficult. One mode of treatment involving the induction of an allergic contact dermatitis has been shown to elicit hair regrowth in AA.2 Chemicals or substances that elicit an allergic contact dermatitis and immunity upon exposure are called topical sensitizers. With subsequent exposures the sensitizer should produce a T-cell memory inflammatory response. Squaric acid dibutylester (SADBE) is a sensitizer found to induce hair growth in patients with AA in 1980.3 SADBE is believed to elicit an inflammatory reaction to disrupt the autoimmune pathogenesis of the disease. Many studies have shown SADBE to be superior to placebo and other treatment options. As no needles or daily oral medication is employed with this regimen, SADBE can be ⁎ Corresponding author. E-mail address: [email protected] (N.D. Hill). http://dx.doi.org/10.1016/j.clindermatol.2014.12.001 0738-081X/© 2015 Elsevier Inc. All rights reserved.
used as a primary treatment for severe refractory AA in adults and children. This contribution reviews the use of SADBE, a topical sensitizing agent, in the treatment of AA. The mechanism of action, its use in the treatment of AA, and the efficacy and safety of SADBE are summarized.
Alopecia areata AA is an autoimmune condition characterized by nonscarring hair loss involving the scalp, body, and nails. The prevalence of AA in the United States is 0.1% to 0.2% with a lifetime risk of 1.7%.4,5 AA classically presents as one or more smooth, hairless patches on the scalp with preserved follicular ostia and no erythema. This localized presentation may progress to involve the entire scalp (alopecia totalis) and body and facial hair (alopecia universalis). The exact cause of AA is unknown, but investigators have implicated a T-cell lymphocytes interaction with autoantigens located in the hair follicles.6 Various studies have demonstrated the immunologic influence on this condition. Skin transferred from the scalp of AA patients can regrow
Use of squaric acid for alopecia areata hair when grafted onto athymic nude mice. Severe combined immunodeficiency (SCID) mice can be induced to develop alopecic patches when injected with scalp-infiltrating CD4 +/ CD8 + T cells from a patient with AA (cite/Bolognia). The hair follicle is an immune-privileged site with a low level of major histocompatibility complex (MHC) expression.7 AA requires an interruption in this immune privilege, allowing infiltration of T cells into the hair follicle.8 Triggering factors cause a predominantly CD8 + driven, Th1 T-cell response against hair follicles, resulting in acute hair loss. Certain proinflammatory molecules, including substance P and interferon γ (IFN-γ), have been shown to upregulate MHC class Ia expression in hair follicles. IFN-γ is the most potent inducer of MHC I expression in murine anagen hair bulbs in vivo, and similarly, IFN-γ was shown to accelerate the development of AA in genetically susceptible mice.9,10 As IFN-γ is mainly produced by activated T lymphocytes, an activation of the immune system may precede the loss of immune privilege.7 Another theory is that AA is an autoimmune response against melanogenesis-related proteins. According to this theory, microtrauma, neurogenic inflammation, or microbial antigens leads to the production of proinflammatory cytokines, which are responsible for the breakdown of MHC I negativity in the proximal anagen hair bulb. This disruption of immune privilege exposes autoantigens from melanogenesis-related proteins and triggers an autoimmune response consisting of 2 phases. First, CD8 + T cells recognize melanogenesis-related proteins abnormally presented by MHC I molecules and initiate the disease on melanocytes and keratinocytes. Second, CD4 + T cells and antigen presenting cells mount an attack against MHC class II presented autoantigens that were exposed by damaged melanocytes and keratinocytes. This second response causes most of the follicular damage.10 AA is thought to be a multifactorial disease, involving elements of autoimmunity, genetics, and several other factors. Acute episodes of the disease are more likely to occur in individuals undergoing times of profound stress, grief, or fear, implying that certain environmental factors contribute to the pathogenesis.11 Infectious pathogens including bacteria and viruses have been implicated in the development of other autoimmune diseases, such as diabetes mellitus and systemic lupus erythematosus, and several theories hold that AA is also the result of an infectious process. It has been postulated that AA results when individuals with a CD8 + T-cell deficiency are infected with Epstein-Barr virus, leading to the production of autoreactive T cells and the formation of ectopic lymphoid follicles.12 Cytomegalovirus has also been theorized to play a role, after researchers13 discovered the presence of cytomegalovirus DNA sequences in scalp biopsies taken from patients with AA. One report14 of 12 patients who self-reported a history of mononucleosis b 6 months before the onset of AA corroborates this theory. The seasonal pattern of AA flares has been described, with relapses mostly occurring in February and March. The relative increase in relapses during these months could possibly be due to the seasonal increase in viral infections.15
301 AA occurs in genetically susceptible individuals, and several genetic markers have been identified that are associated with the development of the disease. Genome-wide association studies, 11 which uses single-nucleotide polymorphism markers to identify regions of linkage disequilibrium, were performed on 1054 unrelated AA patients and 3278 controls. Several AA-associated single-nucleotide polymorphisms were identified, clustered in eight regions across the genome and involving genes implicated in the immune system, as well as genes unique to the hair follicle.11 In particular, the MHC antigens HLA-DQ3, DRB1*0401, and DQB1*0301 have been shown to be associated with severity of disease. Polymorphisms of interleukin 1 receptor antagonist are associated with severe early-onset of AA.34
The development of topical sensitizing agents Topical sensitizers were first reported as a treatment for alopecia areata in 1978,2 and it was found that eliciting an allergic contact dermatitis using dinitrochlorobenzene stimulated hair growth. This agent was later found to be mutagenic in 1980, by the Ames test and genotoxic by sister chromatid exchange in human fibroblasts.16,17 Other topical sensitizers have since replaced dinitrochlorobenzene including diphenylcyclopropenone (DPCP) and SADBE. SADBE is associated with fewer side effects than DPCP, and its absence of mutagenicity by Ames testing is more reassuring than that of DPCP, which has been found to sometimes contain a mutagenic precursor. SADBE is an ideal sensitizing agent, as it is a potent topical sensitizer, is not widely found in the environment, does not cause significant adverse effects, and does not cross-react with other chemicals.18 Of note, SADBE is not as stable in acetone, requires refrigeration, and is more expensive compared with DPCP. Other sensitizers, including urushiol, are known to interrupt the pathologic inflammation of AA, but, due to the uncontrolled reactions, the two previously-mentioned sensitizers are preferred.
