Persistent inhibition of CYP3A4 by ketoconazole in modified Caco-2 cells

The intestinal metabolism of some CYP3A substrates can be altered profoundly by co-administration of the potent inhibitor, ketoconazole. The present research was conducted to test the hypothesis that, unlike the inhibition kinetics observed with isolated microsomes, inhibition of CYP3A4 by ketoconazole in an intestinal cell monolayer is time-dependent and slowly reversible.

Confluent, 1alpha,25-dihydroxy Vitamin D3-treated Caco-2 cells were exposed to 1 microM ketoconazole for two hours (Phase I) and then washed three times with culture medium containing no inhibitor. This was followed by a second incubation period (Phase II) that varied in the composition of the apical and basolateral culture medium: Condition 1. apical/basolateral differentiation medium (DM); Condition 2, apical/ basolateral DM + basolateral 2g/dL Human Serum Albumin (HSA); Condition 3, apical/basolateral DM + apical/basolateral 2 g/dL HSA. After various lengths of time for the second phase (0 to 4 hours), both apical and basolateral medium were exchanged with fresh DM. Midazolam (6 microM) was included in the apical medium for determination of CYP3A4 activity (Phase III).

Two-way ANOVA of the data revealed persistent inhibition of CYP3A4 under Conditions 1 and 2 (p < 0.001). In contrast, cells treated under Condition 3 exhibited rapid reversal of CYP3A4 inhibition. The level of CYP3A4 activity observed was inversely correlated with the amount of ketoconazole remaining in the cell monolayer at the end of Phase II.

These studies provide mechanistic evidence that ketoconazole can be sequestered into the intestinal mucosa after oral administration, producing a persistent inhibition of first-pass CYP3A4 activity.