xcelligence Cardio Symposium 2014 Cardiotoxicity, QT Prolongation, and Arrhythmia Prediction Using icell Cardiomyocytes: Past, Present, and Future

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1 xcelligence Cardio Symposium 2014 Cardiotoxicity, QT Prolongation, and Arrhythmia Prediction Using icell Cardiomyocytes: Past, Present, and Future Blake Anson Ph.D. Oct 18, 2014

2 Outline Proarrhythmia Testing - moving from single channel to holistic assessments Cardiotoxicity Testing - icell Cardiomyocytes and xcelligence RTCA Drug Discovery and Population - Disease modeling and incorporating populations Key manufacturing components

3 In-vitro detection of proarrhythmia The road to in-vitro proarrhythmia testing.. started in a fly

4 Drug induced Electrophysiological Aberrations not a new phenomenon Quinidine Syncope and Delayed Repolarization Syndromes. Reynolds E and Vander Ark C. M Modern Concepts of Cardiovascular Disease. 45: , but took on a new meaning when caused by non-cardiac compounds Davies et al., BMJ 1989:298 Wyosowski and Bacsanyi NEJM 1995:335 Astemizole-induced Arrhythmmia. From Vorperian et al., JACC 1996:15

5 Fruit flies provided insight to arrhythmia EAG Gene: Ether-sedated Drosophila (Fruit Flies) Leg shaking EAG mutant (ether-a-go-go) Wild type B. Ganetzky

6 herg is a member of the EAG superfamily of K + channels EAG ether a go-go gene sequence Library Screen(s) Hippocampal mrna Hit Analysis herg human ether a go-go related gene Heterologous Expression The herg gene is linked to Long QT Syndrome The herg gene encdes lkr

7 Arrhythmogenic drugs block herg channels and prolong the cardiac AP DIA Zhou and January, 1997

8 herg and In-vitro Safety Assessments Highly sensitive with questionable specificity

9 Comprehensive in-vitro Proarrhythmia Testing The Future Comprehensive in-vitro Proarrhythmia Assessment (CiPA) 1. Assess effects on multiple individual ion channels 2. Model effects (if any) on the ventricular action potential and proarrhythmia 3. Verify conclusions with cardiomyocyte recordings Sager, et al. American Heart Journal (2014)

10 Pro-arrhythmia Testing and Beyond

11 Interrogating Biology Electrical and Mechanical Activity Electrical Cardiomyocyte Activity Electrical, biochemical, and mechanical Biochemical Mechanical Three main areas need to be considered for cardiotoxicity

12 Predicting Proarrhythmia Label Free Impedance Measurements Relevant biology and metrics leads to greater predictivity Qualitative Assessment Expanded dataset o ~120 compounds Fine tune metrics o Include beat rate, atypical beats, onset of IB20 o Use concentration thresholds or IB20 rank ordering Guo et al., 2013 Guo et al., 2011 Greater Predictivity ~120 Compounds >90% -- QT prediction >82% -- arrhy. prediction icell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

13 KI-induced Cardiotoxicity Deconvoluting the problem S. Lamore AstraZeneca icell Cardiomyocytes provide a predictive tool for detecting KI toxicity

14 Detecting Effects on Contractility Moving to higher throughput predictive detection Conventional Interrogation IonOptix Good to excellent validation parameters Primary culture from dog heart Low throughput 1 AR Harmer. Tox App Pharm 2012 Screening with icell Cardiomyocytes xcelligence RTCA Good to excellent assay parameters 2 Human cardiomyocytes Medium to high throughput Parameter IonOptix sensitivity 83% specificity 84% accuracy 82% pos predict 90% neg predict 76% Parameter Impedance 3 sensitivity 90% specificity 74% accuracy 84% pos predict 85% neg predict 82% 49 compound validation set with actives and inactives 2 C. Scott (Tox Sci, 2014 ) icell Cardiomyocytes provide a predictive model for detecting contractility

15 icell Cardiomyocytes and xcelligence RTCA: Predictive solutions for multi-modal cardiotoxicity

16 Actin Reorg Cell Size Disease Modeling Case #1: Cardiac Hypertrophy Cellular and Molecular Markers Increased cell size Enhanced protein synthesis/ sarcomeric organization Re-activation of the fetal gene program (BNP, ANP, etc) Normal Untreated + PE Kuwahara K et al. J Pharmacol Sci 2012;119: Protein Synthesis BNP Expression Diseased Lister K et al. Cardiovasc Res 2006;70: Glenn D et al. Hypertension 2009;53:

17 In-Vitro Recapitulation of Hypertrophy icell Cardiomyocytes Cell Size Cytoskeletal Rearrangements Fetal Gene Expression Control ET-1 (10 nm) Control +ET-1 (10 nm) Control +ET-1 (10 nm) 1500 Total Area ( m 2 ) Log [ET-1] (M) icell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

18 icell Cardiomyocyte Hypertrophy Relevance Aggarwal et al., Plos One 2014 Hypertrophic icell Cardiomyocytes share similarities with cardiac samples from LVH patients

19 Case #2: Diabetic Cardiac Myopathy Environmental Induction Application of a diabetic medium (ET-1, cortisol, glucose) to icell CMs induces a hypertrophic phenotype Increases in; Cell and nuclear size Glycolysis Cytoskeletal disorganization Lipid accumulation ROS Accumulation Drawnel, 2014 in press

20 ROS Production Case #3: Diabetic Cardiac Myopathy Environmental Induction ipsc-cms from diabetic patients exhibit markers of hypertrophy under basal conditions S: icell CMs SP: MyCells; Di-CM appearance > 15 yrs post diabetes onset FP : MyCells; Di-CM appearance < 5 yrs post diabetes onset Cytoskeletal disorganization Lipid Accumulation Compounds have been identified that revert the diabetic phenotype present in the ipsc-cms Oxidative Stress Drawnel, 2014 in press

21 ctnt Case study #4: Induced disease models MYH7-R403Q linked hypertrophic cardiomyopathy A B C Cell Type icell CM Viability MyCell MYH7 R403Q CM 98% 96% Plating Efficiency icell Cardiomyocytes (CM) 79% 56% MyCell MYH7 R403Q CM 76% 45% D ET-1 induced icell CM MyCell MYH7 R403Q CM NPPB 5 ACTA1 4 DUSP4 3 ACTC1 2 ACTN1 1 CREB5 0 MYH7-1 NPPA -2 MYH6-3 TRIM63-4 ADM -5 FBXO32 PDCD4 Relative Expression MYH7 R403Q hypertrophic cardiomyopathy (A) (C) Manufacture of custom lines is similar to catalog products (D) MYH7 R403Q show signs of cardiac hypertrophy under basal conditions hipsc-cardiomyocytes mimic induced and innate disease models

22 Case study #4: Induced disease models MYH7-R403Q linked hypertrophic cardiomyopathy icell CM MYH7 R403Q CM BNP / DAPI 10X image in 384-well plate. icell and MYH7 R403QCMs both respond to pathway inhibition icell and MYH7 R403QCMs differ in basal BNP expression but respond similarly to ET-1 induction MYH7 R403Q CMs Carry a hypertrophic phenotype Are inducible via ET1 React to phenotypic reversion screens Represent a disease-based screening model hipsc-cardiomyocytes mimic induced and innate disease models

23 The Power of IPSC Technology What about. populations?

24 Standardization Manufacturing Benchmarks Scale-Up Manufacturing Quality Quantity Purity CDI Manufacturing Benchmarks (cells per day, >95% purity) 2 billion ips cells 1 billion cardiomyocytes 1 billion neurons 0.5 billion endothelial cells 0.4 billion hepatocytes Scale-Out Manufacturing 1000 s of individuals Billions of cells NHLBI Next Generation Genetic Association Studies (RFA-HL ) 250 patient samples - HyperGEN cohort GWAS Left Ventricular Hypertrophy (LVH) Derive ips cells and cardiomyocytes from all 250 individuals Induce hypertrophy phenotype, perform molecular analyses Correlate GWAS findings with in vitro phenotype

25 Progress Report Population genomics and left ventricular hypertrophy NHLBI Next Generation Genetic Association Studies (RFA-HL ) 250 patient samples HyperGEN cohort GWAS Left Ventricular Hypertrophy (LVH) Derive ips cells and cardiomyocytes Induce hypertrophy, perform molecular analyses Correlate GWAS findings with in vitro phenotype Progress as of July 2014: 250 donors reprogrammed Differentiation protocol optimized to work robustly across all lines 128 ips cell lines (1 per donor) are differentiated or in progress Cardiomyocytes from 89 donors cryopreserved & all pass QC 20 batches of cardiomyocytes are in currently being tested in hypertrophy assays Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel, MCOW) CDI s ipsc technology is enabling population studies

