The search for effective mustard therapeutics is moving towards the development of multifunctional drugs to target the various pathways of SM toxicity. Dachir et al. (2004)
mechanically combined a steroid with an NSAID to optimize anti-inflammatory activity and deplete tissues of prostaglandin E. In this study, the combined drugs were more effective than either drug alone. Similarly, Wormser and co-workers (2000
) combined topical iodine with steroidal and nonsteroidal anti-inflammatory drugs to suppress SM-induced necrosis and hemorrhage while enhancing biomarkers of healing. Little work has been done to explore controlled-release SM therapeutics containing individual drugs such as anti-inflammatory agents and anticholinergics covalently linked in one molecule. An earlier study reported that conjugates formed by a covalent linkage of pyridostigmine with an NSAID via a decyl hydrocarbon spacer were as active as commercial NSAID preparations in ameliorating SM-induced toxicity in a mouse ear model (Amitai et al., 2006
). The concept of linking an AChE inhibitor to common NSAIDs was innovative and effective; however, no in vitro
or in vivo
release of the individual pharmacologic components was shown. In the present work, integration of NSAIDs and anticholinergics into a novel scaffold to facilitate gradual drug-release has allowed us to target multiple mechanisms of SM-induced injury in a way that is unique to the field and will expand the current library of anti-mustard candidates.
A great deal of work has gone into the exploration of anticholinergics as potential anti-inflammatory agents. For example, the anticholinergic, galantamine, when administered peripherally, ameliorates systemic inflammation (Pavlov et al., 2009
). Furthermore, maintaining cholinergic balance with these drugs has been reported to alleviate severe inflammation from sepsis, endotoxemia, and arthritis (Hofer et al., 2008
, Pavlov et al., 2009
, and van Maanen et al., 2009
). Regarding the specific effects that SM has on AChE levels, enzyme synthesis has been reported to be increased in neuroblastoma cells following a SM treatment (Lanks et al., 1975
). Steinritz and co-workers (2007)
also observed elevated AChE levels during apoptosis of SM-exposed pulmonary epithelial cells. Our compounds were synthesized based on the emerging role of AChE in inflammation and the work of Amitai et al. (2006)
demonstrating their potential as anti-mustard agents.
In our MEVM screen, high levels of activity against CEES were observed for several of our Class 1 and 2 bifunctionals containing lipophilic choline analogs linked to NSAIDs (). Several of the most promising prodrugs reduced inflammation and edema up to and greater than 90 % (compounds 5
, and 18
). The anti-inflammatory effects observed were directly associated with potent inhibition of AChE, high lipophilicity, and long half-lives in plasma. We have shown previously that all compounds in these classes release their parent NSAIDs in plasma (Young et al., 2010
). It is not yet established whether the therapeutic effect observed is from the intact compounds or from the in vivo
release of both the NSAID and the anticholinergic.
Class 3 compounds structurally designed around potent AChE inhibitors like galantamine, neostigmine, and pyridostigmine also showed very promising results against CEES. Significant suppression of CEES-induced inflammation was observed, with levels exceeding 75 % reduction in some cases (compounds 22 and 27). Most of these compounds are potent anticholinergics and/or release an NSAID in plasma. The stability of Class 3 compounds in human plasma, however, could limit the in vivo application of these agents as prodrugs. In particular, the rapid release of the NSAID (t1/2 < 5 min) for some agents could be detrimental. Galantamine derivatives showed exceptionally long biological half-lives for enzymatic release of the NSAID and the AChEI. Further in vivo investigation is needed to determine the rate of release of the parent drugs following transdermal absorption.
To study the biological activity of the selected agents in-depth, the inhibitory activity against butyrylcholinesterase (BuChE) was also determined. BuChE is a less selective esterase that is highly homologous with AChE and is found principally in the circulation (Glick et al., 2003
). Inhibitory activity against equine serum BuChE (eBuChE) was observed for our compounds with IC50
s ranging from 3.8–55 μM and potencies lower than those observed for AChE (Young et al., unpublished work, 2010
). Thus, our compounds are able to inhibit both AChE and BuChE, paralleling the known cholinesterase inhibitors, tacrine and galantamine (de los Ríos et al., 2010
). Existing literature does not suggest that attenuation of BuChE levels will have an effect on SM dermal injury.
Several compounds of the set were also screened in the mouse ear model against the phorbol ester TPA, a common proinflammatory agent used in this case to assay the dual-action prodrugs for a wider range of anti-inflammatory properties (Akihisa et al., 2010
and Yasukawa et al., 2010
). Many of our compounds in all three classes reduced TPA-induced inflammation and edema by at least 50–75 %. The potent anti-TPA effects of the simple esters suggest that activity is linked to both the rate of NSAID release and skin penetrability. It is important to note that both anti-CEES and anti-TPA activity likely depend upon both the parent NSAIDs and the prodrugs themselves as previous results have shown that NSAIDs have different rates of transdermal absorption (Singh et al., 1994
and Beetge et al., 2000
In general, the simple ester series was found to be more active against TPA while Class 1 agents were more effective against CEES. TPA is a highly potent inflammatory agent which activates protein kinase C and induces the release of histamine, proteases, and proinflammatory mediators (Nakadate, 1989
and Goel et al., 2007
). The rapid rate of NSAID release may explain the effectiveness that Class 2 agents have against TPA. On the other hand, the CEES or SM-induced blister is a slow-developing lesion resulting from DNA-alkylation, protease activation and an inflammatory response (Cowan et al., 2003
). Considering the gradual development of SM lesions (Kehe and Szinicz, 2005
), Class 1 agents seem more appropriate as SM therapeutics because of their slow-release of the parent NSAID and high lipophilicity allowing enhanced skin penetration.
Directly preventing SM-induced DNA modifications has been used as a strategy for decontaminating the skin following exposure to vesicants. Sulfur species such as sodium thiosulfate are commonly explored as decontaminats (Baskin et al., 2000
). It is proposed that these SM scavengers intervene in macromolecular-alkylation either by interacting with nucleic acids or by directly reacting with the highly electrophilic sulfonium ion formed by SM (Baskin et al., 2000
). Therapeutic intervention of DNA-alkylation has not been explored likely because SM penetrates the skin within 30–60 min after exposure (Wormser et al., 2002
). Various groups have instead taken the approach of designing treatments which target the detrimental effects resulting from SM-induced DNA damage. For example, DNA damaging agents activate poly (ADP-ribose) polymerase (PARP) as well as proteases and inhibitors of these enzymes have been reported to reduce epidermal necrosis following SM exposure (Cowan et al., 2003
). Antioxidants such as melatonin have also been explored as potential treatments because of their ability to reduce cellular damage from oxidative and nitrosative stress (Korkmaz et al., 2008
, Laskin et al., 2010
). Considering the structure of our novel bifunctional agents, which are not alkylating, antioxidant or intercalating agents, it is unlikely that they function by directly modulating SM-induced DNA modifications.
In summary, we have synthesized bifunctional compounds that combine an NSAID with an anticholinergic moiety. This combination results in a novel class of lipophilic NSAID prodrugs that possesses considerable anti-inflammatory activity against CEES and/or TPA in the MEVM. In most cases, the edema and inflammation suppression observed for these compounds is higher than that of the NSAIDs alone. Further studies are needed to more precisely understand their mechanism of action and to determine if they mitigate SM-induced skin injury.