U.S. patent application number 17/512794 was filed with the patent office on 2022-02-17 for treatment of cardiac disorders by blocking sk4 potassium channel.
This patent application is currently assigned to Ramot at Tel-Aviv University Ltd.. The applicant listed for this patent is Ramot at Tel-Aviv University Ltd.. Invention is credited to Bernard ATTALI, Asher PERETZ, David WEISBROD.
Application Number | 20220047557 17/512794 |
Document ID | / |
Family ID | 1000005940928 |
Filed Date | 2022-02-17 |
United States Patent
Application |
20220047557 |
Kind Code |
A1 |
WEISBROD; David ; et
al. |
February 17, 2022 |
TREATMENT OF CARDIAC DISORDERS BY BLOCKING SK4 POTASSIUM
CHANNEL
Abstract
Methods of inducing bradycardia (slowing a heart rate) in a
subject in need thereof, treating a medical condition in which
inducing bradycardia (slowing a heart rate) is desirable or
beneficial in a subject in need thereof and/or treating a medical
condition associated with cardiac arrhythmia, are provided. The
methods are effected by blocking SK4 channel in SAN cell of the
subject and/or by administering to the subject a therapeutically
effective amount of a blocker of an SK4 channel. A method of
identifying candidate compounds for treating an arrhythmic cardiac
disorder, by identifying compounds that reduce a pacing rate of the
SAN cells is also provided.
Inventors: |
WEISBROD; David; (Tel-Aviv,
IL) ; ATTALI; Bernard; (Tel-Aviv, IL) ;
PERETZ; Asher; (Tel-Aviv, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ramot at Tel-Aviv University Ltd. |
Tel-Aviv |
|
IL |
|
|
Assignee: |
Ramot at Tel-Aviv University
Ltd.
Tel-Aviv
IL
|
Family ID: |
1000005940928 |
Appl. No.: |
17/512794 |
Filed: |
October 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15850000 |
Dec 21, 2017 |
11166940 |
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17512794 |
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62437783 |
Dec 22, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4174 20130101;
G01N 33/5061 20130101; A61P 9/06 20180101; G01N 33/5044 20130101;
G01N 33/502 20130101 |
International
Class: |
A61K 31/4174 20060101
A61K031/4174; G01N 33/50 20060101 G01N033/50; A61P 9/06 20060101
A61P009/06 |
Claims
1. A method of identifying a candidate compound for treating an
arrhythmic cardiac disorder in a subject, the method comprising:
contacting a compound identified as a blocker of SK4 potassium
channel with SAN cells; and determining if the compound reduces a
pacing rate of said SAN cells, wherein a compound that reduces a
pacing rate of said SAN cells is identified as a candidate compound
for treating an arrhythmic cardiac disorder.
2. The method of claim 1, wherein a compound is identified as a
blocker of SK4 potassium channel by: contacting the compound with
cells expressing SK4 potassium channel; and determining if a SK4
current amplitude is reduced upon said contacting, wherein a
compound that causes a reduction in said SK4 current amplitude upon
said contacting is identified as a blocker of a SK4 channel.
3. The method of claim 2, wherein said cells expressing SK4
potassium channels are transfected cells ectopically expressing
said channels.
4. The method of claim 1, wherein contacting the compound with said
SAN cells is effected in vitro.
5. The method of claim 4, wherein said SAN cells are obtained from
induced pluripotent stem cells-derived pacemaker cells and/or from
a subject suffering from an arrhythmic cardiac disorder.
6. The method of claim 5, wherein said subject is a post-natal
subject.
7. The method of claim 1, wherein a compound identified as a
candidate compound for treating the arrhythmic cardiac disorder is
administered to a subject suffering from an arrhythmic cardiac
disorder to thereby determine an effect of the compound on a heart
rate of the subject.
8. The method of claim 7, wherein said subject is a post-natal
subject.
9. The method of claim 1, wherein the arrhythmic cardiac disorder
is an atrial arrhythmia.
10. The method of claim 1, wherein the arrhythmic cardiac disorder
is a ventricular arrhythmia.
11. The method of claim 1, wherein the arrhythmic cardiac disorder
is associated with CPVT.
12. The method of claim 1, wherein said subject is a human subject.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 15/850,000 filed on Dec. 21, 2017, which claims the
benefit of priority under 35 USC .sctn. 119(e) of U.S. Provisional
Patent Application No. 62/437,783, filed on Dec. 22, 2016. The
contents of the above applications are all incorporated by
reference as if fully set forth herein in their entirety.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention, in some embodiments thereof, relates
to therapy and, more particularly, but not exclusively, to methods
of treating cardiac disorders, such as cardiac arrhythmia, and/or
of inducing bradycardia, by blocking the Ca.sup.2+-activated
potassium channel SK4.
[0003] The cardiac sinoatrial node (SAN) pacemaker arises from its
ability to generate a spontaneous and cyclical electric signal that
is orchestrated by a cohort of different ion channels. The SAN
pacemaker automaticity is essential for the proper heart
contraction.
[0004] Proper function of the cardiac pacemaker is a critical
feature of heart physiology. Around 75 times per minute, the
sinoatrial node (SAN) produces different ionic currents. The result
of those small currents is the generation of an electrical
stimulation, which cyclically and regularly propagates through the
conductive system (atrioventricular node, His bundles, Purkinje
fibers) to the chambers (right and left auricles, right and left
ventricles), leading to the heart contraction.
[0005] Currently known medications for treating cardiac arrhythmia
include .beta.1-adrenergic blockers (also known as .beta.-blockers
or beta blockers) and calcium (Ca.sup.2+) channel blockers. While
.beta.1-adrenergic blockade is a very common strategy used for the
treatment of several types of cardiopathies, including arrhythmia,
the response to .beta.-blockers often declines with time because of
an "adrenergic escape" phenomenon (AE). For instance, between 31 to
39% of the patients suffering from chronic heart failure develop AE
(see, Frankenstein L et al., Eur J Heart Fail. 2009). Ca.sup.2+
channel blockers, although very effective in mice, have a limited
benefit in humans, even when combined with .beta.-blockers.
[0006] While the SAN has been discovered more than a century ago,
the molecular mechanism of the "pacemaker ionic currents" remains
highly controversial and debated.
[0007] For ethical reasons, studies on SAN cells have been
performed mostly in small animals (rodents, rabbits), which display
very different cardiac characteristics compared with human (higher
heart rhythm, different calcium regulations or protein
kinetics).
[0008] KCa3.1 blockers such as clotrimazole, the structurally
related TRAM-34, and others, have been described in the art as
potential candidates for treating a variety of conditions,
including, for example, sickle cell anemia, asthma, autoimmune and
cardiovascular diseases. TRAM-34 was shown to prevent MOG induced
autoimmune encephalomyelitis, anti-collagen antibody induced
arthritis, and trinitrobenzene sulfonic acid-induced colitis in
mice, renal fibrosis following unilateral ureteral obstruction in
mice and rats, angiogenesis in the mouse matrigel plug assay,
atherosclerosis development in ApoE-/- mice84, as well as
angioplasty induced intimal smooth muscle hyperplasia (restenosis)
in rats and pigs. KCa3.1 blockade has further been found to reduce
microglia activation and thus curb inflammatory responses and
reduce neuronal damage in models of ischemic stroke, traumatic
brain injury, optic nerve transection, and traumatic spinal cord
injury. KCa3.1 has been recognized in the art as an attractive
pharmacological target for indications such as post-angioplasty
restenosis, atherosclerosis, inflammatory bowel disease, autoimmune
encephalomyelitis, immunosuppression and ischemic stroke. See, for
example, reviews by Wulff et al. in J Cardiovasc Pharmacol. 2013
February; 61(2): 102-112. doi:10.1097/FJC.0b013e318279ba20; and in
Expert Rev Clin Pharmacol. 2010 May; 3(3): 385-396.
[0009] Weisbrod et al., in Proc Natl Acad Sci USA 110, E1685-1694
(2013), investigated the cardiac pacemaker process in human
embryonic stem cells-derived cardiomyocytes (hESC-CMs), a cellular
model which mimics the cardiac cells of the primitive heart during
development. In those cells, the currents involved in the pacemaker
mechanism were investigated, and, using biochemical,
electrophysiological and pharmacological techniques, the
intermediate Ca.sup.2+-activated potassium channel (IK.sub.Ca/SK4,
KCa3.1) was identified as a target in the heart pacemaker mechanism
(see, Background Art FIGS. 1A-D, further discussed
hereinafter).
[0010] Catecholaminergic polymorphic ventricular tachycardia
(abbreviated herein throughout and in the art as CPVT) is an
inherited arrhythmogenic syndrome characterized by physical or
emotional stress-induced polymorphic ventricular tachycardia in
otherwise structurally normal hearts with a high fatal event rate
in untreated patients. CPVT comprises heterogeneous genetic
diseases, including mutations in ryanodine receptor type 2 (RyR2),
calsequestrin 2 (CASQ2), triadin or calmodulin5-11. The RyR2
mutations (CPVT1) are `gain of function` mutations while CASQ2
mutants (CPVT2) are `loss of function` mutations, which both lead
to diastolic Ca.sup.2+ leakage from the sarcoplasmic reticulum
(SR). This eventually produces local increases in cytosolic
Ca.sup.2+ that is extruded via the Na.sup.+--Ca.sup.2+ exchanger
NCX1 generating local depolarization with early- or
delayed-afterdepolarizations (EADs or DADs) that trigger premature
beats and fatal polymorphic ventricular tachycardia.
[0011] Recent studies performed in human induced pluripotent stem
cell-derived cardiomyocytes (hiPSC-CMs) from CPVT patients bearing
mutations in either CASQ2 (D307H) or RyR2 (M4109R) showed that
.beta.-adrenergic stimulation caused marked elevation in diastolic
Ca.sup.2+, DADs and oscillatory prepotentials [Itzhaki, I., et al.
J Am Coll Cardiol 60, 990-1000 (2012); Novak, A., et al. J Cell Mol
Med 19, 2006-2018 (2015); and Novak, A., et al. J Cell Mol Med 16,
468-482 (2012)]. Sinus bradycardia was also consistently described
in CPVT patients and in CPVT mouse models, suggesting that
sinoatrial node (SAN) dysfunction may reflect another primary
defect caused by CPVT mutations [Leenhardt, A., et al. Circulation
91, 1512-1519 (1995); Faggioni et al., Trends Cardiovasc Med 24,
273-278 (2014); Glukhov, A. V., et al. Eur Heart J 36, 686-697
(2015); Katz, G., et al. Heart Rhythm 7, 1676-1682 (2010); Neco,
P., et al. Circulation 126, 392-401 (2012); and Postma, A. V., et
al. J Med Genet 42, 863-870 (2005)].
[0012] Current therapies for CPVT are phenotype driven and include
exercise prohibition and .beta.1-adrenergic blockade. The options
in unresponsive patients include additional drugs, primarily
flecainide, or implanting a defibrillator (ICD) and sympathetic
denervation.
[0013] Additional Background art includes U.S. Patent Application
having Publication No. 2009/0306159; Ju et al., Cell Physiol
Biochem (2015) 36:1305-1315; Weisbrod et al., Acta Pharmacologica
Sinica (2016) 37: 82-97; Haron-Khun et al., Poster Presentation at
the 60th Annual Meeting of the Biophysical-Society Location: Los
Angeles, Calif. Date: Feb. 27-Mar. 2, 2016; Hanna Bueno et al.,
Poster Presentation at the 2016 ISPP meeting, Tel Aviv; Haron-Khun
et al., Poster Presentation at the 2016 ISPP meeting, Tel Aviv; and
Haron-Khun et al., EMBO Molecular Medicine (2017) 9, 415-429.
SUMMARY OF THE INVENTION
[0014] According to an aspect of some embodiments of the present
invention there is provided a method of inducing bradycardia (e.g.,
slowing a heart rate) in a subject in need thereof, the method
comprising administering to the subject a therapeutically effective
amount of a blocker of an SK4 channel.
[0015] According to an aspect of some embodiments of the present
invention there is provided a method of treating a medical
condition in which inducing bradycardia (e.g., slowing a heart
rate) is desirable or beneficial in a subject in need thereof, the
method comprising blocking a SK4 channel in SAN cells of the
subject.
[0016] According to some of any of the embodiments described
herein, the blocking comprises administering to the subject a
blocker of a SK4 channel.
[0017] According to an aspect of some embodiments of the present
invention there is provided a method of treating a medical
condition in which inducing bradycardia (e.g., slowing a heart
rate) is desirable or beneficial in a subject in need thereof, the
method comprising administering to the subject a blocker of a SK4
channel.
[0018] According to some of any of the embodiments described
herein, the medical condition is associated with cardiac
arrhythmia.
[0019] According to some of any of the embodiments described
herein, the medical condition is a cardiac disease or disorder.
[0020] According to some of any of the embodiments described
herein, the medical condition is an atrial disease or disorder
(e.g., atrial arrhythmia).
[0021] According to some of any of the embodiments described
herein, the medical condition is a ventricular disease or disorder
(e.g., ventricular arrhythmia).
[0022] According to some of any of the embodiments described
herein, the medical condition is CPVT.
[0023] According to an aspect of some embodiments of the present
invention there is provided a method of treating arrhythmia,
including atrial, ventricular and any other arrhythmia) in a
subject in need thereof, the method comprising administering to the
subject a blocker of a SK4 channel.
[0024] According to some of any of the embodiments described
herein, the subject is a human subject.
