P148 Alginate-encapsulated primary human hepatocyte culture system to increase the accuracy of the predicted in vivo half-life of low clearance compounds

Miyoung Yoon , ScitoVation, LLC, Research Triangle Park, NC
Martin B Phillips , ScitoVation, LLC, Research Triangle Park, NC
Jeffrey R Enders , ScitoVation, LLC, Research Triangle Park, NC
Pergentino Balbuena-Venancio , ScitoVation, LLC, Research Triangle Park, NC
David Billings , ScitoVation, LLC, Research Triangle Park, NC
Rachel Norini , ScitoVation, LLC, Research Triangle Park, NC
Ruby Karsten , ScitoVation, LLC, Research Triangle Park, NC
Harvey J Clewell III , ScitoVation, LLC, Research Triangle Park, NC
Sow intrinsic clearance is considered a desirable property of drugs to reduce dosage frequency and magnitude, stabilizing tissue concentrations, and prolonging in vivo half-life. However, it also presents challenges in drug development and safety testing particularly due to the difficulties in estimating in vivo half-life. Emerging new technologies that enable long-term culture based on organotypic 3D culture and/or microfluidic systems-based methods hold promises for increasing accuracy in hepatic clearance estimation and identifying the potential safety concerns related with metabolites early in lead selection/optimization. However, these alternatives can be resource-intensive and often, companied by a challenge in extrapolating the results to in vivo. This study presents a long-lived primary hepatocyte culture method in a relatively simple 3D format based on alginate hydro-gels. We have optimized this 3D hepatocyte culture model for slow clearance investigation for drugs as well as persistent environmental chemicals. The alginate-encapsulated hepatocytes, referred as “alginate beads”, maintain their viability over weeks. They appear to maintain metabolic competence and profiles comparable to those of freshly isolated cells as evidenced by the metabolism assay results for two marker substrates, 7-ethoxy-coumarin and acetaminophen. Then, we determined the intrinsic clearance of S-warfarin, as a case compound for slow clearance, in a series of 7-day alginate-beads human hepatocyte (from 4 donors) cultures. The alginate-beads determined intrinsic clearance of s-warfarin was about a 1/3 of the reported in vivo value. In addition, the estimated Km in our alginate beads culture was around 2 μM, which is consistent with a reported range for S-warfarin’s Km. We have characterized xenobiotic enzyme expression and activity in the human primary hepatocyte-alginate beads. After about a week’s incubation in dynamic condition, both phase I and II enzyme expression levels returned to a level comparable to that of freshly isolated cells, showing promise in application of our model for low clearance prediction. In general, phase II enzymes including UGT, SULT and carboxylesterases recovered faster than phase I enzymes. Among the phase I enzymes, CYP2C9, 1A2, and 2D6 showed a faster recovery than CYP2E1 or 3A4. In its current form, our model can be used to increase the reliability of in vivo clearance predictions for low clearance compounds and yet still be able to provide a cost-effective and easy-to-use method comparable to the current gold standard of human primary hepatocyte suspension culture. Currently, we are testing other dynamic culture conditions that can be used with the alginate beads including flow-based bioreactor systems as well as putting together an in vitro biokinetic model to assist in vitro-to-in vivo extrapolation (IVIVE) of the results. With these further improved long term culture systems with an IVIVE tool, our alginate-beads culture system will provide a flexible and scalable tool to support both initial screening and later phase toxicity testing during preclinical development without relying on or reducing the need for time and resource-intensive animal tests.