U.S. patent application number 15/888317 was filed with the patent office on 2019-05-16 for use of hdac inhibitors for treatment of cardiac rhythm disorders.
This patent application is currently assigned to THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. The applicant listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Jonathan A. EPSTEIN, Vickas PATEL.
Application Number | 20190142773 15/888317 |
Document ID | / |
Family ID | 40002534 |
Filed Date | 2019-05-16 |
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United States Patent
Application |
20190142773 |
Kind Code |
A1 |
PATEL; Vickas ; et
al. |
May 16, 2019 |
USE OF HDAC INHIBITORS FOR TREATMENT OF CARDIAC RHYTHM
DISORDERS
Abstract
The present invention provides methods of ameliorating or
reducing the extent of cardiac arrhythmia disorders, by
administering an inhibitor of histone deacetylase enzyme
(HDAC).
Inventors: |
PATEL; Vickas; (Havertown,
PA) ; EPSTEIN; Jonathan A.; (Radnor, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
PHILADELPHIA |
PA |
US |
|
|
Assignee: |
THE TRUSTEES OF THE UNIVERSITY OF
PENNSYLVANIA
PHILADELPHIA
PA
|
Family ID: |
40002534 |
Appl. No.: |
15/888317 |
Filed: |
February 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12599480 |
Aug 11, 2010 |
9884031 |
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PCT/US2008/005816 |
May 7, 2008 |
|
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15888317 |
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60924326 |
May 9, 2007 |
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Current U.S.
Class: |
514/21.1 ;
514/557; 514/616; 514/619; 514/646 |
Current CPC
Class: |
A61P 9/00 20180101; A61K
31/185 20130101 |
International
Class: |
A61K 31/185 20060101
A61K031/185 |
Claims
1. A method of treating a cardiac rhythm disorder in a subject,
comprising administering to said subject an inhibitor of a histone
deacetylase (HDAC), thereby treating a cardiac rhythm disorder in a
subject.
2. The method of claim 1, wherein said cardiac rhythm disorder
comprises an irregular cardiac rhythm.
3. The method of claim 1, wherein said cardiac rhythm disorder
comprises a rapid cardiac rhythm.
4. The method of claim 1, wherein said cardiac rhythm disorder
comprises a chaotic cardiac rhythm.
5. The method of claim 1, wherein said cardiac rhythm disorder is a
chronic cardiac rhythm disorder.
6. The method of claim 1, wherein said subject is further affected
with a ventricular hypertrophy disorder.
7. The method of claim 1, wherein said subject is further affected
with a diastolic dysfunction.
8. The method of claim 1, wherein said cardiac rhythm disorder is
an atrial fibrillation disorder.
9. The method of claim 1, wherein said inhibitor of an HDAC is
trichostatin A.
10. The method of claim 1, wherein said inhibitor of an HDAC is
selected from: a valproic acid, a suberoylanilide hydroxamic acid
(SAHA), an epoxyketone-containing cyclic tetrapeptide, a
non-epoxyketone-containing cyclic tetrapeptide, a benzamine,
depudecin, or any mixture thereof.
11. The method of claim 1, wherein the step of treating a cardiac
rhythm disorder in said subject, comprises restoring atrial
connexin distribution in said subject.
12. The method of claim 1, wherein the step of treating a cardiac
rhythm disorder in said subject, comprises reducing atrial collagen
content in said subject.
13. A method of reducing the incidence of a cardiac rhythm disorder
in a subject, comprising administering to said subject an inhibitor
of a histone deacetylase (HDAC), thereby reducing said incidence of
a cardiac rhythm disorder in a subject.
14. The method of claim 13, wherein said cardiac rhythm disorder
comprises an irregular cardiac rhythm.
15. The method of claim 13, wherein said cardiac rhythm disorder
comprises a rapid cardiac rhythm.
16. The method of claim 12, wherein said cardiac rhythm disorder
comprises a chaotic cardiac rhythm.
17. The method of claim 13, wherein said cardiac rhythm disorder is
a chronic cardiac rhythm disorder.
18. The method of claim 13, wherein said subject is affected with a
ventricular hypertrophy disorder.
19. The method of claim 13, wherein said subject is affected with a
diastolic dysfunction.
20. The method of claim 13, wherein said cardiac rhythm disorder is
an atrial fibrillation disorder.
21. The method of claim 13, wherein said inhibitor of an HDAC is
trichostatin A.
22. The method of claim 13, wherein said inhibitor of an HDAC is
selected from: a valproic acid, a suberoylanilide hydroxamic acid
(SAHA), an epoxyketone-containing cyclic tetrapeptide, a
non-epoxyketone-containing cyclic tetrapeptide, a benzamine,
depudecin, or any mixture thereof.
23. The method of claim 13, wherein said method comprises the step
of comprises reducing the incidence of atrial arrhythmogenesis in
said subject.
24. The method of claim 13, wherein said method comprises the step
of reducing atrial collagen content in said subject.
25. The method of claim 13, said method comprises the step of
restoring atrial connexin distribution in said subject.
26. A method of reducing atrial collagen content in a subject,
comprising administering to said subject an inhibitor of a histone
deacetylase (HDAC), thereby reducing said atrial collagen content
in a subject.
27. The method of claim 26, wherein said subject is affected with a
ventricular hypertrophy disorder.
28. The method of claim 26, wherein said subject is affected with a
diastolic dysfunction.
29. The method of claim 26, wherein said inhibitor of an HDAC is
trichostatin A.
30. The method of claim 26, wherein said inhibitor of an HDAC is
selected from: a valproic acid, a suberoylanilide hydroxamic acid
(SAHA), an epoxyketone-containing cyclic tetrapeptide, a
non-epoxyketone-containing cyclic tetrapeptide, a benzamine,
depudecin, or any mixture thereof.
31. A method of restoring an atrial connexin distribution in a
subject, comprising administering to said subject an inhibitor of a
histone deacetylase (HDAC), thereby restoring an atrial connexin
distribution in a subject.
32. The method of claim 31, wherein said subject is affected with a
ventricular hypertrophy disorder.
33. The method of claim 31, wherein said subject is affected with a
diastolic dysfunction.
34. The method of claim 31, wherein said inhibitor of an HDAC is
trichostatin A.
35. The method of claim 31, wherein said inhibitor of an HDAC is
selected from: a valproic acid, a suberoylanilide hydroxamic acid
(SAHA), an epoxyketone-containing cyclic tetrapeptide, a
non-epoxyketone-containing cyclic tetrapeptide, a benzamine,
depudecin, or any mixture thereof.
36. A method of reducing the incidence of atrial arrhythmogenesis
in a subject, comprising administering to said subject an inhibitor
of a histone deacetylase (HDAC), thereby reducing the incidence of
atrial arrhythmogenesis in a subject.
37. The method of claim 36, wherein said subject is further
affected with a ventricular hypertrophy disorder.
38. The method of claim 36, wherein said subject is further
affected with a diastolic dysfunction.
39. The method of claim 36, wherein said inhibitor of an HDAC is
trichostatin A.
40. The method of claim 36, wherein said inhibitor of an HDAC is
selected from: a valproic acid, a suberoylanilide hydroxamic acid
(SAHA), an epoxyketone-containing cyclic tetrapeptide, a
non-epoxyketone-containing cyclic tetrapeptide, a benzamine,
depudecin, or any mixture thereof.
41. The method of claim 36, wherein said method comprises the step
of restoring atrial connexin distribution in said subject.
42. The method of claim 36, wherein said method comprises the step
of reducing atrial collagen content in said subject.
Description
FIELD OF THE INVENTION
[0001] The present invention provides methods of ameliorating,
treating or reducing the extent of cardiac rhythm disorders, by
administering an inhibitor of histone deacetylase enzyme
(HDAC).
BACKGROUND OF THE INVENTION
[0002] Atrial fibrillation is an irregular, rapid, chaotic heart
rhythm disorder that is a chronic and debilitating medical
condition. It is the most common cardiac arrhythmia and accounts
for one-third of all hospitalizations for cardiac rhythm disorders
in the United States. Patients with atrial fibrillation experience
increased morbidity including lower exercise tolerance, frequent
palpitations and heart failure. Atrial fibrillation is particularly
prevalent in patients with heart failure and patients with both
diseases are at risk for increased mortality. In particular, left
ventricular hypertrophy and diastolic dysfunction are independent
risk factors for the development of atrial fibrillation and both
are associated with a higher incidence of atrial fibrillation.
Antiarrhythmic drug therapy for atrial fibrillation is relatively
ineffective for restoring and maintaining sinus rhythm and is
associated with multiple side effects. The discovery that ectopic
foci arising from the pulmonary veins trigger atrial fibrillation
has led to catheter-based pulmonary vein isolation as an effective
therapy for atrial fibrillation. Pulmonary vein isolation offers
benefits over antiarrhythmic drugs but present techniques are
associated with procedural risks and are less efficient in patients
with left ventricular hypertrophy.
[0003] In the setting of abnormal ventricular function, atrial
structural changes such as dilatation and fibrosis are prevalent
and promote arrhythmogenesis. Normalization of ventricular
function, by inhibiting angiotensin, appears to partially reverse
atrial structural changes and reduce atrial arrhythmogenesis.
