JPM-06339; No of Pages 12
Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Journal of Pharmacological and Toxicological Methods
journal homepage: www.elsevier.com/locate/jpharmtox

An evaluation of 30 clinical drugs against the comprehensive in vitro
proarrhythmia assay (CiPA) proposed ion channel panel
William J. Crumb Jr. a,⁎, Jose Vicente b, Lars Johannesen b, David G. Strauss bab

Cytocentrics, New Orleans, LA, United States
Ofﬁce of Clinical Pharmacology, Ofﬁce of Translational Sciences, Center for Drug Evaluation and Research, U.S. Food and Drug Association, Silver Spring, MD, United Statesa r t i c l e

i n f oArticle history:
Received 30 January 2016
Received in revised form 28 March 2016
Accepted 30 March 2016
Available online xxxx
Keywords:
CiPA
Electrophysiology
Ion channels
Manual patch clamp
Torsade de pointes

a b s t r a c t
Introduction: The Comprehensive in vitro Proarrhythmia Assay (CiPA) is intended to address the misidentiﬁcation of drug-associated torsade de pointes risk based solely on hERG and QT data. This new paradigm will consistof four interrelated components, one of which is a panel consisting of six ion channels whose currents are important in both depolarization and repolarization of the cardiac action potential. This study examined the effects of
30 clinical drugs on these ion channels.
Methods: Ion currents were evaluated in expression systems using the manual whole cell patch clamp technique.
Currents were elicited using either a ventricular action potential waveform or step-ramp voltage protocols.
Results: Of the seven ion currents studied, hERG was the most often blocked current followed by Nav1.5-late, andCav1.2. Using a 20% reduction in current amplitude as an arbitrary maker, at a free plasma Cmax concentration, no
drug tested blocked Nav1.5-peak, KvLQT1/mink, Kir2.1 and Kv4.3 by that amount. At a 3x free plasma Cmax, every
current except Kir2.1 had at least one drug reduce current amplitude by at least 20%.
Discussion: This is the ﬁrst study of its kind to examine the effects of 30 clinical drugs against the seven ion
currents currently proposed to makeup the CiPA ion channel panel. The results indicate the importance ofdrug-induced block of hERG, Nav1.5-late and Cav1.2 at clinically relevant concentrations, with low risk torsade
drugs having equal or greater Nav1.5-late or Cav1.2 block compared to hERG block. In addition, the results ofthis study provide data which can be used to test the ability of various in silico models to predict drug-induced
arrhythmias.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
The Comprehensive in vitro Proarrhythmia Assay (CiPA) is intended
to address the misidentiﬁcation of drug-associated torsade de pointes
risk based solely on hERG and QT data (Sager, Gintant, Turner, et al.,2014). This new paradigm will consist of four interrelated components:
an ion channel panel, in silico action potential reconstructions of the ionchannel panel activity, and veriﬁcation of results in stem cell derived
human cardiomyocytes and in human phase 1 ECGs. To this end, theSafety Pharmacology Society organized the Ion Channel Working
Group (ICWG) consisting of representatives from the pharmaceuticalindustry, regulatory agencies, contract research organizations, and academia. The ICWG was tasked with selecting the ion channels to be tested (Fermini, Hancox, Abi-Gerges, et al., 2016). The ion channel panel
decided upon consists of six ion channels whose currents are importantin both depolarization and repolarization of the cardiac action potential(IKr, INa, ICa, IKs, Ito and IK1) (Fermini et al., 2016).

⁎ Corresponding author at: Cytocentrics Inc., 1441 Canal St., New Orleans, LA 70112,
United States
E-mail address: w.crumb@cytocentrics.com (W.J. Crumb).

These currents contribute to all components of the cardiac action potential (Grant, 2009; Nerbonne & Kass, 2005). Starting from a resting
membrane potential, determined largely by the inwardly rectifying potassium current (IK1), the upstroke or Vmax of the cardiac action potential is due to an inﬂux of Na+ ions through the Na channel (INa-peak).
This is followed by a rapid phase of repolarization or phase 1 due tothe efﬂux of K+ ions through the transient outward potassium current(Ito). Following this is the plateau of the cardiac action potential carriedby an inﬂux of Ca++ ions through the L-type Ca channel (ICa) and to asmaller extent through an inﬂux of Na+ ions through the Na channel.
This Na current ﬂowing during the plateau period is commonly referred
to as the late Na current (INa-late). Blockade of both the L-type Ca current and INa-late have been associated with a reduction in QTc prolongation and torsade even in the presence of hERG block (Belardinelli et al.,2013; Gintant, Su, Martin, & Cox, 2006; Johannesen et al., 2014). The action potential returns to a resting state via an efﬂux of K+ ions throughboth the rapid and slow components of the delayed rectiﬁer potassium
current (IKr and IKs, respectively). For ease of use across industry and academia, these currents are most often recorded in expression systems
and not primary cardiac myocytes. Therefore, IK1, INa (peak and late),
Ito, ICa, IKr, and IKs are routinely recorded using cell lines expressing
Kir2.1, Nav1.5, Kv4.3, Cav1.2, hERG, and KvLQT1/mink, respectively.

http://dx.doi.org/10.1016/j.vascn.2016.03.009
1056-8719/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

