A New Comprehensive Translational Paradigm Using Human ipsc-derived Cardiomyocytes for Safety Assessment of Drug-Induced Arrhythmias

Transcription

1 A New Comprehensive Translational Paradigm Using Human ipsc-derived Cardiomyocytes for Safety Assessment of Drug-Induced Arrhythmias Armando Lagrutta Safety & Exploratory Pharmacology (SEP) Department Safety Assessment and Laboratory Animal Resources 3D Tissue Models August 29, 2017

2 Takeaways: Highlighting features in 2D and 3D Models BEST SUITED for In Vitro Cardiac Safety Studies 1. Human ipsc-derived cardiomyocyte (hipsc-cm) 2D models developed for acute and long-term assessments of drug-induced proarrhythmic effects in fluorescence- and impedance-based platforms 2. Case studies involving use of hipsc-cm test system for cardiac de-risking of Hepatitis C virus (HCV) NS5b inhibitors BMS-094 cardiac toxicity; Adverse amiodarone drug-drug interaction (DDI) precipitating in clinical bradycardia/bradyarrhythmia 3. Suitable cardiac 3-D models Nanofiber scaffolds for 3-D modeling with High Content Imaging (HCI) endpoints Self-assembling engineered micro-tissues 2

3 Background: Pro-arrhythmic Risk Assessment s: Drug withdrawals due to Torsade de Pointes (TdP) lethal Ventricular Arrhythmias. Recognition of herg K + Channel blockade as primary mechanism 2. Early 2000 s: Harmonization of Requirements for cardiac delayed repolarization and QT prolongation (ICH S7B and E14) Present: Paradigm shift CiPA (Comprehensive In Vitro Pro-Arrhythmia Assay) Consortium involving FDA, ILSI-HESI, global pharmaceutical industry, and global technical stakeholders Similar and complementary efforts in Europe and Japan December Preliminary recommendation by ICH E14/S7B IWG/DG to ICH Assembly regarding whether to re-open TQT / ICH E14 and S7B for complete revision 3

4 Our 2D hipsc-cm Assay Platforms: Key Features hipsc-cms: 1. icell Cardiomyocytes (Cellular Dynamics, Int.) 2. ~ 1.5 million viable cardiomyocytes / vial 3. Spontaneously beating syncytia (30,000 cells /well) FDSS/µCell (Hamamatsu): 1. Integrated fluorescence readout of spontaneously beating hipsc-cm syncytia Fluorescence label. Intracellular Ca 2+ transient, optical membrane potential. Signals well qualified for pro-arrhythmic risk detection 2. Short term (0.5 1 hr) and long term (24 hrs 7 days) effects/time-points Evaluation at predetermined time points Medium or higher throughput: 96-well and 384-well plate available CardioRT and Cardio ECR (xcelligence) : 1. Integrated readout in spontaneously beating hipsc-cm cells Impedance (IMP) signal: label-free, non-invasive, related to mechanical attachment and contractility Simultaneous field potential (FP) signal (ECR only): analogous to ECG signal 2. Short term (0.5 1 hr) and long term (24 hrs 7 day) effect with continuous monitoring 3. Fit-for-purpose investigations 4. Medium throughput: 96-well (CardioRT) or 48-well (ECR) plate limit 4

5 Functional Drug Screening System (FDSS)/ Cell Platform: Ca 2+ Transient Dynamics in hipsc-cms Imaging Platform: - Integrated fluorescence readout of spontaneously beating syncytia of hipsc-cms - Acute and chronic effects 1. Intracellular Calcium transient (Ca 2+ sensitive dye) 2. Optical membrane potential signal (membrane potential dye) hips-cardiomyocytes with Ca 2+ sensitive dyes (Codex ) HAMAMATSU FDSS/ CELL End-points: 96-well plate 1. Peak amplitude and beating rate (peak count) 2. Ca 2+ Transient Duration at 80% 3. EAD-like signals Pre-read Peak amplitude and peak count Ca 2+ Transient Duration at 80% (CTD80) Intracellular Ca 2+ transient dye signal Dofetilide 10 nm 5 Sec Secondary signals, a surrogate of EAD (early-after-depolarization): EAD-like signals. 5

