MEMORANDUM

To: Douglas Throckmorton, M.D., Division Director

HFD 110, Division of Cardio‑Renal Drug Products

From: John Koerner, Ph.D., Pharmacologist

HFD 110, Division of Cardio‑Renal Drug Products

# Through: Charles Resnick, Ph.D., Supervisory Pharmacologist

HFD 110, Division of Cardio‑Renal Drug Products

Subject: Nonclinical Electrophysiological Effects of Ranolazine

Date: 23 October 2003

The sponsor (CV Therapeutics, Inc.) has submitted an amendment to this
NDA[^1] providing nonclinical study reports to address
Pharmacology/Toxicology issues discussed in the discipline review
letter. Among other things, the sponsor attempts to prove that QT
prolongation with ranolazine is not a concern. This memo addresses study
reports describing ranolazine's electrophysiological properties in
nonclinical studies. Other study reports provided in the amendment were
addressed by Dr. Elizabeth Hausner in her memo dated 29 September 2003.

Although some of the evidence provided in this amendment is consistent
with ranolazine induced QT prolongation and repolarization disturbances,
other evidence suggests antiarrhythmic rather than proarrhythmic
potential. Nevertheless, these study results do not alter the previous
conclusion (see original memorandum dated 4 September 2003) that the
nonclinical electrophysiology findings do not preclude human risk, in
large part due to lack of comprehensive information addressing the
sensitivity and specificity of the in vitro assays cited. Findings are
summarized below.

The sponsor addressed effects of ranolazine, its enantiomers and
metabolites on several ionic currents that modulate ventricular
repolarization.

-   Ranolazine did not inhibit the slowly activating delayed rectifier
    potassium current (I~Ks~) through human channels (KvLQT1/minK)
    expressed in *Xenopus* oocytes. Hence, the sponsor concluded that
    ranolazine at concentrations up to 900 μM does not inhibit I~Ks~.
    However, these data are not convincing since the *Xenopus* oocyte
    expression system greatly underestimates potency. The sponsor argued
    that results of previously submitted studies showing that ranolazine
    inhibited native I~Ks~ in isolated canine ventricular myocytes were
    due to current run-down and therefore an artifact of the test
    system. Given the contradictory study reports and assay limitations,
    the effects of ranolazine on I~Ks~ are presently unclear.

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-   Ranolazine enantiomers, like racemic ranolazine, inhibited I~Kr~ and
    late I~Na~ with similar potencies in canine ventricular myocytes.
    Additionally, several ranolazine metabolites inhibited late I~Na~
    when evaluated at a concentration of 10 μM. The sponsor argues that
    late I~Na~ inhibition attenuates ranolazine's effects on action
    potential duration in M-cells and other ventricular tissue, thereby
    preventing proarrhythmic activity. However, the torsadogenic drugs
    terfenadine and cisapride also inhibit late I~Na~, and terfenadine,
    like ranolazine, showed similar potencies on I~Kr~ and late I~Na~.
    Therefore, inhibition of late I~Na~ does not appear to preclude risk
    of drug-induced *torsade de pointes*.

-   Ranolazine increased the decay of I~Ca~ and therefore inhibited what
    the sponsor called late I~Ca~. Although this activity theoretically
    could shorten action potential duration, the actual significance of
    this finding is unknown.

The sponsor addressed proarrhythmic and antiarrhythmic potential of
ranolazine in vitro. The relevance of these findings is unknown since
the sensitivity of these in vitro models has not been sufficiently well
characterized.

-   Ranolazine lengthened epicardial monophasic action potential
    duration in isolated female rabbit hearts. Ranolazine did not
    increase apex-base dispersion of action potential duration nor
    induce ventricular arrhythmias. Additionally, ranolazine, at
    concentrations that inhibit I~Kr~ and late I~Na~ in canine
    ventricular myocytes, exerted actions consistent with antiarrhythmic
    activity, since it prevented pause-dependent ventricular arrhythmias
    induced by positive controls. The specificity of these effects was
    not evaluated with other QT prolonging drugs that inhibit multiple
    ionic currents, e.g. terfenadine and cisapride.

