594589

research-article2015

JBXXXX10.1177/1087057115594589Journal of Biomolecular ScreeningFermini et al.

Review Article

A New Perspective in the Field of Cardiac
Safety Testing through the Comprehensive
In Vitro Proarrhythmia Assay Paradigm

Journal of Biomolecular Screening
﻿1­–11© 2015 Society for Laboratory
Automation and Screening
DOI: 10.1177/1087057115594589
jbx.sagepub.com

Bernard Fermini1, Jules C. Hancox2, Najah Abi-Gerges3*,
Matthew Bridgland-Taylor4, Khuram W. Chaudhary5, Thomas Colatsky6,
Krystle Correll7, William Crumb8, Bruce Damiano9, Gul Erdemli10, Gary Gintant11,
John Imredy12, John Koerner13, James Kramer14, Paul Levesque15, Zhihua Li6,
Anders Lindqvist16, Carlos A. Obejero-Paz14, David Rampe17, Kohei Sawada18,
David G. Strauss19, and Jamie I. Vandenberg20

Abstract
For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation andTorsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to theexisting approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries
a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies
conducted later in drug development. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative is a publicprivate collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia
risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach,incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic
actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human(stem cell–derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA
initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac
safety testing guidelines.
Keywords
CiPA, Comprehensive In Vitro Proarrhythmic Assay, hERG, ICH E14, ICH S7B, QT interval, safety

Introduction
On July 23, 2013, a think-tank meeting sponsored by the
Cardiac Safety Research Consortium (CSRC), Health andEnvironmental Sciences Institute (HESI), and the US Foodand Drug Administration (FDA) was held at the FDA headquarters in Silver Spring, Maryland, to discuss a novel
approach to assess the proarrhythmic potential of drugs that
prolong the QT interval. Prolongation of the QT interval isused as a surrogate marker for predicting the risk of a compound to induce a potentially fatal ventricular cardiac
arrhythmia called Torsade de Pointes (TdP). However, thelink between QT interval prolongation and TdP appears
multifaceted and is influenced by a number of underlying
factors including age, gender, underlying disease state,electrolyte imbalance, concomitant medication, and more.1,2
Although the majority of compounds that can induce TdP
are known to inhibit cardiac potassium channels encodedby human-ether-à-go-go Related Gene (hERG),3–5 block ofionic current (IhERG) carried by recombinant hERG channels
alone is not always predictive of delayed repolarization orproarrhythmic risks. For example, the L-type calcium channel blocker verapamil is a potent blocker of hERG current6
but does not prolong the QT interval or pose a risk of TdP.7
For small chemically manufactured molecules, the current
preclinical (ICH S7B) and clinical (ICH E14) safety guidelines require first a preclinical electrophysiology test against
hERG (or the native cardiac equivalent, the rapid delayed
rectifier current IKr; known also as hERG) and an in vivo
QT measurement followed, for drugs that pass preclinicalReceived Apr 7, 2015, and in revised form Jun 4, 2015. Accepted for
publication Jun 11, 2015.
Corresponding Author:
Bernard Fermini, Global Safety Pharmacology, Pfizer Inc., MS8274-1347
Eastern Point Road, Groton, CT 06340, USA.
Email: Bernard.fermini@pfizer.com

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Journal of Biomolecular Screening ﻿

testing, by a thorough QT (TQT) study.8,9 All of these measures are surrogates for, rather than automatic predicators
of, TdP proarrhythmia, and following a decade of testing
using this approach, it has become clear that the strong initial focus on a single ion channel (hERG) favors a conservative approach that may have led to the attrition of potentially
useful drugs, mainly on the grounds of their activity onhERG rather than due to arrhythmia induction per se. Thereis good reason, therefore, to reconsider approaches to theevaluation of drug-induced arrhythmia.
This article will focus on the preclinical aspects of the
proposed CiPA paradigm and will not address in detail any
potential impact on either the E 14 guidance document or
the dedicated TQT study. To touch on those briefly, collaboration between the Consortium for Innovation and Quality
in Pharmaceutical Development and the CSRC was established to design a clinical study in healthy subjects demonstrating that the TQT study can be replaced by robustelectrocardiogram (ECG) monitoring and exposureresponse analysis of data generated from first-in-humansingle-ascending-dose studies. Results from that study were
presented at a meeting held in Silver Spring, Maryland, in
December 2014, and readers should see Darpo et al.10 for
more information.

