Accepted Manuscript. Stephanie Kao, BA, Alexi Kiss, MD, Tatiana Efimova, PhD, Adam Friedman, MD

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1 Accepted Manuscript An ex-vivo evaluation of cytotoxicity and melanocyte viability after A-101 hydrogen peroxide topical solution 40% or cryosurgery treatment in Seborrheic Keratosis lesions Stephanie Kao, BA, Alexi Kiss, MD, Tatiana Efimova, PhD, Adam Friedman, MD PII: S (18) DOI: /j.jaad Reference: YMJD To appear in: Journal of the American Academy of Dermatology Received Date: 7 January 2018 Revised Date: 21 March 2018 Accepted Date: 21 March 2018 Please cite this article as: Kao S, Kiss A, Efimova T, Friedman A, An ex-vivo evaluation of cytotoxicity and melanocyte viability after A-101 hydrogen peroxide topical solution 40% or cryosurgery treatment in Seborrheic Keratosis lesions, Journal of the American Academy of Dermatology (2018), doi: / j.jaad This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 Title: An ex-vivo evaluation of cytotoxicity and melanocyte viability after A-101 hydrogen peroxide topical solution 40% or cryosurgery treatment in Seborrheic Keratosis lesions Stephanie Kao, BA 1 ; Alexi Kiss, MD 2 ; Tatiana Efimova, PhD 2,3 ; Adam Friedman, MD 3,4 1 The George Washington University School of Medicine & Health Sciences, Washington, DC, USA 2 Department of Anatomy and Cell Biology, The George Washington University School of Medicine & Health Sciences, Washington, DC, USA 3 Department of Dermatology, The George Washington University School of Medicine & Health Sciences, Washington, DC USA 4 Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY USA Corresponding author: Adam Friedman, MD Department of Dermatology George Washington University School of Medicine & Health Sciences 2150 Pennsylvania Ave, NW, Suite 2B-430 Washington, DC U.S.A Phone: ajfriedman@mfa.gwu.edu Funding sources: Aclaris Therapeutics, Inc. Conflicts of interest: Adam Friedman is a consultant for Aclaris Therapeutics Reprint requests: Adam Friedman Manuscript word count: 489 References: 5 Figures: 1 Keywords: A-101; cryosurgery; skin of color; seborrheic keratosis

3 To the Editor Seborrheic keratosis (SK) is one of the most common dermatologic lesions and is therefore the most common skin tumor seen by dermatologists in everyday practice. 1 Currently, cryosurgery is one of the most commonly employed and successful techniques to physically resolve a SK. 2 However, all patients are susceptible to some degree of blistering, infection, scarring, recurrence, and pigmentary changes post-treatment, but darker-skinned individuals are particularly vulnerable to the last three adverse events 3. To help predict the potential toxicological impact of A-101, a recently FDA-approved topical 40% hydrogen peroxide solution for the treatment of SK 4, as compared to the gold standard cryosurgery, we evaluated skin architecture, metabolic activity, and cytotoxicity, with a particular emphasis on melanocytes, utilizing a validated ex-vivo human reconstituted full thickness model derived from Fitzpatrick V skin (MRHE). 5 MRHE were treated with cryosurgery (5 and 10 second freeze cycles) or A-101 (1 and 2 µl) and analyzed by MTT assay, Fontana-Masson, TUNEL, and S100 immunohistochemical staining to determine skin architecture, metabolic activity, and cytotoxicity. Histologic evaluation of untreated and A-101 vehicle-treated tissues revealed a normal stratum corneum, spinous and basal cell layers overlying a well-organized dermis with scattered fibroblasts. MRHE treated with both cryosurgery for 5 and 10 seconds demonstrated overall thinning of the epidermis, with more pyknotic cells noted in the deeper spinous layer of the 10 seconds specimens. In both groups, dermal-epidermal junction separation was noted, though more prominent in the 10 seconds group. In both A-101-treated groups, acanthosis of the epidermis and mild pallor was noted, though not to the extent of the cryosurgery-treated specimens, without epidermal clefting (data not shown). To quantify the degree of epidermal damage incurred by treatment with cryosurgery or A-101, TUNEL staining was utilized to identify apoptotic cells. For cryosurgery 5 and 10 seconds, 16.4

4 and TUNEL-positive cells were identified per HPF, compared to the two doses of A101 used, and positive cells for 1 µl and 2 µl respectively (Figure 1a). Cellular metabolic activity was assessed using MTT assay, showing reduced metabolic activity, indicative of reduced viability, in cryosurgery-treated MRHE samples as compared to A-101-treated groups (Figure 1b). Within HPFs, untreated and vehicle-treated MRHEs were found to have and melanocytes based on S100 staining (Figure 1c). This is compared to and for cryosurgery 5 and 10 seconds, respectively, and A and 2 µls, and , respectively. Together, we show that cryosurgery is more cytotoxic than A-101, with special consideration of melanocyte survival. Our data suggests that A-101 may be a less caustic option for SK removal that reduces the risk of post-treatment pigmentary alterations. However, to best appreciate these findings, a clinical study that examines the risk of hypo or hyperpigmentation in darker skin types after A-101 treatment is warranted and is ongoing (ClinicalTrials.gov Identifier: NCT ).

5 4 73 References Coyne JD. Classification of the Seborrheic Keratosis. Int J Surg Pathol. 2016;24(1): Farhangian ME, Snyder A, Huang KE, Doerfler L, Huang WW, Feldman SR. Cutaneous cryosurgery in the United States. J Dermatolog Treat. 2016;27(1): Andrews MD. Cryosurgery for common skin conditions. Am Fam Physician. 2004;69(10): Draelos ZD KS, Smith SR, Wilson DC, Powala CV, Bradshaw M, Estes E, Shanler SD. Safety and Efficacy of A-101 Hydrogen Peroxide Topical Solution in Adults with Seborrheic Keratosis: Results from the Phase 3, Randomized, Double-Blind, Vehicle- Controlled, Parallel-Group Study. SKIN The Journal of Cutaneous Medicine. 2017;1:s Yoon TJ, Lei TC, Yamaguchi Y, Batzer J, Wolber R, Hearing VJ. Reconstituted 3- dimensional human skin of various ethnic origins as an in vitro model for studies of pigmentation. Anal Biochem. 2003;318(2):

6 Figure Legends Figure 1. Cell viability was assessed via TUNEL immunohistochemical staining (a) and MTT assay (b). 9-millimeter diameter MRHE tissues were fixed in 10% formaldehyde and paraffin embedded. Cell counts to quantify TUNEL-positive cells were performed at high power (40x) from 20 representative fields utilizing randomly selected 5 samples per group. MTT assay data is representative of four independent experiments from four different MRHE tissues. *P<0.05, **P<0.01. S100 immunohistochemistry stains of MRHEs treated with cryosurgery or A-101 were used to assess number of melanocytes, respectively (c). Cell counts were performed at high power (40x) from 20 representative fields utilizing randomly selected 5 samples per group. *P<0.05. HPF, high power field.

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