WO2012014214A2

WO2012014214A2 - Peptides derived from plasminogen activator inhibitor-1 (pai-1) for the treatment of hypertension

Info

Publication number: WO2012014214A2
Application number: PCT/IL2011/000614
Authority: WO
Inventors: Itschak Lamensdorf; Higazi Abd Al-Roof; Abraham Nimrod
Original assignee: Thrombotech Ltd
Current assignee: Thrombotech Ltd
Priority date: 2010-07-28
Filing date: 2011-07-28
Publication date: 2012-02-02
Anticipated expiration: 2013-01-28
Also published as: US20120027558A1; WO2012014214A3

Abstract

The present invention relates to peptides derived from human plasminogen activator inhibitor- 1 (PAI-1) for the treatment of hypertension and complications associated therewith. Particularly, the present invention relates to peptides derived from the amino acid sequence at positions 369-386 of human PAI-1 for the treatment of hypertension.

Description

PEPTIDES DERIVED FROM PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) FOR THE TREATMENT OF HYPERTENSION

FIELD OF THE INVENTION

The present invention relates to peptides derived from human plasminogen activator inhibitor- 1 (PAI-1) useful for the treatment of hypertension and complications associated therewith. Particularly, the present invention relates to peptides derived from the amino acid sequence at positions 369-386 of human PAI-1 for the treatment of hypertension.

BACKGROUND OF THE INVENTION

Hypertension, or high blood pressure, is one of the major risk factors for coronary heart disease and strokes. It can also lead to congestive heart failure, aortic dissection, and renal failure. Over half of patients with angina pectoris, sudden death, stroke, and atherothrombotic occlusion of the abdominal aorta or its branches have hypertension. Greater than 70% of people with dissecting aortic aneurysm, intracerebral hemorrhage, or rupture of the myocardial wall have high blood pressure. It is a major risk factor for atherosclerosis. Screening programs reveal that 25% of the general population is hypertensive. The prevalence of high blood pressure increases with age. However, in older age the disease is usually relatively mild compared to that in young adults where it is often more severe.

According to age, treatment of hypertension must be considered when the systolic arterial pressure is higher than 160-180 mm of mercury and the diastolic pressure higher than 100-110 mm of mercury.

The optimum strategy for the treatment of patients suffering from hypertension is still under discussion. Non-pharmacological treatment (reduction of sodium intake in food, loss of weight, physical exercise, giving up tobacco products, etc.) is a possibility in patients with moderate hypertension. Pharmacological treatment begins with monotherapy, which allows satisfactory blood pressure control in 50-60%) of patients. Changing therapeutic class as well as combination with another class of anti-hypertensive agents represent the alternative treatments in the event of resistance to the first therapy.

Anti-hypertensive agents include aldosterone antagonists; alpha-adrenergic receptor antagonists; potassium channel blockers; beta-adrenergic receptor antagonists; angiotensin-converting enzyme (ACE) inhibitors; angiotensin II receptor antagonists (ARBs); calcium channel antagonists; vasodilators; renin inhibitors; phosphodiesterase inhibitors; and cyclooxygenase-2 (COX-2) inhibitors.

International Patent Application Publication No. WO 97/16170 to Hung discloses treatment for coronary conditions by delivering a therapeutic agent to the pericardial space. According to WO 97/16170, a coronary condition includes hypertension and the therapeutic agent can be a polypeptide such as tPA, urokinase plasminogen activator (uPA), streptokinase and inhibitors of PAI-1.

International Patent Application Publication No. WO 02/00248 to Carmeliet et al. discloses uses of selective inhibitors of urokinase activity for treating cardiac remodeling induced by systemic hypertension. Among the urokinase inhibitors, anti-urokinase monoclonal antibodies and amiloride are listed.