[0025] According to some of any of the embodiments described
herein, the subject is a post-natal subject.
[0026] According to some of any of the embodiments described
herein, the SK-4 channel blocker forms a part of a pharmaceutical
composition which further comprises a carrier.
[0027] According to some of any of the embodiments described
herein, the SK4 channel blocker is selected from the group
consisting of clotrimazole, TRAM-34, Senicapoc, and any of the
other SK4 channel blockers described herein (see, FIG. 11, for
non-limiting examples).
[0028] According to an aspect of some embodiments of the present
invention there is provided a method of identifying a candidate
compound for treating an arrhythmic cardiac disorder, the method
comprising:
[0029] contacting a compound identified as a blocker of SK4
potassium channel with SAN cells; and
[0030] determining if the compound reduces a pacing rate of the SAN
cells,
[0031] wherein a compound that reduces a pacing rate of the SAN
cells is identified as a candidate compound for treating an
arrhythmic cardiac disorder.
[0032] According to some of any of the embodiments described
herein, a compound is identified as a blocker of SK4 potassium
channel by:
[0033] contacting the compound with cells expressing SK4 potassium
channel; and
[0034] determining if a SK4 current amplitude is reduced upon the
contacting,
[0035] wherein a compound that causes a reduction in the SK4
current amplitude upon the contacting is identified as a blocker of
a SK4 channel.
[0036] According to some of any of the embodiments described
herein, the cells expressing SK4 potassium channels are transfected
cells ectopically expressing the channels.
[0037] According to some of any of the embodiments described
herein, contacting the compound with the SAN cells is effected in
vitro.
[0038] According to some of any of the embodiments described
herein, the SAN cells are obtained from induced pluripotent stem
cells-derived pacemaker cells and/or from a subject suffering from
an arrhythmic cardiac disorder.
[0039] According to some of any of the embodiments described
herein, a compound identified as a candidate compound for treating
an arrhythmic disorder is administered to a subject suffering from
an arrhythmic disorder to thereby determine an effect of the
compound on a heart rate of the subject.
[0040] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0041] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0042] In the drawings:
[0043] FIGS. 1A-1D (Background Art) present data obtained in
biochemical experiments revealing the existence of the SK4 protein
on cardiomyocytes (FIG. 1A), with upper panel presenting a Western
blot from young and older hESC-CMs lysates showing a 50 KDa band
corresponding to the SK4 channel and lower panel presenting
immunocytochemistry showing the expression of SK4 and the cardiac
marker .alpha.-actinin in the same single hESC-CMs;
electrophysiological characterization of the SK4 current in a
single cardiac hESC-CM (FIG. 1B); the chemical structure of
clotrimazole (FIG. 1C); and bar graphs showing the pharmacological
effects of clotrimazole on the cardiac pacing, with the spontaneous
electric activity of the cell recorded in the current clamp
configuration of the patch clamp technique before and during
exposure to 2 .mu.M clotrimazole.
[0044] FIGS. 2A-2C present data obtained in experiments conducted
in mice heart samples and human heart biopsia from patients. FIGS.
2A and 2B present biochemical experiments revealing the existence
of the SK4 channel on murine heart (FIG. 2A) and in human right
atrium and ventricle biopsia (FIG. 2B). FIG. 2A, left panel,
presents a reverse transcriptase PCR of the SK4 mRNA
(SAN=sinoatrial node; RA=right appendage; LA=left appendage;
RV=right ventricle, LV=left ventricle); and right panel presents
Western blot on murine lysates from the same heart areas. FIG. 2B
shows the presence of a SK4 channel at the transcript level (left
panel), and at the protein level (right panel). FIG. 2C presents
the pharmacological effects of clotrimazole on the pacemaker
activity of SAN cells, by showing the spontaneous electric activity
of the cell recorded in the current clamp configuration of the
patch clamp technique before and during exposure to 2 .mu.M
clotrimazole.
[0045] FIGS. 3A-3D present representative ECG recording following
intraperitoneal injection of vehicle (upper) and 20 mg/kg
clotrimazole (lower) in norma mice at rest, with sequential vehicle
and clotrimazole injections were performed on the same animal (FIG.
3A); data summary of heart rate at rest (upper; *P=0.0364, n=10)
and PR interval (lower; *P=0.0437, n=10) (FIG. 3B); and
representative ECG recording following intraperitoneal injection of
vehicle (upper) and 20 mg/kg clotrimazole (lower) in mice following
treadmill exercise (FIG. 3C); and data summary of heart rate at
rest (upper) and PR interval (lower) (FIG. 3D).
[0046] FIGS. 4A-4G present representative traces of hiPS-CMs
derived from normal (FIG. 4A) or CPVT2 (CASQ2 D307H) (FIG. 4B)
patients. Cells were held at -20 mV and a voltage ramp of 150 ms
from -90 to +60 mV was applied, as shown in FIG. 2B; the
TRAM-sensitive current calculated as a difference between the
current density measured at +60 mV with solution 1 alone and
solution 1+5 .mu.M TRAM-34 (n=7-9) (FIG. 4C); representative
Western blots of beating EBs lysates from normal and CPVT2 (CASQ2
D307H) patients showing immuno-reactive SK4 protein (about 50 KDa)
(FIG. 4D); representative traces of spontaneous APs recorded in
hiPS-CM derived from a normal individual (FIG. 4E; Left) and bar
graphs presenting data summary of pacing rate (FIG. 4E; Right);
representative traces of spontaneous APs recorded in hiPS-CM
derived from a CPVT2 (CASQ2 D307H) patients (FIG. 4F; Left); bar
graphs showing data summary of pacing rate FIG. 4F (Right); and
representative trace of a voltage-ramp protocol performed in
cardiomyocytes derived from human embryonic stem cells before and
after applying 5 .mu.M TRAM-34 (FIG. 4G), with cells held at -20 mV
and a voltage ramp of 150 ms from -90 to +60 mV was applied.
[0047] FIGS. 5A-5F present representative traces of SAN cells
isolated from WT (FIG. 5A) and CASQ2-D307H KI mice (FIG. 5B), with
cells held at -40 mV and a voltage ramp of 150 ms from -90 to +60
mV was applied; the TRAM-sensitive current calculated as in FIG. 4C
(n=8-12) (FIG. 5C); representative Western blots of SAN lysates
from WT (left) and CASQ2-D307H KI (right) mice showing the
immuno-reactive bands of SK4, CASQ2 and .beta.-actin proteins in
SAN, right and left atrial appendages, right and left ventricle
(FIG. 5D); representative traces of spontaneous APs recorded in
single SAN cell from WT mice (FIG. 5E; Left) and corresponding bar
graphs showing data summary of pacing rate (FIG. 5E; Right);
representative traces of spontaneous APs recorded in single SAN
cell from CASQ2 D307H KI mice (FIG. 5F; Left) and corresponding bar
graphs showing data summary of rate (n=6) (FIG. 5F; Right).
[0048] FIGS. 6A-6D present representative traces of spontaneous
calcium transients recorded ex vivo in intact SAN tissue
preparations from WT mice (FIG. 6A; Left) and a corresponding data
summary of calcium transient rate (FIG. 6A; Right); representative
traces of different types of calcium transient abnormalities
recorded in intact SAN from CASQ2 D307H KI mice, termed as "local
Ca.sup.2+ release" (FIG. 6B; upper left), "double humped
transients" (FIG. 6B; upper right), "large-stored released
Ca.sup.2+ waves" (FIG. 6B; lower left) and "calcium alternans"
(FIG. 6B; lower right); representative trace of spontaneous calcium
transients recorded from intact SAN of CASQ2 D307H KI (FIG. 6C) and
data summary of the arrhythmic calcium transients in SAN from CASQ2
D307H KI under baseline conditions, following exposure to 100 nM
isoproterenol and 100 nM isoproterenol+2 .mu.M TRAM-34 (FIG.
6D).
[0049] FIGS. 7A-7F present representative ECG recording following
intraperitoneal injection of vehicle (upper) and 20 mg/kg TRAM-34
(lower) in WT mice at rest (FIG. 7A) and corresponding data summary
of heart rate (Paired t-test; ***P=0.0003, n=10) and PR interval
(Paired t test; ***P=0.0004, n=10) in WT mice at rest (FIG. 7B);
representative ECG recording following IP injection of vehicle
(upper) and 20 mg/kg TRAM-34 (lower) in CASQ2-D307H KI mice at rest
(FIG. 7C) and corresponding data summary of heart rate (Paired
t-test; ***P<0.0001, n=12) and PR interval (Paired t-test;
***P=<0.0001, n=12) in CASQ2-D307H KI mice at rest (FIG. 7D);
representative ECG recording following IP injection of vehicle
(upper) and 20 mg/kg TRAM-34 (lower) in CASQ2 KO mice at rest
showing that TRAM-34 produced similar effects as in CASQ2-D307H KI
mice (FIG. 7E) and corresponding data summary of heart rate (Paired
t-test; **P=0.004, n=7 mice) and PR interval (Paired t-test;
**P=0.0041, n=7) in CASQ2 KO mice at rest (FIG. 7F).
[0050] FIGS. 8A-8F present representative ECG recording following
intraperitoneal injection of vehicle (FIG. 8A; upper) and 20 mg/kg
TRAM-34 (FIG. 8A; lower) in WT mice during treadmill exercise; and
corresponding data summary of heart rate (Paired t-test;
***P=0.001, n=10) and PR interval (Paired t-test; ***P=0.0005,
n=10) in WT mice during exercise (FIG. 8B); representative ECG
recording following IP injection of vehicle (FIG. 8C; upper) and 20
mg/kg TRAM-34 (FIG. 8C; lower) in CASQ2-D307H KI mice during
treadmill exercise; and corresponding data summary of heart rate
(Paired t-test; ***P=0.0004, n=11) and PR interval (Paired t-test;
**P=0.0099, n=9) in CASQ2-D307H KI mice during exercise (FIG. 8D);
representative ECG recording following IP injection of vehicle
(FIG. 8E; upper) and 20 mg/kg TRAM-34 (FIG. 8E; lower) in CASQ2 KO
mice during exercise. Arrhythmias such as NSVT were suppressed by
TRAM-34 injection; and corresponding data summary of heart rate
(Paired t-test; *P=0.0165, n=7) and PR interval (Paired t-test;
**P=0.0042, n=7) in CASQ2 KO mice during exercise (FIG. 8F).
[0051] FIGS. 9A-9D present representative ECG recording following
IP injection of vehicle (FIG. 9A; upper) and 20 mg/kg clotrimazole
(FIG. 9A; lower) in CASQ2-D307H KI mice at rest; and corresponding
data summary of heart rate (Paired t-test; *P=0.0260, n=7) and PR
interval (n=7) in CASQ2-D307H KI mice at rest (FIG. 9B);
representative ECG recording following IP injection of vehicle
(upper) and 20 mg/kg clotrimazole (FIG. 9C; lower) in CASQ2 KO mice
at rest showing that clotrimazole produced similar effects as in
CASQ2-D307H KI mice; and corresponding data summary of heart rate
(Paired t-test; **P=0.0078, n=7) and PR interval (Paired t-test;
*P=0.0111, n=7) in CASQ2 KO mice at rest (FIG. 9D).
[0052] FIGS. 10A-10D present representative ECG recording following
IP injection of vehicle (FIG. 10A; upper) and 20 mg/kg clotrimazole
(FIG. 10A; lower) in CASQ2-D307H KI mice during treadmill exercise;
and corresponding data summary of heart rate (Paired t-test;
**P=0.004, n=7) and PR interval (Paired t-test; *P=0.0305, n=7) in
CASQ2-D307H KI mice during treadmill exercise (FIG. 10B);
representative ECG recording following IP injection of vehicle
(FIG. 10C; upper) and 20 mg/kg clotrimazole (FIG. 10C; lower) in
CASQ2 KO mice during exercise; and corresponding data summary of
heart rate (Paired t-test; **P=0.0037, n=7) and PR interval (Paired
t-test; *P=0.0394, n=6) in CASQ2 KO mice during exercise (FIG.
10D).
[0053] FIG. 11 presents the chemical structures of exemplary SK4
blockers, taken from Wulff et al., Expert Rev Clin Pharmacol. 2010
May; 3(3): 385-396, which are usable in the context of some
embodiments of the present invention.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0054] The present invention, in some embodiments thereof, relates
to therapy and, more particularly, but not exclusively, to methods
of treating cardiac disorders, such as cardiac arrhythmia, and/or
of inducing bradycardia, by blocking the Ca.sup.2+-activated
potassium channel SK4.
[0055] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0056] Ca.sup.2+-activated potassium channel SK4 (also referred to
herein simply as SK4 channel) was previously identified in the
pacemaker of immature cardiac cells (cardiomyocytes derived from
human Embryonic stem cells) [Weisbrod et al., Proc Natl Acad Sci
USA. 2013; FIGS. 1A-1D].
[0057] The present inventors have uncovered that SK4 channels are
presented also in adult SAN cells and that blockers of SK4 channels
reversibly reduce the pacing rate of isolated SAN cells. In vivo
experiments in normal mice indicated that intraperitoneal injection
of a SK4 blocker produces bradycardic effects, revealed in ECG
recording by a significant increase in the PR interval. In
addition, a prolongation of the PR interval revealed that SK4
K.sup.+ channels also play a role in the heart conduction
system.
[0058] SK4 channels were also identified in human induced
pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) from
healthy and CPVT patients bearing a mutation in calsequestrin 2
(CASQ2-D307H) and in SAN cells from CASQ2-D307H knock-in (KI) mice.
