A Nitric Oxide–Releasing Topical Medication as a Potential Treatment Option for Atopic Dermatitis through Antimicrobial and Anti-Inflammatory Activity

Open AccessPublished:May 15, 2020DOI:https://doi.org/10.1016/j.jid.2020.04.013

      Abbreviations:

      AD (atopic dermatitis), Th (T helper type)
      To the Editor
      Atopic dermatitis (AD) is a common, chronic inflammatory skin condition characterized by intense pruritis and recurrent eczematous lesions (
      • Weidinger S.
      • Novak N.
      Atopic dermatitis.
      ). An unmet medical need exists for a well-tolerated and effective topical treatment (
      • Diaz A.
      • Guttman-Yassky E.
      Topical agents for the treatment of atopic dermatitis.
      ).
      AD pathogenesis involves a complex interplay between genetic predisposition to skin barrier dysfunction and immune dysregulation. Staphylococcus aureus colonization, occurring in >80% of AD lesions, can further alter the skin barrier and stimulate immune responses (
      • Li R.
      • Hadi S.
      • Guttman-Yassky E.
      Current and emerging biologic and small molecule therapies for atopic dermatitis.
      ,
      • Mempel M.
      • Lina G.
      • Hojka M.
      • Schnopp C.
      • Seidl H.P.
      • Schäfer T.
      • et al.
      High prevalence of superantigens associated with the egc locus in Staphylococcus aureus isolates from patients with atopic eczema.
      ,
      • Weidinger S.
      • Novak N.
      Atopic dermatitis.
      ).
      SB414, an investigational topical product, is a cream containing berdazimer sodium (CAS Registry Number 1846565-00-1), a macromolecule composed of a polysiloxane backbone with covalently bound N-diazeniumdiolate nitric oxide donors (
      • Stasko N.
      • McHale K.
      • Hollenbach S.J.
      • Martin M.
      • Doxey R.
      Nitric oxide-releasing macromolecule exhibits broad-spectrum antifungal activity and utility as a topical treatment for superficial fungal infections.
      ).
      Here, we report the effect of SB414 on S. aureus colonization in an FLG-deficient AD murine model (FLGft/ft) and the subsequent cutaneous immune response. A clinical study assessed the mechanism of action of SB414 by analyzing the changes in gene expression of biomarkers indicative of AD-related immune pathways and also evaluated systemic exposure, short-term efficacy, and safety.
      The University of California San Diego Institutional Animal Care and Use Committee reviewed and approved the animal protocol (approval number: S09074). In the AD skin model of ovalbumin-sensitized and tape-stripped FLGft/ft Balb/c mice (
      • Nakatsuji T.
      • Chen T.H.
      • Two A.M.
      • Chun K.A.
      • Narala S.
      • Geha R.S.
      • et al.
      Staphylococcus aureus exploits epidermal barrier defects in atopic dermatitis to trigger cytokine expression.
      ), S. aureus levels in the skin of SB414 6%–treated mice were decreased to 5.3 × 105 colony forming units within 24 hours, representing >90% reduction in S. aureus versus untreated (P = 0.005) and vehicle-treated (P = 0.002) mice (Figure 1a, Supplementary Figure S1). In addition, skin biopsies of S. aureus-colonized lesions revealed that SB414 treatment led to substantial reductions in T helper type (Th) 2–related cytokines, including a 76% reduction in IL-13 gene expression (P = 0.002) and an 87% reduction in IL-4 expression (P = 0.008) versus vehicle treatment (Figure 1b). SB414 treatment also reduced TSLP, IFNγ, and CXCL2, but not IL-17A (data not shown). After confirming modification of the biomarkers in the mouse model, SB414 was advanced to a human clinical trial to prove the mechanism of action.
      Figure thumbnail gr1
