RESEARCH ARTICLE

Electrocardiographic Biomarkers for
Detection of Drug-Induced Late Sodium
Current BlockJose Vicente1,2,3*, Lars Johannesen1, Meisam Hosseini1,2, Jay W. Mason4,5, Philip
T. Sager6, Esther Pueyo3,7, David G. Strauss1*

a11111

1 Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences,
Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, UnitedStates of America, 2 Division of Biomedical Physics, Office of Science and Engineering Laboratories, Center
for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD, United States ofAmerica, 3 BSICoS Group, Aragón Institute for Engineering Research (I3A), IIS Aragón, University of
Zaragoza, Zaragoza, Spain, 4 Cardiology Division, University of Utah, Salt Lake City, UT, United States ofAmerica, 5 Spaulding Clinical Research, West Bend, WI, United States of America, 6 Stanford University,
Palo Alto, CA, United States of America, 7 Biomedical Research Networking Center in Bioengineering,
Biomaterials and Nanomedicine (CIBER-BBN), Zaragoza, Spain* david.strauss@fda.hhs.gov (DGS); jose.vicente@fda.hhs.gov (JV)OPEN ACCESS
Citation: Vicente J, Johannesen L, Hosseini M,
Mason JW, Sager PT, Pueyo E, et al. (2016)
Electrocardiographic Biomarkers for Detection ofDrug-Induced Late Sodium Current Block. PLoS
ONE 11(12): e0163619. doi:10.1371/journal.
pone.0163619
Editor: Randall Lee Rasmusson, University at
Buffalo - The State University of New York, UNITEDSTATES
Received: February 19, 2016
Accepted: September 12, 2016
Published: December 30, 2016
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, orotherwise used by anyone for any lawful purpose.
The work is made available under the Creative
Commons CC0 public domain dedication.
Data Availability Statement: FDA Study 1(NCT01873950) data are available at PhysioNet’s
PhysioBank https://physionet.org/physiobank/database/ecgrdvq/ (doi:10.13026/C2HP45) as well
as at the Telemetric and Holter ECG Warehouse
http://thew-project.org/Database/E-OTH-12-5232020.html. FDA Study 2 (NCT02308748) data are
available at PhysioNet’s PhysioBank https://physionet.org/physiobank/database/ecgdmmld/(doi:10.13026/C2D016).  Please add also thefollowing citation when referencing the Physionet’s

Abstract
Background
Drugs that prolong the heart rate corrected QT interval (QTc) on the electrocardiogram(ECG) by blocking the hERG potassium channel and also block inward currents (latesodium or L-type calcium) are not associated with torsade de pointes (e.g. ranolazine andverapamil). Thus, identifying ECG signs of late sodium current block could aid in the determination of proarrhythmic risk for new drugs. A new cardiac safety paradigm for drug development (the “CiPA” initiative) will involve the preclinical assessment of multiple humancardiac ion channels and ECG biomarkers are needed to determine if there are unexpected
ion channel effects in humans.

Methods and Results
In this study we assess the ability of eight ECG morphology biomarkers to detect latesodium current block in the presence of QTc prolongation by analyzing a clinical trial wherea selective hERG potassium channel blocker (dofetilide) was administered alone and thenin combination with two late sodium current blockers (lidocaine and mexiletine). We demonstrate that late sodium current block has the greatest effect on the heart-rate corrected JTpeak interval (J-Tpeakc), followed by QTc and then T-wave flatness. Furthermore, J-Tpeakc
is the only biomarker that improves detection of the presence of late sodium current blockcompared to using QTc alone (AUC: 0.83 vs. 0.72 respectively, p<0.001).

Conclusions
Analysis of the J-Tpeakc interval can differentiate drug-induced multichannel block
involving the late sodium current from selective hERG potassium channel block. Future

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Detection of Late Sodium Current Block on the ECG

repositories: Goldberger AL, Amaral LAN, Glass L,
Hausdorff JM, Ivanov PCh, Mark RG, Mietus JE,
Moody GB, Peng C-K, Stanley HE. PhysioBank,
PhysioToolkit, and PhysioNet: Components of a
New Research Resource for Complex Physiologic
Signals. Circulation 101(23):e215-e220 [Circulation
Electronic Pages; http://circ.ahajournals.org/cgi/
content/full/101/23/e215]; 2000 (June 13).
PubMed PMID: 10851218.
Funding: This project was supported by FDA’s
Critical Path Initiative, Office of Women’s Healthand appointments to the Research Participation
Programs at the Oak Ridge Institute for Scienceand Education through an interagency agreement
between the Department of Energy and FDA. Pueyo
is funded by Ministerio de Economı́a yCompetitividad (MINECO), Spain, under project
TIN2013-41998-R and by Grupo Consolidado
BSICoS from DGA (Aragón) and European Social
Fund (EU).
Competing Interests: PT Sager has consulting
agreements with Biomedical Systems, Charles
River, and iCardiac. The other authors report noconflicts. This does not alter our adherence to
PLOS ONE policies on sharing data and materials.

methodologies assessing drug effects on cardiac ion channel currents on the ECG should
use J-Tpeakc to detect the presence of late sodium current block.