The mechanism of topical sensitizers The mechanism of action of topical sensitizers, including SADBE, is not completely understood aside from an immune modulation from an allergic contact dermatitis. One theory describes the sensitizing compound as a hapten that binds protein substrates to create a complete antigen, which is the true immune diversion.19 It has been reported that administration of a topical sensitizer decreases the peribulbar CD4 +/CD8 + lymphocytic ratio from 4:1 to 1:13 and reduces the number of intrabulbar CD8 + lymphocytes and Langerhans cells.20 The abnormal expression of class I and II major histocompatibility complex molecules on the proximal follicular epithelium that is implemented in the auto-aggressive response by CD8+ lymphocytes is down-regulated after treatment with a
302 topical sensitizer.19,20 Researchers have proposed a role of antigenic competition3 to explain these findings, whereby the recruited suppressor CD8+ T cells generate an inhibitory effect on the immune process against hair follicles, thus allowing hair growth.
Treatment regimen Patients are usually sensitized using a 2% solution in acetone on a 10 to 16 cm2 area of the scalp with caution to not wash off the solution for 24 hours. One week later, if a severe eczematous reaction has not occurred; a 0.0001% solution is applied to the scalp. If the initial reaction is too severe, it is important to wait 1 week before starting the elicitation phase. The strength of the first eliciting concentration should be inversely proportional to the length of the initial response. The concentration of the solution can be increased weekly up to a maximum of 2% to ensure that an adequate allergic reaction occurs. The patient must be advised to avoid sun and to not wash the treated site for 48 hours to maintain an appropriate level of erythema and pruritus. Once unilateral hair growth has occurred, it is appropriate to initiate treatment of the entire scalp.21–23 Our experience involves sensitizing the patient with a 5% SADBE that is applied with a cotton applicator to a small area of the scalp or upper inner arm. Patients are sent home with a mild topical steroid to use in case of an uncomfortable reaction, such as pruritus or vesicle formation. Patients with adequate sensitization will develop an eczematous patch in the treated area within a few days. One month after sensitization, the patient returns on a 1- to 2-month schedule for application of 5% SADBE to the alopecic patches until maximum hair regrowth. Patients are told to not wash the areas for 24 hours. At follow-up visits, the treated sites are evaluated for hair regrowth. Treatment was deferred for 1 to 2 months in cases of local blistering. If the patient shows response, treatment is continued as tolerated until desired outcome.
Nonresponders One barrier to effective treatment of AA with SADBE is the issue of nonresponse. Van der Steen estimated that between 1% and 2% of patients are unable to be sensitized21 with other estimates as high as 20%.24 Additionally, patients who have been successfully sensitized can develop tolerance and has been reported in 10.8%. A patient can develop tolerance in one bodily location but maintain sensitivity elsewhere. 22 In one study, the histamine antagonist cimetidine has been reported to disrupt tolerance to SADBE, purportedly through augmentation of delayed type hypersensitivity.2,25 Other researchers were not able to reproduce these results.26
N.D. Hill et al.
Adverse effects The most common adverse effects associated with SADBE include eczema, auto-eczematization, blistering, and swelling of regional lymph nodes.18 Other less common side effects include burning sensation after application, persistent allergic contact dermatitis of the primary site of sensitization, severe generalized dermatitis, generalized pruritis, leukoderma, xerosis, scaling, edema of treated skin, scalp folliculitis, and systemic reactions characterized by fever and arthalgias.22 It is reported that most patients tolerate the therapy well and the above reactions may resolve after discontinuation of the treatment or with use of topical corticosteroids and antihistamines.
Safety profile SADBE is a potent sensitizer that is used as an industrial stabilizer and an antifog agent in photographic emulsions. This agent is nonmutagenic in the highly sensitive Salmonella microsome test. Tetrachloro-2-cyclobutene-1-one is an intermediate in the synthesis of SADBE that when injected subcutaneously, caused rhabdomyosarcoma in 1 out of 20 mice. Lifetime subcutaneous injections of SADBE into 20 mice caused one fibrosarcoma. Additional testing on animals is necessary before the safety of SADBE can be established.22 No sensitizer is FDA approved for this therapeutic intervention and caution must be taken when topical sensitizers are recommended as a treatment option for patients.
Efficacy No randomized controlled trials have evaluated the effectiveness of topical sensitizers in AA. Some observational studies have used the half-head model to control for spontaneous regrowth. The success rate of DPCP and SADBE is approximately 50% to 60% with a range of 9% to 87%.22,27 When assessed alone, SADBE had a total response rate (defined as moderate to good regrowth or at least 30% improvement) from 29% to 87%, which correlated with the response rate of 65%.18,22 To date, 13 studies using SADBE and 17 using DPCP have revealed little difference between the two agents,27 which support the notion that these chemicals may elicit the same immunologic mechanism.28 There are several prognostic factors that negatively influence response to topical sensitizers. Van der Steen et al29 found that type and/or extent of alopecia, duration of disease, and the presence of nail factors predicted a poorer response. Extent of alopecia and duration of disease are the most commonly cited negative prognostic factors that have been reported in the literature. Other prognostic factors are age at onset and family history of alopecia areata, followed by presence of nail changes.22,29 In addition, an earlier
Use of squaric acid for alopecia areata development of dermatitis may be a positive prognostic factor. Caserio17 determined that 3 out of 4 successfully treated patients had been sensitized within 3 weeks or less, compared to 10 unsuccessfully treated patients in whom the sensitization took longer to develop.
