In vitro cellular method development for evaluation of cardiac toxicity

1. Evaluation of cardiac liability of drugs by two in vitro functional assays
Shimin Wang, Teddy Lin, Karen Bernards, Yulia Ovechkina, Christine O’Day and Dan Small
MDS Pharma Services- Bothell, WA, USA
Drug induced cardiotoxic effects including delayed cardiac repolarization may induce arrhythmias
such as Torsades of Points or even sudden death. It is a challenge to detect these side effects of
compounds at the early stage of drug development in the pharmaceutical industry. To evaluate a
drug's potential to delay cardiac repolarization, two in vitro functional assays are widely employed: (1)
study of the effects of compounds on hERG K+ channel and (2) measurement of the action potential
duration in cardiac tissue. The hERG K+ channel assay is predominantly employed because most
drugs that reduce hERG K+ current delay cardiac repolarization. To test the cardiac liability of drugs,
these two assays were validated and their results were compared in this study. Eight known hERG K+
channel blockers were tested on HEK-293 cells expressing hERG K+ channel using the automated
voltage clamp technique. The calculated IC50 values of the testing compounds using the hERG K+
channel assay were comparable to those using the manual and automated patch clamp techniques
reported in literature . The effects of three of the eight known hERG K+ channel blockers on rabbit
Purkinje fiber action potential were also tested using the current clamp technique. D, L- Sotalol and
Dofetilide prolonged 50% and 90% action potential duration in a concentration dependent manner.
Quinidine also prolonged 90% action potential duration in a concentration dependent manner but a 10
M concentration of Quinidine reduced the resting potential, slowed the maximum rate of rise of
action potential upstroke, decreased the amplitude of action potential and had no effect on 50% action
potential duration most likely because of its multiple channel inhibitory effects. These results match
those reported in literature. In general, the functional hERG K+ channel assay is a sensitive way to
detect cardiotoxic effect and it is cost effective and high throughput, while the functional action
potential assay provides relatively direct proarrhythmic information on drugs.

2. INTRODUCTION
hERG (human ether-a go-go-related gene) encodes human rapidly activating delayed
rectifier potassium (K+) current (IKr) that is an essential component contributing to the
repolarization of the cardiac myocyte action potential[1]. It is widely recognized that drug-
related inhibition of hERG K+ channel results in a long QT syndrome that may trigger
life-threatening arrhythmias such as Torsades de Pointes (TdPs) and even sudden
death[2].
Since the last decade, many non-antiarrhythmic drugs have been withdrawn from the
market mainly because of their inhibitory effect on the hERG K+ channel. Consequently,
the S7B guideline issued by the International Conference on Harmonization (ICH) in 2002
recommends an in vitro evaluation of the effects of all pharmaceutical compounds that
are targeted for human use on the hERG K+ channel (www.ich.org).
Electrophysiological techniques including voltage- and current- clamp techniques have
provided the opportunity for translating key clinical liabilities into in vitro assays. Recent
advances in the automated patch clamp technique enable broad screening of test
compound effects on ion channels.
Using two different methods we studied (1) the inhibitory effects of eight known hERG K+
channel blockers on hERG K+ channel using PatchXpress 7000A. (2) the effects of three
known hERG K+ channel blockers on Purkinje fiber action potential using current- clamp
technique. (3) the possible relationship between hERG K+ channel blockage and action
potential duration prolongation.