-   Ranolazine prevented isoproterenol-induced delayed
    afterdepolarizations in isolated guinea pig ventricular myocytes.
    This effect is consisted with ranolazine's ability to block β1
    adrenergic receptors at the concentration evaluated.

-   Ranolazine lengthened epicardial monophasic action potential
    duration in isolated guinea pig hearts, while showing effects
    consistent with antiarrhythmic activity. The sensitivity and
    specificity of this model has not been thoroughly characterized.

-   This sponsor argues that ranolazine lacks proarrhythmic activity
    since it does not induce early afterdepolarizations, or increase
    M-cell APD90 and transmural dispersion of repolarization in isolated
    canine cardiac wedge preparations. Ranolazine was additionally
    negative for in vitro proarrhythmic effects in this preparation,
    since *torsade* like arrhythmias did not occur spontaneously and
    could not be elicited with a single extrastimulus in the drug's
    presence. Epicardial stimulation was utilized for these studies
    since stimulation at this site was necessary to capture cisapride's
    proarrhythmic activity, presumably due to increased transmural
    dispersion of repolarization at baseline.

> In contrast to the present study results, a previous study showed
> ranolazine to lengthen M-cell action potential duration and increase
> transmural dispersion of repolarization, but only in the presence of 2
> mM potassium. The lowest potassium concentration evaluated in the
> present studies was 3 mM. The difference in findings can be explained
> by the known enhancement of drug-induced I~Kr~ inhibition by
> hypokalemia.

The individual studies addressed in this memorandum are listed below.

  ------------------------------------------------------------ ----------
  Study Number: Title                                          Page

  CVT303.069-P: Effect of Ranolazine on I~Ks~ in Isolated      3
  Canine Left Ventricle Myocytes                               

  CVT303.063-P: Effects of Ranolazine Enantiomers on I~Ks~,    3
  I~Kr~, and Late I~Na~, and Ranolazine Metabolites on Late    
  I~Na~                                                        

  CVT303.059-P: Electrophysiologic Effects of Ranolazine on    5
  Late I~Ca~ in Isolated Canine Left Ventricular Myocytes      

  CVT303.065-P: Effects of Ranolazine on Ventricular           6
  Repolarization in Isolated Rabbit Hearts                     

  CVT303.070-P: Effects of Ranolazine on lsoproterenol,        7
  Forskolin, and Ouabain Induced Delayed Afterdepolarizations  
  and Triggered Activity of Guinea Pig Ventricular Myocytes    

  CVT303.061-P: Antiarrhythmic Effects of Ranolazine in a      7
  Human LQT Model: The In Vitro Guinea Pig Heart Perfused with 
  the Proarrhythmic Sea Anemone Toxin ATX-II                   

  CVT303.068-P: Electrophysiologic Effects of Ranolazine in    10
  Arterially Perfused Wedge Preparations from the Canine Left  
  Ventricle: A comparison Between Epicardial and Endocardial   
  Stimulation                                                  

  Assessing Predictors of Drug-induced *Torsade de Pointes*    12
  ------------------------------------------------------------ ----------

[CVT303.069-P: Effect of Ranolazine on I~Ks~ in Isolated Canine Left
Ventricle Myocytes]{.underline}

Ranolazine did not inhibit current (I~Ks~) through human channels
(KvLQT1/minK) expressed in *Xenopus* oocytes. Hence, the sponsor
concluded that ranolazine at concentrations up to 900 μM does not
inhibit I~Ks~.

However, these data do not convincingly show lack of effect of
ranolazine on I~Ks~ since the *Xenopus* oocyte expression system greatly
underestimates potency.

![](media/image1.png)

[CVT303.063-P: Effects of Ranolazine Enantiomers on I~Ks~, I~Kr~, and
Late I~Na~, and Ranolazine Metabolites on Late I~Na~]{.underline}

Ranolazine enantiomers were evaluated for effects on ionic currents
modulating ventricular repolarization in isolated canine ventricular
myocytes. Both enantiomers inhibited I~Kr~ and late I~Na~, but only the

S-enantiomer inhibited I~Ks~, and then only weakly.

