Metrion Biosciences: the ion channel specialists

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1 Metrion Biosciences: the ion channel specialists In depth profiling of human ipsc cardiomyocytes: From electrophysiology to phenotypic assays Saïd El-Haou, PhD 30 November

2 Background: The CiPA initiative Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative is a regulatory proposal sponsored by US, European and Japanese safety bodies to replace/augment current cardiac safety testing regimes Drug effects on Multiple Human Cardiac Currents In Silico Reconstruction Human Ventricular Cellular Electrophysiology In Vitro Effects Human Stem-Cell Derived Ventricular Myocytes 1. Multiple cardiac ion channel testing to better assess risk, earlier 2. Use in vitro data for in silico modelling of arrhythmia and Torsade de Pointe (TdP) risk 3. Confirm proarrhythmia and TdP signals in human cardiomyocytes 2

3 Presentation topics 1. Manual Patch Clamp - Current Clamp 2. Axion Maestro - MEA 3. Nanion CardioExcyte96 - Impedance 3

4 hipsc-cm: Axiogenesis vcor.4u New generation of Ncardia ventricular induced pluripotent stem cell derived cardiomyocytes (hipsc-cm) Isolated from 26 year old Caucasian female Day 0 Day 7 Day 7 Spontaneous beating 90 % ventricular and fibroblast free 7 days in culture - fibronectin (10 μg/ml) Cells form syncytial monolayer suitable for electrophysiological and phenotypical assessment Manual Patch Clamp at room temperature Whole-cell configuration for Voltage Clamp (VC) Perforated patch for Current Clamp (CC) Phenotypic assay (MEA and impedance) at 37 C Serum-containing media 5 % CO 2 Methodology 4

5 Action potential recordings: Manual Patch Clamp A. B. C. AP parameters Spontaneous (n=89) Evoked 1 Hz (n=75) MDP (mv) ± ± 0.6 dv/dt max (V.s -1 ) 56.8 ± ± 4.8 APA (mv) ± ± 1.7 APD20 (ms) ± ± 3.0 APD50 (ms) ± ± 3.8 APD90 (ms) 502 ± ± 10.9 Frequency (Hz) 0.43 ± Suitable for recording of spontaneous and evoked activity (0.5-2 Hz) Very stable recordings > 30 min Good resting membrane potential ~ -73 mv Phase 0 upstroke of 50 V.s -1 denoting good functional expression of Na V 1.5 channel Consistent APD90 ~ 450 ms hipsc-cm validation 5

6 Action potential phenotype: Atrial vs Ventricular (1 Hz) A. B. C. D. 1 μm Carbachol K ir 3.1/ nm Tertiapin Q K ir 3.1/ nm ML 365 TASK-1 / K 2P 3.1 We prefer to use pharmacology rather than AP shape to classify ipsc-cm phenotype vcor.4u cells are insensitive to most atrial-selective ion channel blockers 50 μm 4-AP K V 1.5 Small effect of 4-AP suggesting functional expression of K V 1.5 channels K V 1.5 protein is expressed in human ventricle, although functional activity not detected (Mays et al, 1995) Common feature of most hipsc-cm tested so far at Metrion Atrial pharmacology 6

7 Pharmacology: Evoked Action Potentials (1 Hz) A. 100 μm Lidocaine B. 100 nm Nifedipine C. Na V 1.5 Phase 2 Phase 0 Ca V nm Dofetilide herg Phase 3 AP Pharmacology Expected effects of core CiPA reference compounds on action potentials Lidocaine slowed the upstroke velocity by ~60% Nifedipine decreased all APD values Dofetilide prolonged APD90 by ~ 70% but did not trigger EADs at 1Hz 7

8 Presentation topics 1. Current Clamp - Manual Patch Clamp 2. Axion Maestro - MEA 3. Nanion CardioExcyte96 - Impedance 8

