Metabolic Stability Screen for Drug Discovery Using Cassette Analysis and Column Switching

Abstract

In vitro metabolic stability assays are used to screen compounds for stability in the presence of various drug metabolizing enzymes, usually cytochrome P450 in liver preparations (e.g., liver microsomes). High-throughput metabolic stability assays using pooling methods have been developed to keep pace with screening requirements at the lead ADME optimization stage. In our laboratory, we have improved the metabolic stability assay using the cassette analysis method, column switching, and incorporated time saving techniques in method development to yield a robust method which reduces data turnaround time, increases compound throughput, and maximizes mass spectrometer usage. This method can determine metabolic stability using microsomes or hepatocytes from any species. We describe our findings following incubation of 40 different compounds with human liver microsomes and analysis by the cassette and discrete analysis methods. Similar metabolic stability results were obtained using the cassette analysis and discrete analysis method. An overall 70% time savings was achieved by pooling four new compounds into one sample for method development/MS optimization, cassetting four samples into one sample to minimize the number of injections on LC/MS/MS analysis, and using a column switching system to analyze the samples, which results in a two-fold decrease in the LC/MS/MS analysis time.

Metabolite Profiling of Praziquantel and its Analogs During the Analysis of in vitro Metabolic Stability Using Information-Dependent Acquisition on a Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometer

“…1,2) Identifying metabolic properties is a key component of characterizing the ADME profile, and nowadays it is integrated into the early discovery phase. 3) Xenobiotics are biotransformed to less toxic, less active, and more hydrophilic metabolites to enhance their detoxification and elimination.…”

Physiologically Based Pharmacokinetic Modeling to Predict Drug-Drug Interactions Involving Inhibitory Metabolite: A Case Study of Amiodarone

“…The model-based sensitivity analysis was conducted to evaluate the effect of variable f u values on hepatic clearance prediction (see PBPK Model Development section). In vitro metabolic intrinsic clearance (CL int ) of AMIO and MDEA was determined in human liver microsome incubations using an in-house assay (Halladay et al, 2007). The CL int was 28 ml/min per mg for AMIO and 4 ml/min per mg for MDEA incubated at 1 mM of substrate concentration.…”

A Novel In Vitro Experimental System for the Evaluation of Drug Metabolism: Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Cryopreserved Human Hepatocytes)

“…Before the development of cryopreserved hepatocyte technologies, hepatic subcellular fractions such as human liver microsomes and postmitochondrial supernatants (S9 or S10), were used exclusively in drug metabolism studies (Iwatsubo et al, 1997;Gombar et al, 2003). These subcellular systems, especially human liver microsomes, continue to be practical and useful experimental systems in pharmaceutical industrial laboratories, including screening of new chemical entities for metabolic stability (Halladay et al, 2007;Choi et al, 2015), estimation of in vivo hepatic clearance (Obach, 2011;Chen et al, 2017), evaluation of cytochrome P450-related drug properties (Dinger et al, 2014), and UDPGT-mediated drug metabolism (Walsky et al, 2012;Joo et al, 2014). Compared with cryopreserved hepatocytes, the use of subcellular fractions requires relatively simple application procedures, and they are relatively robust and are not readily subjected to functional damages due to handling.…”