**ICH** **S7B** **Guideline**

**Step** **2 Revision**

**The Nonclinical Evaluation of the Potential for Delayed Ventricular
Repolarization (QT Interval Prolongation) by Human Pharmaceuticals**

**June 10, 2004**

This draft guidance, when finalized, will represent the Food and Drug
Administration\'s (FDA\'s) current thinking on this topic. It does not
create or confer any rights for or on any person and does not operate to
bind FDA or the public. You can use an alternative approach if it
satisfies the requirements of the applicable statutes and regulations.
If you want to discuss an alternative approach, contact the FDA staff
responsible for implementing this guidance. If you cannot identify the
appropriate FDA staff, call the appropriate number listed on the title
page of this guidance.

1\. INTRODUCTION [1](#__RefHeading___Toc74687324)

1.1 Objective of the Guideline [1](#__RefHeading___Toc74687325)

1.2 Background [1](#__RefHeading___Toc74687326)

1.3 Scope of the Guideline [2](#__RefHeading___Toc74687327)

1.4 General Principles [2](#general-principles)

2\. Guideline [2](#guideline)

2.1 Objectives of S7B Studies [2](#__RefHeading___Toc74687330)

2.2 Considerations for Selection and Design of Studies
[2](#__RefHeading___Toc74687331)

2.3 Nonclinical Testing Strategy [3](#nonclinical-testing-strategy)

2.3.1 *In vitro* I~kr~ assay [3](#in-vitro-ikr-assay)

2.3.2 *In vivo* QT assay [3](#__RefHeading___Toc74687334)

2.3.3 Chemical/pharmacological class [3](#chemicalpharmacological-class)

2.3.4 Relevant nonclinical and clinical information
[4](#relevant-nonclinical-and-clinical-information)

2.3.5 Follow-up studies [4](#follow-up-studies)

2.3.6 Integrated risk assessment [4](#integrated-risk-assessment)

2.3.7 Evidence of risk [5](#__RefHeading___Toc74687339)

2.4 Timing of S7B Nonclinical Studies and Integrated Risk Assessment in
Relation to Clinical Development [5](#__RefHeading___Toc74687340)

3\. Test Systems [5](#__RefHeading___Toc74687341)

3.1 Considerations for Test Systems [5](#__RefHeading___Toc74687342)

3.1.1 Use of positive control substances and reference compounds
[5](#use-of-positive-control-substances-and-reference-compounds)

3.1.2 *In vitro* electrophysiology studies
[5](#__RefHeading___Toc74687344)

3.1.3 *In vivo* electrophysiology studies
[7](#__RefHeading___Toc74687345)

3.1.4 Simulated pathological conditions and arrhythmias
[8](#__RefHeading___Toc74687346)

**The Nonclinical Evaluation of the Potential for Delayed Ventricular
Repolarization (QT Interval Prolongation) by Human Pharmaceuticals**

# INTRODUCTION

The assessment of the effects of pharmaceuticals on ventricular
repolarization and proarrhythmic risk is the subject of active
investigation. When additional data (nonclinical and clinical) are
accumulated in the future, they will be evaluated and this guideline
might be revised.

## Objective of the Guideline

This guideline describes a nonclinical testing strategy for assessing
the potential of a test substance to delay ventricular repolarization.
This guideline includes information concerning nonclinical assays and an
integrated risk assessment.

## Background

The QT interval (time from the beginning of the QRS complex to the end
of the T wave) of the electrocardiogram (ECG) is a measure of the
duration of ventricular depolarization and repolarization. QT interval
prolongation can be congenital or acquired (e.g.,
pharmaceutical-induced). When ventricular repolarization is delayed and
the QT interval is prolonged, there is an increased risk of ventricular
tachyarrhythmia, including torsade de pointes, particularly when
combined with other risk factors (e.g., hypokalemia, structural heart
disease, bradycardia). Thus, much emphasis has been placed on the
potential proarrhythmic effects of pharmaceuticals that are associated
with QT interval prolongation.

Ventricular repolarization, determined by the duration of the cardiac
action potential, is a complex physiological process. It is the net
result of the activities of many membrane ion channels and transporters.
Under physiological conditions, the functions of these ion channels and
transporters are highly interdependent. The activity of each ion channel
or transporter is affected by multiple factors including, but not
limited to, intracellular and extracellular ion concentrations, membrane
potential, cell-to-cell electrical coupling, heart rate, and autonomic
nervous system activity. The metabolic state (e.g., acid-base balance)
and location and type of cardiac cell are also important. The human
ventricular action potential consists of five sequential phases:

-   phase 0: The upstroke of the action potential is primarily a
    consequence of a rapid, transient influx of Na^+^ (I~Na~) through
    Na^+^ channels.