What Is CiPA?
The new paradigm, termed the Comprehensive In Vitro
Proarrhythmia Assay (CiPA), has the objective to engineer,
early in the drug discovery and development process, assays
allowing the evaluation of the proarrhythmic risk of compounds, instead of concentrating on their ability to inhibit
the hERG current and to prolong the QT interval.11 This
new paradigm is based on the fundamental mechanistic

understanding of the role of ion channels in delayed ventricular repolarization, alterations to which lead to repolarization instability and arrhythmias. It comprises two distinct
series of tests: (1) the in vitro study of drug effects on multiple ion channels (not just hERG) and incorporation ofthese effects in an in silico model of a human ventricularaction potential (AP) in an effort to reconstruct the effects
on ventricular repolarization and identify potential
mechanism-based metrics that can assess proarrhythmia
risk (for example, early afterdepolarizations [EADs] andAP triangulation3,5,12), and (2) confirmation of the ionic current and in silico results using human ventricular myocytes,
likely derived from human-induced pluripotent stem cell
(iPSC) cardiomyocytes. Prolongation of AP duration (APD)
and delayed ventricular repolarization further enables reactivation of calcium channels, resulting in late inflow of calcium ions and contributing to the propensity of developing
EADs (Fig. 1). When generated in the presence of transmural heterogeneity in ventricular repolarization, EADs are
believed to contribute to the generation of extrasystoles that
can trigger TdP.1,3,5,12
The CiPA initiative is being driven by a consortium comprising a number of collaborators including the FDA, HESI,CSRC, Japan National Institute of Health Sciences, Health
Canada, European Medicines Agency, Pharmaceutical andMedical Devices Agency (Japan), Japan iPS Cardiac Safety
Assessment, academics, in silico modelers, and partners
from contract research organizations, the pharmaceuticalindustry, stem cell providers, and device companies.
Because of the complexity of the task proposed, several
work streams have been established in support of CiPA. As
such, the Safety Pharmacology Society (SPS) has positioned itself as an important contributor to this effort byestablishing the Ion Channel Working Group (ICWG). This

1

Global Safety Pharmacology, Pfizer Inc., Groton, CT, USA
School of Physiology and Pharmacology, University of Bristol, Bristol, UK
3Translational Safety, Drug Safety and Metabolism, Innovative Medicines and Early Development, AstraZeneca R&D, Macclesfield, UK
4
Discovery Sciences, Innovative Medicines and Early Development, AstraZeneca R&D, Macclesfield, UK
5
Safety Assessment, GlaxoSmithKline, King of Prussia, PA, USA
6
Division of Applied Regulatory Science, CDER, US Food and Drug Administration, Silver Spring, MD, USA
7
Safety Pharmacology Society, Reston, VA, USA
8
Zenas Technologies LLC, New Orleans, LA, USA
9
Global Safety Pharmacology, Discovery Sciences, Janssen Research & Development LLC, Spring House, PA, USA
10
Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc, Cambridge, MA, USA
11
Department of Integrative Pharmacology, Integrated Sciences & Technology, AbbVie, North Chicago, IL, USA
12
Department of Safety Assessment, Merck & Co, Kenilworth, NJ, USA
13
Division of Cardiovascular and Renal Products, CDER, U.S. Food and Drug Administration, Silver Spring, MD, USA
14
ChanTest, A Charles River Company, Cleveland, OH, USA
15
Bristol Myers Squibb Research & Development, Princeton, NJ, USA
16
Biolin Scientific, Ballerup, DK
17
Preclinical Safety, Sanofi, Bridgewater, NJ, USA
18
Global Cardiovascular Assessment, Eisai Co., Ltd., Ibaraki, Japan
19
Center for Devices and Radiological Health, U.S. Food and Drug Administration, Silver Spring, MD, USA
20
Victor Chang Cardiac Research Institute, St Vincent’s Clinical School, University of NSW, Darlinghurst, NSW, Australia*
Current address: AnaBios Corporation, San Diego, CA, USA
2

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Figure 1. The balance between inward and outward currents determines the morphology and duration of the cardiac action
potential (AP) and, consequently, the duration of the QT interval. Drug-induced inhibition of the IKr/IhERG current can delay
repolarization and prolong the AP duration and the QT interval. Lengthening repolarization enables reactivation of calcium channels,
resulting in late inflow of calcium ions and contributing to the propensity of developing early afterdepolarizations (EADs). Whengenerated in the presence of transmural heterogeneity in ventricular repolarization, EADs are believed to contribute to the generationof extrasystoles that can trigger Torsades de Pointes. The red AP trace highlights delayed repolarization and generation of EADs.
Adapted with permission from ref. 13: Nat. Rev. Drug Discov. 2003, 2, 439–447. © Macmillan Publishers Ltd. All rights reserved.

