U.S. patent application number 13/336264 was filed with the patent office on 2012-04-19 for methods for treatment of cardiovascular disorders and diseases.
This patent application is currently assigned to RAPPAPORT FAMILY INSTITUTE FOR RESEARCH IN THE MEDICAL SCIENCES. Invention is credited to Zaid A. Abassi, Yaron Barac, Ofer Binah, Moussa B.H. YOUDIM.
Application Number | 20120095107 13/336264 |
Document ID | / |
Family ID | 34812068 |
Filed Date | 2012-04-19 |
United States Patent
Application |
20120095107 |
Kind Code |
A1 |
YOUDIM; Moussa B.H. ; et
al. |
April 19, 2012 |
METHODS FOR TREATMENT OF CARDIOVASCULAR DISORDERS AND DISEASES
Abstract
Propargylamine, propargylamine derivatives including
N-propargyl-l-aminoindan, enantiomers and analogs thereof, and
pharmaceutically acceptable salts thereof, are useful for
prevention or treatment of cardiovascular disorders, diseases and
conditions.
Inventors: |
YOUDIM; Moussa B.H.; (Haifa,
IL) ; Binah; Ofer; (Nofit, IL) ; Abassi; Zaid
A.; (Haifa, IL) ; Barac; Yaron; (Kiryat
Motzkin, IL) |
Assignee: |
RAPPAPORT FAMILY INSTITUTE FOR
RESEARCH IN THE MEDICAL SCIENCES
Haifa
IL
TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD.
Technion City
IL
|
Family ID: |
34812068 |
Appl. No.: |
13/336264 |
Filed: |
December 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11449862 |
Jun 9, 2006 |
8097608 |
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13336264 |
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10952367 |
Sep 29, 2004 |
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11449862 |
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60524616 |
Nov 25, 2003 |
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60570496 |
May 13, 2004 |
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Current U.S.
Class: |
514/657 |
Current CPC
Class: |
A61P 9/06 20180101; A61K
31/27 20130101; A61K 31/13 20130101; A61P 9/00 20180101 |
Class at
Publication: |
514/657 |
International
Class: |
A61K 31/135 20060101
A61K031/135; A61P 9/06 20060101 A61P009/06; A61P 9/00 20060101
A61P009/00 |
Claims
1. A method for reducing apoptosis resulting from a cardiovascular
disorder, disease or condition, selected from the group consisting
of congestive heart failure, cardiac hypertrophy including atrial
and ventricular hypertrophy, myocardial ischemia, myocardial
ischemia and reperfusion injury, cardiomyopathies, and arrhythmias
in a subject in need thereof, said method comprising administering
to the subject an effective amount of S(-)-N-propargyl-1-aminoindan
or a pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein said
S(-)--N-propargyl-1-aminoindan or a pharmaceutically acceptable
salt thereof is S(-)--N-propargyl-1-aminoindan.
3. The method of claim 1, wherein said
S(-)--N-propargyl-1-aminoindan or a pharmaceutically acceptable
salt thereof is a pharmaceutically acceptable salt of
S(-)--N-propargyl-1-aminoindan.
4. The method of claim 3, wherein said pharmaceutically acceptable
salt is selected from the group consisting of the mesylate salt;
the esylate salt; the sulfate salt; the hydrochloride salt; the
maleate salt; the fumarate salt, the tartrate salt; the
hydrobromide salt; the p-toluenesulfonate salt; the benzoate salt;
the acetate salt; and the phosphate salt of
S(-)--N-propargyl-1-aminoindan.
5. A method for reducing apoptosis and cardiac myocyte cell death
following onset and as a result of congestive heart failure,
cardiac hypertrophy including atrial and ventricular hypertrophy,
myocardial ischemia, myocardial ischemia and reperfusion injury,
cardiomyopathies, and arrhythmias in a patient in need thereof,
said method comprising administering to the subject an effective
amount of S(-)--N-propargyl-1-aminoindan or a pharmaceutically
acceptable salt thereof.
6. The method of claim 5, wherein said
S(-)--N-propargyl-1-aminoindan or a pharmaceutically acceptable
salt thereof is S(-)--N-propargyl-1-aminoindan.
7. The method of claim 5, wherein said
S(-)--N-propargyl-1-aminoindan or a pharmaceutically acceptable
salt thereof is a pharmaceutically acceptable salt of
S(-)--N-propargyl-1-aminoindan.
8. The method of claim 7, wherein said pharmaceutically acceptable
salt is selected from the group consisting of the mesylate salt;
the esylate salt; the sulfate salt; the hydrochloride salt; the
maleate salt; the fumarate salt, the tartrate salt; the
hydrobromide salt; the p-toluenesulfonate salt; the benzoate salt;
the acetate salt; and the phosphate salt of
S(-)--N-propargyl-1-aminoindan.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of application
Ser. No. 11/449,862, which is a continuation-in-part application of
U.S. patent application Ser. No. 10/952,367, filed Sep. 29, 2004,
and claims the benefit of U.S. Provisional Patent Application No.
60/524,616, filed Nov. 25, 2003, now expired, and U.S. Provisional
Patent Application No. 60/570,496, filed May 13, 2004, now expired,
the entire contents of each and all these applications being
herewith incorporated by reference in their entirety as if fully
disclosed herein.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to compositions and methods
for the treatment of cardiovascular disorders and diseases and,
more particularly, to propargylamine and derivatives thereof for
use in said compositions and methods.
Cardiovascular Disorders and Diseases
[0003] Cardiovascular disorders and diseases and their associated
complications are a principal cause of disabilities and deaths of
individuals in the United States and Western Europe. For example,
in recent years more than 500,000 deaths have occurred annually in
the United States alone as a result of coronary artery disease, and
an additional 700,000 patients have been hospitalized for
myocardial infarction.
[0004] Ischemic heart disease (IHD) is the most common, serious,
chronic, life-threatening illness among the cardiovascular
disorders and diseases. Ischemia, reduced myocardial perfusion,
which causes lack of oxygen (hypoxia) as well as other metabolic
changes, is the most common effect resulting from an inadequate
blood flow through the coronary arteries, which are the blood
suppliers of the heart. The most common cause of myocardial
ischemia is the atherosclerotic disease of epicardial coronary
arteries. The plaques consist of subintimal collections of fat,
cells, and debris, which develop at irregular rates in different
segments of the epicardial coronary tree, and lead eventually to
segmental reductions in cross-sectional area (stenosis). When the
coronary artery cross-section area is reduced by .about.75%, a full
range of increases in flow to meet increased myocardial demand is
not possible. When the luminal area is reduced by more than 80%,
blood flow at rest may be reduced, and further minor decreases in
the stenotic orifice can reduce coronary flow dramatically and
cause myocardial ischemia and infarction. This situation impairs
myocardial contractility during exercise, creating the chest
angina. Critical stenosis of the coronaries can cause chest angina
even at rest, implying that the myocardium is suffering from lack
of perfusion. The most serious complication of ischemic heart
disease is acute myocardial infarction (AMI), which is one of the
most common diagnoses in hospitalized patients. AMI generally
occurs when coronary blood flow decreases abruptly after a
thrombotic occlusion of a coronary artery, previously narrowed by
atherosclerotic plaque. Although the mortality rate after admission
for AMI has declined by about 30% over the last two decades,
approximately 1 of every 25 patients who survives the initial
hospitalization dies in the first year after AMI. The first step is
the dissection of the atherosclerotic plaque, which causes the
exposure of the thrombogenic plaque core to the blood. Because of
its high thrombogenicity, a thrombus consists mainly of fibrin and
activated thrombocyte is rapidly growing from the plaque core.