WO 2007/143099 to Sobel discloses methods of inhibiting cardiac PAI-1 by administering PAI-1 antagonists. According to WO 2007/143099, cardiac PAI-1 content increases in response to age, insulin resistance, and myocardial infarction and it is believed that inhibition of cardiac PAI-1 decreases the risk for cardiovascular disease. Inhibiting cardiac PAI-1 according to WO 2007/143099 could be accomplished by inhibiting the binding of cardiac PAI-1 to one of its targets, such as tPA, or by inhibiting the ability of cardiac PAI-1 to affect one of its targets. PAI-1 antagonists include insulin sensitizing agents, anti-lipidemic agents and ACE inhibitors among others.

Madison et al. (Proc. Natl. Acad. Sci. U.S.A. 87: 3530-3533, 1990) identified a region at positions 350-355 of PAI-1 which contains negatively charged amino acid residues (Glu-Glu-Ile-Ile-Met-Asp), which region was found to be involved in the interaction of PAI-1 with tPA.

U.S. Patent No. 6,750,201 to Cines et al. discloses compositions comprising peptides comprising at least six amino acids derived from PAI-1, particularly disclosed is a peptide of the sequence EEIIMD, and methods of use thereof for promoting internalization and degradation of urokinase-type plasminogen activator.

International Patent Application Publication No. WO 03/095476 to Higazi discloses the peptide EEIIMD and the peptide acetyl-RMAPEEIIMDRPFLYVVR-amide, compositions comprising same and uses thereof either alone or in combination with one or more fibrinolytic agents for enhancing the fibrinolytic activity of the fibrinolytic agents, for reducing the side effects due to vasoactivity caused by the fibrinolytic agents, and/or for prolonging the half lives of the fibrinolytic agents.

International Application Publication No. WO 2008/018084 to Higazi and Cines discloses the use of the peptide EEIIMD and 6-mer peptide analogs thereof for preventing neuronal damage and for treating brain injury.

International Application Publication No. WO 2009/013753 to Higazi discloses 18-mer peptides corresponding to the region of human plasminogen activator inhibitor- 1 that interacts with tPA and uses thereof either alone or in combination with fibrinolytic agents for reducing neurological damage and for fibrinolytic therapy.

International Patent Application Publication No. WO 201 1/086557 to Higazi discloses use of peptides of 7 to 30 amino acid residues derived from the region of human plasminogen activator inhibitor- 1 that interacts with tPA for treating asthma and for inhibiting airway smooth muscle contractility.

There is still an unmet need for improved and highly effective means for treating hypertension and complications associated therewith.

SUMMARY OF THE INVENTION

The present invention provides methods for treating hypertension comprising administering to a subject in need of such treatment an isolated peptide consisting of 18 amino acid residues corresponding to the amino acid sequence at positions 369-386 of human plasminogen activator inhibitor- 1 (PAI-1) or a fragment thereof.

The present invention further provides methods for treating a hypertension-related cardiovascular disease comprising administering to a subject in need of such treatment an isolated peptide consisting of 18 amino acid residues corresponding to the amino acid sequence at positions 369-386 of human plasminogen activator inhibitor- 1 (PAI-1) or a fragment thereof, wherein the cardiovascular disease is not a thrombus-related cardiovascular disease.

The present invention is based in part on the unexpected findings that an 18-mer peptide of the amino acid sequence acetyl-RMAPEEIIMDRPFLYVVR-amide (SEQ ID NO: l) corresponding to the amino acid sequence at positions 369-386 of human PAI-1 was highly effective in decreasing blood pressure in normotensive human subjects. It is disclosed that intravenous administration of the 18-mer peptide decreased the blood pressure of healthy human subjects by 20 mm Hg when measured forty minutes after peptide administration. The effect of the 18-mer peptide was reflected on both the systolic and the diastolic blood pressure.

It is further disclosed that the 18-mer peptide was highly effective in reducing blood pressure in spontaneously hypertensive rats. Intravenous administration of the peptide to the hypertensive rats decreased their blood pressure by about 15% of the initial blood pressure.

It is to be understood that the present invention discloses uses of the 18-mer peptides or fragments thereof for reducing blood pressure in subjects having hypertension as well as in subjects having a hypertension-related cardiovascular disease. The present invention excludes the uses of these peptides for the treatment of thrombus-related cardiovascular diseases. Thus, the subjects to be treated according to the present invention do not undergo therapy with plasminogen activators or with proteins acting as plasminogen activators. As used herein, a "thrombus-related cardiovascular disease" is a disease necessitating administration of fibrinolytic agents including, but not limited to plasminogen activators. The present invention is intended for treatment of hypertension not involving the concurrent need for thrombolysis.