-   phase 1: The termination of the upstroke of the action potential and
    early repolarization phase result from the inactivation of Na^+^
    channels and the transient efflux of K^+^ (I~to~) through K^+^
    channels.

-   phase 2: The plateau of the action potential is a reflection of a
    balance between the influx of Ca^2+^ (I~Ca~) through L-type Ca^2+^
    channels and outward repolarizing K^+^ currents.

-   phase 3: The sustained downward stroke of the action potential and
    the late repolarization phase result from the efflux of K^+^ (I~Kr~
    and I~Ks~) through delayed rectifier K^+^ channels.

-   phase 4: The resting potential is maintained by the inward rectifier
    K^+^ current (I~K1~).

Prolongation of the action potential can result from decreased
inactivation of the inward Na^+^ or Ca^2+^ currents, increased
activation of the Ca^2+^ current, or inhibition of one or more of the
outward K^+^ currents. The rapidly and slowly activating components of
the delayed rectifier potassium current, I~Kr~ and I~Ks~, seem to have
the most influential role in determining the duration of the action
potential and thus the QT interval. The human ether-a-go-go-related gene
(hERG) and KvLQT1 gene encode pore-forming proteins that are thought to
represent the α-subunits of the human potassium channels responsible for
I~Kr~ and I~Ks~, respectively. These α-subunit proteins can form
hetero-oligomeric complexes with auxiliary β-subunits (i.e. MiRP and
MinK gene products), which have been speculated to modulate the gating
properties of the channel proteins. The most common mechanism of QT
interval prolongation by pharmaceuticals is inhibition of the delayed
rectifier potassium channel that is responsible for I~Kr~.

## Scope of the Guideline

This guideline extends and complements the "ICH Guideline on Safety
Pharmacology Studies for Human Pharmaceuticals" (ICH S7A). This
guideline applies to new chemical entities for human use and marketed
pharmaceuticals when appropriate (e.g., when adverse clinical events, a
new patient population, or a new route of administration raises concerns
not previously addressed). Pharmaceuticals for which testing is not
called for are described in ICH S7A.

## General Principles 

Principles and recommendations described in ICH S7A also apply to the
studies conducted in accordance with the present guideline.

*In vitro* and *in vivo* assays are complementary approaches; therefore,
according to current understanding, both assay types should be
conducted.

The investigational approach and evidence of risk should be
individualized for the test substance, depending on its pharmacodynamic,
pharmacokinetic and safety profiles.

# Guideline 

## Objectives of S7B Studies

The objectives of studies are to: 1) identify the potential of a test
substance and its metabolites to delay ventricular repolarization, and
2) relate the extent of delayed ventricular repolarization to the
concentrations of a test substance and its metabolites. The study
results can be used to elucidate the mechanism of action and, when
considered with other information, estimate risk for delayed ventricular
repolarization and QT interval prolongation in humans.

## Considerations for Selection and Design of Studies

Nonclinical methodologies can address the following:

-   Ionic currents measured in isolated animal or human cardiac
    myocytes, cultured cardiac cell lines, or heterologous expression
    systems for cloned human ion channels,

-   Action potential parameters in isolated cardiac preparations or
    specific electrophysiology parameters indicative of action potential
    duration in anesthetized animals,

-   ECG parameters measured in conscious or anesthetized animals,

-   Proarrhythmic effects measured in isolated cardiac preparations or
    animals.

As indicated above, these four functional levels can be investigated by
*in vitro* and/or *in vivo* methods. Findings from the first three
functional levels listed above are considered useful and complementary.
The value of proarrhythmia models is discussed in section 3.1.4.

*In vitro* electrophysiology studies can explore potential cellular
mechanisms that might not be evident from *in vivo* data. Changes in
other cardiovascular parameters or effects on multiple ion channels can
complicate interpretation of data. Complementary assessments in other
systems can address this issue. Although delay of repolarization can
occur through modulation of several types of ion channels, inhibition of
I~Kr~ is the most common mechanism responsible for
pharmaceutical-induced prolongation of QT interval in humans.

Experimental models that possess the full complement of mechanisms can
be more informative with regard to the clinical situation. Carefully
designed and conducted *in vivo* studies allow evaluation of metabolites
and can enable estimation of safety margins. *In vivo* ECG evaluations
provide information on conduction properties and non-cardiac influences
(e.g., autonomic nervous system tone). Studies of action potential
parameters provide information on the integrated activity of multiple
ion channels in the heart.