2

W.J. Crumb Jr. et al. / Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx

The purpose of the present study was to evaluate the ion current
blocking proﬁle of thirty clinical drugs against these expressed ion channels using the manual patch clamp technique and to provide insight into
which ion currents are more frequently blocked at concentrations corresponding to free plasma Cmax and multiples thereof. This could havepreliminary implications for whether all 7 ion channel currents should
be assessed for every drug under CiPA. In addition, the ion channel
blocking patterns of drugs that have high, intermediate and low torsadede pointes risk is compared. Furthermore, the results presented in thisstudy can be used to evaluate various in silico action potential models
for their ability to deﬁne and categorize the arrthymogenic risk of thedrugs tested here.
2. Methods
The cloned equivalent of the human IKr (hERG-HEK), IKs (KvLQT1/
mink-HEK), INa (Nav1.5-HEK), Ito (Kv4.3-HEK), IK1 (Kir2.1-CHO), andthe L-type ICa (Cav1.2-CHO) were used in this study. Cells were obtained
from Cytocentrics Bioscience GmbH (Joachim-Jungius-Straße 9, 18059
Rostock, Germany).
2.1. Internal and external recording solutions
The external (bath) solution for hERG, KvLQT1/minK, Kir2.1, Kv4.3
had a composition of in mM: NaCl 137, KCl 4, MgCl2 1, CaCl2 1.8,
HEPES 10, dextrose 11 with a pH of 7.4 (NaOH). The internal (pipette)
solution had a composition of (in mM): KCl 130, MgCl2 1, NaCl 7,
HEPES 5, EGTA 5 with a pH of 7.2 (KOH). The external solution for
Cav1.2 and Nav1.5 had a composition of in mM: NaCl 137, KCl 4,
MgCl2 1, CaCl2 1.8, HEPES 10, dextrose 11 with a pH of 7.4 (NaOH).
The internal solution was (in mM): CsCl 130, MgCl2 1, NaCl 7, HEPES
5, EGTA 5 with a pH of 7.2 (CsOH). In Cav1.2 experiments, 1.8 mM
CaCl2 was replaced with 4mM BaCl2. Chemicals were obtained fromeither Sigma-Aldrich (MA) or Fisher Scientiﬁc (PA).
2.2. Test compounds
Test compounds were obtained from either Tocris Biosciences (MN)
(dofetilide, quinidine, ranolazine, verapamil, mexiletine, lidocaine, diltiazem) or Sigma-Aldrich (MA) (all others). Compounds were dissolved
in either de-ionized H2O or DMSO to create master stock solutions.
This stock was subdivided into aliquots which were stored frozen
until use. Dilutions of the master stock to create the ﬁnal test concentrations were performed on the day of experimentation. Compounds were
tested at four different concentrations (free plasma Cmax and 3 multiplesof Cmax (see Table 1).
2.3. Data acquisition and analysis
Experiments (n = 3–6) were performed at 36 ± 1 °C with the exception of Kv4.3 which was performed at 22 ± 1 °C. This was done because separation of the Kv4.3 ionic current from the capacitive current
could not be done at 36 °C, making it impossible to measure current amplitude accurately. Currents were measured using the whole-cell variant of the patch clamp method as previously described (Crumb, Pigott,& Clarkson, 1995). Glass pipettes were pulled from borosilicate glassby a horizontal puller (Sutter Instruments, USA). Pipette tip resistance
was approximately 1 to 2 MΩ when ﬁlled with internal solutions. Series
resistance was compensated electronically by approximately 60–80%.
An Axopatch 1-B ampliﬁer (Axon Instruments, Foster City, CA) wasused for whole-cell voltage clamping. Creation of voltage clamp pulses
and data acquisition was controlled by a computer running pClamp
software (ver 9.2 Axon Instruments). After rupture of the cell membrane (entering whole-cell mode), current kinetics and amplitudes
were allowed to stabilize (typically 3–5 min) as the cell was dialyzed
with internal solution and the voltage protocol was applied every 10 s

Table 1
The following test concentrations were used (in nM).
Drug

Free Cmax

Dose 1

Dose 2

Dose 3

Dose 4

Amiodarone
Amitryptiline
Azithromycin
Bepridil
Chloroquine
Chlorpromazine
Cibenzoline
Cisapride
Diltiazem
Dofetilide
Flecainide
Lidocaine
Lopinavir
Mexiletine
Mibefradil
Moxiﬂoxacin
Nilotinib
Ondansetron
Propafenone
Quinidine
QuinineRanolazine
Ritonavir
Ruﬁnamide
Saquinavir
Sertindole
Sotalol
Terfenadine
ToremeﬁneVerapamil

0.7
36.4
1937.0
31.5
249.5
34.5
673.0
2.6
127.5
2.1
752.9
703.7
2560.42503.210.6
3562.5
60.4
358.5
130.9
842.9
3956.7
1948.2436.9
83,126.9
417.2
1.6
14,686.4
0.3
26.3
45.0

0.8
36
2000
30
250
35
600
2.5
20
0.1
200
625
700
625
12
7000
60
200
130
300
4000
575
400
80,000400
1.5
14,000
0.8
25
5

80
360
20,000
180
750
350
1800
7.5
150
1
1000
2500
2100
2500
120
21,000
300
1000
390
900
12,0002300
1200
2400001200
4.5
140,000
8
75
50

800
3600
100,000
900
2500
1750
6000
25
500
2
4000
5000
7000
5000
1200
70,000
1800
4000
1300
2700
40,000
6900
4000
NS
4000
15
700,000
80
250
150

8000
36,000300,000
3000
25,00010,50060,000125
12,5003
20,00010,00035,00010,00012,000350,000
6000
20,0006500
5400
200,000
23,00020,000NS
20,00075
2,100,000
800
1250
500

NS, not soluble
Additional concentrations were added for some of the drugs and some currents as indicated in the Tables in Supplement 1.

