Electrophysiological properties of HBI-3000: a new antiarrhythmic agent with multiple-channel blocking properties in human ventricular myocytes


Guo, D.; Liu, Q.; Liu, T.; Elliott, G.; Gingras, M.; Kowey, P.R.; Yan, G-Xin.

Journal of Cardiovascular Pharmacology 57(1): 79-85

2011


HBI-3000 (sulcardine sulfate) has been shown to suppress various ventricular arrhythmias in animal models. The electrophysiological properties of HBI-3000 were investigated using standard microelectrode and patch-clamp techniques in single human ventricular myocytes. HBI-3000 led to concentration-dependent suppression of dofetilide-induced early afterdepolarizations in single nonfailing human ventricular myocytes and early afterdepolarizations seen in failing ventricular myocytes. The concentration-dependent prolongation of action potential duration (APD) by HBI-3000 was bell shaped with maximum response occurring around 10 μM. Interestingly, HBI-3000 at the concentration of 10 μM modestly prolonged the APD at all 3 basic cycle lengths. The slope of APD-cycle length curve of HBI-3000 was only slightly steeper than that of control (88.8 ± 7.7 ms/s vs. 78.9 ± 5.2 ms/s in control, n = 8, P > 0.05). HBI-3000 only showed a minimal use-dependent prolongation of the APD in human ventricular myocytes. HBI-3000 inhibited fast sodium current (INa-F), late sodium channel (INa-L), L-type calcium current (ICa-L), and rapidly activating delayed rectifier K current (IKr) in single human ventricular myocytes. The estimated half-maximal inhibitory concentration values of INa-F, INa-L, ICa-L, and IKr were 48.3 ± 3.8, 16.5 ± 1.4, 32.2 ± 2.9, and 22.7 ± 2.5 μM, respectively. The ion channel profile and electrophysiological properties of HBI-3000 are similar to those of ranolazine and chronic amiodarone (reduced INa-F, INa-L, ICa-L, and IKr). HBI-3000 may be a promising antiarrhythmic agent with low proarrhythmic risk.

ORIGINAL
ARTICLE
Electrophysiological
Properties
of
HBI-3000:
A
New
Antiarrhythmic
Agent
With
Multiple-channel
Blocking
Properties
in
Human
Ventricular
Myocytes
Donglin
Guo,
MD,
PhD,
*
Que
Liu,
MD,
PhD,
t
Tengxian
Liu,
BS,
*
Gary
Elliott,
PharmD,
PhD,
t
Mireille
Gingras,
PhD,
t
Peter
R.
Kowey,
*t
and
Gan-Xin
Yan,
MD,
PhD*:
Abstract:
HIBI
-
3000
(sulcardine
sulfate)
has
been
shown
to
sup-
press
various
ventricular
arrhythmias
in
animal
models.
The
elec-
trophysiological
properties
of
IIBI-3000
were
investigated
using
standard
microelectrode
and
patch-clamp
techniques
in
single
human
ventricular
myocytes.
I-IBI-3000
led
to
concentration-dependent
suppression
of
dofetilide-induced
early
afterdepolarizations
in
single
nonfailing
human
ventricular
myocytes
and
early
afterdepolarizations
seen
in
failing
ventricular
myocytes.
The
concentration-dependent
prolongation
of
action
potential
duration
(APD)
by
IIBI-3000
was
bell
shaped
with
maximum
response
occurring
around
10
µM.
In-
terestingly,
IIBI-3000
at
the
concentration
of
10
µM
modestly
prolonged
the
APD
at
all
3
basic
cycle
lengths.
The
slope
of
APD—
cycle
length
curve
of
IIBI-3000
was
only
slightly
steeper
than
that
of
control
(88.8
±
7.7
ms/s
vs.
78.9
±
5.2
ms/s
in
control,
n
=
8,
P
>
0.05).
I-IBI-3000
only
showed
a
minimal
use-dependent
pro-
longation
of
the
APD
in
human
ventricular
myocytes.
IIBI-3000
inhibited
fast
sodium
current
a
)
late
sodium
channel
a
)
,-Na-F,
P
,-Na-L,
P
L-type
calcium
current
(I
ca
_
L
),
and
rapidly
activating
delayed
rectifier
IC
E
current
(I
N
)
in
single
human
ventricular
myocytes.
The
estimated
half-maximal
inhibitory
concentration
values
of
I
Na
_F,
INa-L,
ICa-L,
and
I
N
,
were
48.3
±
3.8,
16.5
±
1.4,
32.2
±
2.9,
and
22.7
±
2.5
µM,
respectively.
The
ion
channel
profile
and
electrophysiological
prop-
erties
of
I-IBI-3000
are
similar
to
those
of
ranolazine
and
chronic
amiodarone
(reduced
I
Na
_F,
INa_L,
Ic
a
-L,
and
I
Nr
).
HMI-3000
may
be
a
promising
antiarrhythmic
agent
with
low
proarrhythmic
risk.
Key
Words:
early
afterdepolarizations,
proarrhythmic,
reverse
use-
dependence,
ion
channels
(J
Cardiovasc
PharmacolTM
2011;57:79-85)
Received
for
publication
May
11,
2010;
accepted
October
1,
2010.
From
the
*Lankenau
Institute
for
Medical
Research
&
Main
Line
Health
Heart
Center,
Wynnewood,
PA;
tHUYA
Bioscience
International,
San
Diego,
CA;
and
tJefferson
Medical
College,
Thomas
Jefferson
University,
Philadelphia,
PA.
Supported
by
an
unrestricted
grant
fmm
HUYA
Bioscience
International.
Dr
Que
Liu
is
a
former
employee
of
HUYA
Bioscience
International.
Drs
Gary
Elliott
and
Mireille
Gingras
are
employees
of
HUYA
Bioscience
International.
Dr
Peter
Kowey
has
been
a
consultant
for
HUYA
Bioscience
International
on
matters
unrelated
to
the
subject
of
this
article
and
received
no
remuneration
fmm
the
proceeds
of
this
grant.
D.
Guo
and
Q.
Liu
contributed
equally
to
this
work.