Consequently, blood flow is seriously disturbed until there is no
sufficient blood flow to the myocardium and an infarction begins
due to lack of perfusion of oxygen.
[0005] There are various insults which cause the myocardial damage
during myocardial ischemia and infarction. Lack of adequate
perfusion to the heart tissue may result in: (i) lack of oxygen
(hypoxia); (ii) growth factors and nutrients deprivation (e.g.,
IGF-1, insulin, glucose); (iii) acidosis (lactic acid production);
(iv) hyperkalemia (due to environmental acidosis and cell damage);
and/or (v) during ischemia, but mostly following reperfusion
(resumption of the blood flow to the ischemic tissue), reactive
oxygen species (ROS) such as H.sub.2O.sub.2, O.sub.2..sup.-
OH.sup.- are created by the ischemic cells and by damage to
neighboring cells. Each and every of these insults has been shown
to exert damage to cardiomyocytes both in vivo and in vitro.
[0006] Two main cellular death types occur in nature: necrosis and
apoptosis. While necrosis is a random process, often initiated by
hostile environmental stimuli, apoptosis is a programmed cell death
in which distinct intracellular signaling pathways are activated.
Apoptosis is a fundamental physiological and pathologic mechanism
that allows elimination of no-longer useful cells during
embryogenesis, or of aged or damaged cells during life. Unlike
necrosis, which involves large number of cells, apoptosis usually
affects small number of cells without inflammation. In apoptosis,
the nuclear DNA is "digested" into small fragments by special
DNAses, while cytoskeletal and myofibrillar proteins are degraded
by specialized proteases as well. At the final stages, the cell
dissolves into a characteristic membrane bound vesicles (apoptotic
bodies), which are quickly phagocyted by phagocytic neighboring
cells.
[0007] Cells can undergo apoptosis caused by intrinsic stimuli,
e.g. hypoxia, ROS, chemotherapies, or by extrinsic stimuli, mainly
referred to activation of death receptors such as TNF-.alpha. and
Fas.
[0008] Fas is a ubiquitous cell-surface receptor involved in
apoptosis initiation. Fas belongs to the TNF/NGF superfamily, and
is activated by Fas Ligand (FasL), which may cause apoptosis in
Fas-bearing cells (Berke, 1997). It appears that while healthy
cardiomyocytes are resistant to Fas-mediated apoptosis, during
cardiac pathologies cardiomyocytes become sensitive to Fas-mediated
apoptosis. Recent studies suggest that in several important heart
diseases such as myocarditis, hypertrophy, ischemia,
ischemia/reperfusion and heart failure, Fas activation results in
apoptotic as well as in non-apoptotic effects, both contributing to
cardiac dysfunction (Haunstetter and Izumo, 1998; Binah, 2000).
Recent studies have shown that Fas activation is involved not only
in myocardial pathologies inflicted by immune effectors (CTLs) such
as transplant rejection, myocarditis and the resulting dilated
cardiomyopathy (Binah, 2000; Hershkowitz et al., 1987), but also in
lymphocyte-independent diseases such as ischemia/reperfusion
injuries (Fliss and Gattinger, 1996; Yaoita et al., 1998; Jeremias
et al., 2000). In this regard, it was recently proposed that FasL
can be cleaved by a metalloprotease to form soluble FasL (sFasL),
which can cause apoptosis in susceptible cells. Therefore, sFasL,
which may be secreted from the failing heart and is elevated in
patients with advanced congestive heart failure (Yamaguchi et al.,
1999), is a potential contributor to apoptosis in this wide-spread
heart pathology.
[0009] Programmed cell death (apoptosis) is recognized,
increasingly, as a contributing cause of cardiac myocyte loss with
ischemia/reperfusion injury, myocardial infarction, and
long-standing heart failure.
Propargylamine and Propargylamine Derivatives
[0010] Several propargylamine derivatives have been shown to
selectively inhibit monoamine oxidase (MAO)-B and/or MAO-A activity
and, thus to be suitable for treatment of neurodegenerative
diseases such as Parkinson's and Alzheimer's disease. In addition,
these compounds have been further shown to protect against
neurodegeneration by preventing apoptosis.
[0011] Rasagiline, R(+)--N-propargyl-1-aminoindan, a highly potent
selective irreversible monoamine oxidase (MAO)-B inhibitor, has
been shown to exhibit neuroprotective activity and antiapoptotic
effects against a variety of insults in cell cultures and in
vivo.
[0012] Rasagiline has been developed for Parkinson's disease as
monotherapy or as an adjunct to L-dopa therapy (Youdim et al.,
2001; Parkinson Study Group, 2002; Finberg and Youdim, 2002; Gassen
et al., 2003). Phase III controlled studies have shown that
rasagiline is effective with a dose of as low as 1 mg/kg in
monotherapy (Parkinson Study group, 2002) and as an adjunct to
L-dopa, comparable in its effect to the anti-Parkinson
catechol-O-methyltranferase (COMT) inhibitor, entacapone (Brooks
and Sagar, 2003). Rasagiline has recently finished the phase III
clinical trials and has been approved for treatment of Parkinson's
disease in Europe, Israel, and in the U.S.
[0013] Rasagiline exhibits neuroprotective activities both in vitro
and in vivo (for review see Mandel et al., 2003; Youdim, 2003)
which may contribute to its possible disease modifying activity. It
is metabolized to its major two metabolites: aminoindan (here
designated "TVP-136") and S(-)--N-propargyl-1-aminoindan (here
designated "TVP-1022") (Youdim et al., 2001), which also have
neuroprotective activity against serum deprivation and
1-methamphetamine-induced neurotoxicity in partially differentiated
PC-12 cells (Am et al., 2004).
[0014] Rasagiline [R(+)--N-propargyl-1-aminoindan] and
pharmaceutically acceptable salts thereof were first disclosed in
U.S. Pat. No. 5,387,612, U.S. Pat. No. 5,453,446, U.S. Pat. No.
5,457,133, U.S. Pat. No. 5,576,353, U.S. Pat. No. 5,668,181, U.S.
Pat. No. 5,786,390, U.S. Pat. No. 5,891,923, and U.S. Pat. No.
6,630,514 as useful for the treatment of Parkinson's disease,
memory disorders, dementia of the Alzheimer type, depression, and
the hyperactive syndrome. The 4-fluoro-, 5-fluoro- and
6-fluoro-N-propargyl-1-aminoindan derivatives were disclosed in
U.S. Pat. No. 5,486,541 for the same purposes.
[0015] U.S. Pat. No. 5,519,061, U.S. Pat. No. 5,532,415, U.S. Pat.
No. 5,599,991, U.S. Pat. No. 5,744,500, U.S. Pat. No. 6,277,886,
U.S. Pat. No. 6,316,504, U.S. Pat. No. 5,576,353, U.S. Pat. No.
