Human ips/es Cell Technology and Its Applica;on to Toxicology Tes;ng

Transcription

1 Human ips/es Cell Technology and Its Applica;on to Toxicology Tes;ng - Especially focusing on in vitro cardiac func;on toxicity- Atsushi Sanbuissho (Daiichi Sankyo Co. Ltd.) Kenji Yasuda (Tokyo Medical and Dental Univ.)

2 Outline Preclinical Tes;ng for Cardiac Electrophysiologic Safety - ICH S7A and the following S7B Guidance - herg: Ability and Limita;on - Beyond herg: On- chip Quasi- in vivo Assay - Human Cardiomyocytes delivered from hes/hips Cells - Strategy I: Temporal fluctua;on measurement - Strategy II: Spa;al tachyarrhythmia/fibrilla;on measurement Call for adaptable preclinical strategies to evaluate global cardiac safety

3 Goals for Preclinical Safety Tes;ng Primary Goal: Ensuring The Safety of Phase 1 Volunteers Addi;onal Interests Represent a Mix of Internal and Regulatory Issues - Guide selec;on of Discovery candidates - Provide pharmacodynamic informa;on on metabolites - Minimize azri;on later in Discovery/Development - Guide conduct of early First in Human studies - Enable early Concentra;on- QT(VT/VF) studies - Regulatory Issues Call for adaptable preclinical strategies to evaluate global cardiac safety

4 Preclinical Tes;ng: Regulatory Perspec;ves ICH S7A: Safety Pharmacology Studies for Human Pharmaceu;cals Objec;ves: - - To protect clinical trial par;cipants and pa;ents receiving marketed products from poten;al adverse effects of pharmaceu;cals Avoiding unnecessary use of animals and other resources. Effects of the test substance on the cardiovascular system should be assessed appropriately. Blood pressure, heart rate, and the electrocardiogram should be evaluated. In vivo, in vitro and/or ex vivo evalua;ons, including methods for repolariza;on and conductance abnormali;es, should also be considered. S7B represents minimal approach to addressing repolariza;on safety, not consider global cardiac electrophysiologic effects.

5 herg: Ability and Limita;ons S7B in vitro minimal approachherg assay Excep;ons to link between herg block and TdP - - Most drugs that have been removed from the market block herg current. However, not all drugs that block herg prolong QT or TdP (Verapamil, fluoxe;ne, tolterodine.) - IC50 values must be placed in context. (most cmpds block herg at excessive conc.) Likely that compounds are unduly discarded in Discovery due to early herg screening

6 Necessity to Go Beyond herg U;lity of herg Assay (in isola;on) violates Pharmacology Rule #2 No one assay is 100% Sensi;ve and Specific Elimina;ng herg liability removes one proarrhythmic risk factor Ventricular fibrilla;on might occur without QT prolonga;on herg current assay: A surrogate marker for a surrogate marker herg Mul;ple factors QT Prolonga;on Mul;ple factors Torsades de Pointes Ventricular Tachycardia Ventricular Fibrilla;on Predic;ng VT(TdP), VF Ensuring Global Cardiac Safety

7 Beyond herg: Our Solu;on On- chip Quasi- in vivo Assay Quasi- in vivo assay includes: 1) Using a set of standard human cardiomyocytes prepared from human ips/es cells of different races, sexes and also from pa;ents with various diseases to provide an ideal tes;ng panel plaiorm. 2) To predict lethal arrhythmia (TdP/VT/VF) by evalua;on of (1)temporal fluctua;on of single- cell- level ion channels kine;cs, and by evalua;on of (2) spa;al cell- to- cell conductance fluctua;on using the on- chip cell network loop which can choose different conductance pathways of human cardiomyocytes among neighboring circula;ons. 3) The capacity to quan;ta;vely evaluate the correla;on between calcium release and tension genera;on, and the inhibi;on on the trafficking pathway of ion- channel proteins by its long- term op;cal/electrical simultaneous measurement.

8 Quasi- in vivo Assay: Human Cardiomyocytes delivered from hes/hips Cells FPDC n1 to p1 (ms) Before 10 nm 100 nm Wash-out Prolongation of field potential duration (FPD) caused by E-4031 blocking. 100 µm FP- signal of hips Cardiomyocyte Cluster Field Poten2al (µv) Increasing of the beating rate induced by isoproterenol (ISO) 100 ms Biochem. Biophys. Res. Commun., 385, (2009).

