Scavenging of reactive oxygen and nitrogen species by the prodrug sulfasalazine and its metabolites 5-aminosalicylic acid and sulfapyridine

Sulfasalazine is a prodrug composed by a molecule of 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), linked by an azo bond, which has been shown to be effective in the therapy of inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, as well as of rheumatic diseases, such as rheumatoid arthritis and ankylosing spondylitis. The precise mechanism of action of sulfasalazine and/or its metabolites has not been completely elucidated, though its antioxidant effects are well established and are probably due to its scavenging effects against reactive oxygen and nitrogen species (ROS and RNS), as well as metal chelating properties, in association to its inhibitory effects over neutrophil oxidative burst. The present work was focused on screening and comparing the potential scavenging activity for an array of ROS (O-2(center dot-), H2O2, O-1(2), ROO center dot and HOCl) and RNS ((NO)-N-center dot and ONOO-), mediated by sulfasalazine and its metabolites 5-ASA and SP, using validated in vitro screening systems. The results showed that both 5-ASA and sulfasalazine were able to scavenge all the tested ROS while SP was practically ineffective in all the assays. For HOCl, O-1(2), and ROO center dot, 5-ASA showed the best scavenging effects. A new and important finding of the present study was the strong scavenging effect of 5-ASA against O-1(2). 5-ASA was shown to be a strong scavenger of (NO)-N-center dot and ONOO-. Sulfasalazine was also able to scavenge these RNS, although with a much lower potency than 5-ASA. SP was unable to scavenge (NO)-N-center dot in the tested concentrations but was shown to scavenge ONOO-, with a higher strength when the assay was performed in the presence of 25 mM bicarbonate, suggesting further scavenging of oxidizing carbonate radical. In conclusion, the ROS- and RNS-scavenging effects of sulfasalazine and its metabolites shown in this study may contribute to the anti-inflammatory effects mediated by sulfasalazine through the prevention of the oxidative/nitrative/nitrosative damages caused by these species.