P309 A novel in vitro mrp2 assay to predict in vivo biliary excretion

Federico Colombo , Biological Sciences, Boehringer Ingelheim Canada Ltd, Laval, QC, Canada
Catherine Armstrong , Biological Sciences, Boehringer Ingelheim Canada Ltd, Laval, QC, Canada
Jianmin Duan , Biological Sciences, Boehringer Ingelheim Canada Ltd, Laval, QC, Canada
Nathalie Rioux , Biological Sciences, Boehringer Ingelheim Canada Ltd, Laval, QC, Canada
Purpose: Prediction of biliary excretion in vivo has been one of the challenges for drug discovery scientists due to the lack of an established predictive in vitro assay. The present study explores the possibility of establishing a simple high throughput screen assay to predict in vivo biliary excretion.  Method: Canalicular plasma membrane vesicle (cLPM) were prepared from Sprague-Dawley rat livers and the kinetics of ATP dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) were determined in a 96-well format.  Uptake of CDCF (2 μM) in cLPM (100 μg/mL) was carried out at 37°C for 10 minutes in a buffer containing 250 mM sucrose, 10 mM Tris-Hepes, 10 mM creatine phosphate, 100 µg/mL creatine kinase and 10 mM MgCl2 with or without the inclusion of 3 mM ATP (Controls).  Vesicle-associated CDCF was separated from the matrix by rapid filtration.  The samples were then lysed, filtered and submitted to fluorescent reading.  Assay specificity was verified by competition studies with established mrp2, P-gp, and BSEP modulators (substrates and inhibitors).  The relationship of in vitro IC50 values against ATP-dependent mrp2 uptake and in vivo biliary excretion (expressed as % of the total dose) of nine literature compounds were examined.  Results: The uptake of CDCF in cLPM vesicles was time- and concentration-dependent, with a Km value of 2.2 ± 0.3 μM and a Vmax of 115 ± 26 pmol/mg/min (n=3).  CDCF transport was strongly inhibited by the well-documented mrp2 inhibitors, benzbromarone, MK-571 and cyclosporine A, with IC50 values ≤ 1.2 μM.  In contrast, taurocholate (inhibitor of BSEP), and digoxin (inhibitor of P-gp) had very low potency against the mrp2 mediated uptake (IC50 values of 57 and 101 μM, respectively).  A highly significant linear correlation (R=0.939, P<0.001) between the in vitro IC50 values from the described mrp2 assay and in vivo biliary excretion in rats was observed for the 9 tested mrp2 inhibitors/substrates.  Potent inhibitors (IC50 values <5 μM) of CDCF uptake correlated with >90% of the dose excreted into the rat bile in vivo, whereas weak inhibitors (IC50 values >100 μM) had <5% of the dose excreted into the bile.  Conclusion: The present study describes a novel high throughput assay that selectively identifies potential inhibitors of mrp2 using an in vitro system prepared from normal rat livers.  In addition, the highly significant correlation between in vitro mrp2 IC50 values from this described assay and in vivo biliary excretion suggest its potential use in drug discovery to minimize mrp2-mediated excretion and mrp2-associated drug-drug interactions.