Experimental and clinical evidence support the ability of
angiotensin-inhibition to reduce atrial fibrillation, however
evidence also exists that angiotensin-independent pathways
contribute to atrial structural remodeling and atrial
fibrillation.
SUMMARY OF THE INVENTION
[0004] The present invention provides, in one embodiment, a method
of treating a cardiac rhythm disorder in a subject, comprising
administering to a subject an inhibitor of a histone deacetylase
(HDAC), thereby treating a cardiac rhythm disorder in a
subject.
[0005] In another embodiment, the present invention provides a
method of reducing the incidence of a cardiac rhythm disorder in a
subject, comprising administering to a subject an inhibitor of a
histone deacetylase (HDAC), thereby reducing the incidence of a
cardiac rhythm disorder in a subject.
[0006] In another embodiment, the present invention provides a
method of reducing atrial collagen content in a subject, comprising
administering to a subject an inhibitor of a histone deacetylase
(HDAC), thereby reducing atrial collagen content in a subject.
[0007] In another embodiment, the present invention provides a
method of reducing the incidence of atrial arrhythmogenesis in a
subject, comprising administering to a subject an inhibitor of a
histone deacetylase (HDAC), thereby reducing the incidence of
atrial arrhythmogenesis in a subject.
BRIEF DESCRIPTION OF THE FIGURES
[0008] The foregoing summary, as well as the following detailed
description of the invention, will be better understood better when
read in conjunction with the appended drawings and tables. It
should be understood, however, that the invention is not limited to
the precise arrangements and instrumentalities shown.
[0009] FIGS. 1A-1C show a comparison of Hop transgenic mice
(Hop.sup.Tg) are compared with wild-type controls (WT), and
trichostatin A treated Hop transgenic mice (TSA-Hop). FIG. 1A shows
a micrograph of Mason's trichrome staining of an atrium. Scale Bar:
40.times. images =1 mm; 400.times. images=100 .mu.m. FIGS. 1B and
1C depict bar graphs showing atrial fibrosis (FIG. 1B) and
ventricular fibrosis (FIG. 1C) in the Hop.sup.Tg group compared
with wild-type controls, and trichostatin A Hop transgenic mice
(TSA-Hop). *P<0.05.
[0010] FIGS. 2A-2F depict bar graphs representing the levels of
cardiac angiotensin and atrial cytokines after over-expression of
Hop in the heart. FIG. 2A shows angiotensin II in WT and Hop.sup.Tg
hearts when assessed by enzyme-linked immunoabsorbent assays. FIG.
2B-F shows the relative protein expression of the activated form of
several mitogen-activated protein kinases and cytokines assessed by
Western-blot analysis in the atrium of Hop transgenic mice
(Hop.sup.Tg) compared with wild-type controls (WT) and Hop
transgenic mice treated with trichostatin A (TSA-Hop). Panel (B) is
phospho-JNK (46- and 54-kDa isoforms), panel (C) is
phospho-p38-MAPK, panel (D) is phospho-ERK1/2 (42- and 44-kDa
isoforms), panel (E) is activated TGF-.beta.1 (25-kDa) and panel
(F) is activated IL-1.beta. (28-kDa). Below the blots are
normalized band signal intensities to GAPDH (isoforms of ERK and
JNK were averaged for quantitative analysis). Blots are
representative of three separate experiments. *P<0.05 compared
to WT; .dagger.P<0.05 compared to TSA-Hop.sup.Tg.
[0011] FIGS. 3A-3B depict bar graphs representing connexin40
expression in left Hop transgenic mice and normalized by
trichostatin A (TSA). FIG. 3A shows immunoblot analysis of atrial
connexin40 in wild-type (WT), Hop transgenic (Hop) and trichostatin
A-treated Hop transgenic mice (TSA), while FIG. 3B show parallel
analysis for atrial connexin43 in wild-type , Hop transgenic and
trichostatin A-treated Hop transgenic mice. Blots are
representative of three separate experiments. *P<0.05 compared
to WT; .dagger.P<0.05 compared to TSA-Hop.sup.Tg.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention provides methods of ameliorating or
reducing the extent of cardiac rhythm disorders, by administering
an inhibitor of histone deacetylase enzyme (HDAC).
[0013] In one embodiment, the present invention provides a method
for treating a cardiac rhythm disorder in a subject comprising
administering an inhibitor of an HDAC. In another embodiment, the
present invention provides a method for ameliorating a cardiac
rhythm disorder in a subject comprising administering an inhibitor
of an HDAC. In another embodiment, the present invention provides a
method for reducing the incidence of a cardiac rhythm disorder in a
subject comprising administering an inhibitor of an HDAC.
[0014] In another embodiment, the present invention provides a
method of treating a cardiac rhythm disorder in a subject,
comprising administering to a subject an inhibitor of a histone
deacetylase (HDAC), thereby treating a cardiac rhythm disorder in a
subject. In another embodiment, the cardiac rhythm disorder
comprises an irregular cardiac rhythm. In another embodiment, the
cardiac rhythm disorder comprises a rapid cardiac rhythm. In
another embodiment, the cardiac rhythm disorder comprises a chaotic
cardiac rhythm.
[0015] In another embodiment, the present invention provides a
method of treating a cardiac arrhythmia. In another embodiment,
cardiac arrhythmia of the present invention comprises an
irregularity in the force of the heartbeat. In another embodiment,
cardiac arrhythmia of the present invention comprises an
irregularity in rhythm of the heartbeat. In another embodiment,
cardiac arrhythmia of the present invention is a group of
conditions in which the muscle contraction of the heart is
irregular or is faster or slower than normal.
[0016] In another embodiment, the present invention provides that
some arrhythmias are life-threatening medical emergencies that can
cause cardiac arrest and sudden death. In another embodiment, the
present invention provides that arrhythmias cause aggravating
symptoms, such as an awareness of a different heart beat, or
palpitation, which can be annoying. In another embodiment, the
present invention provides that some arrhythmias are benign and
normal.
[0017] In another embodiment, the present invention comprises sinus
arrhythmia. In another embodiment, sinus arrhythmia is the mild
acceleration followed by slowing of the normal rhythm that occurs
with breathing.
[0018] In another embodiment, the present invention provides that
arrhythmias that are due to fast, abnormal electrical activity can
cause tachycardias that are dangerous. In another embodiment, the
present invention provides that if the ventricles of the heart
experience one of these tachycardias for a long period of time,
there can be deleterious effects. In another embodiment, the
present invention provides that individuals sense a tachycardia as
a pounding sensation of the heart, known as palpitations. In
another embodiment, the present invention provides that if a
tachycardia lowers blood pressure it may cause lightheadedness or
dizziness, or even fainting (syncope). In another embodiment, the
present invention provides that if the tachycardia is too fast, the
pump function of the heart is impeded, which may lead to a sudden
death.
[0019] In another embodiment, the present invention provides that a
serious variety of arrhythmia is known as fibrillation. In another
embodiment, the present invention provides that fibrillation occurs
when the heart muscle begins a quivering motion due to disunity in
contractile cell function. In another embodiment, the present
invention provides that fibrillation affects the atrium (atrial
fibrillation) or the ventricle (ventricular fibrillation). In
another embodiment, the present invention provides that ventricular
fibrillation is imminently life-threatening.
[0020] In another embodiment, the present invention provides that
atrial fibrillation is the quivering, chaotic motion in the upper
chambers of the heart, known as the atria. In another embodiment,
the present invention provides that atrial fibrillation is due to
serious underlying medical conditions, and should be evaluated by a
physician. In another embodiment, the present invention provides
that ventricular fibrillation occurs in the ventricles of the
heart; it is always a medical emergency. In another embodiment, the
present invention provides that ventricular fibrillation (VF, or
V-fib) can lead to death within minutes.
[0021] In another embodiment, the present invention provides that
arrhythmias are often first detected by simple but nonspecific
means: auscultation of the heartbeat with a stethoscope, or feeling
for peripheral pulses. In another embodiment, the present invention
provides that a specific diagnostic test for assessment of heart
rhythm is the electrocardiogram (abbreviated ECG or EKG).
[0022] In another embodiment, the present invention further
comprises SADS, or sudden arrhythmia death syndrome. In another
embodiment, SADS is a term used to describe sudden death due to
cardiac arrest brought on by an arrhythmia. In another embodiment,
causes of SADS in young people are long QT syndrome, Brugada
syndrome, catecholaminergic polymorphic ventricular tachycardia and
hypertrophic cardiomyopathy and arrhythmogenic right ventricular
dysplasia ("arrhythmia"-causing, "right ventricle"-involving,
pre-cancerous malformation (bad-growth)). Each cause of SADS
represents a separate embodiment of the present invention.
[0023] In another embodiment, the present invention comprises
various arrhythmias. In another embodiment, the present invention
comprises atrial arrhythmias. In another embodiment, the present
invention comprises sinus bradycardia. In another embodiment, the
present invention comprises sinus tachycardia. In another
embodiment, the present invention comprises atrial fibrillation. In
another embodiment, the present invention comprises atrial flutter.
