Multiparametric assessment of the effects of chemotherapeutic drugs on the (electro)physiology of Pluricyte Cardiomyocytes
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1 Multiparametric assessment of the effects of chemotherapeutic drugs on the (electro)physiology of Pluricyte Cardiomyocytes Event Presenter Date Ncardia Workshop Tessa de Korte, MSc December 1, 2017 Page 1
2 Ncardia s hipsc-cms and applications Assessment of functional toxicity Assessment of structural toxicity Multiplex assays MEA assays Troponin I Release assays Combined functional and structural toxicity assessment Page 2
3 1 Ncardia s hipsc-cms and assays Page 3
4 Human ipsc-derived cardiomyocytes Fully functional human ipsc-derived cardiomyocytes obtained through in vitro differentiation of transgenic hipscs and puromycin selection technology obtained without genetically modification or purification/selection procedures ü Showing the essential human physiological conditions and pharmacological responses (3R s benefit) Movie of beating monolayer Pluricyte Cardiomyocyte Kit Cor.4U Cardiomyocyte Kit CardioPlate TM Alpha actin (green) Myosin heavy chain 7 (red) Page 4
5 Supported applications for Pluricyte and Cor.4U Cardiomyocytes MEA: ü MCS 96 ü Axion Maestro ü Classic 48 & 96, E-Stim 48, Lumos 48 MEA (and impedance) technology USER GUIDES AND APPLICATION NOTES AVAILABLE HTS Ca 2+ flux technology USER GUIDES AND APPLICATION NOTES AVAILABLE Biomarker assays MEA/Impedance: ü xcelligence Cardio 96 ü xcelligence CardioECR 48 ü CardioExcyte 96 Voltage, Calcium, Contraction Calcium-flux: ü FDSS/µCELL 96 & 384 ü FLIPR Tetra 96 & 384 Pro-BNP ATP Troponin release Metabolite biomarkers Page 5
6 2 Assessment of functional toxicity MEA assays Page 6
7 Assessment of functional toxicity of Pluricyte Cardiomyocytes using MEA technology Functional toxicity: To predict the potential for drug-induced ECG abnormalities. Alterations in the sodium-spike amplitude Axion Maestro system Alterations in FPD and occurrence of arrhythmia CardioECR instrument ECR 1 Chronotropic and inotropic effects CardioECR instrument Impedance ECR (mv) herg K + channel block Na + Channel block ECR (mv) 0 nm E nm E nm E nM E4031 Time (s) nm E nm Isoproterenol 30 nm Isoproterenol Time (s) 60 1 min. Page 7
8 3 independent testing sites confirmed the predictivity of Cor.4U Cardiomyocytes as a human cellular model Electrophysiological analysis of Cor.4U on the MEA System Beat period FPD Pharmacological analysis of Cor.4U on the MEA System Analysis of different parameters by testing site Comparison of different testing sites per compound Page 8
9 3 Assessment of structural toxicity Troponin I Release assays Page 9
10 Assessment of structural toxicity of Pluricyte Cardiomyocytes using Troponin I release assays Structural toxicity: morphological damage to cardiomyocytes, changes to intracellular organelles, or loss of cardiomyocyte viability Cytotoxicity assays Pro-BNP release assays Troponin I release assays 4 [N T -p ro B N P ] O D Basel level N o E T n M E T -1 0 Page 10
11 Troponin I release assay The troponin complex consists of three subunits: troponin C, troponin I, and troponin T, located on the myofibrillar thin (actin) filament of striated (skeletal and cardiac) muscle In some diseases (e.g. pulmunary embolism), transient leakage of cytosolic TnI into the blood stream has been reported. During myocardial infarction which results in serious myocyte damage rapid release of TnI into the blood steam occur and can last up to days The same holds true in case of cardiotoxicity of drugs e.g in chemotherapy Therefore, ctni detection in blood is an important diagnostic marker for several cardiovascular diseases Page 11