6 FDSS Data Correlation: Proarrhytmia Predictivity Assay Sensitivity: 83% Assay Specificity: 85% Assay Predictive Capacity: 84% N= 44 Outcomes No TdP Clinic TdP Clinic No EAD EAD True Negative TN False Negative FN 17 4 False Positive FP True Positive TP 3 20 TN. Amlodipine, diltiazem, verapamil, nifedipine, adenosine, flunarizine, chromanol293, minoxidil, glyburide, amantadine, amiloride, sildenafil, alfuzosin, nicorandil, indapamide, lidocaine, ioxynil. FN. Terfenadine: Evidence of TdP when co-ad with ketoconazole or congenital QTprolongation. Probucol: Chronic effect (evidence of QT and TdP ventricular arrhythmia). Amiodarone: TdP in the clinic. Dronedarone (Amiodarone-like): TdP in the clinic. FP. Ivabradine and tolterodine both caused adverse cardiac effects, although no TdP reported, ranolazine. TP. Dolasetron, ketoconazole, mallotoxin, sertindole, pentamidine, nilotinib, astemizole, cisapride, flecainide, haloperidol, moxifloxacin, sotalol, solifenacin, pimozide, fluoxetine, quinidine, dofetilide, bepridil, terodiline, oubain. Good quantitative correlation for true positives with reported clinical TdP concentrations H. Zeng et al. / J. Pharmacol. Toxicol. Methods 81 (2016)

7 Field Potential Spike Amplitude Beat Amplitude (IMP. fluctuations) Impedance Platforms and End-points: Real-time, Non-invasive Readouts in hipsc-cms Stable monolayer synchronously beating steady state RTCA Cardio: 1.Syncytial beats in 96-plate well format 2.Microelectrodes at the bottom of each well 3.Real-time impedance (IMP) 4.End-points: Beat rate number of full cycles between beginning of the first detected cycle to the end of the last detected cycle during ~1 min recording Beat amplitude (see figure) Cell Index baseline impedance reflecting syncytial attachment and health cell expansion phase 12 Baseline Impedance or Cell Index Following Plating of icell hipsc Cardiomyocytes Fluctuations around Impedance baseline ( ~1% of baseline) Cell Index (Baseline) RTCA CardioECR: 1.Syncytial beats in 48-plate well format 2.Impedance signal as described above 3.Simultaneous real-time measurement of syncytial contractility by IMP and electric activity by field potential (FP) 4.End-points: IMP end-points above Field potential spike amplitude - (see figure) Field potential rate - analogous to beating rate above Full Cycle Full Cycle 7 7

8 % Change Relative to Vehicle Control Case Study: HCV Inhibitors on Impedance Signals Impedance signals at 48 hrs (15 sec. snapshots from representative 96-well plate wells): µm: CTL BMS (INX-08189) MK-0608 Sofosbuvir beating cessation 40% 20% 0% -20% -40% -60% -80% % Change of Beating Rate 48 hr incubation MK-0608 IC50 = 301 µm Sofosbuvir INX % IC50 = 9.8 µm -120% Concentration [µm] MK-0608 EC50 =400 µm Sofosbuvir INX % Control 100% 80% 60% 40% 20% 0% Cell Adherence (Cell Index) 48 hr incubation Concentration [µm] 15 sec MK-0608 IC50 = 447 µm INX IC50 = 4 µm Sofosbuvir IC50 = 417 µm 8

