Microphysiological organ-on-a-chip system for anti-doping research: long-term CYP activity characterization and S107 metabolism in a HepaRG™-based 3D liver model.

Identification of metabolic markers of prohibited and emerging performance-enhancing substances remains a key challenge in anti-doping analysis, where limitations of conventional in vitro models in resembling authentic physiological conditions exist. Organ-on-a-chip (OoC) systems combining 3D human liver-cell cultures with microfluidic perfusion offer a predictive alternative for human-like drug metabolism. In this study, a HepaRG™-based liver OoC model incorporating hepatic stellate cells was characterized over a three-week period with a focus on cytochrome P450 (CYP)-mediated metabolism. CYP activity, gene expression, and inducibility were assessed for major hepatic CYP isoforms, complemented by pharmacogenomic profiling. Metabolic performance was further evaluated using S107, a non-approved and prohibited doping agent, with metabolite formation being determined by liquid chromatography and high-resolution mass spectrometry (LC-HRMS/MS) in comparison with published in vitro data. The OoC system maintained stable viability and hepatic functionality throughout the culture period, demonstrating phase-I metabolic competence. Isoform-specific differences in mRNA expression and enzymatic activity highlighted complex regulatory effects within the microphysiological environment, while rifampicin responses confirmed preserved nuclear receptor-mediated inducibility across the system. Functional limitations were observed for CYP2D6 and CYP2B6, which showed absent or restricted activity despite detectable transcriptional signals. For S107, the model generated both phase-I and phase-II metabolites, consistent with literature data from microsomal incubation experiments. Metabolite formation was stable over the three-week period under dynamic perfusion conditions, demonstrating sustained biotransformation capacity. Overall, the HepaRG™ OoC system represents a physiologically relevant in vitro platform for long-term metabolism and demonstrates potential as a predictive tool for anti-doping applications and for elucidating metabolic pathways of emerging performance-enhancing substances.