SADBE in the literature In 1980,3 application of SADBE was found to be an initially effective treatment for 87% of individuals with alopecia areata, and a persistent response was noted in 70%. Since this initial study, there have been several similar clinical trials assessing the efficacy of SADBE. SADBE has proven to be an effective treatment for long-standing AA. The 21 patients in the study had long-standing, severe disease refractory to previous therapies. Eleven of the 21 had an excellent response consisting of complete hair regrowth. In addition, it was noted that patients with patchy alopecia responded better to treatment than those with alopecia totalis or alopecia universalis (73% and 30%, respectively).24 Analysis of the results of a controlled trial30 involving 119 patients with patchy alopecia treated with SADBE, DPCP, minoxidil, or placebo failed to show a statistically significant difference between the control and treatment groups. It was noted that 18 of the subjects were children (under the age of 14), and there was a significant difference in the rate of complete hair regrowth in children versus adults (38.9% and 71.3%, respectively). In another study on 14 patients with AA who had not responded to conventional treatments, only 28.5% of the patients had persistent hair growth while receiving SADBE treatment. Of these 14 patients, five were 16 years old or younger, and all but one eventually had alopecia totalis or universalis. The younger ages of these patients, as well as their long-standing and extensive disease history, were thought to be responsible for the lower than expected success rate.17 One study18 treated 144 patients with AA of varying degrees with SADBE. Of these patients, 71 had AA affecting less than 50% of the scalp, whereas 73 had a more severe form, including 13 patients with alopecia totalis and two with alopecia universalis. Eighty percent of patients with the less severe type experienced hair regrowth, compared to 49% of the more severe form. Unlike previous studies, Micali et al18 did not find age or duration of alopecia to be factors predictive of response to treatment. In a retrospective case series of 36 AA patients treated with SADBE using medical records,31 about 21% of patients with diffuse hair loss responded to treatment, compared to 58.3% of patients with patchy hair loss. Neither age nor duration of hair loss was found to be a predictor of response. Severity of disease was determined to be a prognostic factor, as those with patchy hair loss benefited more than those with diffuse disease.
303 Other researchers32 followed a group of 54 patients affected by severe AA treated with SADBE for at least 2 years. Collected data were compared to a matched control group of 54 patients who did not receive any treatment. The authors found that 80% of the treatment group was in remission, compared to 50% of the control group. Neither age, gender, nor disease duration were found to be contributing factors. During follow-up, there was not a significant difference in relapse rate between the treatment and control groups (44% and 52%, respectively). Relapses in the treatment group were found to be significantly less severe than that of the control group (P b .001). A study on 60 patients with AA who were unresponsive to conventional therapies19 showed that, overall, 43% experienced successful hair regrowth from SADBE. Subjects with b 50% scalp involvement had a 68% success rate, compared to 29% of those with more diffuse disease. Younger age at time of disease onset, duration of disease, severity of disease, and nail changes were associated with poor response to SADBE. The authors presented a small case study (10 patients) regarding sensitization and treatment with SADBE 5%. The study reported a response usually after 2 to 4 applications with 50% (5/10) graded as good (75%–100%) regrowth, 30% fair (25%–74%) regrowth, and 20% poor (0%–24%) regrowth.33
Conclusions SADBE appears to be a safe and effective treatment for individuals suffering from AA. Several prognostic factors including duration of disease, extent of alopecia, age at onset, and nail changes may be able to predict the level of a patient’s responsiveness to treatment with SADBE. The long-term studies, although demonstrating a significant relapse rate, do suggest immunotherapy has a useful role in managing this condition. Further controlled prospective studies are needed to answer remaining questions about the use of SADBE and other topical sensitizers in AA.
References 1. Gardner S, Freyschmidt-Paul P, Hoffman R, et al. Normalization of hair follicle morphology in C3 H/HeJ alopecia areata mice after treatment with squaric acid dibutylester. Eur J Dermatol. 2000;10: 443-450. 2. Daman LA, Rosenberg EW, Drake L. Treatment of alopecia areata with dinitrochlorobenzene. Arch Dermatol. 1978;114:1036-1038. 3. Happle R, Kalveram KJ, Büchner U, et al. Contact allergy as a therapeutic tool for alopecia areata: Application of squaric acid dibutylester. Dermatologica. 1980;161:289-297. 4. Safavi KH, Muller SA, Suman VJ, et al. Incidence of alopecia areata in Olmsted County, Minnesota, 1975 through 1989. Mayo Clin Proc. 1995;70: 628-633. 5. Safavi K. Prevalence of alopecia areata in the First National Health and Nutrition Examination Survey. Arch Dermatol. 1992;128:702. 6. Norris D. Alopecia areata: Current state of knowledge. J Am Acad Dermatol. 2004;51(1 Suppl):S16-S17.