Trial Registration
NCT02308748 and NCT01873950

Introduction
The current regulatory paradigm for assessing torsade de pointes (torsade) risk of new drugs
focuses on whether the new drug or its metabolites block the hERG potassium channel [1] andprolong the heart rate corrected QT interval (QTc) on the electrocardiogram (ECG) in thethorough QT study [2], or an equivalent ECG analysis. This paradigm can be considered successful because no new marketed drugs have been removed from the market because of unexpected QTc prolongation or torsade risk since its implementation in 2005 [3]. On the other
hand, this approach may have prevented potentially effective medicines from reaching themarket, sometimes inappropriately [3]. This is because there are QTc prolonging drugs thatblock the hERG potassium channel, but have low torsade risk (e.g. ranolazine [4], amiodarone[5], verapamil [6]). In addition to hERG potassium channel block, these drugs block otherinward currents (i.e. late sodium or L-type calcium), which can prevent the occurrence of early
afterdepolarizations, the triggers for torsade [7, 8].
In order to address this, a consortium of multiple global drug regulators (U.S. Food andDrug Administration (FDA), European Medicines Agency, Health Canada, Japan Pharmaceutical and Medical Devices Agency), industry and academia coordinated by multiple public-private partnerships and professional societies (Health and Environmental Sciences Institute,
Safety Pharmacology Society, Cardiac Safety Research Consortium) are developing a new paradigm, the Comprehensive in vitro Proarrhythmia Assay (CiPA), which contains four components [9]. First, all new drugs will be evaluated for the effects on multiple human cardiac ionchannels and currents (e.g. hERG potassium, late sodium, L-type calcium) in overexpression
cell lines and the results integrated together in an in silico computational model of the ventricular cardiomyocyte. Second, the in silico model will output a proarrhythmia score to divide
drugs into very low risk, intermediate risk and high risk categories. Third, in vitro stem cellderived cardiomyocyte assays will be used to confirm the electrophysiologic drug effects fromthe ion channel experiments. Fourth, exposure-response modeling will be applied to human
electrocardiograms (ECGs) from early Phase 1 studies to determine if there are unexpected ionchannel effects compared to preclinical ion channel data, which might occur due to a humanspecific metabolite or protein binding. Under this paradigm, it will be important to differentiate QTc prolonging drugs with balanced ion channel effects, such as ranolazine (i.e. hERG
potassium channel and late sodium current block), that are not associated with torsade fromselective hERG potassium channel blockers, such as dofetilide, that are associated with torsade.
Two FDA-sponsored clinical trials have shown that drug-induced shortening of the heart
rate corrected J-Tpeak interval (J-Tpeakc) is a sign of late sodium current block [10, 11]. In addition, an analysis of 12 T-wave biomarkers from the first FDA-sponsored clinical trial (FDA
study 1: NCT01873950) that included dofetilide, quinidine, ranolazine and verapamil demonstrated that drug-induced changes in multiple T-wave morphology biomarkers are directly
related to the amount of hERG potassium channel block [12]. However, it was not clear if
inward current block affected T-wave morphology biomarkers.

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Detection of Late Sodium Current Block on the ECG

In this study, we assess drug effects on T-wave morphology biomarkers in the second FDAsponsored clinical trial (FDA study 2: NCT02308748) that included two late sodium current
blocking drugs (mexiletine and lidocaine) administered alone and in combination with a selective hERG potassium channel block (dofetilide). In addition, we rank eight ECG biomarkers by
their ability to detect late sodium current block and determine the optimal combination of biomarkers to differentiate selective hERG potassium channel block from multichannel block
involving the late sodium current.

Methods
This study was approved by the US Food and Drug Administration Research Involving
Human Subjects Committee (RIHSC 13-011D and 14-022D) and the local institutional review
board (Chesapeake IRB). All subjects gave written informed consent and the study was performed at a Phase 1 clinic (Spaulding Clinical Research, West Bend, WI, USA).

