Neurotox and Cardiac Safety Assessment: Case Studies Employing iPS - PowerPoint PPT Presentation

Neurotox and Cardiac Safety Assessment: Case Studies Employing iPS Cell lines and Next Generation MEA Technology

Workshop Outline In vitro Assessments of Drug-induced Neuronal Modulation and MEA-based Seizure Prediction Blake Anson, PhD Cellular Dynamics International Please use this link to access the seizure prediction slides Comprehensive In-vitro Proarrhythmia Assay (CiPA) MEA and hiPSC-cardiomyocytes as reproducible and predictive tools for detecting proarrhythmia Hong Shi, PhD Bristol-Myers Squibb Please use this link to access more information on iCell Cardiomyocytes and Cardiomyocytes 2 Get Ready to LEAP TM Mike Clements, PhD A XION Biosystems Please use this link to learn more about the Maestro Edge

In In vitr vitro Asses essments of of Dru Drug-induced Neu euronal l Mod odula lation and MEA-based Seiz eizure Pred ediction Blake Anso Bl nson, , PhD PhD C Cellu lular Dyn Dynamics In Internatio ional Please use use thi this link to o acc access the the slides

Comprehensive In In-vitro Proarrhyt ythmia Assay (C (CiPA) MEA and hiP iPSC-cardiomyocy cytes as s re reproducible and pre redict ctive tools for detecting pro roarrhythmia Hong Shi, MD BMS

Talk Outline Comprehensive In-vitro Proarrhythmia Assay (CiPA) System stability and cross site reproducibility • CiPA Paradigm • MEA Tracings and Drug Effects • Baseline and Control Responses iCell Cardiomyocytes 2 • Data from BMS • CiPA Compound Responses • Data from BMS • Multi-site Correlations • Summary Maestro Multiwell MEA Platform

Short CiPA Overview Comprehensive In-vitro Proarrhythmia Assay (CiPA) Cross site correlations

MEA Tracings Phenotypes of hiPSC-CM Electrophysiology Human iPSC-derived cardiomyocytes and MEA recordings detect electrophysiological phenotypes related to key mechanistic effects.

Baseline Data Safety assays require a consistent phenotype across wells and plates • Baseline histograms were compiled across 14 separate 48-well plates for BP, AMP, FPDc (Fridericia), and BP Coefficient of Variability. • BP CoV and AMP passed the CiPA Phase 2 Protocol inclusion criteria. • BP, BP CoV, and FPDc were highly reliable across plates for the study. Human iPSC-derived cardiomyocytes and MEA recordings provide a stable platform for detect electrophysiological phenotypes

Vehicle and Positive Controls Detection of positive control compounds defines assay sensitivity Negative control - Inter-plate stability 0.5nM Dofetilide - Also used as experimental check IC 50 ~ 12nM 1 - - Used a submaximal concentrations for maximal system sensitivity 1 Snyders and Chaudhary, 1996 The label-free assay supports reliable and minimal responses to vehicle control responses, enabling sensitive detection of the positive control.

CiPA Phase 2 Compounds Detection of positive control compounds defines assay sensitivity Compound selection was determined by experts and designed to cross multiple classifications with concentrations that bracket the therapeutic level

Low Risk Intermediate Risk High Risk Compound Responses BMS Data Low risk compounds generally had little effect on FPDc or elicited a shortening of FPDc. Intermediate risk compounds ranged from minimal to significant prolongation of FPDc. High risk compounds consistently induced significant prolongation. 5/11 compounds 7/8 compounds showed showed FPDc 0/9 compounds showed FPDc FPDc prolongation >50% prolongation prolongation >50% by 30x by 30x Cmax >50% by 30x Cmax Cmax Human iPSC-derived cardiomyocytes and MEA recordings show appropriate compound classification for arrhythmogenic potential Note: EADs included for FPDc calculation

Low Risk Intermediate Risk High Risk Compound Responses Multi-Site Data Data from BMS matched well with that from multiple sites (Eisai, Genentech, Axion) Overall the test system shows good multi-site reproducibility Data divergence generally occurred at >30X Cmax, or upon incidence of EADs Human iPSC-derived cardiomyocytes and MEA recordings show reproducible classification results across multiple sites

Multi-Site Correlations Site-to-site consistency highlights a reliable assay • Each point in the scatter plot represents the percent change in FPDc at two sites for a BMS AXN GNE ESI single compound and concentration. 1 0.94 0.95 0.94 BMS • AXN 0.94 1 0.89 0.92 The data show extremely high correlation for conditions that do not elicit EADs, and good GNE 0.95 0.89 1 0.95 correlation even when EADs are present. 0.94 0.92 0.95 1 ESI • The CiPA Phase 2 data was highly Blinova et al. submitted correlated across all sites using the CDI/Maestro cell-platform combination. Human iPSC-derived cardiomyocytes and MEA recordings show reproducible quantitative data across multiple sites

