Cellular uptake mechanism of amino acid ester prodrugs in Caco-2/hPEPT1 cells overexpressing a human peptide transporter

Purpose: This study characterized the cellular uptake mechanism and hydrolysis of the amino acid ester prodrugs of nucleoside antiviral drugs in the transiently transfected Caco-2 cells overexpressing: a human intestinal peptide transporter, hPEPT1 (Caco-2/hPEPT1 cells). Methods: Amino acid ester prodrugs of acyclovir and AZT were synthesized and their apical membrane permeability and hydrolysis were evaluated in Caco-2/hPEPT1 cells. The cellular uptake mechanism of prodrugs was investigated through the competitive inhibition study in Caco-2/hPEPT1 cells. Results: L-Valyl ester of acyclovir (L-Val-ACV) was approximately ten fold more permeable across the apical membrane than acyclovir and four times more permeable than D-valyl ester of acyclovir (D-Val-ACV). Correspondingly, L-valyl ester of AZT (L- VaI-AZT) exhibited three fold higher cellular uptake than AZT. Therefore, amino acid ester prodrugs significantly increased the cellular uptake of the parent drugs and exhibited the D,L-stereoselectivity. Furthermore, prodrugs were rapidly hydrolyzed to the parent drugs by the intracellular hydrolysis, following. the apical membrane transport. In the inhibition studies, cephalexin and small dipeptides strongly inhibited the cellular uptake of L-Val-ACV while L-valine had no effect, indicating that the peptide transporter is primarily responsible for the apical membrane transport of L-Val-ACV. In addition, the cellular uptake of L-Val-ACV was five rimes higher in Caco-2/hPEPT1 cells than the uptake in the untransfected Caco-2 cells, implying the cellular uptake of L-Var-ACV was related to the enhancement of the peptide transport activity in Caco-2/hPEPT1 cells. Conclusions: Caco-2/hPEPT1 system is an efficient in vitro model for the uptake study of peptidyl derivatives. Amino acid ester prodrugs significantly improved the cellular uptake of the parent drugs via peptide transport mechanism and were rapidly converted to the active parent drugs by the intracellular hydrolysis.