In another embodiment, the present invention comprises
supraventricular tachycardia. In another embodiment, the present
invention comprises premature atrial complex. In another
embodiment, the present invention comprises ventricular
arrhythmias. In another embodiment, the present invention comprises
idioventricular rhythm. In another embodiment, the present
invention comprises accelerated idioventricular rhythm. In another
embodiment, the present invention comprises ventricular
tachycardia. In another embodiment, the present invention comprises
ventricular fibrillation. In another embodiment, the present
invention comprises premature ventricular complex. In another
embodiment, the present invention comprises junctional arrhythmias.
In another embodiment, the present invention comprises junctional
rhythm. In another embodiment, the present invention comprises
junctional tachycardia. In another embodiment, the present
invention comprises premature junctional complex. In another
embodiment, the present invention comprises heart blocks, also
known as AV blocks. In another embodiment, the present invention
comprises first degree heart block, also known as PR prolongation.
In another embodiment, the present invention comprises second
degree heart block. In another embodiment, the present invention
comprises Type 1 second degree heart block, also known as Mobitz I
or Wenckebach. In another embodiment, the present invention
comprises Type 2 second degree heart block, also known as Mobitz
II. In another embodiment, the present invention comprises third
degree heart block, also known as complete heart block. Each
arrhythmia represents a separate embodiment of the present
invention.
[0024] In another embodiment, the present invention provides a
method of treating a cardiac rhythm disorder in a subject,
comprising administering to a subject an inhibitor of a histone
deacetylase (HDAC), thereby treating a cardiac rhythm disorder in a
subject. In another embodiment, the present invention provides a
method of reducing the incidence of a cardiac rhythm disorder in a
subject, comprising administering to a subject an HDAC inhibitor,
thereby reducing the incidence of a cardiac rhythm disorder in a
subject. In another embodiment, the present invention provides a
method of reducing atrial collagen content in a subject, comprising
administering to a subject an HDAC inhibitor, thereby reducing
atrial collagen content in a subject. In another embodiment, the
present invention provides a method of restoring an atrial connexin
distribution in a subject, comprising administering to a subject an
HDAC inhibitor, thereby restoring an atrial connexin distribution
in a subject. In another embodiment, the present invention provides
a method of reducing the incidence of atrial arrhythmogenesis in a
subject, comprising administering to a subject an HDAC inhibitor,
thereby reducing the incidence of atrial arrhythmogenesis in a
subject.
[0025] In another embodiment, the subject of the present invention
is further affected with a ventricular hypertrophy disorder. In
another embodiment, a ventricular hypertrophy disorder of the
present invention is a left ventricular hypertrophy (LVH). In
another embodiment, LVH is the thickening of the myocardium
(muscle) of the left ventricle of the heart. In another embodiment,
LVH is referred to as a pathological reaction to cardiovascular
disease. In another embodiment, LVH is a marker for disease
involving the heart. In another embodiment, LVH can is caused by
aortic stenosis, aortic insufficiency, and hypertension.
[0026] In another embodiment, the subject of the present invention
is further affected with a ventricular hypertrophy disorder. In
another embodiment, a ventricular hypertrophy disorder of the
present invention is a right ventricular hypertrophy (RVH). In
another embodiment, RVH is caused by pulmonary hypertension,
tetralogy of fallot, pulmonary valve stenosis, ventricular septal
defect (VSD), and high altitude.
[0027] In another embodiment, the subject of the present invention
is further affected with a diastolic dysfunction. In another
embodiment, diastolic dysfunction is caused due to the left
ventricle's inability to properly fill with blood during diastole.
In another embodiment, in the setting of a stiff left ventricle, it
is more difficult for blood to flow into it from the left atrium.
In another embodiment, any condition or process that leads to
stiffening of the left ventricle can lead to diastolic dysfunction.
In another embodiment, left ventricular stiffening is caused by
high blood pressure, aortic stenosis, scarred heart muscle,
diabetes, severe systolic dysfunction that has led to ventricular
dilation. In another embodiment, diastolic dysfunction is caused by
mitral stenosis.
[0028] In another embodiment, the cardiac rhythm disorder of the
present invention is an atrial fibrillation disorder also termed
auricular fibrillation. In another embodiment, atrial fibrillation
involves a rapid heart rate, in which the atria are stimulated to
contract in a very disorganized and abnormal manner In another
embodiment, atrial fibrillation is caused by disruption of the
normal functioning of the electrical conduction system of the
heart. In another embodiment, the atria are stimulated to contract
very quickly and differently from the normal activity originating
from the sinoatrial node. In another embodiment, this condition can
be caused by impulses which are transmitted to the ventricles in an
irregular fashion. In another embodiment, this condition can be
caused by impulses failing to be transmitted. In another
embodiment, this condition leads to an irregular (and usually fast)
pulse in atrial fibrillation.
[0029] In another embodiment, atrial fibrillation of the present
invention comprises atrial flutter. In another embodiment, in
atrial flutter the ventricles beat rapidly, but regularly. In
another embodiment, if the atrial fibrillation/flutter is part of a
condition called sick sinus syndrome, the sinus node does not work
properly, and the heart rate may alternate between slow and
fast.
[0030] In another embodiment, underlying causes of atrial
fibrillation and flutter include dysfunction of the sinus node and
a number of heart and lung disorders, including coronary artery
disease, rheumatic heart disease, mitral valve disorders,
pericarditis, and others. In another embodiment, underlying causes
of atrial fibrillation further comprise hyperthyroidism,
hypertension, or heavy alcohol consumption. Each cause of atrial
fibrillation represents a separate embodiment of the present
invention.
[0031] In another embodiment, the cardiac rhythm disorder of the
present invention can be diagnosed by echocardiogram. In another
embodiment, the cardiac rhythm disorder of the present invention
can be diagnosed by nuclear imaging tests. In another embodiment,
the cardiac rhythm disorder of the present invention can be
diagnosed by coronary angiography. In another embodiment, the
cardiac rhythm disorder of the present invention can be diagnosed
by exercise treadmill ECG. In another embodiment, the cardiac
rhythm disorder of the present invention can be diagnosed by
electrophysiologic study (EPS). Each method of diagnosis represents
a separate embodiment of the present invention.
[0032] In another embodiment, treating a cardiac rhythm disorder of
the present invention comprises restoring atrial connexin
distribution in a subject. In another embodiment, disrupted cardiac
connexins alter coordinated electrical activation and conduction
through myocardial tissue. In another embodiment, altered
distribution of connexins is involved in the pathophysiologic
mechanism of atrial fibrillation. In another embodiment,
alterations in the tissue distribution or function of cardiac
connexins predispose to cardiac arrhythmias.
[0033] In another embodiment, treating a cardiac rhythm disorder of
the present invention comprises reducing atrial collagen content in
a subject. In another embodiment, increased collagen deposition is
associated with decreased contractile function, and abnormal
systolic and diastolic Ca.sup.2+ handling. In another embodiment,
abnormalities in action potential propagation and Ca.sup.2+
handling contribute to the initiation of atrial arrhythmias. In
another embodiment, atrial fibrosis and collagen deposition
contribute to the pathogenesis of atrial fibrillation. In another
embodiment, the degree of fibrosis is associated with an increase
in the expression of extracellular matrix proteins.
[0034] In another embodiment, atrial fibrillation is an irregular,
rapid, chaotic heart rhythm disorder that is a chronic and
debilitating medical condition. In another embodiment, patients
with atrial fibrillation experience increased morbidity including
lower exercise tolerance, frequent palpitations and heart failure.
In another embodiment, atrial fibrillation is particularly
prevalent in patients with heart failure and patients with both
diseases are at risk for increased mortality. In another
embodiment, left ventricular hypertrophy and diastolic dysfunction
are independent risk factors for the development of atrial
fibrillation and both are associated with a higher incidence of
atrial fibrillation. In another embodiment, anti-arrhythmic drug
therapy for atrial fibrillation is relatively ineffective for
restoring and maintaining sinus rhythm and is associated with
multiple side effects.
[0035] In another embodiment, in the setting of abnormal
ventricular function, atrial structural changes such as dilatation
and fibrosis are prevalent and promote arrhythmogenesis.
[0036] In another embodiment, the specific histone-deacetylase
inhibitor, trichostatin A (TSA), significantly reduces atrial
fibrillation. In another embodiment, the specific
histone-deacetylase inhibitor, trichostatin A (TSA), significantly
reduces atrial fibrillation inducibility in a mouse model of left
ventricular hypertrophy. In another embodiment, cardiac hypertrophy
induced by Hop over-expression is associated with myocardial
fibrosis and shortening of atrial refractoriness but does not
appear to alter local angiotensin II levels. In another embodiment,
cardiac hypertrophy is characterized by the absence of increased
angiotensin II, TGF-.beta.1 and phosphorylated ERK. In another
embodiment, cardiac hypertrophy is characterized by elevated atrial
collagen content and elevated fibrosis. In another embodiment,
cardiac hypertrophy is characterized by altered connexin
distribution. In another embodiment, atrial fibrillation is
characterized by elevated atrial collagen content. In another
embodiment, atrial fibrillation is characterized by altered
connexin distribution.