      Figure 1Reduction of SA levels and key Th2-related cytokines in skin biopsy specimens in AD mouse model. (a) CFUs averaged 6.8 × 106, 8.9 × 106, and 5.3 × 105 for the SA untreated, vehicle (placebo), and SB414 6%–treated groups, respectively. This represented more than a 90% reduction in SA CFUs recovered from the skin of infected mice following SB414 6% treatment. (b) Inoculation of SA increased IL-13 expression by 3.9-fold and IL-4 expression by 12.7-fold compared with no SA inoculation. SB414 6% treatment reduced IL-13 expression levels by 76% (P = 0.002) and IL-4 by 87% (P = 0.008) compared with vehicle (placebo) 24 hours after treatment. Data represent mean ± SD. P-values based on a 2-tailed t-test. AD, atopic dermatitis; CFU, colony forming unit; Th, T helper type; SA, Staphylococcus aureus.
      An institutional review board (Quorum Review Institutional Review Board, Seattle, WA) reviewed and approved all materials for the clinical study, and patients provided written, informed consent before screening procedures were initiated. A total of 48 adults with mild to moderate AD were randomized to SB414 2% (n = 17), SB414 6% (n = 17), and vehicle (n = 14) applied twice daily. Of five patients who discontinued (SB414 2%, n = 1; SB414 6%, n = 2; and vehicle, n = 2), three were due to adverse events (SB414 6%, n = 2; vehicle, n = 1; all three experienced moderate application-site reactions such as pruritus, pain, or swelling). Mean Eczema Area and Severity Index scores (SD) were 8.2 (5.2) for vehicle, 4.7 (2.0) for SB414 2%, and 7.2 (3.3) for SB414 6%. Mean (SD) Eczema Area and Severity Index scores decreased for all treatment groups from baseline to week 2, with the greatest percent reduction of –23.22% (29.14) for SB414 2%. There were no statistical comparisons of efficacy measures.
      To understand the mechanism of action of SB414, RNA sequencing and RT-PCR were conducted from patient skin biopsy samples. RNA sequencing showed 612 differentially expressed genes (fold change > 2, false discovery rate < 0.05) between lesional and nonlesional skin at baseline (363 upregulated and 249 downregulated). Following 2 weeks of treatment, the study transcriptome showed 65.5% improvement to more closely resemble nonlesional skin for SB414 2% as compared with a worsening of –20.9% for SB414 6% and a minor improvement of 4.5% for vehicle (P < 0.05) (Figure 2a–c).
      Figure thumbnail gr2
      Figure 2Changes in gene expression of lesional skin transcriptome from baseline to week 2 for SB414 2%, SB414 6%, and vehicle. (a) At baseline, there were 612 differentially expressed genes in LS compared with NL skin (363 genes were upregulated and 249 were downregulated) using FCH > 2 and FDR < 0.05 criteria. The heatmap shows reversal of LS transcriptome toward NL skin for the SB414 2% group (red at baseline shifting to blue at W2, and blue at baseline shifting to red at W2). (b) The SB414 2% group achieved a mean percent improvement of 65.5% (P < 0.001 vs. SB414 6% and vehicle). (c) Mean log2 FCH of differentially expressed genes that were upregulated (red boxes) or downregulated (blue boxes) in LS skin compared with NL skin. AD, atopic dermatitis; BL, baseline; FCH, fold change; FDR, false discovery rate; LS, lesional skin; NL, nonlesional skin; W2, week 2.
      Only SB414 2% significantly suppressed the immune signature of AD lesions, whereas exacerbation of the signature for lesions treated with either SB414 6% or vehicle was apparent. Gene set variation analysis, an unsupervised sample-wise enrichment method that produces a score of activity for a gene subset or pathway for each sample (
      • Hänzelmann S.
      • Castelo R.
      • Guinney J.
      GSVA: gene set variation analysis for microarray and RNA-seq data.
      ), also indicated a statistically significant decrease in a subset of immune genes (
      • Bissonnette R.
      • Pavel A.B.
      • Diaz A.
      • Werth J.L.
      • Zang C.
      • Vranic I.
      • et al.
      Crisaborole and atopic dermatitis skin biomarkers: an intrapatient randomized trial.
      ,
      • Ewald D.A.
      • Malajian D.
      • Krueger J.G.
      • Workman C.T.
      • Wang T.
      • Tian S.
      • et al.
      Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
      ,
      • Guttman-Yassky E.
      • Bissonnette R.
      • Ungar B.
