The oxidation of fenamic acid NSAIDs by neutrophil myeloperoxidase produces toxic reactive metabolites that induce leukemic cell death

Fenamic acids are a group of non-steroidal anti-inflammatory drugs (NSAIDs) that are among the most common drugs prescribed globally. However, they have been associated with many adverse effects, such as agranulocytosis, neutropenia, hepatotoxicity, and nephrotoxicity. The interactions between peroxidase enzymes and fenamic acid-like NSAIDs cause the formation of reactive species, potentially involved in side effects. The aim of this study was to investigate the neutrophil myeloperoxidase (MPO)-mediated bioactivation of fenamic acids based on N-phenylanthranilic acid (NPA) and its four drug analogues: flufenamic acid (FFA), mefenamic acid (MFA), meclofenamic acid (MCFA), and tolfenamic acid (TFA). We hypothesized that the enzymatic oxidation of fenamic acids by MPO/hydrogen peroxide (H2O2) would produce reactive metabolites, cause oxidative damage and induce cytotoxicity. We utilized UV–Vis spectrophotometry, liquid chromatography-mass spectrometry (LC-MS), and electron paramagnetic spin resonance (EPR) spectroscopy using purified MPO from human neutrophils. In addition, in vitro studies were performed with MPO-containing human promyelocytic leukemia (HL-60) cells for cytotoxicity and immuno-spin trapping to detect protein-free radicals. UV–Vis spectrophotometry revealed that MPO oxidized the fenamic acids. LC-MS analyses revealed the formation of dimers, hydroxylated, and quinoneimine species, and glutathione (GSH) conjugates. EPR spin trapping with DMPO using GSH revealed that fenamic acids produced glutathionyl radicals in a concentration-dependent manner. We also detected the formation of protein-free radicals in HL-60 cells, which correlated with cytotoxicity. Despite the minor structural differences between the fenamic acids, there were variations in their oxidation potential. These findings revealed a correlation between pro-oxidant metabolite reactivity and cytotoxicity caused by fenamic acid NSAIDs.