[0037] In another embodiment, the methods of the present invention
provide that HDAC inhibitor of the invention reduces atrial
collagen content. In another embodiment, the methods of the present
invention provide that HDAC inhibitor of the invention reduces
fibrosis. In another embodiment, the methods of the present
invention provide that HDAC inhibitor of the invention reduces
arrhythmogenesis. In another embodiment, the methods of the present
invention provide that HDAC inhibitor of the invention restores
atrial connexin distribution. In another embodiment, the methods of
the present invention provide that HDAC inhibitor of the invention
does not affect atrial refractoriness. In another embodiment, the
methods of the present invention provide that HDAC inhibitor of the
invention does not affect action potential duration. In another
embodiment, the methods of the present invention provide that HDAC
inhibitor of the invention reduces atrial arrhythmogenesis in the
setting of left ventricular hypertrophy by reversing atrial
structural remodeling and fibrosis. In another embodiment, the
methods of the present invention provide that administering HDAC
inhibitor to a subject in need reduces the morbidity of cardiac
arrhythmia in a subject. In another embodiment, the methods of the
present invention provide that administering HDAC inhibitor to a
subject in need reduces the morbidity of atrial fibrillation in a
subject.
[0038] In another embodiment, the methods of the present invention
provide that the HDAC inhibitor of the present invention is TSA. In
another embodiment, the methods of the present invention provide
that TSA reduces atrial collagen content. In another embodiment,
the methods of the present invention provide that TSA reduces
fibrosis. In another embodiment, the methods of the present
invention provide that TSA reduces arrhythmogenesis. In another
embodiment, the methods of the present invention provide that TSA
restores atrial connexin distribution. In another embodiment, the
methods of the present invention provide that TSA does not affect
atrial refractoriness. In another embodiment, the methods of the
present invention provide that TSA does not affect action potential
duration. In another embodiment, the methods of the present
invention provide that TSA reduces atrial arrhythmogenesis in the
setting of left ventricular hypertrophy by reversing atrial
structural remodeling and fibrosis. In another embodiment, the
methods of the present invention provide that administering TSA to
a subject in need reduces the morbidity of cardiac arrhythmia in a
subject. In another embodiment, the methods of the present
invention provide that administering TSA to a subject in need
reduces the morbidity of atrial fibrillation in a subject.
[0039] In one embodiment, the HDAC inhibitor used in methods of the
present invention is valproate. In another embodiment, the HDAC
inhibitor is trichostatin. In another embodiment, the HDAC
inhibitor is trichostatin A (TSA). In another embodiment, the HDAC
inhibitor is Scriptaid. In another embodiment, the HDAC inhibitor
is a PXD101. In another embodiment, the HDAC inhibitor is a
benzamine
[0040] In another embodiment, the HDAC inhibitor is a short-chain
fatty acid. In another embodiment, the short-chain fatty acid is a
butyrate. In another embodiment, the short-chain fatty acid is a
phenylbutyrate. In another embodiment, the short-chain fatty acid
is valproate. In another embodiment, the HDAC inhibitor is valproic
acid. In another embodiment, the short-chain fatty acid is any
other short-chain fatty acid that exhibits HDAC inhibitory
activity. Each short-chain fatty acid represents a separate
embodiment of the present invention.
[0041] In another embodiment, the HDAC inhibitor is a hydroxamic
acid. In one embodiment, the hydroxamic acid is a suberoylanilide
hydroxamic acid (SAHA). In another embodiment, the hydroxamic acid
is a derivative of a SAHA. In another embodiment, the hydroxamic
acid is oxamflatin. In another embodiment, the hydroxamic acid is
ABHA. In another embodiment, the hydroxamic acid is pyroxamide. In
another embodiment, the hydroxamic acid is a propenamide In another
embodiment, the hydroxamic acid is any other hydroxamic acid that
exhibits HDAC inhibitory activity. Each hydroxamic acid represents
a separate embodiment of the present invention.
[0042] In another embodiment, the HDAC inhibitor is an
epoxyketone-containing cyclic tetrapeptide. In one embodiment, the
epoxyketone-containing cyclic tetrapeptide is a trapoxin. In
another embodiment, the epoxyketone-containing cyclic tetrapeptide
is an HC-toxin. In another embodiment, the epoxyketone-containing
cyclic tetrapeptide is chlamydocin. In another embodiment, the
epoxyketone-containing cyclic tetrapeptide is ABHA. In another
embodiment, the epoxyketone-containing cyclic tetrapeptide is
pyroxamide. In another embodiment, the epoxyketone-containing
cyclic tetrapeptide is a diheteropeptin. In another embodiment, the
epoxyketone-containing cyclic tetrapeptide is WF-3161. In another
embodiment, the epoxyketone-containing cyclic tetrapeptide is a
Cyl-2. In another embodiment, the epoxyketone-containing cyclic
tetrapeptide is a Cyl-1. In another embodiment, the
epoxyketone-containing cyclic tetrapeptide is any other
epoxyketone-containing cyclic tetrapeptide that exhibits HDAC
inhibitory activity. Each epoxyketone-containing cyclic
tetrapeptide represents a separate embodiment of the present
invention.
[0043] In another embodiment, the HDAC inhibitor is a
non-epoxyketone-containing cyclic tetrapeptide. In one embodiment,
the non-epoxyketone-containing cyclic tetrapeptide is FR901228. In
another embodiment, the non-epoxyketone-containing cyclic
tetrapeptide is an apicidin. In another embodiment, the
non-epoxyketone-containing cyclic tetrapeptide is a
cyclic-hydroxamic-acid-containing peptide (CHAP). In another
embodiment, the non-epoxyketone-containing cyclic tetrapeptide is
any other non-epoxyketone-containing cyclic tetrapeptide that
exhibits HDAC inhibitory activity. Each non-epoxyketone-containing
cyclic tetrapeptide represents a separate embodiment of the present
invention.
[0044] In another embodiment, the HDAC inhibitor is a benzamide. In
one embodiment, the benzamide is MS-275 (MS-27-275). In another
embodiment, the benzamide is CI-994. In another embodiment, the
non-epoxyketone-containing cyclic tetrapeptide is any other
non-epoxyketone-containing cyclic tetrapeptide that exhibits HDAC
inhibitory activity. Each non-epoxyketone-containing cyclic
tetrapeptide represents a separate embodiment of the present
invention.
[0045] In another embodiment, the HDAC inhibitor is a depudecin. In
another embodiment, the HDAC inhibitor is an organosulfur compound.
In another embodiment, the HDAC inhibitor is any other HDAC
inhibitor known in the art. Each possibility represents a separate
embodiment of the present invention.
[0046] In another embodiment, a method of present invention further
comprises administering to the subject an additional
anti-arrhythmia compound. In another embodiment, the HDAC inhibitor
of the present invention and the additional anti-arrhythmia
compound are formulated and/or administered as a combination. In
another embodiment, the combination of an HDAC inhibitor of the
present invention and an additional anti-arrhythmia compound are
administered in a single dosage form. In another embodiment, the
preferred dose of the additional anti-arrhythmia compound is known
to a person of skill in the art. In another embodiment, the
preferred dose of the additional anti-arrhythmia compound is set in
the 2007 Physicians' Desk Reference.RTM. (PDR.RTM.).
[0047] In another embodiment, the additional anti-arrhythmia
compound treats atrial fibrillation. In another embodiment, the
HDAC inhibitors of the present invention are administered while
treating arrhythmia with electrical cardioversion or intravenous
(IV) drugs such as dofetilide, amiodarone, or ibutilide. In another
embodiment, additional anti-arrhythmia compounds are selected from
but not limited to: beta-blockers, calcium channel blockers,
digitalis or other medications (such as anti-arrhythmic drugs). In
another embodiment, additional anti-arrhythmia compounds are
selected from but not limited to: blood thinners, such as heparin
or Coumadin. In another embodiment, additional anti-arrhythmia
treatments include a catheter procedure on the atria called
radiofrequency ablation. In another embodiment, additional
anti-arrhythmia treatments include an atrial pacemaker implanted
under the skin to regulate the heart rhythm. In another embodiment,
additional anti-arrhythmia compounds are selected from but not
limited to: digoxin, atenolol, metoprolol, propranolol, amiodarone,
disopyramide, ibutilide, verapamil, diltiazam, sotalol, flecainide,
procainamide, quinidine, or propafenone.
[0048] In another embodiment of methods of the present invention,
the dose of the HDAC inhibitor is within a range of about 0.1-100
mg/day. In another embodiment, the dose is between about 0.5-50
mg/day. In another embodiment, the dose is between about 1-30
mg/day. In another embodiment, the dose is between about 2-20
mg/day. In another embodiment, the dose is between about 4-15
mg/day. In another embodiment, the dose is between about 6-10
mg/day.
