HTS in Cardiac Safety –hosted by Nanion
Satellite symposium during SPSMaritim Hotel BerlinSunday, 24th September 2017
4:00 – 6:30 PMSalon 15 / Paris (1st floor)Nanion Technologies

HTS in cardiac safety

Dr. Sonja Stölzle-Feix, Director, Scientific Affairs,Nanion Technologies

Nanion participationin CiPA Part of the leadership team in the HTS ion channel screening subteam:
5 cardiac ion channels recorded on automated patch clamp devices(Patchliner & SyncroPatch384 PE and others) Participant in the myocyte phase II study:hiPS cardiomyocytes on impedance/EFP system: the CardioExcyte96

CiPA power tools
SyncroPatch 384/768PE.

The Patchliner.
.

The CardioExcyte 96.

AgendaSyncroPatch 384 PE & cardiac
ion channels in cell lines and iPS CM

CardioExcyte 96 & cardiac
pharmacology in iPS CMSummary

Ion Channels of the CIPA Initiative
Nav1.5 („INa“)Kv11.1 (hERG – „IKr“)
2

1

Cav1.2/b2/a2d („Ica, L“)

Kv7.1-KCNE1 („IKs“)
21

Kv4.3-KChip2.2 („Ito“)Kir2.1 („IK1“)
21233

PEIon Channels of the CIPA Initiative

PEDifferent (iPS CM) cells on one PlateiCell2Cor.4U

CHO Nav1.7

PE

CHO hERG Current Stability

Overlay of the Traces for more than 20 minutes

PE

CiPA Step-Ramp hERG protocol

hERG step-ramp protocol: depolarization for
400 ms at 40 mV followed by a 100 ms ramp
segment where the hERG tail current ismeasured. The protocol runs at 0.2 Hz for 5 min
to establish baseline and 5 min for compoundincubation.

Raw data tracesCurrent vs. timeram p hER
Mi l nes hE
Mean St D ev
N582. 9 417. 2 274127. 2 74. 90 296-4 0 00400800

1200hERG currrent pr. cel l

Neg.

Pos.

Neg.

Pos.

HEK hERG cells kindly provided by Chantest

1600

2000

PE

CiPA Milnes hERG protocol
hERG experiments are also done with theMilnes protocol in order to collect data for thein-silico modeling group.
The protocol is executed 10 times every 30
seconds.

Raw data traces

Current vs. time

ram p hER
Mi l nes hE
Mean St D ev
N582. 9 417. 2 274
127. 2 74. 90 296-4 0 00400

800

1200hERG currrent pr. cel lNeg.

Pos.

Neg.

Pos.

HEK hERG cells kindly provided by Chantest

1600

2000

PE

Pharmacology:
Milnes vs . Step-Ramp hERG protocol

The Milnes and the ramp
protocol where compared interms of IC50’s of Verapamiland Dofetilide.
The IC50´s were found to becomparable between the twomethods.

MilnesStep-Ramp

HEK hERG cells kindly provided by Chantest

PE

CHO hNav1.5
Late Current

The Cell Line was kindly provided byCharles River

PENav1.5 Late – Activation by ATX-II - Current Over Time

Referencephase
ATX-II Activation

Compoundphase
Full Block(here DMSO only)
phase

PE

Nav1.5 Late Raw Traces and Current Over Time
Raw Traces after ATX-II Applicationneg. control(DMSO only)

pos. control(50 µM Tetracaine)

Current Over Time

neg. control(DMSO only)

pos. control(50 µM Tetracaine)PE

CHO hNav1.5 Late Current

10 kHzSamplingInhibition of the late Nav1.5
current by 500 µM Tetracaine.
No leak subtraction wasapplied.

1 kHz
Butterworth
FilterCompleteTrace
10 kHzNo pharmacological tools (ATXII) were used to increase thelate current.
Whole-cell recording
single hole chip

PEInhibition

CHO hNav1.5 Late Current

ControlInhibition

Control

Ratio Peak/Late Current

PECurrent Clamp: iPS derived Cardiomyocytes
Cor.4UThe AP was recorded in 5 mM
Barium. Different shapes andAP lengths can be observed.
5 µM Nifedipine was applied
for Calcium channel inhibition.
An immediate shortening of theAPD can be observed.

PE

APD shortening by Nifedipine

The AP was recorded in 5 mM
Barium.
5 µM Nifedipine was applied
for Calcium channel inhibition.

Online Analysis of the samecell.
The APD90 was calculated
before and after the inhibitionof 5 µM.
Online Analysis of the samecell.
Comparing APD90 and APD50

PEAnalysis Functions for Current Clamp Measurements

AP Duration

Slope

Rise Time

Fall TimePECurrent Clamp: Anode Break Stimulation„Active cellular properties(HCN channels, Ik1
supporting) explain anodebreak stimulation in cardiaccells.“Anodal break in Guinea pig
Ranjan et al. 1998

PLPE

Patchliner and it´s new features
Hardware•External & internal exchange•

Temperature control•

Voltage & current clamp + dynamic clamp•

Single- & multihole chips, produced in-house•

Rseries compensation•Cooled cel hotel

New!

Applications•

Voltage and ligand-gated ion channels•

Whole-cell & perforated patch clamp•

Cell lines, primary cells and stem cells•

Validated for CiPA•Minimized cell consumption•

Easy & customizable analysis tools

The Patchliner offers high flexibility and sophistication.

