Reliable identification of cardiac liability in drug discovery using automated patch
clamp: Considerations and best practices for high throughput recordings of NaV1.5
Maria Giustina Rotordam1*, Alison Obergrussberger1*, Nina Brinkwirth1, Kiyoshi Takasuna2, Nadine Becker1, András Horváth1, Hatsue Furukawa2, Yuka Hasegawa2, Takayuki
Oka3, Niels Fertig1, Sonja Stoelzle-Feix1
1 Nanion Technologies, Munich, Germany; 2 Axcelead Drug Discovery Partners, Inc., Japan, 3 Nanion Technologies Japan K.K., JapanIntroduction
For reliable identification of cardiac
safety risk, compounds should bescreened for effects on cardiac ionchannels in addition to hERG, including
NaV1.5 and CaV1.2. Automated patch
clamp (APC) instruments are increasingly
adoptedfor
cardiacsafetymeasurements but cross-site and crossplatform comparisons of IC50 values has
identified the need for standardized
protocols for reliable pharmacology. In
this study, we identified different
parameters that might affect IC50s ofcompounds on NaV1.5 peak and latecurrents recorded using APC.

Patchliner

4 to 8 recording channels

SyncroPatch 384i

384 recording channels

Effect of voltage protocol on NaV1.5

Fig. 1 A, CiPA step-ramp (left)and double step (right)
voltage protocols. NaV1.5 isreliably recorded (B,C), there
was no rundown of peakcurrent in control (vehicle)
recordings(C).
Voltage
protocol can have an effecton IC50, in particular when
compounds display state
dependence (C,D).

Effect of temperature on biophysical properties and IC50

● www.nanion.de ● ali@nanion.de ●Cultured cells versus frozen stocks and correlation
across platforms

Persistent and late NaV1.5 currentFig. 3 A, Voltage protocol
used to record persistentand late current. B, Example
traces of persistent (left) andlate (right) current activatedby ATXII and blocked byranolazine. C, IC50 of block ofranolazine was similar if
obtained using the persistentor ramp phase of thevoltage protocol. Despite
the relative small current sizeof the late NaV1.5 current
comparedwith
peakcurrent, it can have asignificant effect on action
potential
durationandarrhythmia and therefore is
an important factor toconsiderinsafety
pharmacology.

Fig. 2 A, IC50s for 24 compounds calculated using cultured cells or frozen stocks correlated well witha Pearson’s correlation coefficient, R2 = 0.989 ± 0.031. B, IC50s of 11 compounds obtained on thePatchliner or SyncroPatch 384i correlated well, with an R2 = 0.948 ± 0.106.

Incubation time and reproducibility

Comparison with literature

Fig. 6 IC50 values for 18compounds obtained using theSyncroPatch 384 correlated wellwith literature values except
cisapride shown in red which was
not used for the correlation.

Conclusions• CiPA step-ramp protocol should be used for safety pharmacology testing. This allows effects on
both peak and late current to be studied.
• Temperature affects biophysical properties and pharmacology and, at the very least, should be
carefully controlled and at best, experiments should be performed at physiological temperature(where possible).

• A minimum compound incubation time of 5 min is required for reliable IC50 measurement,
longer is preferred for sticky compounds.
• The use of standardized protocols for APC experiments e.g. voltage protocol, incubation time,
replicate number etc, is critical to be able to compare IC50 values across platforms, sites andwith the literature.

Fig. 4 Effect of temperature on activation and inactivation IV and pharmacology of NaV1.5. A, Vhalf of activation is unchanged bytemperature but the Vhalf of inactivation (B) is shifted by approximately 7 mV to more hyperpolarized potentials (* p<0.05, unpaired
Student’s t test). C – E, Temperature can affect IC50 of mexiletine, IC50 is approximately 2-fold higher at room temperature compared with37°C.

Fig. 5 A, Effect of incubation time on calculated IC50 values for 5 compounds. For mostcompounds, 5 min incubation in compound was sufficient to reach steady state but for
sticky compounds, such as terfenadine, 9 or 10 min incubation was required. B, Data was
reproducible: concentration response curves for lidocaine from different chips and differentdays overlaid almost exactly.

References: Data shown in this poster has been accepted into JPTM
pending revisions.