[0049] In another embodiment, the dose is about 0.1 mg/day. In
another embodiment, the dose is about 0.15 mg/day. In another
embodiment, the dose is about 0.2 mg/day. In another embodiment,
the dose is about 0.3 mg/day. In another embodiment, the dose is
about 0.5 mg/day. In another embodiment, the dose is about 1
mg/day. In another embodiment, the dose is about 1.5 mg/day. In
another embodiment, the dose is about 2 mg/day. In another
embodiment, the dose is about 3 mg/day. In another embodiment, the
dose is about 5 mg/day. In another embodiment, the dose is about 7
mg/day. In another embodiment, the dose is about 10 mg/day. In
another embodiment, the dose is about 15 mg/day. In another
embodiment, the dose is about 20 mg/day. In another embodiment, the
dose is about 30 mg/day. In another embodiment, the dose is about
50 mg/day. In another embodiment, the dose is about 70 mg/day. In
another embodiment, the dose is about 100 mg/day.
[0050] In another embodiment, the dose is about 0.1 mg. In another
embodiment, the dose is about 0.15 mg. In another embodiment, the
dose is about 0.2 mg. In another embodiment, the dose is about 0.3
mg. In another embodiment, the dose is about 0.5 mg. In another
embodiment, the dose is about 1 mg. In another embodiment, the dose
is about 1.5 mg. In another embodiment, the dose is about 2 mg. In
another embodiment, the dose is about 3 mg. In another embodiment,
the dose is about 5 mg. In another embodiment, the dose is about 7
mg. In another embodiment, the dose is about 10 mg. In another
embodiment, the dose is about 15 mg. In another embodiment, the
dose is about 20 mg. In another embodiment, the dose is about 30
mg. In another embodiment, the dose is about 50 mg. In another
embodiment, the dose is about 70 mg. In another embodiment, the
dose is about 100 mg. In another embodiment, the dose administered
the frequency of administration and the duration of the treatment
will vary. In another embodiment, the dose administered the
frequency of administration and the duration of the treatment will
vary as a function of the condition of the patient and is
determined according to standard clinical procedures known to the
practitioner skilled in the relevant art. Each dose or range
thereof represents a separate embodiment of the present
invention.
[0051] "Treating" a disease or disorder refers, in one embodiment,
to arresting the development of the disease or disorder. In another
embodiment, "treating" refers to reversing the development of the
disease or disorder. In another embodiment, "treating" refers to
slowing the development of the disease or disorder. In another
embodiment, "treating" refers to alleviating at least one symptom
of the disease or disorder. Each possibility represents a separate
embodiment of the present invention.
[0052] In one embodiment, an active compound of a method of the
present invention is administered systemically. In another
embodiment, the compound is administered locally.
[0053] The pharmaceutical composition comprising an HDAC inhibitor
of the invention, in one embodiment, administered to a subject by
any method known to a person skilled in the art, such as
parenterally, paracancerally, transmucosally, transdermally,
intramuscularly, intravenously, intradermally, subcutaneously,
intraperitonealy, intraventricularly, intracranially,
intravaginally or intratumorally.
[0054] In another embodiment, the subject of the present invention
is a human subject in need of the methods of the present invention.
In another embodiment, the subject of the present invention is an
animal in need of the methods of the present invention. In another
embodiment, the subject of the present invention is a test animal
wherein the methods of the present invention are applied to.
[0055] In another embodiment, the pharmaceutical compositions are
administered orally, and thus are formulated in a form suitable for
oral administration, i.e. as a solid or a liquid preparation.
Suitable solid oral formulations include, for example, tablets,
capsules, pills, granules, pellets and the like. Suitable liquid
oral formulations include solutions, suspensions, dispersions,
emulsions, oils and the like. In another embodiment of the present
invention, the HDAC inhibitor is formulated in a capsule. In
accordance with this embodiment, the compositions of the present
invention comprise, in addition to the HDAC inhibitor an inert
carrier or diluent, and/or a hard gelating capsule.
[0056] In another embodiment, the pharmaceutical compositions are
administered by intravenous, intraarterial, or intramuscular
injection of a liquid preparation. Suitable liquid formulations
include solutions, suspensions, dispersions, emulsions, oils and
the like. In one embodiment, the pharmaceutical compositions are
administered intravenously, and are thus formulated in a form
suitable for intravenous administration. In another embodiment, the
pharmaceutical compositions are administered intraarterially, and
are thus formulated in a form suitable for intraarterial
administration. In another embodiment, the pharmaceutical
compositions are administered intramuscularly, and are thus
formulated in a form suitable for intramuscular administration.
[0057] In another embodiment, the pharmaceutical compositions are
administered topically to body surfaces, and thus are formulated in
a form suitable for topical administration. Suitable topical
formulations include gels, ointments, creams, lotions, drops and
the like. For topical administration, the HDAC inhibitor of the
present invention and its physiologically tolerated derivatives
such as salts, esters, N-oxides, and the like is prepared and
applied as solutions, suspensions, or emulsions in a
physiologically acceptable diluent with or without a pharmaceutical
carrier.
[0058] Further, in another embodiment, the pharmaceutical
compositions are administered as a suppository, for example a
rectal suppository or a urethral suppository. Further, in another
embodiment, the pharmaceutical compositions are administered by
subcutaneous implantation of a pellet. In a further embodiment, the
pellet provides for controlled release of the HDAC inhibitor over a
period of time.
[0059] Pharmaceutically acceptable carriers or diluents are well
known to those skilled in the art. The carrier or diluent is, in
one embodiment, a solid carrier or diluent for solid formulations,
a liquid carrier or diluent for liquid formulations, or mixtures
thereof.
[0060] Solid carriers/diluents include, but are not limited to, a
gum, a starch (e.g. corn starch, pregeletanized starch), a sugar
(e.g., lactose, mannitol, sucrose, and dextrose), a cellulosic
material (e.g. microcrystalline cellulose), an acrylate (e.g.
polymethylacrylate), calcium carbonate, magnesium oxide, talc, or
mixtures thereof.
[0061] For liquid formulations, pharmaceutically acceptable
carriers are aqueous or non-aqueous solutions, suspensions,
emulsions or oils. Examples of non-aqueous solvents are propylene
glycol, polyethylene glycol, and injectable organic esters such as
ethyl oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Examples of oils are those of petroleum, animal, vegetable,
or synthetic origin, for example, peanut oil, soybean oil, mineral
oil, olive oil, sunflower oil, and fish-liver oil.
[0062] Parenteral vehicles (for subcutaneous, intravenous,
intraarterial, or intramuscular injection) include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's and fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers such as those based
on Ringer's dextrose, and the like. Examples are sterile liquids
such as water and oils, with or without the addition of a
surfactant and other pharmaceutically acceptable adjuvants. In
general, water, saline, aqueous dextrose and related sugar
solutions, and glycols such as propylene glycols or polyethylene
glycol are preferred liquid carriers, particularly for injectable
solutions. Examples of oils are those of petroleum, animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.
[0063] In another embodiment, the compositions further comprise
binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl
cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl
cellulose, povidone), disintegrating s (e.g. cornstarch, potato
starch, alginic acid, silicon dioxide, croscarmelose sodium,
crospovidone, guar gum, sodium starch glycolate), buffers (e.g.,
Tris-HCI., acetate, phosphate) of various pH and ionic strength,
additives such as albumin or gelatin to prevent absorption to
surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile
acid salts), protease inhibitors, surfactants (e.g. sodium lauryl
sulfate), permeation enhancers, solubilizers (e.g., glycerol,
polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite, butylated hydroxyanisole), stabilizers (e.g.
hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity
increasing s(e.g. carbomer, colloidal silicon dioxide, ethyl
cellulose, guar gum), sweetners (e.g. aspartame, citric acid),
preservatives (e.g., Thimerosal, benzyl alcohol, parabens),
lubricants (e.g. stearic acid, magnesium stearate, polyethylene
glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon
dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate),
emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl
sulfate), polymer coatings (e.g., poloxamers or poloxamines),
coating and film forming s (e.g. ethyl cellulose, acrylates,
polymethacrylates) and/or adjuvants.
[0064] In one embodiment, the pharmaceutical compositions provided
herein are controlled release compositions, i.e. compositions in
which the HDAC inhibitor is released over a period of time after
administration. Controlled or sustained release compositions
include formulation in lipophilic depots (e.g. fatty acids, waxes,
oils). In another embodiment, the composition is an immediate
release composition, i.e. a composition in which all of the HDAC
inhibitor is released immediately after administration.
[0065] In another embodiment, the pharmaceutical composition is
delivered in a controlled release system. For example, the
composition is administered using intravenous infusion, an
implantable osmotic pump, a transdermal patch, liposomes, or other
modes of administration. In one embodiment, a pump is used (Langer,
supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald
et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.
321:574 (1989). In another embodiment, polymeric materials can be
used. In yet another embodiment, a controlled release system can be
placed in proximity to the therapeutic target, i.e., the brain,
thus requiring only a fraction of the systemic dose (e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984). Other controlled release systems are discussed in
the review by Langer (Science 249:1527-1533 (1990).