group builds on considerable experience and expertise of its
members in the field of ion channel biophysics, pharmacology, and the understanding of translation from in vitro to invivo models. The ICWG is tasked with bringing together
expertise and resources required to deliver best practice recommendations for generating ion channel data needed forin silico human cardiac AP reconstructions of proarrhythmic liabilities. The SPS also supports the CiPA effort in theform of staged funding of the experimental work to be performed. Other committees critical to the success of CiPA
include the In Silico Working group (ISWG), under the
direction of the FDA, and the Cardiac Stem Cell Working
group (SCWG), sponsored by HESI. The ISWG is responsible for the development and validation of the best in silico
model of human ventricular electrophysiology for the APreconstruction of drug effects on the individual ion channel,
as determined by the work of the ICWG. The role of the
SCWG is to define best practice for experiments using
human stem cell–derived cardiomyocytes in an effort tovalidate the effects observed on ion channels, and/or insilico modeling, and to unmask effects that, for various reasons, were not revealed in either the ion channel or in silico
work. Finally, a subcommittee from the joint HESI/CSRC
Clinical Translation Working Group selected a series of 28
compounds to be tested in the CiPA paradigm that were categorized into three different risk groups based on their torsadogenic potential (high, intermediate, very low, or none;
see Table 1) according to published and publically available data and expert opinion7,14,15 (Credible Meds; FDA

AERS Database, FDA labeling). The intent in the selectionof the compounds is twofold: (1) to provide a battery ofcompounds for training, testing, and validating the in silico
and stem cell CiPA models and (2) to select compounds thatcover a wide spectrum of electrophysiological endpoints
including the degree of torsadogenic risk, actions on ion
channels (with attention to multichannel blockers), varying
levels of block at clinical exposure, and inclusion of somecompounds with non-hERG TdP risk. The complete list of
selected compounds is presented in Table 1.

The Need for a New Paradigm
To appreciate the necessity of modifying the current
strategy, one must revisit the evolution of the regulatory
guidelines in order to appreciate our current position. Indeed,
beginning in the late 1980s, spontaneous case reports of cardiotoxicity related to the use of the nonsedatingH1-antihistamine terfenadine began to appear in the literature. Eventually, this drug was shown to cause TdP and sudden death by prolonging cardiac repolarization following
inhibition of the delayed rectifier potassium channel, IKr.16
As similar reports with other noncardiovascular agentsbegan to appear more frequently, the Committee for
Proprietary Medicinal Products (CPMP) took action andissued a “Points to Consider” document in 1997 that advocated a series of in vitro and in vivo experiments to assess
the risk of QT prolongation (CPMP/986/96). More specifically, the guidance recommended the undertaking of in

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Table 1. List of 28 Comprehensive In Vitro Proarrhythmia Assay (CiPA) compounds and their intended clinical use, categorized into
three different risk groups based on their torsadogenic potential.
CiPA Compound Selection
High Risk
Azimilide (antiarrhythmic)
Bepridil (angina)
Dofetilide (antiarrhythmic)
Ibutilide (antiarrhythmic)
Quinidine (antiarrhythmic)
Vandetanib (oncology)
Methadone (opioid addiction)d,l-sotalol (antiarrhythmic)

Intermediate Risk
Astemizole (antihistamine)
Chlorpromazine (antipsychotic)
Cisapride (gastrokinetic)
Clarithromycin (antibacterial)
Clozapine (antipsychotic)
Domperidone (antiemetic)
Droperidol (antiemetic)
Terfenadine (antihistamine)
Pimozide (antipsychotic)
Risperidone (antipsychotic)
Ondansetron (antiemetic)

vitro electrophysiological studies (effects on AP duration)using cardiac tissue that included ion channels corresponding to those contributing to repolarization in human cardiac
tissue. Following increasing scrutiny by various regulatory
agencies and expert working groups, the International
Conference on Harmonization (ICH) issued two imperative
guidance documents in 2005 that are still the standard
today as safety pharmacology guidelines for the development of new chemical entities: ICH S7B, titled “NonClinical Evaluation of the Potential for Delayed VentricularRepolarization (QT Interval Prolongation) by Human
Pharmaceuticals,” describes a nonclinical testing strategy
for assessing the potential of a test compound to delay ventricular repolarization, and ICH E14, titled “Clinical
Evaluation of the QT/QTc Interval Prolongation andProarrhythmic Potential for Nonantiarrhythmic Drugs,”
requesting that all sponsors submitting new drug applications conduct a thorough QT study to determine if a drug
prolongs the heart rate–corrected QT interval (QTc).8,9
Clearly, the introduction and adoption of these guidance
documents have proven successful: there has been no withdrawal of marketed drugs for concerns of TdP since they
were approved in 2005, while six noncardiac medications
were withdrawn from the U.S. market by the FDA between
1995 and 2003, prior to the adoption of these guidelines.
Nonetheless, current regulatory concern for any candidate
drug to inhibit hERG current and to prolong the QT interval
by a magnitude greater than 5 ms leads to extensive evaluation in phase IIb and III trials at significant costs; since2005, approximately 450 thorough QT/QTc studies havebeen performed at an estimated cost of approximately 1 billion dollars.17,18 Moreover, the major learning from a decadeof clinical studies is that an increase in QTc is a sensitive
but not specific endpoint to predict proarrhythmia.10
Therefore, the current preclinical and clinical strategies are