+----------------------+---------------+---------------+---------------+
| Test Substance       | > IC50 (μM)   |               |               |
+----------------------+---------------+---------------+---------------+
|                      | I~Kr~         | I~Ks~         | Late I~Na~    |
+----------------------+---------------+---------------+---------------+
| R-Ranolazine         | 28            | no            | 8             |
|                      |               | inhibition\^  |               |
+----------------------+---------------+---------------+---------------+
| S-Ranolazine         | 10            | \>100         | 5             |
+----------------------+---------------+---------------+---------------+

\^ tested at concentrations up to 100 μM

The following table summarizes effects of racemic ranolazine and its
enantiomers in the context of positive control drugs. Racemic ranolazine
and positive control drugs and were not evaluated concurrently.

Note that terfenadine and cisapride, both torsadogenic drugs, inhibit
both I~Kr~ and late I~Na~, similar to ranolazine. Furthermore,
terfenadine, like ranolazine, is similarly potent on late I~Na~ and
I~Kr~. Therefore, blockade of late I~Na~ does not appear to preclude
torsadogenic risk.

![](media/image2.png){width="5.059027777777778in"
height="2.6256944444444446in"}

The following figure illustrates effects of several ranolazine
metabolites on late I~Na~. At a concentration of

10 μM, several ranolazine metabolites inhibited late I~Na~. Inhibition
was independent of basic cycle length (BCL).

![](media/image3.png){width="3.86875in" height="3.4125in"}

[CVT303.059-P: Electrophysiologic Effects of Ranolazine on Late I~Ca~ in
Isolated Canine Left Ventricular Myocytes]{.underline}

Ranolazine increased the rate of decay of I~Ca~ in isolated left
ventricular myocytes. Late I~Ca~, defined as current measured 300 ms
after the start of the test pulse, was inhibited with an IC50 of 50 μM.
The figure below, which shows the I~Ca~ current trace vs time,
illustrates that 5 μM ranolazine inhibited late I~Ca~ while only
minimally affecting peak I~Ca~.

![](media/image4.png){width="3.06875in" height="1.8944444444444444in"}

# CVT303.065-P: Effects of Ranolazine on Ventricular Repolarization in Isolated Rabbit Hearts 

Ranolazine and its enantiomers were evaluated for effects on monophasic
action potential duration (MAPD) in isolated female rabbit hearts paced
at a constant rate of 1 Hz. Epicardial MAPDs were monitored in base and
apex of the heart for determination of spatial dispersion. E-4031, which
inhibits I~Kr~, and ATX-II, which enhances the late I~Na~, were utilized
as concurrent control test substances.

Ranolazine at concentrations of 1-100 μM increased MAPD in both base and
apex in a concentration-related manner. Effects were similar in base and
apex, such that the difference between these two sites, or what the
sponsor refers to as dispersion, was not altered. Potencies (EC50s) were
similar for both sites (4.3 and 4.8 μM for base and apex, respectively).
In comparison, E-4031 and ATX-II increased MAPD and dispersion in a
concentration dependent manner. Ranolazine enantiomers also increased
MAPD at a single site in a concentration dependent and manner (the site
at which MAPD was monitored was not provided). The enantiomers'potencies
on MAPD were similar (6.4 and 5.9 M for R and S enantiomers,
respectively).

![](media/image5.png){width="5.6875in" height="4.35625in"}

![](media/image6.png){width="5.514583333333333in"
height="1.2666666666666666in"}

Ranolazine, in contrast to E-4031, did not induce ventricular ectopic
beats at any concentration evaluated (data not shown). Ranolazine at
concentrations of 5 and 10 μM reduced ventricular ectopic beats induced
by E-4031 (data not shown). Additionally, ranolazine at 5 μM attenuated
pause dependent ventricular arrhythmias induced by E-4031 and ATX-II
(data not shown). The specificity of this in vitro antiarrhythmic effect
was not evaluated using other QT prolonging drugs that inhibit multiple
ionic currents, e.g. terfenadine or cisapride.

