Assessing the Hepatic Disposition and Toxicity of Xenobiotics Using Primary Hepatocytes

Over the past decade or more, primary hepatocytes have become a standard in vitro tool to evaluate hepatic drug uptake and metabolism, cytochrome P450 (P450) induction, and drug interactions affecting hepatic metabolism and transport. For studies involving short incubation periods, suspensions of isolated hepatocytes have proved very useful for the study of drug uptake mediated via SLC transporters, metabolism mediated by phase I and phase II drug-metabolizing enzymes and hepatotoxicity, because the incubation medium and cells can be easily separated for analysis of drug metabolites, after which the hepatocytes can be fractionated or extracted. Whole hepatocytes are advantageous for many metabolism-related experiments because these cells do not require the addition of exogenous cofactors and metabolic enzymes such as is the case for microsomes and recombinant enzymes. Moreover, membrane transporters, which could be rate limiting for uptake of certain compounds, are functional in suspension hepatocytes. When longer-term incubations are required, for example, for hepatotoxicity or enzyme regulation studies, the cells need to be maintained in primary culture for several days or weeks, hepatocytes can be cultured in 3D configurations or in sandwich culture, which prolongs the longevity of hepatocyte cultures while retaining essential absorption, distribution, metabolism, excretion (ADME)-related functions. The large number of hepatocytes produced in a single preparation allows for greater data acquisition compared to in situ systems such as isolated perfused liver or in vivo experiments. Improved isolation and cryopreservation strategies, including pooling and freezing of hepatocytes from multiple donors, have provided a reproducible and constant source of cells, allowing for screening studies using the same hepatocyte preparation(s) over extended periods of time. The enzyme and transporter kinetics and response to treatment with drug candidates/toxins are similar in both fresh and cryopreserved hepatocytes and reflect the enzyme specificity and potency observed in vivo in a species-specific manner. Overall, the versatility and biological fidelity of primary hepatocytes makes them an excellent tool for preclinical development and in vitro/in vivo extrapolations for drug metabolism and pharmacokinetics (DMPK). Keywords: hepatocytes; cryopreserved; metabolic stability; induction; inhibition; hepatotoxicity; drug transport