A comparison of the metabolism of midazolam in C57BL/6J and hepatic reductase null (HRN) mice.

The hepatic cytochrome P450 reductase null (HRN) mouse, which has no functional hepatic Cyp P450s, may represent a useful model for examining extra-hepatic P450-related oxidative metabolism. Here the pharmacokinetics and metabolic fate of midazolam, a drug known to undergo such extra-hepatic metabolism, have been investigated in the HRN mouse and compared with a phenotypically normal strain (C57BL/6J). In addition, the effects of co-administration of the pan-P450 inhibitor 1'-aminobenzotriazole (ABT) on the metabolic profile have been compared in both strains. Significant pharmacokinetic differences for midazolam were observed between the two strains of mice with the HRN mice showing lower circulating concentrations of 1'-hydroxymidazolam but higher concentrations of 1'-hydroxymidazolam-O-glucuronide. A significant increase in midazolam exposure was seen upon ABT exposure for both strains of mice, but no differences in the area under the concentration time curves (AUC) for the monitored metabolites were observed. Although oxidative metabolism of midazolam was not abolished, significant decreases in 1'-hydroxymidazolam formation ratios were observed for both strains of mice exposed to ABT. Metabolite profiling of blood and bile showed a number of qualitative and quantitative differences between HRN and normal mice. These differences in midazolam metabolism between the two strains of mice clearly demonstrate the role that liver P450 enzymes play in the murine metabolism of midazolam. The fate of the compound in the HRN mice shows the importance of extrahepatic metabolism and also showed that these mice appear to be more capable of forming circulating phase II glucuronides than the normal strain.

The application of a new microfluidic device for the simultaneous identification and quantitation of midazolam metabolites obtained from a single micro-litre of chimeric mice blood.

RATIONALE: Improvements in the design of low-flow highly sensitive chromatographic ion source interfaces allow the detection and characterisation of drugs and metabolites from smaller sample volumes. This in turn improves the ethical treatment of animals by reducing both the number of animals needed and the blood sampling volumes required. METHODS: A new microfluidic device combining an ultra-high pressure liquid chromatography (UHPLC) analytical column with a nano-flow electrospray source is described. All microfluidic, gas and electrical connections are automatically engaged when the ceramic microfluidic device is inserted into the source enclosure. The system was used in conjunction with a hybrid quadrupole-time-of-flight mass spectrometer. RESULTS: The improved sensitivity of the system is highlighted in its application in the quantification and qualification of midazolam and its metabolites detected in whole blood from chimeric and wild-type mice. Metabolite identification and full pharmacokinetic profiles were obtained from a single micro-litre of whole blood at each sampling time and significant pharmacokinetic differences were observed between the two types of mice. CONCLUSIONS: Improvements in the enhanced ionisation efficiency from the microfluidic device in conjunction with nanoUHPLC/MS was sufficiently sensitive for the identification and quantification of midazolam metabolites from a single micro-litre of whole blood. Detection of metabolites not previously recorded from the chimeric mouse in vivo model was made.

Drug-drug interactions and metabolism in cytochrome P450 2C knockout mice: application to troleandomycin and midazolam.

Drug-drug interactions (DDIs) may cause serious drug toxicity and delay development of candidate drugs. Screening using human liver microsomes and hepatocytes can help predict DDIs but do not always provide the degree of certainty required for confident progression of a candidate drug. Thus a suitable in vivo test system could be of great value. Here a Cyp2c knockout (KO) mouse was investigated for studying DDIs using midazolam (MDZ) a standard human CYP3A4 substrate and troleandomycin (TAO) a potent human CYP3A4 inhibitor. Pharmacokinetics (PK) and biotransformation of MDZ were investigated following dosing to Cyp2c KO and wild type mice before and after TAO treatment. The noteworthy differences in the metabolism of MDZ in Cyp2c KO compared to wild type mice confirms the important role that Cyp2c enzymes play in the murine metabolism of MDZ in vivo. The impact of Cyp3a inhibition produced a further increase in circulating MDZ concentrations in all individuals from both strains of mice though the impact of the elimination of the Cyp2c pathway in the KO mice on the AUC was less than perhaps expected. We have shown that TAO produces an increase in the MDZ concentration and a reduction in the 1'hydroxymidazolam/midazolam formation ratio but the expected difference in the magnitude of this effect between the wild type and the Cyp2c KO mice was not seen. The magnitude of the TAO effect was also smaller than is reported in humans. Hence further work is required before this animal model could be used to predict clinical interactions.