[CVT303.070-P: Effects of Ranolazine on lsoproterenol, Forskolin, and
Ouabain Induced Delayed Afterdepolarizations and Triggered Activity of
Guinea Pig Ventricular Myocytes]{.underline}

Ranolazine at a concentration of 10 μM reduced the amplitude of
isoproterenol-induced delayed afterdepolarizations (DADs) and triggered
activity in isolated guinea pig ventricular myocytes. Ranolazine did not
alter DADs induced by forskolin and ouabain. The effect on
isoproterenol-induced DADs is likely due to ranolazine's β1 adrenergic
receptor blockade at the concentration evaluated.

![](media/image7.png){width="5.351388888888889in"
height="1.2173611111111111in"}

[CVT303.061-P: Antiarrhythmic Effects of Ranolazine in a Human LQT
Model: The In Vitro Guinea Pig Heart Perfused with the Proarrhythmic Sea
Anemone Toxin ATX-II]{.underline}

Ranolazine was evaluated for effects on epicardial monophasic action
potential duration (MAPD~90~) in isolated guinea pig hearts (gender not
provided) paced at a constant rate of 1.5 Hz. Acute AV block was induced
by infusion of N^6^-cyclypentyladenosine, which blocks adenosine
receptors. E-4031, which selectively inhibits I~Kr~, ATX-II, which
selectively enhances the late I~Na~, and chromanol 293, which inhibits
I~Ks~, were utilized as concurrent control test substances. In an
additional experiment, rate dependence of ranolazine was compared to
that of the positive control test substances.

Ranolazine at concentrations of 1-100 μM lengthened epicardial MAPD~90~
in a concentration dependent manner, similar to positive control
substances. Ranolazine's effects on MAPD~90~ were independent of pacing
cycle length, ranging from 400 to 1000 ms (data not shown). In contrast,
E-4031 and ATX-II but not chromanol 293B showed inverse rate dependence,
with greater percentage increases at a basic cycle length of 1000 ms
than at 400 ms.

![](media/image8.png){width="3.377083333333333in"
height="2.7270833333333333in"}

Ranolazine attenuated MAPD lengthening and prevented early
afterdepolarizations and polymorphic ventricular tachycardia induced by
ATX-II in a concentration related manner. Effects of positive control
drugs that inhibit multiple ionic currents (including the late I~Na~),
e.g. terfenadine and cisapride, were not evaluated.

![](media/image9.png){width="3.3506944444444446in"
height="2.597916666666667in"}

![](media/image10.png){width="3.5520833333333335in"
height="2.740972222222222in"}

![](media/image11.png){width="3.4520833333333334in"
height="2.4243055555555557in"}

![](media/image12.png){width="5.950694444444444in"
height="4.540277777777778in"}

[CVT303.068-P: Electrophysiologic Effects of Ranolazine in Arterially
Perfused Wedge Preparations from the Canine Left Ventricle: A comparison
Between Epicardial and Endocardial Stimulation]{.underline}

Endocardial stimulation appears to sensitize the isolated canine left
ventricular wedge preparation to arrhythmias induced by QT prolonging
drugs, since cisapride's proarrhythmic effect in this model was captured
with epicardial, but not with endocardial stimulation. Ranolazine was
previously evaluated using endocardial stimulation; however, effects
with epicardial stimulation have not yet been evaluated.

The purpose of the present study was to evaluate the in vitro
electrophysiologic effects of ranolazine in canine wedges stimulated
from the epicardial surface. Results were compared to those using
endocardial stimulation in the same experiment. Both normokalemic (4 mM
potassium) and hypokalemic (3 mM potassium) conditions were utilized, as
was pacing the preparations at both short and long basic cycle lengths
(500 and 2000 ms) to address rate dependence of effects.

With epicardial stimulation and normokalemia, ranolazine at
concentrations of 1-100 μM did not lengthen M-cell or epicardial action
potential duration (APD90). With epicardial stimulation and hypokalemia,
ranolazine at 100 μM lengthened epicardial but not M-cell APD90.