According to one aspect, the present invention provides a method for treating hypertension comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of an isolated peptide of the amino acid sequence as set forth in SEQ ID N02:

RrArg-Met-Ala-Pro-Xi^-Ile-Ile-Met-Xs-Arg-Pro-Phe-Leu^-Val-Val-Arg-^ or a fragment thereof comprising the amino acid sequence X₁-X₂-Ile-Ile-Met-X₃ as set forth in SEQ ID NO:3,

wherein Rj is selected from the group consisting of a hydrogen, acetyl, alkyl, and an amino blocking group; Xi is selected from the group consisting of Asp, Glu, and Arg;

X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, and a carboxyl blocking group; the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

According to some embodiments, Xj is Glu, X₂ is Glu, and X₃ is Asp. According to additional embodiments, Ri is acetyl and R₂ is amide. According to further embodiments, the peptide to be used for treating hypertension is of the amino acid sequence selected from the group consisting of SEQ ID NOs:l and 4 to 10. According to a certain embodiment, the peptide to be used is of the amino acid sequence as set forth in SEQ ID NO: l. According to another embodiment, the peptide to be used is of the amino acid sequence as set forth in SEQ ID NO:7.

According to another aspect, the present invention provides a method for treating a hypertension-related cardiovascular disease comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of an isolated peptide of the amino acid sequence as set forth in SEQ ID N02:

R₁-Arg-Met-Ala-Pro-X₁-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X₄-Val-Val-Arg-R₂ or a fragment thereof comprising the amino acid sequence

as set forth in SEQ ID NO:3,

wherein Ri is selected from the group consisting of a hydrogen, acetyl, alkyl, and an amino blocking group; X] is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, and a carboxyl blocking group; the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, and wherein the cardiovascular disease is not a thrombus-related cardiovascular disease.

According to some embodiments, X_\ is Glu, X₂ is Glu, and X₃ is Asp. According to additional embodiments, Ri is acetyl and R₂ is amide.

According to further embodiments, the peptide to be used for treating the cardiovascular disease is of the amino acid sequence selected from the group consisting of SEQ ID NOs: l, 4 to 10. According to a certain embodiment, the peptide to be used is of the amino acid sequence as set forth in SEQ ID NO:l . According to another embodiment, the peptide to be used is of the amino acid sequence as set forth in SEQ ID NO:7.

According to additional embodiments, the hypertension-related cardiovascular disease is selected from the group consisting of reduced blood vessel patency, diastolic dysfunction, peripheral vascular disease, coronary artery disease, atherosclerosis, atherogenesis, angina, ischemic heart disease, myocardial infarction, and restenosis. According to some embodiments, the hypertension-related cardiovascular disease is selected from the group consisting of reduced blood patency and myocardial infarction.

According to further embodiments, the pharmaceutical composition is administered by intravenous, subcutaneous, intramuscular, oral, topical, transdermal, intranasal, epidural, ophthalmic, vaginal or rectal administration route. According to a certain embodiment, the pharmaceutical composition is administered intravenously.

According to yet further embodiments, the pharmaceutical composition is formulated in a form selected from the group consisting of a solution, suspension, emulsion, tablet, gel, powder, cream, depot, and a sustained-release formulation.

