Poster: Further characterization of HepatoPac® – non-targeted metabolite-profiling of 7 chemicals including slow clearance chemicals and metabolism-mediated toxicity

Long-term in vitro liver models have been widely explored for human hepatic metabolic clearance prediction and comparison of slow clearance chemicals as NGRA (Next generation risk assessment) in vitro metabolism study model. Recently, several peer-reviewed journals have reported the superiority of the HepatoPac® system compared to other models- HepG2, HepaRG, iPS, PHH in suspension. HepatoPac® is micropatterned co-cultured hepatocytes with 3T3 stromal cells, which has been engineered to possess extended cell viability (typically up to 6 weeks) with retention of in vivo-like hepatocyte functions in culture (e.g. albumin secretion, urea synthesis, drug metabolizing activities, CYP induction, transporter activities). To determine intrinsic clearance of slow clearance compounds, HepatoPac® allows metabolic stability assay of test chemicals over 7 or more days without changing the media. However, there are remaining questions: potential background noise from 3T3 stromal cell and dose responses of HepatoPac in long term culture. Here we report the result of non-targeted metabolite identifications of 7 chemicals (diclofenac, imipramine, dextromethorphan, nicotine, salicylic acid, tolbutamide and R/S-warfarin) incubated in HepatoPac® culture from a single donor over 7 days. In addition, 8 concentrations (Minimum 0.14 µM, maximum 15000 µM) of 7 test chemicals (including amiodarone as a control) were incubated in HepatoPac® over 1, 5 and 7 days without changing the media. The assay was repeated with two donors. Hepatic function (urea, albumin) and cell toxicity (ATP and GSH) were monitored. In brief, the results demonstrate that prominent glucuronidation activity in HepatoPac ® (i.e. salicylic acid metabolism) was observed over time, yet neglectable levels were measured in 3T3 stromal cells. Dose-response data from one of the single donor, showed metabolism-driven toxicity and metabolism driven detoxification in HepatoPac ® in two test chemicals. In summary, HepatoPac® is robust in vitro model to predict clearance rate with confidence for slow clearance chemicals and a potentially useful model to provide information on metabolism-driven effects (such as toxicity).

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