The
author
report
no
conflict
of
interest.
Reprints:
Donglin
Guo,
MD,
PhD,
Lankenau
Institute
for
Medical
Research
&
Main
Line
Health
Heart
Center,
100
Lancaster
Avenue,
Wynnewood,
PA
19096
(e-mail:
guod@m1hs.org
or
Yang@m1hs.org
).
Copyright
©
2011
by
Lippincott
Williams
&
Wilkins
1
Cardiovasc
Pharmacor
Volume
57,
Number
1,
January
2011
INTRODUCTION
Drug-induced
proarrhythmia
remains
a
major
clinical
limitation
of
currently
available
antiarrhythmic
drugs.
These
proarrhythmias
are
most
often
related
to
action
potential
duration
(APD)
and
QT
interval
prolongation.
Blockade
of
rapidly
activating
delayed
rectifier
potassium
current
(k
r
)
by
majority
of
antiarrhythmic
drugs
is
primarily
responsible
for
APD/QT
prolongation.
1
'
2
However,
such
increase
in
APD/QT
interval
is
often
more
pronounced
at
a
slow
versus
fast
heart
rate.
3-5
This
feature
is
called
"reverse
use
dependence."
As
a
result
of
reverse
use
dependence,
these
agents
exert less
or
no
effect
at
fast
heart
rates,
whereas
they
may
exhibit
maximum
prolongation
of
APD/QT
interval
at
slow
heart
rate,
raising
the
risk
of
lethal
ventricular
arrhythmias
such
as
Torsade
de
Points
(TdP).
6
Therefore,
reverse
use
dependence
represents
an
important
cardiac
safety
issue
for
the
clinical
use
of
these
agents.
The
disappointing
results
from
the
SWORD
trial
(survival
with
oral
d-sotalol)
suggest
that
a
pure
potassium
current
blocker
is
associated
with
a
higher
incidence
of
proarrhythmia.
7
Whether
optimal
antiarrhythmic
effects
could
be
conferred
by
an
agent
with
highly
selective
single
channel
blocking
action
or
by
an
agent
that
inhibits
multiple
ion
channel
remains
controversial.
Although
classified
a
Vaugh-
an—Williams
class
III
drug,
studies
have
shown
that
amiodarone
blocks
multiple
cardiac
ion
channels,
including
Ca
t
±
channel,
IC
E
channels,
and
Na
±
channels.
Such
properties
of
amiodarone
have
been
thought
to
contribute
to
its
minimal
reverse
use
dependency
and
low
incidence
of
clinical
TdP,
even
in
patients
who
develop
TdP
on
other
medications.'
A
recent
study
found
that
its
dominant
effect
on
myocardium
is
related
to
late
Na
±
channel
(I
Na4
_,)
blockade."
This
IN
a
d,
blocking
property
of
amiodarone
can
favorably
distinguish
it
from
other
class
III
drugs,
like
pure
or
dominant
IC
E
channel
blockers.
Ranolazine,
another
multiple-channel
blocker
with
the
most
potent
blockade
on
IN
a
-L,
also
possesses
important
antiarrhythmic
activity
without
proarrhythmic
effect.
12
This
suggests
that
agents
with
multiple-channel
(including
I
Na
$
blocking
actions
are
a
viable
approach
to
the
treatment
of
various
arrhythmias,
leading
to
the
hypothesis
that
multiple
ion
channel
blocking
agents
may
be
less
proarrhythmic
than
selective
ion
channel
inhibitors
developed
to
date.
The
novel
compound
HBI-3000
(sulcardine
sulfate)
has
been
shown
to
suppress
ventricular
arrhythmias
in
the
conscious
canine
model
of
sudden
cardiac
death."
In
guinea
pig
patch-clamp
studies,
the
drug
seems
to
inhibit
fast
sodium
www.jcvp.org
I
79
Guo
et
al
1
Cardiovasc
Pharmacor
Volume
57,
Number
1,
January
2011
current
(I
Na
_
F
),
transient
outward
current
(I
(
),
and
L-type
calcium
current
(I
ca
_
L
)
at
concentrations
of
approximately
20-40
IIM.
The
present
study
was
designed
to
access
the
electrophysiological
properties
of
RBI-3000
in
human
ventricular
myocytes.
MATERIALS
AND
METHODS
Human
Ventricular
Myocytes
Isolation
Human
tissue
usage
was
approved
by
the
Institutional
Review
Board
at
the
University
of
Pennsylvania
and
the
Lankenau
Institute
for
Medical
Research.
Human
ventricular
tissue
was
obtained
from
patients
with
severe
heart
failure
at
the
time
of
cardiac
transplantation
and
from
explanted
hearts
of
nonfailing
donors
through
a
nonprofit
organ
procurement
organization
(Gift-of-Life,
Inc,
Philadelphia,
PA).
Prospective
informed
consent
for
research
use
of
heart
tissue
was
obtained
from
transplant
recipients
or
donor's
next
kin.
Explanted
hearts
were
transported
from
the
operating
suite
to
the
laboratory
in
cold
Krebs
solution
(in
millimolar:
glucose
12.5,
KC1
5.4,
lactic
acid
1,
MgSO
4
1.2,
NaCl
130,
NaH
2
PO
4
1.2,
NaHCO
3
25,
and
Na
pyruvate
2,
pH
7.4).
We
have
recently
optimized
our
experimental
method
to
obtain
high-quality
human
ventricular
myocytes
through
an
artery-perfused
wedge
preparation
of
human
left
ventricular
wall.
Myocytes
were
isolated
using
the
enzymatic
digestion
method.
14
'"
Briefly,
a
transmural
wedge
was
dissected
from
the
left
ventricle.
The
wedge
was
initially
perfused
through
one
branch
(Ml)
of
left
circumflex
coronary
artery
with
500
mL
of
Ca
-free
solution
(in
millimolar:
NaC1125,
KC13.5,
KH
2
PO
4
1.5,
MgCl
2
1,
NaHCO
3
20,
and
glucose
10)
gassed
with
95%
0
2
-5%
CO
2
at
37°C
at
a
rate
of
30
mL/min
with
a
peristaltic
pump.
