Abstract

A key requirement in drug discovery is to accurately define intrinsic clearance (CL_int) values of less than 1 µl/min/10⁶ hepatocytes, which requires assays that allow for longer incubation time as a complement to suspended hepatocytes. This study assessed the effectiveness of plated HepaRG cells, plated primary human hepatocytes (PHHs), and the HµREL human hepatocyte/stromal cell co-cultures for determination of low CL_int values. The investigation demonstrated that the systems were capable of providing statistically significant CL_int estimations down to 0.2 µl/min/10⁶ cells. The HµREL assay provided a higher level of reproducibility, with repeat significant CL_int values being defined in a minimum of triplicate consecutive assays for six of seven of the low CL_int compounds compared with four of seven for PHHs and two of seven for HepaRG. The assays were also compared with a suspension assay using drugs with higher CL_int values and diverse enzymology. The CL_int values from the PHH and HµREL assays were similar to those defined by a hepatocyte suspension assay, indicating that they can be used interchangeably alongside a standard assay. Finally, data from these two assays could also predict in vivo hepatic metabolic CL_int to within 3-fold for greater than 70% of the compounds tested, with average fold errors (AFE) of 1.6 and 2.3, respectively, whereas the HepaRG data were predictive to within 3-fold for only 50% of compounds (AFE 2.9). In summary, all systems have utility for low CL_int determination, but the HµREL co-culture appears slightly superior regarding overall assay performance.