CiPA Myocyte study update: Voltage-sensing optical (VSO) probes. Godfrey Smith University of Glasgow Clyde Biosciences UK

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1 CiPA Myocyte study update: Voltage-sensing optical (VSO) probes Godfrey Smith University of Glasgow Clyde Biosciences UK

2 VSOs provide a transmembrane potential signal from monolayers of spontaneously active ips-derived cardiomyocytes Voltage sensitive dye λexc = 47nm λems1 < 585nm λems2 > 585nm Fluorescence <585nm (a.u.) ms PMT1 (<585nm) TRise TU9 TU5+ Ratio (Short/Long) APDs1-9 Fluorescence >585nm (a.u.) ms PMT2 (>585nm) TU1+ 1 ms 2

3 Time course of APD repolarisation is a strong predictor of arrhythmias in whole heart MAP recordings e.g. Triangularisation of AP waveform is associated with beat-to-beat variability and TdeP Unstable non-uniform APD increase Hondeghem et al 213 Stable uniform APD prolongation

4 Recap: VSO Pilot study Cell providers (3): Assay companies/groups (4): Lesley Tung

5 VSO Pilot study 8 compounds (blinded) 4 doses 3 repeats Compound ID Description Conc. 1 Conc. 2 Conc. 3 Conc. 4 no. (µm) (µm) (µm) (µm) Ranolazine 1 I NaL blocker dihydrochloride Quinidine 2 I Na blocker Nifedipine 3 I Ca blocker Moxifloxacin hydrochloride 4 antibiotic Mexiletine 5 I Na blocker hydrochloride JNJ I Ks blocker Flecainide acetate 7 I Na blocker E-431 dihydrochloride 8 I Kr blocker

6 VSO Pilot study 4 different VSO platforms (hardware) 3 different VSO sensors (dyes) 4 different media Site JHU Sensor(s) di-4- ANEPPS Pacing/ spontane ous Electrical Vala FluoVolt Spont Clyde Q- State di-4- ANEPPS QuasAr Spont Electrical /Spon Cell Source (plating density) Media Excitation (nm) Detector Data Acquisition Rate (Hz) CDI (255k/cm 2 ); Axiogenesis (16k/cm 2 ) Tyrode's (1) 488 CMOS 5 CDI (78k/cm 2 ); Axiogenesis (14k/cm 2 ) Tyrode's (2) 488 scmos 3 CDI (78k/cm 2 ); Axiogenesis (78k/cm 2 ) CDI (7k/cm 2 ); Axiogenesis (7k/cm 2 ) Serum free media (1) 488 PMT 1, Serum free media (2) 64 scmos 5

7 VSO Pilot study 2 different dosing protocols (single and cumulative) 2 different stimulation methods (spontaneous & stimulated): Single dose 3min exposure(clyde, Vala, QState) spontaneous and field stimulated Well a Dose 1 Well b Well c Dose 2 Dose 3 Well d 6s baseline recording 3min drug exposure 6s drug recording Dose 4 Cumulative dosing 3min/dose (Johns Hopkins University Tung) field stimulated Dose 4 Dose 3 Dose 2 Dose 1 Well a 6s baseline recording 6s drug recording 6s drug recording 6s drug recording 6s drug recording

8 VSO Pilot study: end-points: Primary end points: Spontaneous beat rate and regularity Action potential rise time (1-9%) APD at 3%, 5% & 9% AP triangulation index (=APD9-APD3) Incidence of triggered activity and tachyarrhythmias 2ms APD3 Baseline Drug APD9

9 VSO Pilot Study 2 ms 2 ms 2 ms % Repolarization Axiogenesis CDI GE-Healthcare mean+sd Action potential shape differs across the 3 different providers (Clyde Biosciences Platform) APD (ms)

10 AP characteristics are different across platforms/sensors and cell types Nifedipine Moxifloxacin

11 Conclusions from pilot study Electrophysiology appears different across different commercial ips- CMs on one platform Electrophysiology of one commercial ips-cm appears different across different platforms (different media) Standardised of protocol and incubation media are important to reduce platform-to-platform and cell-to-cell variability Higher repeat number is essential n>3 (suggest 5/6) Ratio of DMSO controls: drug can be <1 (suggest 1:4) Employ a customised (not standard) APD rate correction option.

