Low-Turnover Drug Molecules: A Current Challenge for Drug Metabolism Scientists

Abstract

In vitro assays using liver subcellular fractions or suspended hepatocytes for characterizing the metabolism of drug candidates play an integral role in the optimization strategy employed by medicinal chemists. However, conventional in vitro assays have limitations in their ability to predict clearance and generate metabolites for lowturnover (slowly metabolized) drug molecules. Due to a rapid loss in the activity of the drug-metabolizing enzymes, in vitro incubations are typically performed for a maximum of 1 hour with liver microsomes to 4 hours with suspended hepatocytes. Such incubations are insufficient to generate a robust metabolic response for compounds that are slowly metabolized. Thus, the challenge of accurately estimating low human clearance with confidence has emerged to be among the top challenges that drug metabolism scientists are confronted with today. In response, investigators have evaluated novel methodologies to extend incubation times and more sufficiently measure metabolism of low-turnover drugs. These methods include plated human hepatocytes in monoculture, and a novel in vitro methodology using a relay of sequential incubations with suspended cryopreserved hepatocytes. In addition, more complex in vitro cellular models, such as HepatoPac (Hepregen, Medford, MA), a micropatterned hepatocyte-fibroblast coculture system, and the HmREL (Beverley Hills, CA) hepatic coculture system, have been developed and characterized that demonstrate prolonged enzyme activity. In this review, the advantages and disadvantages of each of these in vitro methodologies as it relates to the prediction of clearance and metabolite identification will be described in an effort to provide drug metabolism scientists with the most up-to-date experimental options for dealing with the complex issue of low-turnover drug candidates.

Upcyte Human Hepatocytes: a Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds

“…In recent years, considerable efforts were made to provide advanced hepatic in vitro systems mitigating the loss of DME activities to reliably predict human CL of metabolically stable compounds (Di and Obach, 2015;Hutzler et al, 2015). However, limited availability and large variability in DME expression and activity remain shortcomings of PHH, in particular for routine screening applications in drug discovery.…”

“…Primary hepatocyte suspensions are commonly preferred for the in vitro determination of intrinsic CL (CL int ) , representing a fully competent metabolic model (Blanchard et al, 2006;Hewitt et al, 2007) with an expression pattern of drug-metabolizing enzymes (DMEs) comparable to the liver of origin (Richert et al, 2006). However, suspended hepatocytes lose viability and metabolic activity within a few hours (Skett, 1994;Gebhardt et al, 2003), which frequently averts reliable and accurate CL int determination, in particular for slowly cleared compounds (Di and Obach, 2015;Hutzler, et al, 2015).…”

Quantitative Assessment of Population Variability in Hepatic Drug Metabolism Using a Perfused Three-Dimensional Human Liver Microphysiological System

“…Although these systems have been very valuable in drug development (Di et al, 2012) and are easy to use, they lose metabolic activity over time, hence the study of low-clearance compounds is challenging (Di and Obach, 2015; Hutzler et al, 2015). In addition, overall these in vitro systems tend to underpredict in vivo clearance (Hallifax et al, 2010).…”

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

“…However, the activity of drug metabolizing enzymes in these widely used in vitro models declines within hours (3), and therefore, depletion of compounds with low turnover (<10%) cannot be determined with confidence. As the number of lowclearance compounds in pharmaceutical programs increases due to the application of highly effective metabolic clearance reduction strategies in lead optimization to allow once-daily administration of moderate drug quantities (4), more sophisticated in vitro liver models are required (5). For example, models where the metabolic activity is prolonged for up to 2-4 weeks can extend the sensitive range for clearance determination.…”