In vivo ECG recording showed that intraperitoneal injection of an
SK4 channel blocker greatly reduced the ventricular arrhythmic
features of CASQ2-D307H KI and CASQ2 knockout mice at rest and
following exercise.
[0059] FIGS. 2A-C present data showing the existence of SK4 channel
also in adult heart cells, and the pharmacological effects of
clotrimazole on the pacemaker activity of adult SAN cells.
[0060] FIGS. 3A-D present data obtained in in vivo studies in mice,
showing the effect of clotrimazole intraperitoneal injection on the
mice heart rate following treadmill exercise and at rest.
[0061] FIGS. 4A-G present data showing the effect of TRAM-34 on the
pacing rate of hiPS-CMs derived from CPVT2 patients.
[0062] FIGS. 5A-F and 6A-D present data showing the effect of
TRAM-34 on SAN cells isolated from WT and CASQ2-D307H KI mice
[0063] FIGS. 7A-F, 8A-F, 9A-D and 10A-D, and Table 1, present data
obtained in in vivo studies in CASQ2-D307H KI and WT mice, showing
the effect of TRAM-34 intraperitoneal injection on the heart rate
and PR intervals at rest and during exercise.
[0064] The data presented herein demonstrate the pivotal role of
SK4 Ca.sup.2+-activated K.sup.+ channels in adult pacemaker
function, indicating that these channels are therapeutic targets
for the treatment of cardiac ventricular arrhythmias such as CPVT
and other cardiac and/or arrhythmia associated disorders.
[0065] The results presented herein clearly identify the
SK4/IK.sub.Ca channel as a therapeutic target involved in the adult
cardiac pacemaker mechanism.
[0066] The results presented herein show that the exemplary SK4
blockers clotrimazole and TRAM-34 exhibit a bradycardic effect,
while elongating the PR interval and the refractory period between
two heartbeats (effect in the Atrioventricular node manifested by
an increase of the PR interval), like .beta.1-adrenergic or
Ca.sup.2+ channel blockers. This effect was demonstrated at the
cellular level in human and mice cardiac cells and also in
vivo.
[0067] The data presented herein primarily provide the first
evidence that SK4 channels are not only expressed in spontaneously
beating hESC-CMs and hiPS-CMs but in adult SAN cells too.
Inhibition of SK4 K.sup.+ currents by TRAM-34 reduced the intrinsic
SAN firing rate. These data reveal that in SAN cells SK4 channels
are novel regulators of SAN automaticity.
[0068] The in vitro and in vivo data obtained with the SK4 channel
blockers, TRAM-34 and clotrimazole, on the pacing rate of isolated
SAN cells and on ECG parameters of WT mice, as presented herein,
indicate that activation of SK4 channels increases the SAN pacing
rate and their blockade reduces it. Both SK4 channel blockers
produced significant bradycardic effects during rest and following
treadmill exercise, without an indirect impact on autonomic input
to SA and AV nodes.
[0069] The results presented herein further indicate that SK4
channels play a critical role in normal and CPVT diseased pacemaker
function. These data indicate that SK4 channel blockers could be
beneficially utilized in the management of CPVT patients' rhythm
disorders.
[0070] The data presented herein show that inhibition of SK4
K.sup.+ channels rescues in vitro the cardiac arrhythmias exhibited
by hiPS-CMs derived from CPVT2 patients carrying the CASQ2 D307H
mutation and by SAN cells isolated from CASQ2-D307H KI mice. Hence,
TRAM-34 markedly reduced the occurrence of DADs and abnormal
Ca.sup.2+ transients detected following exposure to the
.beta.-adrenergic agonist isoproterenol. SK4 channel blockers can
therefore protect from deleterious ventricular arrhythmic features
revealed by ECG in CASQ2-D307H KI and CASQ2 KO mice at rest and
after treadmill exercise.
[0071] Ventricular premature complexes, non-sustained and sustained
ventricular tachycardia were significantly reduced following a
single IP injection (20 mg/kg) of clotrimazole or TRAM-34. The SK4
channel blockers protected the CASQ2-D307H KI and CASQ2 KO mice
from harmful polymorphic ventricular tachycardia without being
pro-arrhythmic by themselves, since neither sinus arrest nor 2nd
order AV block were recorded in the animals, including WT mice.
[0072] Despite the blockade of SK4 channels, the functional
redundancy of Ca.sup.2+-activated K.sup.+ channels likely preserves
the delicate balance of inward and outward currents necessary for
normal pacemaking.
[0073] Due to their bradycardic effect and slowed AV conduction,
SK4 channel blockers are beneficial for preventing ventricular
tachycardia by prolonging the refractory period, similarly to
.beta.1-adrenergic or Ca.sup.2+ channel blockers, yet without
involving the "adrenergic escape" phenomenon.
[0074] The bradycardic effect and slowed atrioventricular node
conduction exhibited by SK4 channel blockers can therefore be
beneficially utilized for preventing ventricular tachycardia by
prolonging the refractory period, as an alternative to the
currently used .beta.1-adrenergic and Ca.sup.2+ channel blockers,
as well as in treating other cardiac arrhythmias of different
etiologies, non-arrhythmic cardiovascular disorders (cardiac
diseases), ventricular tachyarrhythmias in CPVT and possibly in
other arrhythmic pathologies of different etiologies such as the
long QT syndrome.
[0075] Embodiments of the present invention therefore relate to
methods employing blockers of a SK4 channel. Embodiments of the
present invention also relate to methods of screening and
identifying lead candidates usable in the methods described herein,
by determining blockade of a SK4 channel by the tested
compounds.
[0076] Hereinthroughout, the phrase "SK4 channel" and phrases used
herein interchangeably therewith, describe the
intermediate-conductance calcium-activated potassium channel
K.sub.Ca3.1, which is also referred to in the art as IK1 channel or
SK4 channel.
[0077] SK4 Channel Blockers:
[0078] Herein, the terms "SK4 channel blocker", "blocker of SK4
channel", "an agent that blocks SK4 channel", and "an agent that
inhibits or inactivates SK4 channel", and grammatical diversions
thereof, are used interchangeably, and describe an agent that
blocks the SK4 channel and thus inhibits its function as a channel
of potassium ions (a channel that allows potassium ions to cross
the cell membrane).
[0079] Inhibition and/or inactivation of SK4 channel, as used
herein, can be manifested as reducing the function of the channel
by at least 10%, preferably by at least 20%, or at least 30%, or at
least 40%, or at least 50%, or at least 60%, or at least 70%, or at
least 80%, or at least 90% and in some embodiments, by 05%, 96%,
97%, 98%, 99% or even 100%.
[0080] Reduction in the function of SK4 channel is manifested, for
example, by a reduction in the electrical current produced by the
channel as is further described hereinafter.
[0081] The term "SK4 channel" in the context of blockers is meant
to include SK4 channels as described herein throughout and in the
art.
[0082] Determining if a compound is a blocker of SK4 potassium
channel can be performed using methods known in the art, some are
described hereinafter in the context of the screening method. Other
methods are readily recognized by those skilled in the art.
[0083] Typically, a blocker of SK4 channel is a competitive
antagonist that binds to the channel and prevents it from being
activated by calcium ions, or is an agent that reduces the
concentration of calcium ions that bind to the channel.
[0084] Any agent that blocks an SK4 channel is contemplated
according to the present embodiments. The agent can be a
biomolecule (e.g., a protein, a peptide (such as toxin), a nucleic
acid construct, etc.) or a small molecule, and is preferably a
small molecule.
[0085] In some embodiments, a SK4 blocker is selective towards SK4
channel. In some embodiments, a SK4 blocker is capable of blocking
other calcium ion-activated channel and/or or a potassium
channel.
[0086] In some embodiments, a SK4 blocker binds to the inner pore
of the SK4 channel. Alternatively, the SK4 blocker binds to other
sites of the SK4 channel, for example, the calcium/calmodulin
binding pocket, as well as other sites.
[0087] Representative examples of SK4 channel blockers include, but
are not limited to, the following:
[0088] Clotrimazole (see also FIGS. 1C and 11):
##STR00001##
[0089] TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; a
structural isomer of Clotrimazole; see, FIG. 11):
##STR00002##
[0090] ICA-17043
(4-fluoro-.alpha.-(4-fluorophenyl)-.alpha.-phenyl-benzeneacetamide;
also known as Senicapoc.RTM., see, FIG. 11):
##STR00003##
[0091] Compounds (4)-(10) as depicted in FIG. 11;
[0092] Triarylmethans as described in WO 97/34589;
[0093] Fluorinated triphenyl acetamides as described in
McNaughton-Smith et al., J Med Chem. 2008 Feb. 28;
51(4):976-82;
[0094] 11-Phenyl-diazepines and Diphenylindanones such as described
in U.S. Pat. Nos. 6,992,079 and 7,342,038;
[0095] 4-Phenyl-4H-pyrans and related cyclohexasienes as described
in Urbahns et al. Bioorg Med Chem Lett. 2003 Aug. 18;
13(16):2637-9; and Urbahns et al., Bioorg Med Chem Lett. 2005 Jan.
17; 15(2):401-4;
[0096] Cylcohexadiene lactones as described in DE-9619612645
1997;
[0097] The antimalarial agent quinine;
[0098] the vasodilator cetiedil;
[0099] the L-type Ca channel blockers nifedipine;
[0100] and nitrendipine.
[0101] Exemplary toxins which are known as SK4 blockers include,
but are not limited to, the scorpion toxin charybdotoxin (ChTX);
Maurotoxin (MTX); and the ChTX analog ChTX-Glu.sup.32.
[0102] Any other blockers of SK4 channel as defined herein are also
contemplated.
[0103] It is expected that during the life of a patent maturing
from this application many relevant SK4 blockers will be developed
and the scope of the term "blocker of SK4 channel" is intended to
include all such new technologies a priori.
[0104] Therapeutic Applications:
[0105] According to an aspect of some embodiments of the present
invention there is provided a method of inducing bradycardia
(slowing a heart rate) in a subject in need thereof.
[0106] The term "bradychardia", which is also known as
"bradyarrhythmia", as used herein and in the art, describes a slow
heart rate in a subject compared to a normal, average, heart rate
of a healthy subject of the same age and species, or compared to a
heart rate associated with a subject's medical condition.
[0107] Bradychardia can be determined, for example, by
electrocardiography (ECG).
[0108] The term "bradychardia" encompasses atrioventricular nodal
bradycardia (AV junction rhythm), which usually appears on an ECG
with a normal QRS complex accompanied with an inverted P wave
either before, during, or after the QRS complex, and ventricular
bradycardia, which is manifested by a slow heart rate (e.g., of
less than 50 BPM in human adult), which usually appears as
imbalanced relationship between P waves and QRS complexes in ECG.
By "inducing bradycardia" are encompassed slowing a heart rate of a
subject (e.g., reducing the heart rate of the subject by, for
example, at least 5% or at least 10% or at least 20% or at least
30%, or at least 40% or at least 50%, compared to the heart rate of
the same subject before treatment), and/or regulating an increased
heart rate such that the heart rate of the subject is within the
acceptable range of a healthy subject (e.g., of the same age and
other parameters), and/or decreasing the sinus rate (by, for
example, at least 5% or at least 10% or at least 20% or at least
30%, or at least 40% or at least 50%, compared to the sinus rate of
the same subject before treatment) and/or elongating/prolonging the
PR interval (by, for example, at least 5% or at least 10% or at
least 20% or at least 30%, or at least 40% or at least 50%,
compared to the PR interval of the same subject before
treatment).
[0109] A "Sinus rate", which is also known and referred to in the
art as "sinus rhythm", can be defined by the morphology of P waves
in ECG.
[0110] "PR interval", which is also known and referred to in the
art as "PQ interval" can be defined as the period that extends from
the beginning of the P wave (the onset of atrial depolarization)
until the beginning of the QRS complex (the onset of ventricular
depolarization), in ECG.
[0111] According to some of any of the embodiments described
herein, inducing bradycardia is effected by blocking a SK4 channel
in the subject.
[0112] According to some of any of the embodiments described
herein, inducing bradycardia is effected by blocking a SK4 channel
in SAN cells of the subject.
[0113] According to an aspect of some embodiments of the present
invention there is provided a method of treating a medical
condition in which inducing bradycardia (slowing a heart rate) is
desirable or beneficial in a subject in need thereof, the method
comprising blocking a SK4 channel in SAN cells of the subject.
[0114] In some of any of the embodiments described herein, blocking
the SK4 channel comprises administering to the subject an effective
amount (e.g., a therapeutically effective amount) of a blocker of a
SK4 channel, as defined herein in any of the respective
embodiments.
[0115] In the context of these embodiments, an effective amount is
an amount sufficient to reduce or inhibit a function of a SK4
channel, as defined herein.
[0116] According to an aspect of some embodiments of the present
invention there is provided a method of reducing the firing of SAN
cells, the method comprising contacting SAN cells with a blocker of
SK4 channel.
[0117] In some embodiments, the contacting is effected in vitro,
and the SAN cells are isolated from a subject as described
herein.
[0118] In some embodiments, the contacting is effected in vivo, by
administering to a subject in need of firing SAN cells, an
effective amount (e.g., a therapeutically effective amount) of a
blocker of SK4 channel as defined herein in any of the respective
embodiments.
[0119] In the context of these embodiments, an effective amount is
an amount sufficient to reduce or inhibit a function of a SK4
channel, as defined herein.
