Received for publication July 14, 2006.
Revised September 12, 2006.
Accepted for publication September 14, 2006.

ROFECOXIB IS A POTENT, METABOLISM-DEPENDENT INHIBITOR OF CYP1A2 - IMPLICATIONS FOR IN VITRO PREDICTION OF DRUG INTERACTIONS

Abstract

Rofecoxib was recently found to greatly increase plasma concentrations of the cytochrome P450 1A2 (CYP1A2) substrate drug tizanidine in humans, but there are no published in vitro studies on the CYP1A2 inhibiting effects of rofecoxib. Our objective was to investigate whether rofecoxib is a direct-acting or metabolism-dependent inhibitor of CYP1A2 in vitro. The effect of rofecoxib on the O-deethylation of phenacetin (20 µM) was studied using human liver microsomes. The effect of preincubation time on the inhibitory potential of rofecoxib was also studied, and the inhibitor concentration that supports half the maximal rate of inactivation (K_I) and the maximal rate of inactivation (k_inact) were determined. Rofecoxib moderately inhibited phenacetin O-deethylation (IC₅₀ 23.0 µM), and a 30-min-preincubation with microsomes and NADPH considerably increased its inhibitory effect (IC₅₀ 4.2 µM). Inactivation of CYP1A2 by rofecoxib required NADPH, and was characterized by a K_I of 4.8 µM and a k_inact of 0.07 min^-1. Trapping agents, glutathione, superoxide dismutase, and mannitol, or dialysis could not reverse the inactivation of CYP1A2 caused by rofecoxib. Fluvoxamine concentration-dependently decreased the rofecoxib-caused inactivation of CYP1A2. In conclusion, rofecoxib is a potent, metabolism-dependent inhibitor of CYP1A2, a CYP-form contributing to rofecoxib metabolism. The results provide a mechanistic explanation for the interactions of rofecoxib with CYP1A2 substrates, and may partially explain its nonlinear pharmacokinetics.

Key words: CYP inhibition, drug interactions, in vitro-in vivo prediction, inactivation, liver microsomes, mechanism-based inhibition