The
wedge
was
then
perfused
with
80
mL
of
enzyme
solution
[48
mg
of
collagenase
(type
II),
12
mg
of
hyaluronidase,
80
mg
of
bovine
serum
albumin,
and
300
mg
taurine
to
the
Ca
-free
solution].
At
the
end
of
enzyme
perfusion,
a
thin
layer
of
tissue was
dissected
from
subendocardial
layer.
Cells
were
transferred
to
Tyrode
solution
with
1
mM
Ca'
(in
millimolar:
NaCl
137,
KC1
5,
MgCl
2
1,
CaCl
2
1,
glucose
10,
and
HEPES
10;
pH
7.4)
and
stored
at
10°C.
Only
quiescent
rod-shaped
cells
showing
clear
cross-
striations
were
used.
Single
Myocyte
Action
Potential
Recording
Action
potentials
were
recorded
at
36.0
±
0.3°C
using
the
standard
microelectrode
technique.
The
microelectrode
had
a
resistance
of
25-40
MD,
when
filled
with
3
M
of
KC1.
Cells
were
perfused
with
a
bath
solution
containing
(in
millimolar):
NaCl
137,
KC1
5,
MgCl
2
1,
CaCl
2
2,
glucose
10,
and
HEPES
10
(pH
7.4).
Action
potentials
were
recorded
at
steady
state
using
various
stimulus
frequencies
(0.25, 0.5,
and
1.0
Hz).
Action
potentials
are
frequently
unable
to
be
paced
at
2
Hz
because
the
minimum
APD
is
around
500
milliseconds.
APD
was
measured
at
90%
repolarization
(APD9o)-
Early
afterdepolarizations
(EADs)
are
defined
as
a
depolarizing
afterpotential
that
begins
before
the
completion
of
repolarization
of
an
action
potential.
In
our
experiment,
only
these
have
significant
membrane
potential
oscillations
that
occur
at
potential
plateau
voltages
are
accounted
for
EADs.
Those
that
have
long
APDs
without
significant
potential
oscillations
are
not
EADs.
Membrane
Current
Recording
Aliquots
of
cell-containing
solution
(about
0.1
mL)
were
added
to
a
1.5-mL
bath
chamber
on
a
stage
of
an
inverted
microscope.
Cells
were
superfused
at
2
mL/min
with
perfusion
solution.
Ionic
currents
were
recorded
at
room
temperature
(22-24°C)
using
a
whole-cell
patch-clamp
technique.
Com-
mand
pulses
were
generated
by
a
Digidata
1320A
controlled
by
pClamp
8
software
(Axon
Instruments,
Foster
City,
CA).
Series
resistance
was
compensated
electronically
by
70%-80%.
For
recording
I
Na
_
F
,
the
bath
solution
contained
the
following
(in
millimolar):
NaCl
10,
CsC1
130,
MgCl
2
1.0,
CaCl
2
1.0,
HEPES
5,
glucose
10,
CdC1
0.3,
and
nicardipine
0.002
(pH
was
adjusted
to
7.4
with
CsOH). The
composition
of
the
filling
solution
was
(in
millimolar):
NaCl
10,
CsF
110,
ethylene
glycol
tetraacetic
acid
(EGTA)
5,
HEPES
5,
and
ATP-Mg
5
(pH
adjusted
to
7.2
with
CsOH).
I
Na
_
F
was
recorded
during
an
80-millisesond
depolarizing
voltage
step
from
a
holding
potential
of
-100
mV
to
a
test
potential
of
-30
mV
at
the
stimulus
rate
of
1
seconds.
For
recording
INa-L,
the
bath
solution
contained
(in
millimolar):
NaCl
140,
CsC15,
MgCl
2
2.0,
CaCl
2
1.8,
HEPES
5,
glucose
5,
and
nicardipine
0.002
(pH
was
adjusted
to
7.4
with
NaOH).
The
composition
of
the
filling
solution was
as
follows
(in
millimolar):
NaCl
10,
CsC1
130,
EGTA
5,
HEPES
5,
and
ATP-Mg
5
(pH
adjusted
to
7.2
with
CsOH).
INa-i,
currents
were
recorded
using
a
2000-millisecond
depolarizing
pulse
from
-140
to
-20
mV
at
the
stimulating
rate
of
0.1
Hz
(holding
potential
is
-140
mV).
The
amplitude
of
I
Na
_
L
was
measured
at
200
milliseconds
after
membrane
depolarization.
'
cad
,
was
recorded
with
a
250-millisecond
depolarizing
voltage
step
from
a
holding
potential
(V
h
)
of
-80
mV
to
test
potential
(V
i
)
of
0
mV.
A
5-millisecond
prepulse
from
-80
to
-40
mV
was
used
to
inactivate
f
I
Na
_
F
.
Bath
solution
for
recording
'
cad
,
contained
(in
millimolar):
NaCl
140,
CsC1
5,
MgCl
2
1,
CaCl
2
2,
glucose
10,
and
HEPES
10
(pH
7.4
with
NaOH).
Pipette
solution
contained
(in
millimolar):
CsC1
20,
Cs
aspartate
100,
MgCl
2
1,
tetraethylammonium
chloride
20,
EGTA
10,
HEPES
10,
and
Mg-ATP
5
(pH
7.2
with
CsOH).
For
recording
of
I
mo
,,
the
bath
solution
contained
(in
millimolar):
NaCl
132,
KC1
5,
MgCl
2
1.2,
HEPES
5,
glucose
5,
and
0.002
nicardipine,
pH
was
adjusted
to
7.4
with
NaOH.
The
pipette
solution
(pH
7.2)
contained
(in
millimolar):
K-gluconate
119,
KC1
15,
MgCl
2
2.0,
EGTA
5.0,
HEPES
5,
and
K
2
-ATP
5.
The
peak
tail
current
of
I
Kr
was
determined
as
the
amplitude
of
decaying
current
immediately
after
a
400-millisecond
pulse
from
a
holding
potential
of
-50
to
50
mV.
Drugs
RBI-3000
was
obtained
from
Huya
Bioscience
In-
ternational
(San
Diego,
CA)
and
dissolved
in
dimethylsulfoxide
to
obtain
a
stock
solution
of
0.01
M.
