Predictive Toxicology Advances Through Stem Cells
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The cost of failure of drugs to advance through drug discovery increases exponentially during development and 25% of late stage drug failures are related to cardiotoxicity. In this presentation, we ...

The cost of failure of drugs to advance through drug discovery increases exponentially during development and 25% of late stage drug failures are related to cardiotoxicity. In this presentation, we delve into how cell analysis tools can be used to get an early indication of any cardiotoxic effect.

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Predictive Toxicology Advances Through Stem Cells Presentation Transcript

  • 1. Imagination at work. Applications of human stem cell derived cardiomyocytes for predictive toxicology Predictive toxicology advances through stem cells
  • 2. Drug development & cardiotoxicity Need for earlier toxicity testing and improved prediction businessreview webinar | 19 March 2014 2 Drug withdrawals for safety reasons 1976-2007 Hepatotoxicity Nephrotoxicity Cardiotoxicity Rhabdomyolysis Other Data from: Wilke et al. Nature Reviews Drug Discovery (2007) 6:904-916 Drug Development Time & Cost Research Preclinical NDA Clinical
  • 3. Predicting drug toxicity Where can we do better? businessreview webinar | 19 March 2014 3 Integration of assays & endpoints Kirkland et al. Mutat Res. 2005 584(1-2):1-256  Disparate assays and differing combinations yield varying predictivity  Testing multiple endpoints increases sensitivity but at the expense of specificity Assay Combinations Sensitivity Specificity Relevance of model systems  Animals ≠ Humans  Animal ≠ Animal  Cross species testing may increase false positives  Metabolism & MOA ? Quantity & robustness of cell models  Primary cells/tissues: source variability & scale limitations  Immortalized & engineered cells: more abundant, but limited predictivity
  • 4. Human stem cell derived models A way forward for more predictive toxicity testing businessreview webinar | 19 March 2014 4 Self-renewing Scalable Pluripotent Reproducible Convenient
  • 5. Stem cell derived human heart cells Industrial production of cardiomyocytes (CytivaTM Plus) businessreview webinar | 19 March 2014 5 Functional Performance Metrics (MEA) FPD ISI Amp
  • 6. CytivaTM Plus Cardiomyocytes Differentiated human cells for safety & efficacy testing businessreview webinar | 19 March 2014 6 DNA Troponin I a-ActininSpontaneous contractility
  • 7. Electrophysiology hERG block Na+ & Ca2+ channels QT prolongation contractility Cardiomyocytes in drug safety testing Integrated surveillance across platforms & assays businessreview webinar | 19 March 2014 7 Patch Clamp Multi-Electrode Arrays Impedance High Content Imaging Respiration Biochemical Analysis Functional Integrity mitochondrial function membrane integrity Ca2+ homeostasis morphology
  • 8. Assessing effects in context Cardiac action potential reflects net ion channel function businessreview webinar | 19 March 2014 8 Ca2+ Na+ K+ Planar patch clamp Voltage clamped whole cell currents Verapamil
  • 9. Assessing pro-arrhythmic potential Whole cell patch clamp to assess cardiac AP modulation businessreview webinar | 19 March 2014 9 Terfenadine Antihistamine Cisapride GI motility AP Prolongation 3 – 100 nM AP Prolongation 3 – 30 nM EAD 100n M Withdrawn from market due risk of adverse cardiac events Verapamil Arrhythmia Mixed effects IKr and ICa,L Safe in clinical use
  • 10. Comparative pharmacology Species variation in sensitivity of model systems businessreview webinar | 19 March 2014 10 Compound RABBIT Purkinje Fibre CANINE Purkinje Fibre HUMAN hESC-VM Terfenadine 1.0 mM False Negative 0.03 mM Quinidine 1.0 mM 1.0 mM 0.3 mM Cisapride 0.1 mM 0.1 mM 0.01 mM Sotalol 10 mM 100 mM 10 mM Chromanol 293B False Negative False Negative 300 mM E-4031 N/A 0.1 mM 0.1 mM Nifedipine N/A >10 mM 0.03 mM Most First equal Least None Relative Sensitivity Positive response defined as a change in APD90 >10% Peng, S. et.al., Journal of Pharmacological and Toxicological Methods 61 (2010) 277–286