New!

AgendaSyncroPatch 384 PE & cardiac
ion channels in cell lines and iPS CM

CardioExcyte 96 & cardiac
pharmacology in iPS CMSummary

CE96

Combined Impedance/Extracellular Field Potential (EFP) assay
provides mechanistic electrophysiological endpoints in vitro

 Established SOPs forElectrophysiology

Impedance/EFP measurements
in frame of the CiPA studies
Contractility

Optogeneticstimulation

The CardioExcyte 96•

96 wells – 96 parallel sensors•

Efficient data analysis•

Automated liquid handling robot for cellseeding, medium exchangeCardioExcyte96 experimental view of mean extracellular field potential signals recorded instem cell derived cardiomyocytes. Random selection of multiple wells (black squares) allows
the display of the impedance recordings on the right. Data from cell type 1

CardioExcyte96 experimental view of mean impedance beat signals recorded in stem
cell derived cardiomyocytes. Random selection of multiple wells (black squares) allows
the display of the impedance recordings on the right. Data from cell type 1

CE96

E4031 induces EADs
EADs (Early afterdepolarisations; marked inyellow) detected after
15min (100 nM).

The BRRI dropped down(increasing arrhythmia) to
80 % at 100 nM.

CiPA – Myocyte phase II data

300,0
200,0
100,00,0-100,00.1µM

1µM

10µM

Impedance (Ω)

d, l Sotalol

Voltage (µV)

400,0Control, cell type 1

High Risk
Arrhythmic events, % of

10µM d, l Sotalol, cell type 1

100µM

700,0Arrhythmic events, % of

Disopyramide
500,0300,0100,0-100,0100,00.1µM

1µM

10µM

Vandetanib

80,060,040,020,00,0-20,00.01µM

0.1µM

1µM

10µM Disopyramide, cell type 1

100µM

10µM

Arrhythmic events, % of wells

Repolarisation prolongation Repolarisation prolongation Repolarisation prolongation

CE96

1µM Vandetanib, cell type 1

CE96Long-term and short-term recording for chronicand acute effects

Base ImpedanceDrug application
High resolution impedancecell attachment andnetwork formation
Time (sec)Drug application
cell deathTime (days)
Chronic effects

Acute effects

CE96

Cell adhesion and proliferation assays

Cell proliferation/ Cell QC

Hepatotoxicity

Comp.
addition

Cell proliferation of CHO cells, initiallyseeded cells per well were 2k, 4 k and 16k.
Blue line: medium exchange

Patient specific hepatocyte-like cells treated with
Tween as a control (complete cell death) andincreasing Paracetamol concentrations.
Note the concentration dependent hepatotoxeffect.

CE96

Cell adhesion and proliferation assays

H8N8 for investigations of tumor cell recurrence

NSP®-96 transparent sensor plates allow imaging of seeded
cells and long term recordings over weeks.

Long term monitoring of H8N8 tumor cells. 500 h monitoring of
H8N8 cells, which have been developed as a tool to investigate
tumor cell recurrence, chemoresistance and epithelial-tomesenchymal transition (EMT). Cells were treated with increasing
concentrations of CAF (cyclophosphamide, adriamycin(doxorubicin) and 5-fluouracil) on the CardioExcyte 96 during
impedance measurements.

Data kindly provided by Oliver Reinhardt, MPI Experimentelle Medizin, GöttingenCE96Transparent NSP-96 Sensor plates

Ca2+ imaging (FDSS Hamamatsu) on Cor.4U cells

NSP®-96 transparent sensor plates allow imaging of seeded
cells and long term recordings over weeks.

Ca2+ imaging (FDSS Hamamatsu), Cor.4U
Kindly provided by Ralf Kettenhofen, Axiogenesis AG

AgendaSyncroPatch 384 PE & cardiac
ion channels in cell lines and iPS CM

CardioExcyte 96 & cardiac
pharmacology in iPS CM

Summary

Holistic view on cardiac pharmacology
Myocyte II studyImpedance/ EFP inbeating
networks
Contractility
and electrophysiology

CiPA

Patch clampof single cells

Comprehensive
safety assays –mechanistic
electrophysiologicalComprehensive
understanding
safety assays

In Silico reconstruction
human ventricular cellularelectrophysiology

impedance

Cell linesEFP

HTS study

In silicomodellinggroup

Holistic view on cardiac pharmacology

Impedance/ EFP inbeating
networks
Contractility
and electrophysiology

CiPA
Comprehensive
safety assays –mechanistic
electrophysiologicalComprehensive
understanding
safety assays

Patch clampof single cells

Ca2+Cell linesand iPS CM?

Na+In Silico reconstruction
human ventricular cellularelectrophysiology

Summary We have established SOPs for APC and CardioExcyte 96 measurements inframe of the CiPA studies

 APC: Success rate for those targets were between 80 to 90% after applying theQC filters required from the CiPA guidelines

 Patchliner and SyncroPatch 384PE are equipped with features that are needed
for investigating iPS CM:

 Temperature control
 Low cell consumption, GigaOhm seals
 Current clamp Dynamic clamp (ongoing project)
SyncroPatch 384/768PE.

The Patchliner.
.

The CardioExcyte 96.