3. MATERIALS AND METHODS
hERG K+ current recording:
Cell culture: a HEK-293 cell line stably expressing hERG K+ channel was obtained from Dr. January’s
laboratory[3]. The cells were cultured and harvested to obtain a suspension of single cells in
extracellular solution.
Solutions and reagents: Intracellular solution contains (mM): KCl 130, Na-ATP 5, EGTA 5, MgCl2 5,
HEPES 10, (pH 7.2). Extracellular solution contains (mM): NaCl 137, KCl 4, CaCl2 1.8, MgCl2 1.0, HEPES
10, Glucose 11, (pH 7.4). Eight known hERG K+ channel blockers: Astemizole, Ketoconazole,
Quinidine, Verapamil, Cisapride, Terfenadine, Dofetilide and D,L- Sotalol were tested in this study.
Electrophysiological recording: hERG K+ currents were recorded using PatchXpress 7000A, a system
for automated patch clamp (Molecular Devices Co., Sunnyvale, CA) in a whole-cell configuration. See
Figure 1 and 2 for the pulse protocols. To test the reproducibility of the data, 3 sets of testing data
were generated on 3 different days for 6 individual compounds and 1 set of testing data was produced
from 2 individual reagents. At least 3 cells were obtained for each data set. The experiments were
performed at ambient temperature.
Data analysis: Within each cellular recording, the current responses to test compound addition were
normalized to the vehicle control and the percent of control values were calculated: ([current
response / control peak tail current] x 100%). Means and standard errors were calculated for each test
group. IC50 values were calculated using nonlinear regression to fit data to the Dose-Response, One-
Site Model where: f = A + ((B-A)/(1+((C/x)^D))). Curve-fitting and IC50 calculations were performed
using MathIQ™ software (IDBS).

4. Purkinje fiber action potential recording:
Fiber dissection, solutions and reagents: male rabbits (body weight 1.5 to 2.5 kg) were used for the
experiments. Rabbit Purkinje fibers were dissected surgically from either ventricles in a dissection
Tyrode solution containing (in mM): NaCl 118; KCl 30; CaCl2 1.8; MgCl2 1.0; NaH2PO4 1.8; NaHCO3
25; glucose 55 bubbled with 95% of O2 + 5% of CO2 and heated to 36.5 ± 1oC. The dissected
fibers were moved to a perfusion bath and superfused with normal Tyrode solution containing (in
mM): NaCl 118; KC1 4; CaCl2 1.8; MgCl2 1.0; NaH2PO4 1.8; NaHCO3 25; glucose 11 ( pH 7.40 ~
7.45) bubbled with 95% of O2 + 5% of CO2 and heated to 36.5±1oC. The fibers were continuously
perfused for at least one hour before action potentials were recorded. The microelectrodes were
fabricated and filled with 3M KCl with a resistance of 5 - 45 M . Quinidine, Dofetilide and D, L
Sotalol were used in these experiments.
Action potential recording: Action potentials were recorded in current clamp mode using a Multi-
Clamp 700B amplifier. The Purkinje fiber was triggered by a 2 ms pulse with 2 fold of stimulating
threshold at a frequency of 1Hz or 2Hz.
The perfusion scheme is shown as follows:
Control Vehicle Washout
Concentration 1
Concentration 2
Concentration 3
t=0 t=15 min t=25 min t=35-65 min t=45-75 min t=55-85 min

5. Figure 1. hERG K+ channel current-voltage relationships and steady- state activation. hERG
steady- state activation (B) and deactivation currents (F) induced by stimulating pulses (A) and (E).
(C) Mean I-V relationship, (D) Steady- state activation. Data fit with Boltzmann equation: f =
(1/(1+exp(-(V-V1/2)/k))) and (G) Deactivation I-V relationship.

6. Figure 2. The development of steady- state block of hERG K+ currents by Ketoconazole. (A) Pulse
protocol. (B) hERG K+ currents inhibited by different concentrations of Ketoconazole. (C) Time course of
drug inhibition of hERG K+ currents.

7. Figure 3. Inhibition of hERG K+ currents by Astemizole, Ketoconazole, Quinidine, Verapamil,
Cisapride, Terfenadine, Dofetilide and D, L Sotalol. IC50 values were calculated using a
nonlinear regression to fit data to the Dose-Response, One Site mode: f = [A+((B-
A)/(1+((C/x)^D)))].

