Cardiac Ion Channel Safety Profiling: hERG and beyond G Erdemli Novartis Institutes for Biomedical Research

1. Cardiac Ion Channel Safety Profiling: hERG and beyondG ErdemliNovartis Institutes for Biomedical Research

2. Summary • Introduction to preclinical cardiac ion channel safety profiling • Overview of automated electrophysiology technologies used at Novartis • QPatch 16, HT & HTX • IonWorks Quattro • IonFlux 16 microfluidics system • Cardiac ion channel in vitro assays and case studies • hERG, Nav1.5, Cav1.2 and KCNQ1/minK • Implementation of preclinical in vitro ion channel safety profiling in the integrated risk assessment for cardiac safety • Indirect ion channel modulation, potential mechanisms and implications in preclinical safety assessment G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

3. Ito (hKv 4.2 hKv 4.3) 1 IKs (hminK + hKvLQT1) IKr (KvHERG+hMiRP) QT interval 3 INa 0 2 R ICa T IK1 (hKIR) P RMP -80/90 mV Q S ECG and action potential repolarizationCardiac ion channel profiling cardiac risk assessment Repolarization reserve LQT +25 mV -0 mV G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

4. Genetic basis for LQTS (hERG) Kaufman. Heart rhythm 2009 vol. 6 (8 Suppl) pp. S51-5 Schwartz et al Circulation 2001 G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

5. Cardiac ion channel safety profiling hERG on Qpatch-HT • Routine hERG screening on QPatch-HT • 6 pnt CRC, n=3 • 70% success rate (completed experiments) G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

6. High quality and reproducible results on QPatch-HT G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

7. QPatch data analysis: Reaching equilibrium G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

8. hERG Openers During routine screening hERG channel enhancers from different chemical series are identified Incorporated into automated data analysis for alerts G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

9. Effects of temperature in drug-induced hERG inhibition 22°C 35°C G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

10. hERG current on IonFlux-16Automated patch clamp at physiological temperatures A. C D G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

11. Cardiac ion channel safety profiling Nav1.5 assay on IonWorks Quattro Raw traces pre-cpd Normalized current post-cpd QC Monitor for seals • 8 pnt CRC, n=4 • ~100% success rate • Use-dependency Multi-state IC50 determination Use-dependence Characterization G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

12. hNav1.5 Pharmacology on Quattro Manual patch clamp from literature G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

13. Compound 1 An example of translational value of hNav1.5 assay • Preclinical cardiosafety data • in vitro Nav1.5 IC50 = 15.4 mM • Dose dependent prolongation of P, PQ and QRS in dog telemetry, no/minimal QT interval prolongation • Sudden deaths in 13 week dog toxicity study & polymorphic ventricular tachycardia - consistent with an extreme sodium channel inhibition, with PR prolongation as the first sign • Clinical cardiosafety data • Compound 1 at 640 mg (single dose) caused PR interval prolongation that coincided with the Tmax of the drug’s plasma concentration. NOEL = 1.72 mM (320 mg/kg) • Clinical studiesstudies terminated due to risk of cardiac conduction abnormalities • Therapeutic margin < 0.8 for PR prolongation G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

14. Compound 1 Preclinical Cardiac Safety Data (Dog) • IV/IV Index50: In vitro/In vivo Index: Nav1.5 IC50/Free CMax • IV/IV Index20: In vitro/In vivo Index: Nav1.5 IC20/Free Cmax G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

15. Compound 1 Clinical Cardiac Safety Data NC: No change G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

16. Compound 1 – SAD 320mg, 640mg Maximum PR Interval Increase vs Maximum Plasma Concentration Pre-dose Baseline variability 3 measurements/patient (0.93 µM) (1.9 µM) (3.7 µM) (5.6 µM) (4.7 µM) G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

17. Cardiac Ion Channel Profiling on Automated SystemsA part of preclinical integrated risk assessment Structure G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

18. Indirect modulation of hERG channels • An indirect QTc mechanism is invoked if a drug produces: • An in vivo QTc prolongation despite no in vitro signal indicating direct effect on cardiac ion channels. • A clinical QTc prolongation despite no preclinical signal indicating direct effect on cardiac ion channels • Potential mechanisms • Channel trafficking, maturation and degradation • Hypokalemia - regulation of ventricular repolarization by plasma potassium levels • Furosemide has no effect on hERG current and APD in rabbit purkinje fiber but causes QT interval prolongation • Inverse relationship between the plasma potassium levels and QT and QTc interval durations • Changes in plasma glucose levels • Hypoglycemia-induced hERG channel inhibition - due to decrease in intracellular ATP • Hyperglycemia-induced hERG channel inhibition - due to production of reactive oxygen species • Changes in autonomic tonus • Adrenergic modulation of hERG channel - Functional coupling of α and β adrenoceptors to hERG channel • Adrenergic regulation of IKs channel - functional coupling of β adrenoceptors to IKs • Macromolecular Complexes can produce ↑QTc – Ankyrins, Caveolin-3, Syntrophin - No direct drug-induced examples, but candidates for off-target interactions G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

19. Indirect modulation of cardiac channels Definition & decision tree Negative in Nav1.5, Cav1.2, hERG, KCNQ1 but QTc prolongation in vivo PK/PD relationship Test metabolites Cmax/AUC driven Test on other cardiac ion channels PK/PD disconnect No effect • Delayed disposition (parent/metabolite) to heart tissue • Accumulation (parent/metabolite) in heart tissue • Modulation of ion channel trafficking/maturation/degradation • Modulation of ion channel gene expression • Hypokalemia • Autonomic nervous system • Glucose homeostasis • Structural macromolecular complexes G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

20. Summary • Automated electrophysiology has been implemented in routine cardiac ion channel safety screen in all stages of drug discovery • Allow thousands of compounds profiling with IC50 values and quick turnaround time • Assay validation, optimization and setting QC parameters for automated data analysis are key for successful implementation • Results show very good correlation with conventional electrophysiology in most cases • Common reasons for discrepancies between conventional and automated electrophysiology are differences in • compound application duration • recording temperature • Physicochemical properties of compounds should be taken into consideration for data analysis – solubility, permeability etc Overall in vitro preclinical cardiac ion channel profiling data provide high quality translational information for integrated risk assessment G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010

21. Acknowledgments • Chris Penland • Dan Meyers • TychoHeimbach • Albert Kim • Dmitri Mikhailov • Clayton Springer • Bob Pearlstein Novartis • Xueying Cao • Tony Lee • Mats Holmqvist • A Golden • Steven Whitebread • Karl Chin • Mark Deurinck • Berengere Dumotier • M Traebert • Laszlo Urban • Fluxion • Qin Chen • Nianzhen Li • Tanner Nevill • Juliette Johnson • CristianIonescu-Zanetti • Jeff Jensen • Sophion • Ali Yehia • Millipore • Duncan Jarman G Erdemli, Ion Channel Retreat, Vancouver, June 28-30 2010