Diclofenac-Induced Inactivation of CYP3A4 and Its Stimulation by Quinidine

Abstract

ABSTRACT:Incubation of human liver microsomes with diclofenac in the presence of NADPH resulted in a decrease in testosterone 6␤-hydroxylation activity. The decrease in the activity followed time-and concentration-dependent kinetics, required oxidative metabolism, and was resistant to reduced glutathione, suggesting that diclofenac causes a mechanism-based inactivation of cytochrome P450 (P450) 3A4 (CYP3A4). The inactivation was reproduced by using microsomes from B-lymphoblastoid cell lines expressing CYP3A4 instead of human liver microsomes. No other monooxygenase activities measured as indexes of P450 enzymes; CYP2C8, CYP2C9, or CYP2C19 was inactivated by the same incubation procedure. Quinidine, a stimulant of CYP3A4-mediated diclofenac 5-hydroxylation, did not affect the inactivation of CYP3A4 assessed by testosterone 6␤-hydroxylation activity but accelerated the inactivation assessed by diazepam 3-hydroxylation activity. These results supported the idea that diclofenac 5-hydroxylation is involved in the inactivation of CYP3A4 and described for the first time a stimulation of mechanism-based inactivation attributable to CYP3A4 heterotropic cooperativity. Preincubation of human liver microsomes with 5-hydroxydiclofenac instead of diclofenac did not cause the inactivation of CYP3A4, suggesting that 5-hydroxydiclofenac is not a precursor of a postulated reactive metabolite that inactivates CYP3A4, and thus 5-hydroxylation step is critical to inactivation of CYP3A4.

Increase in covalent binding of 5-hydroxydiclofenac to hepatic tissues in rats co-treated with lipopolysaccharide and diclofenac: involvement in the onset of diclofenac-induced idiosyncratic hepatotoxicity

“…Hepatic CYP enzymes are involved in the generation of DCF reactive metabolites in rats and humans (Masubuchi et al, 2001(Masubuchi et al, , 2002. These enzymes oxidize DCF main- ly into two phenolic metabolites, 4'-OH-DCF and 5-OH-DCF, which are further metabolized into potentially reactive metabolites such as p-benzoquinone imines.…”

“…Quinone imines are well-known reactive metabolites that undergo covalent binding to non-protein or protein sulfhydryl groups due to their thiol reactivity (Boelsterli, 2003). In addition, arene-oxide is reportedly the primary intermediate of 4'-OH-DCF and 5-OH-DCF and may induce irreversible inactivation through its covalent binding to a xenobiotic-metabolizing enzyme cytochrome P450 (CYP) in the liver (Masubuchi et al, 2001(Masubuchi et al, , 2002. Acyl glucuronides of DCF are also potentially protein-reactive intermediates per se because of their rather labile ester bond (Grillo et al, 2003).…”

Automated Screening With Confirmation of Mechanism-Based Inactivation of Cyp3a4, Cyp2c9, Cyp2c19, Cyp2d6, and Cyp1a2 in Pooled Human Liver Microsomes

“…This issue is illustrated by our identification of ritonavir as an MBI of CYP3A4 from the APR screen at a concentration only 10-fold lower than the default concentration range ( Figure SI2). The sensitivity of the screen was underscored by the detection of weak MBIs with k inact /K I Ͻ 1 min Ϫ1 mM Ϫ1 , such as isoniazid for CYP3A4, CYP2C19, and CYP1A2 (Wen et al, 2002), and diclofenac for CYP3A4 (Masubuchi et al, 2002).…”

Evaluation of Time-Dependent Inactivation of Cyp3a in Cryopreserved Human Hepatocytes

“…The HIV protease inhibitor amprenavir was recently shown to inhibit CYP3A by binding to the heme (Ernest et al, 2003). The nonsteroidal anti-inflammatory agent diclofenac was reported to be a time-dependent inactivator of CYP3A with a rather high K I of 1.6 mM (Masubuchi et al, 2002). We did not observe TDI by diclofenac in either HLMs or hepatocytes at concentrations up to 100 M. Further investigation at higher concentrations was not conducted because 100 M is already much higher than free diclofenac exposure observed in humans.…”

“…We did not observe TDI by diclofenac in either HLMs or hepatocytes at concentrations up to 100 M. Further investigation at higher concentrations was not conducted because 100 M is already much higher than free diclofenac exposure observed in humans. Indeed, diclofenac was not likely to cause clinically relevant CYP3A inhibition, given its high microsomal K I and high plasma protein binding (Masubuchi et al, 2002). Thus, diclofenac was excluded from the HLM-hepatocyte prediction exercise.…”