Contribution of the N-Glucuronidation Pathway to the Overall in Vitro Metabolic Clearance of Midazolam in Humans

Abstract

ABSTRACT: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. Because a large number of currently available drugs and future drugs will be metabolized by the members of the CYP3A subfamily, the potential for drug-drug interaction (DDI) is substantial. DDIs involving the inhibition and induction of CYP3A4 are of great scientific and clinical relevance. Indeed, drugs with potent CYP3A4 inhibitory properties have been implicated in significant CYP3A4-mediated DDIs. Interactions of the benzodiazepines with the azole antifungal agents and especially the inhibition of CYP3A-mediated midazolam (MDZ) metabolism by ketoconazole have been widely studied. Concomitant administration of both drugs results in large, variable, and highly significant increases (5-16-fold) in MDZ exposure, depending on the dose regimen of ketoconazole used. Table 1 summarizes available clinical data on the effects of ketoconazole on MDZ plasma levels following p.o. or i.v. administration of MDZ.MDZ is a short-acting water-soluble imidazobenzodiazepine (Fig. 1) extensively used in clinical practice mainly for induction and maintenance of anesthesia, sedation for diagnostic and therapeutic procedures, and also as an oral hypnotic agent (Reves et al., 1985). MDZ is a well known CYP3A substrate because its metabolism has been the focus of many in vitro investigations (Fabre et al., 1988b;Kronbach et al., 1989;Wrighton and Ring, 1994;Ghosal et al., 1996; Maenpaa et al., 1998;Hosea et al., 2000;Wang et al., 2000). MDZ biotransformation is mediated by at least three different CYP3A isoenzymes: CYP3A4, CYP3A5, and CYP3A7 (Gorski et al., 1994;Kuehl et al., 2001). Because CYP3A7 is principally expressed in fetal tissues, CYP3A4 and CYP3A5 represent the main cytochrome P450 (P450) isoforms in adult liver and intestine (Guengerich, 1995).MDZ biotransformation yields two primary hydroxylated metabolites: 1Ј-hydroxy-MDZ (1Ј-OH-MDZ) and 4-hydroxy-MDZ. 1Ј-OH-MDZ represents the main metabolite because it accounts for 95% of net intrinsic clearance of MDZ in human liver microsomes (von Article, publication date, and citation information can be found at

Prediction of Drug Clearance by Glucuronidation from in Vitro Data: Use of Combined Cytochrome P450 and UDP-Glucuronosyltransferase Cofactors in Alamethicin-Activated Human Liver Microsomes

“…However, when BSA is added to the incubation, this trend is reversed, with glucuronidation accounting for 78% of zidovudine metabolism (Table 4), in agreement with the previously reported UGT contribution (Blum et al, 1988). In the case of midazolam, direct glucuronidation accounted for a small proportion of the total clearance (6%), consistent with recent reports on N-glucuronidation via UGT1A4 (Klieber et al, 2008). Therefore, cautious interpretation of the clearance data obtained in the presence of individual cofactors is required, especially if the pathways of metabolism are unknown.…”

Cytochrome P450-Dependent Metabolism in HepaRG Cells Cultured in a Dynamic Three-Dimensional Bioreactor

“…In humans, the clearance of midazolam has been reported to be reduced by approximately 85% by ketoconazole (Tsunoda et al, 1999;Tham et al, 2006;Yong et al, 2008;Krishna et al, 2009), which is comparable with the 69% decrease in the formation rate of 1Ј-hydroxymidazolam seen in the bioreactor in this study using HepaRG cells. A comparable degree of inhibition (83-100%) of the 1Ј-hydroxymidazolam formation rate by 10 M ketoconazole has also been reported in 2D-cultured primary human hepatocytes from 18 donors (Klieber et al, 2008). CYP2C9-dependent inhibition by ketoconazole was not recorded in this experiment.…”

Deoxyschizandrin, a Naturally Occurring Lignan, Is a Specific Probe Substrate of Human Cytochrome P450 3A

“…As reported, erlotinib and panaxytriol, an active component in Shenmai injection (Zeng et al, 2013), both showed significant activation on MDZ 19-hydroxylation, whereas the activation but not inhibition made it difficult for the in vivo extrapolation. Moreover, either glucuronidation of hydroxylmidazolam or direct glucuronidation of MDZ occurs in vitro and in vivo (Klieber et al, 2008;Hyland et al, 2009;Seo et al, 2010). The multiple metabolic pathways may partly compensate for the decrease in MDZ metabolic clearance caused by the addition of the inhibitor and would also make it difficult for the in vivo quantification.…”