5,668,181, U.S. Pat. No. 5,786,390, U.S. Pat. No. 5,891,923, and
U.S. Pat. No. 6,630,514 disclose R(+)--N-propargyl-1-aminoindan and
pharmaceutically acceptable salts thereof as useful for treatment
of additional indications, namely, an affective illness, a
neurological hypoxia or anoxia, neurodegenerative diseases, a
neurotoxic injury, stroke, brain ischemia, a head trauma injury, a
spinal trauma injury, schizophrenia, an attention deficit disorder,
multiple sclerosis, and withdrawal symptoms.
[0016] U.S. Pat. No. 6,251,938 describes
N-propargyl-phenylethylamine compounds, and U.S. Pat. No.
6,303,650, U.S. Pat. No. 6,462,222 and U.S. Pat. No. 6,538,025
describe N-propargyl-1-aminoindan and N-propargyl-1-aminotetralin
compounds, said to be useful for treatment of depression, attention
deficit disorder, attention deficit and hyperactivity disorder,
Tourette's syndrome, Alzheimer's disease and other dementia such as
senile dementia, dementia of the Parkinson's type, vascular
dementia and Lewy body dementia.
[0017] The first compound found to selectively inhibit MAO-B was
R-(-)-N-methyl-N-(prop-2-ynyl)-2-aminophenylpropane, also known as
L-(-)-deprenyl, R-(-)-deprenyl, or selegiline. In addition to
Parkinson's disease, other diseases and conditions for which
selegiline is disclosed as being useful include: drug withdrawal
(WO 92/21333, including withdrawal from psychostimulants, opiates,
narcotics, and barbiturates); depression (U.S. Pat. No. 4,861,800);
Alzheimer's disease and Parkinson's disease, particularly through
the use of transdermal dosage forms, including ointments, creams
and patches; macular degeneration (U.S. Pat. No. 5,242,950);
age-dependent degeneracies, including renal function and cognitive
function as evidenced by spatial learning ability (U.S. Pat. No.
5,151,449); pituitary-dependent Cushing's disease in humans and
nonhumans (U.S. Pat. No. 5,192,808); immune system dysfunction in
both humans (U.S. Pat. No. 5,387,615) and animals (U.S. Pat. No.
5,276,057); age-dependent weight loss in mammals (U.S. Pat. No.
5,225,446); schizophrenia (U.S. Pat. No. 5,151,419); and various
neoplastic conditions including cancers, such as mammary and
pituitary cancers. WO 92/17169 discloses the use of selegiline in
the treatment of neuromuscular and neurodegenerative disease and in
the treatment of CNS injury due to hypoxia, hypoglycemia, ischemic
stroke or trauma. In addition, the biochemical effects of
selegiline on neuronal cells have been extensively studied (e.g.,
see Tatton, et al., 1991 and 1993). U.S. Pat. No. 6,562,365
discloses the use of desmethylselegiline for selegiline-responsive
diseases and conditions.
[0018] Selegiline (1-deprenyl) is a selective MAO-B inhibitor which
is a useful anti-Parkinson drug both in monotherapy (Parkinson
Study Group, 1989) and as an adjunct to L-DOPA therapy, and has
L-DOPA sparing action (Birkmayer et al., 1977; Riederer and Rinne,
1992; Parkinson Study Group, 1989). Selegiline is a propargyl
derivative of 1-methamphetamine and thus its major metabolite is
1-methamphetamine (Szoko et al., 1999; Kraemer and Maurer, 2002;
Shin, 1997), which is neurotoxic (Abu-Raya et al., 2002; Am et al.,
2004). In contrast to aminoindan, a rasagiline metabolite,
L-methamphetamine prevents the neuroprotective activities of
rasagiline and selegiline in partially differentiated cultured
PC-12 cells (Am et al., 2004).
[0019] Selegiline and methamphetamine, unlike rasagiline and
aminoindan, have sympathomimetic activity (Simpson, 1978) that
increases heart rate and blood pressure (Finberg et al., 1990;
Finberg et al., 1999). Recent studies (Glezer and Finberg, 2003)
have indicated that the sympathomimetic action of selegiline can be
attributed to its 1-methamphetamine and amphetamine metabolites.
These properties are absent in rasagiline and in its metabolite
aminoindan. Parkinsonian patients receiving combined treatments
with selegiline plus levodopa have been reported to have a higher
mortality rate than those treated with levodopa alone (Lees, 1995).
This is not related to the MAO-B inhibitory activity of selegiline,
but is rather attributed to its sympathomimetic action and
methamphetamine metabolites (Reynolds et al., 1978; Lavian et al.,
1993).
[0020] Several propargylamine derivatives have been shown to
selectively inhibit MAO-B and/or MAO-A activity and, thus to be
suitable for treatment of neurodegenerative diseases such as
Parkinson's and Alzheimer's disease. In addition, these compounds
have been further shown to protect against neurodegeneration by
preventing apoptosis.
[0021] U.S. Pat. No. 5,169,868, U.S. Pat. No. 5,840,979 and U.S.
Pat. No. 6,251,950 disclose aliphatic propargylamines as selective
MAO-B inhibitors, neuroprotective and cellular rescue agents. The
lead compound, (R)--N-(2-heptyl)methyl-propargylamine (R-2HMP), has
been shown to be a potent MAO-B inhibitor and antiapoptotic agent
(Durden et al., 2000).
[0022] Propargylamine was reported many years ago to be a
mechanism-based inhibitor of the copper-containing bovine plasma
amine oxidase (BPAO), though the potency was modest. U.S. Pat. No.
6,395,780 discloses propargylamine as a weak glycine-cleavage
system inhibitor. Copending U.S. patent application Ser. No.
10/952,379, entitled "Use of propargylamine as neuroprotective
agent", filed on Sep. 29, 2004, discloses that propargylamine
exhibits neuroprotective and anti-apoptotic activities and can,
therefore, be used for all known uses of rasagiline and similar
drugs containing the propargylamine moiety.
[0023] Copending U.S. patent application Ser. No. 11/244,150,
entitled "Methods for treatment of renal failure", filed on Oct. 6,
2005, discloses a method for treatment of a renal failure, either
acute or chronic, which comprises administering to the subject an
amount of an active agent selected from the group consisting of
propargylamine, a propargylamine derivative, and a pharmaceutically
acceptable salt thereof.
[0024] All and each of the above-mentioned US patents and patent
applications are herewith incorporated by reference in their
entirety as if fully disclosed herein.
SUMMARY OF THE INVENTION
[0025] The present invention relates to a method for treatment of a
subject susceptible to or suffering from a cardiovascular disorder,
disease or condition which comprises administering to the subject
an amount of an agent selected from the group consisting of
propargylamine, a propargylamine derivative and a pharmaceutically
acceptable salt thereof, effective to treat the subject.
[0026] In one preferred embodiment of the invention, the agent is
propargylamine or a pharmaceutically acceptable salt thereof. In
another preferred embodiment, the agent is a propargylamine
derivative such as an N-propargyl-1-aminoindan, e.g.
R(+)--N-propargyl-1-aminoindan (rasagiline) or its enantiomer
S(-)--N-propargyl-1-aminoindan (TVP1022), and analog thereof, or a
pharmaceutically acceptable salt thereof.
