Resolving the Distribution–Metabolism Interplay of Eight OATP Substrates in the Standard Clearance Assay with Suspended Human Cryopreserved Hepatocytes

Uptake transporters may act to elevate the intrahepatic exposure of drugs, impacting the route and rate of elimination, as well as the drug–drug interaction potential. We have here extended the assessment of metabolic drug stability in a standard human hepatocyte incubation to allow for elucidation of the distribution–metabolism interplay established for substrates of drug transporters. Cellular concentration–time profiles were obtained from incubations of eight known OATP substrates at 1 μM, each for two different 10-donor batches of suspended cryopreserved human hepatocytes. The kinetic data sets were analyzed using a mechanistic mathematical model that allowed for separate estimation of active uptake, bidirectional diffusion, metabolism and nonspecific extracellular and intracellular binding. The range of intrinsic clearances attributed to active uptake, diffusion and metabolism of the test set spanned more than 2 orders of magnitude each, with median values of 18, 5.3, and 0.5 μL/min/10⁶ cells, respectively. This is to be compared with the values for the apparent clearance from the incubations, which only spanned 1 order of magnitude with a median of 2.6 μL/min/10⁶ cells. The parameter estimates of the two pooled 10-donor hepatocyte batches investigated displayed only small differences in contrast to the variability associated with use of cells from individual donors reported in the literature. The active contribution to the total cellular uptake ranged from 55% (glyburide) to 96% (rosuvastatin), with an unbound intra-to-extracellular concentration ratio at steady state of 2.1 and 17, respectively. Principal component analysis showed that the parameter estimates of the investigated compounds were largely influenced by lipophilicity. Active cellular uptake in hepatocytes was furthermore correlated to pure OATP1B1-mediated uptake as measured in a transfected cell system. The presented approach enables the assessment of the key pathways regulating hepatic disposition of transporter and enzyme substrates from one single, reproducible and generally accessible human <i>in vitro</i> system