Persistent Inhibition of CYP3A4 by Ketoconazole in Modified Caco-2 Cells

Purpose. The intestinal metabolism of some CYP3A substrates canbe altered profoundly by co-administration of the potent inhibitor,ketoconazole. The present research was conducted to test the hypothesisthat, unlike the inhibition kinetics observed with isolated microsomes,inhibition of CYP3A4 by ketoconazole in an intestinal cell monolayeris time-dependent and slowly reversible. Methods. Confluent, 1α,25-dihydroxy Vitamin D3-treated Caco-2 cellswere exposed to 1 μM ketoconazole for two hours (Phase I) and thenwashed three times with culture medium containing no inhibitor. Thiswas followed by a second incubation period (Phase II) that varied inthe composition of the apical and basolateral culture medium: Condition1, 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 μM) was included in the apical medium fordetermination of CYP3A4 activity (Phase III). Results. Two-way ANOVA of the data revealed persistent inhibitionof CYP3A4 under Conditions 1 and 2 (p < 0.001). In contrast, cellstreated under Condition 3 exhibited rapid reversal of CYP3A4inhibition. The level of CYP3A4 activity observed was inversely correlatedwith the amount of ketoconazole remaining in the cell monolayer atthe end of Phase II. Conclusions. These studies provide mechanistic evidence thatketoconazole can be sequestered into the intestinal mucosa after oraladministration, producing a persistent inhibition of first-pass CYP3A4 activity.