Clinical trial design
The study design of this Phase 1 clinical trial (FDA study 2: NCT02308748) has been described
previously [11]. Briefly, this was a five-period, randomized, cross-over trial designed to study
the ability of late sodium or calcium current block to balance the ECG effects of selective hERG
potassium channel block. There was one week between treatment periods. In each treatment
period subjects were dosed three times during the day with placebo, a selective hERG potassium channel blocker (dofetilide [Tikosyn, Pfizer, USA] or moxifloxacin) or a late sodium current blocker (mexiletine [Teva Pharmaceuticals USA Inc.,USA] or lidocaine [B. Braun
Medical, Inc., USA]) alone or in combination (mexiletine combined with dofetilide, lidocaine
combined with dofetilide), or a selective hERG potassium channel blocker (moxifloxacin)
alone or in combination with a calcium channel blocker (diltiazem). Plasma drug concentration was measured using a validated liquid chromatography with tandem mass spectroscopy
method by Frontage Laboratories (Exton, Philadelphia, PA). The present study focuses on theeffects of late sodium current block when combined with selective hERG potassium channel
block on eight ECG biomarkers. Therefore, time-points from the combination of diltiazem
with moxifloxacin were not included in this study. One female was excluded from this analysis
because in the evening time-point of the dofetilide alone arm, plasma dofetilide concentration
was lower despite the evening oral dose, but she still had larger placebo- and baseline-correctedchanges in all ECG biomarkers.

ECG measurement methodology
Continuous ECG recordings were performed using the Mortara Surveyor system (Mortara,
Milwaukee, WI, USA) sampled at 500 Hz with an amplitude resolution of 2.5 μV. Three 10 second non-overlapping 12-lead ECGs were extracted prior to the draw of each pharmacokinetic
sample, based on heart rate stability and signal quality using Antares software (AMPS LLC,
New York, NY, USA). All extracted 10s ECGs were up-sampled to 1000 Hz.
T-wave morphology biomarkers were automatically assessed in the 10s ECGs as previously
described [12]. Briefly, T-wave flatness and asymmetry were automatically assessed with
QTGuard+ (GE Healthcare, Milwaukee, WI, USA). T-wave amplitude as well as 30% of earlyand late repolarization duration (ERD30% and LRD30%) [13] were automatically assessed with
ECGlib [14]. The semi-automatic evaluation of QT, J-Tpeak and Tpeak-Tend subintervals
included in this analysis has been described elsewhere [10, 11].

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Detection of Late Sodium Current Block on the ECG

Analysis and correction of heart rate dependency
T-wave morphology biomarkers dependent on heart rate were corrected for heart rate as previously described [12]. Briefly, a population-based heart rate correction factor was developed
using baseline data from the present study. In all subsequent analyses, the heart rate dependent
biomarkers were corrected for heart rate using an exponential model (biomarkerc = biomarker/RRα), where the values of the α coefficient were 0.50 for T-wave flatness, 0.96 for the
maximum magnitude of the T vector and 1.17 for the ventricular gradient. The heart rate correction was performed using PROC MIXED in SAS 9.3 (SAS Institute, Cary, NC), wherein asignificance level of 0.05 was used to determine if there was a difference by sex.

Statistical methods
We assessed whether there was a mitigation of dofetilide-induced ECG changes by either mexiletine or lidocaine at the population’s maximum plasma drug concentration (Cmax) timepoint during the evening dose (Tmax). Mitigation was assessed using a paired t-test between
the individual placebo-corrected changes from baseline with the combination vs. the individual
predicted placebo-corrected changes from baseline at the individual measured dofetilide concentrations at Tmax in R 3.2.2 (R Foundation for Statistical Computing, Vienna, Austria). We
assessed the magnitude of the mitigation for each ECG biomarker using Cohen’s d [15] effectsize, which we also labeled as "negligible" (|d|<0.2), "small" (|d|<0.5), "medium" (|d|<0.8) or"large" (|d|0.8) [16] in R.3.2.2. The placebo-corrected change from baseline was computed
using PROC MIXED in SAS 9.3, where the change from baseline for each ECG biomarker bytime-point was the dependent variable and sequence, period, time, drug, and an interaction
between treatment and time were included as fixed effects, and subject was included as a random effect. Afterwards, a linear-mixed effects model was used to evaluate the relationshipbetween each of the ECG biomarkers and plasma concentrations for dofetilide and moxifloxacin alone. This was done using PROC MIXED in SAS 9.3 using a random effect on both intercept and slope (i.e., allowing each subject to have his or her own drug concentration-biomarker
relationship). P values <0.05 were considered statistically significant without adjustment for
multiplicity, and should be interpreted with an appropriate level of caution.
ECG signature of selective hERG potassium channel block. We used a previously
described method [12] to assess the ECG signatures of selective hERG potassium channel
block associated with dofetilide and moxifloxacin. Briefly, we used the concentration-responsemodels to predict the individual drug-induced changes for QTc and each ECG biomarker at
25% increments of the population’s Cmax of each drug. Then, predicted changes together with
95% confidence intervals were plotted for each ECG biomarker vs. QTc.
Receiver operating characteristic (ROC) analysis. We developed multiple logistic regression models using one or two ECG biomarkers to classify selective hERG potassium channel
block (dofetilide and moxifloxacin alone arms) vs. multichannel block with late sodium current
inhibition (mexiletine with dofetilide and lidocaine with dofetilide arms) using placebo-corrected changes from baseline in each ECG biomarker pooled from all time-points (afternoon
and evening time-points for dofetilide alone, mexiletine with dofetilide, and lidocaine with
dofetilide; morning and afternoon time-points for moxifloxacin alone). Only biomarkers that
had significant mitigation of dofetilide-induced changes by either mexiletine or lidocaine wereused in the models built with two ECG biomarkers. We computed the receiver operating characteristic (ROC) area under the curve (AUC) to assess the performance of each model and 95%
confidence intervals (CI) of AUC were computed with 2000 stratified bootstrap replicates. We
compared the performances of the models using Delong’s test [17]. This analysis was done in R
3.2.2.