[0066] The preparation of pharmaceutical compositions which contain
an active component is well understood in the art, for example by
mixing, granulating, or tablet-forming processes. The active
therapeutic ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. For oral administration, the HDAC inhibitor or its
physiologically tolerated derivatives such as salts, esters,
N-oxides, and the like are mixed with additives customary for this
purpose, such as vehicles, stabilizers, or inert diluents, and
converted by customary methods into suitable forms for
administration, such as tablets, coated tablets, hard or soft
gelatin capsules, aqueous, alcoholic or oily solutions. For
parenteral administration, the HDAC inhibitor or its
physiologically tolerated derivatives such as salts, esters,
N-oxides, and the like are converted into a solution, suspension,
or emulsion, if desired with the substances customary and suitable
for this purpose, for example, solubilizers or other.
[0067] In another embodiment, the active component is formulated
into the composition as neutralized pharmaceutically acceptable
salt forms. Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of the
polypeptide or antibody molecule), which are formed with inorganic
acids such as, for example, hydrochloric or phosphoric acids, or
such organic acids as acetic, oxalic, tartaric, mandelic, and the
like. Salts formed from the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such
organic bases as isopropylamine, trimethylamine, 2-ethylamino
ethanol, histidine, procaine, and the like.
[0068] In another embodiment, the salts of the HDAC inhibitor are
pharmaceutically acceptable salts. Other salts are, in one
embodiment, useful in the preparation of the compounds according to
the invention or of their pharmaceutically acceptable salts.
Suitable pharmaceutically acceptable salts of the compounds of this
invention include acid addition salts which may, for example, be
formed by mixing a solution of the compound according to the
invention with a solution of a pharmaceutically acceptable acid
such as hydrochloric acid, sulphuric acid, methanesulphonic acid,
fumaric acid, maleic acid, succinic acid, acetic acid, benzoic:
acid, oxalic acid, citric acid, tartaric acid, carbonic acid or
phosphoric acid.
[0069] In one embodiment, the HDAC inhibitor is administered prior
to the arrhythmia or arrhythmia-inducing event. In another
embodiment, the HDAC inhibitor is administered concomitantly with
same. In another embodiment, the HDAC inhibitor is administered
after same.
[0070] In another embodiment, the HDAC inhibitor is administered
within 10 minutes after the arrhythmia or arrhythmia-inducing. In
another embodiment, the interval after same is 20 minutes.
[0071] In another embodiment, the interval is 30 minutes. In
another embodiment, the interval is 40 minutes. In another
embodiment, the interval is 50 minutes. In another embodiment, the
interval is 1 hour. In another embodiment, the interval is 1.5
hours. In another embodiment, the interval is 2 hours. In another
embodiment, the interval is 3 hours.
[0072] Each time of administration of the HDAC inhibitor represents
a separate embodiment of the present invention.
[0073] HDAC inhibition assays are well known in the art, and are
described, e.g. in Kang J et al (Chem Biol Interact. 2004 Jul. 20;
148(3): 115-23) and in Liu C et al (Methods Mol Biol. 2004; 287:
87-97). Each method of assessing inhibition of HDAC represents a
separate embodiment of the present invention.
[0074] Thus, novel compounds having utility in treating and
reducing the incidence of cardiac rhythm disorders can be used
according to the methods of the present invention.
Materials and Experimental Methods
Materials
[0075] Trichostatin A was obtained from Sigma-Aldrich.
Histology and Morphology
[0076] Hearts were excised from, washed in 37.degree. C. Dulbecco's
1.times. PBS, arrested in 50 mmol/L KCl, and fixed in 4% formalin.
Fixed hearts were cut transversely and sectioned serially from
ventricular apex to base. Two sections (5 .mu.m each) were retained
at the beginning of each 50-.mu.m step. Sections were stained with
Masson's trichrome stain for collagen as a marker of fibrosis.
Quantification of the proportion of fibrosis in each heart was then
performed with scientific imaging software (Image-J, National
Institutes of Health)) and high-resolution serial scanning
technique. Histological sections from the entire left ventricle and
both atrium were used for analysis from three different hearts.
Electrophysiology Study
[0077] An in-vivo electrophysiology study was performed in each
mouse to assess atrial and ventricular conduction, refractoriness,
and arrhythmia inducibility. Surface ECG recordings and complete in
vivo electrophysiological studies (EPS) were obtained from
Hop.sup.Tg mice (14-18 weeks old), age-matched non-transgenic
littermates and TSA-treated Hop.sup.Tg mice. Each mouse was
anesthetized with pentobarbital (0.033 mg/kg IP), and multi-lead
ECGs were obtained using 26-gauge subcutaneous electrodes.
Temperature was maintained at 32-33.degree. C. A jugular vein
cutdown was performed and an octapolar 2-French electrode catheter
(CIBer mouse-EP; NuMED, Inc) placed in the right atrium and
ventricle under electrogram guidance to confirm catheter position.
A programmed digital stimulator (DTU-215A, Bloom Associates Ltd)
was used to deliver electrical impulses at approximately twice
diastolic threshold. Surface ECG and intracardiac electrograms were
simultaneously displayed on a multichannel oscilloscope recorder
(Bard Electrophysiology, Inc) at a digitization rate of 2 kHz and
stored on optical media for offline analysis. ECG channels were
filtered from 0.5 to 250 Hz and intracardiac electrograms were
filtered from 5 to 400 Hz. ECG intervals were measured by two
independent observers blinded to the animal's genotype.
ECG Measurements
[0078] The PR, QRS, RR, and QT intervals were measured in 6
surface-limb ECG leads by 2 independent observers, who were both
blinded to the animals' genotype. Intracardiac recordings were
obtained during identical pacing and programmed electrical
stimulation protocols for all mice by experienced
investigators.
Isolation of Adult Atrial Myocytes
[0079] Mice were heparizined, anesthetized with pentobarbital 0.033
milligram [mg]/kilogram [kg] and hearts excised through a
sternotomy. Hearts were mounted on a Langendorf apparatus, perfused
with Tyrode's solution (composition as in Ref. 21) at 3.0-3 5
milliliter [ml]/minute [min] at a temperature of
36.degree.-37.degree. C. Hearts were perfused with Ca.sup.2+-free
Tyrode's solution for 6 min., followed by 12-15 min. of perfusion
with Ca.sup.2+-free Tyrode's solution containing: collagenase B,
collagenase D (Roche Chemical Co.) and protease (Fraction IV, Sigma
Chemical Co.). When the hearts appeared pale and flaccid they were
removed from the Langendorf apparatus, the atria were dissected
away and kept in Ca.sup.2+-free Tyrode's solution with 1 mg/ml of
bovine serum albumin (Fraction XIV, Sigma Chemical Co.). Sections
of atrial tissue were then triturated gently with a Pasteur pipette
to dissociate individual myocytes.
Quantitative Confocal Immunodetection of Atrial Connexins
[0080] The area of immunoreactive signal in discrete spots from
isolated atrial myocytes was quantified using laser scanning
confocal microscopy. Scanning of single optical slices (<1
.mu.m) by confocal laser scanning microscopy was done on the Leica
TCS SP2 system using the spectral detection set for simultaneous
fluorescence imaging of Alexa488 and DIC imaging with transmitted
light to obtain the cell outline. Sections were acquired through
ten randomly selected cells from each group (WT, Hop.sup.Tg and
TSA-Hop.sup.Tg) using the 40.times., 1.25 NA oil immersion
objective at zoom factors ranging from 2 to 3. Areas were
quantified using the count nuclei application in the Metamorph
software package (Molecular Devices). The total area of each atrial
myocyte was calculated from the DIC images and the area of the
connexin immunoreactive spots was calculated from the Alexa488
fluorescent images.
Lysis of Atrial Myocytes and Histone Isolation
[0081] Isolated atrial myocytes were lysed in Triton X/deoxycholate
buffer (20 millimolar [mM] HEPES, pH 7.2, 1% Triton X-100, 1%
sodium deoxycholate, 100 mM sodium chloride, 50 mM sodium fluoride,
5 mM EDTA, 100 .quadrature.M sodium molybdate, with protease
inhibitors), and insoluble material was removed by a 15 minute
(min) centrifugation in a microfuge, and the supernatant was
subjected to 100,000.times.g, 30 min. centrifugation. The pellet
was extracted with 9 molar urea, and the resulting pellet extracted
with 0.3 molar HCl.
Electrophoresis and Western Blotting
[0082] Adult mouse atrium and ventricle were dissected and
homogenized in buffer containing 1% Igepal surfactant, 0.5%
deoxycholate sodium, 2% SDS, 5 mmol EDTA (pH=7.4), 1% protease
inhibitor cocktail tablet (Roche) with and without phosphatase
inhibitor cocktail (Sigma-Aldrich). Samples were cleared of debris
by centrifugation at 5000 g for 10 min. and the protein
concentration was determined using a BCA protein assay kit
(Pierce). Protein samples were run on a 4-12% gradient pre-cast
SDS-PAGE gel (Invitrogen), and after electrophoresis were
transferred onto PVDF membranes (Millipore) using 0.1% acetic
acid/10% methanol as a transfer buffer. Membranes were incubated
with primary antibodies in 5% milk and PBS overnight at 4.degree.