[0120] In some embodiments, the SAN cells are human SAN cells. In
some embodiments, the SAN cells are of a human subject which is a
post-natal subject (e.g., an adult subject).
[0121] According to an aspect of some embodiments of the present
invention there is provided a method of inducing bradycardia
(slowing a heart rate) in a subject in need thereof, the method
comprising administering to the subject a blocker of a SK4 channel,
as defined herein in any of the respective embodiments.
[0122] Subjects in need of induction of bradychardia include, for
example, subjects suffering from a medical condition in which
inducing bradycardia (slowing a heart rate) is desirable or
beneficial, as described herein. According to an aspect of some
embodiments of the present invention there is provided a method of
treating a medical condition in which inducing bradycardia (slowing
a heart rate) is desirable or beneficial in a subject in need
thereof, the method comprising administering to the subject a
blocker of a SK4 channel, as defined herein in any of the
respective embodiments.
[0123] In some embodiments, the medical condition is a cardiac
disease or disorder, and in some embodiments, the medical condition
is a cardiac arrhythmia disease or disorder.
[0124] In some embodiments, the medical condition is associated
with cardiac arrhythmia.
[0125] In some embodiments, the method according any of the
respective embodiments can be used to treat cardiac disorders
characterized by abnormal cardiac rhythm, such as, for example,
cardiac arrhythmia.
[0126] In some embodiments, the medical condition is not directly
associated with cardiac arrhythmia.
[0127] In some embodiments, the medical condition is such that
requires a procedure which is advantageously performed while
slowing a heart rate of the subject, for example, a surgery that
involves interception of an organ or tissue of the cardiovascular
system or any other operation of the cardiovascular system. An
example is an open heart surgery.
[0128] In some embodiments, the medical condition is Myocardial
Infarction (MI).
[0129] Any other cardiac as well as non-cardiac diseases or
disorders or medical conditions in which slowing a heart rate is
beneficial are contemplated.
[0130] According to an aspect of some embodiments of the present
invention there is provided a method of treating cardiac arrhythmia
or a medical condition associated with cardiac arrhythmia in a
subject in need thereof, the method comprising administering to the
subject a blocker of a SK4 channel, as defined herein in any of the
respective embodiments.
[0131] As used herein the phrase "cardiac arrhythmia" refers to a
variation from the normal rhythm of the heart rate, for example,
tachycardia.
[0132] The cardiac arrhythmia can be a ventricular arrhythmia, an
atrial arrhythmia, a junctional arrhythmia and a heart block.
[0133] Medical conditions associated with atrial arrhythmia
include, but are not limited to, Premature atrial contractions
(PACs), Wandering atrial pacemaker, Atrial tachycardia, Multifocal
atrial tachycardia, Supraventricular tachycardia (SVT), Atrial
flutter, and Atrial fibrillation (Afib).
[0134] Medical conditions associated with junctional arrhythmia
include, but are not limited to, AV nodal reentrant tachycardia,
Junctional rhythm, Junctional tachycardia, and Premature junctional
contraction
[0135] Medical conditions associated with ventricular arrhythmia
include, but are not limited to, Premature ventricular contractions
(PVCs), sometimes called ventricular extra beats (VEBs), Premature
ventricular beats occurring after every normal beat are termed
"ventricular bigeminy", Accelerated idioventricular rhythm,
Monomorphic ventricular tachycardia, Polymorphic ventricular
tachycardia, Ventricular fibrillation, and Torsades de pointes.
[0136] Medical conditions associated with heart block include, but
are not limited to, AV heart blocks, which arise from pathology at
the atrioventricular node, including First degree heart block,
which manifests as PR prolongation, Second degree heart block,
including Type 1 Second degree heart block, also known as Mobitz I
or Wenckebach, and Type 2 Second degree heart block, also known as
Mobitz II, and Third degree heart block, also known as complete
heart block.
[0137] Exemplary medical conditions associated with cardiac
arrhythmia include, but are not limited to, atrial fibrillation,
ventricular fibrillation, conduction disorders, premature
contraction, and tachycardia.
[0138] Conduction disorders collectively encompass abnormal or
irregular progression of electrical pulses through the heart, which
cause a change in the heart rhythm. Conductions disorders are not
necessarily associated with arrhythmia but sometimes are the cause
of arrhythmia. Exemplary conductions disorders include, but are not
limited to, Bundle Branch Block, heart block, including first-,
second- and third-degree heart block, and long Q-T syndrome.
[0139] Premature contraction includes premature atrial contractions
and premature ventricular contractions.
[0140] Additional exemplary medical conditions associated with
arrhythmia include Adams-Stokes Disease (also called Stokes-Adams
or Morgangni), atrial flutter, which is usually found in patients
with: Heart failure, Previous heart attack, Valve abnormalities or
congenital defects, High blood pressure, Recent surgery, Thyroid
dysfunction, Alcoholism (especially binge drinking), Chronic lung
disease, Acute (serious) illness, Diabetes, after open-heart
surgery (bypass surgery), or atrial fibrillation; Sick Sinus
syndrome; sinus arrhythmia and Wolff-Parkinson-White (WPW)
syndrome.
[0141] In some of any of the embodiments described herein, the
cardiac disease or disorder is associated with tachycardia.
[0142] In some embodiment, a method as described herein is for
treating or preventing tachycardia.
[0143] The term "tachychardia", which is also known as
"tachyarrhythmia", as used herein and in the art, describes a fast
heart rate in a subject compared to a normal, average, heart rate
of a healthy subject of the same age and species, or compared to a
heart rate associated with a subject's medical condition.
[0144] Tachychardia can be determined, for example, by
electrocardiography (ECG), and encompasses a wide range of
conditions, as listed herein throughout.
[0145] In some embodiments, the tachycardia encompasses atrial and
Supraventricular tachycardia (SVT), including paroxysmal atrial
tachycardia (PAT) or paroxysmal supraventricular tachycardia
(PSVT); Sinus tachycardia, which can be associated with disorders
of that heart which interfere with the normal conduction system of
the heart, including, but not limited to, Lack of oxygen to areas
of the heart due to lack of coronary artery blood flow,
Cardiomyopathy in which the structure of the heart becomes
distorted, Medications, Illicit drugs such as cocaine, and
Sarcoidosis (an inflammatory disease affecting skin or other body
tissues).
[0146] In some embodiments, the tachycardia is a ventricular
tachycardia, a supraventricular tachycardia, atrial fibrillation,
AV nodal reentrant tachycardia (AVNRT), or a AV reentrant
tachycardia (AVRT).
[0147] In some embodiments, the cardiac disease or disorder is
CPVT, as described herein and in the art.
[0148] In some embodiments, the cardiac disease or disorder is a
long QT syndrome.
[0149] The subject to be treated according to some of any of the
embodiments of the present invention can be a mammal, preferably a
human being, including a baby, an infant, and an adult.
[0150] In some of any of the embodiments described herein, the
subject is a post-natal subject.
[0151] In some embodiments, the subject is afflicted by, or suffers
from, any of the medical conditions as described herein.
[0152] Tachycardia and bradycardia are defined in a subject in
accordance with acceptable heart rates defined as normal in
accordance with a subject's age.
[0153] According to an aspect of some embodiments of the present
invention there is provided a blocker of SK4 channel for use in
inducing bradycardia, or in treating any of the medical conditions
described herein.
[0154] According to an aspect of some embodiments of the present
invention there is provided a use of blocker of SK4 channel in the
manufacture of medicament for use in inducing bradycardia, or in
treating any of the medical conditions described herein.
[0155] In some of any of the embodiments described herein, the SK4
blocker can be used in combination with an additional active agent,
for example, an agent usable in treating a medical condition as
described herein.
[0156] In some embodiments the additional agent is a blocker of an
SK channel. In some embodiments, the additional agent is an
anti-arrhythmic agent (e.g., a beta blocker).
[0157] The SK4 blockers according to the present embodiments,
optionally in combination with one or more additional active
agent(s) as described herein, can be used (administered to a
subject) per se or can form a part of a pharmaceutical composition
that further comprises a carrier.
[0158] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0159] Herein the term "active ingredient" refers to the SK4
blocker as described herein.
[0160] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0161] In some embodiments, the agent is a fluid (e.g., liquid)
carrier and in some embodiments, the SK4 blocker is dissolvable,
dispersible or suspendable in the carrier.
[0162] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0163] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0164] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intracardiac, e.g., into the right or left
ventricular cavity, into the common coronary artery, intravenous,
intraperitoneal, intranasal, or intraocular injections.
[0165] Pharmaceutical compositions of some embodiments of the
invention may be manufactured by processes well known in the art,
e.g., by means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0166] Pharmaceutical compositions for use in accordance with some
embodiments of the invention thus may be formulated in conventional
manner using one or more physiologically acceptable carriers
comprising excipients and auxiliaries, which facilitate processing
of the active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0167] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0168] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0169] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0170] For administration by nasal inhalation, the active
ingredients for use according to some embodiments of the invention
are conveniently delivered in the form of an aerosol spray
presentation from a pressurized pack or a nebulizer with the use of
a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichloro-tetrafluoroethane or carbon
dioxide. In the case of a pressurized aerosol, the dosage unit may
be determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin for use in a dispenser
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0171] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0172] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0173] The pharmaceutical composition of some embodiments of the
invention may also be formulated in rectal compositions such as
suppositories or retention enemas, using, e.g., conventional
suppository bases such as cocoa butter or other glycerides.
[0174] Pharmaceutical compositions suitable for use in context of
some embodiments of the invention include compositions wherein the
active ingredients are contained in an amount effective to achieve
the intended purpose. More specifically, a therapeutically
effective amount means an amount of active ingredients effective to
prevent, alleviate or ameliorate symptoms of a medical condition
(e.g., as described herein) or prolong the survival of the subject
being treated.
[0175] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0176] Compositions of some embodiments of the invention may, if
desired, be presented in a pack or dispenser device, such as an FDA
approved kit, which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accommodated by a
notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling
approved by the U.S. Food and Drug Administration for prescription
drugs or of an approved product insert. Compositions comprising a
preparation of the invention formulated in a compatible
pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labeled for treatment of an indicated
condition, as is further detailed above.
[0177] A pharmaceutical composition as described herein can also
include one or more additional active agents as described
herein.
[0178] A pharmaceutical composition as described herein is also
referred to as a medicament.
[0179] Screening Method:
[0180] According to an aspect of some embodiments of the present
invention there is provided a method of identifying a candidate
compound for treating an arrhythmic cardiac disorder (cardiac
arrhythmia, as described herein in any of the respective
embodiments). The method, according to these embodiments,
comprises:
[0181] contacting a compound identified as a blocker of SK4
potassium channel with SAN cells; and determining if the compound
reduces a pacing rate of said SAN cells, wherein a compound that
reduces a pacing rate of said SAN cells is identified as a
candidate compound for treating an arrhythmic cardiac disorder.
[0182] According to some embodiments, identifying a compound as a
blocker of a SK4 potassium channel can be effected by:
[0183] contacting the compound with cells expressing SK4 potassium
channel; and
[0184] determining if a SK4 current amplitude is reduced upon said
contacting,
[0185] wherein a compound that causes a reduction in said SK4
current amplitude upon said contacting is identified as a blocker
of a SK4 channel.
[0186] In some embodiments, the cells expressing SK4 potassium
channels are transfected cells ectopically expressing a SK4
potassium channel (e.g., by means of cDNA encoding SK4 channel).
Alternatively, cells inherently expressing SK4 potassium channels
can be used.
[0187] In some embodiments, the contacting with cells expressing
SK4 potassium channel is effected in vitro.
[0188] In some embodiments, determining if a SK4 current amplitude
is reduced is effected by measuring the SK4 current amplitude, or
preferably, a change in the SK4 current amplitude, upon contacting
the compounds, compared with the SK4 current amplitude without
contacting the compound.
[0189] In some embodiments, identifying a compound as a blocker of
a SK4 channel is effected by screening a plurality of compounds,
and determining their effect on the SK4 channel, for example, by
measuring the SK4 current amplitude, or preferably, a change in the
SK4 current amplitude, upon contacting the compounds, compared with
the SK4 current amplitude without contacting the compound.
[0190] In some embodiments, compounds identified in this screening
as SK4 channel blockers are tested for their effect on the
pacemaking activity of SAN cells.
[0191] In some embodiments, a compound identified as a blocker of
SK4 channel is known as such and its effect on SAN cells is
determined without determining its blocking activity.
[0192] In some of any of the embodiments described herein,
contacting the compound with the SAN cells is effected in
vitro.
[0193] In some embodiments, the SAN cells are obtained from induced
pluripotent stem cells-derived pacemaker cells and/or from a
subject suffering from an arrhythmic cardiac disorder. The subject
can be a human subject, or an animal subject, and can be a
pre-natal or post-natal subject, preferably a post-natal
subject.
[0194] In some embodiments, once a compound is identified as
capable of reducing the pace rate of SAN cells, preferably in in
vitro screening as described herein, it is determined as a
candidate for treating a medical condition associated with
arrhythmia, as described herein.
[0195] In some embodiments, the candidate compound is administered
to a subject suffering from an arrhythmic disorder (e.g., cardiac
arrhythmia as described herein) to thereby determine an effect of
the compound on a heart rate of the subject.
[0196] In an exemplary screening method according to the present
embodiments, a plurality of compounds are tested for their
capability of exerting a blocking activity on SK4 channels
expressed ectopically (heterologous expression) in transfected CHO
cells.
