Development and Characterization of a Human Hepatocyte Low Intrinsic Clearance Assay for Use in Drug Discovery

Abstract

Progression of new chemical entities is a multiparametric process involving a balance of potency; absorption, distribution, metabolism, and excretion; and safety properties. To accurately predict human pharmacokinetics and estimate human efficacious dose, the use of in vitro measures of clearance is often essential. Low metabolic clearance is often targeted to facilitate in vivo exposure and achieve appropriate half-life. Suspension primary human hepatocytes (PHHs) have been successfully used in predictions of clearance. However, incubation times are limited, hindering the limit of quantification. The aims herein were to evaluate the ability of a novel PHH media supplement, HepExtend, in order to maintain cell function, increase culture times, and define the clearance of stable compounds. Cell activity was analyzed with a range of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) substrates, and the mRNA expression of drug disposition and toxicity marker genes was determined. HepExtend and Geltrex were essential to maintain cell activity and viability for 5 days ( = 3 donors). In comparison with CM4000 ± Geltrex, HepExtend + Geltrex displayed a higher level of gene expression on day 1, particularly for the P450s, nuclear receptors, and UGTs. The novel medium, HepExtend + Geltrex, was robust and reproducible in generating statistically significant intrinsic clearance values at 0.1 l/min/10 cells over a 30-hour period ( < 0.05), which was lower than previously demonstrated. Following regression correction, human hepatic in vivo clearance was predicted within 3-fold for 83% of compounds tested for three human donors, with an average fold error of 2.2. The novel PHH medium, HepExtend, with matrix overlay offers significant improvement in determining compounds with low intrinsic clearance when compared with alternative approaches.

Application of New Cellular and Microphysiological Systems to Drug Metabolism Optimization and Their Positioning Respective to In Silico Tools

“…In a challenge to long-term co-culture models, Lancett et al recently showed data indicating that new culture supplements and Matrigel overlay may be employed to sufficiently slow the decay of enzyme activities in monoculture-plated cells to enable multiple-day usage. 39 They reported the Matrigel overlay to enable robust intrinsic clearance predictions over a duration of 30 h, with high gene expression levels for CYP, UGT, and nuclear receptors maintained over this time course. The uncomplicated nature and high cell density (350,000 hepatocytes per well in a 24-well plate) of this system could enable measurement of in vitro intrinsic clearance for highly metabolically stable compounds at lower cost than using the sophisticated co-cultures.…”

“…The uncomplicated nature and high cell density (350,000 hepatocytes per well in a 24-well plate) of this system could enable measurement of in vitro intrinsic clearance for highly metabolically stable compounds at lower cost than using the sophisticated co-cultures. 39 Alternative approaches, such as spheroids or more MPSs, are discussed later.…”

In Vitro Biotransformation Assays Using Liver S9 Fractions and Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): Overcoming Challenges with Difficult to Test Fragrance Chemicals

“…One possible mechanism behind the underprediction could be technical issues. For instance, inappropriate concentrations of assay reactants and of substrates can result in the underestimation of metabolic rates (Wood et al 2017; Lancett et al 2018). Similarly, the use of solvents for hydrophobic chemicals can influence the in vitro clearance rate (Escher et al 2011; Lee et al 2014).…”