95 - Fenamic Acid Analogues Oxidized by Peroxidase Enzymes Cause the Formation of Fibronectin Aggregation and Fibronectin-Derived Radicals

Abstract

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/H2O2; 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/H2O2 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.