Overexpression of human intestinal oligopeptide transporter(hPepT1) via adenoviral transduction: Implication for oral absorption of peptide and peptide-like drugs.
Hsu, Cheng-Pang
1999
Abstract
<italic>Purpose</italic>. Our goal is to evaluate a new approach in improving oral absorption of peptides and peptide-like drugs by overexpression of the human intestinal oligopeptide transporter (hPepT1). The major objectives are to establish an <italic>in vitro</italic> system for screening potential substrates of hPepT1 and to investigate a cell culture system for the evaluation of <italic> in vivo</italic> adenovirus-mediated gene expression in the intestinal epithelium. <italic>Methods</italic>. A recombinant replication-deficient adenovirus, Ad.RSVhPepT1, was constructed and used as a vector for the expression of hPepT1. The increase in the uptake of cephalexin and Gly-Sar in adenovirus-infected cells was determined. The transport of Gly-Sar across both apical and basolateral. membranes of Caco-2 cells was measured after adenoviral transduction as a function of pH, temperature, inhibitors, and substrate concentration. The localization of hPepT1 was examined by immunocytochemistry using confocal laser scanning microscopy. The levels of hPepT1 in Caco 2 cells and in human intestinal biopsy samples were quantitated by computer-aided densitometry following SDS-PAGE and immunoblotting. <italic>Results</italic>. A significant increase in the uptake permeability of Gly-Sar and cephalexin was observed in Caco-2, IEC-18, and HeLa cells after adenoviral transduction. The increase of the hPepT1 level was confirmed by immunoblotting using an antibody against hPepT1. The apical-to-basolateral and basolateral-to-apical transport of Gly-Sar in Caco-2 cells after viral transduction were both increased significantly. This was further confirmed by immunocytochemistry showing that hPepT1 was localized in the apical and basolateral membrane of Caco-2 cells after adenoviral transduction. Substrate permeability was dependent on time of infection, inward pH gradients, temperature, multiplicity of infection (MOI), and substrate concentrations. A good correlation was observed between Gly-Sar uptake and the content of hPepT1 in Caco-2 cells infected at different MOIs. <italic>Conclusions</italic>. Adenoviral infected Hela cells represent a useful tool for screening of potential substrates. Caco-2 cells are an appropriate model to study gene expression in intestinal epithelial cells. hPepT1 is expressed in the basolateral membrane and displays a similar level of transport enhancement after adenovirus mediated hPepT1 gene expression. The success of overexpression of hPepT1 in Caco-2 and IEC-18 cells suggests a new approach to improving oral absorption of peptides and peptidomimetic drugs.Subjects
Adenoviral Hpept1 Human Implication Intestinal Oligopeptide Transporter Oral Absorption Overexpression Peptide-like Drugs Transduction Via
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