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 (N = 3 donors). In comparison with CM4000 6 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 ml/min/106 cells over a 30-hour period (P < 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.