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Clinical studies have revealed that plasma concentrations of midazolam after oral administration are greatly increased by coadministration of erythromycin and clarithromycin, whereas azithromycin has little effect on midazolam concentrations. Several macrolide antibiotics are known to be mechanism-based inhibitors of CYP3A, a cytochrome P450 isoform responsible for midazolam hydroxylation. The aim of the present study was to quantitatively predict in vivo drug interactions in humans involving macrolide antibiotics with different inhibitory potencies based on in vitro studies. alpha- and 4-Hydroxylation of midazolam by human liver microsomes were evaluated as CYP3A-mediated metabolic reactions, and the effect of preincubation with macrolides was examined. The hydroxylation of midazolam was inhibited in a time- and concentration-dependent manner following preincubation with macrolides in the presence of NADPH, whereas almost no inhibition was observed without preincubation. The kinetic parameters for enzyme inactivation (K'app and kinact) involved in midazolam alpha-hydroxylation were 12.6 microM and 0.0240 min-1, respectively, for erythromycin, 41.4 microM and 0.0423 min-1, respectively, for clarithromycin, and 623 microM and 0.0158 min-1, respectively, for azithromycin. Similar results were obtained for the 4-hydroxylation pathway. These parameters and the reported pharmacokinetic parameters of midazolam and macrolides were then used to simulate in vivo interactions based on a physiological flow model. The area under the concentration-time curve (AUC) of midazolam after oral administration was predicted to increase 2.9- or 3.0-fold following pretreatment with erythromycin (500 mg t.i.d. for 5 or 6 days, respectively) and 2.1- or 2.5-fold by clarithromycin (250 mg b.i.d. for 5 days or 500 mg b.i.d. for 7 days, respectively), whereas azithromycin (500 mg o.d. for 3 days) was predicted to have little effect on midazolam AUC. These results agreed well with the reported in vivo observations.

To identify an appropriate animal model for the study of drug interaction via CYP3A4 inhibition, the inhibition of in vitro mexazolam metabolism by various 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors [simvastatin (lactone), simvastatin acid, fluvastatin, atorvastatin, cerivastatin, pravastatin lactone, and pravastatin (acid)] in male and female rat liver microsomes was investigated and compared with that by HMG-CoA reductase inhibitors in human liver microsomes reported previously. The metabolism of mexazolam in male and female rat liver microsomes was inhibited by all the HMG-CoA reductase inhibitors examined except pravastatin (acid). The K(i) values in female rats were lower than those in male rats, demonstrating the presence of a sex difference in the inhibition potency of HMG-CoA reductase inhibitors toward mexazolam. Using anti-cytochrome P450 (P450) antisera, the main P450 isozyme responsible for the metabolism of mexazolam was identified as CYP3A in female rats and CYP2C11 in male rats. Based on these results, we speculate that the sex difference in the inhibition potency of HMG-CoA reductase inhibitors for mexazolam observed in rats is caused by their different inhibition potencies against CYP2C11 and CYP3A isoforms. For mexazolam metabolism, the results obtained in female rats, rather than those in male rats, seem to be a much better reflection of the results in humans. Since species and sex differences were observed in P450 isozymes in the present study, our results show that establishing appropriate experimental conditions, in particular with respect to the P450 isozymes responsible for the drug metabolism in question, is indispensable for the investigation of drug interactions using rats as a model animal for humans.