PT  - JOURNAL ARTICLE
AU  - Dominic P. Williams
AU  - Munir Pirmohamed
AU  - Dean J. Naisbitt
AU  - James L. Maggs
AU  - B. Kevin Park
TI  - Neutrophil Cytotoxicity of the Chemically Reactive Metabolite(s) of Clozapine: Possible Role in Agranulocytosis
DP  - 1997 Dec 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 1375--1382
VI  - 283
IP  - 3
4099  - http://jpet.aspetjournals.org/content/283/3/1375.short
4100  - http://jpet.aspetjournals.org/content/283/3/1375.full
SO  - J Pharmacol Exp Ther1997 Dec 01; 283
AB  - Clozapine is associated with a 0.8% incidence of agranulocytosis. Bioactivation to an unstable protein-reactive metabolite, identified as a nitrenium intermediate, has been implicated in the toxicity. In this study, we investigated whether the reactive metabolite is cytotoxic toward polymorphonuclear leukocytes and mononuclear leukocytes using horseradish peroxidase and H2O2 to generate the metabolite in situ. In the absence of a full metabolizing system (i.e., lack of horseradish peroxidase and/or H2O2), clozapine (0–100 μM) and its stable metabolites were not cytotoxic. With a full metabolizing system, both clozapine (30 μM) and demethylclozapine exhibited cytotoxicity toward polymorphonuclear leukocytes (50.7 ± 7.7% and 17.6 ± 1.2% cell death, respectively) and mononuclear leukocytes (36.6 ± 2.1% and 24.6 ± 4.1%, respectively), whereas clozapine N-oxide was not cytotoxic. Exogenous glutathione (GSH), N-acetylcysteine and ascorbic acid all protected the cells. Bioactivation of clozapine and demethylclozapine, but not the N-oxide, was accompanied by depletion of intracellular GSH. [14C]Clozapine was metabolized to the previously identified C6 and C9 glutathionyl conjugates; GSH conjugates were also detected when demethylclozapine and clozapine N-oxide were bioactivated by horseradish peroxidase and H2O2. In conclusion, using a novel in vitro assay, we have shown that clozapine and its stable metabolites are not cytotoxic per se but are bioactivated to cytotoxic metabolites. The cytotoxic metabolite of clozapine is identical to the protein-reactive metabolite that has been characterized previously. These cytotoxic metabolites may play an important role in the pathogenesis of clozapine agranulocytosis; the mechanism by which this occurs is currently being investigated. The American Society for Pharmacology and Experimental Therapeutics