(applied at 0.1 Hz). For Cav1.2 and KvLQT1/mink, currents which tend
to exhibit more current rundown, the reduction in current amplitude
typically occurred within the ﬁrst minutes after rupture of the cell
membrane. If present (N 20% current reduction), these cells werediscarded. Only cells with a stable ionic current and stable holding
current were used. The voltage protocols used to record the various
currents are given in Panel A of Figs. 1–7. A ventricular action potential waveform was used to elicit hERG, Cav1.2, and KvLQT1/mink(kindly provided by Dr. Gail Robertson, University of Wisconsin).
For Nav1.5 (peak and late), Kv4.3 and Kir2.1, the voltage protocols
used were those proposed by the ICWG. For measurement of
Nav1.5-late, current was elicited by the addition of 50 μM veratridine
to the external solution. Drug was applied to the bath solution via a
rapid perfusion apparatus. Not all four concentrations were examined in every cell. Some currents were also examined for drug blocking
effects by applying the pulse protocol every 1 s (applied at 1 Hz). The
drugs examined (quinidine, moxiﬂoxacin, dofetilide, ranolazine, diltiazem, and verapamil) were those evaluated in recent clinical studies(Johannesen, Vicente, Mason, et al., 2014, 2016). Not all of these drugs
were examined for every current at 1 Hz.
Time-control experiments were performed for each current inwhich cells were exposed to non-drug containing external solution
spiked with 0.1% DMSO for 5–8 min (the time period of a typicalexperiment).
Data are presented as % reduction of current amplitude. This wasmeasured as current reduction after an apparent steady-state effecthas been reached in the presence of drug (or vehicle) relative to current
amplitude before drug (or vehicle) was introduced (control). Each cell
served as its own control. When possible (greater than 50% block inany cell was observed), a nonlinear curve-ﬁtting routine was utilized
to ﬁt a three-parameter Hill equation to the results using R version

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

W.J. Crumb Jr. et al. / Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx

3

Fig. 1. hERG current (IKr) block by the 30 drug panel. A. Action potential waveform used to elicit hERG current at 36 ± 1 °C. B. Example of the effects of sotalol on hERG current. I = 0
represents zero current. Peak outward current was measured. C. Concentration–response relationship for sotalol block of hERG. Fit equation is given in Section 2. D. Amount of hERG
current block produced by 1, 3X multiples of Cmax free plasma concentrations for each member of the 30 drug panel (data derived from Tables in Supplement).

3. Results

the voltage protocol every second. The block produced by quinidine(0.3 μM), moxiﬂoxacin (70 μM), dofetilide (2 nM), ranolazine(6.9 μM), diltiazem (12.5 μM), and verapamil (0.5 μM) at 1 Hz wasless than 6% greater than that observed at 0.1 Hz, suggesting a lack ofrate-dependent block of hERG current by these tested drugs at these
rates. This however does not exclude the possibility that ratedependent changes may be observed with other drugs. Time-control
experiments indicate that over the time course of a typical experiment(5–6 min) hERG current amplitude was reduced by 2.5 ± 1.9% (datanot shown; n = 6).

3.1. hERG (IKr)

3.2. Nav1.5-late (INa-late)

The hERG current is an important current in repolarization of the
myocardium and the most common target for QT prolonging drugs.
The effect of drugs on the amplitude of the hERG current was assessedusing the protocol shown in Fig. 1A. This voltage protocol elicited acurrent that peaked at voltages corresponding to the repolarization
phase of the action potential (Fig. 1B). As an example, addition of QT
prolonging drug sotalol produced a concentration-dependent reductionof hERG current with an IC50 value of 86.4 μM (Fig. 1C). Fig. 1D plots thepercent hERG block associated with all 30 drugs at the free plasma Cmax.
As indicated, drugs commonly associated with QT prolongation produce
a marked blockade of hERG current at therapeutic concentrations (e.g.
quinidine, dofetilide). To assess the rate-dependence of hERG block, a
subset of 6 drugs were evaluated at near IC50 concentrations by applying

The Na current ﬂowing during the plateau of the action potential has
been shown to contribute to action potential duration (APD). This current has been designated INa-late. Blockade of INa-late can shorten APD
and QT, attenuate prolongations resulting from hERG block and prevent
early after depolarizations. At 37 °C, Nav1.5 normally decays rapidly andwithin approximately 3 ms there is little measurable current remaining(Supplemental Figure 1A). In order to observe Nav1.5-late, the normal
process of channel inactivation must be altered. This is typically donewith a toxin such as ATX-II or veratridine (Belardinelli et al., 2013;
Chevalier, Amuzescu, Gawali, et al., 2014). In the present experiments,
Nav1.5-late was elicited by the application of 50 μM veratridine to thebath solution. In the presence of veratridine, INa decays much more
slowly and a measurable late current is easily observed (Supplemental

3.2.2 (R Foundation for Statistical Computing, Vienna, Austria). The
equation is:
y ¼ V maxxnnk þ xnwhere Vmax = 100, k, and n are unconstrained variables. Data
tables with individual values for each drug are given in Supplement 1.

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

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Fig. 2. Nav1.5-late current (INa-late) block by the 30 drug panel. A. Voltage protocol used to elicit current at 36 ± 1 °C. Arrow indicates where current was measured. B. Example of theeffects of ranolazine on Nav1.5-late current. Only current during the step to −15 mV is shown. Current was measured at the end of the step to −15 mV. C. Concentration–response
relationship for ranolazine block of Nav1.5-late. Fit equation is given in Section 2. D. Amount of Nav1.5-late current block produced by 1, 3X multiples of Cmax free plasma
concentrations for each member of the 30 drug panel (data derived from Tables in Supplement).

