Identification of cardiac liability in drug discovery using automated patch clamp: Experimental andtechnical considerations for high throughput recordings of NaV1.5 and hERG
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

The Comprehensive in vitro Proarrhythmia Assay (CiPA) aim is to improve specificity compared to in vitro hERG andin vivo QT studies. Automated patch clamp (APC) instruments are increasingly adopted for cardiac safetymeasurements on hERG, NaV1.5 and CaV1.2, thus requiring standardized experimental protocols and technicalspecifications, e.g. temperature control, accessibility of recording solutions to allow for sample collection or Liquid
Junction Potential (LJP) correction for accurate voltage control. Here, we identified parameters influencing IC50
determination of compounds on hERG and NaV1.5 currents recorded using APC. For example, we found that
although voltage protocol did not affect the IC50 of hERG compounds, both voltage protocol and holding
potential could affect IC50 on NaV1.5 peak currents. Compounds ranolazine and tamoxifen, two blockers known toblock NaV1.5 in the open state, showed more potent IC50s at a holding potential (HP) of -80 mV compared with -95mV (e.g. ranolazine IC50 = 145.64 µM (HP: -95 mV) and 99.75 µM (HP: -80 mV).
Temperature affected the Vhalf of inactivation, shifting Vhalf of inactivation by ~7 mV
to more negative potentials at 37°C vs room temperature (RT), and also affected
the IC50 of mexiletine which was 82.1 ± 19.4 µM (n = 14) at 37°C compared with
181.5 ± 29.0 µM (n = 10) at RT. Given the influence of temperature on NaV1.5 andhERG biophysics and pharmacology, it is critical to maintain a constant recording
temperature throughout the experiment. Experimental parameters such as
incubation time also influenced IC50 values for both hERG and NaV1.5 peakcurrents and whereas compounds such as bepridil reached steady state withinapproximately 5-6 mins, sticky compounds such as terfenadine required longer
incubation times.

SyncroPatch 384

384 recording channels

Patchliner + Dynamite8
4 to 8 recording channels

Effect of temperature on biophysical properties and IC50

Late NaV1.5 current & CiPA protocol
persistentA● www.nanion.de ●Effect of voltage protocol on hERG

Late (ramp)

EBCFig. 2. A, hERG voltage protocols “CiPA” stepramp (left) and “2-s step-pulse” (right) andcorresponding mean data traces (B). C,Normalized peak current in control (grey),vehicle control and increasing verapamilconcentrations (shades of blue). DMSO control(open blue squares) is also shown forcomparison for n = 6 wells for the CiPA stepramp and n = 9 wells for the 2-s step-pulse
protocol. D, Estimated IC50 values gained fromthe CiPA protocol were 828 nM (n =7) and 525
nM (n = 9) when using the 2-s step-pulse
protocol. E Correlation of IC50 values calculated
from block potencies of 27 compounds as
calculated from peak current responses elicitedeither by the CiPA voltage protocol or the 2-s
step-pulse protocol. The R2 was calculated tobe 0.990 ± 0.028 showing excellent correlation.
Experiments were performed as a cumulative 4point concentration application with acompound equilibration time of 5 min, CHO
hERG Duo cells were used.

DFig. 1 A NaV1.5 voltage protocol “CiPA” step-ramp, corresponding meandata traces (B). C Normalized peak current in 60 nM ATXII (grey), a single
concentration of ranolazine (blue) and full block (4 mM lidocaine), n = 25.
D Estimated IC50 value gained for the persistent current was 10.4 µM (n = 25)
and for the late (ramp) current was 6.8 µM (n = 25).

Incubation time and reproducibility
CSample collection The prerequisite for concentration verification

Fig. 5 Sample collection on SyncroPatch 384 allows concentration verification as outlinedby the ICH E14/S7B Implementation Working Group.
From: ICH E14/S7B Implementation Working Group:
Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential- Questions and Answers - Draft Version 2020D••>>Both nominal and measured concentrations should be reported.
Validated analytical method should be applied to solution collected from test chamber
Measured concentrations should be used for IC50 determinations if values differ significantlyConclusions• The NaV1.5 CiPA step-ramp protocol is ideal for safety pharmacology
testing allowing effects on both peak and late current to be studied.
Voltage protocol does not affect IC50 values measured on hERG.

Fig. 3 Effect of temperature on activation and inactivation IV andpharmacology of NaV1.5. A, Vhalf of activation is unchanged bytemperature but the Vhalf of inactivation (B) is shifted byapproximately 7 mV to more hyperpolarized potentials (* p<0.05,unpaired Student’s t test). C – E, Temperature can affect IC50 ofmexiletine, IC50 is approximately 2-fold higher at room temperaturecompared with 37°C.
Recommended reading: https://doi.org/10.1016/j.vascn.2021.107125
and https://doi.org/10.1016/j.vascn.2020.106884• Temperature affects biophysical properties and pharmacology and should
be carefully controlled. Where possible, experiments should be performedat physiological temperature.
Fig. 4 A, Effect of incubation time on calculated IC50 values on NaV1.5 for 5 compounds. For most compounds, 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 anddifferent days overlaid almost exactly. C Compound incubation time plotted against IC50 values for hERG. 4-point IC50values were calculated. Single concentrations were added to each well, compound applications were performed twicewith the 2nd addition after 5 min. All compounds except for dofetilide reached steady state within 7 min, dofetilide
required 10 min. D Correlation of IC50 values gained with 27 compounds that were stored in glass coated 384-well plates
or PP 384-well plates correlated well, the R2 was calculated to be 0.998 ± 0.012. Biggest discrepancies of 2.3–2.9 fold wereseen with astemizole, cisapride, domperidone, droperidol, pimozide and terfenadine (shown as red markers).

• For NaV 1.5 measurements, a minimum compound incubation time of 5 minis required for reliable IC50 measurement, longer is preferred for sticky
compounds. For hERG, we recommend > 6 min.
• The use of standardized protocols for APC experiments e.g. voltage
protocol, incubation time, replicate number etc, is critical to be able tocompare IC50 values across platforms, sites and with literature values.