Dofetilide
was
provided
by
Pfizer
(Groton,
CT)
and
dissolved
in
dimethylsulfoxide.
Dilutions
of
the
stock
solution
were
made
immediately
before
the
experiment
to
obtain
the
desired
concentrations.
80
I
www.jcvp.org
©
2011
Lippincott
Williams
&
Wilkins
1
Cardiovasc
Pharmacor
Volume
57,
Number
1,
January
2011
Electrophysiological
Properties
of
HBI-3000
Data
Analysis
Data
are
expressed
as
mean
±
standard
error
of
the
mean.
Student
t
test
or
2-way
analysis
of
variance
was
used
to
determine
the
statistical
significance
of
differences
between
control
and
test
conditions.
Significance
was
defined
as
a
value
of
P
<
0.05.
RESULTS
Effects
of
HBI-3000
on
EADs
in
Failing
Human
Ventricular
Myocytes
Spontaneous
EADs
were
frequently
observed
in
failing
human
myocytes
that
were
stimulated
at
continuous
low-
frequency
(a
cycle
length
of
4000
milliseconds)
in
the
normal
Tyrode
solution
(Fig.
1).
The
take
off
potentials
of
EADs
ranged
between
—20
and
0
mV.
This
phenomenon
was
observed
in
9
of
12
left
ventricular
myocytes
isolated
from
4
failing
hearts
(3
cells
per
heart).
In
contrast,
recordings
from
left
ventricular
myocytes
of
control
hearts
did
not
register
any
EADs
(0
of
8
cells,
2
cells
per
heart,
P
<
0.01).
To
determine
whether
HBI-3000
could
suppress
the
EADs
seen
in
failing
myocytes,
we
added
HBI-3000
to
the
perfusate
when
EAD
was
elicited.
HBI-3000
exhibits
a
concentration-dependent
suppression
of
EADs
in
failing
myocytes.
HBI-3000
sup-
pressed
EADs
by
12.5%,
62.5%,
and
87.5%
at
the
concentrations
of
3,
10,
and
30
I.LM,
respectively
(Fig.
1,
n
=
8,
2
cells
per
heart).
A
50
Failing
Myocytes
B
50
HBI-3000
(
30
pM)
EADs
---"--
0
0-
>
E
E
>
>.
-50
-50-
-100
-100
400
ms
400
ms
U..,
2
50
7/8
5/8
(5
0
2/8
3
pM
10
pM
30
pM
[HBI-300],
pM
FIGURE
1.
Effect
of
HBI-3000
on
EADs
in
failing
human
myocytes.
Action
potentials
recorded
in
the
absence
(A)
and
presence
(B)
of
HBI-3000
in
a
left
ventricular
myocyte
from
failing
human
heart
at
a
basic
cycle
length
of
4000
milli-
seconds.
C,
Concentration-dependent
suppression
of
EADs
by
HBI-3000
in
failing
human
myocytes.
Effects
of
HBI-3000
on
Dofetilide-Induced
EADs
in
Nonfailing
Human
Ventricular
Myocytes
Figure
2A
shows
typical
recordings
of
action
potentials
in
the
absence
(baseline)
and
presence
of
dofetilide
or
dofetilide
with
HBI-3000
in
ventricular
myocytes
of
non-
failing
human
hearts.
Dofetilide
at
the
concentration
of
0.1
I.LM
caused
a
significant
APD
prolongation
in
human
myocytes
at
the
basic
cycle
length
of
4000
milliseconds.
Spontaneous
EADs
were
frequently
observed
after
addition
of
dofetilide.
HBI-3000
exhibits
a
concentration-dependent
suppression
of
dofetilide-induced
EADs
in
human
myocytes.
As
seen
in
Figure
2C,
HBI-3000
suppressed
EADs
by
12.5%,
62.5%,
and
100%
at
the
concentrations
of
3,
10,
and
30
I.LM,
respectively
(n
=
8,
2
cells
per
heart).
Characteristics
of
HBI-3000
on
APD
90
in
Nonfailing
Human
Ventricular
Myocytes
Action
potentials
were
recorded
in
ventricular
myocytes
isolated
from
subendocardial
layers
of
nonfailing
human
hearts.
The
concentration—response
curves
of
APD
90
to
HBI-
3000
were
bell
shaped
at
the
basic
cycle
length
of
4000
milliseconds.
HBI-3000
led
to
concentration-dependent
prolonged
APD
90
up
to
10
I.LM
in
myocytes.
At
the
concentration
of
30
I.LM,
percentage
increases
of
APD
90
diminished
and
APD
90
was
shortened
at
100
I.LM
versus
the
value
at
30
I.LM
(Fig.
3).
APD
prolongation
with
class
III
antiarrhythmic
com-
pounds
is
typically
more
pronounced
at
slow
stimulating
frequency.
This
property
is
called
reverse
use
dependence.
The
excessive
prolongation
of
APD
at
low
stimulating
frequencies
contributes importantly
to
the
proarrhythmic
effects
of
class
III
antiarrhythmic
agents.
As
seen
in
Figure
4,
dofetilide
(a
specific
I
Kr
blocker)
at
the
concentration
of
10
nM
dramatically
enhanced
the
positive
action
potential
duration—cycle
length
(APD-CL)
relationship
and
exerted
a
significant
reverse
use—
dependent
prolongation
of
the
APD
in
single
ventricular
myocyte
(Figs.
4A,
E).
The
APD-CL
slope
of
dofetilide
was
significantly
steeper
(126.2
±
8.3
ms/s
vs.
71.9
±
4.7
ms/s
in
control,
n
=
8,
P
<
0.05).
In
contrast,
HBI-3000
at
the
concentration
of
10
I.LM
modestly
prolonged
the
APD
at
all
3
frequencies
and
the
APD-CL
slope
was
only
slightly
increased
(88.8
±
7.7
ms/s
vs.
78.9
±
5.2
ms/s
in
control,
n
=
8,
P
>
0.05,
Figs.
4B,
D).