[0197] The compounds identified as blockers of SK4 channels in
transfected cells are then tested in vitro in SAN cells from iPS
(induced pluripotent stem cells-derived pacemaker cells) from
control (healthy) and from CPVT patients or patients afflicted by
another arrhythmia disorder. Compounds which induce a bradycardia
effect on control SAN iPS cells, and which rescue the arrhythmic
features (notably DADs [delayed after depolarization]) in SAN cells
from patients suffering from CPVT or another arrhythmia disorder,
are identified as lead candidates for treating arrhythmia.
[0198] The term "treating" refers to inhibiting, preventing or
arresting the development of a pathology (disease, disorder or
condition) and/or causing the reduction, remission, or regression
of a pathology. Those of skill in the art will understand that
various methodologies and assays can be used to assess the
development of a pathology, and similarly, various methodologies
and assays may be used to assess the reduction, remission or
regression of a pathology.
[0199] As used herein, the term "preventing" refers to keeping a
disease, disorder or condition from occurring in a subject who may
be at risk for the disease, but has not yet been diagnosed as
having the disease.
[0200] As used herein, the term "subject" includes mammals,
preferably human beings at any age which suffer from the pathology.
Preferably, this term encompasses individuals who are at risk to
develop the pathology.
[0201] It is expected that during the life of a patent maturing
from this application many relevant methods for determining SK4
channel activity and/or SAN cells activity will be developed and
the scope of these terms is intended to include all such new
technologies a priori.
[0202] As used herein the term "about" refers to .+-.10% or
.+-.5%.
[0203] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0204] The term "consisting of" means "including and limited
to".
[0205] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0206] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0207] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0208] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0209] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0210] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0211] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0212] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
[0213] Materials and Methods
[0214] Animals:
[0215] SvEv mice (3-6 months old) homozygous for the CASQ2 D307H
mutation [CASQ2 D307H Knock-in (KI)] or for the off-frame exon 9
deletion [CASQ2.DELTA./.DELTA. knock-out (KO)] and matched
wild-type (WT) mice were used. Mice were maintained and bred in a
pathogen-free facility on regular rodent chow with free access to
water and 12-hour light and dark cycles. The procedures followed
for experimentation and maintenance of the animals were approved by
the Animal Research Ethics Committee of Tel Aviv University
(M-14-063) in accordance with Israeli law and in accordance with
the Guide for the Care and Use of Laboratory Animals (1996,
National Academy of Sciences, Washington, D.C.).
[0216] Human Induced-Pluripotent Stem Cell Culture and Cardiac
Differentiation:
[0217] Human induced pluripotent stem cells (hiPS) derived from
normal healthy individuals and from patients bearing the CASQ2
D307H mutation (CPVT2) were grown on mitomycin C-inactivated mouse
embryonic fibroblasts (MEF), in order to maintain them in an
undifferentiated state. The cells were maintained pluripotent in a
culture medium containing 80% DMEM F-12 (Biological Industries),
20% Knock Out SR (Invitrogen), 2 mM L-glutamine, 0.1 mM
.beta.-mercaptoethanol (Gibco), and 1% NEA (Gibco), supplemented
with 4 ng/mL bFGF (Invitrogen). The medium was replaced daily until
the colonies were ready to passage (every 4-5 days). For EBs
induction (d0), hiPS colonies were removed from their MEF feeder by
collagenase IV treatment and collected. After centrifugation, the
cells were resuspended in EBs medium containing 80% DMEM (Gibco),
20% FBS (Biological Industries), 1% NEA, and 1 mM L-Glutamine and
plated on 58-mm Petri dishes. After 7 d of culture in suspension,
EBs were plated on 0.1% gelatin-coated plates and checked daily
until a spontaneous beating activity was visible. Because CASQ2 is
lately expressed in hiPS-CMs, 25 days-old EBs were used. The
beating clusters were mechanically 279 dissected from EBs,
following a three-step dissociation protocol. The hiPS-CMs were
isolated and plated on Matrigel-coated glass coverslips (13 mm
diameter) in 24-well plates. The coverslips were then incubated at
37.degree. C., and a recovery period of 2 d was given before any
electrophysiological experiment was performed.
[0218] Mouse SAN Dissection and Cell Dissociation:
[0219] WT and CASQ2 D307H KI mice were anesthetized with
isofluorane and sacrificed by cervical dislocation. The heart was
rapidly removed and transferred into Tyrode solution containing
heparin. After the atria were pined and the superior and inferior
vena cava localized, the ventricles were removed. The SAN was
anatomically identified between the superior and inferior vena
cava, the crista terminals and the interatrial septum. The area was
cleaned, cut into small strips and washed into a Low Calcium
Solution, containing (in mM): 140 NaCl, 5.4 KCl, 0.5 MgCl.sub.2,
1.2 KH.sub.2PO.sub.4, 5 HEPES-NaOH, 50 taurine, 5.5 glucose (pH
6.9). The osmolarity was adjusted if needed to 315 mOsm.
[0220] The same solution, supplemented with 1 mg/ml albumin, 200
.mu.M CaCl.sub.2, collagenase Type I (Worthington) or liberase TH
(Roche), protease (Sigma) and elastase (Sigma) was used for
enzymatic digestion as previously described [See, Mesirca, P., et
al. Nat Commun 5, 4664 (2014)]. In this step, the tissue was gently
re-suspended with a polished Pasteur pipette in this solution for
9-13 minutes at 37.degree. C. SAN samples were then washed three
times in a modified "Kraftbruhe" solution, containing (in mM): 70
glutamic acid, 80 KOH, 20 KCl, 10 .gamma.-Hydroxybutyric acid
sodium salt, 10 KH.sub.2PO.sub.4, 10 HEPES-KOH, 10 taurine, 1 mg/ml
albumin, 0.1 EGTA-KOH (pH 7.2). The same solution was used to
re-suspend the single cells with a pipet by vigorous up and down,
between 3 to 5 minutes at 37.degree. C. Cells were then gradually
exposed to increasing concentrations of calcium, following a
"Ca.sup.2+ readaptation" protocol [Mesirca et al., 2014, supra].
Experiments were performed the same day at 33.degree. C.
[0221] Drugs:
[0222] Isoproterenol, clotrimazole and E-4031 were 301 purchased
from Sigma, while ZD-7288 and TRAM-34 from Tocris. For in-vivo
telemetric recordings, Tram-34 was solubilized into peanut oil,
while clotrimazole was prepared in peanut oil supplemented with 1%
ethanol.
[0223] Electrophysiology:
[0224] In all experiments, the coverslips were perfused at
33.degree. C. with an external solution containing (in mM): 140
NaCl, 4 KCl, 11 Glucose, 1.2 MgCl.sub.2, 1.8 CaCl.sub.2, 5.5 HEPES
titrated to pH 7.4 with NaOH and adjusted at 320 mOsm with
sucrose.
[0225] Whole-cell patch-clamp recordings were performed with an
Axopatch 700B amplifier (Molecular Devices) and pCLAMP 10.5
software (Molecular Devices).
[0226] Signals were digitized at 5 kHz and filtered at 2 kHz using
microelectrodes with resistances of 4-7 MS/were pulled from
borosilicate glass capillaries (Harvard Apparatus) and filled with
an intracellular solution containing (in mM): 130 KCl, 5 MgATP, 5
EGTA, 10 HEPES titrated to pH 7.3 with KOH and adjusted at 290 mOsm
with sucrose. Unless otherwise stated, internal free calcium
concentrations were 100 nM and 1 .mu.M for current-clamp and
voltage-clamp experiments, respectively and were titrated with EGTA
and CaCl.sub.2 using the MaxChelator software.
[0227] The spontaneous automaticity of isolated SAN cells was
recorded under perforated-patch conditions by adding 30 .mu.M
.beta.-escin49 to the intracellular solution containing (in mM):
130 KCl, 10 NaCl, 10 HEPES, 0.2 EGTA-KOH, 2 MgATP, 6.6
Phosphocreatine, 0.05 cAMP and 1 .mu.M free Ca.sup.2+ (pH 7.2). To
record SK4 K+ current, a voltage ramp protocol was applied. SAN and
hiPS-CMs were held at -40 mV and -20 mV, respectively to
substantially inactivate voltage-gated Na.sup.+ and Ca.sup.2+
currents. Cells were stepped from -90 mV to +60 mV for 150 ms.
Then, a cocktail (solution 1) containing (in mM) 0.3 cadmium, 0.025
ZD-7288 and 0.01 E-4031 was applied extracellularly to inhibit
residual L-type and T-type voltage-gated Ca2+ currents, I.sub.f and
the IKr currents, respectively. Subsequently, TRAM-34 (5 .mu.M) was
added to solution 1 to inhibit SK4 K.sup.+ currents, which were
defined as TRAM-34 sensitive currents. For voltage-clamp recording
of SAN cells, the intracellular solution was the same to that
described above for recording spontaneous automaticity.
[0228] Calcium Transient Measurements:
[0229] SAN tissue preparations were dissected ex vivo from WT and
CASQ2-D307H KI mice as described in Torrente, A. G., et al. Proc
Natl Acad Sci USA 112, 9769-9774 (2015). The dissected whole SAN
tissue was pinned on a hand-made chamber and was incubated in a
Tyrode solution containing 10 .mu.M Fluo-4 AM (Thermo Fisher
Scientific) and pluronic acid for 1 hour at 37.degree. C. in the
dark. The SAN tissue was washed in Tyrode at 37.degree. C. in the
dark for 10 minutes before experiments. Fluorescence of calcium
transients was recorded using a photomultiplier (PTi D-104) at
35.degree. C. and the analog signals were digitized using Digidata
1440 (Molecular Devices) and analyzed with pCLAMP 10.5
software.
[0230] Western Blotting:
[0231] Mouse atrial and ventricular tissues cut in small pieces
(left and right atrial appendages, left and right ventricles,
sinoatrial node) or beating clusters from normal and CASQ2-D307H
hiPS-CMs were resuspended in ice-cold lysis buffer [50 mM Tris.HCl
pH 7.5, 100 mM NaCl, 1% Nonidet P-40, 0.1% SDS, supplemented with
protease cocktail inhibitor (Sigma-Aldrich) and 1 mM
phenylmethylsulfonyl fluoride (Sigma-Aldrich)], incubated on ice
for 45 minutes, shaken by vortex every 2-3 minutes and centrifuged
for 15 minutes at 4.degree. C. at 16,000.times.g. Equal amounts of
proteins (30 .mu.g) of the resulting lysate supernatant were mixed
with Laemmli sample buffer and fractionated by 10% SDS/PAGE. The
resolved proteins were electro-blotted onto a nitrocellulose
membrane. The membrane was incubated with the primary antibodies
followed by horseradish peroxidase-conjugated secondary anti-IgG
antibodies (1:10,000). The primary antibodies were diluted into 5%
skim milk-TBST ((Tris-buffered saline, 0.1% TWEEN.RTM. 20). The
mouse anti-SK4/KCa3.1 (SAB1409264 Sigma 1:1000) was used for rodent
lysates and the rabbit anti-SK4/KCa3.1 (AV35098 Sigma 1:2500) was
used for human hIPS18 CMs lysates. Both SK4 antibodies were 347
incubated overnight at 4.degree. C. The rabbit anti-Casq2
(18422-1-AP proteintech, 1:2500) and the mouse monoclonal anti
.beta.-actin (MP Biomedical clone C4 691001 1:10,000) were
incubated 1 hour at room temperature. Signals were developed using
SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific).
[0232] In Vivo Telemetric Recordings:
[0233] Telemetric ambulatory long-term ECG recordings, analogous to
Holter monitoring in humans, were obtained with implantable
transmitters. WT, CASQ2-D307H KI and CASQ2 KO SvEv mice were
anesthetized with ketamine (75-90 mg/kg) and xylazine (5-8 mg/kg)
intraperitoneally (IP) (Kepro, Holland), and a midline incision was
made along the spine. An implantable 3.5 grams wireless
radiofrequency transmitter (DSI MM USA, device weight 3.8 grams)
was aseptically inserted into a subcutaneous tissue pocket in the
back as described in Katz, G., et al. Heart Rhythm 7, 1676-1682
(2010) and Kurtzwald-Josefson, E., et al. Heart Rhythm 11,
1471-1479 (2014)]. Animals were allowed to recover after surgery
for at least 24 hours before any experiments. Baseline
electrocardiograms (ECG) were obtained 15 minutes after IP
injection of the appropriate vehicle (peanut oil or peanut oil
supplemented with ethanol 1%). For pharmacological experiments, the
same mouse was used a few hours after baseline ECG recordings
(vehicle injection) and for subsequent ECG recordings upon IP
injection of 20 mg/kg clotrimazole or TRAM-34. Telemetered ECG
tracings were obtained in conscious mice at rest for one minute and
during peak exercise (i.e. the first minute of recovery). In the
treadmill exercise, mice were forced to exercise on a rodent
treadmill; gradually increasing the speed up to a maximum of 15
m/minute. Ventricular tachycardia (VT) was defined as four or more
consecutive ventricular beats. If this phenotype was consecutively
observed for more than 15 seconds, it was defined as "sustained"
ventricular tachycardia (SVT). Shorter VTs were characterized as
"non-sustained" (NSVT). All other ventricular arrhythmias, such as
premature beats, ventricular bigeminy, couplets and triplets were
all defined as ventricular premature contractions (VPCs).
[0234] Data Analysis:
[0235] Rate, AP duration at 50% of repolarization (APD50), delayed
afterdepolarizations (DADs), current densities and calcium
transients were analyzed with the Clampfit program (pClamp 10.5;
Molecular Devices). Leak subtraction was performed offline using
the Clampfit software. Sinus rhythm, PR interval, and ECG
arrhythmic features were analyzed with the LabChart 8 Reader
(ADlnstruments).