8. Astemizole Ketoconazol Quinidine Verapamil Cisapride Terfenadine Dofetilide D, L-Sotalol
(nM) e (nM) (nM) (nM) (nM) (nM) (nM)
(nM)
MDS 20 ± 4 * 2439 ± 206 * 1071 ± 89 * 231 ± 32 * 12 ± 1 * 154.3 ± 13 * 15 ±0.14 ψ 417000
PatchXpress ±2085 ψ
Data
PatchXpress 4 – 26A-K 1863–2680A- 745–1300 A-K 239-630A-K 9-85A-K 150D 11-15A,M 76400-
Data K 421000M,N
reported in
literature
Manual 0.9-26B 1700-2000I 300-1000B 140-800B 2-45B 9-350L 10-110L 100000-
Patch Data 810000C,P
reported in
literature
* Mean IC50 ± SEM values derived from three sets of experiments. ψ Mean IC50 ± SEM values derived from one set of experiments (n = 3).
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Table 1. Comparison of our PatchXpress results with those reported in literature.

9. Our Data (n=5) Data from Literature4 (n=11)
Parameter Control (1 Hz) Quinidine 10 uM Control (1 Hz) Quinidine 10 uM
RP (mV) -88.0±1.4 -84.9±0.3* -89±1 -79±5
APA (mV) 125.6±1.9 119.2±1.8* 120±2 105±2*
Vmax (V/s) 460.4±33.0 302.4±14.3* 430±65 245±72*
APD50 (ms) 216.0±18.2 222.0±8.9 248±28 305±45*
APD90 (ms) 306.2±23.3 525.4±66.9* 361±32 604±62*
(100%) (172%) (100%) (167%)
Figure 4. Effects of Quinidine on Purkinje fiber action potential and comparison of our current
clamp results with those reported in literature.

10. Our Data (n=3) Data from Literature5 (n=6)
Parameter Control (1 Hz) D,L-Sotalol 30 uM Control (1 Hz) D,L-Sotalol 30 uM
RP (mV) ±
-91.9±1.2 ±
-86.6±1.5 ±
-91.1±1.4 ±
-87.4±2.2
APA (mV) ±
125.8±2.1 ±
122.5±1.7 ±
124.5±6.1 ±
119.3±13.1
Vmax (V/s) ±
455.0±24.2 ±
401.9±38.3 - -
APD50 (ms) ±
276.8±28.7 ±
502.4±33.5* - -
APD90 (ms) ±
349.0±46.7 ±
666.9±60.7* ±
331.8±34.6 ±
735.7±125.5*
(100%) (190%) (100%) (222%)
Figure 5. Effects of D, L Sotalol on Purkinje fiber action potential and comparison of our current
clamp results with those reported in literature.

11. Our Data (n=5) Data from Literature4 (n=11)
Parameter Control (1 Hz) Dofetilide 10 nM Control (1 Hz) Dofetilide 10 nM
RP (mV) -89.7±3.7 -90.1±3.2 - -
APA (mV) 127.8±2.7 125.3±1.5 125±5 131±6
Vmax (V/s) 440.5±38 420.2±14.6 487±41 511±33
APD50 (ms) 259.7±43.9 420.5±100.9* 198±37 310±30*
(100%) (162%) (100%) (156%)
APD90 (ms) 367.4±37 582.8±105.7* 266±14 444±43*
(100%) (159%) (100%) (167%)
Figure 6. Effects of Dofetilide on Purkinje fiber action potential and comparison of our current
clamp results with those reported in literature.

12. CONCLUSIONS
Eight known hERG K+ channel inhibitors were tested in this study using the automated
PatchXpress platform. Day to day variation of IC50 values obtained from three independent
experiments for 6 individual reagents were below 3-fold suggesting that this assay is consistent
and generates highly reproducible results.
IC50 values derived from this study are similar to the published values using both automated
and manual patch clamp techniques suggesting that the data generated in this study are
accurate and reliable.
Using the current clamp technique, we tested the effects of three known hERG K+ channel
inhibitors on rabbit Purkinje fiber action potential. D,L- Sotalol and Dofetilide prolonged 50%
and 90% action potential durations in a concentration dependent manner. Quinidine at 10 M
reduced action potential amplitude, decreased the resting potential, depressed Vmax and
prolonged 90% action potential duration, but there was no effect on 50% action potential
duration, probably due to its multiple channel inhibitory effect. Our observed effects are
consistent with those reported in literature showing that this assay is reliable and can provide
high quality data.
The drugs that inhibit hERG K+ channel may also prolong action potential duration and
potentially induce long QT syndrome.

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