Very Low Risk
Diltiazem (hypertension/angina)
Loratidine (antihistamine)
Metoprolol (hypertension/angina)
Mexiletine (antiarrhythmic)Nifedipine (hypertension/angina)
Nitrendipine (hypertension/angina)
Ranolazine (angina)
Tamoxifen (oncology)
Verapamil (hypertension/angina)

suitable to predict QT prolongation but are inadequate toassess the risk of TdP and need to be revised.

Drawbacks of the Existing Approachand Predominant Focus on the hERG
ChannelSince the launch of the ICH guidelines, pharmaceuticalcompanies have established a number of preclinical assays
to identify, early in the discovery process, compounds thathave the propensity to block the hERG channel.3,5,12
Arguably, the ideal preclinical assay for drug-induced IKrinhibition would involve electrophysiological recording of
native IKr in ventricular cardiomyocytes from an appropriate model species. However, the requirement for enzymaticand mechanical myocyte dispersion from intact tissue,
together with challenges to effective IKr measurement, makenative IKr measurements impractical as a primary safety
assay, as inevitably such measurements would be of low
throughput.5 In addition, native IKr is of small amplitude,necessitating its pharmacological isolation from other overlapping currents, further complicating its use as a primary
screen.5 Consequently, for more than 10 years, recombinanthERG channel assays have been employed in preclinicaldrug safety testing: as early as 2005, hERG assays wereused by 93% of surveyed respondents from 119 pharmaceutical companies for preclinical evaluation of drug modification of repolarization in line with ICH S7B.19 Mammaliancell lines that express hERG are attractive because they permit moderately high-throughput automated patch clamp
assays, with measurements of a large-amplitude current
compared with native IKr, in the absence of contaminationfrom overlapping conductances.5,20,21 Native IKr may becomposed of hERG1a and hERG1b subunits,22,23 and both
KCNE1 and KCNE2 subunits can associate with hERG

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Fermini et al.
and, potentially, could influence hERG channel pharmacology.24–27 Nonetheless, pharmacological potencies derived
from hERG 1a alone expressed in mammalian cells generally approximate well those for native IKr,28,29 and coexpression of hERG 1a and 1b or hERG 1a with KCNE1 orKCNE2 subunits is not routinely needed for screening.
The ICH 7SB guidelines stipulate that radioligand competition binding assays are not a viable alternative to anelectrophysiological assay, as they provide no information
on a compound’s ability to modify the current itself.8
However, beyond indicating that IKr/hERG ionic current
should be assayed, ICH S7B guidelines are not prescriptive.8 This has the advantage of permitting flexibility of user
approach, both in measurement platform and experimental
protocol. However, the potential disadvantage of this flexibility is variability of data due to a lack of standardized
approach. The potency of some hERG blockers shows
marked sensitivity to stimulus protocol and/or measurement
temperature.30–32 The extent to which measurement conditions/protocol influence half-maximal inhibitory effect concentration (IC50) may vary significantly between drugs: onecomparative analysis of cisapride and dofetilide using manual patch reported variability of IhERG IC50 for dofetilide
between 4 and 46 nM and for cisapride between 7 and 240
nM in published literature derived from mammalian celllines.32 This further correlated with greater variability forcisapride than for dofetilide IC50 (7–72 nM versus 4–15nM) in experiments at a single temperature in a single study,
between step, step-ramp, and AP voltage commands.32 A
difficulty that this issue poses for the study of novel chemical entities (NCEs) is that protocol dependence of hERG
block is not possible to predict in advance. At present, there
is no consensus on a single best protocol for studying IhERG
sensitivity to NCEs, although adoption of a standardized
approach could reduce variability: a comparative blinded
patch-clamp investigation of 12 hERG inhibitors between 2
contract research organizations employing similar measurement methods and standardized solutions, temperature, andprotocol showed an IC50 variability between laboratories
that was in the order of approximately threefold or less forall but one compound.33.
The ICH S7B guidelines recommend that, where possible, full concentration-response relations should be obtainedfor compounds under examination, except where physicochemical properties limit the maximal concentration that canbe tested.8 Given the hERG channel’s now well-recognizedpharmacological promiscuity,5,12,34 this poses problems in theassessment of NCEs: up to ~70% of compounds may interact with hERG at some concentration.35 Automaticallyeliminating from further development all drugs that inhibit
hERG would be costly and may be unjustified in somecases. Consequently, issues arise as to how “safe” and “dangerous” hERG blockers can be reliably distinguished fromone another. Considerable effort has been expended todefine a safety index or safety margin that takes into accountboth potency of an agent against hERG and its effective
therapeutic concentration against its intended target(reviewed in refs. 5, 7, and 12). However, hERG safety margins alone may not suffice for decisions as to whether or not
to proceed with development or approval of an NCE. First,
a sole focus on hERG may miss rare instances in which QT
prolongation/TdP could arise from compound effects onionic currents other than hERG. Second, drug discovery
projects may not have available the measured Cmax values
necessary for accurate safety margin assessment and may
have to rely on Cmax estimates.12 Third, relating hERG
safety margin values to QTc prolongation and risk can becomplex. A comparison of hERG block potency with TQT
study data for 39 drugs found that a hERG safety margin of
45 optimally linked safety margin to QTc interval prolongation but that QTc prolonging drugs were only five- to sevenfold more likely to exhibit safety margins between 1 and 30
than drugs that do not prolong the QTc interval.36.
Additional preclinical in vitro repolarization screens are
not currently required, with multiple different approaches
used, ranging from single Purkinje fibers to ventricularwedge and perfused heart preparations—each of which has
its own strengths and potential disadvantages.3,5 Althoughsuch additional tests are not mandatory according to ICH
S7B,8 when used, they are likely to result in variability intype and comprehensiveness of these complementary data.