[0236] Data were analyzed with Excel (Microsoft) and Prism 5.0
(GraphPad Software) and are expressed as mean.+-.SEM. Statistical
analysis was performed using the two-tailed paired Student t test
and the linear regression for correlation or by one way ANOVA
followed by Tukey's Multiple Comparison Test. P values of less than
0.05 were assumed significant.
Example 1
SK4/IK.sub.Ca Channels as a Therapeutic Target in the Management of
Cardiac Diseases
[0237] Weisbrod et al. (2013, supra) studied the cardiac pacemaker
process in human embryonic stem cells-derived cardiomyocytes
(hESC-CMs), a cellular model which mimics the cardiac cells of the
primitive heart during development. The currents involved in the
pacemaker mechanism in these cells were investigated and, using
biochemistry, electrophysiological and pharmacological techniques,
the intermediate Ca.sup.2+-activated potassium channel
(IK.sub.Ca/SK4, KCa3.1) was identified as a new target in the heart
pacemaker mechanism.
[0238] The data obtained in these studies is presented in FIGS.
1A-D (Background Art). FIG. 1A presents data obtained in
biochemical experiments revealing the existence of the SK4 protein
on cardiomyocytes. Upper panel presents a Western blot from young
and older hESC-CMs lysates showing a 50 KDa band corresponding to
the SK4 channel. Lower panel presents immunocytochemistry showing
the expression of SK4 and the cardiac marker .alpha.-actinin in the
same single hESC-CMs. FIG. 1B presents electrophysiological
characterization of the SK4 current in a single cardiac hESC-CM.
Following a voltage-ramp protocol (electric stimulation from -90 to
+40 mV), the whole conductances from the cell were recorded. Then,
a solution 1 containing several pharmacological blockers was
applied in order to neutralize the dominant cardiac currents. As a
consequence, the amplitudes of the currents were decreased until
linearization as shown on the respective trace (solution 1
contained 10 .mu.M zatebradine, 1 .mu.M nifedipine and 10 .mu.M
E-4031, which are HCN blocker, Ca.sup.2+ channel blocker and
IK.sub.r blocker, respectively). Adding 2 .mu.M of the SK4 blocker
clotrimazole to the same solution 1 decreased the linear voltage
trace in both sides (inward and outward), confirming the existence
of a "clotrimazole-sensitive"/SK4 current in those pacemaker cells.
The chemical structure of clotrimazole is presented in FIG. 1C.
FIG. 1D presents the pharmacological effects of clotrimazole on the
cardiac pacing. The spontaneous electric activity of the cell was
recorded in the current clamp configuration of the patch clamp
technique before and during exposure to 2 .mu.M clotrimazole.
Clotrimazole decreased the firing rate of the cells, depolarized
the membrane of the cell (MDP) until a pacing arrest. It did not
significantly affect the duration of the action potentials (APD50).
Thus, exposing the cells to 2 .mu.M clotrimazole in the external
solution, dramatically decreases the rate of the spontaneous
electrical pulses (action potentials) of the cardiac cells,
culminating by a depolarization of the maximal depolarization
potential (MDP) and arrest of the pacing. The absence of elongation
of the action potential (no changes in the action potential
duration "APD50") discards the possibility of a role of the SK4
current in the repolarization (phase 3) of the action
potential.
[0239] Because hESC-CMs display immature phenotypes, it was
required to confirm the observations in adult heart cells. To this
end, mice heart samples and human heart biopsia from patients were
used. The obtained data is presented in FIGS. 2A-C. FIGS. 2A and 2B
present biochemical experiments revealing the existence of the SK4
channel on murine heart (FIG. 2A) and in human right atrium and
ventricle biopsia (FIG. 2B). In FIG. 2A, left panel presents a
reverse transcriptase PCR of the SK4 mRNA showing a 286 bp band
corresponding to the amplification of the channel transcript in
different heart regions (SAN=sinoatrial node; RA=right appendage;
LA=left appendage; RV=right ventricle, LV=left ventricle); and
right panel presents Western blot on murine lysates from the same
heart areas, showing a specific 50 KDa band corresponding to the
SK4 channel. Thus, similarly to the observations in hESC-CMs,
biochemical experiments performed in the right and left appendages,
the right and left ventricles and the sinoatrial node isolated from
mice showed a very clear expression of SK4 in those areas of the
heart at the transcript and protein level.
[0240] In FIG. 2B it is shown that while the SK4 channel is
observed at the transcript level in the right atrium and ventricle
of several patients (left panel), it is only expressed at the
protein level in the right atrium. Those results were confirmed in
more than 64 right atrium biopsia from different patients (right
panel). These data points towards the right atrium being the
anatomical region of the heart that includes the SAN.
[0241] Cells from the mice SAN were successfully isolated, and
electophysiological experiments were performed on their spontaneous
pacing rate before and after applying clotrimazole. FIG. 2C
presents the pharmacological effects of clotrimazole on the
pacemaker activity of SAN cells. The spontaneous electric activity
of the cell was recorded in the current clamp configuration of the
patch clamp technique before and during exposure to 2 .mu.M
clotrimazole. Clotrimazole strongly decreases the firing rate of
the cells, leading to a bradycardic effect, which is reversible
during washout (blue trace). These data show that clotrimazole led
to a strong reduction of the pacing rate of the cells, pointing
towards the importance of the SK4 channel in the pacemaker
physiological mechanism. Importantly, it is shown that these
effects are reversible when the cells are washed.
Example 2
Clotrimazole Induces Bradycardia
[0242] In order to demonstrate the importance of SK4 as a new
therapeutic target in adult heart, the actions of clotrimazole were
evaluated in vivo. For this, a heart telemetry device was implanted
in norma mice for continuous ECG recording at rest and during
treadmill exercise. For each session, continuous ECG recording was
performed with the same animals receiving first intraperitoneal
(IP) injection of vehicle (peanut oil) and then 20 mg/kg
clotrimazole. The obtained data is presented in FIGS. 3A-D.
[0243] FIG. 3A presents representative ECG recording following
intraperitoneal injection of vehicle (upper) and 20 mg/kg
clotrimazole (lower) in norma mice at rest. Sequential vehicle and
clotrimazole injections were performed on the same animal.
Clotrimazole produced bradycardia (PP) and prolongation of the PR
interval (grey arrows). FIG. 3B presents data summary of heart rate
at rest (upper; *P=0.0364, n=10) and PR interval (lower; *P=0.0437,
n=10).
[0244] FIG. 3C presents representative ECG recording following
intraperitoneal injection of vehicle (upper) and 20 mg/kg
clotrimazole (lower) in mice following treadmill exercise.
Similarly to what happened at rest, clotrimazole produced a
negative chronotropic effect (PP elongation). Despite the visible
similar trend on the PR interval prolongation, the clotrimazole
effect did not reach significance following treadmill exercise
(n=10). FIG. 3D presents data summary of heart rate (upper;
*P=0.0229, n=10) and PR interval (lower), which was 29.27.+-.0.94
ms vs Clotrimazole 37.44.+-.4.03 ms.
[0245] As shown in FIGS. 3A-B, a single injection of clotrimazole
(20 mg/kg, IP) reduced the resting heart rate by 16.1.+-.6.1% (from
718.+-.16 bpm to 604.+-.43 bpm, *P=0.0364, n=10) and prolonged the
PR interval as it can be seen with the grey arrows (from
29.4.+-.0.9 ms to 37.0.+-.3.5 ms, *P=0.0437, n=10). These results
confirm the importance of the SK4 channels not only in the SAN, but
also in the whole cardiac conduction system. As shown in FIGS.
3C-D, a similar trend was observed during treadmill exercise.
Injection of clotrimazole produced a similar reduction of the sinus
rate, from 724.+-.18 bpm to 605.+-.42 bpm (*P=0.0229, n=10).
[0246] These results indicate that SK4 blockade by clotrimazole
leads to sinus bradycardia and to an elongation of the refractoric
period in normal mice and that clotrimazole can be used as an
alternative to .beta.-blocker or Ca.sup.2+ channels blockers
therapies to reduce the heart rate.
Example 3
SK4 Blockade for Treating CPVT
[0247] Decreasing the heartbeat with bradychardic medications such
as .beta.-blockers is a common and widely accepted therapeutic
approach used in order to reduce the incidence of arrhythmia in
several cardiopathies. By reducing the heartbeats, those compounds
extend the refractoric period between two contractions, thus
decreasing the risks of arrhythmia.
[0248] Catecholaminergic polymorphic ventricular tachycardia (CPVT)
is an inherited arrhythmogenic syndrome characterized by physical
or emotional stress-induced polymorphic ventricular tachycardia in
otherwise structurally normal hearts with a high fatal event rate
in untreated patients. In a cellular level, ventricular cells but
also SAN cells and other conductive pacemaker cells display
abnormal cytoplasmic Ca.sup.2+ levels. During exercise or stress
(activation of the sympathetic system), those calcium events
generate small and local depolarizations called "delayed
afterdepolarizations" (DADs), which trigger an uncoupling between
the normal sinoatrial rhythm and the ventricular activity. The
consequence is the observation of a "ventricular tachycardia"
(absence of P waves before the QRS complexes) observable during ECG
recordings, which lead to cardiac arrest and sudden death if the
patients are not treated or implanted with an implantable
cardioverter defibrillator.
[0249] Studies were conducted in order to explore whether SK4
channels are expressed in SAN and play a role in CPVT.
[0250] Clotrimazole and TRAM-34 (an isomer of clotrimazole as
depicted hereinabove) were tested on a model of ventricular
disorder in order to see if SK4 blockade decreases the arrhythmic
features.
[0251] Single spontaneously beating hiPS-CMs (25 days-old EBs)
derived from normal (healthy) and CPVT2 patients carrying the CASQ2
D307H mutation were used and investigated for their spontaneous
firing and ionic currents. A voltage ramp was applied as previously
described [Wiesbrod et al., 2013, supra] and cells were held at -20
mV to substantially inactivate voltage-gated Na.sup.+ and Ca.sup.2+
currents. The data obtained in these studies is presented in FIGS.
4A-G.
[0252] FIGS. 4A-B presents representative traces of hiPS-CMs
derived from normal (FIG. 4A) or CPVT2 (CASQ2 D307H) (FIG. 4B)
patients. Cells were held at -20 mV and a voltage ramp of 150 ms
from -90 to +60 mV was applied, as shown in FIG. 2B. The respective
traces show the various currents recorded with 1 .mu.M internal
free Ca.sup.2+ and without blockers in the extracellular solution.
Other respective traces indicate that exposing cells to blocker
solution 1 (300 .mu.M CdCl.sub.2, 25 .mu.M ZD7288, and 10 .mu.M
E-4031), markedly depressed the inward humps and the currents in
the inward and outward directions. Other respective traces reflect
the addition of the selective SK4 channel blocker TRAM-34 (5 .mu.M)
to solution 1. Other respective traces show the currents obtained
when the cell was washed out with solution 1 alone.
[0253] As shown in FIGS. 4A-B, in the absence of blockers, the
voltage ramp revealed the presence of two inward humps peaking at
about -40 mV and -5 mV and reflecting activation of residual T type
and L-type Ca2+ currents, respectively. These inward humps
disappeared following exposure to 300 .mu.M CdCl.sub.2. Exposing
cells to Solution 1 (300 .mu.M CdCl.sub.2, 25 .mu.M ZD7288, and 10
.mu.M E-4031), suppressed the inward humps and markedly depressed
inward and outward currents. Addition of the selective SK4 channel
blocker TRAM-34 (5 .mu.M) to solution 1 reversibly decreased the
currents in both inward and outward directions. This
TRAM-34-sensitive current was never detected in zero internal free
Ca.sup.2+.
[0254] The TRAM-sensitive current was calculated as a difference
between the current density measured at +60 mV with solution 1
alone and solution 1+5 .mu.M TRAM-34 (n=7-9), and is shown in FIG.
4C. Following TRAM-34 exposure, residual inward and outward
currents could still be detected and likely correspond to
non-selective cationic conductances, which shifted the reversal
potential away from EK. TRAM-34 sensitive currents were observed in
7 out of 15 normal hiPS-CMs and in 9 out of 13 CPVT2 hiPSC-CMs. No
significant differences were found in TRAM-34-sensitive current
densities of normal and CPVT2 hiPSC-CMs.
[0255] FIG. 4D presents representative Western blots of beating EBs
lysates from normal and CPVT2 (CASQ2 D307H) patients showing
immuno-reactive SK4 protein (about 50 KDa). SK4 channel expression
was thus confirmed at the protein level, where an SK4
immunoreactive band of about 50 kDa was identified in Western blots
from beating cluster lysates of normal and CPVT2 hiPSC-CMs.
[0256] FIG. 4E (Left) presents representative traces of spontaneous
APs recorded in hiPS-CM derived from a normal individual. Baseline
pacing (control) was significantly increased following exposure of
normal hiPSC532 CMs to 3 .mu.M isoproterenol. Adding 5 .mu.M
TRAM-34 depolarized the MDP and decreased the DD slope, which
eventually culminated by a suppression of the pacing. The TRAM-34
effect was reversible by washout. FIG. 4E (Right) are bar graphs
presenting data summary of pacing rate (normalized to Ctrl; one-way
ANOVA **P=0.0071, n=14), DD slope (paired t-test, ***P<0.0001,
n=14) and APD50 (one-way ANOVA *P=0.0243, n=18). These data show
that exposure of normal hiPSC-CMs to 3 .mu.M isoproterenol
significantly increased their firing rate and reduced their APD50.
