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Fenamic acids form the substructure of many non-steroidal anti-inflammatory drugs (NSAIDs); they are amongst the most commonly prescribed drugs globally. Interactions between fenamic acid NSAIDs and peroxidases have shown that reactive metabolites can be produced, although detailed studies of these metabolites or their relative reactivity have not been studied. Adverse drug reactions may be catalyzed by peroxidase enzymes in off-target tissues. Myeloperoxidase (MPO) is a peroxidase enzyme found abundantly in neutrophils, which are recruited during inflammation and it is well known that MPO can catalyze drug oxidation. Moreover, it has been demonstrated that MPO binds to fibronectin (FN); the latter is an important multi-functional protein. The objective of this study was to examine the differential reactivity of fenamic acid NSAIDs when oxidized by MPO and the consequent effects on FN. Studies using ESR spin trapping with DMPO revealed that N-phenanthranilic acid (N-PA) produced a stable radical when oxidized by MPO/H 2 O 2 ; diphenylamine (DPA) and 3-methyldiphenylamine (3-MDA) produced ● OH (DMPO/ ● OH). In addition, the analogs showed the formation of GS ● after the addition of GSH to the MPO/H 2 O 2 system, with 3-MDA producing the most intense DMPO/GS ● spectrum. To determine the cytotoxic potential of these radical metabolites, we used HL-60 promyelocytic leukemia cells which contain MPO. 3-MDA, DPA and N-PA demonstrated MPO-catalyzed cytotoxicity in these cells, but not mefenamic acid (MFA). LC/MS analysis of the DPA product formation showed the formation of a hydroxydiphenylamine and a quinoneimine product, indicating MPO oxidation resulted in a hydroxylation reaction. The quinoneimine products appeared to react with GSH, forming a GSH conjugate. Immuno-spin trapping analysis demonstrated that MFA was the most potent in producing FN-protein free radical formation in a MPO-system. However, FN aggregation analysis by silver staining was most prominent with DPA. MFA and its analogues showed the formation of free radicals in a peroxidase-system which interferes with normal cell functioning. Future studies are needed to determine the functional impact of fenamic acid NSAID mediated damage to FN. [ABSTRACT FROM AUTHOR]

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H 2 O 2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 μM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV–Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 μM H 2 O 2 or a flux of H 2 O 2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H 2 O 2 -induced toxicity was ameliorated at high edaravone concentrations (100–200 μM). In contrast, low concentrations of edaravone (1–10 μM) exacerbated the H 2 O 2 -induced toxicity. However, the effect of edaravone at low concentration (0–10 μM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug. Image 1 • Edaravone is a substrate for myeloperoxidase. • Edaravone (≤10 μM) induced HOCl formation but attenuated at ≥ 30 μM. • The edaravone free radical metabolite was characterized, and polymers were observed. • Edaravone increased or decreased H 2 O 2 cytotoxicity depending on its concentration. [ABSTRACT FROM AUTHOR]