9 MEA platform: Axion Maestro APD A. B. C. Control Environment control unit allows long term recording 37 C / 5 % CO 2 Acute / chronic pharmacology High success rate using 96 MEA well plate 100 % wells recorded MEA signal >95 % of the 784 electrodes FPD QT interval Action Potential CM-MEA Field Potential Clinical ECG MEA parameters vcor.4u Spike Amplitude Mean (mv) 8.6 ± 1.6 Spike Slope Mean (V.s -1 ) ± 3 FPD Mean (ms) ± 16.3 FPDcF Mean (ms) ± 14.9 Beat Rate (bpm) 40.9 ± 1.2 Conduction Velocity Mean (mm.ms -1 ) 0.22 ± 0.1 Beat Period Irregularity (%) 0.3 ± 0.2 Microelectrode array 9

10 2 mv MEA: Na + channel blocker Lidocaine A. B. C. 1 ms 0.1 % DMSO 10 μm Lidocaine 30 μm Lidocaine 100 μm Lidocaine Lidocaine increased inter-spike interval and prolonged FPDc at the highest concentration Spike amplitude, slope and conduction velocity were decreased as expected No arrhythmic or EAD-like events 1 s Pre-drug 100 μm Lidocaine (30 min) D. Spike amplitude Pre-drug 100 μm Lidocaine Microelectrode array 10 Conduction velocity

11 500 μv MEA: Ca 2+ channel blocker Nifedipine A. B. C. Pre-drug Nifedipine decreased FPDc and inter-spike interval Effects were observable after five minutes and stable over time 1 s D. 300 nm Nifedipine (30 min) Pre-drug Microelectrode array No effects on the spike amplitude and slope Beat Rate nm NIF 300 nm NIF 1 μm NIF

12 500 μv MEA: herg channel blocker Dofetilide A. B. C. Pre-drug Dofetilide increased inter-spike interval and FPDc It also reduced the spike amplitude and slope 2 s 10 nm Dofetilide (5 min) 10 nm Dofetilide (30 min) Microelectrode array EAD were observed after five minutes and degenerated into fibrillation at higher concentrations 12

13 MEA: safe CiPA relevant compounds FPDc Arrhythmia Clinical TdP Risk JiCSA icell (1) FDA icell (2) FDA Cor.4U (2) FDA icell (3) Metrion vcor.4u JiCSA icell (1) FDA icell (2) FDA Cor.4U (2) Diltiazem Lidocaine = = = = Mexilitine = = = Nifedipine - - Ranolazine x FP x FP - x FP - Verapamil : Increase FPDc : Decrease FPDc = : No change FDA icell (3) x : Arrhythmia / EAD events - : No arrhythmic events FP: False positive FN: False negative Metrion vcor.4u All clinically safe drugs were correctly identified as non-proarrhythmic in Metrion s MEA assay Microelectrode array 13 (1) Ando et al. 2017; Nozaki et al. 2016, 2017 (2) Blinova et al (3) Schocken et al. 2017

14 MEA: high risk CiPA relevant compounds FPDc Arrhythmia Clinical TdP Risk JiCSA icell (1) FDA icell (2) FDA Cor.4U (2) FDA icell (3) Metrion vcor.4u 14 JiCSA icell (1) FDA icell (2) FDA Cor.4U (2) FDA icell (3) Metrion vcor.4u Bepridil = - FN - FN - FN - FN - FN Chlorpromazine = = x - FN - FN x x Cisapride x x - FN x x D,L-Sotalol x x Dofetilide x x x x x Ondansetron x x Quinidine x - x x x Terfenadine = - FN - FN - FN x x : Increase FPDc : Decrease FPDc = : No change x : Arrhythmia / EAD events - : No arrhythmic events All high risk compounds showed a significant prolongation of FPDc FP: False positive FN: False negative Serum-containing media was important for correctly identifying high risk compounds Only Bepridil did not generate EAD / arrhythmic events Also observed by FDA and JiCSA Chronic effects (trafficking?) (1) Ando et al. 2017; Nozaki et al. 2016, 2017 (2) Blinova et al (3) Schocken et al Microelectrode array

15 Presentation topics 1. Current Clamp - Manual Patch Clamp 2. Axion Maestro - MEA 3. Nanion CardioExcyte96 - Impedance 15

