Midazolam α‐Hydroxylation by Human Liver Microsomes in vitro : Inhibition by Calcium Channel Blockers, Itraconazole and Ketoconazole

The inhibitory effects of five calcium channel blockers (diltiazem, isradipine, mibefradil, nifedipine and verapamil) and three azole antifungal agents (itraconazole, hydroxyitraconazole and ketoconazole) on the α‐hydroxylation of midazolam, a probe drug for CYP3A4‐mediated interactions in humans, were studied in vitro using human liver microsomes. IC 50 and K i values were determined for each inhibitor. The kinetics of the formation of α‐hydroxymidazolam were best described by simple Michaelis‐Menten kinetics. The estimated values of V max and K m were 696 pmol min. −1 mg −1 and 7.46 μmol l −1 , respectively. All the compounds studied inhibited midazolam α‐hydroxylation activity in a concentration‐dependent manner, but there were marked differences in their relative inhibitory potency. Ketoconazole was the most potent inhibitor of midazolam α‐hydroxylation (IC 50 0.12 μmol l −1 ), being 10 times more potent than itraconazole (IC 50 1.2 μmol l −1 ). The inhibitory effect of hydroxyitraconazole (IC 50 2.3 μmol l −1 ) was almost as large as that of itraconazole. Among the calcium channel blockers, mibefradil was the most potent inhibitor of the α‐hydroxylation of midazolam, with an IC 50 value (1.6 μmol l −1 ) similar to that of itraconazole. The other calcium channel blockers were much weaker inhibitors than mibefradil: verapamil exhibited a modest inhibitory effect with an IC 50 of 23 μmol l −1 , while isradipine, nifedipine and diltiazem, with IC 50 values ranging from 57 to >100 μmol l −1 , were weak inhibitors. This rank order of potency against the α‐hydroxylation of midazolam was verified by the K i values. With the exception of diltiazem, these in vitro results conform with the observed interaction potential of these agents with midazolam and many other CYP3A4 substrates in vivo in man.