Zafirlukast Metabolism by Cytochrome P450 3A4 Produces an Electrophilic α,β-Unsaturated Iminium Species That Results in the Selective Mechanism-Based Inactivation of the Enzyme

Abstract

Zafirlukast is a leukotriene antagonist indicated for the treatment of mild to moderate asthma, but the drug has been associated with occasional idiosyncratic hepatotoxicity. Structurally, zafirlukast is similar to 3-methylindole because it contains an N-methylindole moiety that has a 3-alkyl substituent on the indole ring. The results presented here describe the metabolic activation of zafirlukast via a similar mechanism to that described for 3-methylindole. NADP(H)-dependent biotransformation of zafirlukast by hepatic microsomes from rats and humans afforded a reactive metabolite, which was detected as its GSH adduct. Mass spectrometry and NMR data indicated that the GSH adduct was formed by the addition of GSH to the methylene carbon between the indole- and methoxy-substituted phenyl rings of zafirlukast. The formation of this reactive metabolite in human liver microsomes was shown to be exclusively catalyzed by CYP3A enzymes. Evidence for in vivo metabolic activation of zafirlukast was obtained when the same GSH adduct was detected in bile of rats given an iv or oral dose of the drug. On the basis of results with model peroxidases and of the structures of product alcohols from incubations containing H2(18)O, it appeared that zafirlukast underwent dehydrogenation by two sequential one-electron oxidations. In addition, zafirlukast proved to be a mechanism-based inhibitor of CYP3A4 activity in human liver microsomes and in microsomes containing cDNA-expressed CYP3A4. The enzyme inhibitory property of zafirlukast was selective for this enzyme among all of the P450 enzymes that were tested in human liver microsomes. The inactivation was characterized by a K(I) of 13.4 microM and k(inact) of 0.026 min(-1). In summary, zafirlukast dehydrogenation to an electrophilic alpha,beta-unsaturated iminium intermediate may be associated with idiosyncratic hepatotoxicity and/or cause drug-drug interactions through inactivation of CYP3A4.

Bioactivation of a Novel 2-Methylindole-Containing Dual Chemoattractant Receptor-Homologous Molecule Expressed on T-Helper Type-2 Cells/d-Prostanoid Receptor Antagonist Leads to Mechanism-Based CYP3A Inactivation: Glutathione Adduct Characterization and Prediction of In Vivo Drug-Drug Interaction

“…These 2-alkylpyrrole derivatives are bioactivated via an analogous mechanism involving an epoxide intermediate, and a GSH adduct has been identified with the glutathionyl moiety on the 3-position of the pyrrole ring (Chen et al, 2006b). With respect to pathway B, a mechanism involving two sequential 1-electron abstractions is consistent with the previously reported HRP-mediated bioactivation of zafirlukast (Kassahun et al, 2005). The efficient conversion of 2MI to G1 by HRP in the present study implies that this mechanism (B), proceeding through the diimine, may predominate for the peroxidase enzyme system.…”

Covalent Binding and Tissue Distribution/Retention Assessment of Drugs Associated with Idiosyncratic Drug Toxicity

“…Most of the drugs used in the present study are known to form various reactive metabolites (Kalgutkar et al, 2005), as shown in Table 1. This includes several types of reactive metabolites, such as quinone imine derived from amodiaquine (Christie et al, 1989); ortho-quinone from benzbromarone (McDonald and Rettie, 2007); quinone methide from tacrine (Madden et al, 1993); nitrenium ion from clozapine (Pirmohamed et al, 1995); iminium ion from nevirapine (Kalgutkar et al, 2005) and zafirlukast (Kassahun et al, 2005); nitroso species from erythromycin (Larrey et al, 1983), phenacetin (Koymans et al, 1990), procainamide (Uetrecht, 1985), and sulfamethoxazole (Naisbitt et al, 2002); arene oxide from imipramine (Masubuchi et al, 1996); acyl glucuronide from valproic acid (Baillie, 1988) and zomepirac (Wang et al, 2001); and radical species from aminopyrine (Uetrecht et al, 1995), flutamide (Kang et al, 2007), and phenytoin (Cuttle et al, 2000). Quinone imine formation from amodiaquine was reported to be caused by autoxidation (Maggs et al, 1987); thus, the high levels of covalent binding without NADPH observed in this study seem to be reasonable.…”

Mechanism-Based Inactivation of Lung-Selective Cytochrome P450 CYP2F Enzymes

“…Another example is the dehydrogenation of zafirlukast, a leukotriene receptor antagonist (Kassahun et al, 2005) that contains a 3-alkylindole moiety. The dehydrogenation of the 3-alkylindole moiety of zafirlukast formed the same type of imine methide electrophilic intermediate as 870.…”