DOI: 10.5061/dryad.gqnk98shs
title: A targeted metabolomics-based assay using human induced pluripotent stem cell-derived cardiomyocytes identifies structural and functional cardiotoxicity potential

description: Implementing screening assays that identify functional and structural
 cardiotoxicity earlier in the drug development pipeline has the potential
 to improve safety and the cost and time required to bring new drugs to
 market. In this study, a metabolic biomarker-based assay was developed
 that predicts the cardiotoxicity potential of a drug based on changes in
 the metabolism and viability of human induced pluripotent stem
 cell-derived cardiomyocytes (hiPSC-CM). Assay development and testing was
 conducted in two phases: (1) biomarker identification and (2) targeted
 assay development. In the first phase, metabolomic data from hiPSC-CM
 spent media following exposure to 66 drugs was used to identify biomarkers
 that identified both functional and structural cardiotoxicants. Four
 metabolites that represent different metabolic pathways (arachidonic acid,
 lactic acid, 2’-deoxycytidine, and thymidine) were identified as
 indicators of cardiotoxicity. In phase two, a targeted, exposure-based
 biomarker assay was developed that measured these metabolites and hiPSC-CM
 viability across an eight-point concentration curve. Metabolite-specific
 predictive thresholds for identifying the cardiotoxicity potential of a
 drug were established and optimized for balanced accuracy or sensitivity.
 When predictive thresholds were optimized for balanced accuracy, the assay
 predicted the cardiotoxicity potential of 81 drugs with 86% balanced
 accuracy, 83% sensitivity, and 90% specificity. Alternatively, optimizing
 the thresholds for sensitivity yields a balanced accuracy of 85%, 90%
 sensitivity, and 79% specificity. This new hiPSC-CM-based assay provides a
 paradigm that can identify structural and functional cardiotoxic drugs
 that could be used in conjunction with other endpoints to provide a more
 comprehensive evaluation of a drug’s cardiotoxicity potential.
description: A 60% methanol/40% acetonitrile solution cooled to -20°C was
 added to each cardiomyocyte cell culture media sample to precipitate out
 the proteins present in the sample. The precipitation solution  contained
 three internal standards, L-citrulline-4,4,5,5-d<sub>4</sub>,
 thymidine-α,α,α,6-d<sub>4</sub> (C/D/N Isotopes Inc.,
 Pointe-Claire, Quebec, Canada), and sodium
 L-lactate-3,3,3-d<sub>3</sub> (CIL). Samples were centrifuged
 at 2,000 x <i>g</i> at 4°C for 10 minutes and the supernatant
 was transferred to a UPLC-HRMS injection plate for
 analysis.Ultra-Performance Liquid Chromatography-High Resolution Mass
 Spectrometry<i> </i>UPLC-MS data was acquired on an Agilent
 1290 Infinity LC system interfaced with an Agilent G6530A QTOF HRMS using
 the Agilent MassHunter Acquisition software. Chromatographic separation of
 metabolites was performed with an ACQUITY BEH amide column (Waters,
 Milford, Massachusetts) with dimensions 2.1×150 mm and 1.7 μm particle
 size and maintained at 40°C.
description: The file contains the raw peak apex information from each analyte
 and spiked-in internal standard for each sample analyzed in development of
 the final predictive model.