[0027] The methods and compositions of the invention are suitable
for preventing and/or treating congestive heart failure (CHF),
cardiac hypertrophy including both atrial and ventricular
hypertrophy, myocardial infarction, myocardial ischemia, myocardial
ischemia and reperfusion, cardiomyopathies, or arrhythmias.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIGS. 1A-1B depict apoptosis induced in H9c2 rat heart cells
by means of recombinant Fas ligand (rFasL). The apoptotic cells
detected by DAPI staining are marked by the arrows (FIG. 1B). FIG.
1A--control.
[0029] FIGS. 2A-2C show that rasagiline,
S(-)--N-propargyl-1-aminoindan (TVP1022) and propargylamine block
Fas-mediated apoptosis in H9c2 cells. Maximal apoptotic effect of
Fas activation, attained at 10 hours incubation with rFasL, was
completely prevented by 10 .mu.M rasagiline (2A). Apoptotic effect
of Fas activation, attained at .about.10 hours incubation with
rFasL, was completely prevented by both TVP1022 (0.1 or 1.0 .mu.M)
(2B) and propargylamine (0.1 or 1.0 .mu.M) (2C).
[0030] FIGS. 3A-3E show that rasagiline, propargylamine and
S(-)--N-propargyl-1-aminoindan (TVP1022) protect against serum
starvation-induced apoptosis in H9c2 cells: (3A) maximal apoptotic
effect, induced by 9 hours serum starvation, was completely
prevented by 10 .mu.M rasagiline; (3B-3D) anti-apoptotic effects
obtained by either 0.1-10 .mu.M rasagiline, 0.01-1 .mu.M
propargylamine or 0.01-1 .mu.M TVP1022, respectively; (3E)
anti-apoptotic effect obtained by 0.1-10 .mu.M TVP1022, using the
MTT staining assay as a measure for apoptosis.
[0031] FIG. 4 shows that rasagiline protects against serum
starvation-mediated but not H.sub.2O.sub.2-- induced apoptosis in
H9c2 cells (n=4 experiments, .about.2000 cells counted). * compared
to control. ** compared to serum starvation (p<0.05).
[0032] FIG. 5 shows that both propargylamine and
S(-)--N-propargyl-1-aminoindan (TVP1022) block Fas-mediated
hypertrophy in cultured neonatal rat ventricular myocytes. The top
panel depicts representative atrial natriuretic peptide (ANP) mRNA
blots in control, rFasL, rFasL+propargylamine, and in
rFasL+TVP1022. The lower panel depicts the summary of three
experiments performed with each one of these drugs. Hypertrophy was
expressed as the ratio between ANP and actin. *P<0.05 vs.
control.
[0033] FIGS. 6A-6C show the effect of serum starvation (SS) in
cultures of neonatal rat ventricular myocytes (NEVM) on apoptosis
induction, indicated by the level of caspase-3 cleavage, and the
effect of propargylamine (PA) thereon. (6A) serum starvation causes
apoptosis, represented by a marked increase in caspase-3 cleavage.
(6B) 0.1 .mu.M propargylamine attenuates serum starvation-induced
apoptosis as indicated by decreased level of caspase-3 cleavage
(n=3, P<0.01 compared to SS). (6C) 0.1 .mu.M propargylamine
attenuates serum starvation-induced apoptosis as indicated by
increased expression of Bcl-2 (n=3, P<0.05 compared to SS).
[0034] FIG. 7 shows that both S(-)--N-propargyl-1-aminoindan
(TVP1022) and propargylamine, at a concentration of either 1 or 10
.mu.M, significantly attenuate the doxorubicin-induced apoptosis
effect in cultured neonatal rat ventricular myocytes, as indicated
by the drug-induced decrease in caspase-3 cleavage.
[0035] FIGS. 8A-8D show the effect of intravenous administration of
S(-)--N-propargyl-1-aminoindan (TVP1022) (either 1 or 10 mg/kg) on
the cardiac function in rats: (8A) cardiac output (ml/min); (8B)
cardiac index (ml/min*100 gr body weight); (8C) heart rate
(beats/min); and (8D) mean arterial pressure (mm/Hg).
Recovery=after washout period.
[0036] FIGS. 9A-9E show the effects of propargylamine and
S(-)--N-propargyl-1-aminoindan (TVP1022) (5 mg/kg/day), orally
administered for 21 days, on the expression of mitochondrial Bax, a
pro-apoptotic protein, and of mitochondrial Bcl-2 and
PKC-.epsilon., both anti-apoptotic proteins. Propargylamine does
not affect Bax expression (9A) but increases Bcl-2 expression (9B),
resulting in marked increase in the ratio Bcl-2/Bax expression
(9C). Propargylamine increases PKC-.epsilon. expression (9D).
TVP1022 increases PKC-.epsilon. expression (9E).
[0037] FIGS. 10A-10B show that both caspase-3 (10A) and cytochrome
C (10B) markedly increase following induction of volume overload,
indicating that volume overload-induced CHF is associated with
increased expression of these two proteins. Sham-operated rats
served as controls.
[0038] FIGS. 11A-11B show that both S(-)--N-propargyl-1-aminoindan
(TVP1022) and propargylamine significantly reduce CHF-induced
increase in caspase-3 and cytosolic cytochrome C, both
pro-apoptotic proteins. (11A) Effect of TVP1022 (7.5 mg/kg/day,
orally administered for 21 days) on caspase-3 expression in
CHF-induced rats (vehicle=untreated CHF rats). (11B) Effect of
TVP1022 (1 mg/kg/day) and propargylamine (5 mg/kg/day), orally
administered for 21 days, on cytochrome C expression in CHF-induced
rats (vehicle=untreated CHF rats).
[0039] FIGS. 12A-12C show that S(-)--N-propargyl-1-aminoindan
(TVP1022) completely prevents the hypertrophic increase in the
diastolic area seen in CHF rats at days 10 and 21 of the treatment
protocol, as described in Material and Methods hereinafter.
[0040] FIGS. 13A-13C show that S(-)--N-propargyl-1-aminoindan
(TVP1022) completely prevents the hypertrophic increase in the
systolic area seen in CHF rats at days 10 and 21 of the treatment
protocol, as described in Material and Methods hereinafter.
[0041] FIGS. 14A-14C show that the fractional shortening in the CHF
rats, 14 days post surgical creation of an aorto-caval fistula
(AVF), is significantly reduced, but completely prevented by
administration of S(-)--N-propargyl-1-aminoindan (TVP1022), as
described in Material and Methods hereinafter.
[0042] FIGS. 15A-15C show that the administration of propargylamine
as described in Material and Methods hereinafter completely
prevents the hypertrophic increase in the diastolic (15A) and
systolic (15B) areas seen in the CHF rats, 14 days post surgical
creation of aortocaval fistula (AVF), as well as a significant
reduction in the fractional shortening.
DETAILED DESCRIPTION OF THE INVENTION
[0043] As described in detail in the Examples section hereinafter,
propargylamine and propargylamine derivatives such as rasagiline
and its enantiomer S(-)--N-propargyl-1-aminoindan (also designated
TVP1022) provide protection against apoptosis induced by several
means in the embryonic cardiac cell line H9c2 or in neonatal rat
ventricular myocytes (NRVM), as well as against hypertrophy induced
by Fas receptor activation.
