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.

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.

BACKGROUND OF THE INVENTION

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 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.

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 ˜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.

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 H2O2, O2.− OH− 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.

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.

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-α and Fas.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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).

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).

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.

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).

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.

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.

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

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.

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.

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.

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.

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 μM rasagiline (2A). Apoptotic effect of Fas activation, attained at ˜10 hours incubation with rFasL, was completely prevented by both TVP1022 (0.1 or 1.0 μM) (2B) and propargylamine (0.1 or 1.0 μM) (2C).

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 μM rasagiline; (3B-3D) anti-apoptotic effects obtained by either 0.1-10 μM rasagiline, 0.01-1 μM propargylamine or 0.01-1 μM TVP1022, respectively; (3E) anti-apoptotic effect obtained by 0.1-10 μM TVP1022, using the MTT staining assay as a measure for apoptosis.

FIG. 4 shows that rasagiline protects against serum starvation-mediated but not H2O2— induced apoptosis in H9c2 cells (n=4 experiments, ˜2000 cells counted). * compared to control. ** compared to serum starvation (p<0.05).

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.

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 μM 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 μM propargylamine attenuates serum starvation-induced apoptosis as indicated by increased expression of Bcl-2 (n=3, P<0.05 compared to SS).

FIG. 7 shows that both S(−)—N-propargyl-1-aminoindan (TVP1022) and propargylamine, at a concentration of either 1 or 10 μM, significantly attenuate the doxorubicin-induced apoptosis effect in cultured neonatal rat ventricular myocytes, as indicated by the drug-induced decrease in caspase-3 cleavage.

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.

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-ε, 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-ε expression (9D). TVP1022 increases PKC-ε expression (9E).

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.

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).

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.

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.

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.

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

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.

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-ε, 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 naïve 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.

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.

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.

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.

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.

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.

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)-α-methyl-N′-propargyl phenethylamine HCl; (rac)-3-(N,N-dimethyl-carbamyloxy)-α-methyl-N′-methyl, N-propargyl phenethylamine HCl; (rac)-3-(N-methyl, N-hexyl-carbamyloxy)-α-methyl-N′-methyl, N′-propargyl phenethylamine mesylate; (rac)-3-(N-methyl, N-cyclohexyl-carbamyloxy)-α-methyl-N′-methyl, N′-propargylphenethyl HCl; and (S)-3-(N-methyl, N-hexyl-carbamyloxy)-α-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.

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.

In yet another embodiment, the active agent is selegiline, desmethylselegiline or norprenyl, pargyline or chlorgyline.

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).

All the US patents and other publications mentioned hereinabove are hereby incorporated by reference in their entirety as if fully disclosed herein.

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.

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.

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.

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.

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.

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.

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).

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.

The following examples illustrate certain features of the present invention but are not intended to limit the scope of the present invention.

(i) Materials.

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).

(ii) Cell Line H9c2.

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 μg/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 5×104 cells/ml with DMEM (high glucose) and cultured at 0.5 ml/well on sterile glass cover slips in 24-well plates.

(iii) Protocols Inducing Apoptosis

(a) H2O2 Incubation protocol—To induce apoptosis, H9c2 cultures were exposed to H2O2 (0.5 μM) for 7 hours.

(b) Serum starvation—To induce apoptosis, H9c2 cultures were incubated in the culture medium containing 0% FCS for the indicated times.

(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 μg/ml) for the indicated times, according to the manufacturer\'s recommendations (Alexis Biochemicals, San Diego, Calif.).

(iv) Determination of Apoptosis by DAPI.

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.

(v) Animals.

Studies were conducted on male Sprague Dawley rats (Harlan Laboratories Ltd., Jerusalem, Israel), weighing ˜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.

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).

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.

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 μM) 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 (˜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.

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 μM 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 ˜10%, attained at ˜10 hours incubation with rFasL, and it was completely prevented by both S(−)—N-propargyl-1-aminoindan (2B) and propargylamine (2C).

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 μM) 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.

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 μM) 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 μM), respectively, and FIG. 3E shows the anti-apoptotic effect obtained by S(−)—N-propargyl-1-aminoindan (0.1-10 μM) using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) staining assay as a measure for apoptosis.

In another experiment, we repeated the serum starvation protocol, and also tested in the same cultures whether rasagiline can protect against H2O2-induced apoptosis. Rasagiline was introduced to the culture medium 2 hours before inducing serum starvation or adding H2O2, and was present throughout the apoptosis-inducing protocol (n=4 experiments; ˜2000 cells counted). As clearly shown in FIG. 4, rasagiline prevented the apoptosis induced by serum starvation (green bar), but not by H2O2 (gray bar).

In neonatal rat ventricular myocytes (NRVM), activation of the Fas receptor does not cause apoptosis, but induces marked hypertrophy.

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 μg/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 μM/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).

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 μM) 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).

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.

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.

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 μM 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).

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.

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 μM) were introduced to the culture medium 24 hours before the incubation (24 hours) with 1 μM doxorubicin.

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.

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 naïve 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.

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.

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).

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 naïve rats.

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-ε (FIGS. 9D-9E, respectively).

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.

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.

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.

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.

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.

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.

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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