304 7. McElwee KJ, Gilhar A, Tobin DJ, et al. What causes alopecia areata? Exp Dermatol. 2013;22:609-626. 8. Gilhar A, Paus R, Kalish R. Lymphocytes, neuropeptides, and genes involved in alopecia areata. J Clin Investig. 2007;117:2019-2027. 9. Rückert R, Hofmann U, van der Veen C, et al. MHC class I expression in murine skin: Developmentally controlled and strikingly restricted intraepithelial expression during hair follicle morphogenesis and cycling, and response to cytokine treatment in vivo. J Invest Dermatol. 1998;111:25-30. 10. Paus R, Slominski A, Czarnetzki B. Is alopecia areata an autoimmuneresponse against melanogenesis-related proteins, exposed by abnormal MHC class I expression in the anagen hair bulb? Yale J Biol Med. 1994;66:541-554. 11. Jabbari A, Petukhova L, Cabral RM, et al. Genetic basis of alopecia areata: A roadmap for translational research. Dermatol Clin. 2013;31: 109-117. 12. Pender MP. CD8 + T-cell deficiency, Epstein-Barr virus infection, Vitamin D deficiency, and steps to autoimmunity: A unifying hypothesis. Autoimmun Dis. 2012:189096. 13. Skinner Jr RB, Light WH, Leonardi C, et al. PCR evidence of cytomegalovirus in alopecia areata. J Invest Dermatol. 1995;104:686. 14. Rodriguez TA, Duvic M. Onset of alopecia areata after Epstein-Barr virus infectious mononucleosis. J Am Acad Dermatol. 2008;59:137-139. 15. D’Ovidio R. d’Ovidio F. Recidive stagionali dell’alopecia areata. G Ital Dermatol Venereol. 1995;295:130-134. 16. DeLeve LD. Dinitrochlorobenzene is genotoxic by sister chromatid exchange in human skin fibroblasts. Mutat Res. 1996;371:105-108. 17. Caserio RJ. Treatment of alopecia areata with squaric acid dibutylester. Arch Dermatol. 1987;123:1036-1041. 18. Micali G, Cicero RL, Nasca MR, et al. Treatment of alopecia areata with squaric acid dibutylester. Int J Dermatol. 1996;35:52-56. 19. Ajith C, Gupta S, Kanwar AJ. Efficacy and safety of the topical sensitizer squaric acid dibutylester in alopecia areata and factors influencing the outcome. J Drugs Dermatol. 2006;5:262-266. 20. Brocker EB, Echternacht-Happle K, Hamm H. Abnormal expression of class I and class II major histocompatibility antigens in alopecia areata modulation by topical immunotherapy. J Invest Dermatol. 1987;88: 564-568.
N.D. Hill et al. 21. Van der Steen PH, Happle R. Topical immunotherapy of alopecia areata. Dermatol Clin. 1993;11:619-622. 22. Rokshar CK, Shupack JL, Vafai JJ, et al. Efficacy of topical sensitizers in the treatment of alopecia areata. J Am Acad Dermatol. 1998;39:751-761. 23. Shapiro J. Topical immunotherapy in the treatment of chronic severe alopecia areata. Dermatol Clin. 1993;11:661. 24. Case PC, Mitchel AJ, Swanson NA, et al. Topical therapy of alopecia areata with squaric acid dibutylester. J Am Acad Dermatol. 1984;10:447-451. 25. Avella J, Madsen JE, Binder HJ, et al. Effect of histamine H2-receptor antagonists on delayed hypersensitivity. Lancet. 1978;1:624-626. 26. Van der Steen PH, van Baar HM, Happle R, et al. Prognostic factors in the treatment of alopecia areata with diphenylcyclopropenone. J Am Acad Dermatol. 1991;24:227-230. 27. Harries MJ, Sun J, Paus R, et al. Management of alopecia areata. BMJ. 2010;341:c3671. 28. Happle R, Klein H, Macher E. Topical immunotherapy changes in the composition of the peribulbar infiltrate in alopecia areata. Arch Dermatol Res. 1986;278:214-218. 29. Van der Steen PH, van Baar HM, Perret CM, et al. Treatment of alopecia areata with diphenylcyclopropenone. J Am Acad Dermatol. 1991;24:253-257. 30. Tosti A, De Padova MP, Minghetti G, et al. Therapies versus placebo in the treatment of patchy alopecia areata. J Am Acad Dermatol. 1986;15:209-210. 31. Pardasani AG, Turner E, McMichael AJ. Squaric acid dibutylester: Indications for use and efficacy in alopecia areata. Arch Dermatol. 2001;137:970-972. 32. Dall’oglio F, Nasca MR, Musumeci ML, et al. Topical immunomodulator therapy with squaric acid dibutylester (SADBE) is effective treatment for severe alopecia areata (AA): Results of an open-label, paired-comparison, clinical trial. J Dermatolog Treat. 2005;16:10-14. 33. Diaz L, Morgan L, Pacha O, et al. Treatment of alopecia areata in children with topical 5% squaric acid dibutylester. Poster session presentation. 38th Annual Meeting of the Society for Pediatric Dermatology; 2012 July 11-1; Monterey, CA. 34. Tarlow JK, Clay FE, Cork MJ. Severity of alopecia areata is associated with a polymorphism in the interleukin-1 receptor antagonist gene. J Invest Dermatol. 1994;103:387-390.
Treatment of alopecia areata with squaric acid dibutylester Nikki D. Hill, MD a,⁎, Kristin Bunata, BBA b , Adelaide A. Hebert, MD a a
Department of Dermatology University of Texas at Houston Health Science Center, 6655 Travis Street #600, Houston, TX 77030 University of Texas at Houston Medical School, 6431 Fannin Street, Houston, TX 77030
b
Abstract Squaric acid dibutylester is a topical sensitizing agent utilized for the treatment of alopecia areata. The mechanism of action is not fully understood, but is believed to redirect the inflammatory response by invoking allergic contact dermatitis. Several studies have compared the efficacy of squaric acid dibutylester to other treatments vs placebo with favorable results. This contribution reviews the history of the topical sensitizing agent squaric acid dibutylester and discusses the mechanism of action, the use in alopecia areata, and the efficacy and safety of this therapeutic agent. © 2015 Elsevier Inc. All rights reserved.