[0044] In particular, it has been found in accordance with the
present invention that propargylamine and TVP1022, which do not
inhibit monoamine oxidase, decrease the expression of key
pro-apoptotic proteins such as caspase-3 and cytosolic cytochrome
C, and increase the expression of anti-apoptotic proteins such as
mitochondrial Bcl-2 and PKC-.epsilon., thus shifting the balance
between the anti- and the pro-apoptotic proteins towards the former
and generating anti-apoptotic effect. These studies have been
conducted both in in vitro and in vivo experiments, in which both
naive and volume overload-induced congestive heart failure (CHF)
rats have been used. Furthermore, as clearly demonstrated in
Example 10, pretreatment with propargylamine or TVP1022 blocks the
volume overload induced hypertrophy in CHF rats and the reduction
in ventricular mechanical function as derived from
echocardiological parameters.
[0045] The present invention thus relates to a method for treatment
of a subject susceptible to or suffering from a cardiovascular
disorder, disease or condition which comprises administering to the
subject an amount of an agent selected from the group consisting of
propargylamine, a propargylamine derivative, or a pharmaceutically
acceptable salt thereof, effective to treat the subject.
[0046] The present invention further relates to a method for
treatment of a subject susceptible to or suffering from a
cardiovascular disorder, disease or condition which comprises
administering to the subject an amount of an agent selected from
the group consisting of propargylamine, a propargylamine
derivative, or a pharmaceutically acceptable salt thereof,
effective to protect ventricular muscle from apoptosis,
particularly Fas-mediated apoptosis, wherein said cardiovascular
disorder or disease is ischemia/reperfusion injury, myocardial
infarction, and long-standing heart failure.
[0047] In one preferred embodiment, the active agent used in the
present invention is propargylamine or a pharmaceutically
acceptable salt thereof. The use of any physiologically acceptable
salt of propargylamine is encompassed by the present invention such
as the hydrochloride, hydrobromide, sulfate, mesylate, esylate,
tosylate, sulfonate, phosphate, or carboxylate salt. In more
preferred embodiments, propargylamine hydrochloride and
propargylamine mesylate are used according to the invention.
[0048] In another preferred embodiment, the active agent used in
the present invention is N-propargyl-1-aminoindan, either in its
racemic form (described, for example, in U.S. Pat. No. 6,630,514)
or as the R-enantiomer R(+)--N-propargyl-1-aminoindan (rasagiline,
described, for example, in U.S. Pat. No. 5,387,612) or as the
S-enantiomer S-(-)--N-propargyl-1-aminoindan (TVP1022, described,
for example, in U.S. Pat. No. 6,277,886). In a more preferred
embodiment of the invention, the active agent is rasagiline, the
R(+)--N-propargyl-1-aminoindan, or its enantiomer
S(-)--N-propargyl-1-aminoindan.
[0049] In another preferred embodiment, the active agent is a
pharmaceutically acceptable salt of N-propargyl-1-aminoindan or of
an enantiomer thereof including, but not limited to, the mesylate,
maleate, fumarate, tartrate, hydrochloride, hydrobromide, esylate,
p-toluenesulfonate, benzoate, acetate, phosphate and sulfate salts.
In preferred embodiments, the salt is a pharmaceutically acceptable
salt of R(+)--N-propargyl-1-aminoindan such as, but not limited to,
the mesylate salt (described, for example, in U.S. Pat. No.
5,532,415), the esylate and the sulfate salts (both described, for
example, in U.S. Pat. No. 5,599,991), and the hydrochloride salt
(described, for example, in U.S. Pat. No. 6,630,514) of
R(+)--N-propargyl-1-aminoindan or
S(-)--N-propargyl-1-aminoindan.
[0050] In a further embodiment, the active agent is an analog of
N-propargyl-1-aminoindan, an enantiomer or a pharmaceutically
acceptable salt thereof. In one embodiment, the analogs are the
compounds described in U.S. Pat. No. 5,486,541 such as, but not
limited to, the compounds 4-fluoro-N-propargyl-1-aminoindan,
5-fluoro-N-propargyl-1-aminoindan,
6-fluoro-N-propargyl-1-aminoindan, an enantiomer thereof and
pharmaceutically acceptable addition salts thereof. In another
embodiment, the analogs are the compounds described in U.S. Pat.
No. 6,251,938 such as, but not limited to, the compounds
(rac)-3-(N-methyl,
N-propyl-carbamyloxy)-.alpha.-methyl-N'-propargyl phenethylamine
HCl; (rac)-3-(N,N-dimethyl-carbamyloxy)-.alpha.-methyl-N'-methyl,
N-propargyl phenethylamine HCl; (rac)-3-(N-methyl,
N-hexyl-carbamyloxy)-.alpha.-methyl-N'-methyl, N'-propargyl
phenethylamine mesylate; (rac)-3-(N-methyl,
N-cyclohexyl-carbamyloxy)-.alpha.-methyl-N'-methyl,
N'-propargylphenethyl HCl; and (S)-3-(N-methyl,
N-hexyl-carbamyloxy)-.alpha.-methyl-N'-methyl, N'-propargyl
phenethylamine ethane-sulfonate. In a further embodiment, the
analogs are the compounds described in U.S. Pat. No. 6,303,650 such
as, but not limited to, the compounds (rac) 6-(N-methyl,
N-ethyl-carbamyloxy)-N'-propargyl-1-aminoindan HCl; (rac)
6-(N,N-dimethyl, carbamyloxy)-N'-methyl-N'-propargyl-1-aminoindan
HCl; (rac) 6-(N-methyl,
N-ethyl-carbamyloxy-N'-propargyl-1-aminotetralin HCl; (rac)
6-(N,N-dimethyl-thiocarbamyloxy)-1-aminoindan HCl; (rac)
6-(N-propyl-carbamyloxy-N'-propargyl-1-aminoindan HCl; (rac)
5-chloro-6-(N-methyl,
N-propyl-carbamyloxy)-N'-propargyl-1-aminoindan HCl;
(S)-6-(N-methyl), N-propyl-carbamyloxy)-N'-propargyl-1-aminoindan
HCl; and (R)-6-(N-methyl,
N-ethyl-carbamyloxy)-N'-propargyl-1-aminoindan hemi-(L)-tartrate,
and 6-(N-methyl, N-ethyl-carbamyloxy)-N'-methyl,
N'-propargyl-1-aminoindan described in U.S. Pat. No. 6,462,222.
[0051] In a still further embodiment, the active agent is an
aliphatic propargylamine described in U.S. Pat. No. 5,169,868, U.S.
Pat. No. 5,840,979 and U.S. Pat. No. 6,251,950 such as, but not
limited to, the compounds N-(1-heptyl)propargylamine;
N-(1-octyl)propargylamine; N-(1-nonyl)propargylamine;
N-(1-decyl)propargylamine; N-(1-undecyl)propargylamine:
N-(1-dodecyl)propargylamine; R--N-(2-butyl)propargylamine;
R--N-(2-pentyl)propargylamine; R--N-(2-hexyl)propargylamine;
R--N-(2-heptyl)propargylamine; R--N-(2-octyl)propargylamine;
R--N-(2-nonyl)propargylamine; R--N-(2-decyl)propargylamine,
R--N-(2-undecyl)propargylamine; R--N-(2-dodecyl)propargylamine:
N-(1-butyl)-N-methylpropargylamine;
N-(2-butyl)-N-methylpropargylamine; N-(2-pentyl)-N-methylpropargyl
amine; N-(1-pentyl)-N-methylpropargylamine;
N-(2-hexyl)-N-methylpropargylamine;
N-(2-heptyl)-N-methylpropargylamine;
N-(2-decyl)-N-methylpropargylamine;
N-(2-dodecyl)-N-methylpropargylamine;
R(-)--N-(2-butyl)-N-methylpropargylamine; or a pharmaceutically
acceptable salt thereof.
