A high throughput in vitro mrp2 assay to predict in vivo biliary excretion

1. Prediction of biliary excretion is a challenge for drug discovery scientists due to the lack of in vitro assays. This study explores the possibility of establishing a simple assay to predict in vivo biliary excretion via the mrp2 transport system.

2. In vitro mrp2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) in canalicular plasma membrane vesicles (cLPM) from rat livers. The CDCF uptake was time- and concentration-dependent (K_m of 2.2?±?0.3 µM and V_max of 115?±?26 pmol/mg/min) and strongly inhibited by the mrp2 inhibitors, benzbromarone, MK-571, and cyclosporine A, with IC₅₀ values ≤ 1.1 µM.

3. Low inhibition of CDCF uptake by taurocholate (BSEP inhibitor; 57 µM) and digoxin (P-gp inhibitor; 101 µM) demonstrated assay specificity towards mrp2.

4. A highly significant correlation (r²?=?0.959) between the in vitro IC₅₀ values from the described mrp2 assay and in vivo biliary excretion in rats was observed using 10 literature compounds.

5. This study demonstrated, for the first time, that a high throughput assay could be established with the capability of predicting biliary excretion in the rat using CDCF as a substrate.