+2+
Drug Effects on human Na /Ca Exchanger and Implications

for Drug Development.
Maria Barthmes, Andre Bazzone, Marta Lemme, Michael George, Sonja Stoelzle-Feix, Niels Fertig, Andrea Bruggemann
Nanion Technologies GmbH, Ganghoferstr. 70A, 80339 Munich, Germany, contact: Info@nanion.de

Introduction

Methods

Development of an electrophysiological screening assay
1A

Background.

BABCD2• The Na+/Ca2+-exchanger (NCX) plays an important role in cellular calcium homeostasis underphysiological but also pathological conditions.
• The pharmacological potential and risks of NCX have been debated for many years. The
consequences of changes in activity and mode of operation of NCX are still discussed.
Mostly a potential beneficial effect of NCX reverse mode inhibition under ischemic conditions
has been suggested.

Objectives.
• Investigate and compare NCX pharmacology in iPSC derived cardiomyocytes and inoverexpressing HEK293 cells using different assay types with medium to high throughput.
• Develop a novel 96-well based electrophysiological assay to investigate compound effects
on NCX. Validate the assay and test performance with a showcase screening using 28
compounds with different cardiac risk profile.

Solid Supported Membrane (SSM)-based electrophysiology (SURFE2R
N1 and SURFE2R 96SE)
is a sensor-based method, which allows the resolution of low amplitude electric events inbiological membranes, bringing the advantages of electrophysiology to the membranetransporter field. A large capacitive sensor coated with a high amount of membrane vesicles
or proteoliposomes is used to detect changes in electrical potential that are generated byelectrogenic transport processes with high amplification. The method is applied inmechanistic and pharmacological transporter research.

Literature.
•Amran et al., Cardiovascular Drug Reviews, Vol. 21, No. 4, pp. 255–276•Antoons et al., Pharmacology & Therapeutics 134 (2012) 26–42•

Herrmann et al., Cardiovascular Research (2013) 99, 780–788•

Huag, JPTM 87 (2017) 11–23•

Lee and Hryshko, Cardiovascular Drug Reviews, Vol. 22, No. 4, pp. 334–347•

Watanabe, et al., J. Pharmacol.Sci.102, 7 – 16 (2006)•Matsuda, et al., 2001. JPET. 298(1): 249-256•

Watanabe & Kimura., 2001. Jpn. J. Pharmacol. 85: 370-375•Gintant, G., Sager, P. T., & Stockbridge, N. (2016). Nature Reviews. Drug Discovery, 15(7), 457–471.

D
Compound 5 C1Compound 6 C1Compound 7 C1Compound 8 C1Compound 9 C1

Compound 5 C2Compound 6 C2Compound 7 C2Compound 8 C2Compound 9 C2Compound 10 C2 Compound 10 C1

Compound 11 C2 Compound 11 C1

GHF% Change Beat Rate

D3456789101112

Compound 2 C5Compound 4 C1
Compound 4 C2

FDMSO 1%E2

Compound 2 C4Compound 3 C1

D1

Compound 2 C3

12

Compound 2 C2

11

Compound 2 C1

10DMSO 0.1%9

Compound 1 C58

Compound 1 C47

Compound 1 C36

Compound 1 C25

Compound 1 C1

4DMSO 0.1%

3

Compound 3 C2C2

Compound 2 C1

B

1

Compound 2 C2ACompound 1 C1

EShowcase screening of cardiac drugs

Compound 1 C23EFG5ns**

28 drugs categorized as high, intermediate, and low proarrhythmic risk weretested – several showed inhibiting effects on NCX currents.

-5100nM 300nM

1µM

iPSC derived CMs have large NCX specific currents - revealed by SSM-based
electrophysiology.
SSM-based electrophysiology (SURFE2R N1) was applied to detect NCX activity in human iPSC-CMs. The
current characteristics and pharmacology was compared to HEK 293 cell expressing NCX1 (A-D). We
measured significant and specific NCX activity, indicating a high expression level of NCX in these cells. Thisfunctional evidence for high NCX activity in a human model system points to the relevance of inadvertentand intended pharmacological effects on NCX in the human heart.

Investigating physiological effects of NCX inhibitors using FLEXcyte 96technology.
hiPSC-CMs were seeded on the FLEXcyte 96 well plate and the beat rate was determined via impedance
recordings. Addition of SEA0400 significantly increased the beat rate in a dose dependent manner as along-term effect (E+F). Although reproducible, this finding is contradictive to published research, therefore
further evaluation is necessary for a comprehensive interpretation.

0C_low

C_highPerformance of the assay was validated by means ofplate-to-plate reproducibility, the success rate and SEA
0400 pharmacology.

