mesc Derived Cardiomyocytes and the RTCA Cardio Instrument - The Perfect Match for Screening Cardiac Effects

Transcription

1 mesc Derived Cardiomyocytes and the RTCA Cardio Instrument - The Perfect Match for Screening Cardiac Effects Dr. Kristina Tressat, Axiogenesis AG ELRIG Drug Discovery II 7-8 Sep 2011, Manchester

2 Challange of Drug Discovery Substantial amounts of all drugs are withdrawn due to cardiac side effects, mainly detected in late clinical development Great demand for new in vitro methods to detect cardiac side effects early in drug discovery Solution: xcelligence RTCA Cardio Instrument Approach: spontaneously beating cardiomyocytes (mouse embyonic stem (mes) cell derived, Cor.At ) for cardiac effect screening in the xcelligence Cardio Instrument Result: Reference drugs displays expected effects on cardiomyocyte function Attractive novel method for cardiac safety profiling in early drug discovery

3 Content xcelligence RTCA Cardio Instrument Description of the RTCA Cardio Instrument Principle Advantages Cor.At Cardiomyocytes Selection of cardiomyocytes from Differentiated Mouse ES Cells Characterisation Multi Electrod Array (MEA) data of reference drugs Cor.At in the xcelligence RTCA Cardio Instrument- Validation Examples of detectable effects Protocol and experimental set up Summary and Conclusion

4 The RTCA (Real-Time Cell Analyser) Cardio Instrument (by Roche Applied Science and ACEA Biosciences) Instrument for monitoring adherent contracting cells, based on microelectronic sensor technology Fast sampling rate of resistance measurement enables the detection of the cardiomyocytes beating cycle in real time

5 Principle of Cell-Based Impedance Measurement The presence of cell culture medium or buffer and application of low voltage create an electric field between the electrodes, which can be impeded by the presence of cells. Cell Index (CI) -parameter for relative change in electrical impedance. CI -influenced by cell number, cell morphology and the attachment qualities Abassi et al.2008 The contraction of cardiomyocytes involve cyclic modulation of cell morphology and adhesion to the substrate, thus inducing change in impedance. From the cyclic changes in impedance the beating frequency and the beating pattern is calculated.

6 Advantages of the RTCA Cardio Instrument Label-free, non-invasive detection 96-well format Real-time monitoring and long term measurments Automated measurement Easy setup, operation and High information content High sensitivity and accuracy

7 Production of Cor.At Cardiomyocytes from Genetically Engineered Mouse Embryonic Stem Cells Transgene integrated in ES cells Cardiac-specific promotor PuroR IRES EGFP ES Cell Aggregation Embryoid Bodies d0-2 Transfer of EBs in Cell Spinner Bottles d2 Puromycin-selected Cardiac Bodies Initiation of Puromycin Selection d9 Dissociation of Cardiac Bodies d Million Cor.At cardiomyocytes per week Freezing of Cardiomyocytes d12 Quality Control Transfer into LN2

8 Characterization of Cor.At cells demonstrate full Cardiomyocyte functionality IK ICa Cardiomyocytes beat spontaneously. Clusters of coupled cardiomyocytes show synchronous calcium flux. INa Cardiomyocytes transplanted into a cryoinfarcted area of a mouse heart fully integrate and restore function (Kolossov et al. 2006) Cardiomyocytes form syncitium. Staining for actinin and gap junction protein CX43. The functionality of the three ion currents IK, ICa and INa was demonstrated by voltage clamp experiments

9 Functionality of Ion Channels/Receptors in Cor.At Cardiomyocytes analysed by Multi Electrode Array (MEA) Induction of arrythmic beating by herg blocker Dofetilide Negative chronotropic effect of the INa blocker TTX Positive chronotropic effect of the ß-adrenergic receptor agonist Isoproterenol Positive chronotropic effect of the ICa blocker Verapamil intervall: 30 s Induction of arrythmic beating starting at 100 nm decrease of beating frequence at 0.1µM, beating arrest at 10µM increase of beating frequence at 1nM increase of beating frequence at 0.1µM, beating arrest at 10µM

