Application of osmotic pumps for sustained release of 1-aminobenzotriazole and inhibition of cytochrome P450 enzymes in mice: Model comparison with the hepatic P450 reductase null mouse

The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor

Defining the in Vivo Role for Cytochrome b(5) in Cytochrome P450 Function through the Conditional Hepatic Deletion of Microsomal Cytochrome b(5)

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner

Deletion of Microsomal Cytochrome b(5) Profoundly Affects Hepatic and Extrahepatic Drug Metabolism

We recently demonstrated that cytochrome b5 plays an important in vivo role in hepatic cytochrome P450 (P450) function (Finn et al., J. Biol. Chem. (2008)). We have now generated a model where cytochrome b5 has been deleted in all tissues (BCN; cytochrome b5 complete null), which surprisingly results in a viable mouse in spite of the putative in vivo roles of this protein in lipid and steroid hormone metabolism, and the reduction of methemoglobin. In contrast to the liver-specific deletion, complete deletion of cytochrome b5 leads to a neonatal increase in the expression of many hepatic P450s, at both the protein and mRNA level. In extra-hepatic tissues, some changes in P450 expression were also observed, which were isoform-dependent. In vitro cytochrome P450 activities in liver, kidney, lung and small intestine of BCN mice were determined for a range of model substrates and probe drugs; a profound reduction in the metabolism of some substrates, particularly in lung, kidney and small intestine was observed. In vivo, the metabolism of metoprolol was significantly altered in BCN mice, in contrast to the previous finding in the liver-specific cytochrome b5 deletion, suggesting that extra-hepatic cytochrome b5 plays a significant role in its disposition. Testicular Cyp-17 hydroxylase and lyase activities were also significantly reduced by cytochrome b5 deletion, leading to significantly lower levels of testicular testosterone. The BCN mouse provides an additional model system with which to further investigate the functions of cytochrome b5, particularly in extra-hepatic tissue

A Role for Cytochrome b5 in the In Vivo Disposition of Anticancer and Cytochrome P450 Probe Drugs in Mice

The role of microsomal cytochrome b5 (Cyb5) in defining the rate of drug metabolism and disposition has been intensely debated for several decades. Recently we described mouse models involving the hepatic or global deletion of Cyb5, demonstrating its central role in in vivo drug disposition. We have now used the cytochrome b5 complete null (BCN) model to determine the role of Cyb5 in the metabolism of ten pharmaceuticals metabolized by a range of cytochrome P450s, including five anticancer drugs, in vivo and in vitro. The extent to which metabolism was significantly affected by the absence of Cyb5 was substrate-dependent; AUC increased (75–245%) and clearance decreased (35–72%) for phenacetin, metoprolol, and chlorzoxazone. Tolbutamide disposition was not significantly altered by Cyb5 deletion, while for midazolam clearance was decreased by 66%. The absence of Cyb5 had no effect on gefitinib and paclitaxel disposition, while significant changes in the in vivo pharmacokinetics were measured for: cyclophosphamide [maximum plasma concentration (Cmax) and terminal half-life increased 55% and 40%, respectively], tamoxifen (AUClast and Cmax increased 370% and 233%, respectively), and anastrozole (AUC and terminal half-life increased 125% and 62%, respectively; clearance down 80%). These data provide strong evidence that both hepatic and extrahepatic Cyb5 levels are an important determinant of in vivo drug disposition catalyzed by a range of cytochrome P450s, including currently prescribed anticancer agents, and that individuality in Cyb5 expression could be a significant determinant in rates of drug disposition in man