Figure 1 A & B). Furthermore, when measuring Nav1.5-late current
40 ms after initiation of a step to −15 mV, there is a clear distinction between the peak and late components of Nav1.5 (Supplemental
Figure 1B). As indicated in Fig. 2, the most well described blocker ofINa-late, ranolazine, concentration-dependently blocked Nav1.5-latewith an IC50 of 7.9μM. At free Cmax plasma concentrations, 5 drugs(quinine, ranolazine, ﬂecainide, lidocaine and mexiletine) exhibited approximately a 10% or greater blockade of Nav1.5-late and between approximately 25% to 50% blockade at 3x Cmax (Fig. 2D). To further
verify that the current being measured 40 ms after the start of a pulse
to −15 mV was indeed Nav1.5-late, for 3 drugs the current amplitude
was measured at − 15 mV during the voltage ramp which is 342 ms
after pulse initiation (see Fig. 2A). The IC50 values for ranolazine, lidocaine, and mexiletine were 10.1 μM, 8.4 μM, and 8.4 μM when measured
342 ms after pulse initiation, respectively. This compares very well withvalues obtained 40 ms after pulse initiation (ranolazine: 7.9 μM,
lidocaine: 10.8 μM, mexiletine: 8.9 μM). Time-control experiments of
Nav1.5-late showed little change in current amplitude (0.4 ± 0.3% reduction, n = 8).
3.3. Cav1.2 (ICaL)
Cav1.2, which encodes the L-type Ca++ channel, plays a critical rolein the contraction of the myocardium and block of this current can

attenuate the APD and QT prolonging effects of hERG blocking drugs.
Initial experiments in which Cav1.2 was measured using 1.8mM Ca++
indicated that a small amplitude current (typically b200 pA), was elicited by the action potential waveform shown in Supplement Figure 2. For
the purposes of the experiments described here, Ca++ was replacedwith Ba++ to increase current amplitude and more easily allow pharmacological evaluation. This does not suggest that Ba++ will be chosen
as the charge carrier in the ﬁnal CiPA assay. Using Ba++ as a charge
carrier, the inward current recorded was blocked by verapamil andexhibited a characteristic Cav1.2 current voltage (I–V) relationship(Supplement Figure 2). An example of the pharmacology of thischannel is shown in Fig. 3. As illustrated, diltiazem blocked Cav1.2
with an IC 50 of 112nM. Fig. 3D illustrates that classic L-type Ca++channel blockers such as diltiazem and verapamil block Cav1.2
by approximately 20% or more at free plasma Cmax . The ratedependence (1 Hz) of Cav1.2 block was determined for the 2 L-type
calcium channel blocking drugs, verapamil (150 nM) and diltiazem(150 nM). In the absence of drug, pacing at 1 Hz reduced Cav1.2 current amplitude by 29.2 ± 6.7% (n = 6). In the presence of either diltiazem or verapamil, there was no additional relative block observed
at a pacing rate of 1 Hz as compared to 0.1 Hz (diltiazem: 1.8 ± 1.1%,
verapamil: 0 ± 0%, n = 3). Time-control experiments indicate thatover the time course of a typical experiment Cav1.2 current amplitude was reduced by 2.0 ± 1.2% (n = 4).

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

W.J. Crumb Jr. et al. / Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx

5

3.4. Nav1.5-peak (INa-peak)

3.5. Kv4.3 (Ito)The rapidly activating component of the sodium current (INa-peak)plays a critical role in depolarization of cardiac cells and conduction
through the myocardium. Nav1.5-peak was recorded at physiologic
temperatures (36 ± 1 °C) and using 137 mM NaCl in the external solution. To evaluate whether proper voltage control was present underthese conditions, I–V relationships were constructed (Supplement
Figure 2D). As indicated, the I–V relationship under the present recording conditions exhibited typical characteristics for well controlled INa
with approximately 25–30 mV between current activation and peakcurrent amplitude and a reversal potential which appears to be very
close to the predicted + 79 mV. The I–V relationship after addition of
1 μM tetrodotoxin (TTX) is also shown. Flecainide reduced Nav1.5peak with an IC50 of 6.7 μM (Fig. 4). At Cmax free plasma concentrations,
all of the drugs examined blocked Nav1.5-peak by less than 20%(Fig. 4D). Time controls were associated with a 2.3 ± 2.3% reductionin Nav1.5-peak (n = 4). Rate-dependence at 1 Hz was determined forranolazine (23 μM), diltiazem (12.5 μM), lidocaine (10 μM), mexiletine(10 μM), dofetilide (6 nM), verapamil (1 μM), quinidine (5.4 μM), andmoxiﬂoxacin (350 μM). There was less than a 10% additional reductionin Nav1.5-peak current when paced at 1 Hz compared to 0.1 Hz for anyof the drugs tested (range: 9.7 ± 6.1% for quinidine, 1.2 ± 1.2% for
dofetilide, n = 3).

Kv4.3 encodes the transient outward current (Ito) which plays animportant role in the rapid phase (phase 1) of action potential repolarization. The current elicited by the voltage protocol shown in Fig. 5A, activated to a peak and then decayed characteristic of the Ito recorded inhuman cardiac cells (Crumb et al., 1995). As indicated, quinine exhibited concentration-dependent block of Kv4.3 with an IC50 of 79.3 μM(Fig. 5B & C). Of the 30 drugs tested, amitriptyline was associated withthe greatest amount of Kv4.3 block at free plasma Cmax concentrations(Fig. 5D). Time-control experiments showed a 2.3 ± 2.0% reduction(n = 4) in current amplitude.
3.6. KvLQT1/mink (IKs)
KvLQT1/mink encodes the slow component of the delayed rectiﬁer(IKs) and plays a role in repolarization of the action potential. Fig. 6Bshows an example of KvLQT1/mink block. As indicated, the outward
current elicited with the action potential waveform was blocked by quinine with an IC50 of 37.4 μM (Fig. 6C). All of the drugs tested blocked
KvLQT1/minK by less than 20% at free plasma Cmax concentrations(Fig. 6D). The rate-dependence (1 Hz) of KvLQT1/minK block was determined for the 2 blocking drugs, quinidine (5.4 μM) and moxiﬂoxacin(70 μM). With either drug, there was little additional block observed

Fig. 3. Cav1.2 current (L-type ICa) block by the 30 drug panel. A. Action potential waveform used to elicit Cav1.2 current at 36 ± 1 °C. B. Example of the effects of diltiazem on Cav1.2 current.
I = 0 represents zero current. Peak inward current was measured. C. Concentration–response relationship for diltiazem block of Cav1.2. Fit equation is given in Section 2. D. Amount of
Cav1.2 current block produced by 1, 3X multiples of Cmax free plasma concentrations for each member of the 30 drug panel (data derived from Tables in Supplement).