12 Validation study 28 drugs (low; intermediate; high TdP risk) Cell providers (2): Assay companies (3):

13 Validation study 1 incubation media (serum free)* 1 protocol (single drug 3 min incubation) only spontaneous activity (not stimulated) Site Sensor(s ) Pacing/ spontane ous Vala FluoVolt Spont Clyde di-4- ANEPPS Spont Q-State QuasAr Spont Cell Source (plating density) CDI (78k/cm 2 ); Axiogenesis (93k/cm 2 ) CDI (78k/cm 2 ); Axiogenesis (78k/cm 2 ) CDI (7k/cm 2 ); Axiogenesis (7k/cm 2 ) Media Excitation (nm) Detector Data Acquisition Rate (Hz) Serum free media 488 scmos 3 Serum free media 488 PMT 1, Serum free media 64/488 scmos 5 * The two cell providers supply their own SFM Technical controls to be added each plate: Dofetilide (compulsory) Quality control standard: Nifedipine (optional) Coefficient of variation on beat Lidocaine (optional) interval.5 (5%)

14 VALIDATION STUDY VSO END-POINTS: Primary end points: Spontaneous beat rate and regularity Action potential rise time (1-9%) APD at 3%, 5% & 9% AP triangulation index (=APD9-APD3) Assessment of arrhythmia type event: 6 categories 4 ms Type PreT arrhythmia 2 s Quiescent (Q)

15 icell 2 (Batch#CMC11515) vs Cor4U (Batch#CB434CL) Events N. Events N Mean = 1.24ms SD =.19ms n= 924 Mean = 4.4ms SD = 1.5ms n= 888 Mean = 5.9ms SD = 1.9ms n= TRise (ms) Mean = 1.66ms SD =.36ms n= Cycle Length (s) VSO Validation study Clyde Biosciences baseline data (serum free media) Events N. Events N Mean = 1.4% SD = 1.2% n= % is >2% Mean = 1.5% SD = 1.6% n= % is >2% CiPA Threshold cv CL (%) Mean = 484ms SD = 78.4ms n= 888 Mean = 313.7ms SD = 3.1ms n= APD 9 (ms)

16 Validation study: Clyde Biosciences Technical controls icell 2 (Batch#CMC11515) vs Cor4U (Batch#CB434CL) Dofetilide (C+) and Vehicle Control (C-) change of baseline 2 2 APD9 (%) ±.6% 5±5% APD9 (%) ±.6% 87±15.2% Triangularization (%) Control Dofetilide.5nM 128±15% 4.7±1.6% Control Dofetilide.5nM Triangularization (%) Control Dofetilide 3nM 24.9±48% 4.2±1.6% Control Dofetilide 3nM mean±sem

17 Validation study: (Clyde Biosciences) Rate correction of APD9 to 1 Hz (6bpm) APD 9 (ms) [13/1/216 1:6 "/Graph1" ( )] Linear Regression for DataCDI_APD9: Y = A + B * X Parameter Value Error A B R SD N P < Events N Raw Mean = 484ms SD = 78.4ms CoV = 16% n= Cycle Length (s) rapd9 capd APD 9 (ms) APD 9 (ms) Corrected Mean = 369ms SD = 33ms CoV = 9% n= [13/1/216 1:7 "/Graph1" ( )] Linear Regression for DataAxio_APD9: Y = A + B * X Parameter Value Error A B R SD N P < Events N. Cycle Length (s) Raw Mean = 313.7ms SD = 3.1ms CoV = 9.6% n= 924 rapd9 capd APD 9 (ms) Corrected Mean = 288ms SD = 2ms CoV = 6.9% n= 924

18 Progress: VSO Validation study: Clyde Biosciences: completed and data submitted (Sept 216) Vala Sciences: studies ongoing Qstate: studies ongoing Intermediate conclusions (1) Repeat number (n= 6) gives high resolution of APD shape. (2) Technical controls show uniform behaviour across plates/time. Statistical analysis will address: Detection of concentration-dependant APD prolongation (threshold etc). Does AP shape (e.g. Triangularisation) correlate with severity of TdeP drug? (How does this compare with in silico reconstruction?) Does arrhythmia-like behaviour and what type (e.g. EAD) correlate with severity and known severity of the drug? (How does this compare with in silico reconstruction?)