[0052] In yet another embodiment, the active agent is selegiline,
desmethylselegiline or norprenyl, pargyline or chlorgyline.
[0053] In still another embodiment, the active agent is the
compound N-methyl-N-propargyl-10-aminomethyl-dibenzo[b,f]oxepin
(known as CGP 3466, described in Zimmermann et al., 1999).
[0054] All the US patents and other publications mentioned
hereinabove are hereby incorporated by reference in their entirety
as if fully disclosed herein.
[0055] In another aspect, the present invention provides a
pharmaceutical composition for prevention and/or treatment of a
cardiovascular disorder, disease or condition comprising a
pharmaceutically acceptable carrier and an agent selected from the
group consisting of propargylamine, a propargylamine derivative, or
a pharmaceutically acceptable salt thereof as described above.
[0056] The pharmaceutical composition provided by the present
invention may be in solid, semisolid or liquid form and may further
include pharmaceutically acceptable fillers, carriers or diluents,
and other inert ingredients and excipients. The composition can be
administered by any suitable route, e.g. intravenously, orally,
parenterally, rectally, or transdermally. The dosage will depend on
the state of the patient and severity of the disease and will be
determined as deemed appropriate by the practitioner.
[0057] In one embodiment, the pharmaceutically acceptable carrier
is a solid and the pharmaceutical composition is in a suitable form
for oral administration including tablets, compressed or coated
pills, dragees, sachets, hard or soft gelatin capsules, and
sublingual tablets. In a more preferred embodiment, the
pharmaceutical composition is a tablet containing an amount of the
active agent in the range of about 0.1-100 mg, preferably from
about 1 mg to about 10 mg.
[0058] In another embodiment, the pharmaceutically acceptable
carrier is a liquid and the pharmaceutical composition is an
injectable solution. The amount of the active agent in the
injectable solution is in the range of from about 0.1 mg/kg to
about 100 mg/kg, more preferably 1 mg/kg to about 10 mg/kg.
[0059] For parenteral administration the invention provides
ampoules or vials that include an aqueous or non-aqueous solution
or emulsion. For rectal administration there are provided
suppositories with hydrophilic or hydrophobic (gel) vehicles.
[0060] The methods and compositions of the invention are for
preventing and/or treating congestive heart failure, cardiac
hypertrophy including both atrial and ventricular hypertrophy,
myocardial infarction, myocardial ischemia, myocardial ischemia and
reperfusion, arrhythmias, or long-standing heart failure. In
preferred embodiments, the cardiovascular disorder is congestive
heart failure, and/or cardiac hypertrophy, and/or ischemia and/or
arrhythmias.
[0061] The dosage and frequency of administration of the drug will
depend from the age and condition of the patient, type of disorder
and its severity, and will be determined according to the
physician's judgment. It can be presumed that for preventive
treatment of subjects susceptible to a cardiovascular disorder or
disease lower doses will be needed while higher doses will be
administered in acute cases. The susceptibility to cardiovascular
disorder or disease may derive from diseases or disorders such as
diabetes and obesity, or from genetic or ethnic factors. It has
been reported that people with ancestry in South Asia are highly
susceptible to cardiovascular diseases (BMJ, 2002, 324:
625-626).
[0062] In one embodiment of the invention, the active agent is
administered alone. In other embodiments of the invention, the
active agent is administered in combination with another known
cardiovascular drug, either before, simultaneously or after said
other cardiovascular drug.
[0063] The following examples illustrate certain features of the
present invention but are not intended to limit the scope of the
present invention.
EXAMPLES
Materials and Methods
[0064] (i) Materials.
[0065] Rasagiline, its enentiomer S(-)--N-propargyl-1-aminoindan
(also designated here TVP1022), and propargylamine were kindly
donated by Teva Pharmaceutical Industries Ltd. (Petach Tikva,
Israel).
[0066] (ii) Cell Line H9c2.
[0067] Experiments were performed on the embryonic rat heart cell
line H9c2. H9c2 cells were cultured in DMEM (Biological Industries,
Beit-Haemek, Israel) supplemented with 10% fetal calf serum (FCS),
50 units/ml penicillin G, 50 .mu.g/ml streptomycin sulfate, 2 mg/ml
L-glutamine and sodium pyruvate. H9c2 cells were harvested by
trypsinization, washed with PBS, diluted to a concentration of
5.times.10.sup.4 cells/ml with DMEM (high glucose) and cultured at
0.5 ml/well on sterile glass cover slips in 24-well plates.
[0068] (iii) Protocols Inducing Apoptosis
[0069] (a) H.sub.2O.sub.2 Incubation protocol--To induce apoptosis,
H9c2 cultures were exposed to H.sub.2O.sub.2 (0.5 .mu.M) for 7
hours.
[0070] (b) Serum starvation--To induce apoptosis, H9c2 cultures
were incubated in the culture medium containing 0% FCS for the
indicated times.
[0071] (c) Activation of the Fas receptor--Fas activation was
induced by incubating the cultures with recombinant human Fas
Ligand (rFasL; 10 ng/ml) plus the enhancing antibody (1 .mu.g/ml)
for the indicated times, according to the manufacturer's
recommendations (Alexis Biochemicals, San Diego, Calif.).
[0072] (iv) Determination of Apoptosis by DAPI.
[0073] Cultures were counterstained with
4',6-diamidino-2-phenylindole (DAPI) to visualize the nuclear
morphology. Cells were scored as apoptotic, only if they exhibited
unequivocal nuclear chromatin condensation and fragmentation.
[0074] (v) Animals.
[0075] Studies were conducted on male Sprague Dawley rats (Harlan
Laboratories Ltd., Jerusalem, Israel), weighing .about.300 g. The
animals were kept in a temperature-controlled room and maintained
on standard rat diet (0.5% NaCl). All experiments were performed
according to the guidelines of the Technion Committee for
Supervision of Animal Experiments (Haifa, Israel). Heart failure
was induced by surgical creation of an aortocaval fistula (AVF)
between the abdominal aorta and the inferior vena cava (side to
side, outer diameter 1-1.2 mm), which is a well established model
of volume-overload induced heart failure, featuring many of the
clinical symptoms of heart failure and dilated cardiomyopathy in
humans. Sham-operated rats served as controls. Drugs (or saline as
control) were orally administered, starting 7 days prior to surgery
(day 0) and were continued for 21 days. Surgery was performed on
day 7 and animals sacrificed 14 days post-surgery (day 21). Cardiac
function was determined by echocardiography on days 0, 10 (3 days
post-surgery) and 21 (before sacrifice). After the last
echocardiography measurement, rats were sacrificed and hearts were
analyzed.