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

6

W.J. Crumb Jr. et al. / Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx

Fig. 4. Nav1.5-peak current (INa-peak) block by the 30 drug panel. A. Voltage protocol used to elicit current at 36 ± 1 °C. Arrow indicates where current was measured. B. Example of theeffects of ﬂecainide on Nav1.5-peak current. Only current during the step to −15 mV is shown. Peak inward current at the beginning of the step to − 15 mV was measured. C.
Concentration–response relationship for ﬂecainide block of Nav1.5-peak. Fit equation is given in Section 2. D. Amount of Nav1.5-peak current block produced by 1, 3X multiples ofCmax free plasma concentrations for each member of the 30 drug panel (data derived from Tables in Supplement).

at a pacing rate of 1 Hz as compared to 0.1 Hz (quinidine: 3.8 ± 2.2%,
moxiﬂoxacin: 2.6 ± 2.6%, n = 3). Time-control experiments showed
an 8.2 ± 0.4% reduction (n = 4) in current amplitude.
3.7. Kir2.1 (IK1)Kir2.1 encodes the inwardly rectifying potassium current (IK1)
which helps to set the resting membrane potential in cardiac cells andalso contributes to the terminal phase of repolarization of the action potential. Using the voltage protocol shown in Fig. 7A, an inward current is
activated that is blocked by chlorpromazine with an IC50 of 9.3 μM. As
illustrated, none of the drugs tested reached a 10% mean blockade of
Kir2.1 at free plasma Cmax concentrations (Fig. 7D).
3.8. Relative incidence of channel block
It is important to gain insight into the relative frequency with which
the ion currents which have been suggested to be included in routine
screening are affected by these 30 drugs. Taking a 20% reduction in current amplitude as an arbitrary marker, the relative frequency withwhich the tested ion currents were blocked at any concentration, 1x
therapeutic free plasma Cmax, and 3x Cmax is shown in Fig. 8. As expected, hERG is the most frequently blocked current. Nav1.5-late and Cav1.2

were the next most frequently blocked currents with approximately 4times fewer drugs blocking by 20% or more at 1x and 3x free plasma
Cmax. In contrast, at 1x therapeutic Cmax, the other currents tested
were not blocked by 20% or more by any of the drugs tested. At a 3x
multiple of free plasma Cmax, Kv4.3 was blocked approximately as frequently as Nav1.5-late and Cav1.2, while Nav1.5-peak and KvLQT1/
mink were less frequently blocked. Kir2.1 was not blocked by any
drug tested by 20% or more at a 3x multiple of free plasma Cmax.
3.9. Relationship between channel block and arrhythmic risk
The relationship between ion channel block and proarrhythmic risk
was examined for a subset of 12 drugs that were rated by the CiPA clinical working group to have low, intermediate or high risk of torsade depointes. As indicated in Figs. 9 and 10, at both a 1X and a 3X free plasma
Cmax concentration, drugs which are classiﬁed as possessing a high riskfor pro-arrhythmic events are associated with a large and rather selective block of hERG. In contrast, drugs in the low risk category block either Cav1.2 or Nav1.5-late more so or to a similar degree as hERG at
Cmax. Drugs in the intermediate category block hERG more than othercurrents but on average are associated with less hERG block at Cmax
than members of the high risk category. Of note, the 40% hERG blockshown in Fig. 9 for terfenadine (intermediate risk drug) is associatedPlease cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

W.J. Crumb Jr. et al. / Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx

7Fig. 5. Kv4.3 current (Ito) block by the 30 drug panel. A. Voltage protocol used to elicit current at 22 ± 1 °C. Arrow indicates where current was measured. B. Example of the effects ofquinine on Kv4.3-peak current. Peak outward current was measured. C. Concentration–response relationship for quinine block of Kv4.3. Fit equation is given in Section 2. D. Amount of
Kv4.3 current block produced by 1, 3X multiples of Cmax free plasma concentrations for each member of the 30 drug panel (data derived from Tables in Supplement).