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Detection of Late Sodium Current Block on the ECG

Decision tree. Lastly, we developed a C4.5 decision tree [18] to assess the relationshipbetween the ECG biomarkers and selective hERG potassium channel block vs. multichannel
block. The decision tree was developed using 10-fold cross-validation on all data from selectivehERG potassium channel block (dofetilide [afternoon and evening time-points] and moxifloxacin [morning and afternoon] alone) vs. multichannel block (afternoon and evening timepoints of mexiletine with dofetilide and lidocaine with dofetilide) (training set). We assessed
the performance of the decision tree in the training set, but also using all data from our previous clinical study (FDA study 1: NCT01873950, validation set) [10], where each drug waslabeled according to its corresponding ion channel current block at the population’s Cmax
achieved in the clinical trial [12]. More specifically, dofetilide and quinidine were labeled as
predominant hERG potassium channel blockers and ranolazine and verapamil as multichannel
blockers. The decision tree was computed using the J48 algorithm in WEKA data mining software [19] version 3.6. The “positive” class for all classification methods (ROC-AUC and decision tree) was “multichannel block”.

Results
The study included 21 healthy subjects (8 female). No serious adverse events were observed[11]. Table 1 reports the baseline characteristics of the subjects included in this analysis.

Selective hERG potassium channel block: dofetilide and moxifloxacin
At equivalent drug-induced QTc prolongation, T-wave morphology changes induced by moxifloxacin were similar to those caused by dofetilide. Moreover, the ECG “signature” of dofetilide
was consistent with the ECG “signature” observed in our previous clinical study [12] (Fig 1).
Table 1. Baseline characteristics.
All subjects(N = 21)
Demographic
Age (years)

26 ± 5

Female8 (38%)

Body mass index (kg/m2)

23.7 ± 2.3Heart rate (beats per minute)60.7 ± 6.3

QT and subintervals
QTc (ms)

397.1 ± 14.0

JTpeakc (ms)228.2 ± 18.2

Tpeak-Tend (ms)82.2 ± 6.4

T-wave morphology
T-wave flatness (d.u.)

0.43 ± 0.05

T-wave asymmetry (d.u.)

0.20 ± 0.00

ERD30% (ms)50.1 ± 7.4

LRD30% (ms)T-wave amplitude (μV)

31.1 ± 5.3
569.0 ± 149.0

Continuous variables are represented as mean ± SD. QTc, Fridericia’s heart rate corrected QT interval;
J-Tpeakc, heart rate corrected J-Tpeak interval; EDR30%, 30% of early repolarization duration; LRD30%, 30% oflate repolarization duration; d.u., dimensionless units.
doi:10.1371/journal.pone.0163619.t001

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Detection of Late Sodium Current Block on the ECG

Fig 1. ECG “signature” of selective hERG potassium channel block. The ECG “signature” of selectivehERG potassium channel block is shown as the relationship between predicted drug-induced placebocorrected changes from baseline in QTc and (a) J-Tpeakc, (b) Tpeak-Tend, (c) 30% of early repolarization
duration, (d) 30% of late repolarization duration, (e) T-wave flatness, (f) T-wave asymmetry and (g) T-wave
amplitude. Average predictions (dots) and 95% confidence intervals (horizontal and vertical lines) are fromPLOS ONE | DOI:10.1371/journal.pone.0163619 December 30, 2016

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Detection of Late Sodium Current Block on the ECG

the concentration-dependent models for QTc (x axis) and the different T-wave morphology biomarkers (yaxis) at 25% increments of the population’s Cmax for dofetilide alone (light gray) and moxifloxacin (yellow)
arms in this study together with the dofetilide arm from previous clinical study (Dofetilide—Study 1) [10, 12].
QTc, Fridericia’s heart rate corrected QT; J-Tpeakc, heart rate corrected J-Tpeakc interval.
doi:10.1371/journal.pone.0163619.g001

Mitigation of ECG changes by mexiletine and lidocaine
When administered in combination with dofetilide, Cmax of mexiletine and lidocaine wereachieved after the third dose of the corresponding treatment period (Table 2). Fig 2 shows the
ECG signatures of dofetilide alone, mexiletine + dofetilide and lidocaine + dofetilide together
with the ECG “signature” of ranolazine from our previous clinical study [12]. Late sodium current block by both mexiletine and lidocaine caused a numerical reduction of the ECG changes
induced by dofetilide alone for all assessed ECG biomarkers except for 30% of late repolarization duration (Table 3). However, the changes were statistically significant only for QTc(p<0.001), J-Tpeakc (p<0.001) and T-wave flatness (p<0.001 and p = 0.015 for mexiletine+ dofetilide and lidocaine + dofetilide, respectively).