Example 1
Rasagiline, S(-)--N-propargyl-1-aminoindan and Propargylamine
Protect H9c2 Heart Cells Against Apoptosis Induced by Fas
Activation
[0076] The first apoptosis-inducing protocol tested was activation
of the Fas receptor with recombinant Fas Ligand (rFasL) plus the
enhancing antibody (Yaniv et al., 2002).
[0077] Cultures of embryonic rat heart cell line H9c2 were
incubated with rFasL (10 ng/ml) and an enhancing antibody for
periods of time of 9, 10 and 24 hours, and apoptosis measured
thereafter. As shown in FIG. 1B, Fas activation caused prominent
apoptosis in H9c2 cells, as detected by the DAPI assay.
[0078] In order to determine whether rasagiline can prevent
Fas-mediated apoptosis, the Fas receptor was activated for 9, 10
and 24 hours as described above. Rasagiline (10 .mu.M) was
introduced to the culture medium 16 hours before, and was present
throughout the apoptosis-inducing protocol (n=3 wells). As seen in
FIG. 2A, the maximal apoptotic effect (.about.20% apoptosis) of Fas
activation was attained at 10 hours incubation with rFasL. This
apoptotic effect was completely prevented by rasagiline,
demonstrating that rasagiline blocks Fas-mediated apoptosis.
[0079] Similar results were obtained using the S-enantiomer,
S(-)--N-propargyl-1-aminoindan, and propargylamine. Each one of the
drugs, at a concentration of either 0.1 or 1.0 .mu.M was introduced
to the culture medium 16 hours before, and was presented throughout
the apoptosis-inducing protocol (n=3 wells). As shown in FIGS.
2B-2C, the Fas-mediated apoptosis was .about.10%, attained at
.about.10 hours incubation with rFasL, and it was completely
prevented by both S(-)--N-propargyl-1-aminoindan (2B) and
propargylamine (2C).
Example 2
Rasagiline, S(-)--N-propargyl-1-aminoindan and Propargylamine
Protect H9c2 Heart Cells Against Apoptosis Induced by Serum
Starvation
[0080] The next apoptosis-inducing stimulus tested was serum
starvation (24 hrs, 0% serum in the culture medium). To induce
apoptosis, H9c2 cells were incubated in the culture medium
containing 0% FCS for 6, 7, 8 or 9 hours. Rasagiline (10 .mu.M) was
introduced to the culture medium 2 hours before inducing serum
starvation and was present throughout the apoptosis-inducing
protocol (n=3 wells). As seen in FIG. 3A, the most effective
protocol was 9 hrs serum starvation, which caused 12% apoptosis.
This effect was completely prevented by rasagiline.
[0081] In the next stage, H9c2 cells were incubated in the culture
medium containing 0% FCS for 24 hours, and the anti-apoptotic
effect obtained by various concentrations of rasagiline,
S(-)--N-propargyl-1-aminoindan and propargylamine was measured.
FIG. 3B shows the anti-apoptotic effect obtained by rasagiline
(0.1-10 .mu.M) introduced to the culture medium 2 hours before
serum starvation, FIGS. 3C-3D show that similar anti-apoptotic
effects were obtained by either S(-)--N-propargyl-1-aminoindan or
propargylamine (0.01-1 .mu.M), respectively, and FIG. 3E shows the
anti-apoptotic effect obtained by S(-)--N-propargyl-1-aminoindan
(0.1-10 .mu.M) using the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
staining assay as a measure for apoptosis.
Example 3
Rasagiline Protects H9c2 Heart Cells Against Apoptosis Induced by
Serum Starvation but not H.sub.2O.sub.2-Induced Apoptosis
[0082] In another experiment, we repeated the serum starvation
protocol, and also tested in the same cultures whether rasagiline
can protect against H.sub.2O.sub.2-induced apoptosis. Rasagiline
was introduced to the culture medium 2 hours before inducing serum
starvation or adding H.sub.2O.sub.2, and was present throughout the
apoptosis-inducing protocol (n=4 experiments; .about.2000 cells
counted). As clearly shown in FIG. 4, rasagiline prevented the
apoptosis induced by serum starvation (green bar), but not by
H.sub.2O.sub.2 (gray bar).
Example 4
Rasagiline, S(-)--N-propargyl-1-aminoindan and Propargylamine Block
Hypertrophy Induced by Activation of the Fas Receptor in Cultures
of Neonatal Rat Ventricular Myocytes
[0083] In neonatal rat ventricular myocytes (NRVM), activation of
the Fas receptor does not cause apoptosis, but induces marked
hypertrophy.
[0084] In order to test whether rasagiline can prevent the marked
hypertrophy induced in cultured neonatal rat ventricular myocytes
(for methods, see Yaniv et al., 2002), Fas was activated for 24
hours by incubation with rFasL (10 ng/ml plus 1 .mu.g/ml of the
enhancer antibody). Hypertrophy was assessed by determining the
mRNA levels (by means of RT-PCR) of the atrial natriuretic peptide
(ANP), which is a most common molecular marker of hypertrophy.
Rasagiline (10 .mu.M/ml) was added to the culture 1 hour before Fas
activation and remained in the medium throughout the 24 hours
exposure to rFasL. In these preliminary experiments we have found
that rasagiline prevented Fas-mediated hypertrophy (data not
shown).
[0085] In order to test whether S(-)--N-propargyl-1-aminoindan and
propargylamine have the same effect on marked hypertrophy induced
in cultured neonatal rat ventricular myocytes, similar experiments
were performed using either propargylamin or
S(-)--N-propargyl-1-aminoindan (both at a concentration of 10
.mu.M) instead of rasagiline. As shown in FIG. 5, the marked ANP
mRNA elevation induced by Fas activation for 24 hours was
completely blocked by both S(-)--N-propargyl-1-aminoindan and
propargylamine (3 experiments per each drug).
[0086] Based on these experiments we conclude that rasagiline,
S(-)--N-propargyl-1-aminoindan and propargylamine protect
ventricular myocytes against hypertrophy caused by activation of
the Fas receptor, a finding which may have an important clinical
significance.
Example 5
Propargylamine Protects Cultured Neonatal Rat Ventricular Myocytes
Against Serum Starvation-Induced Apoptosis
[0087] Caspase-3 is a protein of the cysteine-aspartic acid
protease (caspase) family, known as a key pro-apoptotic protein and
therefore as a common marker of apoptosis. It exists as inactive
proenzymes that undergo proteolytic processing at conserved
aspartic residues to produce 2 subunits, large and small, that
dimerize to form the active enzyme. FIG. 6A shows that serum
starvation (0% FCS, 24 hours) in cultures of neonatal rat
ventricular myocytes (NRVM) causes apoptosis, represented by a
marked increase in caspase-3 cleavage.
[0088] In order to test whether propargylamine can prevent serum
starvation-induced apoptosis in cultured neonatal rat ventricular
myocytes, we repeated the serum starvation protocol and 0.1 .mu.M
propargylamine was introduced to the culture medium 1 hour before
serum starvation. As shown in FIGS. 6B-6C, propargylamine
attenuated serum starvation-induced apoptosis in neonatal rat
ventricular myocytes as indicated both by the drug-induced decrease
in caspase 3 cleavage (FIG. 6B) and increase in the expression of
mitochondrial Bcl-2, known as an anti-apoptotic protein (FIG.
6C).
