Induction of Metabolism-Dependent and -Independent Neutrophil Apoptosis by Clozapine

Abstract

Clozapine, an atypical antipsychotic used in the treatment of refractory schizophrenia, causes neutropenia and agranulocytosis in 3 and 0.8% of patients, respectively. Clozapine undergoes bioactivation to a chemically reactive nitrenium ion, which has been shown to cause neutrophil cytotoxicity. To define further the mechanism of cell death, we have investigated the toxicity of clozapine, its stable metabolites, and its chemically reactive nitrenium ion to neutrophils and lymphocytes. Clozapine was able to induce neutrophil apoptosis at therapeutic concentrations (1-3 microM) only when it was bioactivated to the nitrenium ion. The parent drug caused apoptosis at supratherapeutic concentrations (100-300 microM) only. Neutrophil apoptosis induced by the nitrenium ion, but not by the parent drug itself, was inhibited by antioxidants and genistein and was accompanied by cell surface haptenation (assessed by flow cytometry) and glutathione depletion. Dual-color flow cytometry showed that neutrophils that were haptenated were the same cells that underwent apoptosis. No apoptosis of lymphocytes was evident with the nitrenium ion or the parent drug, despite the fact that the former caused cell surface haptenation, glutathione depletion, and loss of membrane integrity. Demethylclozapine, the major stable metabolite in vivo, showed a profile that was similar to, although less marked than that observed with clozapine. N-oxidation of clozapine or replacement of the nitrogen (at position 5) by sulfur produced compounds that were entirely nontoxic to neutrophils. In conclusion, the findings of the study expand on potential mechanisms of clozapine-induced cytotoxicity, which may be of relevance to the major forms of toxicity encountered in patients taking this drug.

Potential clozapine target sites on peripheral hematopoietic cells and stromal cells of the bone marrow

“…First-strand cDNA reactions for each cell type primed with either oligo(dT) [12][13][14][15][16][17][18] or random hexamers, essentially gave similar results with cDNA species of up to 8 kb synthesized (data not shown). Subsequent amplifications of cDNA prepared using either priming method gave identical results for each receptor targeted (Figure 1a-d).…”

“…The integrity of the RNA isolated was assessed [12][13][14][15][16][17][18] and random hexamers. To increase the sensitivity of PCR amplification of cDNA targets, the RNA template was removed by digestion with 2.0 U RNase H after completion of first-strand synthesis.…”

“…15 Covalent modification of neutrophil/bone marrow proteins by such reactive species could cause direct cell death, or alternatively, hapten formation and immune-mediated hypersensitivity. 12 Disruption to stromal cells may also arrest neutrophil development given their anatomical and functional link to hematopoietic precursor cells.…”

Clozapine and its Major Stable Metabolites, N-desmethylclozapine and Clozapine N-oxide do not Affect Human Bone Marrow Stromal Cells in vitro

“…Furthermore, the metabolite can potentially be metabolized back to clozapine [21] which may in part explain the variability in profiles of primary stromal and HAS303 cell viability across clozapine N-oxide concentrations. Studies with the neutrophil have also shown no concentration dependent induction of cell death in the presence of up to 300 µM clozapine Noxide [22] . It is noteworthy that LP101 cells in the presence of clozapine and its metabolites showed limited fluctuations in viability across drug concentrations, compared with that seen with primary stromal and HAS303 cells.…”

Clozapine inhibits cell survival-related genes in bone marrow cells

“…2 The mechanism of clozapine-induced agranulocytosis is not fully explained; however, it has been postulated as immunemediated mechanism and direct toxic effects. 3,4 It has been demonstrated that the reactive nitrenium ion, a toxic metabolite, is produced from clozapine through myeloperoxidase-H 2 O 2 -dependent reaction, and then induces the apoptosis of neutrophils in vitro. 4 In order to determine whether clozapine-treated bone marrow cells were susceptible to cell death, we compared the expression profiles of bone marrow cells between clozapine-and vehicle (saline)-treated mice using cDNA microarray analysis.…”