Effects of reference compounds on impedance signals from stem cellderived human cardiomyocytes Herbert M. Himmel, Safety Pharmacology, Bayer Pharma AG, Wuppertal, Germany SPS Webinar Cardiac Safety Testing Models 20 NOV 2014 Page 1
Disclaimer The thoughts expressed here are those of the speaker and do not necessarily reflect those of the speakers employer Bayer HealthCare AG. Page 2
Agenda introduction stem cell-derived cardiomyocytes impedance assay: principle & examples summary & conclusion Page 3
Introduction (1) 1990s: market withdrawal and late stage failure of drugs due to sudden cardiac death associated with QT prolongation and herg K + channel block 2005: ICH guidelines S7B and E14 cardiac risk assessment using at the very least herg assay, animal QT assay, and human thorough QT study 2010s: limited ability to predict risk of cardiotoxicity at substantial costs Recent technological developments: human induced pluripotent stem (hips) cell-derived cardiomyocytes measurement of impedance signals ± field potentials improved prediction of cardiotoxicity and proarrhythmia? Revolution dawning in cardiotoxicity testing Nature Rev. Drug Discov. 2013; 12:565-567 Page 4
Introduction (2) compound failure due to cardiotoxicity ~25% electrophysiology ~75% Ca homeostasis mitochondria Nature Rev. Drug Discov. 2013; 12:565-567 [Ca 2+ ] i handling coordinated activity of ion channels electrical activity mechanical activity energy supply mitochondria How can impedance signals from hipsc-derived cardiomyocytes improve early safety assessment and prediction of drug-induced cardiotoxicity?? Page 5
Agenda introduction stem cell-derived cardiomyocytes impedance assay: principle & examples summary & conclusion Page 6
Stem cell-derived vs adult cardiomyocytes: phenotype similar, but different slow (ultra)structural maturation 25 µm cardiac markers striation pattern cell shape alignment cell shortening Kattman et al. (2011) J CV Trans Res 4:66 (icells ) adult human VM (failing heart) Harding et al. (2007) Pharmacol Ther 113:341 Kamakura et al. (2013) Circ. J. 77:1307 Page 7
Human SC-derived cardiomyocytes: partially immature gene expression pattern Ca 2+ handling & ion channel genes Synnergren et al. (2012) Physiol. Genomics 44:245 (hesc cell line SA002) 0- (UD), 3- and 7-weeks post-differentiation versus fetal/adult heart (FH/AH) Liang et al. (2013) Circulation 127:1677 adult LV, hesc-cm, hipsc-cm [healthy, LQT, HCM, DCM] ion channels: I Na, I to, I Ca.L, herg (I Kr ), KvLQT1 (I Ks ), I K1 Page 8
Agenda introduction stem cell-derived cardiomyocytes impedance assay: principle & examples summary & conclusion Page 9
SC-derived cardiomyocytes: impedance-based contraction monitoring (1) measurement principle: impedance signals 96-well plate-based systems rhythmic contractions of spontaneously beating cardiomyocytes short-/long-term ms s min hrs - d early assay for contractility? low-voltage signal current cell spreading impedance (cell index) cell shape impedance Himmel (2013) JPTM 68:97 Page 10
SC-derived cardiomyocytes: impedance-based contraction monitoring (2) CO 2 incubator ACEA xcelligence CardioECR Nanion CardioExcyte 96 Page 11
SC-derived cardiomyocytes: impedance-based contraction monitoring (3) Impedance signal analysis: amplitude duration rise/fall time beating rate irregularity Guo et al. (2011) Toxicol Sci 123:281 Page 12
Human ipsc cardiomyocytes: impedance-based arrhythmia detection E-4031 Guo et al. (2011) Toxicol Sci 123:281 (icells ) Himmel (2013) JPTM (icells ) impedance-based contractions: amplitude( ), rate /, arrhythmia guinea-pig ventricular myocytes and LA/RA: cell shortening force (+20%), rate (-25%) (Wettwer et al., 1991) Page 13
Human ipsc cardiomyocytes: impedance-based contraction monitoring (4) ATX-II verapamil Himmel (2013) JPTM (icells ) impedance-based contractions: amplitude ( ), rate, AUC guinea-pig/rat ventric. myocytes & pap. m.: cell shortening, force (Hoey et al., 1994; Isenberg & Ravens, 1984) impedance-based contractions: amplitude ( ), rate / dog VM, gp RA, human PM: cell shortening, rate, force (Harmer, 2012; Tanaka, 1996; Schwinger, 1990) Page 14
Human ipsc cardiomyocytes: impedance-based contraction monitoring (5) 0.36 µm human ipsc-cm 10.8 µm Jonsson et al. (2011) Assay DDT 9:589 impedance-based contractions (e.g. iso [ampl ( ), rate ], carb [ampl, rate ( )]) positive/negative ino-/chronotropic effects in multicellular cardiac tissue preparations due to negative amplitude-frequency relation in hipsc cardiomyocytes Peters et al. (2014) Cardiovasc. Toxicol. DOI 10.1007/s12012-014-9268-9 impedance-based signals: proarrhythmia contractility Page 15
SC cardiomyocytes: impedance-based monitoring of delayed cardiotoxicity doxorubicin pentamidine Abassi et al. (2012) BJP 165:1424; mouse esc-cm Similar results: kinase inhibitor-mediated delayed cardiotoxicity + biomarkers (e.g. ctnt injury, cellular ATP) (Lamore et al. (2013) Toxicol. Sci. 135:402) Page 16
Agenda introduction stem cell-derived cardiomyocytes impedance assay: principle & examples summary & conclusion Page 17
Summary & Conclusion impedance assays in human SC-derived cardiomyocytes: strenghts, weaknesses, opportunities stem cell-derived cardiomyocytes impedance assay strength weakness opportunity accessible immature human disease reproducible heterogenous many techniques low anisotropy micropatterns simple throughput time scale imcompletely understood pacing field potentials biomarkers impedance assays in human SC-derived cardiomyocytes offer a lot of potential for early detection of drug-induced cardiotoxicity full leverage of this potential in combination with pacing and field potentials Page 18
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