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WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Midazolam is extensively metabolized and is eliminated in urine predominantly as conjugates of 1′-hydroxymidazolam. • Midazolam is known to undergo N-glucuronidation in vitro by UGT1A4. • In vitro 1′-hydroxymidazolam is O-glucuronidated by UGT2B4 and UGT2B7, and N-glucuronidated by UGT1A4. 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. AIMS Midazolam (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. METHODS An 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 1H-13C long range correlation experiments. The metabolite was quantified in vivo in human urine samples. Enzyme kinetic behaviour of the pathway was investigated in HLM and recombinant UGT (rUGT) enzymes. Additionally, preliminary experiments were performed with 1′-OH midazolam (1′-OH MDZ) and 4-OH-midazolam (4-OH MDZ) to investigate N-glucuronidation. RESULTS NMR data confirmed conjugation of midazolam N-glucuronide (MDZG) standard to be on the α-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 µM, 64 µM), Vmax (445 pmol min−1 mg−1, 427 pmol min−1 mg−1) and Ki (58 µM, 79 µM) 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. CONCLUSIONS A 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.