Adding 5 .mu.M TRAM-34 depolarized the maximal diastolic potential
(MDP), and significantly decreased the slope of diastolic
depolarization (DD), which eventually culminated by a suppression
of the pacing (in 10 out of 14 cells).
[0257] Similar experiments were performed on CASQ2 D307H hiPS-CMs
and the obtained data is shown in FIG. 4F.
[0258] FIG. 4F (Left) presents representative traces of spontaneous
APs recorded in hiPS-CM derived from a CPVT2 (CASQ2 D307H)
patients. From a regular pacing, DADs appeared when the cell was
exposed to 3 .mu.M isoproterenol (arrows). Adding 5 .mu.M TRAM-34
to isoproterenol markedly reduced the DADs (upper trace) until the
SK4 blockade led to the pacing arrest (lower trace). The
suppression of cell 540 automaticity was reversible upon washout.
FIG. 4F (Right) present bar graphs showing data summary of pacing
rate (n=17), on APD50 (n=17) and DADs (Normalized to Ctrl; one-way
ANOVA***P=0.0001, n=15).
[0259] These data show that Isoproterenol did not produce positive
chronotropic effect on CPVT2 hiPSC-CMs. Instead, isoproterenol
triggered DADs (see, FIG. 4F, arrows). Adding TRAM-34 to the
isoproterenol solution drastically reduced the number of DADs and
led to subsequent and reversible cessation of the spontaneous
activity.
[0260] For selectivity purposes, it was examined whether TRAM-34
interfered with major pacemaker currents in hESC-CMs. It has been
previously showed that If and INCX currents were unaffected by 5
.mu.M TRAM-34 [Weisbrod et al., 2013, supra].
[0261] FIG. 4G presents representative trace of a voltage-ramp
protocol performed in cardiomyocytes derived from human embryonic
stem cells before and after applying 5 .mu.M TRAM-34. Cells were
held at -20 mV and a voltage ramp of 150 ms from -90 to +60 mV was
applied. The experiments were performed in zero free Ca2+ in the
pipet solution. In the absence of TRAM-34, the voltage ramp
revealed the presence of two inward humps peaking at about -40 mV
and -5 mV and reflecting the activation of residual T type and
L-type Ca2+ currents, respectively. Results show that 5 .mu.M
TRAM-34 does not alter the voltage-gated Ca2+ currents (n=7). Thus,
it was shown that 5 .mu.M 90 TRAM-34 did not alter the T type and L
type Ca2+ currents measured by the two inward humps (see, zero free
Ca.sup.2+ in pipet solution in FIG. 4G).
Example 4
Studies in SAN Cells from CASQ2-D307H KI Mice
[0262] Individual SAN cells were isolated from WT and CASQ2-D307H
homozygous KI mice and recorded as described above, except that
cells were held at -40 mV to improve their stability.
[0263] FIGS. 5A-B presents representative traces of SAN cells
isolated from WT and CASQ2-D307H KI mice. Cells were held at -40 mV
and a voltage ramp of 150 ms from -90 to +60 mV was applied. The
respective traces show the various currents recorded with 1 .mu.M
internal free Ca.sup.2+ and without blockers in the extracellular
solution. The respective traces indicate that cells were exposed to
blocker solution 1. The respective traces reflect the addition of
TRAM-34 (5 .mu.M) to solution 1. In the absence of blockers, the
voltage ramp revealed the presence of only one inward hump peaking
at about -40 mV and reflecting activation of T type Ca.sup.2+
currents with minor contribution of L108 type Ca.sup.2+ currents.
The inward hump and substantial inward and outward currents
disappeared upon exposure of cells to solution 1.
[0264] In FIG. 5C, the TRAM-sensitive current was calculated as in
FIG. 4C (n=8-12). TRAM-34-sensitive currents with similar densities
were isolated in SAN cells from both WT and CASQ2-D307H KI
mice.
[0265] FIG. 5D presents representative Western blots of SAN lysates
from WT and CASQ2-D307H KI mice showing the immuno-reactive bands
of SK4, CASQ2 and .beta.-actin proteins in SAN, right and left
atrial appendages, right and left ventricle. The expression of SK4
channels and CASQ2 in adult mouse heart of WT and CASQ2-D307H KI
mice was confirmed. Western blots of lysates from SAN, right and
left atrial appendages, right and left ventricles showed specific
immunoreactive bands corresponding to SK4 channel and to CASQ2
protein.
[0266] FIG. 5E (Left) presents representative traces of spontaneous
APs recorded in single SAN cell from WT mice. Baseline pacing
(control) was increased following exposure to 50 nM isoproterenol.
Adding 2 .mu.M TRAM-34 depolarized the MDP and decreased the DD
slope and eventually suppressed the pacing. FIG. 5E (Right)
presents bar graphs showing data summary of pacing rate (paired
t-test *P<0.049; n=4), DD slope (one-way ANOVA, ***P=0.0087,
n=7) and APD50 (n=7). Isoproterenol (50 nM) significantly increased
the pacing of SAN cells from WT mice with an increased DD slope.
Adding 2 .mu.M TRAM-34 to isoproterenol, depolarized the MDP,
markedly reduced the DD slope, decreased the beating rate and
eventually stopped the pacing activity in 3 out of 7 cells.
[0267] FIG. 5F (Left) presents representative traces of spontaneous
APs recorded in single SAN cell from CASQ2 D307H KI mice. From a
regular pacing, the rate increased and DADs appeared following
addition of 50 nM isoproterenol (arrows). Adding 2 .mu.M TRAM-34 to
isoproterenol noticeably reduced DADs occurrence. FIG. 5F (Right)
presents bar graphs showing data summary of rate (n=6), APD50 (n=6)
and DADs (Normalized to Ctrl: one-way ANOVA,**P=0.0025, n=5).
[0268] In SAN cells from CASQ2-D307H KI mice, addition of 50 nM
isoproterenol initially produced a positive chronotropic effect.
However, after 1-2 minutes isoproterenol led to DADs (FIG. 5F,
arrows). When 2 .mu.M TRAM-34 were added to the isoproterenol
solution the occurrence of DADs was drastically reduced.
[0269] To investigate the spontaneous calcium transients of the
SAN, intact SAN tissue preparations dissected ex vivo from WT and
CASQ2-D307H KI mice were exposed to Fluo-4 AM as previously
described [Torrente, A. G., et al. Proc Natl Acad Sci USA 112,
9769-9774 (2015)].
[0270] FIG. 6A (Left) presents representative traces of spontaneous
calcium transients recorded ex vivo in intact SAN tissue
preparations from WT mice. The baseline rate of calcium transients
(control) was significantly increased in presence of 100 nM
isoproterenol and the additional exposure of 2 .mu.M TRAM-34 did
not alter the pattern of the Ca.sup.2+ waves. Right: data summary
of calcium transient rate (one-way-ANOVA***P=0.0003; n=12).
[0271] In SAN from WT mice, the rate of calcium transients was
significantly increased in presence of 100 nM isoproterenol and the
additional exposure of 2 .mu.M TRAM-34 did not alter the pattern of
the Ca.sup.2+ waves.
[0272] Consistent with previous studies in different CPVT1 and
CPVT2 mouse models and hiPSC-CMs, exposing SANs from CASQ2-D307H KI
mice to 100 nM isoproterenol produced various Ca.sup.2+ transient
abnormalities, which were classified according to their degree of
severity.
[0273] FIG. 6B presents representative traces of different types of
calcium transient abnormalities recorded in intact SAN from CASQ2
D307H KI mice, termed as "local Ca.sup.2+ release" (upper left),
"double humped transients" (upper right), "large-stored released
Ca.sup.2+ waves" (lower left) and "calcium alternans" (lower
right).
[0274] FIG. 6C presents representative trace of spontaneous calcium
transients recorded from intact SAN of CASQ2 D307H KI. The baseline
rate of calcium transients (control) yielded chaotic calcium
transients following incubation of the SAN with 100 nM
isoproterenol. Subsequent addition of 2 .mu.M TRAM-34 to the
solution drastically improved the arrhythmic features of the
calcium transients. FIG. 6D presents data summary of the arrhythmic
calcium transients in SAN from CASQ2 D307H KI under baseline
conditions, following exposure to 100 nM isoproterenol and 100 nM
isoproterenol+2 .mu.M TRAM-34.
[0275] Adding 2 .mu.M TRAM-34 normalized the shapes of
isoproterenol induced aberrant calcium waves in SAN from
CASQ2-D307H KI mice. For instance, TRAM-34 brought back to zero the
number of SANs displaying double humped transients or large-stored
released Ca.sup.2+ waves.
Example 5
In Vivo Studies
[0276] A heart telemetry device was implanted in WT, CASQ2-D307H KI
and CASQ2 KO mice for continuous ECG recording at rest and during
treadmill exercise. For each session, continuous ECG recording was
performed with the same animals receiving first intraperitoneal
(IP) injection of vehicle (peanut oil) and then the SK4 channel
blocker. The obtained data is presented in FIGS. 7A-10D.
[0277] FIG. 7A presents Representative ECG recording following
intraperitoneal injection of vehicle (upper) and 20 mg/kg TRAM-34
(lower) in WT mice at rest. Sequential vehicle and TRAM-34
injections were performed on the same animal. TRAM-34 produced
bradycardia (PP) and prolongation of the PR interval (grey arrows).
FIG. 7B presents data summary of heart rate (Paired t-test;
***P=0.0003, n=10) and PR interval (Paired t test; ***P=0.0004,
n=10) in WT mice at rest.
[0278] FIG. 7C presents representative ECG recording following IP
injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in
CASQ2-D307H KI mice at rest. TRAM-34 produced bradycardia and
markedly reduced arrhythmic features such as ventricular premature
complexes. FIG. 7D presents data summary of heart rate (Paired
t-test; ***P<0.0001, n=12) and PR interval (Paired t-test;
***P=<0.0001, n=12) in CASQ2-D307H KI mice at rest.
[0279] FIG. 7E presents representative ECG recording following IP
injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in CASQ2
KO mice at rest showing that TRAM-34 produced similar effects as in
CASQ2-D307H KI mice. FIG. 7F presents data summary of heart rate
(Paired t-test; **P=0.004, n=7 mice) and PR interval (Paired
t-test; **P=0.0041, n=7) in CASQ2 KO mice at rest.
[0280] FIG. 8A presents representative ECG recording following
intraperitoneal injection of vehicle (upper) and 20 mg/kg TRAM-34
(lower) in WT mice during treadmill exercise. TRAM-34 produced
bradycardia (PP) and prolongation of the PR interval (grey arrows).
FIG. 8B presents data summary of heart rate (Paired t-test;
***P=0.001, n=10) and PR interval (Paired t-test; ***P=0.0005,
n=10) in WT mice during exercise.
[0281] FIG. 8C presents representative ECG recording following IP
injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in
CASQ2-D307H KI mice during treadmill exercise. TRAM-34 produced
bradycardia and markedly reduced arrhythmic features such as
ventricular tachycardia. FIG. 8C presents data summary of heart
rate (Paired t-test; ***P=0.0004, n=11) and PR interval (Paired
t-test; **P=0.0099, n=9) in CASQ2-D307H KI mice during
exercise.
[0282] FIG. 8E presents representative ECG recording following IP
injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in CASQ2
KO mice during exercise. Arrhythmias such as NSVT were suppressed
by TRAM-34 injection. FIG. 8F presents data summary of heart rate
(Paired t-test; *P=0.0165, n=7) and PR interval (Paired t-test;
**P=0.0042, n=7) in CASQ2 KO mice during exercise.
[0283] FIG. 9A presents representative ECG recording following IP
injection of vehicle (upper) and 20 mg/kg clotrimazole (lower) in
CASQ2-D307H KI mice at rest. Clotrimazole produced bradycardia and
markedly reduced arrhythmic features such as ventricular premature
complexes. FIG. 9B presents data summary of heart rate (Paired
t-test; *P=0.0260, n=7) and PR interval (n=7) in CASQ2-D307H KI
mice at rest.
[0284] FIG. 9C presents representative ECG recording following IP
injection of vehicle (upper) and 20 mg/kg clotrimazole (lower) in
CASQ2 KO mice at rest showing that clotrimazole produced similar
effects as in CASQ2-D307H KI mice. FIG. 9D presents data summary of
heart rate (Paired t-test; **P=0.0078, n=7) and PR interval (Paired
t-test; *P=0.0111, n=7) in CASQ2 KO mice at rest.
[0285] FIG. 10A presents representative ECG recording following IP
injection of vehicle (upper) and 20 mg/kg clotrimazole (lower) in
CASQ2-D307H KI mice during treadmill exercise. Clotrimazole changed
the non-sustained ventricular tachycardia (NSVT) into ventricular
premature complex (VPC). FIG. 10B presents data summary of heart
rate (Paired t-test; **P=0.004, n=7) and PR interval (Paired
t-test; *P=0.0305, n=7) in CASQ2-D307H KI mice during treadmill
exercise.
[0286] FIG. 10C presents representative ECG recording following IP
injection of vehicle (upper) and 20 mg/kg clotrimazole (lower) in
CASQ2 KO mice during exercise. Typical arrhythmic features such as
NSVT were improved by clotrimazole treatment. FIG. 10D presents
data summary of heart rate (Paired t-test; **P=0.0037, n=7) and PR
interval (Paired t-test; *P=0.0394, n=6) in CASQ2 KO mice during
exercise.