## Nonclinical Testing Strategy 

The following sections describe a general nonclinical testing strategy
for assessing risk for delayed ventricular repolarization and QT
interval prolongation that is pragmatic and based on currently available
information. The figure illustrates the component elements of the
testing strategy, but not specific test systems or their designs.

![](media/image1.wmf){width="6.133333333333334in" height="4.35in"}

### *In vitro* I~kr~ assay 

Results from an assay that evaluates effects on I~Kr~ or the ionic
current through a native or expressed I~Kr~ channel protein, such as
that encoded by hERG (see section 3.1.2).

### *In vivo* QT assay

Results from an *in vivo* assay that measures indices of ventricular
repolarization such as QT interval (see section 3.1.3).

### Chemical/pharmacological class 

Consideration should be given to whether the test substance belongs to a
chemical/pharmacological class in which some members have been shown to
induce QT interval prolongation in humans (e.g., antipsychotics,
histamine H-1 receptor antagonists, fluoroquinolones). This should,
where appropriate, influence the choice of reference compound(s) and be
included in the integrated risk assessment.

### Relevant nonclinical and clinical information 

Additional information for the integrated risk assessment can include
results from:

-   Pharmacodynamic studies,

-   Toxicology/safety studies,

-   Pharmacokinetic studies, including plasma levels of parent substance
    and metabolites (including human data if available),

-   Drug interaction studies,

-   Tissue distribution and accumulation studies,

-   Post-marketing surveillance.

### Follow-up studies 

Follow-up studies are intended to provide greater depth of understanding
or additional knowledge regarding the potential of test substance for
delayed ventricular repolarization and QT interval prolongation in
humans. Such studies can provide additional information concerning
potency, mechanism of action, slope of the dose-response curve, or
magnitude of the response. Follow-up studies are designed to address
specific issues, and, as a result, various *in vivo* or *in vitro* study
designs can be applicable.

In circumstances where results among nonclinical studies are
inconsistent and/or results of clinical studies differ from those for
nonclinical studies, retrospective evaluation and follow-up nonclinical
studies can be used to understand the basis for the discrepancies.
Results from follow-up studies can be a significant component of an
integrated risk assessment.

Relevant nonclinical and clinical information along with the following
should be considered in the selection and design of follow-up studies:

-   Use of ventricular repolarization assays that measure action
    potential parameters in isolated cardiac preparations (see section
    3.1.2),

-   Use of specific electrophysiological parameters indicative of action
    potential duration in anesthetized animals (see section 3.1.3),

```{=html}
<!-- -->
```
-   Repeated administration of test substance,

-   Selection of animal species and gender(s),

-   Use of metabolic inducers or inhibitors,

-   Use of concurrent positive control substances and reference
    compounds (see section 3.1.1),

-   Inhibition of other channels not previously evaluated,

-   Measurement of electrophysiological parameters at multiple time
    points,

-   Confounding effects in conscious animals that limit the
    interpretation of data such as test substance-induced effects on
    heart rate or autonomic tone, or toxicities such as tremor,
    convulsion, or emesis.

### Integrated risk assessment 

The integrated risk assessment is the evaluation of non-clinical study
results including the results from follow-up studies and other relevant
information. The integrated risk assessment should be scientifically
based and individualized for the test substance. Such an assessment can
contribute to the design of clinical investigations and interpretation
of their results. The integrated risk assessment should be provided for
the Investigator's Brochure and the Nonclinical Overview (ICH M4). The
integrated risk assessment should also consider:

-   Potencies of test substance in S7B assays relative to reference
    compound(s),

-   Safety margins from *in vivo* QT assays,

-   Assay sensitivity and specificity,

-   Contribution of metabolites to QT interval prolongation as well as
    metabolic differences between humans and animals.

### Evidence of risk

Evidence of risk is the overall conclusion from the integrated risk
assessment for a test substance to delay ventricular repolarization and
prolong QT interval in humans.

## Timing of S7B Nonclinical Studies and Integrated Risk Assessment in Relation to Clinical Development

Results from S7B nonclinical studies assessing the risk for delayed
ventricular repolarization and QT interval prolongation generally do not
need to be available prior to first administration in humans. However,
these results, as part of an integrated risk assessment, can support the
planning and interpretation of subsequent clinical studies. The early
availability of these data is considered valuable.

