Role of basolateral efflux transporter MRP4 in the intestinal absorption of the antiviral drug adefovir dipivoxil

Abstract

Adefovir dipivoxil is a diester prodrug of the antiviral drug adefovir, with much greater oral bioavailability than adefovir. Evidence shows that the prodrug is metabolized to adefovir in the enterocytes during intestinal absorption. However, it is unknown how the highly charged and hydrophilic adefovir crosses the basolateral membrane in the intestine. This study determines the role of specific basolateral transporter(s) in the egress of adefovir across the basolateral membrane when formed from adefovir dipivoxil in Caco-2 cells, a model for intestinal epithelium. Multidrug resistance-associated protein 4 (MRP4) plays an important role in renal secretion of adefovir. Immunofluorescence images showed that MRP4 is localized in the basolateral membrane of Caco-2 cells. This localization was further confirmed by Western blotting of the apical and basolateral membrane fractions that were isolated by a novel method involving biotinylation of respective membrane proteins and affinity enrichment. MRP4-knockdown Caco-2 cells were produced by stable transfection with MRP4-specific siRNA expression plasmid. These cells showed reduced MRP4 protein expression and corresponding reduction in the basolateral egress of adefovir when adefovir dipivoxil was dosed on the apical side. A comparison of these data with the reduction in the basolateral egress of adefovir by the general MRP inhibitor indomethacin established that MRP4, among MRPs, plays a predominant role in the basolateral egress of adefovir in Caco-2 cells. The results highlight the importance of MRP4 in oral absorption of adefovir dipivoxil, and suggest that significant drug–drug interactions can occur if an MRP4 inhibitor is co-administered with adefovir dipivoxil.