12 Cardio-oncology: the intersection of cardiovascular disease and cancer Incidence of drug-induced structural cardiotoxicity à associated with the use of anthracycline anti-cancer drugs, but has also been shown to occur following treatment with the new generation of targeted anti-cancer agents: tyrosine-kinase inhibitors Prognosis for many cancers improving + aging of the population à increased the focus on the cardiovascular risks of cancer patients (Sanjeev A. et al, 2015, Albini et al, 2010) Cardiotoxicity associated with anticancer drugs: Direct cytotoxic effects and associated cardiac systolic dysfunction Left ventricular (LV) dysfunction Cardiac ischemia QT-prolongation Thrombosis Metabolic abnormalities Arrhythmias Pericarditis Repolarization abnormalities Normal heart Heart Failure Page 12
13 ctni release assay to assess potential structural toxicity of TKI s Setup: Specimen Compounds Harvest Readout à Pluricyte Cardiomyocytes on Matrigel coated plates (384 W plates) à lapatinib, sunitinib and staurosporine (10, 5, 2,5, 1,25, 0,6 and 0.1 µm) à 16 and 24 hr post-treatment à ctni release assay Timeline: D0 D1 D3 D6 D7 (16hr and 24 hr) ctni release assay Seed PCs in PCM Med ref Med ref Med ref + Cpd addition Harvest sup + Lyse cells Page 13
14 Sunitinib and staurosporine induce structural toxicity in Pluricyte Cardiomyocytes after long-term incubation Lapatinib Sunitinib Staurosporine Page 14
15 4 Multiplex assays Functional and structural toxicity assessment Page 15
16 Multiplexing ctni release assay with CardioECR assay (Impedance and ECR) to assess potential structural and functional toxicity Setup: Specimen Compounds Harvest Readout à Pluricyte Cardiomyocytes on Fibronectin coated E-plates (48 W plates) à Ponatinib, doxorubicin, lapatinib, nilotinib (0.3, 3 and 10 µm) and nitrendipine (0.01, 0.1 and 1µM) à Acute, 1, 16, 24, 40, 48 and 64 hr post-treatment à ctni release and CardioECR (Impedance and ECR) Timeline: CardioECR assay Acute 64hr D0 D1/D3/D5 D8 D11 (64hr) D10 (40hr and 48hr) D9 (16hr and 24hr) ctni release assay Seed PCs in PCM Med ref Cpd addition Harvest sup Page 16
17 Multiplexing Impedance/MEA and Troponin I release CI over time Troponin I release Impedance amplitude Ponatinib Ponatinib Ponatinib counts u M u M n M C trl CI/Impedance peak amplitude h r 2 4 h r 4 0 h r 4 8 h r 6 4 h r Doxorubicin Doxorubicin Doxorubicin counts u M u M n M C trl h r 2 4 h r 4 0 h r 4 8 h r 6 4 h r Nitrendipine Nitrendipine Nitrendipine C a rd io A m p litu te (m V ) b a s e lin e a c u te 1 h r 1 6 h r 2 4 h r 4 0 h r 4 8 h r 6 4 h r 1 0 u M 3 u M n M C trl CI/impedance over time well-connected monolayer of cells à High impedance e.g. drug addition counts Disturbed cell monolayer à deattachement and cell loss à Low impedance Page 17
18 Multiplexing Impedance/MEA and Troponin I release CI over time Troponin I release Impedance and ECR Lapatinib Lapatinib 0 µm lapatinib 10 µm lapatinib Nilotinib counts counts Nilotinib 0 µm Nilotinib 10 µm Nilotinib Page 18
19 Concluding remarks Troponin I Immunoassay allows us to detect small amount of ctni (around 10 pg/ml) using small sample volume (5 µl) W formats It is possible to perform kinetic measurement of ctni release in a 384W format and in combination with other functional assays Multiplexing the ctni release with impedance and MEA technology leads to better prediction of drug-induced cardiotoxicity Short term vs long term, structural vs functional toxicity could be assessed simultaneously from a single well Page 19
20 Safety Services Integrated cardiovascular services to enhance the assessment of cardiac safety and toxicity of drug candidates. Based on fully functional and validated hipsc-derived cardiomyocytes that are manufactured under strict quality assurance. Ø Ø Ø Ø Ø Ø Ø Ø Cardio.Acute Cardio.Long Cardio.Plex Cardio.Effect Cardio.Force Cardo.Flux Cardio.Tox Troponin I Customized Project Service Benefits of Ncardia s safety services: ü High quality our safety pharmacology experts perform the studies on validated platforms ü Save time - no assay optimization and data analysis needed let our experts do this for you ü Reliable assays are based on fully functional and validated hipsc-derived cardiomyocytes ü Versatile we offer a broad range of optimized assays on various platforms ü Flexible we will make sure to meet your specific needs Page 20
21 Thank you! Tessa de Korte, MSc Page 21
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