Multi-Well Array Culture of Primary Human Hepatocyte Spheroids for Clearance Extrapolation of Slowly Metabolized Compounds

Accurate prediction of human pharmacokinetics using in vitro tools is an important task during drug development. Albeit, currently used in vitro systems for clearance extrapolation such as microsomes and primary human hepatocytes in suspension culture show reproducible turnover, the utility of these systems is limited by a rapid decline of activity of drug metabolizing enzymes. In this study, a multi-well array culture of primary human hepatocyte spheroids was compared to suspension and single spheroid cultures from the same donor. Multi-well spheroids remained viable and functional over the incubation time of 3 days, showing physiological excretion of albumin and alpha-AGP. Their metabolic activity was similar compared to suspension and single spheroid cultures. This physiological activity, the high cell concentration, and the prolonged incubation time resulted in significant turnover of all tested low clearance compounds (n = 8). In stark contrast, only one or none of the compounds showed significant turnover when single spheroid or suspension cultures were used. Using multi-well spheroids and a regression offset approach (log(CLint) = 1.1 x + 0.85), clearance was predicted within 3-fold for 93% (13/14) of the tested compounds. Thus, multiwell spheroids represent a novel and valuable addition to the ADME in vitro tool kit for the determination of low clearance and overall clearance prediction.

Automated summary

In this study, multi-well spheroids of primary human hepatocytes were compared to suspension and single spheroid cultures. The multi-well spheroids showed sustained physiological function and metabolic activity, resulting in significant turnover of low clearance compounds. Compared to single spheroid or suspension cultures, multi-well spheroids provided more accurate predictions of clearance.