Research Articles
Kinetic Analyses for Species Differences in P-glycoprotein-Mediated Drug Transport

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Abstract

P-glycoprotein (P-gp) plays an important role in the pharmacokinetics of drugs. There is little information on the species differences in P-gp-mediated drug transport activity. The purpose of the present study was to clarify the differences in the kinetic parameters and the existence of species differences in the P-gp-mediated drug transport activity using seven multidrug resistence1 (MDR1) transfected cell lines, in which the cDNA was from human, monkey, canine, rat (MDR1a and MDR1b), and mouse (mdr1a and mdr1b). The transcellular transport of diltiazem, cyclosporin A, and dexamethasone across monolayers of MDR1 transfected cells. The apparent Km values of diltiazem exhibited approximately 16.5-fold differences among the seven cell lines. Concerning the diltiazem transport, the Vmax/Km value of human P-gp corrected by the P-gp expression level was similar to that of monkey P-gp, but was 5.6-fold higher than that of canine P-gp. On the other hand, the corrected Vmax/Km value of human P-gp for cyclosporin A transport was 3.8-fold higher than that of monkey P-gp. The present study would be valuable to evaluate the P-gp function of various animals in the same experimental condition. It was clarified that the species differences in P-gp-mediated drug transport activity evaluated by the corrected Vmax/Km value differed according to the substrate.

Section snippets

INTRODUCTION

P-glycoprotein (P-gp) is a product of multidrug resistant 1 (MDR1) gene and is one of the most important transporters affecting the pharmacokinetics of drugs in normal cells as well as in cancer cells. A rational approach is needed to determine whether a compound is a P-gp substrate or not, especially in drug development. The Caco-2 cells and human MDR1 transfected cell lines such as LLC-GA5-COL150 and L-MDR1 established by Ueda et al.1 and Schinkel et al.,2 respectively, are frequently used

Materials

[mebmt-β-3H]Cyclosporin A (333.0 GBq/mmol) and [1,2,4-3H]dexamethasone (1480.0 GBq/mmol) were obtained from Amersham Biosciences (Buckinghamshire, UK). cis-(+)-[N-methyl-3H]Diltiazem (3145.0 GBq/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Colchicine, cyclosporin A, dexamethasone, and diltiazem hydrochloride were obtained from Wako Pure Chemical Industries (Osaka, Japan). The mouse monoclonal antibodies (C219) and rabbit polyclonal antibodies (H241) against MDR1-encoded P-gp

Immunoblot Analysis of MDR1 Transfected Cells

The results of the immunoblot analysis using anti-P-gp antibodies, C219 and H241, are shown in Figure 1. In the case of the C219 antibodies, the densities of the bands from pMDR1, cMDR1, rMDR1b, and mmdr1a were higher than that from hMDR1 (Fig. 1A). The relative densities of hMDR1, pMDR1, cMDR1, rMDR1a, rMDR1b, mmdr1a, and mmdr1b were 1.0, 1.6, 2.6, 0.9, 1.6, 1.6, and 1.1, respectively. On the other hand, in the case of the H241 antibodies, the densities of the bands from pMDR1 and cMDR1 were

DISCUSSION

Absorption, distribution, metabolism, and excretion are important factors in the pharmacokinetics of a drug and are influenced by the effects of ATP-binding cassette proteins such as P-gp. In vitro studies using MDR1 transfected cells and in vivo studies using mdr1 knockout mice are frequently performed for drug discovery and the assessment of drug interactions.13,14 To estimate and extrapolate the pharmacokinetics and bioavailability of a drug in human, information about species differences is

ACKNOWLEDGMENTS

We acknowledge Brent Bell for reviewing the manuscript.

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