with a Cmax of 9 nM, a concentration only achieved after metabolic inhibition. However, a more standard Cmax of 0.3 nM is only associated with
8% hERG block, which could contribute to it being an intermediate versus high risk drug. Other factors such as the kinetics of drug binding to
the hERG channel may also be important for differentiating intermediate from high risk drugs.
4. Discussion
For more than a decade, the cardiac potassium channel hERG has
been the focus of both regulatory agencies and drug developers. While
this attention has been warranted since drug induced blockade of thischannel has been associated with QT prolongation and in rare cases torsade de pointes, the singular focus on this channel has likely led to unwarranted drug attrition and labeling (Stockbridge, Morganroth, Shah,et al., 2013). In virtually all cases of drug-associated torsade de pointes,
the offending drug has been shown to selectively block hERG at relevant
plasma concentrations. However, there are drugs which block hERG anddespite this activity are associated with little or no QT prolongation andminimal torsade de pointes risk, sometimes due to block of the L-type
ICa (e.g. verapamil) or INa-late (e.g. ranolazine). In the present study,
we observed that the most frequently blocked channels at clinical free
Cmax concentrations were hERG, Nav1.5-late and Cav1.2. While thesubstantial hit rate for hERG is expected, results from the present
study using the manual patch clamp technique under physiologic conditions highlight for the ﬁrst time that a large number of drugs also
block Nav1.5-late and/or Cav1.2 currents, which can reduce the risk of
torsade de pointes caused by hERG block (Antoons, Oros, Beelman,
et al., 2010; Antzelevitch, Belardinelli, Zygmunt, et al., 2004; Fauchier,Babuty, Autret, et al., 1999; Gintant et al., 2006; January & Riddle,1989). This is consistent with the four CiPA low risk drugs in thisstudy having equal or greater Nav1.5-late block (ranolazine andmexiletine) or Cav1.2 block (verapamil, diltiazem) than hERG block,while the intermediate and high risk drugs had greater hERG block
than other channels.
4.1. Comparison to published data
The results obtained in the present study were compared to previously published results (Kramer, Obejero-Paz, Myatt, et al., 2013).
While many of the drugs evaluated in the present study have publisheddata for various ion channels, the Kramer et al. dataset has many of thedrugs tested here and contains data on hERG, Nav1.5-peak, and Cav1.2.
As indicated in Table 2, with few exceptions, the IC50 values from the 2studies are very similar. One notable exception is dofetilide, which has
an IC50 value for block of hERG in the present study (2 nM) which is

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

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W.J. Crumb Jr. et al. / Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx

Fig. 6. KvLQT1/minK current (IKs) block by the 30 drug panel. A. Voltage protocol used to elicit current at 36 ± 1 °C. B. Example of the effects of quinine on KvLQT1/minK-peak current. Peakoutward current was measured. C. Concentration–response relationship for quinine block of KvLQT1/minK. Fit equation is given in Section 2. D. Amount of KvLQT1/minK current block
produced by 1, 3X multiples of Cmax free plasma concentrations for each member of the 30 drug panel (data derived from Tables in Supplement).

approximately 15 times lower than in the Kramer et al. study (30 nM).
The results obtained from the present study are consistent with the freeplasma levels of dofetilide (2.7 nM) associated with an approximately
75 ms increase in QTc (Johannesen et al., 2014).
The overall similarity in results observed for the 16 drugs in common
between the 2 studies is interesting given the very different experimental methods. In the present study, all 3 currents were measured using
the manual patch clamp technique at approximately 36 °C using either
an action potential waveform (hERG, Cav1.2) or a step-pulse (Nav1.5peak). In the Kramer et al. study, data was obtained using an automatedpatch clamp platform at room temperature and using step-pulse voltage
protocols. These results suggest that for most of the drugs in Table 2, experimental parameters such as temperature, patch clamp platform, andvoltage protocols do not exert a marked effect on IC50 values. This is
somewhat comforting since ultimately the ion channel portion of the
CiPA paradigm is expected to be performed via automated patch platforms to accommodate large numbers of compounds and multiple
channels. However, there could be greater differences when evaluating
drug blocking kinetics.
Four antihypertensive/antianginal drugs with primary effects on the
L-type Ca channel (Cav1.2) were evaluated in this study. Two of the four
drugs exhibited substantial block of Cav1.2 at free plasma Cmax concentrations. For instance, diltiazem and verapamil had Cav1.2 IC50 values of

112 nM and 202 nM (respectively) and free plasma Cmax values of approximately 128 nM and 45 nM, respectively. In contrast, mibefradil
was associated with virtually no block of Cav1.2 at 12 nM and an IC50
of 652 nM (free plasma Cmax = 10.6 nM). This is consistent with previous reports in myocytes in which mibefradil exerts a far greater effecton T-type calcium current than on the L-type (Leuranguer, Mangoni,
Nargeot, et al., 2001)). Bepridil showed only a 4.2% reduction in
Cav1.2 at 30 nM and an IC50 of 2.8 μM (free plasma Cmax = 31.5 nM).
In guinea pig ventricular myocytes, bepridil blocks the L-type Ca current
with an IC50 of 500 nM (Yatani, Brown, & Schwartz, 1986). These results
suggest that a characterization of Cav1.2 to deﬁne the cardiac effects of
L-type Ca++ channel blockers is an appropriate surrogate for acutely
isolated ventricular myocytes.
4.2. Frequency of hits on ion currents
To determine the frequency of blockade for the tested ion channel
currents, the number of drugs producing a 20% reduction of a given current at any tested concentration was plotted (see Fig. 8). The 20% reduction value is an arbitrary number but one commonly referred to in theliterature (Crumb, Ekins, Sarazan, et al., 2006; Redfern, Carlsson, Davis,
et al., 2003). The results indicate that hERG was the current most frequently blocked, followed by Nav1.5-late and Cav1.2 each being blocked

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

W.J. Crumb Jr. et al. / Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx

9Fig. 7. Kir2.1 current (IK1) block by the 30 drug panel. A. Voltage protocol used to elicit current at 36 ± 1 °C. Arrow indicates where current was measured. B. Example of the effects ofchlorpromazine on Kir2.1 current. Trace is truncated to show current during ramp to − 120 mV. Peak inward current was measured at −120 mV at the end of the ramp. C.
Concentration–response relationship for chlorpromazine block of Kir2.1. Fit equation is given in Section 2. D. Amount of Kir2.1 current block produced by 1, 3X multiples of Cmax freeplasma concentrations for each member of the 30 drug panel (data derived from Tables in Supplement).

by greater than 60% of the drugs tested. Kir2.1 was the least frequently
blocked current. In addition, the frequency of block at 1x and 3x the freeplasma Cmax was plotted. This showed a very different proﬁle with hERG
being blocked by 20% or more by 37% of the drugs and all other currents
being blocked by less than 10% of the drugs tested. No drugs blocked

Nav1.5-peak, Kv4.3, LvLQT1/mink, or Kir2.1 by 20% or more at a 1x
Cmax concentration. At a 3x Cmax concentration, hERG was blocked by
70% of the drugs tested whereas all other currents were blocked by
20% or less of the drugs tested. None of the drugs tested blocked
Kir2.1 by 20% or more at a 3x Cmax concentration.