While ranolazine prolonged transmural QT interval and increased Tpeak
--Tend in a concentration dependent manner, it did not lengthen M-cell
action potential duration or transmural dispersion of repolarization
(TDR) under any conditions. Indeed, ranolazine decreased TDR when
evaluated with hypokalemia, likely due to a proportionately greater
increase in epicardial vs M-cell APD90.

![](media/image13.png){width="6.854166666666667in"
height="2.1354166666666665in"}

![](media/image14.png){width="6.745138888888889in"
height="2.3868055555555556in"}

With endocardial stimulation, ranolazine at concentrations of 1-100 μM
did not lengthen M-cell or epicardial action potential duration (APD90).
Effects were independent of potassium concentration.

Ranolazine prolonged transmural QT interval in a biphasic manner, with
peak effects at 5-10 μM.

Ranolazine increased Tpeak --Tend over control values, but only at the
highest concentration evaluated. Ranolazine did not significantly
lengthen M-cell action potential duration or increase transmural
dispersion of repolarization (TDR). Ranolazine's electrophysiologic
effects were similar at basic cycle lengths of 500 and 2000 ms (only
effects at 2000 ms are shown).

![](media/image15.png){width="6.548611111111111in"
height="2.4916666666666667in"}

![](media/image16.png){width="6.548611111111111in"
height="2.4805555555555556in"}

Ranolazine did not induce *torsade de pointes* - type arrhythmias in
vitro in canine left ventricular wedges using epicardial stimulation.

+------------------+----------+-------------------+-------------------+
| Test Substance   | P        | Spontaneous       | St                |
|                  | otassium |                   | imulation-induced |
|                  | (μM)     | Arrhythmia        | Arrhythmia\^      |
+------------------+----------+-------------------+-------------------+
| Ranolazine       | 4        | 0/5               | 0/5               |
| (1-100 μM)       |          |                   |                   |
+------------------+----------+-------------------+-------------------+
| Ranolazine       | 3        | 0/5               | 0/5               |
| (1-100 μM)       |          |                   |                   |
+------------------+----------+-------------------+-------------------+

\^ The sponsor attempted to induce ventricular arrhythmias using a
single extrastimulus applied to the

epicardial surface at progressively shorter intervals until
refractoriness was reached. This methodology was

successful in eliciting ventricular arrhythmias in 2 of 6 wedge
preparations exposed to cisapride (0.2 μM).

Spontaneous arrhythmias were not observed with cisapride. Cisapride's
proarrhythmic effects were limited to a

single concentration, with slightly higher and lower concentrations
yielding no arrhythmias.

[Assessing Predictors of Drug-Induced *Torsade de Pointes*]{.underline}

The sponsor provided a manuscript arguing that delayed ventricular
repolarization and proarrhythmia are separable, i.e. drugs that do not
induce early afterdepolarizations or increase dispersion of
repolarization are unlikely to cause TdP, even in the setting of
prolonged QT.

[Sponsor's Abstract]{.underline}

Torsades de Pointes (TdP) is a malignant polymorphic ventricular
tachyarrhythmia that can be caused by drugs that induce
electrophysiological changes. Although the number of drugs known to
cause TdP has increased in recent years, there is no cell-based assay,
*in vitro* heart preparation, or animal model that predicts a drug's
potential to induce TdP in humans. Nevertheless, certain
electrophysiologic events are known to be associated with the
development of TdP. A drug that prolongs action potential duration,
induces early afterdepolarizations and ectopic beats, and increases
dispersion of ventricular repolarization is very likely to cause TdP. By
contrast, a drug that does not induce these changes is unlikely to cause
TdP. The exact relationship between prolonged action potential duration,
early afterdepolarizations, ectopic beats, increased dispersion of
ventricular repolarization, and the development of TdP has not been
defined, but the potential of a drug to elicit these events might
predict its pro-arrhythmic risk.

The sponsor supports the hypothesis that torsadogenic drugs are always
associated with early afterdepolarizations and spatial dispersion of
repolarization with data from several in vitro models. The sponsor did
not provide a comprehensive assessment of the sensitivity and
specificity of any single model.

[^1]: Amendment dated 13 September 2003; received by Center on 15
    September 2003.