26 CIRM Award ips Cell Manufacture & Banking California Institute for Regenerative Medicine (CIRM) Human ips Cell Initiative 3 Awards Sample Collection (7 awardees) ips Cell Derivation (CDI) ips Cell Banking (Coriell; CDI primary subcontractor) ips Cell Derivation 3000 donors (healthy & disease phenotypes) 3 ips cell clones per donor Disease categories: epilepsy, autism, cerebral palsy, cardiomyopathy, Alzheimer s disease, eye diseases, hepatitis (HCV), non-alcoholic steatohepatitis (NASH), pulmonary fibrosis Derived from peripheral blood (preferred) or skin fibroblasts Episomal footprint-free method CDI Coriell Partnership Extensive collaboration to bring together expertise in electronic record-keeping, sample tracking, ips cell derivation & characterization, cell banking & distribution Joint facility located within the Buck Institute, Novato, CA

27 Will this potential be realized? Yes But it is not easy

28 Cell Purity CDI Commitment Quality, Quantity, Purity Exhibit key cellular characteristics Recapitulate normal human biology Reproducible Known and relevant genotype Quality Target Cell (non proliferating) Non-Target Cell (proliferating) Days in Culture Quantity Purity Sufficient to support HTP drug screening and safety testing Currently 1Bn icell Cardiomyocytes/day

29 QMS Framework Overview Key Systems QA/QC Standard Operating Procedures Calibration/Qual/Val Change Management CAPA Supplier Qual & Mgmt Materials Management Training Complaint Handling New Product Introduction Objectives Compliance and product consistency Consistent procedures Equipment/facilities/processes fit for intended use Changes are documented, assessed for risk, and tested Report, correct, and prevent product quality issues Quality and reliability of raw materials Control, trace, and monitor stock inventory Education and proficiency Customer satisfaction and continuous improvement Improve likelihood that product meets market need An ISO / GMP hybrid QMS system ensures customer safety and satisfaction

