Mitigation of nitrogen mustard mediated skin injury by a novel indomethacin bifunctional prodrug

Abstract

Nitrogen mustard (NM) is a bifunctional alkylating agent that is highly reactive in the skin causing extensive tissue damage and blistering. In the present studies, a modified cutaneous murine patch model was developed to characterize NM-induced injury and to evaluate the efficacy of an indomethacin pro-drug in mitigating toxicity. NM (20 µmol) or vehicle control was applied onto 6 mm glass microfiber filters affixed to the shaved dorsal skin of CD-1 mice for 6 min. This resulted in absorption of approximately 4 µmol of NM. NM caused localized skin damage within 1 d, progressing to an eschar within 2–3 d, followed by wound healing after 4–5 d. NM-induced injury was associated with increases in skin thickness, inflammatory cell infiltration, reduced numbers of sebocytes, basal keratinocyte double stranded DNA breaks, as measured by phospho-histone 2A.X expression, mast cell degranulation and increases in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Wound healing was characterized by epidermal hyperplasia and marked increases in basal cells expressing proliferating cell nuclear antigen. A novel indomethacin-anticholinergic prodrug (4338) designed to target cyclooxygenases and acetylcholinesterase (AChE), was found to markedly suppress NM toxicity, decreasing wound thickness and eschar formation. The prodrug also inhibited mast cell degranulation, suppressed keratinocyte expression of iNOS and COX-2, as well as markers of epidermal proliferation. These findings indicate that a novel bifunctional pro-drug is effective in limiting NM mediated dermal injury. Moreover, our newly developed cutaneous patch model is a sensitive and reproducible method to assess the mechanism of action of countermeasures.

Sulfur mustard induced mast cell degranulation in mouse skin is inhibited by a novel anti-inflammatory and anticholinergic bifunctional prodrug

“…Of particular interest were our findings that throughout the pathogenic response to SM, extensive mast cell degranulation occurred (Joseph et al, 2011). Similar degranulation of mast cells in mouse skin has been reported following exposure to nitrogen mustard (Composto et al, 2016; Jain et al, 2014). Mast cells are known to release a variety of mediators that control hemostasis and inflammation during the early stages of tissue injury, and cellular proliferation and remodeling during later stages of wound repair suggesting that they are key to both processes (Joseph et al, 2016; Kennelly et al, 2011; Theoharides et al, 2012; Weller et al, 2006).…”

“…1), was synthesized as previously reported (Young et al, 2010). Mice (male SKH1-Hr mice, 5–8 weeks of age, Charles River Laboratories, Wilmington, MA) were treated topically with an ointment containing 1% NDH4338 twice per day beginning 1 h after SM exposure (Composto et al, 2016). For analysis of mast cells, full thickness skin punch biopsies were collected 1, 3, 7, and 14 days post exposure.…”

“…Six micron histological sections were prepared and stained with toluidine blue O (Sigma Chemical, St. Louis MO) to visualize mast cell metachromatic/basophilic granules. Extrusion of basophilic toluidine blue stained granules was evidence of mast cell degranulation (Composto et al, 2016; Joseph et al, 2016; Joseph et al, 2011; Puebla-Osorio et al, 2017; Tewari-Singh et al, 2009). Mast cell number and the percentage of degranulated mast cells at the dermal/epidermal junction were analyzed in tissue sections as previously described (Composto et al, 2016; Joseph et al, 2011).…”

“…Mast cells are known to release a variety of mediators that control hemostasis and inflammation during the early stages of tissue injury, and cellular proliferation and remodeling during later stages of wound repair suggesting that they are key to both processes (Joseph et al, 2016; Kennelly et al, 2011; Theoharides et al, 2012; Weller et al, 2006). In studies with nitrogen mustard, we found that a novel bifunctional prodrug consisting of the non-steroidal anti-inflammatory drug (NSAID) indomethacin covalently linked to a choline bioisostere that targets cyclooxygenase enzymes and acetylcholinesterase, effectively inhibited mast cell degranulation (see Figure 1 for structure) (Composto et al, 2016). This NSAID prodrug was also effective in reducing nitrogen mustard-induced skin injury in mouse skin suggesting a link between inhibition of mast cell degranulation and inhibition of vesicant-induced skin toxicity.…”

Tissue injury and repair following cutaneous exposure of mice to sulfur mustard

“…As many of these mediators play roles in mustard‐induced skin injury, inhibitors of mast cell degranulation have been proposed as potential countermeasures . In this regard, we have shown that an indomethacin prodrug that stabilizes mast cells suppresses mustard‐induced injury in mouse skin …”