9 Case Study: Reported Clinical Cardiac Drug-Drug Interaction (DDI) impacting HCV-NS5B NI programs Field potential rate (normalized) Prodrugs, cleavage intermediate metabolites, and NTP metabolites studied Species Uridine Nucleoside Analogs Diastereoisomers L-Ala,S P D-Ala,R P Prodrug R-Group Designation Prodrug Name F sofosbuvir MNI-2 C C-H MNI-1 MNI-4 Cl MNI-3 MK-3682 Cleavage Intermediate Metabolite Nucleoside Tri-Phosphate (NTP) A.. Lagrutta et al. / Nat. Sci. Rep. 7 (2017) 44820, doi: /srep Studies with hipsc-cm syncytia in CardioECR helped identify diastereoisomeric specificity of DDI and critical role of prodrug vs. metabolites Ebel 10µM + SOF 3µM + Amio 0.3µM Ebel 10µM Ebel 3µM + SOF 3µM + Amio 0.3µM Ebel 3µM SOF 3µM + Amio 0.3µM SOF 3µM DMSO 0.1% Amio 0.3µM Time (hrs) 9

10 Optimizing Cardiac Tissue Models: Shortcomings of 2D Models and Solutions Offered Shortcomings: 1. Lack of architectural or functional anisotropy 2. Immature calcium handling 3. Negative force-frequency relationship 4. Immature repolarization reserve with exaggerated proarrhythmic effect of herg channel inhibition Solutions offered: 1. Enabling substrates (e.g., improved 2D structure, self-assembling 3D) 2. Long-term electrical or mechanical stimulation 3. Extracellular matrix mimetics (e.g., aligned nanofiber scaffolds) 4. Optimization of tension / stiffness / afterload (e.g., muscular thin film, cell drum ) 5. Modulation by other cell types (e.g., fibroblasts, mesenchymal stem cells) GOALS: FULLY FUNCTIONAL, DIFFERENTIATED CARDIAC PHENOTYPE NOT NECESSARILY 2D vs 3D QUESTION 10 10

11 3D Cardiac Models: From 2D-Anisotropy to Engineered Tissues Engineered Heart Tissues (Eschenhagen s laboratory) I. Mannhardt et al. / Stem Cell Reports 7 (2016) K. Breckwoldt et al. / Nat. Protoc. 12 (2016) Thin Muscular Film / Heart-on-a-Chip (Parker s laboratory) A.W. Feinberg et al. / Science 317 (2007) A. Grosberg et al. / Lab Chip 11 (2011) J.U. Lind et al. / Nat. Mater. 16 (2017) mm 50 µm Spheroid microtissues (Cross s laboratory) S.M. Ravenscroft et al. / Tox. Sci. 152 (2016) hcmp: human cardiac muscle patch (Ogle s and Zhang s laboratories) L. Gao et al. / Circ. 7 (2017)

12 Multiparametric In Vitro Cardiac Safety Prediction: Workflow for Compound Testing 1. Collect multiple parameters from several platforms Ion Channel (patch clamp) FDSS: Ca 2+ i RTCA Cardio: Impedance; Field Potential High Content: Imaging cell, mitochondrial health Other platforms 2. Integrate data from different platforms and from Pharmapendium Clinical PK 3. Create a multi-parametric compound profile ( fingerprint ) based on effects on most critical parameters 4. Assign safety risk based on comparison with compounds with known cardiac effects in clinic and in vivo (Principal Component Analysis; Unsupervised clustering) Physico Chemical properties Types and Frequency of Adverse Events 12 12

13 hipsc-cms on 3D-Aligned Nanofibers: Structural Features and Cardiotoxicity Assessment by HCI 3D Mimetix aligned nanofiber plates (top) vs. regular 2D tissue culture plates (bottom). f-actin (orange red); integrin beta-1 (green); Hoechst (blue). Image: GE InCell 2000 Analyzer, 20X obj. Scale bar = 50 µm. 13 Effect of µm conc. of sunitinib on mitochondrial intensity. Averaged single cell data (3 wells/plate per concentration and time point and 3 different plates). Haloperidol, also tested, showed no significant effects. Positive controls: antimycin (0.1 µm); staurosporine (3 µm). 13

14 hipsc-cms on 3D-Aligned Nanofibers: HCI vs 2D Assessment of Cell Viability Providing more favorable conditions to detect drug-induced changes in hipsc-cm with HCI Effect of µm conc. of sunitinib and haloperidol on cell viability. (Left) Averaged single cell data (3 wells/plate per concentration and time point and 3 different plates). Positive controls: antimycin (0.1 µm); staurosporine (3 µm). (Right) Loss of baseline impedance (cell index) in 2D hipsc-cm syncytia with concentration-dependence and timecourse for sunitinib (r. top) and haloperidol (r. bottom) 14 14

15 hipsc-cms on 3D-Aligned Nanofibers: Contractility readouts and pharmacology (FDSS Ca 2+ i) Matrix stiffness does not necessarily translate to effective tension 15 15

16 hipsc-cms on 3D-Aligned Nanofibers Effects of Substrate Stiffness - Straight vs Wavy Fibers Straight (top) and wavy (bottom) Mimetix PLLA nano-fibers. Scale bar = 500 µm. Analyzed HCI features - nucleus: intensity, morphology, texture; mitochondria: intensity, texture. Scale of mitochondrial changes (both 4 and 48 hr) and some nuclear changes (48 hr) between the sunitinib and DMSO was larger for the wavy nano-fibers 16

17 Cardiac Microtissues: Modelling Afterload on Sunitinib-Induced Cardiotoxicity Pillar stiffness vs collagen content on effective tension Impact of pillar stiffness on sunitinib effects Margulies laboratory: T. Boudou et al. / Tissue Engineering Part A; 18: (2012) R. Truitt et al. / manuscript submitted 17

18 Conclusions: Suitability of 2D or 3D Models 1. hipsc-cms grown on 3D-aligned nano-fiber plates resembled native human cardiac tissue, and the inner cell structures shared closer similarity to the native CMs, with myofibrils aligned along the long cell axis and clearly visible Z-lines. 2. No clear advantage was observed on contractility readouts, or on the effect of sunitinib or haloperidol on cell viability monitored in 2D model by impedance or in 3D model by HCI. 3. The 3D model DOES provide the means to conduct HCI assessments of cardiotoxicity (technically difficult in the 2D model). 4. Tension differences impact cardiotoxicity detection in two separate 3D models: Engineered microtissues: stiff vs soft pillars Nano-fiber aligned hipsc-cms: wavy vs. straight PLLA 5. Matrix stiffness may not translate into effective tension (confounding impact on remodelling, functionally-enabling anisotropy, etc.) Nano-fiber aligned hipsc-cms: PLLA (500 MPa) vs Bionate (7 MPa) Engineered microtissues: pillar stiffness vs. collagen content

19 Points for Discussion: A Balanced View Regarding Cardiac Tissue Models Model fidelity: 1. Spontaneously beating syncytium vs. mature adult heart (specialized conducting tissues + excitable yet quiescent contractile tissues) 2. Isotropy vs. anisotropy 3. Species relevant (e.g., focus on human ipsc-derived tissues) 4. Translatability 5. Component cells (e.g., differentiated state, optimal ratio) Practical considerations: 1. Robustness (e.g. well-to-well reproducibility, ability to maintain / test for weeks) 2. Graded vs Quantal gains (e.g., inotropy; calcium handling and T-tubules) 3. Access by pharmacological agents, nutrients, etc. (e.g., diffusion limitations, vascularization ) 4. Similar end achieved by different means (e.g., gels, native extracellular matrix, synthetic scaffolds) 19

20 THANK YOU Acknowledgments: Merck (SEP, SALAR) John Imredy Elena Trepakova Haoyu Zeng Jing Chen Bharathi Balasubramanian Holly Clouse Frederick Sannajust University of Pennsylvania Kenneth B. Margulies Elise Corbin Rachel Truitt