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

1. The quantitative prediction of the pharmacokinetic parameters of a drug from data obtained using human in vitro systems remains a significant challenge i.e. prediction of metabolic clearance in humans and estimation of the relative contribution of enzymes involved in the clearance. This has become particularly problematic for low turnover compounds. 2. Having human hepatocytes with stable cellular function over several days that adequately mimic the complexity of the physiological environment would be a major advance. Thus, we evaluated human hepatocytes, maintained in culture during 7 days in the microfluidic LiverChip™ system, in terms of morphological appearance, relative mRNA expression of phase I and II enzymes and transporters as a function of time, and metabolic capacity using probe substrates. 3. The results showed that mRNA levels of the major genes for enzymes involved in drug metabolism were well-maintained over a 7-day period of culture. Furthermore, after 4 days of culture, in the Liverchip™ device, human hepatocytes exhibited higher or similar CYPs activities compared to 1 day of culture in 2D-static conditions. 4. The functional data were supported by light/electron microscopies and immunohistochemistry showing viable tissue structure and well-differentiated human hepatocytes: presence of cell junctions, glycogen storage, and bile canaliculi.

The in vitro metabolism of dronedarone and its major metabolites has been studied in human liver microsomes and cryopreserved hepatocytes in primary culture through the use of specific or total cytochrome P450 (CYP) and monoamine oxidase (MAO) inhibitors. The identification of the main metabolites and enzymes participating in their metabolism was also elucidated by using rhCYP, rhMAO, flavin monooxygenases (rhFMO) and UDP-glucuronosyltransferases (rhUGT) and liquid chromatography/tandem mass spectrometry (LC/MS-MS) analysis. Dronedarone was extensively metabolized in human hepatocytes with a metabolic clearance being almost completely inhibited (98 ± 2%) by 1-aminobenzotriazole. Ketoconazole also inhibited dronedarone metabolism by 89 ± 7%, demonstrating the crucial role of CYP3A in its metabolism. CYP3A isoforms mostly contributed to N-debutylation while hydroxylation on the butyl-benzofuran moiety was catalyzed by CYP2D6. However, hydroxylation on the dibutylamine moiety did not appear to be CYP-dependent. N-debutyl-dronedarone was less rapidly metabolized than dronedarone, the major metabolic pathway being catalyzed by MAO-A to form propanoic acid-dronedarone and phenol-dronedarone. Propanoic acid-dronedarone was metabolized at a similar rate to that of N-debutyl-dronedarone and was predominantly hydroxylated by CYP2C8 and CYP1A1. Phenol-dronedarone was extensively glucuronidated while C-dealkyl-dronedarone was metabolized at a slow rate. The evaluation of the systemic clearance of each metabolic process together with the identification of both the major metabolites and predominant enzyme systems and isoforms involved in the formation and subsequent metabolism of these metabolites has enhanced the overall understanding of metabolism of dronedarone in humans.

Prediction of drug-drug interactions due to cytochrome P450 isoform 3A4 (CYP3A4) overexpression is important because this CYP isoform is involved in the metabolism of about 30% of clinically used drugs from almost all therapeutic categories. Therefore, it is mandatory to attempt to predict the potential of a new compound to induce CYP3A4. Among several in vitro-in vivo extrapolation methods recently proposed in the literature, an approach using a scaling factor, called a d factor, for a given hepatocyte batch to provide extrapolation between in vitro induction data and clinical outcome has been adopted by leading health authorities. We challenged the relevance of the calibration factor determined using a set of 15 well-known clinical CYP3A4 inducers or the potent CYP3A4 inducer rifampicin only. These investigations were conducted using six batches of human hepatocytes and an established HepaRG cell line. Our findings show that use of a calibration factor is preferable for clinical predictions, as shown previously by other investigators. Moreover, the present results also suggest that the accuracy of prediction through calculation of this factor is sufficient when rifampicin is considered alone, and the use of a larger set of fully characterized CYP3A4 clinical inducers is not required. For the established HepaRG cell line, the findings obtained in three experiments using a single batch of cells show a good prediction accuracy with or without the d factor. Additional investigations with different batches of HepaRG cell lines are needed to confirm these results.

Midazolam is one of the most commonly used in vivo and in vitro CYP3A4 probe substrate for drug‐drug interactions studies in humans based on the CYP3A4‐mediation of its 1′‐hydroxylation, a major metabolic pathway. In the present work, the in vitro metabolic pathways of midazolam in humans were studied in details in order to elucidate whether or not an alternative metabolic route such as direct N‐glucuronidation could be responsible for a metabolic shift between CYP3A4/5 and UGT. Indeed, following incubation of midazolam with human liver microsomes supplemented with UDPGA, two metabolites were identified as quaternary direct β‐N‐glucuronides of midazolam. Molecular modelling experiments showed that both glucuronide derivatives were in an atropoisomerism equilibrium. Only isoform UGT1A4 can catalyze this direct N‐glucuronidation which follows Michaelis‐Menten kinetics. Midazolam‐N‐glucuronide was formed in human hepatocytes incubated with and without ketoconazole. Direct midazolam‐N‐glucuronidation may partly compensate for the decrease in midazolam metabolic clearance caused by ketoconazole thus potentially leading to a certain extent of misinterpretation of CYP3A4‐mediated DDI study results both in vitro and in vivo.