title: Peptide transporter substrate identification during permeability screening in drug discovery: comparison of transfected MDCK-hPepT1 cells to Caco-2 cells.
abstract: The purpose of this study was to investigate the utility of stably transfected MDCK-hPepT1 cells for identifying peptide transporter substrates in early drug discovery and compare the characteristics of this cell line with Caco-2 cells. MDCK-hPepT1, MDCK-mock, and Caco-2 cells grown to confluence on 24-well Transwell were used for this study. Expression levels of different transporter proteins (PepT1, PepT2, P-gp) in these cell lines were assessed by qRT-PCR. Permeability studies were conducted in parallel in all the cells with a diverse set of peptide substrates using the optimized experimental condition: 100 microM, apical pH 6.0, basolateral pH 7.4, 2 hr incubation at 37 degrees C. Permeability studies were also conducted with classical P-gp substrates (tested in bi-directional mode) and paracellularly absorbed probes to investigate the differences between the cell lines. As expected, MDCK-hPepT1 cells express significantly higher level of PepT1 mRNA compared to both Caco-2 and MDCK-mock cells. Efflux transporter, P-gp, was expressed adequately in all the cell lines. Permeability studies demonstrated that classical peptide substrates had significantly higher permeability in stably transfected MDCK-hPepT1 cells compared to MDCK-mock and Caco-2 cells. The transfected MDCK-hPepT1 cells were qualitatively similar to Caco-2 cells with respect to functional P-gp efflux activity and paracellular pore activity. Stably transfected MDCK-hPepT1 cells have been demonstrated as a viable alternative to Caco-2 cells for estimating the human absorption potential of peptide transporter substrates. These cells behave similar to Caco-2 cells with regards to P-gp efflux and paracellular pore activity but demonstrate greater predictability of absorption values for classical peptide substrates (for which Caco-2 cells under-estimate oral absorption).
authors.affiliation: Metabolism and Pharmacokinetics, Bristol-Myers Squibb, Princeton, NJ 08543, USA. Praveen.balimane@bms.com
authors.forename: Praveen V
authors.initials: PV
authors.lastname: Balimane
authors.forename: Saeho
authors.initials: S
authors.lastname: Chong
authors.forename: Karishma
authors.initials: K
authors.lastname: Patel
authors.forename: Yong
authors.initials: Y
authors.lastname: Quan
authors.forename: Julita
authors.initials: J
authors.lastname: Timoszyk
authors.forename: Yong-Hae
authors.initials: YH
authors.lastname: Han
authors.forename: Bonnie
authors.initials: B
authors.lastname: Wang
authors.forename: Balvinder
authors.initials: B
authors.lastname: Vig
authors.forename: Teresa N
authors.initials: TN
authors.lastname: Faria
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D018938:Caco-2 Cells; D004285:Dogs; D006801:Humans; D007408:Intestinal Absorption; D000074011:Peptide Transporter 1; D010539:Permeability; D027981:Symporters / Q000378:metabolism*; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters