Assessment of Drug Effects on Cardiomyocyte Function:
Comprehensive In Vitro Proarrhythmia Assay (CiPA) Results
Corina. T. Bot1, Krisztina Juhasz2,3, Liudmila Polonchuk4, Martin Traebert5, Niels Fertig2, and Sonja StölzleFeix2
1Nanion Technologies Inc, Livingston, NJ

2Nanion Technologies GMBH, Munich, Germany

3Institute for Nanoelectronics, Technische Universität München, Munich, Germany

4F. Hoffmann-La Roche Ltd, Roche Pharma Research and Early Development, Basel, Switzerland
5Safety Pharmacology, Novartis Pharma AG, Basel, Switzerland

Abstract

Label-free toxicity and cardiac safety screening

The on-going comprehensive in vitro pro-arrhythmia assay (CiPA) initiative
aims to improve drug safety testing and help more beneficial chemical
entities reach the market. The project consists of three main parts, which
include 1) electrophysiological investigation of drug effects on humanventricular ion channels, 2) characterization of the in-silico cardiosafetymodel predictions, 3) assessment of discrepancies and gaps in fully
integrated biological systems (human induced pluripotent stem-cell derived
cardiac myocytes (iPSC- CMs) and the human ECG). To assess and validate
drug effects on hiPSC-CMs in a combined measurement of cardiac
excitation and contraction, a subset of compounds which are part of thePhase II CiPA study were tested. A prospective comparison study using
combined extracellular field potential (EFP) and impedance technology
was conducted across 4 independent laboratories testing 12 referencecompounds in 4 independent commercially available hiPSC-CMs. Our data
demonstrates that hERG channel blockers, such as Sotalol and Dofetilide
prolonged field potential duration (FPD) at low concentration and induced
arrhythmia as measured by field potential (FP) recording and impedance(IMP) recordings at higher concentrations. The cross-cell comparison
displayed different minimal effective concentrations regarding FPD
prolongation and cessation of beating. On the contrary, Diltiazem, acalcium channel inhibitor, weakened cell contractile activity and shortened
FPD. Multichannel inhibitors, such Quinidine increased FPD, weakened thecontraction force and induced arrhythmia. Furthermore, validation on low
risk proarrhythmia compounds such as Tamoxifen and intermediate risk
compounds such as Droperidol will be presented. Comparison of thecompound effects across the four different sites showed the consistent
trend of the effect. Taken together, commercially available hiPSC-CMs inconjunction with combined measurements of ion channel activity andcontractility is a reliable approach for risk assessment of proarrhythmic
compounds.

Transparent NSP®-96
sensor plates

Automated Field Potential Duration and Arrhythmia Detection

AB
CElectrophysiology

ContractilityVehicle

60.0
40.0
20.0
0.0
-20.0
-40.0
-60.0

CLS Site 4200

CDI Site 1Dofetilide

CDI Site 3

150

Figure 5: Primary
endpoint in drug-freebaseline conditions,FPDmax , is shown
across the different
cell types.

CDI Site 3
AXG Site 1
AXG Site 4100

CDI Site 2
AXG Site 2
CLS Site 250

PLR Site 20-80.00.5nM 0.5nM 3nM

5nM

3nM

3nM

10nM

10nM 10nM 10nM-50

Figure 5600

iCell Cardiomyocytes
489 ±79 (16%)

Average FPDmax (ms)Cor.4U
Cellartis
247 ± 64 (26%)
333 ± 98 (29%)

2600

iCell Cardiomyocytes2500600

Cor.4U

500300200100400300
20010000.30.4

0.5

0.6

0.7

FPDmax (s)

500

300
200
1000.1

0.2

0.3

0.4

0.5

0.2

FPDmax (s)

400
300
200
1000More

Pluricyte

600

4000More

Cellartis

500

Frequency

400

Pluricyte
548 ± 163 (29%)

Frequency-100.0Figure 4: A. No significant changes arepresent in % change of FPDmax whencomparing vehicle control across sites.
B. An increase in repolarization prolongation
is observed post application of 3, 5 or 10nM
Dofetilide, when compared to vehicle
control.

BB

Frequency

Reference electrode

80.0

CDI Site 1
CDI Site 2CDI Site 3
AXG Site 1
AXG Site 2
AXG Site 4
CLS Site 2
CLS Site 4
PLR Site 2Frequency

Figure 2: Development of beat rate(A, impedance) and EFP amplitude (B) ofiCell® Cardiomyocytes (CDI). The mean
beat is shown for days 1, 4 and 6 days
after plating.

AA 100.0% change FPD(max)Figure 1: The CardioExcyte 96 combines impedance and EFP recordings toassess contractility and ion channel activity:• Electrical and Optical Stimulation• Independent system – no incubator required• Automated liquid handling robot for cell seeding, medium exchange and compound addition(Integra Viaflo Assist)• Long-tem recording capabilities
Impedance /EFP electrode

Figure 3: A. Secondary beat detection allows quantification of number of arrhythmic events for each recorded sweep. B. Automated detection of T-wave in
an interval specified by user. C. Field potential duration maximum (FPDmax) is measured from initial spike depolarization to the maximum value of therepolarization (left). Automated detection of beat period, impedance amplitude in case of monophasic (left figure) and biphasic (middle) impedance traces.

% change FPD(max)

Optogenetic
stimulation

0.3

0.4

0.5

More

FPDmax (s)

00.3

0.4

0.6

0.7

0.8

MoreFPDmax (s)Test article concentrations (µM)

Disopyramide M.I.C.

Extracellular Field Potential and Arrhythmia
1.6

Impedance (Ω)

Figure 7: FPDmax
prolongation androlling EAD-like events
are observed in thecase of Pluricyte
cardiomyocytes
treated with 0.3µM
Droperidol (orangearrows). Beat rate
coefficient ofvariation (Br CoV)shows an increase
post addition ofDroperidol, indicativeof arrhythmia.

DisopyramideCor.4U

2.1

Extracellular Field Potential (Voltage)

0.6
0.1

-0.4

0.2

0.4

0.6

Time (s)Cor.4U® hiPSC-derived CMs

Figure 8: Disopyramide affects contractility and has multiple ion channel
effects (MIC). Example raw impedance and EFP waveforms (Cor.4U®,
AXG cells) (left) are presented before (red) and after (shades of blue)
dosing with increasing concentrations of Disopyramide. Most prominent
effects of Disopyramide were decreased contractility together with areduction of EFP spike amplitude across all cell types.

Figure 10: Effect of Nifedipine on the EFP signals of iCell cardiomyocytes2: mean

beat view of overlaid voltage traces (left graph). Effect of Nifedipine on EFP
parameters: amplitude, field potential duration (FPDmax), beat rate and beat rate
regularity shown as a spider chart (right graph).

Impedance

Dose 2

0.1 1
Dose1 2
Dose

type

Dose103†
type1004
Dose††
typeSite 2, CDI

0.7

0.1

1-

10100

Disopyramide

Site 1,3, CDI

0.7

0.1

11--

1010

Disopyramide

Site 2,1, CDI
AXG

0.7

0.1

11--

1010

Disopyramide

Site 3,2, CDIAXG

0.70.111--

1010

Disopyramide

Site 1,4, AXG

0.70.111--1010†††††††

Disopyramide

Site 2, AXG
CLS

0.70.1

11--1010--

100100

Disopyramide

Site 2,4, CLS

0.70.1

11--

1010-

100100

Disopyramide Site 2, PLR
INTERMEDIATE RISK:

0.70.111--1010††††100100††††
††
††
††
†††
††
†
††
††
††
††

Domperidone

Site 2, CDI

0.020.003

0.03-

0.33

Domperidone

Site 2, AXG

0.020.003

0.03-

0.3

Domperidone

Site 2, CLS

0.020.003

0.03-0.3

Domperidone
LOW RISK

Site 2, PLR

0.02

0.003

0.03-

0.3

††
††††††

Tamoxifen

Site 1, CDI

0.02

0.1

0.3-

0.95-

3

Tamoxifen
Tamoxifen

Site 2, CDI
Site 3, CDI

0.02
0.02

0.1
0.1

0.3
0.3

-

0.95
0.95-

3
3

Tamoxifen

Site 1, AXG

0.02

0.1

0.3

-

0.95-

3

Tamoxifen
Tamoxifen
Tamoxifen

Site 2, AXG
Site 2, CLS
Site 2, PLR

0.02
0.02
0.02

0.1
0.1
0.1

0.3
0.3
0.3

-

0.95
0.95
0.95-

33
3

Site / Cell
typeTrend

Site / CellSitetype1, CDITrend

Test article
concentrations(µM)
type Dose 3
type Dose 4100100100100100100100100333typeTrend = illustrates trend of average FPD
data with min and max values pointed out

FPD>10%

FPD>30%

FPD> - 10% FPD > -30%

††††
††
††
--Figure 12: A summary of % FPDmax changes at 30min post compound addition compared to baseline is summarized forone compound from each risk category, all cell types and across all participating sites. "Trend” column displays actualaverage data per compound, n= 4 or 5 replicates each, with min (blue dot) and max (red dot) values pointed out.
Arrhythmia classification was simplified by marking the occurrence of EAD-like events with † (type A arrhythmia listed
under arrhythmia category specified in the HESI Myocyte Phase II Validation Study Protocol), next to each test article
concentration. All other types of / combinations of arrhythmia observed are marked with †† (type B, C, D arrhythmias
listed in Myocyte Phase II Validation Study Protocol)

Figure 6: Addition of 10 µM Sotalol
produced EAD-like events and ectopic
beats in iCell cardiomyocytes2(orange arrows).
A decrease in beat rate
is observed post
addition of 10µM Sotalol,
while impedance
amplitude remains
unchanged (top panel).
Beat rate coefficient ofvariation shows a large
increase post
addition of Sotalol,
indicative of arrhythmia.

Dose 1

CompoundHIGH RISK
Disopyramide
CompoundDisopyramide
HIGH RISK

Control
0.1µM
1µM
10µM
100µM

1.10

ETPCFree(uM)
ETPCFree0.7(uM)

SummaryiCell® Cardiomyocytes2

Figure 9: Disopyramide demonstrates expected electrophysiologicalresponses at 0.1, 1, 10, 100µM. The primary endpoints (right) arequantified across replicates at x sites (n=4 or 5 replicates). Error bars
indicate +/- standard error across the replicates.
iCell® Cardiomyocytes2

Figure 11: Effect of increasing concentrations of Nifedipine on the impedance
signals of iCell cardiomyocytes2: mean beat view of overlaid impedance traces(left graph). Effect of Nifedipine on impedance parameters: amplitude, Pulse
width 20, beat rate and beat rate regularity shown as a spider chart (rightgraph).

Impedance data confirms arrhythmia detected in EFP modes. Arrhythmia occurrence was consistent withprevious literature findings, and hiPSC-CMs tested exhibited prolongation of FPDmax in agreement withmechanism of action reported for compounds under testing. Altogether, these results demonstrate thepotential of predicting torsadogenic risk with hiPSC-CMs using the CardioExcyte 96 system.
Acknowledgements: Special thank you to Sarah Tahir for assistance with poster formatting.
We thank Cellular Dynamics International, a FujiFilm Company, for providing us with cardiomyocytes
iCell cardiomyocytes2) and NCardia, for providing us with cardiomyocytes (Cor.4U®, Pluricyte®).
We thank Takara Bio Europe AB for providing Cellartis® hiPS-CMs.