According to another aspect, the present invention provides an isolated peptide of the amino acid sequence as set forth in SEQ ID NO:2:

R₁-Arg-Met-Ala-Pro-X₁-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X₄-Val-Val-Arg- ₂ or a fragment thereof comprising the amino acid sequence Xi-X₂-Ile-Ile-Met-X₃ as set forth in SEQ ID NO: 3 for the manufacture of a medicament for the treatment of hypertension, wherein is selected from the group consisting of a hydrogen, acetyl, alkyl, and an amino blocking group;

is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, and a carboxyl blocking group.,

R₁-Arg-Met-Ala-Pro-Xi-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X4-Val-Val-Arg-R₂ or a fragment thereof comprising the amino acid sequence X₁-X₂-Ile-Ile-Met-X₃ as set forth in SEQ ID NO: 3 for the preparation of a medicament for the treatment of a hypertension-related cardiovascular disease, wherein Ri is selected from the group consisting of a hydrogen, acetyl, alkyl, and an amino blocking group; Xi is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, and a carboxyl blocking group, and wherein the cardiovascular disease is not thrombus-related cardiovascular disease. These and other embodiments of the present invention will be better understood in relation to the figures, description, examples, and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of the 18-mer peptide designated THR-18 on blood pressure of hypertensive rats.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the findings that an 18-mer peptide corresponding to the amino acid sequence at positions 369-386 of human PAI-1 was capable of decreasing blood pressure in animals and human subjects.

The present invention provides isolated peptides derived from PAI-1 for the treatment of hypertension and cardiovascular diseases associated therewith.

Specifically, the present invention relates to the following peptides:

1) Ac-RMAPEEIIMDRPFLYVVPv-amide (SEQ ID NO: 1 )

2) RMAPEEIIMDRPFLYVVR (SEQ ID NO:4)

3) Ac-RMAPEEIIMDRPFLYVVR (SEQ ID NO:5)

4) RMAPEEIIMDRPFLYVVR-amide (SEQ ID NO:6)

5) Ac-RMAPEEIIMDRPFLFVVR-amide (SEQ ID NO:7)

6) RMAPEEIIMDRPFLFVVR (SEQ ID NO:8)

7) Ac-RMAPEEIIMDRPFLFVVR (SEQ ID NO:9)

8) RMAPEEIIMDRPFLFVVR-amide (SEQ ID NO: 10)

9) EEIIMD (SEQ ID NO: 1 1)

10) Full-length amino acid sequence of human PAI-1 (SEQ ID NO: 12)

It should be appreciated that tissue-type plasminogen activator (tPA) is approved by the U.S. Food and Drug Administration for the treatment of acute ischemic stroke. tPA, like urokinase (uPA), converts plasminogen to the active protease plasmin, the latter dissolves fibrin clots by a process called fibrinolysis. In addition to its salutary role in lysing intravascular clots and hence in reperfusion, tPA mediates cerebrovasodilation which leads to intracerebral hemorrhage.

Previous studies have demonstrated that the 6-mer peptide of the amino acid sequence EEIIMD (SEQ ID NO:l 1) and the 18-mer peptide of the amino acid sequence Ac-RMAPEEIIMDRPFLYVVR-amide (SEQ ID NO: l) were capable of inhibiting the vasoactivity of tPA and uPA without inhibiting their fibrinolytic activity in subjects suffering from thromboembolic diseases (see, for example, Nassar et al. Blood 103:897- 902, 2004; WO 2008/018084; and WO 2009/013753). The 6-mer and 18-mer peptides were shown to reduce the deleterious effects of intracerebral hemorrhage induced by the fibrinolytic agent tPA in these subjects. Due to the fact that the 6-mer and 18-mer peptides inhibited tPA-induced cerebrovasodilation and cerebral hemorrhage, and without being bound by any mechanism of action, it is assumed that the effect of these peptides does not bring to cerebrovasodilation. The effect of these peptides seems to culminate in cerebrovasoconstriction. In addition, the 6-mer and 18-mer peptides were shown to prevent neuronal damage in subjects suffering from such damage who had not been treated with tPA. Nowhere in the art is it disclosed or suggested that peptides corresponding to the amino acid sequence of PAI-1 which is involved in the interaction of PAI-1 with tPA, can reduce blood pressure and as such can be used as hypotensive agents.

The present invention discloses for the first time that the 18-mer peptide of SEQ ID NO:l or analogs or fragments thereof when administered in the absence of fibrinolytic agents such as tPA or uPA, can exert significant antihypertensive effect. Thus, by virtue of their hypotensive activity, these peptides as well as their analogs and fragments are useful for treating hypertension and cardiovascular diseases attributed to hypertension.

