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 hy drolysis of the amino acid eater prodrugs of nucleoside antiviral drug s 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 t heir apical membrane permeability and hydrolysis were evaluated in Cac o-2/hPEPT1 cells. The cellular uptake mechanism of prodrugs was invest igated through the competitive inhibition study in Caco-2/hPEPT1 cells . Results. L-Valyl ester of acyclovir (L-Val-ACV) was approximately te n fold more permeable across the apical membrane than acyclovir and fo ur times more permeable than D-valyl ester of acyclovir (D-Val-ACV). C orrespondingly, L-valyl ester of AZT (L- VaI-AZT) exhibited three fold higher cellular uptake than AZT. Therefore, amino acid eater prodrugs significantly increased the cellular uptake of the parent drugs and e xhibited the D,L-stereoselectivity. Furthermore, prodrugs were rapidly hydrolyzed to the parent drugs by the intracellular hydrolysis, follo wing. the apical membrane transport. In the inhibition studies, cephal exin and small dipeptides strongly inhibited the cellular uptake of L- Val-ACV while L-valine had no effect, indicating that the peptide tran sporter is primarily responsible for the apical membrane transport of L-Val-ACV. In addition, the cellular uptake of L-Val-ACV was five rime s higher in Caco-2/hPEPT1 cells than the uptake in the untransfected C aco-2 cells, implying the cellular uptake of L-Var-ACV was related to the enhancement of the peptide transport activity in Caco-2/hPEPT1 cel ls. Conclusions, Caco-2/hPEPT1 system is an efficient in vitro model f or the uptake study of peptidyl derivatives. Amino acid eater prodrugs significantly improved the cellular uptake of the parent drugs via pe ptide transport mechanism and were rapidly converted to the active par ent drugs by the intracellular hydrolysis.