Introduction The treatments for alopecia areata (AA) are numerous, and many of them are quite ineffective.1 The lack of randomized double-blind controlled trials and the incidence of spontaneous hair regrowth render validating a therapy difficult. One mode of treatment involving the induction of an allergic contact dermatitis has been shown to elicit hair regrowth in AA.2 Chemicals or substances that elicit an allergic contact dermatitis and immunity upon exposure are called topical sensitizers. With subsequent exposures the sensitizer should produce a T-cell memory inflammatory response. Squaric acid dibutylester (SADBE) is a sensitizer found to induce hair growth in patients with AA in 1980.3 SADBE is believed to elicit an inflammatory reaction to disrupt the autoimmune pathogenesis of the disease. Many studies have shown SADBE to be superior to placebo and other treatment options. As no needles or daily oral medication is employed with this regimen, SADBE can be ⁎ Corresponding author. E-mail address: [email protected] (N.D. Hill). http://dx.doi.org/10.1016/j.clindermatol.2014.12.001 0738-081X/© 2015 Elsevier Inc. All rights reserved.
used as a primary treatment for severe refractory AA in adults and children. This contribution reviews the use of SADBE, a topical sensitizing agent, in the treatment of AA. The mechanism of action, its use in the treatment of AA, and the efficacy and safety of SADBE are summarized.
Alopecia areata AA is an autoimmune condition characterized by nonscarring hair loss involving the scalp, body, and nails. The prevalence of AA in the United States is 0.1% to 0.2% with a lifetime risk of 1.7%.4,5 AA classically presents as one or more smooth, hairless patches on the scalp with preserved follicular ostia and no erythema. This localized presentation may progress to involve the entire scalp (alopecia totalis) and body and facial hair (alopecia universalis). The exact cause of AA is unknown, but investigators have implicated a T-cell lymphocytes interaction with autoantigens located in the hair follicles.6 Various studies have demonstrated the immunologic influence on this condition. Skin transferred from the scalp of AA patients can regrow
Use of squaric acid for alopecia areata hair when grafted onto athymic nude mice. Severe combined immunodeficiency (SCID) mice can be induced to develop alopecic patches when injected with scalp-infiltrating CD4 +/ CD8 + T cells from a patient with AA (cite/Bolognia). The hair follicle is an immune-privileged site with a low level of major histocompatibility complex (MHC) expression.7 AA requires an interruption in this immune privilege, allowing infiltration of T cells into the hair follicle.8 Triggering factors cause a predominantly CD8 + driven, Th1 T-cell response against hair follicles, resulting in acute hair loss. Certain proinflammatory molecules, including substance P and interferon γ (IFN-γ), have been shown to upregulate MHC class Ia expression in hair follicles. IFN-γ is the most potent inducer of MHC I expression in murine anagen hair bulbs in vivo, and similarly, IFN-γ was shown to accelerate the development of AA in genetically susceptible mice.9,10 As IFN-γ is mainly produced by activated T lymphocytes, an activation of the immune system may precede the loss of immune privilege.7 Another theory is that AA is an autoimmune response against melanogenesis-related proteins. According to this theory, microtrauma, neurogenic inflammation, or microbial antigens leads to the production of proinflammatory cytokines, which are responsible for the breakdown of MHC I negativity in the proximal anagen hair bulb. This disruption of immune privilege exposes autoantigens from melanogenesis-related proteins and triggers an autoimmune response consisting of 2 phases. First, CD8 + T cells recognize melanogenesis-related proteins abnormally presented by MHC I molecules and initiate the disease on melanocytes and keratinocytes. Second, CD4 + T cells and antigen presenting cells mount an attack against MHC class II presented autoantigens that were exposed by damaged melanocytes and keratinocytes. This second response causes most of the follicular damage.10 AA is thought to be a multifactorial disease, involving elements of autoimmunity, genetics, and several other factors. Acute episodes of the disease are more likely to occur in individuals undergoing times of profound stress, grief, or fear, implying that certain environmental factors contribute to the pathogenesis.11 Infectious pathogens including bacteria and viruses have been implicated in the development of other autoimmune diseases, such as diabetes mellitus and systemic lupus erythematosus, and several theories hold that AA is also the result of an infectious process. It has been postulated that AA results when individuals with a CD8 + T-cell deficiency are infected with Epstein-Barr virus, leading to the production of autoreactive T cells and the formation of ectopic lymphoid follicles.12 Cytomegalovirus has also been theorized to play a role, after researchers13 discovered the presence of cytomegalovirus DNA sequences in scalp biopsies taken from patients with AA. One report14 of 12 patients who self-reported a history of mononucleosis b 6 months before the onset of AA corroborates this theory. The seasonal pattern of AA flares has been described, with relapses mostly occurring in February and March. The relative increase in relapses during these months could possibly be due to the seasonal increase in viral infections.15
301 AA occurs in genetically susceptible individuals, and several genetic markers have been identified that are associated with the development of the disease. Genome-wide association studies, 11 which uses single-nucleotide polymorphism markers to identify regions of linkage disequilibrium, were performed on 1054 unrelated AA patients and 3278 controls. Several AA-associated single-nucleotide polymorphisms were identified, clustered in eight regions across the genome and involving genes implicated in the immune system, as well as genes unique to the hair follicle.11 In particular, the MHC antigens HLA-DQ3, DRB1*0401, and DQB1*0301 have been shown to be associated with severity of disease. Polymorphisms of interleukin 1 receptor antagonist are associated with severe early-onset of AA.34
The development of topical sensitizing agents Topical sensitizers were first reported as a treatment for alopecia areata in 1978,2 and it was found that eliciting an allergic contact dermatitis using dinitrochlorobenzene stimulated hair growth. This agent was later found to be mutagenic in 1980, by the Ames test and genotoxic by sister chromatid exchange in human fibroblasts.16,17 Other topical sensitizers have since replaced dinitrochlorobenzene including diphenylcyclopropenone (DPCP) and SADBE. SADBE is associated with fewer side effects than DPCP, and its absence of mutagenicity by Ames testing is more reassuring than that of DPCP, which has been found to sometimes contain a mutagenic precursor. SADBE is an ideal sensitizing agent, as it is a potent topical sensitizer, is not widely found in the environment, does not cause significant adverse effects, and does not cross-react with other chemicals.18 Of note, SADBE is not as stable in acetone, requires refrigeration, and is more expensive compared with DPCP. Other sensitizers, including urushiol, are known to interrupt the pathologic inflammation of AA, but, due to the uncontrolled reactions, the two previously-mentioned sensitizers are preferred.
