Midazolam alpha-hydroxylation by human liver microsomes in vitro: inhibition by calcium channel blockers, itraconazole and ketoconazole

Pharmacol Toxicol. 1999 Oct;85(4):157-61. doi: 10.1111/j.1600-0773.1999.tb00085.x.


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 alpha-hydroxylation of midazolam, a probe drug for CYP3A4-mediated interactions in humans, were studied in vitro using human liver microsomes. IC50 and Ki values were determined for each inhibitor. The kinetics of the formation of alpha-hydroxymidazolam were best described by simple Michaelis-Menten kinetics. The estimated values of Vmax and Km were 696 pmol min.-(1) mg(-1) and 7.46 micromol l(-1), respectively. All the compounds studied inhibited midazolam alpha-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 alpha-hydroxylation (IC50 0.12 micromol l (-1)), being 10 times more potent than itraconazole (IC50 1.2 micromol l(-1)). The inhibitory effect of hydroxyitraconazole (IC50 2.3 micromol l (-1) was almost as large as that of itraconazole. Among the calcium channel blockers, mibefradil was the most potent inhibitor of the alpha-hydroxylation of midazolam, with an IC50 value (1.6 micromol 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 IC50 of 23 micromol l(-1), while isradipine, nifedipine and diltiazem, with IC50 values ranging from 57 to >100 micromol l (-1), were weak inhibitors. This rank order of potency against the alpha-hydroxylation Qf midazolam was verified by the Ki 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antifungal Agents / pharmacology*
  • Biotransformation
  • Calcium Channel Blockers / pharmacology*
  • GABA Modulators / metabolism*
  • Humans
  • Hydroxylation / drug effects
  • Itraconazole / pharmacology*
  • Ketoconazole / pharmacology*
  • Male
  • Microsomes, Liver / drug effects*
  • Microsomes, Liver / metabolism*
  • Midazolam / metabolism*


  • Antifungal Agents
  • Calcium Channel Blockers
  • GABA Modulators
  • Itraconazole
  • Midazolam
  • Ketoconazole