  • 11. Increasing throughput of electrophysiological tests businessreview webinar | 19 March 2014 11 Higher throughput Multi-well formats >> screening ‘ECG-like’ traces, data-rich FPD (“QT”), beat rate, amplitude & slope, conduction velocity, etc. Non-disruptive label-free recording Preserves cell function and cellular connectivity. Chronic vs. acute effects Multiple recording sites in parallel Action potential propagation Ease of use Less skill required compared to manual patch clamp Monolayer culture High success rate Spontaneous & Consistent beat rate Expected shape & pharmacology Multi-Electrode Arrays (MEA) Cardiomyocyte requirements
  • 12. FPD (‘QT’) prolongation & contractility Effect of quinidine assessed by multi-electrode arrays businessreview webinar | 19 March 2014 12 [Quinidine] 0.0 mM 0.3 mM 1.0 mM 3.0 mM FPD (“QT”) Prolongation Contractility Changes Beat Rate (bpm) Interval (ms) Q T
  • 13. Electrophysiology hERG block Na+ & Ca2+ channels QT prolongation contractility Cardiomyocytes in drug safety testing Integrated surveillance across platforms & assays businessreview webinar | 19 March 2014 13 Patch Clamp Multi-Electrode Arrays Impedance High Content Imaging Respiration Biochemical Analysis 25% Functional Integrity mitochondria plasma membrane intracellular calcium morphology
  • 14. Functional integrity High Content Analysis (HCA) businessreview webinar | 19 March 2014 14 Membrane Integrity Dissipation of gradients, organelle disruption, loss of homeostasis Biochemical Integrity Disruption of signal transduction, synthesis, metabolism, cytoskeletal machinery 75%
  • 15. High Content Analysis (HCA) & screening businessreview webinar | 19 March 2014 15 Extracting and interpreting multi-parameter data obtained from high-throughput sub-cellular imaging [cells + sensors] [images + data] [information + knowledge]
  • 16. Why choose High Content Analysis? Cellular detail extracted rapidly in situ and in context businessreview webinar | 19 March 2014 16 Position-dependent expression Changes in orientation Colonies, tissues, whole organisms Spatial Relationships Structure, Location, Shape Structural detail (e.g. sarcomere formation) Localization (e.g. Golgi trafficking) Morphology classification (e.g. apoptosis) Temporal Information Drug induced changes in heart cell beat rate Cell migration & division tracked over 37 hours
  • 17. High content analysis solutions Automated microscopy and analysis for high throughput, high content cell imaging businessreview webinar | 19 March 2014 17 IN Cell Analyzer 2200 Faster, Brighter, Better IN Cell Analyzer 6000 Cell analysis redefined Flexible modular wide field imaging On-board image restoration 7-wavelength solid state illumination Scientific grade CMOS camera Laser based confocal imaging Iris variable aperture technology 405, 488, 561 & 642 nm laser illumination Scientific grade CMOS camera
  • 18. CytivaTM HCA kits: cell health & integrity Live interrogation of multiple cell health markers businessreview webinar | 19 March 2014 18 Amiodarone Nifedipine Amiodarone Nifedipine Characteristic “signature” for each compound Cellular Parameters nuclei mitochondria cell viability calcium or apoptosis
  • 19. Cardiotoxic potential of selective kinase inhibitors Using stem cell derived cardiomyocytes & High Content Analysis (HCA) businessreview webinar | 19 March 2014 19 In collaboration with Hirdesh Uppal Ariel Kauss
  • 20. Cancer & cardiotoxicity Tyrosine kinase inhibitors 20 Drug TK Target Indication s Cardiotoxicity Imatinib Bcr-Abl, c-kit, PDGFR CML, PhALL, GIST, CMML, CEL, DFSP CHF, LVEF depression Dasatinib Bcr-Abl, c-kit, PDGFR, Src CML QT prolongation, oedema Nilotinib Bcr-Abl, c-kit, PDGFR CML QT prolongation Sunitinib VEGFR, RET, PDGFR, c-kit RCC, GIST Hypertension, LVEF depression, CHF, MI Sorafeni b VEGFR, c-kit, PDGFR, FLT3, RAF1 RCC, HCC Acute coronary syndrome, MI, Hypertension Lapatinib EGFR, ERBB2 Breast Ca Asymptomatic LVEF depression Data from: Orphanos G.S. et.al. Cardiotoxicity induced by tyrosine kinase inhibitors 2009; Acta Oncologica, 48: 964-970
