Edaravone as an antioxidant adjuvant to attenuate clozapine toxicity in vitro.

Despite being the gold-standard therapy for treatment-resistant schizophrenia, clozapine use remains constrained by the risk of rare but serious adverse drug reactions, including severe neutropenia and agranulocytosis. Neutrophil myeloperoxidase (MPO)-mediated bioactivation of clozapine to reactive metabolites has long been implicated in agranulocytosis. Although clozapine is metabolized by MPO, there are numerous other compounds that can also be metabolized by this enzyme. Edaravone is a drug used to treat amyotrophic lateral sclerosis and has potent antioxidant activity; we have previously shown that edaravone is metabolized by MPO. Therefore, we hypothesized that edaravone can attenuate early toxicological events associated with clozapine through MPO modulation. Using purified human MPO, MPO-rich human promyelocytic leukemia (HL-60) cells, and isolated human neutrophils, we evaluated clozapine oxidation, competitive intracellular uptake, protein adduct formation, and cytotoxicity. UV-visible spectroscopy demonstrated concentration-dependent inhibition of MPO-catalyzed clozapine oxidation by edaravone, with 100µM edaravone producing suppression comparable to 100µM 4-aminobenzoic acid hydrazide. LC-MS analysis showed that MPO/H₂O₂ reduced clozapine levels to 26.7 ± 2.9% of control, whereas co-incubation with edaravone preserved clozapine to 67.9 ± 16.2%. Under the same conditions, edaravone was almost completely consumed (0.03 ± 0.02% remaining), consistent with its sacrificial antioxidant activity. In HL-60 cells, edaravone attenuated MPO activity and did not significantly alter clozapine uptake after 30minutes (97.2 ± 7.6% of clozapine alone). Immunoblotting revealed that edaravone reduced clozapine-protein adduct formation in purified MPO, HL-60 cells, and neutrophils. Clozapine (75µM) induced marked HL-60 cytotoxicity after 24hours, whereas edaravone concentration-dependently restored viability, with 200µM fully preventing toxicity. These findings provide proof-of-concept that edaravone mitigates multiple mechanistic events linked to clozapine-induced hematological toxicity and may represent a potential strategy to improve clozapine safety.