A CARDIOMYOCTYE-MEA PLATFORM FOR PRO-ARRHYTHMIA DETECTION MARCH 11, 2014 Jim Ross, PhD CTO, Axion Biosystems CiPA Objective Ion Channel Panel In Silico Simulations Proarrhythmia Score Mechanism Based, Continuous Scale, Rank Ordered, Contextual Data Clinical Assessment Human ECG Integrated Human Cellular Studies Myocyte Objective Ion Channel Panel 1. Repolarization 2. Arrhythmia In Silico Simulations Integrated Human Cellular Studies 3. Scalability/Reproducibility 4. Risk Assessment Clinical Assessment Human ECG 3 Myocyte Objective Ion Channel Panel 1. Repolarization In Silico Simulations 2. Arrhythmia CM-MEA Field Potential 3. Scalability/Reproducibility 4. Risk Assessment Clinical Assessment Human ECG 4 CM-MEA bridges the gap between AP and ECG Ion Channel Panel Action Potential In Silico Simulations Na Ca K Integrated Human Cellular Studies CM-MEA Field Potential Clinical Assessment Clinical ECG Human ECG 5 CM-MEA bridges the gap between AP and ECG Action Potential Na Ca K CM-MEA Field Potential Clinical ECG 6 CM-MEA Assay A Potential Comprehensive Pro-A Solution A grid of microelectrodes interfaces with electro-active tissue, modeling complex, human systems in a dish 7 CM-MEA Assay Capturing field potentials of beating cardiomyocytes Movie Frame 1 of 3 CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates 8 CM-MEA Assay Capturing field potentials of beating cardiomyocytes Movie Frame 2 of 3 CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates 9 CM-MEA Assay Capturing field potentials of beating cardiomyocytes Movie Frame 3 of 3 CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates 10 CM-MEA Assay Field potentials derive from AP propagation Movie Frame 1 of 6 Membrane Potential Field Potential CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates CM-MEA Assay Field potentials derive from AP propagation Movie Frame 2 of 6 Membrane Potential Field Potential CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates CM-MEA Assay Field potentials derive from AP propagation Movie Frame 3 of 6 Membrane Potential Field Potential CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates CM-MEA Assay Field potentials derive from AP propagation Movie Frame 4 of 6 Membrane Potential Field Potential CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates CM-MEA Assay Field potentials derive from AP propagation Movie Frame 5 of 6 Membrane Potential Field Potential CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates CM-MEA Assay Field potentials derive from AP propagation Movie Frame 6 of 6 Membrane Potential Field Potential CM-MEA assay directly measures cardiac activity, capturing the sum of the underlying ion channel/receptor activity while providing relevant in-vivo correlates CM-MEA Assay Direct measure of depolarization & repolarization CM-MEA Assay Direct measure of depolarization & repolarization CM-MEA Assay Direct measure of depolarization & repolarization CM-MEA Assay Literature support for cardiomyocyte assessment Myocyte Objectives CM-MEA publications per year A. Repolarization B. Arrhythmia Characterization C. Scalability/Reproducibility D. Risk Assessment E. Additional/Future Features A growing body of literature demonstrates that the CM-MEA assay meets the Myocyte objectives 20 CM-MEA Assay A: Repolarization E-4031 hERG K+ channel block increases FPD CM-MEA Assay captures and quantifies relevant ion channel and receptor activity. GE SPS 2013 CM-MEA Assay A: Repolarization E-4031 hERG K+ channel block increases FPD Mexiletine Na+ channel block decreases amplitude CM-MEA Assay captures and quantifies relevant ion channel and receptor activity. GE SPS 2013 CM-MEA Assay A: Repolarization E-4031 hERG K+ channel block increases FPD Nifedipine L-type Ca2+ channel block decreases FPD Mexiletine Na+ channel block decreases amplitude CM-MEA Assay captures and quantifies relevant ion channel and receptor activity. GE SPS 2013 CM-MEA Assay A: Repolarization E-4031 hERG K+ channel block increases FPD Mexiletine Na+ channel block decreases amplitude Nifedipine L-type Ca2+ channel block decreases FPD Isoproterenol β-adrenergic receptor agonist increases BP CM-MEA