Vol. 289, Issue 2, 1143-1150, May 1999

Midazolam Metabolism by Modified Caco-2 Monolayers: Effects of Extracellular Protein Binding¹

Jeannine M. Fisher, Steven A. Wrighton, Justina C. Calamia, Danny D. Shen, Kent L. Kunze and Kenneth E. Thummel

Departments of Pharmaceutics (J.C.C., D.D.S., K.E.T.) and Medicinal Chemistry (K.L.K.), University of Washington, Seattle, Washington; and Department of Drug Disposition, Eli Lilly & Co., Indianapolis, Indiana (J.M.F., S.A.W.)

It has been suggested that the binding of a drug to plasma proteins will influence the intestinal extraction efficiency when drug is delivered to the mucosal epithelium via either the gut lumen or vasculature. We evaluated this hypothesis using cytochrome P-450 (CYP)3A4-expressing Caco-2 monolayers as a model for the intestinal epithelial barrier and midazolam as a CYP3A-specific enzyme probe. The rate of 1'-hydroxylation was measured following apical or basolateral midazolam administration to monolayers incubated in the presence or absence of 4 g/dl of human serum albumin (HSA) in the basolateral compartment medium. The midazolam-free fraction in culture medium containing HSA was 3.3%. Inclusion of HSA in the basolateral medium decreased peak intracellular midazolam accumulation after an apical midazolam dose (3 µM) by 35% and reduced the 1'-hydroxymidazolam formation rate by ~20%. Because of the accelerated diffusion of midazolam through the cell monolayer and into the basolateral compartment, there was a 61% reduction in the first-pass metabolic extraction ratio: 13.3 ± 0.12% for control versus 5.2 ± 1% with HSA. Compared with control, addition of HSA resulted in a 91% decrease in the peak intracellular midazolam level and a 86% decrease in the rate of 1'-hydroxylation after the administration of midazolam into basolateral medium. These findings suggest that, in vivo, binding of a drug to plasma proteins will impact both first-pass and systemic intestinal midazolam extraction efficiency. Furthermore, the effect will be more pronounced for a drug that is delivered to mucosal enterocytes by way of arterial blood, compared with oral drug delivery.