Purpose To evaluate and optimize the use of Caco-2 cell monolayers to predict thein vivo absorption of a broad range of compounds in man

In Vitro Permeability Across Caco-2 Cells (Colonic) Can Predict In Vivo (Small Intestinal) Absorption in Man—Fact or Myth

MDCK (Madin-Darby canine kidney) cells : a tool for membrane permeability screening

Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells.

Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers.

Cloning and Functional Expression of a Human Na+and Cl−-dependent Neutral and Cationic Amino Acid Transporter B0+

Various processes involved in the transcellular transport (TT) of loracarbef (LOR) were studied in the Caco-2 cell monolayer, a cell culture model of the small intestinal epithelium. The results provide support for presence of two AP to BL peptide TT pathways in the intestinal epithelial cell monolayer (Caco-2). The H+ gradient-dependent pathway (Km = 0.789 mM, and Jmax = 163 pmol/min per cm2) is relatively "high affinity" and "low capacity" compared to H+ gradient-independent pathway (Km = 8.28 mM, and Jmax = 316 pmol/min per cm2). In addition, TT of LOR in the presence of a H+ gradient was inhibited 77% to 88% (p < 0.05) by 10 mM of cephalexin, enalapril, Gly-Pro and Phe-Pro, while TT of LOR in the absence of a H+ gradient was only inhibited 42% to 48% (p < 0.05) by 10 mM of Gly-Pro and Phe-Pro. Since AP uptake is H+ gradient-dependent and saturable while the BL efflux is mostly nonsaturable and not driven by a H+ gradient, these two transmembrane transport processes must be different, which could be the result of two different peptide carriers. In vivo, these two transport processes must have worked in concert to produce transcellular flux of loracarbef. To explain the differences between kinetic characteristics of AP uptake and TT transport, a cellular pharmacokinetic (PK) model was developed and the results indicate that the PK model appropriately described the kinetics of LOR TT. The use of this PK model may provide an additional advantage to the use of the cell culture model because kinetic parameters at both sides of the intestinal epithelial membrane may be obtained using the same preparation. Taken together, the Caco-2 model system represents an excellent model system for the study of carrier-mediated processes involved in the TT of peptides and peptide-like drugs.

Mechanism and Kinetics of Transcellular Transport of a New β-Lactam Antibiotic Loracarbef Across an Intestinal Epithelial Membrane Model System (Caco-2)

The metabolism of Phe-Pro was investigated in Caco-2 cell monolayers, a model of small intestinal epithelium. The results indicate that the majority of Phe-Pro was hydrolyzed during passage from the apical (AP) to basolateral (BL) side. The enzyme responsible for the hydrolysis is prolidase, a cytosolic enzyme. Through kinetic studies of a supernatant enzyme preparation, a Km of 30.4 μ M and Vmax of 38.9 nmol/min per mg of protein were obtained. The enzyme catalyzed hydrolysis was inhibited by proline (66%), Zn++ (86%), Cu ++ (100%), Fe+++ (100%), PCMB (89%), and captopril (66%), but not by leucine. We also studied the transcellular transport of Phe-Pro by measuring the amount of Phe in the receiver media. In the presence of a proton gradient (AP pH6, BL pH7.4), the appearance rate of Phe in the BL media after Phe-Pro was loaded apically was at least 100 times faster than that in the AP media after Phe-Pro was loaded basolaterally. The former is also higher than the appearance rate of Phe without ...

The Caco-2 Cell Monolayers as an Intestinal Metabolism Model: Metabolism of Dipeptide Phe-Pro

Mechanism and kinetics of uptake and efflux of L-methionine in a human intestinal epithelial model system (Caco-2) were studied to understand the transcellular transport process and to determine its rate-limiting step. The kinetic studies indicated that uptakes from both the apical and basolateral sides were saturable [for apical uptake: Km = 0.96 mmol/L, maximum flux = 673 pmol/(min.cm2); for basolateral uptake: Km = 3.46 mmol/L, maximum flux = 3480 pmol/(min.cm2)], whereas the efflux from these two membranes was apparently linear for intracellular concentrations < 6.5 mmol/L [for apical efflux, the apparent first-order rate constant = 1.01 x 10(-4) cm/min; for basolateral efflux, the rate constant = 1.78 x 10(-4) cm/min]. The results of inhibition studies indicate the apical uptake is partially active and Na(+)-dependent via a combination of amino acid transport systems B0,+ and ASC, which is somewhat different from the less energy- and Na(+)-dependent basolateral uptake. The basolateral uptake is also more dependent on system L and exhibits high counter-exchange capability. Finally, the rate-limiting step in the apical to basolateral transport of methionine is determined to be the basolateral efflux.

Mechanisms and kinetics of uptake and efflux of L-methionine in an intestinal epithelial model (Caco-2)

Purpose. To determine the transport mechanisms of quinapril and cephalexin in Caco-2 cell monolayers, a cell culture model of the human small intestinal epithelium. 
Methods. Uptake, transepithelial transport and intracellular accumulations of these two drugs were measured using Caco-2 cell monolayers grown onto Millicells™ and magnetically stirred diffusion chambers. 
Results. Transepithelial transport, apical (AP) uptake and intracellular accumulation of both drugs depended on the maintenance of a transepithelial proton gradient and temperature of the medium. However, quinapril transport and accumulation, which did not display a maximum at approximately pH 6, was more sensitive to proton gradient change, whereas cephalexin transport was more sensitive to concentration change (range 0.5-5 mM). In addition, quinapril (1 mM) transport was decreased significantly (p<0.05) by 10 mM cephalexin, loracarbef, Gly-Pro and Phe-Pro, but not by enalapril; whereas cephalexin (0.1 mM) transport was decreased significantly (p<0.05) by all four compounds. Similarly, AP quinapril (1 mM) uptake was also decreased by 10 mM loracarbef, Gly-Pro, cephalexin, and enalapril, but these inhibitory effects (20-50%) were quantitatively less than their inhibitory effects on cephalexin uptake (50-90%). Finally, the AP uptake of quinapril was also significantly (p<0.05) inhibited by FCCP (10 µg/ml), amiloride (0.5 mM), DEP (0.5 mM), and staurosporine (5 nM). 
Conclusions. The transport of quinapril in the Caco-2 cells is via a combination of the carrier-mediated proton gradient-dependent peptide transporter and passive diffusion.

Jiyue Chen

Papers

Mechanism and Kinetics of Transcellular Transport of a New β-Lactam Antibiotic Loracarbef Across an Intestinal Epithelial Membrane Model System (Caco-2)

The Caco-2 Cell Monolayers as an Intestinal Metabolism Model: Metabolism of Dipeptide Phe-Pro

Mechanisms and kinetics of uptake and efflux of L-methionine in an intestinal epithelial model (Caco-2)

Mechanisms of transport of quinapril in Caco-2 cell monolayers: comparison with cephalexin.

Determination of Absorption Characteristics of AG337, a Novel Thymidylate Synthase Inhibitor, Using a Perfused Rat Intestinal Model