In vitro and in vivo glucuronidation of midazolam in humans

Abstract

Keywords 1′-hydroxymidazolam, 4-hydroxymidazolam, glucuronidation, in vitro metabolism, in vivo metabolism, midazolam ---------------------------------------------------------------------- Received WHAT THIS STUDY ADDS• N-glucuronide of midazolam has been quantified in human urine, indicating for the first time that this route of metabolism occurs in vivo.• Metabolism of 4-hydroxymidazolam has been compared with that of 1′-hydroxymidazolam in vitro.• This study provides further evidence, in vitro and in vivo, of the importance of N-glucuronidation in the metabolism of midazolam and its metabolites. AIMSMidazolam (MDZ) is a benzodiazepine used as a CYP3A4 probe in clinical and in vitro studies. A glucuronide metabolite of MDZ has been identified in vitro in human liver microsome (HLM) incubations. The primary aim of this study was to understand the in vivo relevance of this pathway. METHODSAn authentic standard of N-glucuronide was generated from microsomal incubations and isolated using solid-phase extraction. The structure was confirmed using proton nuclear magnetic resonance (NMR) and RESULTSNMR data confirmed conjugation of midazolam N-glucuronide (MDZG) standard to be on the a-nitrogen of the imidazole ring. In vivo, MDZG in the urine accounted for 1-2% of the administered dose. In vitro incubations confirmed UGT1A4 as the enzyme of interest. The pathway exhibited atypical kinetics and a substrate inhibitory cooperative binding model was applied to determine Km (46 mM, 64 mM), Vmax (445 pmol min -1 mg -1, 427 pmol min -1 mg -1) and Ki (58 mM, 79 mM) in HLM and rUGT1A4, respectively. From incubations with HLM and rUGT enzymes, N-glucuronidation of 1′-OH MDZ and 4-OH MDZ is also inferred. CONCLUSIONSA more complete picture of MDZ metabolism and the enzymes involved has been elucidated. Direct N-glucuronidation of MDZ occurs in vivo. Pharmacokinetic modelling using Simcyp™ illustrates an increased role for UGT1A4 under CYP3A inhibited conditions.

Conjugation of Synthetic Cannabinoids JWH-018 and JWH-073, Metabolites by Human UDP-Glucuronosyltransferases

“…The majority of glucuronidation reactions exhibited classical Michaelis- Menten kinetics; however, there were a few cases in which atypical kinetic profiles were found. There is growing evidence in the literature that UGT-catalyzed reactions, especially at high substrate concentrations, exhibit several different kinetic profiles (Iwuchukwu and Nagar, 2008;Hyland et al, 2009;Iwuchukwu et al, 2009;Aprile et al, 2010;Zhou et al, 2010), and our data are in agreement with these earlier reports. These atypical kinetic parameters indicate that, at higher concentrations, clearance by specific UGTs could be altered.…”

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.…”

Physiologically Based Pharmacokinetic Model of Mechanism-Based Inhibition of CYP3A by Clarithromycin

“…Midazolam is a commonly used in vivo probe for CYP3A activity that undergoes CYP3A-mediated hydroxylation to form 1Ј-hydroxymidazolam and 4-hydroxymidazolam (Kronbach et al, 1989;Gorski et al, 1994). A small proportion, 1 to 2% of the dose, is glucuronidated (Hyland et al, 2009) and less than 1% of the administered dose is excreted unchanged in the urine. In contrast with other CYP3A substrates, midazolam does not appear to undergo P-glycoprotein-mediated transport (Schmiedlin-Ren et al, 1997;Kim et al, 1999).…”