# Test Systems

## Considerations for Test Systems

This section provides an overview of methodologies currently used to
assess the potential for a test substance to delay ventricular
repolarization and to prolong QT interval. The following criteria should
be considered in selecting the most appropriate test systems:

-   Assay methodology and experimental endpoints are scientifically
    valid and robust,

-   Assays and preparations are standardized,

-   Results are reproducible,

-   Endpoints/parameters of the assays are relevant for assessing human
    risk.

### Use of positive control substances and reference compounds 

Positive control substances should be used to establish the sensitivity
of *in vitro* preparations for ion channel and action potential duration
assays. In the case of *in vivo* studies, positive control substances
should be used to validate and define the sensitivity of the test
system, but need not be included in every experiment.

For test substances belonging to a chemical/pharmacological class that
is associated with QT interval prolongation in humans, the use of
concurrent reference compound(s) (member(s) of the same class) in *in*
*vitro* and *in vivo* studies should be considered to facilitate ranking
the potency of the test substance in relation to its comparators.

Whether or not positive control substances or reference compounds are
used in experiments should be justified.

### *In* *vitro* electrophysiology studies

*In vitro* electrophysiology studies can provide valuable information
concerning the effect of a test substance on action potential duration
and/or cardiac ionic currents. These assays have an important role in
assessing the potential for QT interval prolongation and elucidating
cellular mechanisms affecting repolarization. *In vitro*
electrophysiology studies employ either single cell (e.g., heterologous
expression systems, disaggregated cardiomyocytes) or multicellular
(e.g., Purkinje fiber; papillary muscle; trabeculae; perfused
myocardium; intact heart) preparations. Multicellular preparations are
stable test systems to study action potential duration. While more
fragile, single cell preparations minimize diffusional barriers to the
site of action. The analysis of parameters for each phase of the action
potential such as V~max~ for phase 0 (I~Na~), APD~30~ for phase 2
(I~Ca~) and "triangulation" for phase 3 (I~K~) can be useful to
investigate the effects on specific channels responsible for these
phases. In addition, some parameters derived from the Langendorff
preparation have been reported to provide information regarding
proarrhythmia. Heterologous expression systems, where human ion channel
protein(s) are expressed in noncardiac cell lines, are used to assess
the effects of a test substance on a specific ion channel. Disaggregated
myocytes are technically more challenging than the expression systems
but have the advantage of being suitable for assessing effects on both
action potential duration and ionic currents.

Tissue and cell preparations for *in vitro* assays are obtained from
different laboratory animal species including rabbit, ferret, guinea
pig, dog, swine, and occasionally from humans. The ionic mechanisms of
repolarization in adult rats and mice differ from larger species,
including humans (the primary ion currents controlling repolarization in
adult rats and mice is I~to~); therefore, use of tissues from these
species is not considered appropriate. Species differences in terms of
which cardiac ion channels contribute to cardiac repolarization and to
the duration of the action potential should be considered in selecting a
test system. When native cardiac tissues or cells are used, the
characteristics and source of the preparation should be considered
because the distribution of ion channel types varies according to the
region and type of cell.

Test substance concentrations for *in vitro* studies should span a broad
range, covering and exceeding the anticipated maximal therapeutic plasma
concentration. Ascending concentrations should be tested until a
concentration-response curve has been characterized or physicochemical
effects become concentration-limiting. Ideally, the duration of exposure
should be sufficient to obtain steady-state electrophysiological
effects, unless precluded by the viability of the cell or tissue
preparation. The duration of exposure should be indicated. Appropriate
positive control substances should be used to establish the sensitivity
of the *in vitro* assay system as well as to confirm that the ion
channels of interest are present and stable.

Factors that can confound or limit the interpretation of *in vitro*
electrophysiology studies include the following:

-   The testing of high concentrations of the test substance can be
    precluded by limited solubility in aqueous physiological salt
    solutions,

-   Adsorption to glass or plastic or non-specific binding to the test
    matrix can reduce the concentration of the test substance in the
    incubation or perfusion medium,

-   Test substance concentrations can be limited by cytotoxic or
    physicochemical attributes of the test substance that disrupt cell
    membrane integrity so that electrophysiological endpoints cannot be
    obtained,

-   Cardiac cells and tissues have limited capacity for drug metabolism
    and therefore *in vitro* studies using the parent substance do not
    provide information on the effects of metabolites. When *in vivo*
    nonclinical or clinical studies reveal QT interval prolongation that
    is not corroborated by *in vitro* studies using the parent
    substance, testing metabolites in the *in vitro* test systems should
    be considered.

