Showing papers by "Dennis C. Dean published in 1999"

Studies on Cytochrome P-450-Mediated Bioactivation of Diclofenac in Rats and in Human Hepatocytes: Identification of Glutathione Conjugated Metabolites

Abstract: The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.

Interaction of diclofenac and quinidine in monkeys: stimulation of diclofenac metabolism.

Abstract: The cytochrome P-450 (CYP)3A4-mediated metabolism of diclofenac is stimulated in vitro by quinidine. A similar effect is observed in incubations with monkey liver microsomes. We describe an in vivo interaction of diclofenac and quinidine that leads to enhanced clearance of diclofenac in monkeys. After a dose of diclofenac via portal vein infusion at 0.055 mg/kg/h, steady-state systemic plasma drug concentrations in three male rhesus monkeys were 87, 104, and 32 ng/ml, respectively (control). When diclofenac was coadministered with quinidine (0.25 mg/kg/h) via the same route, the corresponding plasma diclofenac concentrations were 50, 59, and 18 ng/ml, representing 57, 56, and 56% of control values, respectively. In contrast, steady-state systemic diclofenac concentrations in the same three monkeys were elevated 1.4 to 2.5 times when the monkeys were pretreated with L-754,394 (10 mg/kg i.v.), an inhibitor of CYP3A. Further investigation indicated that the plasma protein binding (>99%) and blood/plasma ratio (0.7) of diclofenac remained unchanged in the presence of quinidine. Therefore, the decreases in plasma concentrations of diclofenac after a combined dose of diclofenac and quinidine are taken to reflect increased hepatic clearance of the drug, presumably resulting from the stimulation of CYP3A-catalyzed oxidative metabolism. Consistent with this proposed mechanism, a 2-fold increase in the formation of 5-hydroxydiclofenac derivatives was observed in monkey hepatocyte suspensions containing diclofenac and quinidine. Stimulation of diclofenac metabolism by quinidine was diminished when monkey liver microsomes were pretreated with antibodies against CYP3A. Subsequent kinetic studies indicated that the K m value for the CYP-mediated conversion of diclofenac to its 5-hydroxy derivatives was little changed (75 versus 59 μM), whereas V max increased 2.5-fold in the presence of quinidine. These data suggest that the catalytic capacity of monkey hepatic CYP3A toward diclofenac metabolism is enhanced by quinidine.

Suzuki cross‐coupling for the incorporation of labeled methyl groups onto aryl halides. A synthesis of [14C]tosyl chloride and its use in the synthesis of [14C]L‐738,167

Abstract: A synthesis of [4-methyl]-14C]tosyl chloride (2) has been developed which utilizes a Suzuki Cross-Coupling reaction between 4-iodophenylsulfonic acid and a labeled methyl borinate as the key step. This process avoids the poor regioselectivity typically attendant with aromatic sulfonation procedures. We now describe the use of this [14C]tosyl chloride in the synthesis of the orally active fibrinogen receptor antagonist L-738,167 (1). Copyright © 1999 John Wiley & Sons, Ltd.