As
seen
in
Figure
4E,
HBI-3000
produced
a
largely
rate-independent
prolongation
of
APD
in
human
ventricular
myocytes.
Effects
of
HBI-3000
on
Ionic
Currents
in
Human
Myocytes
Figure
5
shows
the
superimposed
concentration—
response
curves
of
HBI-3000
on
4
major
inward
or
outward
currents
in
human
ventricular
myocytes.
HBI-3000
led
to
concentration-dependent
inhibition
of
INa-F,
INa-L,
Ica-L,
and
I
Kr
currents.
The
calculated half-maximal
inhibitory
concentra-
tion
(IC
50
)
values
for
HBI-3000
blocking
IN,F,
IN
a
d,
Ic
a
d,
and
I
Kr
were
48.3
±
3.8,
16.5
±
1.4,
32.2
±
2.9,
and
22.7
±
2.5
I.LM
(n
=
8
cells
from
4
control
hearts,
2
cells
per
heart),
©
2011
Lippincott
Williams
&
Wilkins
www.jcvp.org
I
81
400
ms
EADs
C
loo
50
3
pM
10pM
30
pM
[HBI-3000],
pM
5/8
8/8
2/8
**
Baseline
Dof
Dof+HB1-3000
A
B
30
0_
"6
15
a)
zn
8.9
0
50
-
10pM
3
pM
30
pM
1
M
control
100
pM
-100
-
200
ms
FIGURE
3.
Effects
of
HBI-3000
on
action
potentials
of
single
human
myocytes.
A,
Action
potential
traces
recorded
in
a
myocyte
isolated
from
human
left
ventricle
at
the
basic
cycle
length
of
4000
milliseconds.
HBI-3000
displayed
a
bimodal
effect
on
action
potential,
prolonging
the
action
potential
at
1,
3,
and
10
µM,
reducing
the
degree
of
prolonga-
tion
at
30
µM
versus
at
10
µM,
and
inducing
an
absolute
shortening
of
action
potential
at
100
µM.
B,
Per-
centage
changes
of
APD
90
in
human
ventricular
myocyte.
Mean
±
stan-
dard
error
of
the
mean,
n
=
8.
\
10
100
[HBI-3000],
pM
Guo
et
al
Cardiovasc
Pharmacor
Volume
57,
Number
1,
January
2011
Baseline
Dof
(0.1
pM)
Dof+HBI-3000
(30
pM)
A
50
E
0
cs)
0
>
-50
-
100
g
1200
FIGURE
2.
Effect
of
HBI-3000
on
dofetilide-induced
EADs
in
nonfail-
ing
human
myocyte.
A,
Action
potentials
recorded
in
the
absence
(baseline)
and
presences
of
dofeti-
lide,
and
dofetilide
with
HBI-3000
from
a
left
ventricular
myocyte
of
nonfailing
human
heart
at
a
basic
cycle
length
of
4000
milliseconds.
B,
APD
90
in
the
absence
and
presence
of
dofetilide,
and
dofetilide
with
HBI-3000
in
control
myocytes.
C,
Concentration-dependent
suppres-
sion
of
EADs
by
HBI-3000
in
control
human
myocytes.
Mean
±
standard
error
of
the
mean,
n
=
6,
**P
<
0.01
versus
baseline.
E
800
0
400
respectively.
Interestingly,
HBI-3000
exhibited
a
strong
in-
hibition
on
I
Nad
,
current
over
other
currents
(Fig.
5E).
DISCUSSION
In
agreement
with
half-maximal
inhibitory
concentra-
tion
measurements,
it
can
be
hypothesized
that
the
pro-
longation
of
APD
90
by
HBI-3000
at
lower
concentrations
is
mainly
due
to
blockade
of
I.
Blockade
of
I
Na4
_,
and
Ic
a
-
L
,
by
HBI-3000
attenuated
APD
prolongation
or
even
shortened
APD
at
high
concentrations.
The
net
effect
of
inhibitions
of
these
currents
by
HBI-3000
resulted
in
a
bell-shaped
curve
of
concentration
response
of
APD
90
in
human
ventricular
myocytes.
HBI-3000
is
similar
to
ranolazine
and
amiodarone
with
inhibitory
effects
on
multiple
cardiac
ion
channels,
such
as
I
Na4
_,,
I
Kr
,
and
'
Cad
,.
HBI-3000
differs
significantly
from
dofetilide
that
blocks
I
Kr
and
induces
EADs
in
human
ven-
tricular
myocytes.
HBI-3000—induced
prolongation
of
the
APD
shows
little
reverse
use
dependence
and
is
not
associated
with
EADs
and
other
abnormal
activities
in
isolated
human
ventricular
myocytes.
Indeed,
HBI-3000
at
the
concentrations
of
3-30
I.LM
displayed
concentration-dependent
suppres-
sions
of
dofetilide-induced
EADs
in
normal
ventricular
myocytes
and
in
spontaneously
developing
EADs
seen
in
failing
ventricular
myocytes.
Excessive
prolongation
of
QT
interval
or
APD
by
medicinal
compounds
has
become
a
major
concern
among
medical
professionals
and
the
pharmaceutical
industry
82
I
www.jcvp.org
©
2011
Lippincott
Williams
&
Wilkins
A
4D
ai
cs)
0
>
-40
1
Cardiovasc
Pharmacor
Volume
57,
Number
1,
January
2011
Electrophysiological
Properties
of
HBI-3000
Control
80
r
D
ofetilide
1
0
nM
4000
m
s
2000 ms
1000
m
s
-80
-80
200
m
s
200
m
s
40
cti
0
>
-4
0
-.4000
m
s
2000
m
s
1000
m
s
B
80
C
ontrol
80
r
r
H
B
I
-
3
0
00 10
uM
C
(Li
co
40
40D
0
m
s
s
0
>
-40
-4000
m
s
ms
2000
2000
m
1000
s
m
s
1000
m
200
m
s
-80
200
ms
Dofetilide
D
HBI-3000
Dofetilide
10
nM
1
1
0 0
0
—41.—
10
pM
HBI-3000
-CD-Control
—0—
Control
E
a.
