The chronic use of procainamide is associated with a high incidence of drug-induced lupus and also agranulocytosis. We have previously demonstrated that procainamide is metabolized in the liver to reactive hydroxylamine (PAHA) and nitroso (nitroso-PA) metabolites which covalently bind to protein and are toxic to lymphocytes. We proposed that these metabolites were responsible for the toxicities of procainamide. However, PAHA and nitroso-PA do not appear to escape the liver in significant concentrations. In this paper we describe the metabolism of procainamide to a reactive hydroxylamine by neutrophils and mononuclear leukocytes. Such metabolism only occurs if the cells have been stimulated to have a respiratory burst. These observations have obvious possible implications for the mechanism of procainamide-induced agranulocytosis (formation of a reactive metabolite by neutrophils) and procainamide-induced lupus (formation of a reactive metabolite by monocytes). The metabolism of drugs to reactive metabolites by monocytes may be a general mechanism for hypersensitivity reactions because monocytes play a key role in the processing of antigen and stimulation of antibody synthesis.
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.
Diclofenac is associated with a low, but significant, incidence of hepatotoxicity and bone marrow toxicity. It has been suggested that this could be due to a reactive acyl glucuronide. An alternative hypothesis is that an oxidative reactive metabolite could be responsible for such reactions and such metabolites formed by the enzymes present in neutrophils could be responsible for bone marrow toxicity. Others had reported the formation of 2,2'-dihydroxyazobenzene during the oxidation of diclofenac by myeloperoxidase/hydrogen peroxide. In contrast, in similar experiments we did not find evidence for the formation of 2,2'-dihydroxyazobenzene, but we did find several products, including a reactive iminoquinone. The same iminoquinone was formed by the oxidation of 5-hydroxydiclofenac. This iminoquinone was also formed by oxidation of diclofenac by HOCl or by activated neutrophils. It reacted with glutathione to form a conjugate. 5-Hydroxydiclofenac is also a major hepatic metabolite of diclofenac, and we found that rat hepatic microsomes oxidized 5-hydroxydiclofenac to the iminoquinone which was trapped with glutathione. This reactive metabolite represents another possible cause of the idiosyncratic reactions associated with the use of diclofenac.
In previous studies we had shown that procainamide is metabolized to reactive metabolites by activated leukocytes, and evidence pointed to involvement of myeloperoxidase (MPO). In this study we examine the metabolism of procainamide by MPO/H2O2, in the presence and absence of chloride ion. In the absence of chloride ion, the metabolism was very similar to that seen with activated leukocytes. The major metabolite was formed by oxidation of the arylamine group to a hydroxylamine. In the presence of chloride ion, a much greater degree of metabolism occurred, and the major product (40% of the starting procainamide) was a reactive species that could not be isolated. This metabolite spontaneously rearranged to 3-chloroprocainamide, and from its mass spectrum and chemical reactions, we deduce its structure to be N-chloroprocainamide. The N-chloroprocainamide metabolite reacted very rapidly with reducing agents, such as ascorbate, and also reacted with protein such as albumin, the major product in both cases being procainamide. This metabolite also chlorinated phenylbutazone. When radiolabeled procainamide was oxidized by MPO/H2O2 in the presence of albumin, covalent binding of the radiolabel to albumin occurred, and binding was greater under conditions in which N-chloroprocainamide was formed. It is probable that the failure to observe N-chloroprocainamide, when procainamide is oxidized by activated leukocytes, is due to its rapid reaction with the cells. We propose that modification of neutrophils (or neutrophil precursors in the bone marrow) by these reactive metabolites is responsible for procainamide-induced agranulocytosis. In a similar manner, procainamide-induced lupus could be due to modification of monocytes by monocyte-generated reactive metabolites.
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