Midazolam metabolism by modified Caco-2 monolayers: Effects of extracellular protein binding

It has been suggested that the binding of a drug to plasma proteins will in fluence the intestinal extraction efficiency when drug is delivered to the mucosal epithelium via either the gut lumen or vasculature. We evaluated th is hypothesis using cytochrome P-450 (CYP)3A4-expressing Caco-2 monolayers as a model for the intestinal epithelial barrier and midazolam as a CYP3A-s pecific enzyme probe. The rate of 1'-hydroxylation was measured following a pical or basolateral midazolam administration to monolayers incubated in th e presence or absence of 4 g/dl of human serum albumin (HSA) in the basolat eral compartment medium. The midazolam-free fraction in culture medium cont aining HSA was 3.3%. Inclusion of HSA in the basolateral medium decreased p eak intracellular midazolam accumulation after an apical midazolam dose (3 mu M) by 35% and reduced the 1'-hydroxymidazolam formation rate by similar to 20%. Because of the accelerated diffusion of midazolam through the cell monolayer and into the basolateral compartment, there was a 61% reduction i n the first-pass metabolic extraction ratio: 13.3 +/- 0.12% for control ver sus 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% decreas e in the rate of 1'-hydroxylation after the administration of midazolam int o basolateral medium. These findings suggest that, in vivo, binding of a dr ug to plasma proteins will impact both first-pass and systemic intestinal m idazolam extraction efficiency. Furthermore, the effect will be more pronou nced for a drug that is delivered to mucosal enterocytes by way of arterial blood, compared with oral drug delivery.