10 The Optimal Cellular Model Physiological properties Highly Pure Ready-to-use availability Lot-to-lot reproducibility Relevant and predictive Services offered: Mel.COR- MEA Patch Clamp C.Tox-Cardiotox CardioEffect- RTCA Cardio

11 Cor.At in the RTCA Cardio Instrument: Optimized Experimental protocol Cor.At cardiomyocytes /96-well medium exchange twice a day cultivation for 40-46h prior addition of compounds For addition of compound and vehicle, remove half volume of the media and add of 2x conc. compound Wash out of compound after 24h

12 Cor.At Cardiomyocytes in the RTCA Cardio Instrument-Spontaneously beating is required Cardio 96 well plate prior compound addition 10s Excluded Excluded Excluded Excluded Every well can be monitored at any time point in parallel The data are normalized to the baseline measured in each well prior compound addition. Wells not fulfilling the quality criteria (beating rate, amplitude) are excluded

13 sts Different Beating Pattern are induced by different Compounds Control after compound addition 10s Fibrillation like Bursts frequency decrease Arrythmic beating Beating arrest Amplitude decrease

14 Validation Compounds analysed ß-adrenergic receptor agonist Isoproterenol Activator of L-type calcium channels (S)-(-) Bay K8644 Cardiac voltage-gated Na + channel blocker Tetrodotoxin (TTX) L-type calcium channel blocker Nifedipine Isradipine herg/ikr blocker E-4031 Dofetilide Cisapride Astemizol herg trafficking blocker Pentamidine Inhibitor of the myosin heavy chain ATPase Blebbistatin - inhibit cardiomyocyte contraction

15 Isoproterenol: Experimental Setup Total Cell Index during experimental time Adhering phase M M Compound M wash out M Cell Index (CI) Experimental time in hours Isoproterenol M= Medium change Vehicle

16 Isoproterenol: The ß-adrenergic Receptor Agonist induces an increase in beating Frequency Baseline 20s Time: 30 min 10µM 1µM 100nM 10nM 1nM Time: 12h 10µM 1µM 100nM 10nM 1nM 10µM 1µM 100nM 10nM 1nM Time: 24h wash out Concentration dependent increase in beating frequency with the maximal effect reached at 100nM and the lowest effective at 1nM. After 12h this results in irregular beating and bursts. Effects can be washed out

17 Tetrodotoxin (TTX): A Cardiac Voltage-gated Na + Channel Blocker induces a reduction in beating Frequency 30 min 20s 10µM 1µM 100nM 10nM 1nM TTX shows a significant negative chronotropic effect after 30 min at 1µM

18 L-type Calcium Channel Blocker induce beating arrest of Cor.At cardiomyocytes Time: 30 min Isradipine 20s Time: 30 min Nifedipine 20s 10µM 1µM 100nM 10nM 1nM 10µM 1µM 100nM 10nM 1nM Time: 24h, wash out Time: 24h, wash out Beating arrest at 100nM, Effects can partly be washed out Beating arrest at 1µM, Effects can partly be washed out

19 (S)-(-) Bay K8644: Activator of L-type Calcium Channels, induce Bursts in Cor.At Cardiomyocytes 10µM 1µM 100nM 10nM 1nM Time: 30 min 20s beating frequency increase and arrythmic beating can be detected at concentration 100nM, after 30 min 10µM 1µM 100nM 10nM 1nM Time: 6h Bursts can be detected at concentration 6h at 100nM Time: 24h, washout Effects can be washed out at the lower concentration

20 All tested herg/ikr Blocker induce arrythmic beating in Cor.At Cardiomyocytes E4031 Dofetilide Time: 30 min 20s Time: 30 min 20s 1µM 250nM 100nM 1µM 100nM 10nM Time: 4h Time: 4h 1µM 250nM 100nM 1µM 100nM 10nM Induce arrythmic beating at 250nM, beating pattern changed with time Induce arrythmic beating at 100nM, beating pattern changed with time

