Evaluation of small interfering RNA–dependent knockdowns of drug-metabolizing enzymes in multiwell array culture of primary human hepatocyte spheroids for estimation of fraction metabolized

The determination of the relative contribution of different drug-metabolizing enzymes to the metabolism of slowly metabolized compounds is a challenging task. The quantification of low compound turnover in standard in vitro systems, such as liver microsomes or hepatocyte suspension cultures, can be difficult. Thus, the use of long-term liver models, such as HepatoPac (BioIVT) or liver spheroids, has been suggested. Inhibitors of cytochrome P450 (P450) enzymes, the most important group of drug-metabolizing enzymes, represent the current standard to evaluate the route of drug metabolism. However, a long-term inhibition in systems such as spheroid models may be technically challenging due to limited stability of some of the commonly used inhibitors. Small interfering RNA (siRNA)-dependent knockdown of P450 enzymes in spheroid cultures of primary human hepatocytes represents a novel alternative to the established methods. In the current study, we report the successful attenuation of the CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoforms via siRNA on gene expression, as well as on the functional level, for at least 7 days. The analysis revealed that the knockdowns had only minor effects on the overall transcriptome. They also showed acceptable selectivity towards drug-metabolizing enzymes, except for the knockdown of CYP2C19. Applicability of the system for the determination of the fraction metabolized of low clearance substances was examined using 6 compounds metabolized by different P450s. By introducing siRNA-dependent knockdowns in phenotypically relevant primary human hepatocyte spheroid cultures, we hope to provide a novel alternative to standard systems to elucidate clearance pathways in vitro. SIGNIFICANCE STATEMENT: Small interfering RNA-mediated gene knockdowns of different cytochrome P450 enzymes were shown to be effective in long-term primary human hepatocyte spheroid cultures, representing a novel alternative for reaction phenotyping. This method has the potential to improve the assessment of pharmacokinetic variability and victim drug-drug interaction risks due to enzyme polymorphism or inhibition/induction with more confidence, particularly for low clearance drug candidates. Furthermore, minor effects of the small interfering RNA-mediated gene knockdowns for different cytochrome P450 enzymes on cell viability and the transcriptome were observed which implies that this system may be useful in deconvoluting toxicity caused by metabolites.