800
600
400
2000 3000
4000 1000 2000 3000 4000
Cycle
Lengths,
ms
Cycle
Lengths,
ms
E
300
**
Dofetilide
(10
nM)
200
**
100
HBI-3000
(10
pM)
p=ns
0
1000 2000 3000 4000
Cycle
Lengths,
ms
FIGURE
4.
Frequency-dependent
effects
of
dofetilide
and
HBI-3000
on
human
ventricular
myocytes.
A,
Action
potential
traces
of
single
myocytes
in
the
absence
and
presence
of
10
nM
dofetilide
at
various
cycle
lengths.
B,
Action
potential
traces
of
single
myocytes
in
the
absence
and
presence
of
10µM
H
BI-3000
at
various
cycle
lengths.
C,
Effects
of
dofetilide
on
APD
90
at
various
cycle
lengths.
D,
Effect
of
HBI-3000
on
APD
90
at
various
cycle
lengths.
E,
Features
of
frequency-dependent
prolongation
of
APD
by
dofetilide
and
HBI-3000.
Mean
±
standard
error
of
the
mean,
n
=
8,
**P
<
0.01
versus
baseline
(1000
milliseconds).
E
413
ai
Lo
-4
0
8
0
E
o
0
o_
a
800
600
400
1000
because
of
their
proarrhythmic
effects.
16'17
The
use
of
specific
I
Kr
blockers
such
as
d-sotalol,
almokalant,
and
E-4031
in
the
treatment
of
various
atrial
and
ventricular
arrhythmias
is
complicated
by
the
likelihood
of
proarrhythmic
episodes,
typically
TdP.
18
'
19
It
has
been
shown
that
combination
therapy
with
quinidine
I
Kr
,
and
blocker)
and
mexiletine
('Na
blocker)
is
more
effective
in
the
prevention
of
ventricular
tachycardia
and
ventricular
fibrillation
in
animal
models
and
in
humans
as
opposed
to
monodrug
therapy.
20,21
Therefore,
multiple-channel
inhibition
has
been
suggested
to
underlie
the
safety
and
efficacy
of
I
Kr
blockers
that
prolong
APD
or
QT
without
inducing
EADs
or
triggering
of
TdP.
Indeed,
these
features
contribute
importantly
to
the
suppression
of
EADs
and
other
arrhythmias.
The
efficacy
of
amiodarone
and
its
low
incidence
of
proarrhythmic
effect
may
be
attributable
to
this
complex
multiple-channel
inhibition.'
Similar
compounds
also
include
ranolazine
and
sodium
pentobarbita1.
12
'
23
Our
data
suggest
that
HBI-3000
fits
this
electrophysiological
profile
as
well.
HBI-3000
exerted
multiple-channel
blocking
effects
and
did
not
produce
any
EADs
or
other
electrical
abnormalities.
It
was
antiarrhythmic
against
both
heart
failure—associated
and
dofetilide-induced
EADs
in
human
ventricular
myocytes,
©
2011
Lippincott
Williams
&
Wilkins
www.jcvp.org
I
83
0
pM
H
BI-3000
Control
100
m
s
10
30 100
[H
BI-300],
pM
E
1
.0
2
a)
TI
0.5
O
0
-
l„.„„
(IC
,=
48.3
pM)
A.
l
c
,.
(IC
,
n
=
3
0
.2
pM)
'•
I
x
,
(1C,.=22.7
pM)
I.,,
(IC
so
=16.5
pM)
D
U-
0
.4
0.2
0.0
2
0
0
m
s
C
0
u_
-3
-6
-9
Guo
et
al
Cardiovasc
Pharmacor
Volume
57,
Number
1,
January
2011
N
a
-F
30
pM
HBI-3000
Control
B
0
.0
-
LL
-0
.5
-
—z
-1.0-
N
a
-L
10
tr
pM
HBI-3
0 0
0
C
ontrol
500
m
s
A
0
1
8_
-30
10
m
s
-1
.5
-
FIGURE
5.
Concentration-depen-
dent
inhibitions
of
INa-Fi
INa-Le
iCa-Le
and
I
Kr
by
HBI-3000
in
human
ventricular
myocytes.
A,
Represen-
tative
currents
of
I
Na
_
F
from
a
single
myocyte
in
the
absence
and
pres-
ence
of
30
µM
HBI-3000.
B,
Repre-
sentative
recordings
from
a
single
myocyte
of
I
Na
-
L
in
the
absence
and
presence
of
10
µM
HBI-3000.
C,
Representative
recordings
from
a
single
myocyte
of
I
ca
_ L
in
the
absence
and
presence
of
30
HBI-3000.
D,
Representative
record-
ings
from
a
single
myocyte
of
I
Kr
in
the
absence
and
presence
of
10
HBI-3000.
E,
Summarized
concen-
tration—response
relationships
for
inhibitions
of
I
N
,
F
,
I
ca
-
L
,
and
I
Kr
in
human
ventricular
myocytes
(n
=
6
for
each).
indicating
that
it
may
be
a
safe
antiarrhythmic
drug
despite
I
Kr
blocking
effect.
However,
the
exact
ionic
mechanism
of
the
beneficial
effect
of
multiple-channel
blockers
such
as
amiodarone
and
ranolazine
is
not
clear.
A
recent
study
found
that
the
dominant
effect
on
myocardium
seems
to
be
related
to
I
Na4
_,
blockade.
11
This
I
Na4
_,
blocking
property
of
amiodarone
and
ranolazine
can
favorably
distinguish
these
drugs
from
other
class
III
drugs,
such
as
pure
or
dominant
IC
E
channel
blockers.
Our
recent
studies
have
demonstrated
that
I
Na
.-
L
,
is
sensitive
to
a
rate
change
due
to
its
slow
inactivation
and
recovery
kinetics.
Thus,
compounds
with
a
dominant
inhibitory
effect
on
the
channel
may
decrease
or
even
diminish
the
reverse
use
dependence
of
APD/QT
prolongation.
Ranolazine
is
a
multiple
current
inhibitor
with
a
dominant
inhibitory
effect
on
the
I
Nad
,
current.
Antzelevitch
et
al
reported
that
ranolazine-induced
prolongation
of
the
APD
is
rate
independent.
