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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on May 18, 2005; DOI: 10.1124/jpet.105.087148

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Received for publication March 31, 2005.
Revised May 11, 2005.
Accepted for publication May 11, 2005.

Delineation of HPEPT1 Meditated Uptake and Transport of Substrates with Varying Transporter Affinities Utilizing Stably Transfected HPEPT1/MDCK Clones and Caco-2 Cells

Rajinder K Bhardwaj 1, Dea Herrera-Ruiz 2, Patrick J Sinko 1, Olafur S. Gudmundsson 3, Gregory Knipp 4*

1 Rutgers, the State University of New Jersey 2 Universidad Autonoma del Estado de Morelos 3 Bristol-Myers Squibb 4 Rutgers University

* Address correspondence to: E-mail: gknipp{at}cop.rutgers.edu

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 MDCK/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 dependency 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 vs. 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 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, as compared to mock-corrected hPepT1-MDCK cells suggested the multi-transporters involvement in Caco-2 cells. Thus hPepT1-MDCK cells corrected for endogenous transporter expression may be more appropriate model for screening compounds for their affinity to hPepT1.


Key words: Caco-2 cells, MDCK cells, PepT1, Transport, Uptake, stable transfection


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Drug Metab. Dispos., January 1, 2008; 36(1): 102 - 123.
[Abstract] [Full Text] [PDF]


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