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 [Ca2+]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 ♦ Ca2+ 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) Page 8 • Liang et al. (2013) Circulation 127:1677 • adult LV, hESC-CM, hiPSC-CM [healthy, LQT, HCM, DCM] • ion channels: INa, Ito, ICa.L, hERG (IKr), KvLQT1 (IKs), IK1 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 CO2 incubator SC-derived cardiomyocytes: impedance-based contraction monitoring (2) ACEA xCELLigence CardioECR Page 11 Nanion CardioExcyte 96 SC-derived cardiomyocytes: impedance-based contraction monitoring (3) Page 12 Guo et al. (2011) Toxicol Sci 123:281 Impedance signal analysis: amplitude duration rise/fall time beating rate irregularity E-4031 Human iPSC cardiomyocytes: impedance-based arrhythmia detection Guo et al. (2011) Toxicol Sci 123:281 (iCells) Himmel (2013) JPTM (iCells) Page 13 • impedance-based contractions: amplitude(↓), rate↑/↓, arrhythmia • guinea-pig ventricular myocytes and LA/RA: cell shortening ↑ force (+20%), rate (-25%) (Wettwer et al., 1991) Human iPSC cardiomyocytes: impedance-based contraction monitoring (4) Himmel (2013) JPTM (iCells) ATX-II • impedance-based contractions: amplitude (→), rate ↓, AUC ↑↑ • impedance-based contractions: amplitude (↓), rate ↑/↓ • guinea-pig/rat ventric. myocytes & pap. m.: cell shortening ↑, force ↑ • dog VM, gp RA, human PM: cell shortening ↓, rate ↓, force ↓ (Hoey et al., 1994; Isenberg & Ravens, 1984) Page 14 verapamil (Harmer, 2012; Tanaka, 1996; Schwinger, 1990) Human iPSC cardiomyocytes: impedance-based contraction monitoring (5) 0.36 µM human iPSC-CM 10.8 µM • 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 ⇒ impedance-based signals: proarrhythmia Page 15 contractility Peters et al. (2014) Cardiovasc. Toxicol. DOI 10.1007/s12012-014-9268-9 Jonsson et al. (2011) Assay DDT 9:589 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 accessible reproducible many techniques simple throughput time scale weakness immature heterogenous low anisotropy imcompletely understood opportunity human disease micropatterns 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 Thank you! Page 19
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