Fig. 8. Relative ion current panel activity. Plot of the % of drugs out of 30 which blocked the indicated ion currents by at least 20% at any of the concentrations tested (A), at 1X the freeplasma Cmax (B), and at 3X the free plasma Cmax (C). Reference data was obtained from tables in the Supplement.

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
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Fig. 9. Plot of the ion channel blocking proﬁle of members of the high (top row), intermediate (middle row), and low (bottom row) CiPA proarrhythmic risk categories. % block represents
block at 1X free plasma Cmax. Data was derived from Tables in the Supplement. The Cmax chosen for terfenadine was 9 nM, a concentration achieved after metabolic inhibition. A more
standard Cmax of 0.3 nM is only associated with 8% hERG block.

Fig. 10. Plot of the ion channel blocking proﬁle of members of the high (top row), intermediate (middle row), and low (bottom row) CiPA proarrhythmic risk categories. % block represents
block at 3X free plasma Cmax. Data was derived from Tables in the Supplement.

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009

W.J. Crumb Jr. et al. / Journal of Pharmacological and Toxicological Methods xxx (2016) xxx–xxx
Table 2Comparison of IC50 values.
Drug

hERG

Cav1.2

Nav1.5-peak

Amiodarone
Bepridil
Chlorpromazine
Cisapride
Diltiazem
Dofetilide
Flecainide
Mibefradil
Moxiﬂoxacin
Nilotinib
Quinidine
Saquinavir
Sertindole
Sotalol
Terfenadine
Verapamil

0.9/0.9
0.2/0.2
1.1/1.5
0.01/0.02
6.6/53.2
0.002/0.03
0.7/1.5
0.3/1.7
92.7/86.2
0.1/1.0
0.3/0.7
3.4/16.9
0.01/0.03
86.3/111.4
0.02/0.05
0.7/0.3

1.3/1.9
2.8/1.0
8.2/3.4
N0.13/11.8
0.1/0.8
N0.006/26.7
N20/27.1
0.7/0.5
N350/173.0
N6/17.5
N5.4/6.4
3.2/1.9
N0.08/6.3
N2100/193.3
0.7/0.9
0.1/0.2

4.6/15.9
2.9/2.3
4.5/3.0
N0.13/337.0
N12.5/22.4
N0.006/54.2
6.7/6.2
8.5/5.6
N350/1112.0
N6/13.3
N5.4/14.6
15.6/12.1
N0.08/6.9
N2100/7013.9
N0.8/2.0
N1.0/32.5

All values are IC50 values or the highest concentration tested (in μM). First value in eachpair is from the present study, second value from Kramer et al., 2013. In some cases in
the present study and IC50 value was not reached. In those cases, the IC50 value is designated as being N than the highest concentration tested.

4.3. Stratiﬁcation of safety risk
Included in the drugs tested were 4 members from each of the CiPArisk categories (high, intermediate and low). These categories werebased upon the risk of drug-associated torsade de pointes. We compared ion channel block against all tested ion currents at free plasma
Cmax values of 1x and 3x. This method has been used before on hERG,
Cav1.2, and peak Nav1.5 using relative IC50 values. As indicated in
Figs. 9 & 10, members of the high risk category blocked hERG at therapeutic free plasma Cmax either exclusively or to a much greater extent
than any other current examined. In the intermediate category, drug
block of hERG was on average less than in the high risk category,
supporting a lower risk of torsade de pointes. In the low risk category,the members were associated with a greater or equal block of either
Cav1.2 or Nav1.5-late when compared to block of hERG. These results
clearly indicate the need for testing drug candidates against a panel ofion channels.
As indicated in Figs. 9 and 10, the ion current blocking proﬁle forsome members of the intermediate torsade de pointes risk category issimilar to that observed with some members of the high risk category.
There are several possible reasons for this including (1) pharmacokinetic
or drug interaction properties which may lead to higher plasma concentrations some in patients, (2) differences in risk factors in patient populations receiving each drug, (3) the assignment of drugs into high andintermediate risk categories may be imperfect, and (4) the kinetics ofthe drug block, particularly for hERG, may make some drugs moreproarrhythmic than others. The kinetics of drug binding to the hERG
channel and its ability to improve risk stratiﬁcation is currently being investigated further by the CiPA ion channel and in silico working groups(Fermini et al., 2016).
In a recent clinical study of quinidine and dofetilide, the free plasma
concentrations associated with the approximately 70% and 55% reduction in hERG current amplitude, respectively (see Fig. 9) was associatedwith a79 ms and 74 ms increase in QTc (Johannesen et al., 2014) and Twave morphology changes (Vicente, Johannesen, Mason, et al., 2015).
Furthermore, in the same study, ranolazine was associated with a
12 ms increase in QTc and verapamil was associated with no increase
in QTc. The ion channel proﬁle for verapamil supports the clinical resultsin that there is more block of Cav1.2 than hERG at therapeutic concentrations. The observation of a modest increase in QTc upon exposure
to therapeutic concentrations of ranolazine suggests that the hERG
block induced QTc prolongation is not completely counteracted byblock of Nav1.5-late, but is enough to counteract torsade de pointes

