Contribution of the N-glucuronidation pathway to the overall in vitro metabolic clearance of midazolam in humans.

Midazolam (MDZ) is one of the most commonly used in vivo and in vitro CYP3A4 probe substrates for drug-drug interactions (DDI) studies. The major metabolic pathway of MDZ in humans consists of the CYP3A4-mediated 1'-hydroxylation followed by urinary excretion as 1'-O-glucuronide derivative. In the present study, following incubation of MDZ with human liver microsomes supplemented with UDP-glucuronic acid, two major high-performance liquid chromatography (HPLC) peaks were isolated. HPLC and liquid chromatography/tandem mass spectrometry analyses identified these two metabolites as quaternary direct N-glucuronides of MDZ, thus revealing an additional metabolic pathway for MDZ. (1)H NMR spectrometry studies were performed showing that these two glucuronides were beta-N-glucuronides, which could be considered as two different conformers of the same molecule. According to molecular modeling experiments, the two glucuronide derivatives could be involved in atropoisomerism equilibrium. The formation of MDZ N-glucuronide exhibited moderate intersubject variability (at most 4.5-fold difference, n = 10). Among the recombinant human UDP glucuronosyltransferase (UGT) isoforms tested, only isoform UGT1A4 catalyzed the N-glucuronidation of MDZ fitting a Michaelis-Menten model. K(m) and V(max) values were 29.9 +/- 2.4 microM and 659.6 +/- 19.0 pmol/min/mg protein, respectively. The N-glucuronide derivative was found in human hepatocytes incubated under control conditions but also in the presence of the well known CYP3A4 inhibitor, ketoconazole. In the context of the in vitro study of CYP3A4-mediated DDI using MDZ and ketoconazole, direct MDZ N-glucuronidation may partly compensate the decrease in MDZ metabolic clearance caused by the addition of the inhibitor, thus potentially leading to underestimation, at least in vitro, of the extent of DDI.

In vitro metabolism of ferroquine (SSR97193) in animal and human hepatic models and antimalarial activity of major metabolites on Plasmodium falciparum.

Ferroquine (SSR97193) has been shown to be a promising antimalarial, both on laboratory clones and on field isolates. So far, no resistance was documented in Plasmodium falciparum. In the present work, the metabolic pathway of ferroquine, based on experiments using animal and human hepatic models, is proposed. Ferroquine is metabolized mainly via an oxidative pathway into the major metabolite mono-N-demethyl ferroquine and then into di-N,N-demethyl ferroquine. Some other minor metabolic pathways were also identified. Cytochrome P450 isoforms 2C9, 2C19, and 3A4 and, possibly in some patients, isoform 2D6, are mainly involved in ferroquine oxidation. The metabolites were synthesized and tested against the 3D7 (chloroquine-sensitive) and W2 (chloroquine-resistant) P. falciparum strains. According to the results, the activity of the two main metabolites decreased compared with that of ferroquine; however, the activity of the mono-N-demethyl derivative is significantly higher than that of chloroquine on both strains, and the di-N-demethyl derivative remains more active than chloroquine on the chloroquine-resistant strain. These results further support the potential use of ferroquine against human malaria.