Further Assessment of the Relay Hepatocyte Assay for Determination of Intrinsic Clearance of Slowly Metabolised Compounds Using Radioactivity Monitoring and LC–MS Methods

Abstract

Background and Objectives: Low-clearance drugs are widely used by industry mostly because of their often longer half-life, allowing for lower or less frequent dosing. Nevertheless, prediction of human clearance for these molecules from in vitro models presents a great challenge for pharmaceutical scientists. The objective of this study was to further characterise the predictive accuracy of the relay hepatocyte assay using 14C and 3H labelled proprietary compounds with a low extraction ratio and the known clearance mechanism in rats. Highly permeable compounds cleared by metabolism as well as rate limitation by transport were included in this study. Methods: Blood clearance was determined from concentration–time profiles following intravenous dosing to rats. In vitro clearance was determined from the single concentration parent depletion-time profiles throughout the incubation period of up to 20 h (five relays) using radioactivity monitoring in tandem with mass spectrometry. A new approach was proposed to correct concentrations for loss and dilution during the relay steps. Clearance was predicted with a standard well-stirred model for the liver and predicted values were then compared with observed data to evaluate method accuracy. Results: The results showed that intrinsic clearance values predicted using the relay hepatocyte assay from either radioactivity or mass spectrometry concentration data were comparable. A significant difference in prediction accuracy between the permeable compounds cleared by hepatic metabolism (about 2-fold) and the compound that was the hepatic uptake substrate (5- to 6-fold of actual) was demonstrated. Conclusions: The relay method is effective in predicting in vivo clearance for the compounds that are cleared via hepatic metabolism but tends to be notably underpredictive for drugs that rely on uptake transport. Consistent with the overall trend toward underprediction of hepatic clearance from the in vitro models prevalently used in the pharmaceutical industry, all values predicted from the hepatocyte relay method were lower than observed.