Ability of ECG biomarkers to detect late sodium current block
Fig 3 shows Cohen’s d effect sizes for each ECG biomarker for mexiletine + dofetilide and lidocaine + dofetilide vs. dofetilide alone assessed at Tmax. While QTc, J-Tpeakc and T-wave flatness
had “large” effect sizes, the effect sizes of QTc and J-Tpeakc were around two fold the effect sizeof T-wave flatness (Fig 3). All other five ECG biomarkers had “medium” or smaller effect sizes.
In the ROC-AUC analysis, J-Tpeakc had the largest AUC (0.83 [95% CI 0.80 to 0.87]) of allassessed ECG biomarkers (Fig 4). QTc had smaller AUC than J-Tpeakc (p<0.001) but greaterthan T-wave flatness (p<0.001). AUC of the model including both J-Tpeakc and QTc was notdifferent than the AUC of J-Tpeakc alone (p = 0.63). Lastly, AUC of the model including bothT-wave flatness and QTc was not different from QTc alone (p = 0.47). Table 4 summarizes
AUC and the highest combined sensitivity + specificity from the ROC curve for all assessed
models.
Decision tree analysis selected the ECG biomarkers in the same order as the ROC-AUC
analysis (J-Tpeakc, QTc and T-wave flatness). Fig 5 shows the decision tree developed using thebest features in the ROC-AUC analysis (QTc and J-Tpeakc). The accuracy of the decision tree
differentiating multichannel block from hERG potassium channel block was 0.80 [0.76 to 0.83](sensitivity 0.85, specificity 0.77) in the training set and 0.83 [0.81 to 0.85] in the validation set(sensitivity 0.83, specificity 0.82).

Discussion
In this study we assessed the effects of drug-induced late sodium current block on ECG changes
caused by drug-induced selective hERG potassium channel block. The ECG signature of
Table 2. Cmax time-points (Tmax) and plasma drug concentrations of mexiletine and lidocaine when given in combination with dofetilide.
Arm
Mexiletine + Dofetilide
Lidocaine + Dofetilide

Tmax (hours)

Drug

13

Dofetilide

Concentration (ng/mL)
1.83 [1.71 to 1.95]

13

Mexiletine

1426 [1312 to 1540]

12.5

Dofetilide

1.72 [1.52 to 1.91]

12.5

Lidocaine

2261 [2072 to 2450]

Time: hours after first dose of day; Concentration: mean and 95% confidence intervals.
doi:10.1371/journal.pone.0163619.t002

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Fig 2. ECG “signatures” of dofetilide, ranolazine, mexiletine + dofetilide and lidocaine + dofetilide.
The ECG “signatures” of dofetilide (light gray), ranolazine (blue), mexiletine + dofetilide (orange) andlidocaine + dofetilide (green) are shown as the relationship between predicted drug-induced placebocorrected changes from baseline in QTc and (a) J-Tpeakc, (b) Tpeak-Tend, (c) 30% of early repolarization
duration, (d) 30% of late repolarization duration, (e) T-wave flatness, (f) T-wave asymmetry and (g) T-wave

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Detection of Late Sodium Current Block on the ECG

amplitude. Average predictions (dots) and 95% confidence intervals (horizontal and vertical lines) are shown
from the concentration-dependent models for QTc (x axis) and the different T-wave morphology biomarkers(y axis) at 25% increments of the population’s Cmax for dofetilide alone (light gray) arm in FDA study 2 andranolazine (blue) arm from FDA study 1 [10, 12]. Average (dots) and 95% confidence intervals (vertical andhorizontal bars) of placebo-corrected changes from baseline are shown for mexiletine + dofetilide (orange)
and lidocaine + dofetilide (green). QTc, Fridericia’s heart rate corrected QT; J-Tpeakc, heart rate corrected
J-Tpeakc interval.
doi:10.1371/journal.pone.0163619.g002

selective hERG potassium channel block was consistent between dofetilide and moxifloxacin
and reproducible across studies for dofetilide. QTc, J-Tpeakc and T-wave flatness were the only
three ECG biomarkers for which late sodium current block (mexiletine, lidocaine) caused statistically significant mitigation of changes induced by selective hERG potassium channel block(dofetilide). ROC-AUC analysis showed that J-Tpeakc was the best ECG biomarker for detecting late sodium current block, followed by QTc and then T-wave flatness. While the specificthresholds may not carry over to other studies, decision tree analysis showed that J-Tpeakc and
QTc can be used to detect late sodium current block in the presence of QTc prolongation
caused by selective hERG potassium channel block. Future methodologies assessing drug
effects on the ECG, which could be applied in early Phase 1 studies under the CiPA initiative,
should use J-Tpeakc to detect drug-induced late sodium current block.
Table 3. Placebo- and baseline-corrected drug-induced ECG changes at Tmax.
Biomarker

