A Reactive Metabolite of Clozapine Induces Hematopoietic Toxicity in HL-60 Cells Undergoing Granulocytic Differentiation through Its Effect on Glutathione Metabolism.

Clozapine is an atypical antipsychotic with several advantages over conventional antipsychotics, in addition to its well-known efficacy in treatment-resistant schizophrenia. However, the high risk of agranulocytosis associated with clozapine therapy limits its clinical application. Clozapine bioactivation to an unstable protein-reactive metabolite, identified as a nitrenium intermediate, has been implicated in cytotoxicity toward neutrophils. Clozapine affects myeloid precursor cells rather than neutrophils; however, the impact of its reactive metabolite on myeloid precursor cells undergoing granulocytic differentiation remains unclear. Herein, we used hydrogen peroxide (H2O2) to generate the reactive metabolite and compared reactive metabolite-induced cytotoxicity between HL-60 cells undergoing granulocytic differentiation and differentiated HL-60 cells. In addition, we examined the role of oxidative stress in this type of cytotoxicity. The reactive metabolite of clozapine induced rapid cytotoxicity in HL-60 cells undergoing granulocytic differentiation, but not in differentiated HL-60 cells, with the metabolite exhibiting more potent cytotoxicity than clozapine. No cytotoxicity was observed following incubation with olanzapine, a structural analog of clozapine, even after exposure of the drug to H2O2. The reactive metabolite of clozapine decreased the levels of reduced glutathione, while addition of reduced glutathione attenuated the reactive metabolite-induced cytotoxicity. These findings indicate that glutathione metabolism plays a role in the hematopoietic toxicity induced by the reactive metabolite of clozapine. Oxidative stress may potentially increase susceptibility to the hematopoietic toxicity induced by the reactive metabolite of clozapine.

Human neutrophils show decreased survival upon long-term exposure to clozapine.

OBJECTIVE: Clozapine is an atypical antipsychotic prescribed for treatment-resistant schizophrenic patients, but treatment with clozapine is strictly limited because it can induce lethal-hematologic side effects. We investigated the effects of short- and long-term exposure of human neutrophils derived from healthy subjects to clozapine and compared them with the effects of reactive metabolite of clozapine, olanzapine, and doxorubicin. METHODS: Neutrophils were exposed to clozapine and olanzapine (1, 10, 50, or 100 µM), reactive metabolite of clozapine (50 or 100 µM), or doxorubicin (0.2 µM) and cultured for a short (2 hr) or long (24 or 48 hr) duration, and then the survival rate of neutrophils was calculated. RESULTS: Decreased human neutrophil survival was observed in short-term exposure to clozapine (100 µM) and long-term exposure to clozapine even at a lower concentration (50 µM). A similar phenomenon was observed in reactive metabolite of clozapine and long-term exposure to doxorubicin (0.2 µM), but not to olanzapine (1-100 µM). CONCLUSIONS: The effect of long-term exposure to clozapine on neutrophil survival is plausibly associated with delayed onset of agranulocytosis after initial exposure. Our results suggest that human neutrophils are vulnerable to clozapine and its reactive metabolite in a concentration- and time-dependent manner.

Involvement of the histamine H4 receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells.

Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100µM) and doxorubicin (0.2µM) decreased the cell survival rate, but olanzapine (1-100µM) did not. Under granulocytic differentiation for 5days, clozapine, even at a concentration of 25µM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H4 receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H4 receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H4 receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H4 receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation.