The liver is the major drug-metabolizing organ which is responsible for the clearance of approximately 60% of drugs available on the market through cytochrome P450 (CYP)-mediated metabolism. Therefore, hepatic clearance is undeniably one of the most critical pharmacokinetic parameters. In the early stages of drug discovery, the in vitro hepatic metabolic stability assay carries great importance as it provides a time- and cost-effective route for measuring the metabolic clearance in a high-throughput fashion and predicts in vivo hepatic clearance through in vitro-in vivo extrapolation. This valuable information permits effective and critical decision-making before the in vivo studies take place. The hepatic metabolic stability assay measures the disappearance of a test compound over time in the presence of an in vitro system, for example, liver microsomes, S9, recombinant CYP450 enzymes, hepatocyte suspensions, and hepatocyte cocultures. Hepatocyte suspensions are extensively used among various available in vitro systems, as it is a more representative and integrated system with a full complement of metabolic enzymes and retained cellular compartments. Compound percent remaining is quantified by liquid chromatography tandem mass spectrometry (LC/MS/MS) with an individually tuned, condition optimized, signal quality high-throughput balanced method. Half-life is calculated from the natural logarithm of the compound remaining in percent over time. Intrinsic clearance is calculated from the half-life combined with known scaling factors. Intrinsic clearance, together with blood protein binding (fub) and liver blood flow (QH), are used in the prediction of in vivo hepatic clearance. Predicted in vivo clearance facilitates a better understanding of in vitro-in vivo correlations and guides the best use of in vitro pharmacokinetics data. This chapter offers a detailed high-throughput in vitro method to assess metabolic clearance of test compounds using hepatocyte suspensions.
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