11risk. However, the QTc prolongation associated with ranolazine has a
speciﬁc ‘signature’ that involves Tpeak-Tend prolongation without prolongation of the heart rate corrected J-Tpeak (J-Tpeakc) interval(Johannesen et al., 2014). Additional recent clinical study suggests that
the J-Tpeakc interval is a better biomarker (than QTc) in assessing thebalance of inward and outward current block and predictor of low torsade risk drugs (Johannesen et al., 2014). The CiPA clinical phase 1
ECG working group is expanding this analysis to additional drugs andwill propose a framework for potential implementation of this new approach at an April 2016 Cardiac Safety Research Consortium meeting.
4.4. Limitations
The following are limitations of the present study. 1). The action potential waveform used was recorded from rabbit, and not human, ventricle. While this waveform has been used previously to characterizehERG (Zhou, Gong, Ye, et al., 1998), we are cognizant that the data
may be more relevant if a human ventricular AP was used instead. 2).
Kv4.3 current was measured at room temperature and not physiologic
temperature, as with the other ion currents. This was done to allow separation of the capacitive transient from peak Kv4.3 current. It is notknown if the Kv4.3-drug interaction, for the drugs tested here, is temperature sensitive. 3). The drug effects on ion currents were tested at apacing rate of 0.1 Hz. This pacing rate was chosen because it is often
used in the literature and it represents a common pacing rate for all currents tested that was not associated with signiﬁcant rundown. However, currents were evaluated against some drugs at a pacing rate of 1 Hz,
which showed limited differences from the 0.1 Hz data. 4). Nav1.5-latewas elicited by the use of veratridine. Although sodium channel inactivation modiﬁers such as veratridine and ATX-II are commonly used toincrease the amplitude of late-INa, it is not known if they may interfere/alter the effects of late INa blockers. 5). In the present study, drugeffects were not “washed out” to provide insight into whether the reduction in current amplitude was due to actual drug block or current
rundown. However, time controls suggest that any reduction in current
amplitude was due primarily to drug-related ion current block. Furthermore, not all drug blocking effects, particularly at higher concentrations,are able to be “washed out”. 6). The Ca++ and Mg++ in the external solution can have blocking effects on hERG current (Ho, Kim, Lee, & Earm,
1998; Po, Wang, Yang, et al., 1999). This may make it difﬁcult in calculating the actual drug-hERG current blocking effects. However, the ion
concentrations were physiologic and it is unlikely that Ca++ andMg++ will be absent in the plasma of individuals taking these drugs.
The in silico and ion channel working group for the CiPA initiative
are currently studying voltage clamp protocols that model dynamic
drug–ion channel interactions for hERG, and potentially for other channels (Fermini et al., 2016). Thus, ﬁnal CiPA voltage clamp protocols for
hERG, and potentially other channels, may differ substantially fromthose used in this study. However, a critical ﬁrst step is to identify IC50
values or the maximal percent block at clinically relevant drug concentrations to determine which channels are the most important channels
to be assessed under CiPA. While this ion channel data could also be entered into in silico models, such as the O'Hara-Rudy model (O'Hara,
Virág, Varró, & Rudy, 2011), we elected to not do that here because it
is beyond the scope of this manuscript and because the in silico
proarrhythmia metrics have not been ﬁnalized. Rapidly publishing thisdata will allow any investigators to use it for in silico modeling. Finally,
drugs were selected with a stratiﬁed risk of torsade de pointes, existing
clinical ECG data, and with a range of ion channel effects. However, thehit rates for block of the different channels are only reﬂective of the 30
drugs in this study.
4.5. Conclusion
This is the ﬁrst study of its kind to examine the effects of 30 clinicaldrugs against the 7 ion channel currents currently proposed to makeup

Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
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the CiPA ion channel panel. The results of the present study highlightthe importance of drug-induced block of hERG, Nav1.5-late andCav1.2 at clinically relevant concentrations. These were the most frequently blocked currents and are likely the most important in terms ofincreased risk for torsade de pointes (blocking hERG) vs. decreasing
risk (blocking Nav1.5-late and/or Cav1.2). This was consistent withthe 12 CiPA drugs, where high and intermediate risk drugs had greaterhERG block than other channels, while low risk drugs had equivalent or
greater Nav1.5 block (ranolazine and mexiletine) or Cav1.2 block(verapamil, diltiazem) than hERG block. Nav1.5-peak block was thenext most commonly blocked channel, and its importance in conduction and potential for use-dependent block that was not thoroughly
assessed here, suggest that it also is important to be assessed under
CiPA. Based upon the 30 drugs tested in this study, the less frequent
hit rates for Kv4.3, KvLQT1/mink and Kir2.1 suggest that these channels
may be less critical for assessment of all drugs under CiPA. This should
be further evaluated with consideration of potential false positive
hit rates along with the impact of block of these channels has onproarrhythmic risk in the CiPA in silico action potential model.
Conﬂict of interest statement
This work was funded by the US Food and Drug Administration
Critical Path Initiative.
Acknowledgments
The step ramp protocols used in this study were developed by the
CiPA ion channel working group. The authors would like to thank Drs.
Bernard Fermini, Najah Abi Gerges, Jules Hancox, Adam Hill, Jamie
Vandenberg and Norman Stockbridge for their expert opinions and review of the manuscript. This project was supported by the FDA Critical
Path Initiative and appointments to the Research Participation Programs at the Oak Ridge Institute for Science and Education through aninteragency agreement between the Department of Energy and FDA.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.vascn.2016.03.009.
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Please cite this article as: Crumb, W.J., et al., An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA)
proposed ion channel panel, Journal of Pharmacological and Toxicological Methods (2016), http://dx.doi.org/10.1016/j.vascn.2016.03.009