title: A comparison of the covalent binding of clozapine, procainamide, and vesnarinone to human neutrophils in vitro and rat tissues in vitro and in vivo.
abstract: Covalent binding of drug reactive metabolites to neutrophils or their precursors is thought to play a role in the development of drug-induced agranulocytosis. In this study, we used immunochemical techniques to compare the covalent binding of clozapine, vesnarinone, and procainamide (three drugs associated with agranulocytosis) to phorbol-12,13-myristate acetate (PMA)-activated human neutrophils in vitro and rat tissues in vivo. In PMA-activated human neutrophils in vitro, clozapine and procainamide modified neutrophil proteins with molecular masses ranging from 30 to 200 kDa, while vesnarinone predominately formed adducts with molecular masses greater than 70 kDa. All three drugs formed adducts at 126, 98, and 58 kDa, and they all covalently bound to human myeloperoxidase when incubated with this enzyme and H2O2 in vitro. Covalent binding to PMA-activated neutrophils was inhibited by nucleophiles, such as glutathione and N-acetylcysteine, but not by N-acetyllysine. In the presence of the PMA, all three drugs covalently bound to activated rat bone marrow cells in vitro, while in its absence only clozapine did. Covalently modified liver proteins were observed in rats treated for 6 weeks with clozapine (25 or 50 mg/kg/day), vesnarinone (300 mg/kg/day), or procainamide (50 mg/kg/day). Clozapine extensively modified proteins in all subcellular fractions; procainamide formed a 99 kDa adduct in a membrane-containing fraction and 57, 47, and 36 kDa adducts in a cytosolic fraction, while vesnarinone formed liver-protein adducts with molecular masses of 82, 62, 49, and 40 kDa in membrane, cytosolic, and S9 fractions. In addition, clozapine and procainamide, but not vesnarinone, formed a 49 kDa drug-protein adduct in the bone marrow of treated rats. Furthermore, procainamide covalently bound to a 58 kDa protein in neutrophils of a patient treated with the drug. We suspect that covalent modification of common targets in the neutrophils by these three drugs plays a role in the development of drug-induced agranulocytosis.
authors.affiliation: Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 2S2, Canada.
authors.forename: Iain
authors.initials: I
authors.lastname: Gardner
authors.forename: Marija
authors.initials: M
authors.lastname: Popović
authors.forename: Nasir
authors.initials: N
authors.lastname: Zahid
authors.forename: Jack P
authors.initials: JP
authors.lastname: Uetrecht
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000378:metabolism; D003024:Clozapine / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006433:Hemocyanins / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D015394:Molecular Structure; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011342:Procainamide / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011719:Pyrazines; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013696:Temperature
chemical_list: D007106:Immune Sera; D010703:Phorbol Esters; D011719:Pyrazines; D011804:Quinolines; C041667:vesnarinone; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D003024:Clozapine; D011342:Procainamide