Investigation of MDR1-overexpressing cell lines to derive a quantitative prediction approach for brain disposition using in vitro efflux activities.

MDR1-overexpressing Lilly Laboratories cell porcine kidney 1 cells (LLC-PK1-MDR1) and Madin-Darby canine kidney cells (MDCK-MDR1) are widely used in drug discovery to evaluate the in vivo relevance of MDR1-mediated efflux. However, as the in vitro efflux ratio (ER) of these cell lines are variable among research facilities, the in vitro ER of these cell lines that would affect quantitative predictivity of brain disposition has not been fully clarified. The aim of this study was to examine the effect of ER on the quantitative predictivity of brain disposition toward compounds with MDR1 and/or breast cancer resistant protein (BCRP) liabilities. Test compounds including internal molecules and five typical substrates of MDR1 and/or BCRP were assessed via an in vitro transporter assay to determine the corrected flux ratio (CFR) and an in vivo animal study using wild-type (WT) and Mdr1a (-/-)/Bcrp(-/-) (dual KO) rats. To assess the in vivo ER for MDR1, the two cell lines LLC-PK1-MDR1 and MDCK-MDR1 were used. After intravenously administering 29 test compounds to rats, the K ratio (ratio of K to K), which is considered to be the unbound plasma-to-brain ratio (K) that does not require correction for protein binding in both plasma and brain, was determined by measuring their concentrations in the plasma and brain. The K ratio of these compounds was predicted by fitting scaling factor that was extrapolated from the in vitro to in vivo ER for MDR1 and BCRP, defined as α and β, respectively. K ratio values of 83% and 68% of compounds were predicted by using MDCK-MDR1 and LLC-PK1-MDR1, respectively, within a 2-fold range of the actual corresponding values. The α predicted from CFRs of MDCK-MDR1 was 47-fold smaller than that of LLC-PK1-MDR1; however, a dramatic change in β was not observed. This result appears to be consistent with the data of in vitro transport activity of MDR1, which was estimated to be ~28-fold higher in MDCK-MDR1 than in LLC-PK1-MDR1 by correlation analysis with CFR. Through this study, we revealed that 1) brain disposition in rats was well-predicted by considering the in vitro efflux activities for both MDR1 and BCRP, and 2) MDCK-MDR1 was the superior cell line for the quantitative prediction of brain disposition.