According to one aspect, the present invention provides a peptide having the amino acid sequence of general formula I:

R Arg-Met-Ala-Pro-XrXi-Ile-Ile-Met-Xs-Arg-Pro-Phe-Leu-^-Val-Val-Arg-^ or a fragment thereof comprising the amino acid sequence X₁-X₂-Ile-Ile-Met-X₃, wherein Ri is selected from the group consisting of a hydrogen, acetyl, alkyl, and an amino blocking group; Xj is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, and a carboxyl blocking group. Thus, the present invention provides uses of 6 to 18-mer peptides comprising in a preferred embodiment the sequence EEIIMD, for the treatment of hypertension or cardiovascular diseases attributed to hypertension, with the proviso that the treatment excludes co-treatment with plasminogen activators or with proteins acting as plasminogen activators. According to some embodiments, the peptide is an 18- mer peptide of the amino acid sequence Ac-RMAPEEIIMDRPFLYVVR-amide as set forth in SEQ ID NO:l, or a fragment thereof comprising the sequence EEIIMD. According to a certain embodiment, the peptide is of the amino acid sequence as set forth in SEQ ID NO: 1.

The term "peptide" as used throughout the specification and claims designates a linear series of amino acid residues connected one to the other by peptide bonds. The amino acid residues are represented throughout the specification and claims by one-letter or three-letter codes according to IUPAC conventions.

The term "amino acid" or "amino acid residue" is understood to include the 20 naturally occurring amino acids.

The peptides of the present invention consist of 6 to 18 amino acid residues. The peptides of the present invention can be isolated by any protein purification method known in the art. For example, PAI-1 can be subjected to one or more proteolytic enzymes to yield a mixture of peptides, which can further be purified by any protein purification method known in the art to obtain the isolated peptides. Alternatively or additionally, PAI-1 can be cleaved by chemical agents such as, for example, CNBr to yield a mixture of peptides that can be further purified to obtain isolated peptides.

The peptides of the present invention can also be prepared by methods well known in the art including chemical synthesis or recombinant DNA technology. A preferred method of synthesizing the peptides of the present invention involves solid- phase peptide synthesis utilizing a solid support as described by Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). Large-scale peptide synthesis is described, for example, by Andersson et al. (Biopolymers 55(3): 227-50, 2000). Examples of solid phase peptide synthesis methods include the BOC method, which utilizes tert-butyloxcarbonyl as the a- amino protecting group, and the FMOC method, which utilizes 9- fluorenylmethyloxcarbonyl to protect the a-amino of the amino acid residues, both methods are well-known by those of skill in the art. Alternatively, the peptides of the present invention can be synthesized by standard solution synthesis methods (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984).

The peptides useful for practicing the present invention need not consist of an amino acid sequence identical to RMAPEEIIMDRPFLYVVR set forth in SEQ ID NO:4 or RMAPEEIIMDRPFLFVVR set forth in SEQ ID NO:8 so long as the peptide analogs are capable of treating and/or preventing hypertension or a cardiovascular disease attributed to hypertension. The term "analog" includes any peptide comprising altered sequence by amino acid substitutions, deletions, or chemical modifications of the peptides listed herein above and which displays anti-hypertensive activity. By using "amino acid substitutions", it is meant that functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are known as conservative substitutions. Additionally, a non-conservative substitution may be made so long as the anti- hypertension activity of the peptide is maintained. It will be appreciated that the present invention encompasses peptide analogs, wherein one or more amino acids are substituted by another amino acid to produce a peptide analog having increased stability or longer half-life as compared to the peptides listed herein above.

While the amino acid residues of the peptide sequences set forth herein above are all in the "L" isomeric form, residues in the "D" isomeric form can substitute any L- amino acid residue so long as the peptide analog retains anti-hypertension activity. Production of a retro-inverso D-amino acid peptide analog where the peptide is made with the same amino acids as disclosed, but at least one amino acid, and perhaps all amino acids are D-amino acids is well known in the art. When all of the amino acids in the peptide analog are D-amino acids, and the N- and C-terminals of the peptide analog are reversed, the result is an analog having the same structural groups being at the same positions as in the L-amino acid form of the peptide. However, the peptide analog is more stable to proteolytic degradation and is therefore useful in many of the applications recited herein.