16 Impedance assay: Nanion CardioExcyte 96 Dual mode platform: impedance & microelectrode array (MEA) High success rate ~ 100 % well display regular beating vcor.4u cells start to beat after six hours Base Impedance which represent the growth phase, is stable after five days BR stable after 72 hours CardioExcyte 96 parameters vcor.4u Base impedance (Ω) ± 4.1 Beat Rate (bpm) ± 1.0 Amplitude (Ω) 3.6 ± 0.1 PW30 (ms) ± 1.0 BR CoV 0.04 ± 0.0 CardioExcyte 96 16

17 1 Ω 1 Ω 1 Ω Impedance assay: Contractility & proarrhythmic effects Pre-drug A. B. C. 100 μm Lidocaine 300 nm Nifedipine 50 nm Dofetilide 5 min 30 min 1 s # # # 1 s 1 s Acute impedance Drug effects on contractility mirrored electrophysiological pharmacology Lidocaine stopped contraction but recovered with a decreased BR and amplitude Nifedipine increased BR but decreased amplitude Dofetilide decreased amplitude and BR. Secondary beats degenerated into fibrillation 17

18 Impedance assay: Chronic cardiotoxicity A. B. C. Chronic cardiotoxicity is a useful readout to assess safety of clinical candidates with varied mechanismsof-action Base impedance is a good indicator of cell viability and allows possible cardiotoxicity effects to be evaluated vcor.4u cells are stable over 72 hours in control Suitable for acute / chronic cardiotoxicity and safety screen experiments Lidocaine had no effect on cell viability Other ion channel modulators did show concentration-dependent cardiotoxic effects High concentrations of Nifedipine or Dofetilide decreased base impedance Chronic impedance 18

19 10 Ω Cardiotoxicity assay: Amiodarone A. 1 μm Amiodarone B. C. Predrug 1 h 24 h 48 h 72 h 1 s Class III antiarrhythmic widely used for treatment of atrial fibrillation Known cardiac side effects: Arrhythmia: Torsade de Pointe, VF and sinus bradycardia Congestive Heart Failure Base impedance decreased only after five hours drug exposure Chronic impedance 19

20 10 Ω Cardiotoxicity assay: Doxorubicin A. 1 μm Doxorubicin B. C. Predrug 1 h 24 h 48 h 72 h 1 s Broad spectrum anti-cancer medication belonging to Anthracycline family DNA intercalating agent Known cardiac side effects: Acute atrial and ventricular arrhythmia Chronic cardiomyopathy Congestive heart failure Concentration-dependent decrease of base impedance after 24 hours Chronic impedance 20

21 10 Ω Cardiotoxicity assay: Bortezomib A. 100 nm Bortezomib B. C. Predrug 1 h 24 h 48 h 72 h 1 s Bortezomib is a proteasome inhibitor used for treating relapsed multiple myeloma and mantle cell lymphoma Known cardiac side effects: Acute heart failure Atrioventricular block Base impedance increased during the first 24 hours (transient swelling?) Contraction activity was impaired after 24 hours followed by a concentration-dependent decrease of base impedance 21 Chronic impedance

22 Summary vcor.4u ipsc-cm are suitable for use in a range of ion channel and cardiac safety assays: Manual Patch Clamp: current clamp & voltage clamp Impedance: cardiotoxicity assay using CardioExcyte 96 platform Microelectrode array using the Axion Maestro vcor.4u cardiomyocytes display ventricular-like pharmacology: Mostly insensitive to atrial-specific agents Expected responses to core pharmacological tools Able to generate EAD and arrhythmic events (fibrillation) hipsc-cm validation 22

23 Acknowledgment Metrion Biosciences Sarah Williams John Ridley Louise Webdale Kathy Sutton Robert Kirby Nanion technologies Sonja Stölzle-Feix Krisztina Juhasz Thanks This project received funding from the Eurostars-2 joint program with co-funding from the European Union Horizon 2010 research and innovation program 23

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