Abstract
In the present investigation, the uptake and transport kinetics of valacyclovir (VACV), 5-aminolevulinic acid (5-ALA), and benzylpenicillin (BENZ) were studied in stably transfected Madin-Darby canine kidney (MDCK)/human peptide transporter 1 (hPepT1)-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein (low, medium, and high expression) and in Caco-2 cells to delineate hPepT1-mediated transport kinetics. These compounds were selected due to the fact that they are known PepT1 substrates, yet also have affinity for other transporters. Caco-2 cells, traditionally used for studying peptide-based drug transport, were included for comparison purposes. The time, pH, sodium, and concentration dependence of cellular uptake and permeability were measured using mock, clonal hPepT1-MDCK, and Caco-2 cells. A pH-dependent effect was observed in the hPepT1-expressing clones and Caco-2 cells, with an increase of 1.96-, 1.84-, and 2.05-fold for VACV, 5-ALA, and BENZ uptake, respectively, at pH 6 versus 7.4 in the high-expressing hPepT1 cells. BENZ uptake was significantly decreased in Caco-2 and MDCK cells in Na+-depleted buffer, whereas VACV uptake only decreased in Caco-2 cells. Concentration-dependent uptake studies in the mock-corrected hPepT1-MDCK and Caco-2 cells demonstrated hPepT1 affinity ranking of VACV > 5-ALA > BENZ. The apical-to-basal apparent permeability coefficient (Papp) values of VACV, 5-ALA, and BENZ in mock-corrected hPepT1-MDCK cells showed solely hPepT1-mediated transport in contrast to Caco-2 cells. Lower Km values and higher Papp in Caco-2 cells compared with hPepT1-MDCK cells suggested the involvement of multiple transporters in Caco-2 cells. Thus, hPepT1-MDCK cells corrected for endogenous transporter expression may be a more appropriate model for screening compounds for their affinity to hPepT1.
Footnotes
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Funding for this research was provided by the National Institute of General Medical Sciences (NIGMS) (RO1-GM65448) and Rutgers University, Ernest Mario School of Pharmacy.
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doi:10.1124/jpet.105.087148.
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ABBREVIATIONS: hPepT1, human peptide transporter 1; MDCK, Madin-Darby canine kidney; VACV, valacyclovir; 5-ALA, 5-aminolevulinic acid; BENZ, benzylpenicillin; FBS, fetal bovine serum; MES, 2-(4-morpholino)-ethanesulfonic acid; PIPES, 1,4-piperazine-bis(2-ethanosulfonic acid); AP, apical; BL, basolateral; TEER, transepithelial electrical resistance.
- Received March 30, 2005.
- Accepted May 11, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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