Human Hepatic Stability Screening: a Simplified Higher Throughput Assay and Evaluation of Different Hepatocyte Preparations

In the early stages of drug discovery, hepatic stability screening is an important and widely used method to assess the metabolic stability as well as predict the in vivo hepatic clearance of new chemical entities. Here we present a simplified higher throughput method for rapid stability screening using hepatocytes in a 96-well format for LC-MS determinations. A set of 24 known drugs was chosen that act as substrates for the major human hepatic P450 and UGT isoforms. The predicted human hepatic clearance for these known drugs was determined using cryopreserved, single donor hepatocyte preparations (In Vitro Technologies), and data were compared to literature values for in vivo systemic clearance. We were able to rank accurately compounds based on high, medium and low stability by determining the loss of parent compound at a single time point of 2 hours in comparison to multiple time point assessments. This has resulted in profound savings of compound, time and hepatocytes. Comparative studies with both single and pooled donor hepatocytes, as well as with fresh human hepatocytes, are underway currently. In recent years, freshly isolated hepatocytes, as well as cryopreserved hepatocytes, have been used for the prediction of metabolic clearance.1-6 To date, most of th