Development of an automated mass spectrometry system for the quantitative analysis of liver microsomal incubation samples: a tool for rapid screening of new compounds for metabolic stability

Korfmacher, Walter A.; Palmer, Cynthia A.; Nardo, Cymbelene; Dunn-Meynell, Kimberly; Grotz, Diane E.; Cox, Kathleen; Lin, Chin‐Chung; Elicone, Chris; Liu, Charles; Duchoslav, Eva

doi:10.1002/(sici)1097-0231(19990530)13:10<901::aid-rcm583>3.0.co;2-5

Cited by 78 publications

(20 citation statements)

References 10 publications

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“…The concentration of the test compound reported in the literature for microsomal stability assays varies from 0.5 to 15 µM. 4,[6][7][8][11][12][13] Microsomal stability, based on percent remaining of parent, is dependent on the test concentration due to nonlinear kinetics. Higher test concentrations (e.g., 10 µM) produce results indicating apparently higher stability.…”

Section: Discussionmentioning

confidence: 99%

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

Di¹,

Kerns²,

Hong³

et al. 2003

SLAS Discovery

127

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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)

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Section: Discussionmentioning

confidence: 99%

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

Di¹,

Kerns²,

Hong³

et al. 2003

SLAS Discovery

127

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“…All tested compounds showed low CYP inhibition towards CYP 2C19, 2D6 and 2C9 isoforms, but high CYP 1A2 and 3A4 inhibition were detected for compounds such as linezolid, excluding exo-alcohol 2a for CYP 3A4. The selected compounds were stable based on human microsomal incubation tests 37) with a minimum of 95.2% activity after 30 min. Based on monoamine oxidase (MAO) inhibition assays using Amplex ® Red Monoamine Oxidase Assay Kit (A12214, Invitrogen, Carlsbad, CA, U.S.A.), the compounds showed significantly reduced MAO-A and -B inhibition compared to linezolid, which is a weak reversible inhibitor of MAO that can interact with tyramine-containing food and should be used with caution in pediatrics.…”

Section: Synthesis and In Vitro Evaluation Of The Antitubercular And mentioning

confidence: 99%

Synthesis and <i>in Vitro</i> Evaluation of the Antitubercular and Antibacterial Activity of Novel Oxazolidinones Bearing Octahydrocyclopenta[<i>c</i>]pyrrol-2-yl Moieties

Bhattarai

Lee

Seo

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…These assays typically use 96-well technologies for incubations and sample preparation and ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) for data acquisition (Korfmacher et al, 1999;Di et al, 2003), which would require more than 50 separate analyses for each enzyme. More recently, high-resolution mass spectrometers have been used for analysis; however, data extraction and data analysis remain cumbersome (O'Connor et al, 2006;Shui et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software

Shah

Kerns

Nguyễn³

et al. 2016

Drug Metabolism and Disposition

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Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CL int ) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CL int for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t 1/2 ) method] was chosen to determine CL int . The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t 1/2 compounds, carbamazepine and antipyrine (t 1/2 > 30 minutes); one moderate t 1/2 compound, ketoconazole (10 < t 1/2 < 30 minutes); and two short t 1/2 compounds, loperamide and buspirone (t ½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CL int and t 1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes.

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Development of an automated mass spectrometry system for the quantitative analysis of liver microsomal incubation samples: a tool for rapid screening of new compounds for metabolic stability

Cited by 78 publications

References 10 publications

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

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

Synthesis and <i>in Vitro</i> Evaluation of the Antitubercular and Antibacterial Activity of Novel Oxazolidinones Bearing Octahydrocyclopenta[<i>c</i>]pyrrol-2-yl Moieties

An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software

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