High-Throughput Microsomal Stability Assay for Screening New Chemical Entities in Drug Discovery

Abstract

In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities. (Journal of Biomolecular Screening 2008:862-869)

Development of a High-Throughput and Miniaturized Cytokinesis-Block Micronucleus Assay for Use as a Biological Dosimetry Population Triage Tool

“…Currently, to track the demanding throughput required for screening in drug discovery, pharmaceutical and biotechnology companies are applying automation and robotic technologies such as liquid handling systems. They are being used for multiple sample preparations to increase the number of molecules that can be biologically assayed (40–42). The RABiT is an example of an advanced throughput system developed at Columbia University Medical Center for the purpose of biological dosimetry applications (2, 5, 43, 44).…”

Novel benzidine and diaminofluorene prolinamide derivatives as potent hepatitis C virus NS5A inhibitors

“…Compounds 26, 27, 42, and 43 had lower levels of degradation after 0.5 h (Table 7, entries 2, 3, 4, and 5, respectively). These results indicate that these promising compounds had high microsomal stability in the human liver [102,103].…”

“…5.2.7. Method to determine stability in rat and human liver microsomes [103] The metabolic stability of the compounds was evaluated in pooled rat (BD Gentest ® Cat No. 452501) or human livers (BD GentestCat.…”

Recent development in high-throughput bioanalytical support forin vitroADMET profiling

“…Just as in the case of cassette dosing, data interpretation could sometimes be problematic with this approach, due to the potential interaction between the pooled compounds and the biological system. The sample pooling approach does not suffer from this potential pitfall, and it has been used extensively by groups supporting ADMET profiling in assays such as metabolic stability [101,102], Caco-2 [103], protein binding [104] and CYP inhibition [64,105]. However, sample dilution, as well as potential cross interference and matrix suppression of co-eluting compounds, could still be a challenge.…”