      • Suárez-Fariñas M.
      • Ardeleanu M.
      • Esaki H.
      • et al.
      Dupilumab progressively improves systemic and cutaneous abnormalities in patients with atopic dermatitis.
      ,
      • Pavel A.B.
      • Song T.
      • Kim H.J.
      • Del Duca E.
      • Krueger J.G.
      • Dubin C.
      • et al.
      Oral Janus kinase/SYK inhibition (ASN002) suppresses inflammation and improves epidermal barrier markers in patients with atopic dermatitis.
      ), including genes related to innate immunity (IL-6), Th2 pathway (IL-4R and CCL11), Th17/Th22 (IL-36G; S100A7, 8, 9,12; IL-22), Th9 (IL-9), and Th1 pathway (CXCL10 and CXCL11) with SB414 2% at week 2 (data not shown).
      Based on gene set variation analysis, SB414 2% was associated with statistically significantly greater suppression of Th17-related gene expression versus both vehicle and baseline (P < 0.01 for both comparisons). Other AD-related pathways showed similar changes using gene set variation analysis, including the meta-analysis–derived AD transcriptome and Th1-, Th2-, and Th22-specific pathways (data not shown).
      Changes in expression of key AD-related biomarkers identified by RNA sequencing data were validated using quantitative RT-PCR analysis of 45 immune response genes. Overall, changes in AD-related biomarkers were observed with the SB414 2% treatment after 2 weeks (Supplementary Figure S2). Although all immune axes showed significant modulation with treatment, the most robust changes were observed in Th17/Th22 genes such as S100A8, 9, and 12; STAT3; IL-6; IL-6R; CXCL2 (P < 0.01); and IL-22 (P < 0.05).
      Only descriptive pharmacokinetic analyses were performed owing to limited systemic exposure to SB414. No SB414 2% patient had quantifiable concentrations of hydrolyzed N-methylaminopropyl-trimethoxysilane, a backbone of the polymeric drug substance, postdose on day 1 or 15. Only five SB414 6% patients had quantifiable hydrolyzed N-methylaminopropyl-trimethoxysilane concentrations at 1 hour postdose on day 1 or 15 (highest concentration, 34.46 ng/ml; lower limit of quantitation, 5 ng/ml); levels then declined, becoming undetectable in all but two samples (14.3%) by 12 hours postdose.
      Adverse events were reported by one SB414 2% patient (headache), two SB414 6% patients (application-site reactions), and three vehicle patients (application-site reactions, accidental product exposure, and AD), with no correlation between adverse events and systemic hydrolyzed N-methylaminopropyl-trimethoxysilane concentrations.
      The exacerbation in gene expression of AD-related biomarkers observed following SB414 6% treatment may be due to skin irritation. In patients who reported irritation (SB414 2%, n = 2; SB414 6%, n = 4; vehicle, n = 2), a post hoc analysis revealed that SB414 6% was associated with greater expression of Th1 and innate cytokines, including IL-1β, IFNγ, CXCL9, CXCL10, and CCL3 in lesional skin, compatible with a possible irritant contact dermatitis profile (
      • Nosbaum A.
      • Vocanson M.
      • Rozieres A.
      • Hennino A.
      • Nicolas J.F.
      Allergic and irritant contact dermatitis.
      ). TSLP did not change significantly following treatment with either 2% or 6% SB414 twice daily. In a toxicology study of minipigs, SB414 10% increased skin pH and irritation over time, whereas SB414 0.5% and 2% did not (data not shown), supporting the tolerability concern with SB414 6%.
      Although this study is preliminary, evaluating short-term immunomodulatory effects of SB414 on AD lesions, topical nitric oxide–releasing SB414 2% showed significant reductions in Th2-, Th22-, Th1-, and Th17-related biomarker expression and may help treat the various clinical and molecular endotypes of AD (
      • Czarnowicki T.
      • He H.
      • Krueger J.G.
      • Guttman-Yassky E.
      Atopic dermatitis endotypes and implications for targeted therapeutics.
      ;
      • Renert-Yuval Y.
      • Guttman-Yassky E.
      What's new in atopic dermatitis.
      ).