Assay captures and quantifies relevant ion channel and receptor activity. GE SPS 2013 CM-MEA Assay Field Potential Duration A: Repolarization Drug Concentration (nm) CM-MEA Assay is highly sensitive to changes in repolarization across a diverse set of compounds GE SPS 2013 CM-MEA Assay A: Repolarization CM-MEA Assay shows strong concordance and equal/greater sensitivity than the wedge preparation Harris et al 2013 CM-MEA Assay B: Arrhythmia Characterization Repolarization Voltage (V) Control 300 nM Cisapride Arrhythmic Event 1s CM-MEA assay captures and quantifies aberrant activity. Preliminary Data CM-MEA Assay Quinidine 1µM B: Arrhythmia Characterization Navarrete 2013 CM-MEA assay captures and quantifies arrhythmic activity. CM-MEA Assay Sotalol 500 µM B: Arrhythmia Characterization Navarrete 2013 CM-MEA assay captures and quantifies arrhythmic activity. CM-MEA Assay Cisapride B: Arrhythmia Characterization Triggered Activity Harris 2013 CM-MEA assay captures and quantifies arrhythmic activity. CM-MEA Assay C: Physiological Reliability/Repeatability 31 CM-MEA Assay C: Physiological Reliability/Repeatability CM-MEA field potential demonstrates high reliability across time and space in a single well How does this extend across wells? 32 CM-MEA Assay C: Physiological Reliability/Repeatability Clinical TQTc Malik (2011) MEA FPD (ms) Intra- vs. Inter-Well Variability 650 630 610 590 570 550 530 510 490 470 450 Preliminary Data 0 10 20 30 40 CM-MEA baseline reliability mirrors the clinic CM-MEA Assay C: Pharmacological Reliability/Repeatability FPD responses of Quinidine & Verapamil from three independent studies Braam (2010) Qunidine Navarrete (2013) Harris (2013) Navarrete (2013) Verapamil Braam (2010) Navarrete (2013) Harris (2013) Navarrete (2013) Cross-site and multi-sourced SC-CM MEA recordings show reproducibility within a half-log CM-MEA Assay D: Risk assessment compiled from literature Drug Name Free Drug (mM) FPD20 (mM) Safety Margin TdP Risk QUINIDINE 3.237 1.41 0.44 K PROCAINAMIDE 54.18 35 0.65 K MOXIFLOXACIN 3.887 2.6 0.67 K FLECAINIDE 0.753 1 1.33 K TERODILINE 0.145 0.23 1.59 K SOTALOL 14.69 29.7 2.02 K E-4031 0.013 0.0351 2.70 K 0.001056 0.003 2.84 K 0.01 0.03 3.00 K TOLTERODINE 0.001625 0.005 3.08 P ASTEMIZOLE 0.0008 0.003 3.75 K SPARFLOXACIN 1.766 10 5.66 K TERFENADINE 0.009 0.095 10.56 K CISAPRIDE 0.003 0.11 36.67 K SERTINDOLE 0.002 0.1 50.00 P SUNITINIB 0.013 1 76.92 P ALFUZOSIN 0.0033475 0.325 97.09 P K = Known 10 ---- ---- 0 P = Possible NIFEDIPINE 0.008 ---- ---- 0 VERAPAMIL 0.088 ---- ---- 0 0.1 ---- ---- 0 DOFETILIDE DOMPERIDONE ASPIRIN KETOCONAZOLE * FPD 20 calculations based on averages across studies from literature Preliminary CM-MEA literature results demonstrate clinical concordance 35 0 = No Risk CM-MEA Assay D: Risk Assessment +TDP Preliminary Data *Induces EADs at concentrations > 100X ETPC ChanTest Axiogenesis CM-MEA detects arrhythmia events and quantifies risk 36 CM-MEA: Bridging the gap 37 CM-MEA Assay E: Future Opportunities ms 6 4 2 Propagation Delay 8 GE SPS 2013 0 Propagation Pacing Prediction • Propagation – Capturing conduction effects • Pacing – Assessing reverse rate dependencies • Prediction– Identifying mechanisms of action CM-MEA assay provides high-content information 38 CM-MEA Assay E: Future Opportunities– Ion Channel Modelling Direct relationship between AP and FP links the CiPA Objectives 39 Next Steps Training & Optimization Validation Study Blinded Confirmation 1. Optimize protocol, generating final assay with clear endpoints, procedures, & controls. 2. Verify assays with a validation set of compounds. 3. Perform blinded study. Report sensitivity and specificity. Provide comprehensive review of gaps. 40 CM-MEA ASSAY • Direct – Multi-site field potentials derive from electrophysiological activity in a cardiac network, tracking the events that lead to arrhythmia. • Comprehensive – Unbiased assay captures unknown effects from sub-cellular to network- and organ-level. • Expandable – CM-MEA assay provides a pathway for precise mechanism identification and functional manipulation. 41
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