Dofetilide

Combination

P

45.2 [37.8 to 52.6]

17.3 [11.6 to 23]<0.001

Mexiletine + Dofetilide
QT and subintervals
QTcF (ms)J-Tpeakc (ms)29.1 [20.2 to 38]

1.1 [-5.5 to 7.7]

<0.001

Tpeak-Tend (ms)17.9 [9.0 to 26.7]

14.6 [10.0 to 19.2]

0.36

T-wave flatness (d.u.)

0.11 [0.08 to 0.14]

0.06 [0.04 to 0.08]<0.001

T-wave asymmetry (d.u.)

0.20 [0.11 to 0.29]

0.09 [0.00 to 0.19]

0.056

ERD30% (ms)19.8 [11.5 to 28.1]

11.1 [4.5 to 17.7]

0.12

LRD30% (ms)

9.8 [5.0 to 14.6]

11.3 [5.8 to 16.7]

0.57-87.9 [-106.3 to -69.5]-55.1 [-98.3 to -11.9]

0.16

T-wave morphology

T-wave amplitude (μV)
Lidocaine + Dofetilide
QT and subintervals
QTcF (ms)45 [35.5 to 54.6]

17.9 [11.7 to 24.1]<0.001

J-Tpeakc (ms)28.3 [18.2 to 38.3]

4.1 [-1.9 to 10.2]<0.001

Tpeak-Tend (ms)18.4 [8.2 to 28.7]

12.9 [7.6 to 18.2]

0.23

T-wave flatness (d.u.)

0.11 [0.07 to 0.14]

0.06 [0.05 to 0.08]

0.015

T-wave asymmetry (d.u.)

0.19 [0.09 to 0.29]

0.1 [0.02 to 0.18]

0.14

ERD30% (ms)18.4 [8.8 to 27.9]

11.2 [6.8 to 15.6]

0.19

10.0 [4.5 to 15.4]

9.8 [4.6 to 15.0]

0.90-82.5 [-107.6 to -57.5]-57.6 [-99.8 to -15.4]

0.27

T-wave morphology

LRD30% (ms)T-wave amplitude (μV)

Values reported as mean and 95% confidence intervals. QTc, Fridericia’s heart rate corrected QT interval; J-Tpeakc, heart rate corrected J-Tpeak interval; Twave flatness, heart rate corrected T-wave flatness; EDR30%, 30% of early repolarization duration; LRD30%, 30% of late repolarization duration; T-wave
amplitude, heart rate corrected T-wave amplitude; d.u., dimensionless units.
doi:10.1371/journal.pone.0163619.t003

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Fig 3. Cohen’s d effect size for each ECG biomarker. Cohen’s d effect size for each ECG biomarker for mexiletine + dofetilide and lidocaine+ dofetilide vs. dofetilide alone.
doi:10.1371/journal.pone.0163619.g003

The ECG signatures of selective hERG block were consistent between drugs (dofetilide andmoxifloxacin) and reproducible for dofetilide across studies (Fig 1). Moxifloxacin is commonlyused as a positive control in thorough QT studies [2] because it is a weak hERG potassiumchannel blocker at clinical concentrations, prolonging QTc by 10 to 14 ms (between 2 and 4
hours after 400mg oral dose), and has minimal torsade risk [20–22]. The similarity between
moxifloxacin and dofetilide ECG “signatures” (Fig 1) suggests that the low torsade risk of moxifloxacin may be because moxifloxacin therapeutic doses cause limited hERG potassium channel block [11]. However, rare cases of moxifloxacin-induced torsade have been reported inpatients at higher risk for torsade [23–26]. Therefore, in patients exposed to higher moxifloxacin concentrations with reduced “repolarization reserve” [27] or other comorbidities that
increase risk of torsade [28], moxifloxacin may exhibit an ECG signature similar to dofetilidewith larger QTc prolongation, larger T-wave morphology changes and higher risk for torsade.
There are multiple drugs that prolong QTc with minimal torsade risk [4–6], likely because
they block inward currents [7, 29] in addition to the hERG potassium channel. We have previously shown that shortening of J-Tpeakc is a sign of inward (late sodium or L-type calcium) current block [10, 11, 30]. We have also shown that drug-induced changes in T-wave morphology
biomarkers are directly related to the amount of hERG potassium channel block, and that multichannel blockers caused greater T-wave morphology changes than selective hERG potassium