       Data availability statement

      Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141571, hosted at the National Center for Biotechnology Information’s Gene Expression Omnibus (accession number: GSE141571; secure reviewer's token: qpwzewmadpuvdcx).

      ORCIDs

      Conflict of Interest

      EGY is an employee of Mount Sinai and has received research funds (grants paid to the institution) from Abbvie, Almirall, Amgen, AnaptysBio, Asana Biosciences, Boerhinger-Ingelhiem, Celgene, Dermavant, DS Biopharma, Eli Lilly, Glenmark, Galderma, Innovaderm, Janssen, Kiniska, Kyowa Kirin, Leo Pharma, Novan, Pfizer, Ralexar, Regeneron, Sienna Biopharma, UCB, and Union Therapeutics. EGY is also a consultant for Abbvie, Almirall, Amgen, Asana Biosciences, Boerhinger-Ingelhiem, Cara Therapeutics, Celgene, Concert, DBV, Dermira, DS Biopharma, Eli Lilly, EMD Serono, Escalier, Galderma, Glenmark, Kyowa Kirin, Leo Pharma, Mitsubishi Tanabe, Pfizer, RAPT Therapeutics, Regeneron, Sanofi, Sienna Biopharma, and Union Therapeutics.
      RLG has received research funding from Novan. RLG is a cofounder of and has equity interest in MatriSys.
      EM and TMC are employees of and stockholders in Novan, Inc.

      Acknowledgments

      Research was sponsored by Novan, Inc, ClinicalTrials.gov Identifier: NCT03431610. The authors are grateful to the patients who gave the gift of clinical research participation. In addition, the authors thank all the principal investigators for their contributions to the study: David M. Pariser, MD, Norfolk, VA; Michael A. Hassman, DO, Berlin, NJ; Mae Gutierrez, MD, Coral Gables, FL; Diane O. McConnehey, DO, CPI, Boise, ID; Phoebe Rich, MD, Portland, OR; Scott A. Fretzin, MD, Indianapolis, IN; Edward L. Lain, MD, MBA, Pflugerville, TX; Ana M. Elosegui, MD, Sweetwater, FL; and Stephen M. Schleicher, MD, Hazleton, PA. The authors also thank Carolyn Enloe of Novan, Inc for operational support of the phase 1b study. Medical writing assistance was provided by Dana L. Randall, MS, PharmD, Intuitive Graphite, Inc and funded by Novan , Inc.
      Data included in this manuscript were presented in part as a poster at the International Investigative Dermatology 2018 Meeting (Hollenbach S, Nakatsuji T, Gallo R, Stasko N. Effects of SB414 cream on S. aureus and tissue cytokines in an atopic dermatitis mouse model. May 16–19, 2018; Orlando, FL) and at the 3rd Inflammatory Skin Disease Summit in December 2018 in Vienna, Austria (Maeda-Chubachi T, Durham T, Schleicher S, Rich P, Guttman-Yassky E. Abstract 30. A topical nitric oxide-releasing cream SB414: results of a phases 1b double-blind, randomized, vehicle-controlled study in patients with mild-to-moderate atopic dermatitis. Exp Dermatol. 2018;27(Suppl 2):14).

      Disclaimer

      Novan, Inc was involved in the study design; collection, analysis, and interpretation of data; writing of the report; and the decision to submit the article for publication.

      Author Contributions

      Conceptualization: EGY, RG, EM, TMC; Data Curation: ABP, TN; Formal Analysis: ABP, TN; Funding Acquisition: TMC; Investigation: RL, NZ, TN; Methodology: EGY, RG, TN, TMC; Project Administration: EGY, RG, TMC; Resources: EGY, ABP, TN; Software: ABP, TN; Supervision: EGY, RG, TMC, EM; Validation: ABP, TN; Visualization: ABP, TN; Writing - Original Draft Preparation: ABP; Writing - Review and Editing: EGY, RG, ABP, EM, TMC

      Supplementary Material

      Figure thumbnail fx1
      Supplementary Figure S1Effects of SB414 6% on Staphylococcus aureus colonization in an FLG-defect atopic dermatitis mouse module (FLGft/ft mice). (a) Groups 1, 2, and 3: 48 hours with no S. aureus inoculation. (b) Groups 4, 5, and 6: 48 hours after S. aureus inoculation, CFUs averaged 6.8 × 106 for the untreated, 8.9 × 106 for the vehicle, and 5.3 × 105 for the SB414 groups. S. aureus levels in the skin of SB414-treated mice represented more than a 90% reduction in S. aureus compared with untreated (P = 0.005) and vehicle-treated (P = 0.002) mice. P-values based on a 2-tailed, equal variance, t-test. CFU, colony forming unit.
      Figure thumbnail fx2
      Supplementary Figure S2Changes in RT-PCR gene expression of atopic dermatitis–related inflammatory biomarkers and associated fold changes between baseline and W2. A heatmap demonstrating the visual change in genomic expression of atopic dermatitis–related biomarkers after application of vehicle, SB414 2%, or SB414 6% b.i.d. to target lesions for 2 weeks is shown on the left. For each treatment group, results from skin biopsies of LS at BL, LS at W2, and NL skin at baseline are shown. Blue indicates downregulation of gene expression and red indicates upregulation. On the right, fold changes in biomarker gene expression for W2 versus baseline are shown for LS. P-values are based on a linear mixed-effect model with fixed factors for tissue type (LS vs. NL skin), visit, and treatment and a random intercept for each patient were used to estimate the within-treatment group adjusted means and fold changes (i.e., differences) between LS samples pre- and post-treatment. b.i.d., twice daily; BL, baseline; LS, lesional skin; NL, nonlesional skin; Th, T helper type; W2, week 2.

