3D hepatic spheroid arrays for clearance assessment of small and bRo5 molecules

Accurately predicting hepatic clearance remains a critical hurdle in preclinical pharmacokinetics, especially for metabolically stable compounds and emerging modalities such as proteolysis targeting chimeras (PROTACs) that fall in beyond-rule-of-five chemical space. In this study, we developed and evaluated a 3-dimensional hepatic spheroid-array system that improves clearance prediction for both small molecules and PROTACs. Using a panel of 15 small molecules and 9 PROTACs, we compared in vitro clearance predictions from the spheroid-array model with conventional suspension hepatocytes. The spheroid-array predicted human hepatic clearance within a 2-fold range for 9 of 15 and within a 3-fold range for 12 of 15 small molecules. The average absolute fold error was lower for the spheroid system than the suspension system (1.74 vs 3.52). Notably, unlike the suspension system, the spheroid-array quantified clearance for several low-clearance compounds such as warfarin and theophylline. Regression correction improved prediction accuracy for both, with a significant improvement for the suspension model. For several PROTACs, metabolic turnover could not be measured in the suspension system, highlighting its limited suitability for highly stable compounds. In contrast, the spheroid system demonstrated significant metabolic activity, enabling characterization of hepatic clearance for these otherwise stable PROTACs. Together, these findings establish the spheroid-array system as a sensitive and physiologically relevant tool for preclinical clearance assessment across traditional and emerging therapeutic modalities. SIGNIFICANCE STATEMENT: We developed a 3-dimensional hepatic spheroid-array that shows excellent viability and functional activity for 3 weeks. The system showed better hepatic clearance predictivity with a lower average absolute fold error than the suspension system. Our work also first time validates the spheroid-array system for beyond-rule-of-five molecules showing the possibility for evaluating clearance of proteolysis targeting chimeras. This work establishes spheroid as a reliable tool for preclinical hepatic clearance assessment.