PLOS ONE | DOI:10.1371/journal.pone.0163619 December 30, 201610 / 16

Detection of Late Sodium Current Block on the ECG

Fig 4. ROC curves for multiple logistic regression models. QTc, Fridericia’s heart rate corrected QT interval; J-Tpeakc, heart rate corrected
J-Tpeakc interval; T-wave flatness, heart rate corrected T-wave flatness. Area under the curve of each model reported in parenthesis.
doi:10.1371/journal.pone.0163619.g004

channel blockers at equivalent amount of QTc prolongation [12]. In this study we assessedwhich of all these ECG biomarkers can differentiate multichannel block (late sodium current
block + hERG potassium channel block) from selective hERG potassium channel block on the
ECG and therefore add value to QTc assessment. While there was a numerical reduction of

PLOS ONE | DOI:10.1371/journal.pone.0163619 December 30, 201611 / 16

Detection of Late Sodium Current Block on the ECG

Table 4. Logistic regression by performance.
model

AUC

Sensitivity

Specificity

J-Tpeakc

0.83 [0.80 to 0.87]

0.82

0.77

QTc + J-Tpeakc

0.83 [0.80 to 0.87]

0.84

0.74

QTc + T-wave flatness

0.73 [0.69 to 0.77]

0.58

0.77

QTc

0.72 [0.68 to 0.77]

0.62

0.74

T-wave flatness

0.60 [0.56 to 0.65]

0.60

0.57

LRD30%

0.60 [0.56 to 0.65]

0.89

0.26

Tpeak-Tend

0.59 [0.55 to 0.64]

0.86

0.28

T-wave asymmetry

0.57 [0.52 to 0.62]

0.88

0.32

T-wave amplitude

0.53 [0.48 to 0.58]

0.89

0.23

ERD30%

0.49 [0.45 to 0.54]

0.54

0.49

Performance reported as area under the curve [AUC] to predict when additional inward current block is present. AUC brackets show 95% confidence
intervals. Sensitivity and specificity are the highest combination of sensitivity + specificity on the ROC curve. QTc, Fridericia’s heart rate corrected QTinterval; J-Tpeakc, heart rate corrected J-Tpeak interval; EDR30%, 30% of early repolarization duration; LRD30%, 30% of late repolarization duration.
doi:10.1371/journal.pone.0163619.t004

hERG potassium channel induced changes for almost all ECG biomarkers after addition of latesodium current block, the effect was statistically significant only for QTc, J-Tpeakc and T-wave
flatness (Table 3). This may have been due to larger inter-subject variability in the druginduced changes in the T-wave morphology biomarkers compared to the more consistent
response in QTc, J-Tpeakc and T-wave flatness (assessed as Cohen’s d effect size [15], Fig 5).
When comparing the ability of ECG biomarkers to detect the presence of late sodium current block, J-Tpeakc had the largest AUC in the ROC-AUC analysis. T-wave flatness did not
add value to QTc assessment alone. Moreover, the J-Tpeakc model performed better than QTc
alone or in combination with either J-Tpeakc or T-wave flatness (Table 4). The performance of
the C4.5 decision tree developed with J-Tpeakc and QTc identifying inward current block using
ECG data from FDA study 2 was similar to the performance achieved with ECG data of drugs
from FDA study 1 [10] (accuracy of 0.80 [0.76 to 0.83] vs. 0.83 [0.81 to 0.84]). This suggests
that, while appropriate thresholds still have to be established, a methodology using J-Tpeakc
and QTc can identify balanced inward current (late sodium or calcium) and hERG potassiumchannel block vs. selective hERG potassium channel block, in particular in the presence of QTc
prolongation.
The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative [9] proposes an assessment of actual torsade risk beyond hERG potassium channel block and QTc prolongation.
CiPA utilizes a mechanistically driven assessment of in vitro drug effects on multiple ion channels coupled with an in silico model of human cardiomyocytes and verification of predicted
responses in human induced pluripotent stem cell derived cardiomyocytes. CiPA would
include a clinical confirmation using Phase 1 ECGs to determine that no unanticipated clinical
ECG changes are found compared to the preclinical ion channel data. Results of this study suggest that J-Tpeakc should be considered as a biomarker to detect late sodium current block in
Phase 1 clinical studies under CiPA.
In addition to CiPA, there is another initiative that may affect the future of the current regulatory paradigm of proarrhythmic assessment of drugs. The Consortium for Innovation andQuality in Pharmaceutical Development and the Cardiac Safety Research Consortium(IQ-CSRC) recently demonstrated that exposure-response analysis of ECG and pharmacokinetic data from first-in-human studies can be used to assess drug-induced QTc prolongation