The mechanism of topical sensitizers The mechanism of action of topical sensitizers, including SADBE, is not completely understood aside from an immune modulation from an allergic contact dermatitis. One theory describes the sensitizing compound as a hapten that binds protein substrates to create a complete antigen, which is the true immune diversion.19 It has been reported that administration of a topical sensitizer decreases the peribulbar CD4 +/CD8 + lymphocytic ratio from 4:1 to 1:13 and reduces the number of intrabulbar CD8 + lymphocytes and Langerhans cells.20 The abnormal expression of class I and II major histocompatibility complex molecules on the proximal follicular epithelium that is implemented in the auto-aggressive response by CD8+ lymphocytes is down-regulated after treatment with a
302 topical sensitizer.19,20 Researchers have proposed a role of antigenic competition3 to explain these findings, whereby the recruited suppressor CD8+ T cells generate an inhibitory effect on the immune process against hair follicles, thus allowing hair growth.
Treatment regimen Patients are usually sensitized using a 2% solution in acetone on a 10 to 16 cm2 area of the scalp with caution to not wash off the solution for 24 hours. One week later, if a severe eczematous reaction has not occurred; a 0.0001% solution is applied to the scalp. If the initial reaction is too severe, it is important to wait 1 week before starting the elicitation phase. The strength of the first eliciting concentration should be inversely proportional to the length of the initial response. The concentration of the solution can be increased weekly up to a maximum of 2% to ensure that an adequate allergic reaction occurs. The patient must be advised to avoid sun and to not wash the treated site for 48 hours to maintain an appropriate level of erythema and pruritus. Once unilateral hair growth has occurred, it is appropriate to initiate treatment of the entire scalp.21–23 Our experience involves sensitizing the patient with a 5% SADBE that is applied with a cotton applicator to a small area of the scalp or upper inner arm. Patients are sent home with a mild topical steroid to use in case of an uncomfortable reaction, such as pruritus or vesicle formation. Patients with adequate sensitization will develop an eczematous patch in the treated area within a few days. One month after sensitization, the patient returns on a 1- to 2-month schedule for application of 5% SADBE to the alopecic patches until maximum hair regrowth. Patients are told to not wash the areas for 24 hours. At follow-up visits, the treated sites are evaluated for hair regrowth. Treatment was deferred for 1 to 2 months in cases of local blistering. If the patient shows response, treatment is continued as tolerated until desired outcome.
Nonresponders One barrier to effective treatment of AA with SADBE is the issue of nonresponse. Van der Steen estimated that between 1% and 2% of patients are unable to be sensitized21 with other estimates as high as 20%.24 Additionally, patients who have been successfully sensitized can develop tolerance and has been reported in 10.8%. A patient can develop tolerance in one bodily location but maintain sensitivity elsewhere. 22 In one study, the histamine antagonist cimetidine has been reported to disrupt tolerance to SADBE, purportedly through augmentation of delayed type hypersensitivity.2,25 Other researchers were not able to reproduce these results.26
N.D. Hill et al.
Adverse effects The most common adverse effects associated with SADBE include eczema, auto-eczematization, blistering, and swelling of regional lymph nodes.18 Other less common side effects include burning sensation after application, persistent allergic contact dermatitis of the primary site of sensitization, severe generalized dermatitis, generalized pruritis, leukoderma, xerosis, scaling, edema of treated skin, scalp folliculitis, and systemic reactions characterized by fever and arthalgias.22 It is reported that most patients tolerate the therapy well and the above reactions may resolve after discontinuation of the treatment or with use of topical corticosteroids and antihistamines.
Safety profile SADBE is a potent sensitizer that is used as an industrial stabilizer and an antifog agent in photographic emulsions. This agent is nonmutagenic in the highly sensitive Salmonella microsome test. Tetrachloro-2-cyclobutene-1-one is an intermediate in the synthesis of SADBE that when injected subcutaneously, caused rhabdomyosarcoma in 1 out of 20 mice. Lifetime subcutaneous injections of SADBE into 20 mice caused one fibrosarcoma. Additional testing on animals is necessary before the safety of SADBE can be established.22 No sensitizer is FDA approved for this therapeutic intervention and caution must be taken when topical sensitizers are recommended as a treatment option for patients.