The present invention further encompasses peptide derivatives of the peptides listed herein above. The term "derivative" refers to a peptide having an amino acid sequence that comprises the amino acid sequence of the peptide of the invention, in which one or more of the amino acid residues is subjected to chemical derivatizations by a reaction of side chains or functional groups, where such derivatizations do not destroy the anti-hypertension activity of the peptide derivative. Chemical derivatization of amino acid residues include, but are not limited to, acetylation, amidation, glycosylation, oxidation, reduction, myristylation, sulfation, acylation, ADP-ribosylation, cyclization, disulfide bond formation, hydroxylation, iodination, and methylation.

The peptide derivatives according to the principles of the present invention also include bond modifications, including but not limited to CH₂-NH, CH₂-S, CH₂-S=0, 0=C-NH, CH₂-0, CH₂-CH₂, S=C-NH, CH=CH, and CF=CH and backbone modifications. Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO-); ester bonds (-C(R)H-C-0-0-C(R)-N); ketomethylene bonds (-CO-CH2-); oc-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl group, e.g., methyl; carba bonds (-CH2-NH-); hydroxyethylene bonds (-CH(OH)-CH2-); thioamide bonds (-CS-NH-); olefmic double bonds (-CH=CH-); and peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.

Blocking groups are well known to those of skill in the art as are methods of coupling such groups to the appropriate residue(s) of the peptides of the present invention (see, e.g., Greene et al., (1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc. Somerset, N.J.).

In certain embodiments, the terminal amino acids of the peptides of the invention are blocked with a protecting or blocking group. A wide number of blocking groups are suitable for this purpose. Such groups include, but are not limited to, acetyl, alkyl and amide groups with acetyl and alkyl groups being particularly preferred for N-terminal protection and amide groups being preferred for carboxyl terminal protection. In certain embodiments, an acetyl group is used to protect the amino terminus and an amide group is used to protect the carboxyl terminus. In certain embodiments, the protecting groups can include alkyl chains as in fatty acids, propeonyl, formyl, or others. Particularly preferred carboxyl blocking groups include amides, esters, and ether-forming blocking groups. Other blocking groups include, but are not limited to Fmoc, t-butoxycarbonyl (t- BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9- fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4- methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4- methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2- pyridinesulphenyl (Npys), l-(4,4-dimentyl-2,6-diaxocyclohexylid-ene)ethyl (Dde), 2,6- dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-C1--Z), 2- bromobenzyloxycarbonyl (2~Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy (cHxO), t- butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5 -hydroxy lysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. The peptides may also contain non-natural amino acids. Examples of non-natural amino acids are norleucine, ornithine, citrulline, diaminobutyric acid, homoserine, isopropyl Lys, 3-(2'-naphtyl)-Ala, nicotinyl Lys, amino isobutyric acid, and 3-(3'-pyridyl-Ala). The peptides may also contain non-protein side chains or one or more non-amino acid monomers (e.g., fatty acids, complex carbohydrates, and the like).

The present invention includes conjugates of the peptides of the invention. The term "conjugate" is meant to define a peptide of the present invention coupled to or conjugated with another protein or polypeptide. Such conjugates may have advantages over the peptides themselves. Such conjugates can be made by protein synthesis, e. g., by use of a peptide synthesizer, or by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric protein by methods commonly known in the art.

A peptide of the present invention may also be conjugated to itself or aggregated in such a way as to produce a large complex containing the peptide. Such large complexes may be advantageous because they may have new biological properties such as longer half-life in circulation or greater activity.

