Clozapine, an atypical
antipsychotic drug effective in treatment of
refractory schizophrenia causes potentially life-threatening
agranulocytosis. The
drug undergoes bioactivation to a toxic, chemically reactive intermediate with capacity to target stromal cells, central components of the bone marrow microenvironment implicated in neutrophil development. To identify possible mechanisms underpinning disruption of stroma as a site of
drug bioactivation, toxicity was induced in vitro. Therefore metabolite generation procedures utilizing HOCl or HRP-H(2)O(2) as primary components involved in
clozapine metabolism were adapted for stromal culture and coupled with viability determinations.
Drug oxidation by HOCl was less toxic to stromal cells than HRP-H(2)O(2) based methods. More specifically,
clozapine bioactivation by HRP-H(2)O(2) caused dose-dependent inhibition of stromal viability at therapeutically relevant concentrations. Differences in susceptibility of HAS303 and LP101 cells to the
clozapine nitrenium ion were also evident. Stromal cell death was attributed to
clozapine in the presence of a complete metabolising system comprising HRP and H(2)O(2). In the absence of a complete metabolising system
clozapine was not cytotoxic. For LP101 cells,
drug plus HRP (minus H(2)O(2)) also induced toxicity. Importantly, other
antipsychotic drugs including
risperidone,
olanzapine and
haloperidol when bioactivated, were not cytotoxic, indicating system specificity for
clozapine. Exogenous GSH,
N-acetylcysteine,
l-ascorbic acid,
catalase, and
sodium azide afforded protection to cells whereas S-methylGSH,
GSSG,
ketoprofen and
proadifen did not. Thus functional data derived from the in vitro stromal system defined in these studies may enable further investigation of the mechanisms subserving stromal impairment in
clozapine-induced
agranulocytosis and direct attention to improved methods for its prevention.