[0287] TRAM-34 (20 mg/kg, IP) significantly decreased the resting
heart rate of WT mice by 16.+-.3% as measured by the PP interval
(FIGS. 7A-B). A significant prolongation of 20% in the PR interval
was also seen on the ECG traces of WT mice (FIGS. 7A-B). TRAM-34
produced similar bradycardic effects and PR interval prolongation
during treadmill exercise of WT mice (FIGS. 8A-B).
[0288] The SK4 channel blocker clotrimazole (20 mg/kg, IP)
significantly reduced the resting heart rate by 16.+-.6% and
prolonged by 27% the PR interval (data not shown). A similar trend
was noticeable during treadmill exercise (data not shown).
[0289] CASQ2-D307H KI and CASQ2 KO mice displayed lower basal heart
rates compared to WT mice but also irregular sinus rhythm and
ventricular premature complexes as shown on the ECG traces (FIGS.
7C-D). TRAM-34 injection (20 mg/kg, IP) to these mice produced like
in WT animals significant bradycardic effects (FIGS. 7E-F; 24.+-.4%
and 34.+-.7% heart rate decrease in 12 KI and 7 KO mice
respectively; p<0.005) and PR prolongation (KI mice: 23%;
P=0.0001, n=12; KO mice: 46%; P=0.0041, n=7). TRAM-34 injection
improved the ECG arrhythmic features observed under resting
conditions and totally suppressed them in 9 out of 12 KI mice.
[0290] During treadmill exercise, the ECG cardiac abnormalities
were aggravated with "non-sustained" and even "sustained"
ventricular tachycardia (FIGS. 8C and 8E). Under these conditions,
TRAM-34 injection decreased the prevalence and severity of
arrhythmias (see, Table 1).
[0291] During treadmill exercise, TRAM-34 also produced significant
sinus bradycardia and PR interval prolongation in KI and KO mice
(FIGS. 8C-F).
[0292] Clotrimazole (20 mg/kg, IP) elicited similar effects to
those observed with TRAM-34. Under basal conditions (FIGS. 9A-D)
and during treadmill exercise (FIGS. 10A-D), bradycardia and PR
prolongation were noticed in CASQ2-D307H KI and CASQ2 KO mice
following clotrimazole injection. Clotrimazole improved the ECG
arrhythmic features observed at rest and following treadmill
exercise and even succeeded to convert them to normal sinus rhythm
in 3 out of 5 KI mice and 4 of out 6 KO mice at rest (See, FIGS.
9A-D and 10A-D and Table 1).
[0293] The types of arrhythmic features were classified following
their seriousness: sinusal rhythm (normal), ventricular premature
contractions (VPC), non-sustained ventricular tachycardia (NSVT)
and sustained ventricular tachycardia (SVT). For each mouse was
considered the most severe form of arrhythmia recorded under ECG.
Table 1 below presents the arrhythmogenic features at rest or
during exercise in CPVT2 CASQ2-D307H KI and CASQ2 KO mice after IP
injection of the SK4 blockers TRAM-34 or Clotrimazole.
TABLE-US-00001 TABLE 1 Clotrimazole TRAM-34 KI at rest Vehicle 20
mg/kg Vehicle 20 mg/kg Number of 5 5 12 12 mice (n) Normal 0 3 3 9
VPC 4 2 6 3 NSVT 1 0 3 0 SVT 0 0 0 0 KI during Clotrimazole TRAM-34
Exercise Vehicle 20 mg/kg Vehicle 20 mg/kg Number of 5 5 12 12 mice
(n) Normal 0 0 1 4 VPC 1 2 2 4 NSVT 4 2 9 4 SVT 0 1 0 0
Clotrimazole TRAM-34 KO at rest Vehicle 20 mg/kg Vehicle 20 mg/kg
Number of 6 6 6 6 mice (n) Normal 0 4 1 6 VPC 4 2 5 0 NSVT 2 0 0 0
SVT 0 0 0 0 KO during Clotrimazole TRAM-34 exercise Vehicle 20
mg/kg Vehicle 20 mg/kg Number of 6 6 6 6 mice (n) Normal 0 0 0 3
VPC 0 2 0 0 NSVT 5 3 3 3 SVT 1 1 3 0
[0294] As it can be seen in Table 1, the number of mice suffering
from severe forms of arrhythmia under IP vehicle injection was
decreased following treatments with the SK4 blockers clotrimazole
and TRAM-34 both at rest and following exercise.
Example 6
Concluding Remarks
[0295] The data presented herein demonstrate the pivotal role of
SK4 Ca.sup.2+-activated K.sup.+ channels in adult pacemaker
function, making them promising therapeutic targets for the
treatment of cardiac ventricular arrhythmias such as CPVT and other
cardiac disorders.
[0296] The results presented herein clearly identify the
SK4/IK.sub.Ca channel as a therapeutic target involved in the adult
cardiac pacemaker mechanism.
[0297] The results presented herein show that the SK4 blockers
clotrimazole and TRAM-34 exhibit a bradycardic effect, while
elongating the PP interval and the refractory period between two
heartbeats (effect in the Atrioventricular node manifested by an
increase of the PR interval), like .beta.1-adrenergic or Ca.sup.2+
channel blockers. This effect was demonstrated at the cellular
level in human and mice cardiac cells and also in vivo.
[0298] The data presented herein primarily provide the first
evidence that SK4 channels are not only expressed in spontaneously
beating hESC-CMs and hiPS-CMs but in SAN cells too. Inhibition of
SK4 K.sup.+ currents by TRAM-34 reduced the intrinsic SAN firing
rate. These data reveal that in SAN cells SK4 channels are novel
regulators of mouse SAN automaticity.
[0299] Cardiac automaticity is achieved by the integration of
voltage-gated currents ("membrane clock") with rhythmic Ca.sup.2+
release from internal Ca.sup.2+ stores ("Ca.sup.2+ clock"). See,
e.g., Brown, H. F. Electrophysiology of the sinoatrial node.
Physiol Rev 62, 505-530 (1982). SAN pacemaker activity is due to
the ability to generate DD, where a cohort of inward currents
slowly depolarize the membrane potential until reaching the
threshold of a next action potential (AP) mainly triggered by
opening of voltage-gated Ca.sup.2+ channels. These include funny
currents (I.sub.f), T-type Ca.sup.2+ currents and the
Na.sup.+/Ca.sup.2+ exchanger NCX1 that is activated in its forward
mode by cyclical SR Ca.sup.2+ release via RyR232,39. Outward
K.sup.+ currents can affect very differently murine SAN
excitability. While IKR, SK2 and Ito repolarize AP, IKACh (GIRK4)
can act during DD to dampen SAN firing rate [Mangoni, M. E. &
Nargeot, J. Physiol Rev 88, 919-982 (2008); Li, N., et al. J
Physiol 587, 1087-1100 (2009). Mahida, S. Heart Rhythm 11,
1233-1238 (2014); Xu, Y., et al. J Biol Chem 278, 49085-49094
(2003)].
[0300] The data presented herein clearly indicate that SK4 channels
do not significantly alter AP duration but affect the MDP and the
DD slope. In all SK channels, activation results from Ca.sup.2+
binding to calmodulin followed by conformational changes that open
the pore. The time constant (.tau.=5 ms) of this activation process
is strongly dependent on intracellular Ca.sup.2+. SK channel
deactivation, initiated by dissociation of Ca.sup.2+, is
independent of intracellular Ca.sup.2+ and occurs on a much slower
time scale (.tau.=15-60 ms). SK channels can remain active for more
than 100 ms after [Ca.sup.2+]i has returned to resting levels. See,
for example, Berkefeld et al. Physiol Rev 90, 1437-1459 (2010).
[0301] Because of this slow channel deactivation, it has been
suggested herein that SK4 channel contribution becomes significant
only at the late repolarization, thereby contributing to the MDP
hyperpolarization, which facilitates activation of I.sub.f and
recovery from inactivation of voltage-gated Ca.sup.2+ channels.
Thus, the net effect of SK4 channel activation is an increase in
the firing rate. SK4 channels may act in SAN like BKCa channels in
hippocampal neurons, where their activation counterintuitively
increases excitability, while their inhibition reduces firing.
[0302] The in vitro and in vivo data obtained with the SK4 channel
blockers, TRAM-34 and clotrimazole, on the pacing rate of isolated
SAN cells and on ECG parameters of WT mice, as presented herein,
indicate that activation of SK4 channels increases the SAN pacing
rate and their blockade reduces it. Both blockers produced
significant bradycardic effects during rest and following treadmill
exercise. An indirect impact of TRAM-34 or clotrimazole on
autonomic input to SA and AV nodes in vivo can be excluded because
both blockers exert similar effects on isolated SAN cells. In line
with these data, RA-2, a structurally different molecule from
TRAM-34 and clotrimazole, with a mixed blocker activity toward SK4
and SK2 channels, induced bradycardia in mice, an effect abolished
in SK4 knockout mice [Olivan-Viguera, A., et al. Mol Pharmacol 87,
338-348 (2015)]. The prolongation of the PR interval is related to
either AV node and/or the HisPurkinje system and suggests that SK4
channels are likely expressed in the conduction system.
[0303] Previous transcriptional analysis showed a 9-fold
upregulation of SK4 in the developing conduction system compared to
SK1-346. Reflecting functional redundancy among SAN ionic
conductances, it is noted that additional Ca.sup.2+-activated
K.sup.+ channels have been characterized in the murine cardiac
pacemaker. Blockade of SK2 channels prolonged the AP duration in
atrioventricular nodal cells and knockout of SK2 channels in mice
resulted in bradycardia and prolongation of the PR interval.
Conversely, overexpression of SK2 channels decreased AP duration,
increased spontaneous firing rate of atrioventricular nodal cells
and reduced PR and RR intervals in ECG. See, for example, Zhang,
Q., et al. Circ Res 102, 465-471 (2008)]. More recently, Ca.sup.2+-
and voltage-activated BK K.sup.+ channels were also identified in
murine SAN cells. Genetic ablation or pharmacological inhibition of
BK channels were associated with reduced heart rate in ECG and
slowed SAN cells pacing without alteration of AP duration. See, for
example, Lai, M. H., et al. Am J Physiol Heart Circ Physio 307,
H1327-1338 (2014)]. This apparent redundancy of Ca.sup.2+-activated
K.sup.+ currents indicates that they share similar properties such
as bradycardia upon channel blockade (SK2, SK4 and BK) but they
also exhibit subtle differences notably regarding their impact on
AP duration (e.g., SK2 versus SK4).
[0304] The data presented herein show that inhibition of SK4
K.sup.+ channels rescues in vitro the cardiac arrhythmias exhibited
by hiPS-CMs derived from CPVT2 patients carrying the CASQ2 D307H
mutation and by SAN cells isolated from CASQ2-D307H KI mice. Hence,
TRAM-34 markedly reduced the occurrence of DADs and abnormal
Ca.sup.2+ transients detected following exposure to the
.beta.-adrenergic agonist isoproterenol. SK4 channel blockers can
therefore protect in vivo the animals from deleterious ventricular
arrhythmic features revealed by ECG in CASQ2-D307H KI and CASQ2 KO
mice at rest and after treadmill exercise.
[0305] Ventricular premature complexes, non-sustained and sustained
ventricular tachycardia were significantly reduced following a
single IP injection (20 mg/kg) of clotrimazole or TRAM-34. The SK4
channel blockers protected the CASQ2-D307H KI and CASQ2 KO mice
from harmful polymorphic ventricular tachycardia without being
pro-arrhythmic by themselves, since neither sinus arrest nor 2nd
order AV block were recorded in the animals, including WT mice.
[0306] Despite the blockade of SK4 channels, the functional
redundancy of Ca.sup.2+-activated K.sup.+ channels likely preserves
the delicate balance of inward and outward currents necessary for
normal pacemaking. Along the same line, recent studies showed that
cardiac SAN arrhythmias induced by silencing either HCN4 (I.sub.f
current) or Cav1.3 (L-type Ca.sup.2+ currents) could be rescued by
genetic deletion or pharmacological inhibition of GIRK4 channels
(IKACh currents) [Lai, M. H., et al. (2014), supra; Mesirca, P., et
al. Nat Commun 5, 4664 (2014); Mesirca, P., et al. Proc Natl Acad
Sci USA 113, E932-941 (2016)].
[0307] Due to their bradycardic effect and slowed AV conduction,
SK4 channel blockers, very much like .beta.1-adrenergic or
Ca.sup.2+ channel blockers, are beneficial for preventing
ventricular tachycardia by prolonging the refractory period.
[0308] These data indicate that the therapeutic indications of SK4
channel blockers could be extended to non arrhythmic cardiovascular
disorders, ventricular tachyarrhythmias in CPVT and possibly in
other arrhythmic pathologies of different etiologies such as the
long QT syndrome.
[0309] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0310] It is the intent of the Applicant(s) that all publications,
patents and patent applications referred to in this specification
are to be incorporated in their entirety by reference into the
specification, as if each individual publication, patent or patent
application was specifically and individually noted when referenced
that it is to be incorporated herein by reference. In addition,
citation or identification of any reference in this application
shall not be construed as an admission that such reference is
available as prior art to the present invention. To the extent that
section headings are used, they should not be construed as
necessarily limiting. In addition, any priority document(s) of this
application is/are hereby incorporated herein by reference in
its/their entirety.
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