• Applying the FLEXcyte 96 technology to investigate CM
beating behavior we found a suitable tool to observe
physiological effects of NCX inhibition.

030nM0.2• Our findings demonstrate the presence of functional NCX inhiPSC-CMs which until now has been poorly investigated. Thisemphasizes the relevance of cardiac NCX pharmacology.

***

100.4

H

***

150.6

• SSM-based electrophysiology is a suitable tool to investigate
NCX currents in iPSC-CMs and can also be upscaled to aformat that allows screening
compound effects withreasonable throughput. Unlike in other screening assays, theNCX directly and isolated from secondary effects.

B20

Bepridil

4BD

SEA 0400

Conclusions

AC

0.8

DMSO Control

A: Amplitudes of the full activation were homogenously distributed andreproducible. Wells with very low amplitude were not used for analysis. B:The success rate was high (>95%), and the signal-to noise ratio stable andsufficient. C+D: Applying the activation sequence introduced in the rightpanel, it is possible to analyze compound effects as single point (C) or
dose-dependent manner (D). Applying these two modes we determined
either 2 IC50s (n=8) per plate or 11 compounds in two concentrations(n=4). DMSO vehicle control showed a stable base-line and the current
was inhibited in the expected concentration range by SEA 0400.

is an impedance-based technology, which allows recordings from human iPSC derived
cardiomyocytes to be performed on a pro-maturation flexible substrate. Characterization ofelectric and mechanical properties of the beating cells allows the assessment of efficacy andsafety of new drug candidates on a human model system.

ABD

FLEXcyte 96 Technology

DMSO 0.1%

CCell line was provided by AXXAM S.p.A.. NCX activity was triggered byapplication of Ca2+ to Na+ loaded membrane vesicles on the sensor.
Currents can be triggered repetitively with a stable amplitude. Nanion’s
SURFE2R 96SE platform was used for application and recording. A:
Individual NCX current response in a single well. B: Overview of a full 96
well plate in the instrument software. C: Heatmap view of a 96 well sensor
displaying the maximum current amplitude before (top) and after(bottom) addition of different compounds. D: Current amplitude
maximum in an individual well over time: Baseline, maximum current (2x),inhibitor (2x), wash out (2x).

C

NCX in human iPSC derived cardiomyocytesA

NCX was overexpressed in HEK293 cells, the plasma
membrane was purified and applied to a SSM sensor.

1% Inhibition• There is ongoing effort to find specific and direction-specific NCX inhibitors, but inadvertent
NCX inhibition is usually not tested during drug development.

CHigh TdP Risk
Bepridil
Vandetanib
Dofetilide
Quinidine
AzimilideI butilide
Disopyramide
SotalolIntermediate TdP Risk
AstemizoleChlorpromazine
Terfenadine
Pimozide
Clozapine
Droperidol
Domperidone
Risperidon
Ondansetron
Clarithromycin
Cisaprid

No/very low TdP Risk
Tamoxifen
Diltiazem
Loratidine
Ranolazine
Metoprolol
Mexiletine
Nifedipin
Nitrendipin
Verapamil

The drugs were picked and separated into risk categories by the CIPA initiative. We chose these compounds
because of their cardiac context and good availability. The list contained bepridil, which is known as
unspecific NCX inhibitor. Initially each compound was tested at a low and a high concentration, depending
on solubility (A left). B: Several of the drugs did have an effect on NCX activity, tough usually only at highconcentrations. Fife compounds caused a disturbance of the measurement (marked grey). IC50’s wereestimated using the two concentration datapoints and assuming that a full inhibition can be accomplished.
The drugs were ranked by the estimated IC50’s. There was no connection between the risk categories andthe effect on NCX (C). In a second step the drugs that showed activity plus the NCX inhibitor KBR as control
were further characterizes by a more detailed IC50 estimation applying a different addition scheme (A right).
For most of the drugs the estimated IC50 was confirmed.

Assessment of impact
This small screen was performed as a default test scenario and the data must be interpreted with care. The
validity of the results is supported by the reliable detection of known NCX inhibitors. However, whether theobserved inhibition effects are NCX specific, in particular at high concentrations, should be evaluated using
complementary techniques. Physiological implications are unlikely given the high concentrations - unlessaccumulation effects occur, as described e.g. for Bepridil.

• From a set of compounds with cardiac activity, a surprising
25% had a significant effect on NCX activity in the low
micromolar range. Although these experiments must be
refined in order to be able to make assumptions about thephysiological implications, this is an intriguing observation
hinting to a potential role of NCX in drug safety.

→At this point we can state that we could put someperspective on a possible role of NCX in safety
pharmacology. We outlined the relevance forfurther investigations of the electrogenic transporterand demonstrated that novel technologies canhelp break new ground also in well establishedresearch areas.