C. and then washed for 5 min, 4 times with TBS-T solution. The
following primary antibodies were all used at a concentration of
1:1000 and consisted of: mouse anti-GAPDH monoclonal antibody
(Chemicon), mouse anti-TGF.beta.1 monoclonal antibody (Sigma),
rabbit anti-phospho-ERK1/2 polyclonal antibody (Cell Signaling),
mouse anti-phospho-JNK monoclonal antibody (Santa Cruz), rabbit
anti-phospho-p38-MAPK polyclonal antibody (Santa Cruz), goat
anti-IL-1.beta. polyclonal antibody (Santa Cruz), goat
anti-connexin40 polyclonal antibody (Santa Cruz), rabbit
anti-connexin43 polyclonal antibody (Zymed) and anti-acetyl H3
antibody (Upstate Charlottesville, Va.) which recognizes
acetyl-Lys9 H3 peptides. Membranes were then incubated with
secondary antibody (1:250, Western Breeze, Invitrogen) for 45 min.
at room temperature and subsequently washed in TBS-T for 5 min. 4
times. Results were visualized by enhanced chemiluminescence using
a commercially available kit (Western Breeze, Invitrogen) and data
were recorded on BioMax-MR film (Kodak, Rochester, N.Y.).
Densitometry was determined using the Image-J software package
(National Institutes of Health).
Enzyme-Linked Immunosorbent Assay
[0083] Whole hearts were suspended in 1 molar acetic acid,
homogenized and centrifuged at 15,000.times.g for 30 minutes at
4.degree. C. Supernatants were dried, reconstituted with 0.1%
trifluoroacetic acid and purified on a C18 Sep-Pak column (Waters
Associates). This fraction was eluted from the column with 30%
acetonitrile in 5 mL of 0.1% trifluoroacetic acid, dried and
dissolved in 0.25 mL Tris-buffered saline plus 0.1% Tween 80 (TBST)
solution. Protein concentration was determined by the Bradford
method and 200 .mu.g samples were analyzed in a microtiter plate
using an anti-angiotensin II antibody (Peninsula ELISA) and
biotinylated angiotensin II as a tracer. The microtiter plate was
washed 5 times with TBST and treated with streptavidin-horseradish
peroxidase. The color reaction was developed with 100 .mu.L of
tetramethylbenzidine substrate and terminated by addition of 2
normal HCl. The absorbance was recorded at 450 nanometers and
angiotensin II concentration was calculated from the standard curve
generated each time.
Invasive Hemodynamic Measurements
[0084] Hop.sup.Tg mice 14-18 weeks old were compared with wild-type
littermates and TSA treated Hop.sup.Tg mice. Anesthesia was induced
with 3% isoflurane and maintained by ventilation with 0.75%
isoflurane. Temperature was maintained at 36-37.degree. C.
Intracardiac pressures were recorded using a 1.4 French
micromanometer catheter (Micro-Tip SPR-671; Millar Instruments)
that was zeroed prior to making measurements on each animal in a
saline bath. The catheter was inserted via the right carotid artery
to record intracardiac pressures and digitized at 2-kHz using a
PowerLab/16 SP A/D converter (ADInstruments Ltd.).
Statistical Analyses
[0085] All continuous variables, were compared with those of sex-
and age-matched control mice, with data presented as the
mean.+-.SD. Continuous variables, such as ECG intervals, cardiac
conduction properties, intracardiac pressures and area of fibrosis
were compared by two-way ANOVA using a commercially available
statistical software package (SPSS 12.0). Test of significance
between groups was performed using Bonferroni's multiple
comparisons tests. The numbers of arrhythmic episodes were assumed
to have a Poisson distribution and the Kolmogorov-Smirnov test was
used to assess statistical significance between groups. A value of
P<0.05 was considered statistically significant.
EXAMPLE 1
Trichostatin a Reverses Inducible Atrial Arrhythmia
[0086] Trichostatin A 0.6 mg/kg daily in a volume of 0.2 mL was
given by intraperitoneal injection for 14 consecutive days prior to
cardiac electrical stimulation in Hop transgenic mice (Hop.sup.Tg)
compared with wild-type controls (WT).
[0087] Invasive electrophysiology studies revealed Hop.sup.Tg mice
had significantly more and longer episodes of inducible atrial
fibrillation than trichostatin A treated Hop.sup.Tg or control
mice. Trichostatin A treatment of Hop.sup.Tg mice reduced the
duration and episodes of atrial fibrillation, atrial fibrosis and
restored the distribution of connexin40 and connexin43 in the
atrium.
[0088] Table 1 shows the results of invasive cardiac electrical
stimulation in Hop transgenic mice (Hop.sup.Tg) compared with
wild-type controls (WT), while treatment with trichostatin A of Hop
transgenic mice (TSA-Hop) resulted in complete reversal of atrial
arrhythmia inducibility. Atrial programmed electrical stimulation
induced significantly more and longer episodes of atrial arrhythmia
in 9 of 11 Hop.sup.Tg mice compared to control littermates where
atrial arrhythmias were induced in 2 of 12 mice (P<0.01).
Ventricular programmed electrical stimulation induced similar
numbers and durations of ventricular tachycardia in 3 of 11
Hop.sup.Tg mice compared to 4 of 12 control mice (P=NS). Following
treatment with trichostatin A from a separate cohort of Hop.sup.Tg
mice (TSA-Hop.sup.Tg) atrial programmed electrical stimulation
induced significantly fewer and shorter episodes of atrial
arrhythmia in only 2 of 10 TSA-Hop.sup.Tg mice compared to the
Hop.sup.Tg mice (P<0.01) Similarly, ventricular programmed
electrical stimulation also induced fewer and shorter episodes of
ventricular tachycardia in 2 of 10 TSA-Hop.sup.Tg mice compared to
Hop.sup.Tg mice (P<0.05). *P<0.05 compared to WT;
.sup..dagger.P<0.05 compared to TSA-Hop.sup.Tg.
TABLE-US-00001 TABLE 1 Summary of Inducible Arrhythmias in Hop
Transgenic Mice. Hop.sup.Tg WT TSA-Hop.sup.Tg (n = 11) (n = 12) (n
= 10) Episodes AT 48*.sup..dagger. 9 5 Duration AT (s) .sup. 1.307
.+-. 0.289*.sup..dagger. 0.167 .+-. 0.114 0.148 .+-. 0.110 AT CL
(ms) 46.2 .+-. 10.0 45.4 .+-. 5.5 47.0 .+-. 7.2 Mice with AT
13/15*.sup..dagger. 2/15 2/15 Episodes VT 11.sup..dagger.
10.sup..dagger. 4 Duration VT (s) 3.31 .+-. 3.08 2.77 .+-. 1.69
0.89 .+-. 1.07 VT CL (ms) 50.7 .+-. 9.9 51.5 .+-. 7.8 50.4 .+-. 8.6
Mice with VT 3/15.sup. 4/15 2/15 AT = Atrial tachycardia; AT CL =
atrial tachycardia cycle length; VT = Ventricular tachycardia; VT
CL = ventricular tachycardia cycle length.
EXAMPLE 2
Trichostatin a has Minimal Effects Upon Cardiac Structure and
Function in Hop Transgenic Mice
[0089] Trichostatin A 0.6 mg/kg daily in a volume of 0.2 mL was
given by intraperitoneal injection for 14 consecutive days upon
inducible atrial arrhythmias and atrial structurally remodeling in
an engineered mouse model with left ventricular hypertrophy and
inducible atrial fibrillation. Transgenic mice over-expressing the
homeodomain-only protein (Hop.sup.Tg), which promotes cardiac
hypertrophy and interstitial fibrosis, were divided into three
groups: control, Hop.sup.Tg mice and Hop.sup.Tg mice treated with
trichostatin A.
[0090] Table 2 shows the results of invasive hemodynamics
measurements and morphometeric analysis of hearts from Hop
transgenic mice (Hop.sup.Tg) compared with wild-type controls (WT)
and Hop transgenic mice treated with trichostatin A (TSA-Hop).
Hop.sup.Tg mice had higher left ventricular end-diastolic pressure
(LVEDP), and reduced peak systolic contraction and diastolic
relaxation compared to WT mice. However, trichostatin A had no
effect upon the LVEDP, systolic contraction or diastolic relaxation
when treatment was initiated in animals aged 12-14 weeks of age.
These parameters were similar between Hop.sup.Tg and TSA-Hop.sup.Tg
mice, though still abnormal when compared to WT mice. In addition,
the heart weight to tibia length ratio (HW/TL) was higher in
Hop.sup.Tg and TSA-Hop.sup.Tg mice compared to WT, but there was no
difference in the HW/TL between Hop.sup.Tg and TSA-Hop.sup.Tg mice.
*P<0.05 compared to WT; .dagger.P<0.05 compared to
TSA-Hop.sup.Tg.
TABLE-US-00002 TABLE 2 Invasive Hemodynamic and Morphometric
Parameters in Hop Transgenic Mice. Hop.sup.Tg WT TSA-Hop.sup.Tg (n
= 8) (n = 9) (n = 8) Heart rate (bpm) 378 .+-. 50.4 407 .+-. 61.2
392 .+-. 63.5 LV systolic 94.6 .+-. 12.4 90.9 .+-. 15.8 90.6 .+-.