It
does
not
display
reverse
use-dependent
prolongation
of
APD
in
canine
ventricular
myocytes.
12
Like
ranolazine,
chronic
amiodarone
inhibits
multiple
currents
in
cardiac
cell
membrane
with
a
strong
blockade
on
I
Na4
_,
in
human
ventricular
myocytes.
11
Clinical
data
demonstrated
that
chronic
administration
of
amiodarone
shows
a
minimal
use-dependent
prolongation
of
QT
in
humans.'
These
electrophysiological
features
of
ranolazine
and
amiodarone
may
account
in
part
for
their
high
efficacy
of
the
drugs
and
their
low
propensity
to
cause
TdP.
In
the
present
study,
we
found
that
HBI-3000
has
a
prominent
blocking
effect
over
other
cardiac
currents
(ie,
I
Na
-
B
I
K
,
and
I
ca
$
and
shows
a
minimal
reverse
use—dependent
prolongation
of
APD
in
human
ventricular
myocytes.
The
electrophysiological
feature
of
HBI-3000
may
also
explain
its
antifibrillatory
actions
in
the
conscious
canine
model
of
sudden
cardiac death
and
its
EADs
suppression
effect
in
human
ventricular
myocytes.
However,
the
potency
for
INa_F
would
probably
be
significantly
enhanced
if
the
voltage
clamp
protocols
were
modified
to
reflect
a
cardiac-like
duty
cycle
when
frequencies
are
achieved
during
exercise.
Most
class
III
agents
have
been
shown
to
be
proarrhythmic
due
to
the
blockade
of
I
Kr
current
in
cardiac
cell
membrane.
These
agents
cause
greater
APD/QT
length-
ening
when
heart
rate
is
slow.
Selective
block
of
I
Kr
by
class
III
agents
in
ventricular
myocytes
resulted
in
an
unopposed
I
Na
_
L
,
which
would
prolong
the
APD/QT
at
slow
heart
rate
and
depolarize
the
membrane
sufficiently
to
disturb
the
balance
in
favor
of
EAD
formation.
As
seen
in
Figure
2,
dofetilide
at
the
concentration
of
0.1
µM
could
induce
excessive
APD
prolongation
and
EAD
formation
in
human
ventricular
myocytes.
HBI-3000
concentration
dependently
suppressed
these
EADs.
These
results
support
the
hypothesis
that
'
Nag
,
plays
a
critical
role
in
EADs
formation
in
human
ventricular
myocytes.
Selective
blockade
of
I
Nad
,
may
be
a
promising
target
for
antiarrhythmic
therapy.
A
chief
function
of
I
Na
_
F
is
to
initiate
cardiac
impulse
conduction.
Blockade
of
the
I
Na
_
F
by
HBI-3000,
especially
at
a
high
concentration,
may
cause
cardiac
conduct
deficit
and
slow
the
cardiac
rhythm.
Similarly,
HBI-3000
blocks
'
cad
,
at
high
concentration,
which
indicates
that
it
may
have
a
potential
negative
inotropic
effect
to
the
cardiac
muscle.
Therefore,
84
I
www.jcvp.org
©
2011
Lippincott
Williams
&
Wilkins
1
Cardiovasc
Pharmacor
Volume
57,
Number
1,
January
2011
Electrophysiological
Properties
of
HBI-3000
further
studies
are
required
to
elucidate
the
effects
of
HBI-
9.
3000
in
the
cardiac
conducting
function
and
contractility.
Another
limitation
is
that
terfenadine
is
known
to
be
proarrhythmic
and
it
inhibits
I
Na
-
B
'
Cad
,
and
IK
r
.
25
'
26
Therefore,
multiple-channel
block
does
not
always
result
in
a
compound
to.
with
low
risk
of
proarrhythmic.
CONCLUSIONS
HBI-3000
modestly
increases
APD
mainly
by
I
Kr
current
inhibition
in
human
ventricular
myocytes.
These
effects
are
self-limited
due
to
their
dominant
I
Nad
,
and
I
ca
-
L
,
blockade
actions,
which
may
explain
why
HBI-3000
exerts
a
minimal
use-dependent
prolongation
of
APD
and
antiar-
rhythmic
action
with
possible
low
proarrhythmic
risk.
REFERENCES
1.
Jurkiewicz
NK,
Sanguinetti
MC.
Rate-dependent
prolongation
of
cardiac
action
potentials
by
a
methanesulfonanilide
class
DI
antiarrhythmic
agent.
Specific
block
of
rapidly
activating
delayed
rectifier
K+
current
by
dofetilide.
Circ
Res.
1993;72:75-83.
2.
Singh
BN,
Wadhani
N.
Antiarrhythmic
and
proarrhythmic
properties
of
QT-prolonging
antianginal
drugs.
J
Cardiovasc
Pharmacol
Ther.
2004;
9(Suppl
1):S85-S97.
3.
Hafner
D,
Berger
F,
Borchard
U,
et
al.
Electrophysiological
character-
ization
of
the
class
HI
activity
of
sotalol
and
its
enantiomers.
New
interpretation
of
use-dependent
effects.
Arzneimittelforschung.
1988;38:
231-236.
4.
Tande
PM,
Bjornstad
H,
Yang
T,
et
al.
Rate-dependent
class
HI
antiarrhythmic
action,
negative
chronotropy,
and
positive
inotropy
of
a
novel
lk
blocking
drug,
UK-68,798:
potent
in
guinea
pig
but
no
effect
in
rat
myocardium.
J
Cardiovasc
Pharmacol.
1990;16:401-410.
5.
Gwilt
M,
Arrowsmith
JE,
Blackburn
KJ,
et
al.
UK-68,798:
a
novel,
potent
and
highly
selective
class
11I
antiarrhythmic
agent
which
blocks
potassium
channels
in
cardiac
cells.
.1
-
Pharmacol
Exp
Ther.
1991;256:318-324.
6.
Hondeghem
LM,
Snyders
DJ.
Class
DI
antiarrhythmic
agents
have
a
lot
of
potential
but
a
long
way
to
go.
Reduced
effectiveness
and
dangers
of
reverse
use
dependence.