      References

        • Bissonnette R.
        • Pavel A.B.
        • Diaz A.
        • Werth J.L.
        • Zang C.
        • Vranic I.
        • et al.
        Crisaborole and atopic dermatitis skin biomarkers: an intrapatient randomized trial.
        J Allergy Clin Immunol. 2019; 144: 1274-1289
        • Czarnowicki T.
        • He H.
        • Krueger J.G.
        • Guttman-Yassky E.
        Atopic dermatitis endotypes and implications for targeted therapeutics.
        J Allergy Clin Immunol. 2019; 143: 1-11
        • Diaz A.
        • Guttman-Yassky E.
        Topical agents for the treatment of atopic dermatitis.
        Expert Rev Clin Immunol. 2019; 15: 369-382
        • Ewald D.A.
        • Malajian D.
        • Krueger J.G.
        • Workman C.T.
        • Wang T.
        • Tian S.
        • et al.
        Meta-analysis derived atopic dermatitis (MADAD) transcriptome defines a robust AD signature highlighting the involvement of atherosclerosis and lipid metabolism pathways.
        BMC Med Genomics. 2015; 8: 60
        • Guttman-Yassky E.
        • Bissonnette R.
        • Ungar B.
        • Suárez-Fariñas M.
        • Ardeleanu M.
        • Esaki H.
        • et al.
        Dupilumab progressively improves systemic and cutaneous abnormalities in patients with atopic dermatitis.
        J Allergy Clin Immunol. 2019; 143: 155-172
        • Hänzelmann S.
        • Castelo R.
        • Guinney J.
        GSVA: gene set variation analysis for microarray and RNA-seq data.
        BMC Bioinformatics. 2013; 14: 7
        • Li R.
        • Hadi S.
        • Guttman-Yassky E.
        Current and emerging biologic and small molecule therapies for atopic dermatitis.
        Expert Opin Biol Ther. 2019; 19: 367-380
        • Mempel M.
        • Lina G.
        • Hojka M.
        • Schnopp C.
        • Seidl H.P.
        • Schäfer T.
        • et al.
        High prevalence of superantigens associated with the egc locus in Staphylococcus aureus isolates from patients with atopic eczema.
        Eur J Clin Microbiol Infect Dis. 2003; 22: 306-309
        • Nakatsuji T.
        • Chen T.H.
        • Two A.M.
        • Chun K.A.
        • Narala S.
        • Geha R.S.
        • et al.
        Staphylococcus aureus exploits epidermal barrier defects in atopic dermatitis to trigger cytokine expression.
        J Invest Dermatol. 2016; 136: 2192-2200
        • Nosbaum A.
        • Vocanson M.
        • Rozieres A.
        • Hennino A.
        • Nicolas J.F.
        Allergic and irritant contact dermatitis.
        Eur J Dermatol. 2009; 19: 325-332
        • Pavel A.B.
        • Song T.
        • Kim H.J.
        • Del Duca E.
        • Krueger J.G.
        • Dubin C.
        • et al.
        Oral Janus kinase/SYK inhibition (ASN002) suppresses inflammation and improves epidermal barrier markers in patients with atopic dermatitis.
        J Allergy Clin Immunol. 2019; 144: 1011-1024
        • Renert-Yuval Y.
        • Guttman-Yassky E.
        What's new in atopic dermatitis.
        Dermatol Clin. 2019; 37: 205-213
        • Stasko N.
        • McHale K.
        • Hollenbach S.J.
        • Martin M.
        • Doxey R.
        Nitric oxide-releasing macromolecule exhibits broad-spectrum antifungal activity and utility as a topical treatment for superficial fungal infections.
        Antimicrob Agents Chemother. 2018; 62 (e01026-17)
        • Weidinger S.
        • Novak N.
        Atopic dermatitis.
        Lancet. 2016; 387: 1109-1122