  • 21. Cardiotoxicity of anticancer drugs Overlapping cardiac signalling & oncology targets 21 Hypertrophic Stimuli Physiological Stimuli Energy Stress Growth Factors Cell Proliferation Hypertrophy Autophagy Cell Death AKT GSK3 ERK RAS RAF AKT BAD AKT LKB AMPK Pathological Stimuli [Ca2+] PLK JNK GSK3 NFAT RTK RTK PI3K PI3K mTO R MEK1 Adapted from; Force T & Kolaja K.L. Nature Reviews Drug Discovery (2011) 10, 111-126
  • 22. Cardiotoxicity study compounds Panel of 134 compounds, kinase inhibitor focus, range of classes businessreview webinar | 19 March 2014 22 Nifedipine Neg ControlAmiodarone Pos Control
  • 23. Cardiotoxicity screen workflow High content analysis (HCA) 384-well format, 7-point dose curves 24h, 48h, 72h time points n=3 wells per treatment condition, 4 images per well businessreview webinar | 19 March 2014 23 Cells Imaging Image Analysis Data Analysis Compounds Fluorescent probes
  • 24. Data analysis & interpretation Multi-parameter high content data businessreview webinar | 19 March 2014 24 Pharmacodynamics Phenotypic profiling Mitochondrial Count ATP 100μM0.05μM
  • 25. PD 325901 (MEK1) D [Ca2+] only businessreview webinar | 19 March 2014 25 Mitochondrial Integrity Calcium Viability -
  • 26. Entinostat (HDAC) Mitochondrial count and D [Ca2+] businessreview webinar | 19 March 2014 26 - Mitochondrial Integrity Calcium Viability
  • 27. Imatinib/Gleevec (TK) Mitochondrial count & morphology, D [Ca2+], viability businessreview webinar | 19 March 2014 27 - Mitochondrial Integrity Calcium Viability
  • 28. Profile clustering Self organizing maps businessreview webinar | 19 March 2014 28 - +
  • 29. Clustering results businessreview webinar | 19 March 2014 See tutorial regarding confidentiality disclosures. 29 * * * * *reported clinical cardiotoxicity - + *
  • 30. Stem cell models in toxicology businessreview webinar | 19 March 2014 30 Stem cell technology offers a way forward for abundant and reproducible supply of human cell models Assessing drug effects in electrophysiological context may reduce likelihood of false negative & positive results ESC-Cardiomyocytes (CytivaTM Plus) provide a relevant model for integrated cardiotoxicity assessment Complementary high content imaging approaches yield mechanistic insights that aid informed decision making
  • 31. Stem cell models in toxicology Vision for future development businessreview webinar | 19 March 2014 31 o Connectivity in cell models o Integration of interrogation methods o Comprehensive liability surveillance
  • 32. Learn more at the Drug Discovery Knowledge Center The Drug Discovery Knowledge Center gives you access to a wide variety of information to help you achieve deeper insights at every stage of drug discovery from target identification to lead optimization
  • 33. CytivaTM Cardiomyocytes are sold under licence from Geron Corporation and Wisconsin Alumni Research Foundation under US patent and publication numbers : US 7,425,448, US 2009/0017465, US 6,800,480, US 5,843,780, US 6,200,806, US 7,029,913, US 7,582,479, US 7,413,902, US 7,297,539, US 2009/0047739 and US 2007/0010012 and equivalent patent and patent applications in other countries. The IN Cell Analyzer system and the IN Cell Investigator software are sold under use license from Cellomics Inc. under US patent numbers US 5989835, 6365367, 6416959, 6573039, 6620591, 6671624, 6716588, 6727071, 6759206, 6875578, 6902883, 6917884, 6970789, 6986993, 7060445, 7085765, 7117098, 7160687, 7235373, 7476510 ; Canadian patent numbers CA 2282658, 2328194, 2362117, 2381344; Australian patent number AU 730100; European patent numbers EP 0983498, 1095277, 1155304, 1203214, 1348124, 1368689; Japanese patent numbers JP 3466568, 3576491, 3683591, 4011936 and equivalent patents and patent applications in other countries. Notice to purchaser: Important license information. © 2014 General Electric Company – All rights reserved. www.gelifesciences.com, GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK Presented as a businessreview webinar, Predictive toxicology advances through stem cells, 19 March 2014. Thank You !