Example 6
Propargylamine and S(-)--N-propargyl-1-aminoindan Protect Cultured
Neonatal Rat Ventricular Myocytes Against Doxorubicin-Induced
Apoptosis
[0089] Adriamycin (doxorubicin) is a commonly used, highly
effective anti cancer drug. However, its clinical efficacy is
limited by severe acute cardiotoxic side effects, e.g., apoptosis,
that limit the total dose of the medicine that may be used safely.
Therefore, finding a drug that will attenuate the cardiotoxic
effects of doxorubicin is of prime importance.
[0090] In order to test whether propargylamine or
S(-)--N-propargyl-1-aminoindan can prevent doxorubicin-induced
apoptosis in cultured neonatal rat ventricular myocytes, these
drugs (at a concentration of either 1 or 10 .mu.M) were introduced
to the culture medium 24 hours before the incubation (24 hours)
with 1 .mu.M doxorubicin.
[0091] As shown in FIG. 7, doxorubicin (Dox) induced marked
apoptosis, as indicated by the marked increase in caspase-3
cleavage, whereas the doxorubicin-induced apoptosis effect was
significantly attenuated by propargylamine and
S(-)--N-propargyl-1-aminoindan, as indicated by the drug-induced
decrease in caspase-3 cleavage.
Example 7
S(-)--N-propargyl-1-aminoindan Improves Cardiac Function
[0092] As the first step in testing the beneficial in vivo efficacy
of the propargylamine derivatives on the cardiac function, we
measured key cardiovascular hemodynamic parameters in control naive
rats, and in rats administered IV with a bolus of 1 mg/kg
S(-)--N-propargyl-1-aminoindan, followed with a bolus of 10 mg/kg
S(-)--N-propargyl-1-aminoindan (Sprague Dawley rats were used, n=3
rats in each group). Measurements were made at baseline, 30 minutes
after each drug administration, and 1 hour (recovery) after drug
administration.
[0093] As shown in FIGS. 8A-8D, intravenous administration of 10
mg/kg S(-)--N-propargyl-1-aminoindan had prominent beneficial
effect on cardiac function. In particular,
S(-)--N-propargyl-1-aminoindan markedly increased cardiac output
(8A) and cardiac index (8B), but did not affect heart rate (8C) or
mean arterial pressure (MAP) (8D). The above-described effect was
reversible during the washout period.
Example 8
Propargylamine and S(-)--N-propargyl-1-aminoindan Increase
Anti-Apoptotic Proteins in Naive Rats
[0094] The major goal of the experiments described in the following
Examples was to examine whether pre-treatment with a propargylamine
derivative can confer protection against "future" stressful cardiac
insults. The clinical implication of this question is whether it
will be able to protect patients at risk. In particular, we
investigated whether propargylamine and
S(-)--N-propargyl-1-aminoindan can attenuate the cardiac
dysfunction in rats with congestive heart failure (CHF) caused by
volume overload induced by aortocaval fistula (AVF).
[0095] In this experiment we tested the effects of propargylamine
and S(-)--N-propargyl-1-aminoindan on several key anti-apoptotic
and pro-apoptotic proteins in hearts of naive rats.
[0096] The drugs (5 mg/kg/day) were orally administered to rats for
21 days (n=4-6 rats in each group), and measurements were made
after sacrifice. These experiments showed that propargylamine did
not affect the expression of mitochondrial pro-apoptotic protein
Bax (FIG. 9A), whereas it markedly increased the expression of the
mitochondrial anti-apoptotic protein Bcl-2 (FIG. 9B), resulting in
marked increase in the ratio Bcl-2/Bax (FIG. 9C), thus generating
an anti-apoptotic effect. Furthermore, both propargylamine and
S(-)--N-propargyl-1-aminoindan increased the expression of the key
anti-apoptotic PKC-.epsilon. (FIGS. 9D-9E, respectively).
Example 9
Propargylamine and S(-)--N-propargyl-1-aminoindan Generate an
Anti-Apoptotic Effect in CHF Rats
[0097] Rats were treated as described in Materials and Methods
hereinabove and volume overload was induced by surgical creation of
an aortocaval fistula (AVF). Sham-operated rats served as controls.
14 days after induction of volume-overload, caspase-3 cleavage and
cytosolic cytochrome C, both pro-apoptotic proteins, were analyzed.
As shown in FIGS. 10A-10B, both caspase-3 and cytochrome C were
markedly increased, indicating that volume overload-induced
congestive heart failure (CHF) is associated with increased
expression of these two proteins.
[0098] In the following experiment we tested whether propargylamine
or S(-)--N-propargyl-1-aminoindan can reduce CHF-induced increase
in caspase-3 and cytochrome C. Rats were treated and drugs were
administered (1 or 7.5 mg/kg/day S(-)--N-propargyl-1-aminoindan, or
5 mg/kg/day propargylamine) as described in Materials and Methods
hereinabove. As shown in FIGS. 11A-11B, both drugs significantly
reduced CHF-induced increase in caspase-3 and cytochrome C,
suggesting that propargylamine derivatives produce an
anti-apoptotic effect both in control and CHF rats, by shifting the
balance between the anti-apoptotic proteins and the pro-apoptotic
proteins towards the former.
Example 10
Propargylamine and S(-)--N-propargyl-1-aminoindan Prevent
Ventricular Hypertrophy and the Decline Ventricular Function in CHF
Rats
[0099] In this set of experiments we determined the ability of
pre-treatment with propargylamine or S(-)--N-propargyl-1-aminoindan
to prevent ventricular hypertrophy and the decline in ventricular
function in CHF rats.
[0100] Rats were treated as described in Materials and Methods
hereinabove and volume overload was induced by surgical creation of
an aortocaval fistula (AVF). Drugs (7.5 mg/kg/day) were
administered according to the protocol described above, starting 7
days prior to surgery (day 0) and during 21 days. Cardiac function
was determined by echocardiography, from which two principle
parameters, namely, diastolic area and systolic area, were
calculated. These parameters were used for calculating the
fractional shortening, which is an established measure of the
ventricular contraction capacity, according to the equation:
Fractional shortening=(diastolic area-systolic area)/diastolic
area.
[0101] As shown in FIGS. 12 and 13, respectively, the treatment
with S(-)--N-propargyl-1-aminoindan completely prevented the
hypertrophic increase in the diastolic and systolic areas seen in
the CHF group (n=3) at days 10 (3 days post-surgery) and 21 (14
days post-surgery). Furthermore, as shown in FIG. 14, the
fractional shortening in the CHF rats on day 21 was significantly
reduced compared to the control rats, but
S(-)--N-propargyl-1-aminoindan completely prevented this
reduction.
[0102] Similar results were obtained with propargylamine using
identical experimental and drug administration protocols. As shown
in FIGS. 15A-15B, the treatment with propargylamine completely
prevented the hypertrophic increase in the diastolic and systolic
areas seen in the CHF rats 14 days post-surgery. FIG. 15C shows
that the fractional shortening in the CHF rats, 14 days
post-surgery was significantly reduced, however, this reduction was
completely prevented by the propargylamine.
[0103] These in vivo experiments are of prime importance since they
demonstrate that both S(-)--N-propargyl-1-aminoindan and
propargylamine block the volume-overload induced hypertrophy and
the reduction in ventricular mechanical function in CHF rats.
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