Functional Effects on Multiple Human
Cardiac Ionic Channels
As a first measure, CiPA proposes to evaluate the proarrhythmic risk of compounds by studying the effects on multiple human ventricular ionic channels. Because the studyof native currents from primary human cardiac tissue is nota viable approach (due to the same limitations as discussed
above in respect of IKr), the core strategy will consist ofmeasuring key ionic currents from recombinant humanchannels expressed in various heterologous systems. The
initial voltage clamp work will be performed manually bothat physiologic temperature (~35–37 °C) and room temperature in an effort to establish biophysical and pharmacological characterization of the effects of selected compounds oneach current using the gold standard approach. These results
will then be used as reference when the work is transitioned
to automated high-throughput methods in order to adapt
CiPA to the current screening environment of most pharmaceutical companies.
To begin with, it is essential to define which ionic currents
play a significant role in conferring proarrhythmic properties
to compounds. The duration of the QT interval on the electrocardiogram is defined as the period between the beginning of
the QRS complex (depolarization of the ventricles) and theend of the T wave (repolarization of the ventricles; see Fig.

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Figure 2. Overview of the different
ion channels and their contribution
to the morphology and duration of
the cardiac action potential (AP). The
duration of the QT interval on theelectrocardiogram is defined as theduration between the beginning of the
QRS complex and the end of the T
wave. It is a reflection of ventricular
AP duration and represents thetime during which the ventricles
depolarize and repolarize. Numerous
overlapping ionic currents contributeto determining the morphology
and duration of the ventricular AP.
Adapted with permission from ref.
13: Nat. Rev. Drug Discov. 2003, 2,
439–447. © Macmillan Publishers Ltd.
All rights reserved.

2).37 It is a reflection of ventricular APD, and several distinct
ionic channels contribute to defining the morphology andduration of APs, including the rapid inward sodium current(INa) responsible for the rising phase of the AP (phase 0), thelate sodium (INa-Late) and L-type calcium channel (ICa-L) in
control of the plateau phase (phase 2), and multiple overlapping outward repolarizing potassium currents comprising thetransient outward current (Ito; phase 1), the slow (IKs) and
rapid (IKr) components of the delayed rectifier potassiumchannels (phase 3), and the inward rectifier channel (IK1;phase 4), the latter responsible for setting the resting potentialof working cardiac myocytes.38 Based on their fundamentalrole in defining human APD, and together with the information obtained from a Safety Pharmacology Society survey, the
ICWG selected these seven different human ionic channels as
initial working material for the CiPA assays. More specifically, as it will be recombinant rather than native channels thatare viable for safety testing assays, experiments will focus on
the following human recombinant channels expressed in
mammalian cell lines: Nav1.5 (rapid INa), toxin-modified
Nav1.5 (INa,Late), Cav1.2 (ICa,L), Kv4.3+KChIP2 (Ito), hERG
(IKr), KCNQ1+KCNE1 (IKs), and Kir2.1 (IK1). Currents will
be studied individually using protocols that will not only
assess the potency of block (IC50 determination) but also evaluate kinetics of block that might prove critical in understanding the potential proarrhythmic potential of compounds.39
Ultimately, only the most informative protocols will be
retained as final, and it is likely that the list of seven targetedchannels will narrow once their role as proarrhythmic markers

are confirmed, or not, based on experimental outcomes. A significant benefit to be expected from this effort will be theestablishment of best practices for ion channel studies used tocharacterize drug effects, such that standardized protocols andmethods are developed and adopted in an effort to minimize
intra- and interlaboratory variability and allow a more levelplaying field across the pharmaceutical industry.

