NEUTROPHIL CYTOTOXICITY OF THE CHEMICALLY REACTIVE METABOLITE(S) OF CLOZAPINE - POSSIBLE ROLE IN AGRANULOCYTOSIS

Clozapine is associated with a 0.8% incidence of agranulocytosis. Bioa ctivation to an unstable protein-reactive metabolite, identified as a nitrenium intermediate, has been implicated in the toxicity, In this s tudy, we investigated whether the reactive metabolite is cytotoxic tow ard polymorphonuclear leukocytes and mononuclear leukocytes using hors eradish peroxidase and H2O2 to generate the metabolite in situ. In the absence of a full metabolizing system (i.e., lack of horseradish pero xidase and/or H2O2), clozapine (0-100 mu M) and its stable metabolites were not cytotoxic, With a full metabolizing system, both clozapine ( 30 mu M) and demethylclozapine exhibited cytotoxicity toward polymorph onuclear leukocytes (50.7 +/- 7.7% and 17.6 +/- 1.2% cell death, respe ctively) 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 th e cells. Bioactivation of clozapine and demethylclozapine, but not the N-oxide, was accompanied by depletion of intracellular GSH, [C-14]Clo zapine was metabolized to the previously identified C6 and C9 glutathi onyl conjugates; GSH conjugates were also detected when demethylclozap ine and clozapine N-oxide were bioactivated by horseradish peroxidase and H2O2. In conclusion, using a novel in vitro assay, we have shown t hat 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 be en characterized previously. These cytotoxic metabolites may play an i mportant role in the pathogenesis of clozapine agranulocytosis; the me chanism by which this occurs is currently being investigated.