Circulation.
1990;81:686-690.
7.
Waldo
AL,
Camm
AJ,
deRuyter
H,
et
al.
Effect
of
d-sotalol
on
mortality
in
patients
with
left
ventricular
dysfunction
after
recent
and
remote
myocardial
infarction.
The
SWORD
Investigators.
Survival
With
Oral
d-Sotalol.
Lancet.
1996;348:7-12.
8.
Singh
SN,
Fletcher
RD,
Fisher
SG,
et
al.
Amiodarone
in
patients
with
congestive
heart
failure
and
asymptomatic
ventricular
arrhythmia.
Survival
Trial
of
Antiarrhythmic
Therapy
in
Congestive
Heart
Failure.
N
Engl
.1
-
Med.
1995;333:77-82.
Cairns
JA,
Connolly
SJ,
Roberts
R,
et
al.
Randomised
trial
of
outcome
after
myocardial
infarction
in
patients
with
frequent
or
repetitive
ventricular
premature
depolarisations:
CAMIAT.
Canadian
Amiodarone
Myocardial
Infarction
Arrhythmia
Trial
Investigators.
Lancet.
1997;349:
675-682.
Julian
DG,
Camm
AJ,
Frangin
G,
et
al.
Randomised
trial
of
effect
of
amiodarone
on
mortality
in
patients with
left-ventricular
dysfunction
after
recent
myocardial
infarction:
EMIAT.
European
Myocardial
Infarct
Amiodarone
Trial
Investigators.
Lancet.
1997;349:667-674.
11.
Maltsev
VA,
Sabbah
FIN,
Undtxwinas
AI.
Late
sodium
current
is
a
novel
target
for
amiodarone:
studies
in
failing
human
myocardium.
.1
-
Mol
Cell
Cardiol.
2001;33:923-932.
12.
Antzelevitch
C,
Belardinelli
L,
Zygmunt
AC,
et
al.
Electrophysiological
effects
of
ranolazine,
a
novel
antianginal
agent
with
antiarrhythmic
properties.
Circulation.
2004;110:904-910.
13.
Lee
JY,
Lucchesi
BR.
Antifibrillatory
actions
of
HBI-3000
in
the
conscious
canine
model
of
sudden
cardiac
death.
FASEB
J
2009;23:613.
14.
Guo
D,
Zhao
X,
Wu
Y,
et
al.
L-type
calcium
current
reactivation
contributes
to
arrhythmogenesis
associated
with
action
potential
tri-
angulation.
J
Cardiovasc
Electrophysiol.
2007;18:196-203.
Guo
D,
Young
L,
Patel
C,
et
al.
Calcium-activated
chloride
current
contributes
to
action
potential
alternations
in
left
ventricular
hypertrophy
rabbit.
Am
.1
-
Physiol
Heart
Circ
Physiol.
2008;295:H97-H104.
Haverkamp
W,
Breithardt
G,
Camm
AJ,
et
al.
The
potential
for
QT
prolongation
and
pro-arrhythmia
by
non-anti-arrhythmic
drugs:
clinical
and
regulatory
implications.
Report
on
a
Policy
Conference
of
the
European
Society
of
Cardiology.
Cardiovasc
Res.
2000;47:219-233.
17.
Antzelevitch
C,
Shimizu
W.
Cellular
mechanisms
underlying
the
long
QT
syndrome.
Curr
Opin
Cardiol.
2002;17:43-51.
18.
Yap
YG,
Camm
AJ.
Drug
induced
QT
prolongation
and
torsades
de
pointes.
Heart.
2003;89:1363-1372.
19.
Farkas
A,
Lepran
I,
Papp
JG.
Proarrhythmic
effects
of
intravenous
quinidine,
amiodarone,
d-sotalol,
and
almokalant
in
the
anesthetized
rabbit
model
of
torsade
de
pointes.
J
Cardiovasc
Pharmacol.
2002;39:
287-297.
20.
Duff
HJ,
Gault
NJ.
Mexiletine
and
quinidine
in
combination
in
an
ischemic
model:
supra-additive
antiarrhythmic
and
electrophysiologic
actions.
J
Cardiovasc
Pharmacol.
1986;8:847-857.
21.
Duff
HJ,
Mitchell
LB,
Manyari
D,
et
al.
Mexiletine-quinidine
combination:
electrophysiologic
correlates
of
a
favorable
antiarrhythmic
interaction
in
humans.
.1
-
Am
Coll
Cardiol.
1987;10:1149-1156.
22.
Hohnloser
SH,
Klingenheben
T,
Singh
BN.
Amiodarone-associated
proarrhythmic
effects.
A
review
with
special
reference
to
torsade
de
pointes
tachycardia.
Ann
Intern
Med.
1994;121:529-535.
23.
Shimizu
W,
McMahon
B,
Antzelevitch
C.
Sodium
pentobarbital
reduces
transmural
dispersion
of
repolarization
and
prevents
torsades
de
Pointes
in
models
of
acquired
and
congenital
long
QT
syndrome.
J
Cardiovasc
Electrophysiol.
1999;10:154-164.
24.
Sager
PT,
Uppal
P,
Follmer
C,
et
al.
Frequency-dependent
electrophys-
iologic
effects
of
amiodarone
in
humans.
Circulation.
1993;88:
1063-1071.
25.
Crumb
WJ,
Wible
B,
Arnold
DJ,
et
al.
Blockade
of
multiple
human
cardiac
potassium
currents
by
the
antihistamine
terfenadine:
possible
mechanism
for
terfenadine-associated
cardiotoxicity.
Mol
Pharmacol.
1995;47:181-190.
26.
Lu
Y,
Wang
Z.
Terfenadine
block
of
sodium
current
in
canine
atrial
myocytes.
J
Cardiovasc
Pharmacol.
1999;33:507-513.
ACKNOWLEDGMENTS
15.
The
authors
thank
Dr.
Kenneth
Margulies,
Department
of
Medicine,
University
of
Pennsylvania,
for
his
support
and
16.
for
providing
the
human
tissues.
©
2011
Lippincott
Williams
&
Wilkins
www.jcvp.org
I
85