In Silico Cellular Simulations
The integration of in silico modeling into the CiPA effort
offers the potential to provide integrative, cost-effective,
and high-throughput solutions to predict drug-inducedchanges in AP duration by combining the ICWG’s results
on individual ion channels. The O’Hara-Rudy (OHR) modelof the human ventricle was selected by leading in silico
modelers at a consensus meeting in July 2013. The OHR
model offers several advantages, including the fact that it isopen source (it can be accessed on the Rudy Laboratory
research section of the website http://rudylab.wustl.edu); allconstants (extracellular ionic concentrations, cell geometry,ionic conductance) and all the initial conditions for statevariable and scaling factors have been determined; and,
finally, it is fully validated with ionic data described for ahuman ventricular AP model.40
The ISWG is focused on evaluating and extending themodel so that it can provide assessment of ion pharmacology and clinical risk of TdP. The philosophy behind the useof the model is to use the data generated by the ICWG

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Fermini et al.
iteratively to parametrize and validate the performance ofthe model at each step in the development process while
keeping the process as simple as possible by using patchclamp protocols that are not overly complicated or challenging to apply experimentally. Pilot patch-clamp protocols
to parametrize the endogenous IKr current (hERG) are being
developed, and at the time of writing, preliminary manual
voltage clamp data have been generated for three drugs(dofetilide, cisapride, and verapamil) both at room andphysiological temperature. These data will be used to assess
the adequacy of the protocol in providing the parameters
needed to model dynamic drug block of IKr.
A guiding principle of the in silico effort is to introduce
additional complexities into the OHR model only whennecessary to increase its predictive accuracy. Initial testingof the model was done using newly generated IC50s for eachof the seven targeted channels at physiological temperature
for a subset of 12 drugs with well-characterized clinical risk
profiles. Comparing the results of these simulations toknown clinical responses will help to inform decisions on
which candidate channels are needed in the assay andwhether the model can be improved. Pilot simulation studies using the OHR model and data for dofetilide suggest
that, at least for hERG, IC50s alone may not be adequate inassessing the potential for a drug to prolong the AP and
induce EADs. Once the final set of targeted channels andtypes of pharmacology data needed have been determined
and the most promising candidate risk metric(s) identified,
the performance of the model and the boundary conditions
for using it to predict TdP risk will be defined using newly
generated pharmacology data for an independent set of 18
well-characterized drugs.

Stem Cell–Derived Human Myocyte
Studies
The primary goal of using human iPSC-derived cardiomyocytes is to identify repolarization effects that are not anticipated from either ion channel studies or in silico reconstruction
efforts. Such effects may provide insight on the modulationof cardiac APs by mechanisms that do not directly affect ion
channels or may not manifest themselves in the present version of the in silico model. Although iPSC-derived cardiomyocytes offer clear advantages over isolated primary humancardiac cells or cardiac tissue preparations, both in terms ofavailability and ease of use, numerous aspects of the biology
and pharmacology of these cells remain to be determinedwith certainty (see the Limitations and Challenges section).
In an effort to reach their goal, the SCWG has formed asubgroup under the auspices of HESI. Two technologicalstudy platforms were suggested for evaluation, namely, themultielectrode array (MEA) and the voltage-sensing optical(VSO) AP platforms; two-dimensional cell preparations
used for the study were prepared according to vendor
instructions. A pilot study was initiated in 2014 that consisted of volunteer efforts across 12 work sites and four
stem cell providers. This pilot study evaluated the effects ofeight blinded compounds selected to test for assay sensitivity to specific ion channel blockers (mexiletine for INa, nifedipine for ICaL, E-4031 for IKr/hERG, and JNJ303 for IKs;
across multiple sites and platforms) using changes in either
the field potential duration (for MEA) or AP (for VSO) as
one primary endpoint. A preliminary comparison of theeffects of three drugs across two sites for one stem cellsource suggests a good concordance when results were presented as percentage change versus baseline values.
Additional experiments and validation work are needed todetermine best practices and to reach definitive conclusions
on the functional utility of human stem cell–derived cardiomyocytes for determining the proarrhythmic risk of established and future drugs. Toward this goal, a validation study
with cardiomyocytes is to commence in the third quarter of2015.