8.8 pressure (mmHg) LV end diastolic 7.0 .+-. 1.6* 3.9 .+-. 1.5 7.2
.+-. 2.4* pressure (mmHg) Peak positive 7001 .+-. 2337 6815 .+-.
3434 6465 .+-. 1613 dP/dt (mmHg/s) Peak negative -2051 .+-. 1268*
-4060 .+-. 808 -2267 .+-. 1320* dP/dt (mmHg/s) HopX.sup.Tg WT
TSA-HopX.sup.Tg (n = 11) (n = 15) (n = 13) Heart weight (mg) .sup.
201 .+-. 17.0* 121.4 .+-. 11.5 192.8 .+-. 15.5* Heart weight/ 11.0
.+-. 5.5* 8.1 .+-. 6.1 7.4 .+-. 0.67* body weight (mg/g) LV = left
ventricular.
[0091] Mason's trichrome staining revealed increased atrial
interstitial fibrosis in Hop transgenic mice (Hop.sup.Tg) compared
with wild-type controls (WT), while treatment with trichostatin A
of Hop transgenic mice (TSA-Hop) resulted in complete reversal of
atrial fibrosis. There was no obvious ventricular interstitial
fibrosis in the three groups shown (FIG. 1).
[0092] To better characterize the effects of TSA upon ventricular
structure and function, echocardiographic analysis of HopX.sup.Tg,
TSA-HopX.sup.Tg and WT mice was performed. This analysis revealed
that HopX.sup.Tg mice do have increased ventricular wall thickness
with enhanced ventricular systolic function compare to their WT
littermates (Table 4). However, TSA had no effect upon these
parameters as ventricular wall thickness and systolic function were
similar between HopX.sup.Tg and TSA-HopX.sup.Tg mice (Table 4),
consistent with the hemodynamic analysis of these animals.
Table 3 shows the effects of TSA upon ventricular function in Hop
mice as assessed by echocardiography.
TABLE-US-00003 HopX.sup.Tg WT TSA-HopX.sup.Tg (n = 4) (n = 4) (n =
4) Left Ventricle 1.18 .+-. 0.26* 0.69 .+-. 0.07 1.10 .+-. 0.14*
Posterior Wall (mm) Intra-ventricle 1.2 .+-. 0.24* 0.71 .+-. 0.05
1.13 .+-. 0.18* Septum (mm) LV Fractional 59.9 .+-. 15.3* 31.50
.+-. 3.31 53.9 .+-. 0.50* Shortening (%) LV Ejection 87.9 .+-.
13.0* 59.3 .+-. 3.51* 87.7 .+-. 7.00* Fraction (%) *P < 0.05
compared to WT; .sup..dagger.P < 0.05 compared to
TSA-HopX.sup.Tg. LV = left ventricular.
EXAMPLE 3
Levels of Cardiac Angiotensin Remain Unchanged in Hop and Control
Mice
[0093] Trichostatin A 0.6 mg/kg daily in a volume of 0.2 mL was
given by intraperitoneal injection for 14 days upon inducible
atrial arrhythmias and atrial structurally remodeling in an
engineered mouse model with left ventricular hypertrophy and
inducible atrial fibrillation. Transgenic mice over-expressing the
homeodomain-only protein (Hop.sup.Tg), which promotes cardiac
hypertrophy and interstitial fibrosis, were divided into three
groups: control, Hop.sup.Tg mice and Hop.sup.Tg mice treated with
trichostatin A.
[0094] Enzyme-linked immunosorbent assay showed that myocardial
angiotensin II levels were similar between control and Hop.sup.Tg
mice, suggesting the effects of trichostatin A are independent of
angiotensin II (FIG. 2A). These results suggest that
histone-deacetylase inhibition reverses atrial substrate remodeling
and atrial fibrillation vulnerability in an intact in vivo mouse
model of left ventricular hypertrophy. Furthermore, the levels of
atrial cytokines were assessed in Western blot analysis in mice
over-expressing Hop in the heart and WT, control, mice (FIGS.
2B-F). Specifically, FIG. 2B-F shows the relative protein
expression of the activated form of several mitogen-activated
protein kinases and cytokines and in the atrium of Hop transgenic
mice (Hop.sup.Tg) compared with wild-type controls (WT) and Hop
transgenic mice treated with trichostatin A (TSA-Hop). Activated
TGF-.beta.1 is lower in Hop.sup.Tg mice relative to control and
further decreased in TSA-Hop.sup.Tg mice. Phospho-ERK1/2 is also
lower in Hop.sup.Tg mice relative to control but increased in
TSA-Hop.sup.Tg mice. Phospho-JNK and phospho-p38-MAPK were not
different between Hop.sup.Tg , TSA-Hop.sup.Tg or control mice.
EXAMPLE 4
Connexin40 and Connexin43 Expression in Hop Transgenic Mice
[0095] Trichostatin A 0.6 mg/kg daily in a volume of 0.2 mL was
given by intraperitoneal injection for 14 days upon inducible
atrial arrhythmias and atrial structurally remodeling in an
engineered mouse model with left ventricular hypertrophy and
inducible atrial fibrillation. Transgenic mice over-expressing the
homeodomain-only protein (Hop.sup.Tg), which promotes cardiac
hypertrophy and interstitial fibrosis, were divided into three
groups: control, Hop.sup.Tg mice and Hop.sup.Tg mice treated with
trichostatin A.
[0096] Connexin40 expression was reduced with left ventricular
hypertrophy in Hop transgenic mice and normalized by trichostatin
A. FIG. 3A shows an immunoblot analysis of atrial connexin40 which
was found to be lower in Hop.sup.Tg mice and normalized by TSA.
FIG. 3B shows that no changes in connexin43 expression were evident
between wild-type (WT), Hop transgenic (Hop) and trichostatin A
treated Hop transgenic mice (TSA-Hop).
EXAMPLE 5
Testing of Compounds for Inhibition of HDAC Activity in Cardiac
Myocyte Extracts
[0097] HDAC activity of cardiac myocyte extracts are assessed, in
the presence and absence of a test compound.
[0098] This assay is used to identify novel agents to treat and
reduce cardiac arrhythmia, as inhibition of HDAC activity is in the
present invention leads to reduction in cardiac arrhythmia.
EXAMPLE 6
Effects of TSA Upon Atrial Weight to Body Weight and Tibia
Length
[0099] With regards to mechanical function, TSA has been shown to
improve myocardial mechanical function in models of
pressure-overload and angiotensin-induced hypertrophy. Therefore,
it is possible that TSA reduces atrial arrhythmogenesis by
improving myocardial mechanical function and normalizing
intracardiac pressures. However, no significant changes were
observed in the HW/TL, AW/TL, LVEDP, peak positive dP/dt or peak
negative dP/dt in HopX.sup.Tg mice treated with TSA compared to
HopX.sup.Tg mice without TSA treatment.
[0100] Echocardiographic, MRI and histological analysis did not
reveal any evidence of mitral valve abnormalities in HopX.sup.Tg
mice, so the LVEDP accurately reflects left atrial pressure. While
TSA treatment partially reduces cardiac hypertrophy in 3 week-old
HopX.sup.Tg mice, this does not appear to be the case in the older
14-18 week-old mice used in the examples described herein since no
change were observed in the heart weight/tibial length ratio,
atrial weight/tibial length ratio or left ventricular wall
dimensions. While myocardial dysfunction and altered hemodynamics
probably do contribute to atrial arrhythmogenesis in this model, it
appears HDACi does not target these factors in reversing atrial
fibrosis and arrhythmias.
[0101] The apparent discrepancy observed in the effects of HDACi
upon cardiac hypertrophy in 14-18 week-old HopX.sup.Tg mice,
compared to its effect in models of hypertrophy induced by
pressure-overload or angiotensin-infusion, are related to
differences in the downstream pathways evoked by HopX
over-expression. Specifically, cardiac restricted over-expression
of HopX does not affect myocardial angiotensin II, and this may be
another pathway HDACi affects but is not active in this model. A
relatively low dose of TSA was selected in this example (0.6
mg/kg/day) for a short treatment period (2 weeks) to see if is
possible to selectively target atrial pathology and
arrhythmogenesis, without inducing significant effects upon
ventricular function or morphology. This turned out to be the case
in this model, and previous studies have shown that TSA used at a
dosage of 0.6 mg/kg/day does augment histone acetylation in the
heart. In addition, it is possible once cardiac hypertrophy is
established, and temporally removed from the embryonic gene
programs responsible for its development, HDAC inhibition will no
longer have significant effects upon regression of hypertrophy.
Class I HDACs also complex with and regulate the function of
non-histone proteins, in particular transcription factors through
alterations in acetylation. The dramatic reversal of established
atrial structural changes and fibrosis observed in this model
indicates these effects of HDAC inhibition are less dependent upon
global alterations in transcription induced by chromatin
winding/relaxation, and are related to regulation of specific
transcription factors involved with fibrosis and inflammation, such
as Sp1 and p65.
* * * * *