9 Quasi- in vivo Assay: Strategy I. Temporal fluctua;on of single- cell- level ion channels kine;cs Field Poten;al Recording Currents recorded from a single cell on a microelectrode Long- term measurement during on- chip cul;va;on System Monitor for imaging DV Recorder D/A s2mulator Display for MEA measurement A/D Converter Display for MEA control Amplifica2on Sampling rate: Max 100 khz (10 µsec) Gain: Max 50,000 LPF: 2-10 khz, HPF: Hz Voltage Field Poten2al Signal Time: 1 sec influx 2 3 efflux Ca 2+ K + Ca 2+ K + 0 mv MEA Chip 1 influx Insula2ng Na + Electrode Na + Agorose layer Collagen layer 2 cm Type IIII 16 4 MEA Bar = 0.1 mm 10μm ITO (Indium- Tin oxiside)- Pt Block Electrode 64 channel Reference electrode: Pt

10 Quasi- in vivo Assay: Strategy I. Temporal fluctua;on of single- cell- level ion channels kine;cs Fluc;a;on measurement of ion channels for TdP predic;on X- axismean FPD value (Similar index of APD) IC 50 (7.7 nm) ** n=24 n=25 Ctrl 100 pm 1 nm 10 nm 100 nm 1 um ns n=19 E (n=27) Y- axisfpd Fluctua;on 2.0 (New index) Field Poten;al Signals Field Poten;al Dura;ons (FPDs) FPD n-1 (sec) Control 40 nm 400 nm FPD n (sec)

11 Quasi- in vivo Assay: Strategy I. Temporal fluctua;on of single- cell- level ion channels kine;cs on-chip FPD & STV assay using hes-cmcs in vitro assay and clinical 100 Category in vitro assay herg APD Clinical FPD Fluctuation Index (%) Tamoxifen Tamiflu Citalopram Terfenadine Terfenadine Bepridil Verapamil PBS Ebastine Amiodarone Paroxetin Famotidine Amiodarone Moxifloxacin Verapamil Bepridil Paroxetin Famotidine Citalopram Cisapride Astemizole FPD Prolongation Index (%) E-4031 Cisapride d-sotarol D-Sotalol E-4031 Astemizole Positive APD false negative herg false negative Not detectable False positive herg false positive APD false positive Unknown??? Negative Drug conc.: hold C Max of plasma conc. at clinical use : Guinea-pig papillary muscle 11

12 Quasi- in vivo Assay: Strategy II. Spa;al cell- to- cell conductance fluctua;on Evaluate spa;al dispersion of repolariza;on (a proposed mechanism of proarrhythmia: ion- channel fluctua;on + electrical conduc;vity change ) Measure changes of QT (ST) intervals, TdP and VT/VF Action potential recordings Field potential recordings (each single electrode) Quasi ECG (Sum of electrodes) A B C D In vivo ECG Integration Differential AP prolonga;on Action potential duration (APD) Established in vitro assay (Patch clamp assay) FP prolonga;on Field potential duration (FPD) Single cell informa;on Sum On-chip cell network assay (Field potential recordings) Pseudo ST interval QT interval QT prolonga;on Established in vivo assay (ECG) Cell- to- cell conductance informa;on

13 Quasi- in vivo Assay: Strategy II. Spa;al cell- to- cell conductance fluctua;on We can acquire the following three indices simultaneously: 2D Sheet Based Network Bleu: Nuclear Green: Mitochondria (Cardiomyocyte) (FPD prolonga;on) Myocyte (ST interval?) Fibroblast MEA chip (VT/VF) 50 µm ITO electrode with PtB Microfabrication by IR laser 6 Myocytes & 2 Fibroblasts in yellow box 100 µm Collagen coating Spin coating of agarose Collagen layer Agarose layer 1 mm

14 Cell network Quasi- in vivo Assay: Strategy II. Spa;al cell- to- cell conductance fluctua;on (b) (c) 0.1 mv 10 s Ring electrode Before addition of drug (Astemizole) 10 nm 100 nm (d) Abnormal bea;ng Fibrilla;on 2 s 1 µm 500 µv ST (ms) * STV of ST (ms) * Control Control [Drug] (nm) [Drug] (nm) Informa;onal Content Much Greater than Simply QT(ST) Prolonga;on

15 Conclusion: On- chip quasi- in vivo assay has strong poten;al to go beyond herg Quasi- in vivo assay includes: Human Cardiomyocytes + On- chip Cell Network Tech. 1) A set of standard human cardiomyocytes prepared from human ips/es cells of different races, sexes and also from pa;ents with various diseases to provide an ideal tes;ng panel plaiorm. 2) Predic;on of lethal arrhythmia (TdP/VT/VF) by evalua;on of temporal fluctua;on of single- cell- level ion channels kine;cs, and by evalua;on of spa;al cell- to- cell conductance fluctua;on using the on- chip cell network loop which can choose different conductance pathways of human cardiomyocytes among neighboring circula;ons. 3) Correla;on between calcium release and tension genera;on, and Inhibi;on on the trafficking pathway of ion- channel proteins.