Optimization of a Higher Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates

Abstract

Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects. (Journal of Biomolecular Screening 2003:453-462)

Metabolic stability optimization and metabolite identification of 2,5-thiophene amide 17β-hydroxysteroid dehydrogenase type 2 inhibitors

“…Compounds 7a, 8a, 16a, 19a, 22a, 27a and 27 were tested according to established method [34][35][36] For evaluation of phase I and II metabolic stability 1 µM compound was incubated with 1 mg/ml pooled mammalian liver S9 fraction (BD Gentest), 2 mM NADPH regenerating system, 1 mM UDPGA and 0.1 mM PAPS at 37°C for 0, 5, 15 and 60 minutes at a final volume of 100 µL. The 20 incubation was stopped by precipitation of S9 enzymes with 2 volumes of cold acetonitrile containing internal standard.…”

The Conduct of Drug Metabolism Studies Considered Good Practice (II): In Vitro Experiments

“…If the concentrations have not been determined, a range of 1-10 μM of the final drug concentration can generally be used to closely mimic in vivo levels of the drug. However, literature reported that the incubating concentrations of the test drugs (0.5-15 μM) affected the stability results of these drugs in the same microsomal preparation [18]. In general, a test drug at its higher concentration (e.g., 10 μM) shows more stable in the microsomes than at its lower concentration.…”

“…However, it has been shown that the activity of the commercial microsomes varied significantly from batch to batch, and from vendor to vendor. For instance, the rat liver microsomes from two vendors showed great activity in metabolizing drugs buspirone and loperamide, while those from the third vendor exhibited no activity at all; and three different batches of rat liver microsomes from the same vendor resulted in different activity in metabolizing buspirone and loperamide [18]. The differences in the microsomal activity may be due to inherent animal-to-animal variation and differences in preparation processes among the vendors.…”

“…This thawing-restoring flexibility allows us to use microsomal aliquots from a single batch for different experiments to facilitate normalization or comparison of different experiment data, and thus avoid confusing results introduced from different batches and vendors. Di et al [18] examined activity of different microsomal aliquots from the same commercial batch every week for 50 days for their activity inhibited by midazolam, buspirone and loperamide, and found good reproducibility of the microsomal activity inhibited by the drugs across all aliquots. However, microsomes that have been incubated at 37°C should not be re-used or refrozen.…”

“…For example, the percent remaining of propanolol at 10 μM was 76-fold higher than that at 1 μM. Some drugs considered CYP substrates did not show the concentration-related stability under the same condition [18]. Drugs that are not the substrates of CYPs are usually stable in microsomal incubation and their stability will not be affected by their incubating concentrations [25].…”