title: Bioactivation and covalent binding of hydroxyfluperlapine in human neutrophils: implications for fluperlapine-induced agranulocytosis.
abstract: The use of fluperlapine and the structurally related clozapine has been associated with the induction of agranulocytosis in humans. Unlike clozapine, fluperlapine is relatively resistant to chemical and biochemical oxidations. In this study we demonstrated that 7-hydroxyfluperlapine, the major metabolite of fluperlapine in humans, is oxidized to a reactive intermediate by HOCl and by myeloperoxidase in the presence of H(2)O(2) and Cl(-). This reactive intermediate was identified as an iminoquinone species with a M + 1 ion at m/z 324 by mass spectrometry. The iminoquinone intermediate was trapped by N-acetyl-L-cysteine (NAC) as well as GSH. NMR spectra of the NAC adducts indicated that the NAC was bound to the 6 and 9 positions of the aromatic ring. This is the same orientation as the binding of nucleophiles to the reactive metabolite of clozapine. We were able to use an antibody against clozapine to demonstrate that 7-hydroxyfluperlapine, but not fluperlapine itself, covalently modifies human myeloperoxidase. Furthermore, we demonstrated that 7-hydroxyfluperlapine is metabolized by activated neutrophils to a reactive intermediate that covalently binds to neutrophils. In the presence of NAC or GSH, such covalent binding was inhibited and the NAC or GSH adducts were formed. Thus, the reactivity and even the orientation of the binding of the reactive metabolite of 7-hydroxyfluperlapine is very similar to that of clozapine. These results provide a mechanism for the formation of a reactive metabolite of fluperlapine similar to clozapine that may explain its ability to induce agranulocytosis.
authors.affiliation: Faculties of Pharmacy, University of Toronto, Toronto, Canada.
authors.forename: W G
authors.initials: WG
authors.lastname: Lai
authors.forename: I
authors.initials: I
authors.lastname: Gardner
authors.forename: N
authors.initials: N
authors.lastname: Zahid
authors.forename: J P
authors.initials: JP
authors.lastname: Uetrecht
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D000380:Agranulocytosis / Q000139:chemically induced; D001667:Binding, Competitive; D003024:Clozapine / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D003984:Dibenzazepines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D006433:Hemocyanins / Q000737:chemistry / Q000276:immunology; D006801:Humans; D006997:Hypochlorous Acid / Q000378:metabolism; D007106:Immune Sera / Q000737:chemistry; D015151:Immunoblotting; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism; D011485:Protein Binding
chemical_list: D003984:Dibenzazepines; D007106:Immune Sera; D006997:Hypochlorous Acid; D006433:Hemocyanins; D009195:Peroxidase; C041528:fluperlapine; C032808:keyhole-limpet hemocyanin; D005978:Glutathione; D003024:Clozapine; D000111:Acetylcysteine