New Methods to Evaluate Intestinal Drug Absorption Mediated by Oligopeptide Transporter from In vitro Study using Caco-2 Cells

Abstract

The aim of the present work is to develop a convenient and rapid screening system in vitro for intestinal drug absorption mediated by oligopeptide transporter (PepT1). In this study, (1) Transports of cephalexin (CEX) and L-phenylalanine (L-Phe) across Caco-2 monolayers were measured and compared with those of passively transported drugs, (2) Inhibitory effects of various drugs on the transport of [(14)C]glycylsarcosine (Gly-Sar) across Caco-2 monolayers were measured and correlated with their in vivo permeability to rat small intestine, (3) Intracellular pH-change induced by co-transport of drugs with proton into Caco-2 cells was monitored by using Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices Corp.). Concentration-dependent transport was observed in Caco-2 monolayers for CEX and L-Phe, although their permeability was relatively low compared to those of passively transported drugs. Inhibitory effects of various drugs including beta-lactam antibiotics and angiotensin converting enzyme-inhibitors on the transport of Gly-Sar correlated well with their in vivo permeability to rat small intestine. It was demonstrated that CEX, but not cefazolin, induced gradual decrease in the intracellular pH of Caco-2 cells. The degree of intracellular pH-change caused by various drugs showed a sigmoidal or saturable relationship with their permeability to rat small intestine. These in vitro approaches with Caco-2 cells should be useful to evaluate in vivo intestinal permeability of drugs mediated by PepT1, suggesting a possibility of high throughput screening of drug absorption.

Absorption of lisdexamfetamine dimesylate and its enzymatic conversion to d-amphetamine

“…To further investigate the involvement of the PEPT1 transporter in absorption of LDX, permeability experiments in Caco-2 cells were conducted, as PEPT1 has been shown to be expressed in this cell line 14–16. LDX flux across Caco-2 cells was similar in magnitude to the measured flux of cephalexin, a known substrate for the PEPT1 transporter15,17,18 and was partially inhibited by Gly-Sar, an established competitive substrate for PEPT1.…”

Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

“…Caco-2 cells, a human colorectal adenocarcinoma cell line, have a epithelial-like morphology with a microvilli structure similar to that of the human small intestine, and form a tight junction by culturing on a cell culture insert 5 , 6 . Caco-2 cells express various drug transporters expressed in the human small intestine, thus they are widely used as an in vitro assay system for predicting gastrointestinal absorption ( Fa ) of orally administered drugs 7 , 8 . However, Caco-2 cells rarely express the drug-metabolizing enzyme, cytochrome P450 (CYP) 9 .…”