High throughput potassium channel assays are being developed. While
novel ion channel activity assays can be useful in preliminary screening
of test substances to identify lead candidates for further
electrophysiological testing, more experience will establish whether
they have sufficient predictive value to be an alternative to voltage
clamp assays.

Another screening approach is the use of competition binding protocols
in which test substances are studied for their ability to displace a
radiolabeled hERG channel blocker from a cell line expressing hERG.
However, competition for radioligand-binding sites provides no
information on agonistic or antagonistic effects of the test substance
on I~Kr~. Moreover, this assay will not identify test substances that
bind to hERG at sites other than the radioligand binding sites. Based
upon these potential limitations, this assay is not considered a
substitute for voltage clamp assays described above.

### *In vivo* electrophysiology studies

Intact animal models allow investigation of ventricular repolarization
or associated arrhythmias where integrated effects on the full
complement of ion channel and cell types are assessed. Also, potential
neuronal and hormonal influences on the pharmacodynamic effect of the
pharmaceuticals are present in animals.

The QT interval of the ECG is the most commonly used endpoint to gauge
effects of a test substance on ventricular repolarization. In
specialized electrophysiology studies, regional information regarding
the ventricular repolarization (e.g., monophasic action potential
duration and effective refractory period) can also be obtained from *in
vivo* models. Additional safety parameters of interest, including blood
pressure, heart rate, PR interval, QRS duration, the presence of U
waves, and arrhythmias, can be assessed simultaneously.

The QT interval and heart rate have an inverse, non-linear relationship,
which varies among species, between animals, or even within the same
animal at different heart rates. Thus, a change in heart rate exerts an
effect on QT interval, which can confound the assessment of the effect
of the test substance on ventricular repolarization and the QT interval.
There are two important situations where there is variability in heart
rate among animals: one is due to difference in autonomic tone, and the
other is due to effects of test substances on heart rate. Therefore, the
interpretation of data from *in vivo* test systems should take into
account the effect of coincident changes in heart rate. Ideally, QT
interval data obtained after administration of a test substance should
be compared with control and baseline data at similar heart rates. When
the variability is not due to the test substance, it can be reduced by
training, or the use of anesthetized animal models. When the effects are
due to test substances, the most common approach is to correct the QT
interval for heart rate (QTc) using formulae such as Bazett or
Fridericia; however, these corrections can yield misleading data,
especially when differences in heart rate between treatment and control
are large. An alternative approach is to maintain a constant heart rate
using cardiac pacing.

Laboratory animal species used for *in vivo* electrophysiology studies
include dog, monkey, swine, rabbit, ferret, and guinea pig. The ionic
mechanisms of repolarization in adult rats and mice differ from larger
species, including humans (the primary ion currents controlling
repolarization in adult rats and mice is I~to~); therefore, use of these
species is not considered appropriate. The most appropriate *in vivo*
test systems and species should be selected and justified.

The dose range should be in accord with that discussed in ICH S7A and,
whenever feasible, should include and exceed the anticipated human
exposure. The dose range can be limited by animal intolerance to the
test substance, e.g., emesis, tremor, or hyperactivity. For studies
designed to relate the extent of delayed ventricular repolarization to
concentrations of the parent test substance and its metabolites,
controlled exposure via constant intravenous infusion can be used.
Monitoring exposure to the test substance and metabolites (see ICH S3A)
provides opportunities to interpret dose- and concentration-response
data and to design follow-up studies, if appropriate.

Factors that should be considered in conducting studies and interpreting
the results include the following:

-   Data acquisition and analysis methods,

-   Sensitivity and reproducibility of the test systems,

-   Dosing period and measurement points,

-   Heart rate and other cardiovascular effects that confound
    interpretation of QT interval data,

-   Inter-species and gender differences, e.g., cardiac
    electrophysiology, hemodynamics, or metabolism of pharmaceuticals,

-   Pharmaceuticals that have effects on several ion channels can yield
    complex dose-response relationships that could be difficult to
    interpret.

### Simulated pathological conditions and arrhythmias

The precise relationship between test substance-induced delay of
ventricular repolarization and risk of proarrhythmia is not known.
Directly assessing the proarrhythmic risk of pharmaceuticals that
prolong the QT interval would be a logical undertaking; however,
modeling of the clinical condition where pharmaceuticals elicit
arrhythmia is complicated. Indices of proarrhythmic activity (e.g.
electrical instability and temporal and/or spatial dispersion of
refractoriness, reverse use-dependency, changes in action potential
configuration) and animal models might have utility in assessing
proarrhythmia. Interested parties are encouraged to develop these models
and test their usefulness in predicting risk in humans.