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.

An initial animal proof-of-concept study for central administration of clozapine to schizophrenia patients

“…When clozapine was formulated with HPBCD and added to the cells, significant toxicity was observed at final clozapine concentrations of 10 μg/ml (30 μM) or higher. This toxicity is similar to that reported to occur to human neutrophils and monocytes (Gardner et al 1998). The 0.1 μg clozapine in 1.0 μl injection of clozapine into the anterior lateral ventricle of male DBA/2 mice did not significantly alter any of the auditory gating variables tested.…”

Procainamide, but notN-Acetylprocainamide, Induces Protein Free Radical Formation on Myeloperoxidase: A Potential Mechanism of Agranulocytosis

“…Uetrecht's group has shown, through the use of an anti-PA antibody, that PA itself became bound to MPO after metabolism by MPO/ H 2 O 2 . The neutrophils from a patient undergoing PA therapy exhibited a 58 kDa protein that was reactive towards anti-PA (63). This protein is believed to be covalently bound to PA through an electrophilic metabolite of PA (e.g., N-nitroso-PA, N-chloro-PA), or possibly via a free radical metabolite.…”

Idiosyncratic Drug Reactions: Past, Present, and Future

“…Another example is the comparison of the pattern of covalent binding of procainamide, vesnarinone, and clozapine, all of which are associated with a relatively high incidence of agranulocytosis (57). In particular, the reactive metabolites of vesnarinone and clozapine are similar in that they are formed by the same enzyme (myeloperoxidase) and have approximately the same half-life, and both react preferentially with thiol nucleophiles.…”