21 All tested herg/ikr Blocker induce arrythmic beating in Cor.At Cardiomyocytes Time: 30 min Cisapride Astemizole 20s 20s Time: 30 min 10µM 2.5µM 0.5µM 1µM 100nM 10nM Time: 4h Time: 4h 10µM 2.5µM 0.5µM 1µM 100nM 10nM Induce fibrillation like beating at the lowest conc. tested 0.5µM and arrythmic beating at 2.5µM Induce arrythmic beating at 100nM, beating pattern changed with time

22 Pentamidine: The herg trafficking Blocker induce Beating Irregularities first after 12h Induction Time: 30 min 20s 200µM 20µM 2µM No beating irregularities seen after 30min Time: 12h 200µM 20µM 2µM First beating irregularities seen after 12h with 20µM Time: 24h 200µM 20µM Long term effects 2µM

23 Blebbistatin: An Inhibitor of the Myosin Heavy Chain ATPase induce Beating Arrest Time: 30 min 20s 100µM 10µM 1µM At the lowest concentration tested 1µM, the amplitude is clearly reduced. Concentrations 10µM induce beating arrest Time: 24h wash out Effects can be washed out with one medium change at the lower concentrations Field potential recording (Multi-Electrode Arrays) Blebbistatin inhibits cardiomyocyte contraction, an effect which can not be detected using electrophysiology Abassi et al.,2011

24 Conclusions: An in vitro system monitoring combined effects on different targets involved in cardiac contraction All reference drugs used showed expected effects/beating profiles Early and late effects on cardiac contraction can be detected Compounds not detected by electrophysiological methods can be identified High predictability Potential use for herg effects Can be used for safety pharmacology as well as identification of new APIs effecting cardiac contractility

25 Publication Br J Pharmacol Aug 12. [Epub ahead of print] Dynamic Monitoring of Beating Periodicity of Stem Cell Derived Cardiomyocytes as a Predictive Tool for Preclinical Safety Assessment. Abassi YA, Xi B, Li N, Ouyang W, Seiler A, Watzele M, Kettenhofen R, Bohlen H, Ehlich A, Kolossov E, Wang X, Xu X. Source ACEA Biosciences Inc., San Diego, California, USA; Roche Applied Science, Penzberg, Germany; Axiogenesis AG, Cologne, Germany; Hangzhou High Throughput Screening Center, Hangzhou District, Zhejiang Province, China.

26 Axiogenesis AG Service Today Screening Service of compounds on the RTCA Cardio Instrument using Cor.At Service package for safety screening: CardioEffect (RTCA Cardio), Mel.COR (MEA), Patch Clamp and C.Tox (Cardio toxcicity assay) Future Human ips derived cardiomyocytes Induced cardiac disease model for drug discovery Hypertrophic cardiomyopathy model

27 Acknowledgment Axiogenesis AG: Dr. Heribert Bohlen Dr. Ralf Kettenhofen Anika Duenbostell Roche Diagnostics GmbH: Dr. Markus Schmitz Distributors: world wide: Japan:

28 Thank you for your attention!

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30 Why is it possible to identify herg blocker effects in mouse ES cell-derived cardiomyocytes?

31 Developmental Changes of Mouse Cardiac Repolarization Change of the Dofetilide Sensitivity of Mouse Cardiac Repolarization Wang et al. Developmental changes in the delayed rectifier K + channels in mouse heart. Circ Res Jul;79(1):79-85.

32 Summary Compound class Compound Effect conc. RTCA Effect conc. MEA herg/ikr blocker E-4031 IB 250 nm fadp 50nM, IB 250nM Cisapride IB 500 nm fadp 100nM, IB 1000nM Dofetilide IB 100 nm fadp 10nM, IB 100nM herg trafficking blocker Astemizole IB 100nM ND Pentamidine IB 2000nM ND Na + channel blocker Teterodoxin (TTX) NC 1000nM NC 100nM, BA1000nM L-type calcium channel blocker Nifedipine BA 1000nM BA 200nM Isradipine BA 100nM ND L-type calcium channel activator (s)-(-) Bay K8644 IB 100nM PC 100nM ß-adrenergic receptor agonist Isoproterenol PC 1nM 1 nm ATPase inhibitor Blebbistatin 1000nM No effect IB- irregular beating PC- positive chronotropic NC- negative chronotropic BA-beating arrest fadp- field action potential duration ND- not determined