title: Delineation of human peptide transporter 1 (hPepT1)-mediated uptake and transport of substrates with varying transporter affinities utilizing stably transfected hPepT1/Madin-Darby canine kidney clones and Caco-2 cells.
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 (P(app)) 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 K(m) values and higher P(app) 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.
authors.affiliation: Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, NJ 08854-8022, USA.
authors.forename: Rajinder K
authors.initials: RK
authors.lastname: Bhardwaj
authors.forename: Dea
authors.initials: D
authors.lastname: Herrera-Ruiz
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Sinko
authors.forename: Olafur S
authors.initials: OS
authors.lastname: Gudmundsson
authors.forename: Gregory
authors.initials: G
authors.lastname: Knipp
mesh_terms: D000212:Acyclovir / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D000622:Aminolevulinic Acid / Q000493:pharmacokinetics; D000818:Animals; D001692:Biological Transport; D018938:Caco-2 Cells; D015820:Cadherins / Q000502:physiology*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010400:Penicillin G / Q000493:pharmacokinetics; D012964:Sodium / Q000502:physiology; D014162:Transfection; D000077483:Valacyclovir; D014633:Valine / Q000031:analogs & derivatives / Q000493:pharmacokinetics
chemical_list: C490584:CDH17 protein, human; D015820:Cadherins; D000622:Aminolevulinic Acid; D012964:Sodium; D014633:Valine; D000077483:Valacyclovir; D010400:Penicillin G; D000212:Acyclovir