title: Involvement of myeloperoxidase and NADPH oxidase in the covalent binding of amodiaquine and clozapine to neutrophils: implications for drug-induced agranulocytosis.
abstract: Amodiaquine (AQ) and clozapine (CLZ) are associated with a relatively high incidence of idiosyncratic agranulocytosis, a reaction that is suspected to involve covalent binding of reactive metabolites to neutrophils. Previous studies have shown that both AQ and CLZ are oxidized to reactive intermediates in vitro by activated neutrophils or by the combination of hydrogen peroxide and myeloperoxidase (MPO). Neutrophil activation leads to an oxidative burst with activation of NADPH oxidase and the production of hydrogen peroxide. However, the importance of this pathway in covalent binding in vivo has not been examined. In this study, we found that the binding of both AQ and CLZ to neutrophils from MPO knockout mice ex vivo decreased approximately 2-fold compared to neutrophils from wild-type mice, whereas binding to activated neutrophils from gp91 knockout (NADPH oxidase null) mice decreased 6-7-fold. When the AQ studies were performed in vivo, again the binding was decreased in MPO knockout mice to about 50% of the binding in wild-type mice; however, covalent binding was significant in the absence of MPO. Surprisingly, there was no significant decrease in covalent binding of AQ to neutrophils in vivo in gp91 knockout mice. In addition, there was extensive binding of AQ to many types of bone marrow cells and to peripheral lymphocytes. These results indicate that MPO is not the only neutrophil enzyme involved in the oxidation of AQ and that NADPH oxidase is not the major source of peroxide. There was also no decrease in AQ binding to neutrophils in COX-1 or COX-2 knockout mice. We were not able to readily reproduce the AQ in vivo studies with CLZ because of its acute toxicity in mice. These are the first studies to examine the enzymes involved in the bioactivation of AQ by neutrophils in vivo.
authors.affiliation: Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.
authors.forename: Alexandra R
authors.initials: AR
authors.lastname: Lobach
authors.forename: Jack
authors.initials: J
authors.lastname: Uetrecht
mesh_terms: D000380:Agranulocytosis / Q000139:chemically induced*; D000655:Amodiaquine / Q000378:metabolism*; D000818:Animals; D003024:Clozapine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000655:Amodiaquine; D009195:Peroxidase; D019255:NADPH Oxidases; D003024:Clozapine