30 icell Cardiomyocytes Market Validation (8//2014) 1. Nakamura Y 1, Matsuo J (2014) Assessment of testing methods for drug-induced repolarization delay and arrhythmias in an ips cellderived cardiomyocyte sheet: multi-site validation study. J Pharmacol Sci. 124(4): Eldridge S, Guo L, et al. (2014) Examining the Protective Role of ErbB2 Modulation in Human Induced Pluripotent Stem Cell- Derived Cardiomyocytes. Toxicol Sci Jul 23. pii: kfu150. [Epub ahead of print] 3. Kolaja K. (2014) Stem cells and stem cell-derived tissues and their use in safety assessment. J Biol Chem Feb 21;289(8): Uesugi M, Ojima A, et al. (2014) Low-density plating is sufficient to induce cardiac hypertrophy and electrical remodeling in highly purified human ips cell-derived cardiomyocytes. J Pharmacol Toxicol Methods. 69(2): Cameron BJ, Gerry AB, et al. (2013) Identification of a Titinderived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells. Sci Transl Med. 5(197):197ra Carlson C, Koonce C, et al. (2013) Phenotypic screening with human ips cell-derived cardiomyocytes: HTS-compatible assays for interrogating cardiac hypertrophy. J Biomol Screen 18(10): Doherty, K., Wappel, R. et al. (2013) Multiparameter in vitro toxicity testing of crizotinib, sunitinib, erlotinib, and nilotinib in human cardiomyocytes. Toxicol Appl Pharmacol. 272(1): Fine M, Lu F, et al. (2013) Human Induced Pluripotent Stem Cellderived Cardiomyocytes for Studies of Cardiac Ion Transporters. Am J Physiol Cell Physiol. 305(5):C Guo L, Coyle l, et al. (2013) Refining the Human ipsc- Cardiomyocyte Arrhythmic Risk Assessment Model. Toxicol Sci 136(2): Harris K, Aylott M, et al. (2013) Comparison of Electrophysiological Data from Human Induced Pluripotent Stem Cell Derived Cardiomyoyctes (hipsc-cms) to Functional Preclinical Safety Assays. Toxicol Sci. 134(2): Ivashchenko CY 1, Pipes GC, et al. (2013). Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype. Am J Physiol Heart Circ Physiol. 305(6):H Jehle J, Ficker E, et al. (2013) Mechanisms of Zolpidem-induced Long QT Ayndrome: Acute Inhibition of Recombinant herg K+ Channels and Action Potential Prolongation in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells. British J Pharm 168: Puppala D, Collis LP, et al. (2013) Comparative Gene Expression Profiling in Human Induced Pluripotent Stem Cell Derived Cardiocytes and Human and Cynomolgus Heart Tissue. Toxicol Sci 131: Rao C, Prodromakis T, et al. (2013) The effect of microgrooved culture substrates on calcium cycling of cardiac myocytes derived from human induced pluripotent stem cells. Biomaterials 34(10): Schweikart K, Guo L, et al. (2013) The Effects of Jaspamide on Human Cardiomyocyte Function and Cardiac Ion Channel Activity. Toxicol in Vitro 27: Sirenko O, Crittenden C, et al. (2013) Multiparameter In Vitro Assessment of Compound Effects on Cardiomyocyte Physiology Using ips Cells. J Biomol Screening 18: Sirenko O, Cromwell EF, et al. (2013) Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity. Toxicol Appl Pharmacol 273(3): Babiarz JE, Ravon M, et al. (2012). Determination of the Human Cardiomyocyte mrna and mirna Differentiation Network by Fine-scale Profiling. Stem Cells Dev 21: Cerignoli R, Charlot D, et al. (2012) High Throughput Measurement of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cellderived Cardiomyocytes by Kinetic Image Cytometry. J Pharmacol Toxicol Methods 66: Lee P, Kloss M, et al. (2012) Simultaneous Voltage and Calcium Mapping of Genetically Purified Human Induced Pluripotent Stem Cell-derived Cardiac Myocyte Monolayers. Circ Res 110: Mioulane M, Foldes G, et al. (2012) Development of High Content Imaging Methods for Cell Death Detection in Human Pluripotent Stem Cell-derived Cardiomyocytes. J of Cardiovasc Trans Res 5: Rana P, Anson BD, et al. (2012) Characterization of Humaninduced Pluripotent Stem Cell-derived Cardiomyocytes: Bioenergetics and Utilization in Safety Screening. Toxicol Sci 130: Reynolds JG, Geretti E, et al. (2012) HER2-targeted Liposomal Doxorubicin Displays Enhanced Anti-tumorigenic Effects without Associated Cardiotoxicity. Toxicol Appl Pharmacol 262: Wei H, Zhang G, et al. (2012) Hydrogen Sulfide Suppresses Outward Rectifier Potassium Currents in Human Pluripotent Stem Cell-Derived Cardiomyocytes. Plos One 7(11):e Zhi D, Irvin MR, et al. (2012) Whole-exome Sequencing and an ipsc-derived Cardiomyocyte Model Provides a Powerful Platform for Gene Discovery in Left Ventricular Hypertrophy. Frontiers in Genetics 3: Cohen JD, Babiarz JE, et al. (2011) Use of Human Stem Cellderived Cardiomyocytes to Examine Sunitinib Mediated Cardiotoxicity and Electrophysiological Alterations. Toxicol Appl Pharmacol 257: Guo L, Qian JY, et al. (2011) The Electrophysiological Effects of Cardiac Glycosides in Human ipsc-derived Cardiomyocytes and in Guinea Pig Isolated Hearts. Cell Physiol Biochem 27: Guo L, Abrams RM, et al. (2011) Estimating the Risk of Druginduced Proarrhythmia Using Human Induced Pluripotent Stem Cell-derived Cardiomyocytes. Toxicol Sci 123: Jonsson MKB, Wang QD, et al. (2011) Impedance-based Detection of Beating Rhythm and Proarrhythmic Effects of Compounds on Stem Cell-derived Cardiomyocytes. Assay and Drug Dev Tech 9: Ma J, Guo L, et al. (2011) High Purity Human-induced Pluripotent Stem Cell-derived Cardiomyocytes: Electrophysiological Properties of Action Potentials and Ionic Currents. Am J Physiol Heart Circ Physiol 301:H2006-H2017. ~40 Peer-reviewed Publications (10/2014): Characterization Toxicity testing Disease modeling

31 Summary Proarrhythmia Testing - moving toward a cellular, mechanistic approach that may take advantage of stem cell cardiomyocytes Cardiotoxicity Testing - icell Cardiomyocytes and xcelligence RTCA provide predictive solutions Drug Discovery and Population - CDI products exhibit induced and innate disease phenotypes for drug discovery Key manufacturing components - Quality is king!

32 Product Portfolio icell Cardiomyocytes icell Endothelial Cells icell Neurons icell Hepatocytes icell Astrocytes icell Cardiac Progenitor Cells icell DopaNeurons icell Products icell Cardiomyocytes icell Cardiac Progenitor Cells (New) icell Hematopoietic Progenitor Cells icell Endothelial Cells icell Hepatocytes icell Neurons icell Astrocytes icell DopaNeurons (New) icell Skeletal Myoblasts MyCell Products ips Cell Reprogramming ips Cell Genetic Engineering ips Cell Differentiation MyCell Disease and Diversity Panel (New) MyCell Products Essential 8 Medium Episomal Reprogramming Kit Vitronectin icell Hematopoietic Progenitor Cells icell Skeletal Myoblasts