ImpactIf successful, CiPA will have a significant impact on themanner in which cardiac safety assessment is performed in
the pharmaceutical industry. Although this is a consortium
effort, some general rules around data sharing and use havebeen developed and compiled under the CiPA Guiding
Principles.18 By moving the evaluation of proarrhythmic
risks earlier in the development process, it will allow for theremoval of compounds with undesirable effects on cardiac
repolarization, alleviating the risk and the costs of unmasking a QT signal of concern in clinical trials.
From a preclinical perspective, the CiPA paradigm willallow standardization of all in vitro ion channel assays tocharacterize the drug effects on cardiac repolarization. It isalso envisioned that it will make available a single, common,and fully validated in silico model to quantify the risk ofarrhythmia based on the ion channel data. Finally, it will alsoestablish best practices for the use of stem cell–derived cardiomyocytes, in an effort to confirm the translation between
ion channels, in silico modeling, and human cardiac tissue. A
concise summary of the similarities and differences between
S7B/E14 and CiPA is presented in Table 2.
To a certain extent, CiPA will accomplish what the S7B
guidance document has failed to achieve since its inception:
standardization of the way preclinical assays are conductedacross the pharmaceutical industry in order to remove bias
and to provide a reliable and reproducible data set allowing
complete assessment of the potential effects of drugs onhuman cardiac electrophysiology.

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Table 2. Comparison between S7B/E14 and evolving Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm.
S7B/E14

CiPA

Preclinical/clinical strategy for assessing the potential of a test
substance to delay ventricular repolarization
Focused on the ability of a test substance to inhibit IKr/IhERG (S7B)
Additional preclinical in vitro repolarization screens
are not prescribed, resulting in variability in type andcomprehensiveness of complementary data
Promulgates the idea that small effects on hERG will lead toadverse regulatory outcome
Guidelines are not prescriptive, permitting flexibility ofuser approach, leading to variability of data due to lack ofstandardized approach
Guidelines do not suggest in silico modeling
Does not directly address the endpoint of clinical concern(i.e., proarrhythmic risk)
Supports well-controlled in vivo electrocardiogram (ECG)
assessment in preclinical studies
Supports careful clinical assessment of electrophysiologicaleffects of test substance in phase 1 ECG studies before
progressing to thorough QT (TQT) study
Requests that all sponsors submitting a new drug application
conduct a TQT study to determine if a drug prolongs QTc

Moreover, an early and more complete assessment ofproarrhythmic risks, rather than the determination of QT prolongation alone, will reduce the number of false-positive (highsensitivity) results based on activity at the hERG channel andallow a more accurate determination of true-negative (highspecificity) compounds. As a consequence, it is expected that
CiPA will lead to a reduction in the need to conduct TQT studies. Given an individual cost of approximately US $2M per
TQT study, CiPA has the potential to make a significant positive impact on the cost of developing new medicines. Moreover,it should lead to an increase in drugs reaching phase 1, as wellas improve the accuracy of current or future labeling and allow
more drugs to reach the market to treat unmet medical needs.
In addition, unlike the approach adopted for the S7B guidance
document, CiPA is a consortium effort, allowing sharing of
knowledge and expertise to support the project. By doing so, it
lends itself to the sharing of resources with the common goalof defining best practice in developing safer drugs and inreevaluating the true cardiac risk of drugs that have been discarded because of their activity on the hERG channel or thatare currently marketed but carry a warning for QT prolongation in their label.

Limitations and Challenges
The CiPA initiative is the next logical step following thereview and analysis of a decade of data obtained using the

Preclinical strategy to evaluate the proarrhythmic risk of a test
substance that delays ventricular repolarization, or not
Composed of two distinct series of tests:
In vitro study of drug effects on multiple cardiac ion channels (notjust IKr/IhERG) and incorporation of these effects in an in silico
model of human ventricular action potential
Confirmation of the in silico results using human ventricular
myocytes derived from human induced pluripotent stem cells
Prevents unwarranted attrition due to hERG liability based onmechanistic understanding of proarrhythmic risk
Establishes best practices for ion channel and stem cellcardiomyocyte studies; standardized protocols and methods aredeveloped and adopted to minimize intra- and interlaboratory
variability
Makes available a single, common, and fully validated in silico model
to quantify the risk of arrhythmia based on ion channel data
Provides a more complete assessment of proarrhythmic risk earlyin the drug development process
Supports well-controlled in vivo ECG assessment in preclinicalstudies
Supports robust QT assessment of test substance in early-phaseclinical studies (phase 1); test substances with a safe CiPA profile
do not require TQT study
Scheduled to replace TQT studies

