Evaluation of transporter-mediated hepatic uptake in a non-radioactive high-throughput assay: a study of kinetics, species difference and plasma protein effect.

1. In this manuscript we describe a non-radioactive, high-throughput method to evaluate hepatic uptake using cryopreserved hepatocytes. We have validated the uptake of pravastatin with different amounts of hepatocytes and the impact of the oil layer used in separation. The time- and concentration-dependent uptake profiles of several anionic and cationic charged drugs were evaluated. The results with our method compare favourably with the literature for pravastatin, atorvastatin and estrone 3-sulfate. 2. Two approaches for kinetic determination (temperature difference and fitting the linear and non-saturable passive diffusion rate in the equation, i.e. V = (V(max) × S)/(K(m) + S) + P(dif) × S) have been evaluated. Kinetic studies indicate that the different approaches for determining passive diffusion can affect K(m) and V(max), but not the clearance of active uptake (V(max)/K(m)). 3. Using pravastatin as a probe substrate, species differences were observed in the organic anion-transporting polypeptide (OATP) 1B1 and 1B3 activities. Plasma protein significantly reduced the uptake of atorvastatin, but not pravastatin. 4. Our data suggests that evaluation of the role of active uptake in hepatic clearance in humans should consider the relative ratio of active uptake to passive diffusion, species differences and plasma protein binding when applying in vitro uptake data.