Enzyme Kinetics for the Formation of 3-Hydroxyquinine and Three New Metabolites of Quinine in Vitro; 3-Hydroxylation by CYP3A4 is Indeed the Major Metabolic Pathway

Abstract

ABSTRACT:The formation kinetics of 3-hydroxyquinine, 2-quininone, (10S)-11-dihydroxydihydroquinine, and (10R)-11-dihydroxydihydroquinine were investigated in human liver microsomes and in human recombinant-expressed CYP3A4. The inhibition profile was studied by the use of different concentrations of ketoconazole, troleandomycin, and fluvoxamine. In addition, formation rates of the metabolites were correlated to different enzyme probe activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in microsomes from 20 human livers. Formation of 3-hydroxyquinine had the highest intrinsic clearance in human liver microsomes (mean ؎ S.D.) of 11.0 ؎ 4.6 l/min/mg. A markedly lower intrinsic clearance, 1.4 ؎ 0.7, 0.5 ؎ 0.1, and 1.1 ؎ 0.2 l/min/mg was measured for 2-quininone, (10R)-11-dihydroxydihydroquinine and (10S)-11-dihydroxydihydroquinine, respectively. Incubation with human recombinant CYP3A4 resulted in a 20-fold higher intrinsic clearance for 3-hydroxyquinine compared with 2-quininone formation whereas no other metabolites were detected. The formation rate of 3-hydroxyquinine was completely inhibited by ketoconazole (1 M) and troleandomycin (80 M). Strong inhibition was observed on the formation of 2-quininone whereas the formation of (10S)-11-dihydroxydihydroquinine was partly inhibited by these two inhibitors. No inhibition on the formation of (10R)-11-dihydroxydihydroquinine was observed. There was a significant correlation between the formation rates of quinine metabolites and activities of the CYP3A4 selected marker probes. This in vitro study demonstrates that 3-hydroxyquinine is the principal metabolite of quinine and CYP3A4 is the major enzyme involved in this metabolic pathway.Quinine is one of the Cinchona alkaloids used in the treatment of severe forms of malaria (White, 1992). Although the drug has been used for more than 300 years, its metabolism and elimination in humans is not well elucidated. Even though several metabolites have been identified (Bannon et al., 1998;Mirghani et al., 2001), the relative roles of these metabolites in quinine elimination and their antimalarial activity have not been investigated.The major metabolite of quinine has been reported to be 3-hydroxyquinine just by comparing the peak heights with that of other metabolites in the chromatograms without quantification (Wanwimolruk et al., 1995). Cytochrome P450 3A4 (CYP3A4) is the most abundant isoform in human liver (up to 30%) and involved in the metabolism of more than 50% of clinically used drugs (Kuehl et al., 2001). CYP3A4 is reported to catalyze the formation of 3-hydroxyquinine both in vitro (Zhang et al., 1997) and in vivo (Mirghani et al., 1999). In a previous in vivo study, we found that about 30% of a single oral dose was excreted as quinine and 3-hydroxyquinine in human urine (Mirghani et al., 1999). Since quinine is extensively metabolized in the liver, elimination via other metabolic pathways may partly explain this low recovery. Four of the quinine metabolites, 3-hydroxyquinine, 2Ј-quininone, (10S)-11-dihydroxydih...

In vitro metabolism of testosterone in the horse liver and involvement of equine CYPs 3A89, 3A94 and 3A95

“…Inhibition experiments were carried out using 1 μ m ketoconazole, 100 μ m troleandomycin, 10 μ m clopidogrel, 5 μ m sulfaphenazole or 25 μ m quercetin. High inhibitor concentrations were chosen according to the literature because inhibitors have not been extensively evaluated for the horse yet (Newton et al ., ; Li et al ., ; Mirghani et al ., ; Richter et al ., ; Sanchez et al ., ; Ghosal et al ., ; Polasek & Miners, ; Nebot et al ., ; Greenblatt et al ., ). Inhibitors were pre‐incubated for 30 min prior to addition of 100 μ m TES in order to account for possible mechanism‐based inhibition.…”

The karolinska cocktail for phenotyping of five human cytochrome P450 enzymes

“…The 8‐hour urinary ratio of debrisoquin/4‐hydroxydebrisoquin was used as the phenotype measure of CYP2D6 activity 28 . The activity of CYP3A4 was estimated by the hydroxylation of quinine to the 3‐hydroxy metabolite in a 16‐hour plasma sample 29 , 30 …”

Genetic epidemiology of induced CYP3A4 activity

“…We measure CYP3A4 activity indirectly through the exogenously administered probe drug quinine [17], which is primarily metabolized by CYP3A4 -and to a lesser extent by CYP3A5 -into 3-hydroxy quinine [18,19]. Previous studies have shown that quinine 3-hydroxylation reaction measured in plasma and urine samples in humans is a stable measure of baseline and induced CYP3A4 activity [20][21][22].…”