Interactions of a nonpeptidic drug, valacyclovir, with the human intestinal peptide transporter (hPEPT1) expressed in a mammalian cell line

The results of previous work performed in our laboratory using an in situ p erfusion technique in rats and rabbit apical brush border membrane Vesicles have suggested that the intestinal uptake of valacyclovir (VACV) appears t o be mediated by multiple membrane transporters. Using these techniques, it is difficult to characterize the transport kinetics of VACV with each indi vidual transporter in the presence of multiple known or unknown transporter s. The purpose of this study was to characterize the interaction of VACV an d the human intestinal peptide transporter using Chinese hamster ovary (CHO ) cells that overexpress the human intestinal peptide transporter (hPEPT1) gene. VACV uptake was significantly greater in CHO cells transfected with h PEPT1 than in cells transfected with only the vector, pcDNA3. The optimum p H for VACV uptake was determined to occur at pH 7.5. Proton cotransport was not observed in hPEPT1/CHO cells, consistent with previously observed resu lts in tissues and Caco-2 cells. VACV uptake was concentration dependent an d saturable with a Michaelis-Menten constant and maximum velocity of 1.64 /- 0.06 mM and 23.34 +/- 0.36 nmol/mg protein/5 min, respectively. A very s imilar K-m value was obtained in hPEPT1/CHO cells and in rat and rabbit tis sues and Caco-2 cells; suggesting that hPEPT1 dominates the intestinal tran sport properties of VACV in vitro. VACV uptake was markedly inhibited by va rious dipeptides and beta-lactam antibiotics, and K-i values of 12.8 +/- 2. 7 and 9.1 +/- 1.2 mM were obtained for Gly-Sar and cefadroxil at pM 7.5, re spectively. The present results demonstrate that VACV is a sub;strafe for t he human intestinal peptide transporter in hPEPT1/CHO cells and that althou gh transport is pH dependent, proton cotransport is not apparent. Also, the results demonstrate that the hPEPT1/CHO cell system has use in investigati ng the transport kinetics of drugs with the human intestinal peptide transp orter hPEPT1; however, the extrapolation of these transport properties to t he in vivo situation requires further investigation.