Data Interpretation Risk classification of Ando et al, 2016 Current results can be mapped onto existing scheme(s) - TdP risk (y-axis) based on  FPDc AXION FPDc BMS FPDc - Ratio (x-axis) based on concentration ratio of in-vitro effect/ clinical level Ando et al., 2016 J Pharm Tox Meth Red = high risk Yellow = intermediate Green = low risk Sensitivity = 0.79 Sensitivity = 0.84 Specificity = 0.78 Specificity = 0.67 Accuracy = 0.79 Accuracy = 0.79 • Current data set is consistent with previous classification results Smaller data set, unequal grouping • Additional assessment paradigms will continue to emerge Human iPSC-derived cardiomyocytes and MEA datasets will enable the generation and refinement of in-vitro tools to predict proarrhythmia

Summary Site-to-site consistency highlights a reliable assay • • Baseline data was reproducible Positive Controls • • Across MEA plates Reproducible (see left) • Experimental days • Were used at sub-maximal concentrations • Experimental sites (0.5nM Dofetilide) to demonstrate detection of sensitive effects • Vehicle controls • Compound Responses • Were highly reproducible (see above) • Highly correlated across sites ( ≥ 0.89) • Acted as an internal system check • Risk Classification was robust across sites • Translated well to available clinical data Human iPSC-derived cardiomyocytes and MEA recordings provide an efficient, robust, and translatable in-vitro paradigm for predicting proarrhythmia

03.12.18 Society of Toxicology

Field Potential Signal for the CM-MEA Assay Relationship to the Cardiomyocyte Signal Spectrum LEAP Action Potential Field Potential Field Potential Clinical ECG

Local Extracellular Action Potential (LEAP) How does it work? Transmembrane Potential Recorded Amplitude (mV) LEAP LEAP Field Potential Field Potential G Ω Sealing Resistance Adapted from Borkholder, 1998 Patent Pending

Maestro Pro & Edge World’s most advanced MEA platforms BioCore v4 Maestro Pro & Edge

Local Extracellular Action Potential (LEAP) Signal Specifications FP at Same Scale 5-20 mV SNR ~1000+ Peak-to-Peak 10 mV Stable for 10-20+ minutes after induction

The LEAP Advantage #1 FP to AP “Translation” FP and LEAP Signals from the Same Wells, 10x Zoom on the FP 500uV 5mV 5mV Depolarization Repolarization EADs The LEAP signal provides a direct mapping from field potential to action potential morphology

The LEAP Advantage #2 Arrhythmia/EAD Detection The LEAP signal improves the accuracy of automated analysis, and allows automation of EAD detection

The LEAP Advantage #2 Arrhythmia/EAD Detection The updated CiPA Analysis Tool provides automated EAD detection for LEAP signals, as well as other LEAP endpoints.

The LEAP Advantage #3 LEAP Morphology LEAP Duration (LEAPD) 5mV The LEAP signal provides additional and complementary metrics to the standard CM-MEA field potential assay

The LEAP Advantage #4 LEAP does not disrupt the underlying biology The induction of LEAP does not affect the underlying electrophysiologic properties of the cardiomyocyte syncytium.

Implementing the LEAP Assay Baseline Dosed LEAP 100uV 2mV 800m s

LEAP Case Study on Selective Blockers L-Type Calcium Block hERG Potassium Block Multi-Ion Channel Block (Nifedipine) (E-4031) (Verapamil)

LEAP Case Study on Selective Blockers

LEAP Case Study on Tolterodine vs. Terodiline Tolterodine Terodiline Martin et al, 2006

“Next Steps” Combining LEAP with Other Maestro Technology Electrical Pacing CytoView MEA 24-well

Maestro Pro Enables Next Generation Applications • The Local Extracellular Action Potential (LEAP) signal adds a new dimension to the standard CM-MEA assay • A simple, label-free induction phase produces the LEAP signal, which is characterized by: • An action potential-like waveform with high amplitude • Stability on the time scale of 10-20+ minutes • LEAP enables: • Clear translation of field potential signals to action potential signals • Improvements in automated EAD detection • Analysis of LEAP morphology to provide additional end points • LEAP does not disrupt the underlying biology Announcing the LEAP Assay for use in Spring 2018!

Acknowledgments Daniel Millard Heather Hayes Anthony Nicolini Colin Arrowood Jim Ross Visit www.axionbio.com For more information on the Maestro Pro TM and Edge TM

Learn more about LEAP Tuesday March 13 | 1:30 - 3:30pm Booth #1342 Poster P691