ICH-S7B and ICH E14 guidance documents.8,9 It has been
touted as “revolutionary,” and to some extent, it is, at theleast, a visionary initiative. That said, although it is an
attractive proposal, it will require a significant amount ofwork and likely a few years of testing before it comes tofruition to the point at which regulatory guidelines can be
updated definitively. The technology is currently available
to perform the ion channel work in a high-throughput environment, albeit most likely at ambient rather than physiological temperature. However, although recording of ioniccurrents from recombinant channels expressed in various celllines offers significant advantages over recording endogenous currents in human cardiac myocytes, the underlying
properties of these channels may not fully recapitulate thatof endogenous channels. For example, when expressed incell lines, the cardiac calcium channel Cav1.2 displays very
slow rates of recovery from inactivation at room temperature and therefore can be recorded only at very slow pacing
rates. Given that one important aspect of the CiPA paradigm
is to understand not only the potency of compounds but also
the kinetics of block, such properties will potentially limit
the ability to perform experiments at a more physiologic
pacing rate. Therefore, full validation of the ion channel
assays for the targets chosen will require time, effort, andfunding from sources that may need to go beyond SPS but
have not yet been clearly identified. In addition, although
the particular ion channels chosen are those collectively

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Fermini et al.
most likely to influence APD and arrhythmia susceptibility,
other proteins including the Na-Ca exchanger, Na-K pumpin the sarcolemmal membrane, and ryanodine receptors inthe sarcoplasmic reticulum can contribute directly or (in thecase of the RyR) indirectly to electrogenesis; in principle,
NCEs might, albeit rarely, influence these processes.
The predictive ability of the ventricular cell model(s)used will depend substantially on the current formulas used,and vigilance will be required to limit the model dependency of the results. Simulations that allow TdP to manifest(rather than simply cellular precursors such as EADs) will
require the use of tissue as well as cell models.
Stem cell–derived human cardiomyocytes offer numerous advantages over isolated primary cardiac cells (theiruse circumvents the use of animals, they can be cultured forextended periods of time allowing for chronic exposure to
test compounds, and, potentially most importantly, derivefrom humans). However, the commercial cells currently
available are composed of a mixed phenotype of atrial,nodal, and ventricular cells (often enriched for the latter)that is closer to an immature phenotype and may not fully
recapitulate the (electro)physiological properties of adultventricular cardiac myocytes (e.g., refs. 41–46). Also, there
have been some reports in which the pharmacology of standard compounds has proven inconsistent with cardiac cells,raising the question of the reliability of the assay relative tohuman cardiac tissue.47-50 It should be noted that recordings
from stem cell–derived myocytes do not fully recapitulate
ECG measurements from the intact heart in situ, although
they may reveal the propensity of a compound to delay
repolarization and favor EAD generation. In addition, CiPA
will not address the issue of compounds that prolong the QTinterval via changes in autonomic tone or blood pressure.
Relatively important issues remain to be addressed by
the CiPA Steering Committee. For example, at what stageof preclinical drug discovery and development will this newparadigm be applied (lead identification? lead optimization? candidate drug selection?) and at what cost? One canenvision, for example, that testing an additional number ofion channels at the lead identification/optimization stage
has the propensity to mitigate any potential costs savings
consequent to the elimination of TQT studies. Anotherimportant issue is whether testing will need to be conductedin compliance with good laboratory practice or good laboratory science. Thus, there may be a significant impact on
costs and timelines, depending on the stage at which thisnew paradigm is implemented. These and other significant
issues will need to be addressed before CiPA is ready to be
fully implemented.
Although the initial timelines that were proposed to complete the in vitro series of tests for this effort were very ambitious (i.e., revision of S7B by June 2016), in reality the CiPAeffort is likely to go through multiple iterations before it
reaches a point of applicability. The issues addressed are

extremely complex and will require the international consensus of protocols and data from all three core assays, as well as
the consensus of regulatory bodies across the globe, in order
to be accepted internationally. It is an evolving initiative withevolving workflows that will require scientific, intellectual,and practical contributions from multiple parties and, in consequence, will require time to be successful.
It is evident that the time has come to consider new, morecomprehensive ways of preclinical testing for proarrhythmic risk of evolving drugs. The hope is that this new CiPA
approach will prevent inappropriate compound attrition due
to hERG liability, provide a complete assessment of proarrhythmic risk, reduce animal and clinical work, rescue
drugs labeled with cardiac warnings based on small degrees
of QT prolongation, and help bring complete and standardized submission packages to regulatory agencies. This will
enable new drugs to address unmet medical needs and toreach patients more quickly.
Acknowledgment
The authors would like to thank Drs. Hugo Vargas, Derek
Leishman, and Sian Ratcliffe for carefully reviewing the manuscript and providing valued suggestions.

Declaration of Conflicting Interests
This publication reflects the views of the authors and should not be
construed to represent the FDA’s views or policies. The authors
declare that there are no conflicts of interest.

Funding
The authors received no financial support for the research, authorship, and/or publication of this article.

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