A Novel Relay Method for Determining Low-Clearance Values

Abstract

ABSTRACT:A novel relay method has been developed using cryopreserved human hepatocytes to measure intrinsic clearance of low-clearance compounds. The relay method involved transferring the supernatant from hepatocyte incubations to freshly thawed hepatocytes at the end of the 4-h incubation to prolong the exposure time to active enzymes in hepatocytes. An accumulative incubation time of 20 h or longer in hepatoctyes can be achieved using the method. The relay method was validated using seven commercial drugs (diazepam, disopyramide, theophylline, timolol, tolbutamide, S-warfarin, and zolmitriptan) that were metabolized by various cytochrome P450s with low human in vivo intrinsic clearance at approximately 2 to 15 ml ⅐ min ؊1 ⅐ kg ؊1. The results showed that the relay method produced excellent predictions of human in vivo clearance. The difference between in vitro and in vivo intrinsic clearance was within 2-fold for most compounds, which is similar to the standard prediction accuracy for moderate to high clearance compounds using hepatocytes. The relay method is a straightforward, relatively low cost, and easy-to-use new tool to address the challenges of low clearance in drug discovery and development.

Addressing the Challenges of Low Clearance in Drug Research

“…The assay has a straightforward format and can be utilized by typical drug metabolism laboratories with a minimal cost. The hepatocyte relay method is being used for intrinsic clearance determination (12,13) and metabolite identification (14) for humans and laboratory animal species. The method has been developed to conduct reaction phenotyping experiments with Transfer Supernatant Fig.…”

“…1. Hepatocyte relay method (12) P450 selective inactivators in order to estimate the fraction of metabolic clearance catalyzed by specific P450 enzymes for low-CL int compounds. For compounds with high nonspecific binding to plastics, buffer controls can have a significant decline.…”

“…2. IVIVC in Human, rat, and dog using the hepatocyte relay method (12,13). Open circles represent compounds having significant contribution of extra-hepatic metabolism or transporter-mediated clearance clearance pathway, since there was no turnover of the compound in the standard human liver microsome or hepatocyte suspension assays.…”

In Vitro–In Vivo Correlation for Low-Clearance Compounds Using Hepatocyte Relay Method

“…In drug discovery, low-intrinsic-clearance compounds have increased significantly over the years due to the effective use of highthroughput absorption, distribution, metabolism, excretion, and toxicity screening and early metabolite identification, which enable a rapid structure-activity relationship to address high-intrinsic clearance as a liability (Di et al, 2012). In addition, new chemical space of drug discovery is being explored, such as liver targeting (Oballa et al, 2011;Pfefferkorn et al, 2012) and once-weekly dosing (Rangan et al, 2007), and all of these require low clearance.…”

“…This frequently leads to inaccurate prediction of human clearance, half-life, and dose. To address low clearance challenges, the hepatocyte relay method was recently developed to predict human intrinsic clearance for low-intrinsic-clearance compounds (Di et al, 2012). In this study, we expanded the hepatocyte relay method to predict rat and dog intrinsic clearance to establish IVIVC in three preclinical species to support high-confidence translation of human IVIVC for low-clearance compounds.…”