PLOS ONE | DOI:10.1371/journal.pone.0163619 December 30, 201612 / 16

Detection of Late Sodium Current Block on the ECG

Fig 5. Decision tree. The decision tree classifies the drug effects on any ECG from any time point based on the time-matched placebo-correctedchanges from baseline (ΔΔ) as selective of predominant hERG potassium channel block (right bin), multichannel block (left bin) or inconclusive(middle bin). More specifically, if ΔΔJ-Tpeakc is greater than 9ms the ECG is classified as predominant or selective hERG potassium channel block. If
ΔΔ J-Tpeakc is less than or equal to 9ms and ΔΔQTc less than or equal to 29ms, then the ECG is classified as multichannel block. Otherwise theclassification is inconclusive. The number of ECGs from each class in the training set is reported in parenthesis on top of each class bin. The
percentage of ECGs correctly classified within each class is reported below each class bin. See text for more details on classification performance inboth training (hERG [dofetilide and moxifloxacin] vs. multichannel [dofetilide + mexiletine and dofetilide + lidocaine]) and validation (hERG [dofetilide

PLOS ONE | DOI:10.1371/journal.pone.0163619 December 30, 201613 / 16

Detection of Late Sodium Current Block on the ECG

and quinidine] vs. multichannel [ranolazine and verapamil]) sets. QTc, Fridericia’s heart rate corrected QT interval; J-Tpeakc, heart rate corrected
J-Tpeak interval; hERG, selective or strong hERG potassium channel block; Multichannel, inward (late sodium or calcium) current and hERG
potassium channel block.
doi:10.1371/journal.pone.0163619.g005

with high confidence. The ICH E14 questions and answers (Q&A) were revised recently withharmonized guidance on how to use exposure-response modeling of QTc data (Q&A # 5.1)[31]. This ‘Early QT assessment’ could be used in lieu of thorough QT studies in some cases[32], which could potentially reduce drug development costs. However, this approach does notaddress the distinction between QTc prolonging drugs that selectively block the hERG potassium channel (high torsade risk) from QTc prolonging drugs that block inward currents inaddition to the hERG potassium channel (low torsade risk) [9]. Thus, the ability of J-Tpeakc todetect inward current block in small sample sizes should be assessed and considered as a potential enhancement of the IQ-CSRC proposed paradigm.
Validation and implementation strategies for confirming CiPA preclinical findings using
ECG data in small sample size Phase 1 clinical studies were discussed in a public workshop
sponsored by the CSRC in April 2016. There is ongoing validation work on a large number ofprior clinical studies with drugs that block individual and multiple ion channels. In addition, astatistical framework that can be applied in exposure-response analysis similar to the IQ-CSRC
paradigm is being developed. Lastly, we are designing a confirmatory prospective clinical study
with a small sample size design similar to first-in-human single or multiple ascending dosestudies. It is anticipated that this prospective study will include a combination of selectivehERG potassium channel blockers, multichannel blockers and no ion channel effect drugs[33].
In order to facilitate J-Tpeakc interval assessment in clinical drug trials, we have developed
an automated algorithm for assessment of the J-Tpeakc interval. The automated algorithm canreproduce the semi-automated measurements of 8 drugs and 3 drug combinations from thetwo FDA-sponsored clinical trials. An implementation of this algorithm is being released as
open-source code [34].

Limitations
Classes in the training set were defined as ‘selective hERG potassium channel block’ vs. ‘late
sodium current + hERG potassium channel block’, while in the validation set, the corresponding classes are predominant hERG block (dofetilide, quinidine) and multichannel block (ranolazine [late sodium current + hERG potassium channel block] and verapamil [strong calcium
current block with some hERG potassium channel block]). This results in a division of thedrugs in the test set into those with high torsade risk and predominant hERG block (dofetilideand quinidine) vs. low torsade risk and balanced inward and outward current block (ranolazine
and verapamil). Thresholds of the decision tree should be interpreted with caution because differences in statistical methods may result in different values.

Conclusion
J-Tpeakc was the best of the eight studied ECG biomarkers for detecting late sodium current
block. ROC-AUC and decision tree analysis showed that an integrated assessment of J-Tpeakc
and QTc can differentiate drug-induced multichannel block from selective hERG potassiumchannel block. Future methodologies assessing drug effects on cardiac ion channel currents on
the ECG should consider J-Tpeakc to detect the presence of late sodium current block.

PLOS ONE | DOI:10.1371/journal.pone.0163619 December 30, 201614 / 16

Detection of Late Sodium Current Block on the ECG

Acknowledgments
The authors would like to acknowledge the valuable suggestions made by Dr. Norman Stockbridge. QTGuard+ was provided by GE Healthcare through a material transfer agreement. The
mention of commercial products, their sources, or their use in connection with material
reported herein is not to be construed as either an actual or implied endorsement of such products by the U.S. Department of Health and Human Services. This article reflects the views of
the authors and should not be construed to represent FDA’s views or policies.

Author Contributions
Conceived and designed the experiments: JV LJ JWM DGS.
Performed the experiments: JV LJ JWM DGS.
Analyzed the data: JV LJ MH JWM PTS EP DGS.
Contributed reagents/materials/analysis tools: JV LJ MH.
Wrote the paper: JV LJ MH JWM PTS EP DGS.

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