Evidence for overlapping substrate specificity between large neutral amino acid (LNAA) and dipeptide (hPEPT1) transporters for PD 158473, an NMDA antagonist.

Purpose: The objective of this research was to investigate the substrate specificity of large neutral amino acid carrier (LNAA) and di/tripeptide (hPEPT1) transporters with respect to PD 158473, an NMDA antagonist.

Methods: Cellular uptake studies were carried out using two types of Chinese Hamster Ovary (CHO). CHO-K1 cells represent the wild type with inherent large neutral amino acid (LNAA) activity. CHO-PEPT1 cells were generated by stable transfection of hPEPT1 gene into CHO cells. Therefore, these cells possess both LNAA activity and di/tripeptide transporter activities as a result of the transfection. Cellular uptake of PD 158473 was quantified using a HPLC method previously developed in our laboratory.

Results: The utility of the CHO-PEPT1 cell model was demonstrated by determining the uptake kinetics of Gly-Sar, a prototypical dipeptide transporter substrate. Uptake kinetics of PD 158473 displayed two carrier-mediated transport components in CHO-PEPT1 cells, while in CHO-K1 cells the relationship was consistent with classic one component Michaelis-Menten kinetics. These results confirmed the affinity of PD 158473 for both LNAA and di/tripeptide transporters. Further, results from inhibition experiments using these two cell types indicate that the high affinity-low capacity system was the LNAA carrier and the low affinity-high capacity carrier was the di/tripeptide transporter.

Conclusions: This study demonstrates overlapping substrate specificity between LNAA carrier and di/tripeptide transporter (hPEPT1) for PD 158473, an amino acid analog. Establishing Structure Transport Relationship (STR) for this overlap will aid in a design strategy for increasing oral absorption or targeting specific drugs to selected tissues.