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KMID : 0043320070300040507
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Archives of Pharmacal Research
2007 Volume.30 No. 4 p.507 ~ p.518
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Peptide Transporter Substrate Identification during Permeability Screening in Drug Discovery: Comparison of Transfected MDCK-hPepT1 Cells to Caco-2 Cells
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Balimane Praveen. V.
Chong Saeho
Patel Karishma
Quan Yong
Timoszyk Julita
Han Yong-Hae
Wang Bonnie
Vig Balvinder
Faria Teresa N.
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Abstract
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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 qRTPCR. Permeability studies were conducted in parallel in all the cells with a diverse set of peptide substrates using the optimized experimental condition: 100 ?, apical pH 6.0, basolateral pH 7.4, 2 hr incubation at 37캜. 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 MDCKhPepT1 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 Pgp 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).
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KEYWORD
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hPepT1, MDCK cells, Caco-2 cells, High-throughput, P-gp, Drug discovery, Permeability, Transporters
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