Efficacy No randomized controlled trials have evaluated the effectiveness of topical sensitizers in AA. Some observational studies have used the half-head model to control for spontaneous regrowth. The success rate of DPCP and SADBE is approximately 50% to 60% with a range of 9% to 87%.22,27 When assessed alone, SADBE had a total response rate (defined as moderate to good regrowth or at least 30% improvement) from 29% to 87%, which correlated with the response rate of 65%.18,22 To date, 13 studies using SADBE and 17 using DPCP have revealed little difference between the two agents,27 which support the notion that these chemicals may elicit the same immunologic mechanism.28 There are several prognostic factors that negatively influence response to topical sensitizers. Van der Steen et al29 found that type and/or extent of alopecia, duration of disease, and the presence of nail factors predicted a poorer response. Extent of alopecia and duration of disease are the most commonly cited negative prognostic factors that have been reported in the literature. Other prognostic factors are age at onset and family history of alopecia areata, followed by presence of nail changes.22,29 In addition, an earlier
Use of squaric acid for alopecia areata development of dermatitis may be a positive prognostic factor. Caserio17 determined that 3 out of 4 successfully treated patients had been sensitized within 3 weeks or less, compared to 10 unsuccessfully treated patients in whom the sensitization took longer to develop.
SADBE in the literature In 1980,3 application of SADBE was found to be an initially effective treatment for 87% of individuals with alopecia areata, and a persistent response was noted in 70%. Since this initial study, there have been several similar clinical trials assessing the efficacy of SADBE. SADBE has proven to be an effective treatment for long-standing AA. The 21 patients in the study had long-standing, severe disease refractory to previous therapies. Eleven of the 21 had an excellent response consisting of complete hair regrowth. In addition, it was noted that patients with patchy alopecia responded better to treatment than those with alopecia totalis or alopecia universalis (73% and 30%, respectively).24 Analysis of the results of a controlled trial30 involving 119 patients with patchy alopecia treated with SADBE, DPCP, minoxidil, or placebo failed to show a statistically significant difference between the control and treatment groups. It was noted that 18 of the subjects were children (under the age of 14), and there was a significant difference in the rate of complete hair regrowth in children versus adults (38.9% and 71.3%, respectively). In another study on 14 patients with AA who had not responded to conventional treatments, only 28.5% of the patients had persistent hair growth while receiving SADBE treatment. Of these 14 patients, five were 16 years old or younger, and all but one eventually had alopecia totalis or universalis. The younger ages of these patients, as well as their long-standing and extensive disease history, were thought to be responsible for the lower than expected success rate.17 One study18 treated 144 patients with AA of varying degrees with SADBE. Of these patients, 71 had AA affecting less than 50% of the scalp, whereas 73 had a more severe form, including 13 patients with alopecia totalis and two with alopecia universalis. Eighty percent of patients with the less severe type experienced hair regrowth, compared to 49% of the more severe form. Unlike previous studies, Micali et al18 did not find age or duration of alopecia to be factors predictive of response to treatment. In a retrospective case series of 36 AA patients treated with SADBE using medical records,31 about 21% of patients with diffuse hair loss responded to treatment, compared to 58.3% of patients with patchy hair loss. Neither age nor duration of hair loss was found to be a predictor of response. Severity of disease was determined to be a prognostic factor, as those with patchy hair loss benefited more than those with diffuse disease.
303 Other researchers32 followed a group of 54 patients affected by severe AA treated with SADBE for at least 2 years. Collected data were compared to a matched control group of 54 patients who did not receive any treatment. The authors found that 80% of the treatment group was in remission, compared to 50% of the control group. Neither age, gender, nor disease duration were found to be contributing factors. During follow-up, there was not a significant difference in relapse rate between the treatment and control groups (44% and 52%, respectively). Relapses in the treatment group were found to be significantly less severe than that of the control group (P b .001). A study on 60 patients with AA who were unresponsive to conventional therapies19 showed that, overall, 43% experienced successful hair regrowth from SADBE. Subjects with b 50% scalp involvement had a 68% success rate, compared to 29% of those with more diffuse disease. Younger age at time of disease onset, duration of disease, severity of disease, and nail changes were associated with poor response to SADBE. The authors presented a small case study (10 patients) regarding sensitization and treatment with SADBE 5%. The study reported a response usually after 2 to 4 applications with 50% (5/10) graded as good (75%–100%) regrowth, 30% fair (25%–74%) regrowth, and 20% poor (0%–24%) regrowth.33
Conclusions SADBE appears to be a safe and effective treatment for individuals suffering from AA. Several prognostic factors including duration of disease, extent of alopecia, age at onset, and nail changes may be able to predict the level of a patient’s responsiveness to treatment with SADBE. The long-term studies, although demonstrating a significant relapse rate, do suggest immunotherapy has a useful role in managing this condition. Further controlled prospective studies are needed to answer remaining questions about the use of SADBE and other topical sensitizers in AA.
References 1. Gardner S, Freyschmidt-Paul P, Hoffman R, et al. Normalization of hair follicle morphology in C3 H/HeJ alopecia areata mice after treatment with squaric acid dibutylester. Eur J Dermatol. 2000;10: 443-450. 2. Daman LA, Rosenberg EW, Drake L. Treatment of alopecia areata with dinitrochlorobenzene. Arch Dermatol. 1978;114:1036-1038. 3. Happle R, Kalveram KJ, Büchner U, et al. Contact allergy as a therapeutic tool for alopecia areata: Application of squaric acid dibutylester. Dermatologica. 1980;161:289-297. 4. Safavi KH, Muller SA, Suman VJ, et al. Incidence of alopecia areata in Olmsted County, Minnesota, 1975 through 1989. Mayo Clin Proc. 1995;70: 628-633. 5. Safavi K. Prevalence of alopecia areata in the First National Health and Nutrition Examination Survey. Arch Dermatol. 1992;128:702. 6. Norris D. Alopecia areata: Current state of knowledge. J Am Acad Dermatol. 2004;51(1 Suppl):S16-S17.
304 7. McElwee KJ, Gilhar A, Tobin DJ, et al. What causes alopecia areata? Exp Dermatol. 2013;22:609-626. 8. Gilhar A, Paus R, Kalish R. Lymphocytes, neuropeptides, and genes involved in alopecia areata. J Clin Investig. 2007;117:2019-2027. 9. Rückert R, Hofmann U, van der Veen C, et al. MHC class I expression in murine skin: Developmentally controlled and strikingly restricted intraepithelial expression during hair follicle morphogenesis and cycling, and response to cytokine treatment in vivo. J Invest Dermatol. 1998;111:25-30. 10. Paus R, Slominski A, Czarnetzki B. Is alopecia areata an autoimmuneresponse against melanogenesis-related proteins, exposed by abnormal MHC class I expression in the anagen hair bulb? Yale J Biol Med. 1994;66:541-554. 11. Jabbari A, Petukhova L, Cabral RM, et al. Genetic basis of alopecia areata: A roadmap for translational research. Dermatol Clin. 2013;31: 109-117. 12. Pender MP. CD8 + T-cell deficiency, Epstein-Barr virus infection, Vitamin D deficiency, and steps to autoimmunity: A unifying hypothesis. Autoimmun Dis. 2012:189096. 13. Skinner Jr RB, Light WH, Leonardi C, et al. PCR evidence of cytomegalovirus in alopecia areata. J Invest Dermatol. 1995;104:686. 14. Rodriguez TA, Duvic M. Onset of alopecia areata after Epstein-Barr virus infectious mononucleosis. J Am Acad Dermatol. 2008;59:137-139. 15. D’Ovidio R. d’Ovidio F. Recidive stagionali dell’alopecia areata. G Ital Dermatol Venereol. 1995;295:130-134. 16. DeLeve LD. Dinitrochlorobenzene is genotoxic by sister chromatid exchange in human skin fibroblasts. Mutat Res. 1996;371:105-108. 17. Caserio RJ. Treatment of alopecia areata with squaric acid dibutylester. Arch Dermatol. 1987;123:1036-1041. 18. Micali G, Cicero RL, Nasca MR, et al. Treatment of alopecia areata with squaric acid dibutylester. Int J Dermatol. 1996;35:52-56. 19. Ajith C, Gupta S, Kanwar AJ. Efficacy and safety of the topical sensitizer squaric acid dibutylester in alopecia areata and factors influencing the outcome. J Drugs Dermatol. 2006;5:262-266. 20. Brocker EB, Echternacht-Happle K, Hamm H. Abnormal expression of class I and class II major histocompatibility antigens in alopecia areata modulation by topical immunotherapy. J Invest Dermatol. 1987;88: 564-568.
N.D. Hill et al. 21. Van der Steen PH, Happle R. Topical immunotherapy of alopecia areata. Dermatol Clin. 1993;11:619-622. 22. Rokshar CK, Shupack JL, Vafai JJ, et al. Efficacy of topical sensitizers in the treatment of alopecia areata. J Am Acad Dermatol. 1998;39:751-761. 23. Shapiro J. Topical immunotherapy in the treatment of chronic severe alopecia areata. Dermatol Clin. 1993;11:661. 24. Case PC, Mitchel AJ, Swanson NA, et al. Topical therapy of alopecia areata with squaric acid dibutylester. J Am Acad Dermatol. 1984;10:447-451. 25. Avella J, Madsen JE, Binder HJ, et al. Effect of histamine H2-receptor antagonists on delayed hypersensitivity. Lancet. 1978;1:624-626. 26. Van der Steen PH, van Baar HM, Happle R, et al. Prognostic factors in the treatment of alopecia areata with diphenylcyclopropenone. J Am Acad Dermatol. 1991;24:227-230. 27. Harries MJ, Sun J, Paus R, et al. Management of alopecia areata. BMJ. 2010;341:c3671. 28. Happle R, Klein H, Macher E. Topical immunotherapy changes in the composition of the peribulbar infiltrate in alopecia areata. Arch Dermatol Res. 1986;278:214-218. 29. Van der Steen PH, van Baar HM, Perret CM, et al. Treatment of alopecia areata with diphenylcyclopropenone. J Am Acad Dermatol. 1991;24:253-257. 30. Tosti A, De Padova MP, Minghetti G, et al. Therapies versus placebo in the treatment of patchy alopecia areata. J Am Acad Dermatol. 1986;15:209-210. 31. Pardasani AG, Turner E, McMichael AJ. Squaric acid dibutylester: Indications for use and efficacy in alopecia areata. Arch Dermatol. 2001;137:970-972. 32. Dall’oglio F, Nasca MR, Musumeci ML, et al. Topical immunomodulator therapy with squaric acid dibutylester (SADBE) is effective treatment for severe alopecia areata (AA): Results of an open-label, paired-comparison, clinical trial. J Dermatolog Treat. 2005;16:10-14. 33. Diaz L, Morgan L, Pacha O, et al. Treatment of alopecia areata in children with topical 5% squaric acid dibutylester. Poster session presentation. 38th Annual Meeting of the Society for Pediatric Dermatology; 2012 July 11-1; Monterey, CA. 34. Tarlow JK, Clay FE, Cork MJ. Severity of alopecia areata is associated with a polymorphism in the interleukin-1 receptor antagonist gene. J Invest Dermatol. 1994;103:387-390.