Pharmaceutical compositions and administration routes

The present invention provides methods for treating or preventing hypertension or a cardiovascular disease due to hypertension comprising administering to a subject having hypertension or the hypertension-related cardiovascular disease a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the invention and a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutical composition" refers to a preparation of one or more of the peptides described herein with other chemical components such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the one or more peptides to an organism.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The pharmaceutical compositions of the present invention can be formulated as pharmaceutically acceptable salts of the peptides of the present invention. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, Ν,Ν'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

When the peptide of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, trifluoroacetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids. The term "carrier" refers to a diluent or vehicle that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

The pharmaceutical compositions of the invention can further comprise an excipient. Herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Suitable pharmaceutical excipients include mannitol, starch, glucose, lactose, sucrose, trehalose, gelatin, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, propylene glycol, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents; pH buffering agents such as acetates, citrates or phosphates; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethyl enediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

Typically, pharmaceutical compositions, which contain peptides as active ingredients are prepared as injectable, either as liquid solutions or suspensions, however, solid forms, which can be suspended or solubilized prior to injection, can also be prepared. The compositions can also take the form of emulsions, tablets, gels, slurries, powders, creams, depots, sustained-release formulations and the like.

Methods of introduction of a pharmaceutical composition comprising a peptide of the invention include, but are not limited to, intravenous, subcutaneous, intramuscular, intraperitoneal, oral, topical, transdermal, intranasal, epidural, ophthalmic, vaginal and rectal routes. The pharmaceutical compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other therapeutically active agents. The administration may be localized, or may be systemic. Pulmonary administration can also be employed, e.g., by use of an inhaler or neubilizer. According to a certain embodiment, the pharmaceutical composition is administered by bolus injection.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers further comprising excipients which facilitate processing of the active ingredients into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, dragees, liquids, gels, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable excipients as desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

All formulations should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a neubilizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.

For directed internal applications, the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to the materials of the above type, a liquid carrier such as fatty oil. In addition, dosage unit forms can contain other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

A peptide of the invention can be delivered in a controlled release system. For example, the peptide can be administered in combination with a biodegradable, biocompatible polymeric implant, which releases the peptide over a controlled period of time at a selected site. Examples of preferred polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.). A controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of a systemic dose.

Uses of the peptides

The present invention provides peptides for the treatment of hypertension. The present invention further provides peptides for the treatment of cardiovascular diseases attributed to hypertension with the proviso that the treatment of the cardiovascular disease excludes a combination therapy with plasminogen activators or with proteins acting as plasminogen activators.

It will be appreciated that the term "treatment" as used herein includes both treatment and/or prophylactic use of the pharmaceutical compositions comprising the peptides of the invention. In the present invention prophylactic use of the pharmaceutical compositions comprises administering to a subject in heed of such treatment a therapeutically effective amount of the pharmaceutical composition disclosed herein above to prevent the onset of hypertension or a cardiovascular disease attributed to hypertension; and to prevent the progression of hypertension or the cardiovascular disease, wherein the subject having or at risk of having the cardiovascular disease is not co-treated with plasminogen activators or with proteins acting as plasminogen activators.

The term a "therapeutically effective amount" means an amount of a peptide effective to prevent, alleviate, or ameliorate hypertension or symptoms of a cardiovascular disease attributed to hypertension in the subject being treated.

The term "hypertension" refers to chronic hypertension, systolic hypertension, diabetic hypertension, pulmonary hypertension, acute severe hypertension, ocular hypertension and any combination thereof. According to a certain embodiment, hypertension is acute severe hypertension.

The term "hypertension-related cardiovascular disease" refers to any cardiovascular disease or disorder known in the art which is attributed to hypertension, including, but not limited to, diastolic dysfunction, reduced blood vessel patency, peripheral vascular disease (including vascular ischemia), coronary artery disease, myocardial infarction, atherosclerosis, atherogenesis, angina (including chronic, stable, unstable and variant (Prinzmetal) angina pectoris), myocardial ischemia, and restenosis. It is to be understood that thrombus-related cardiovascular diseases are excluded from the present invention. The term a "thrombus-related cardiovascular disease" as used herein refers to a cardiovascular disease which is attributed to thrombi formation and which disease requires administering of fibrinolytic agents such as, for example, plasminogen activators in order to treat the disease.

The term "plasminogen activator" refers to any protein capable of activating plasminogen and as such to be useful in fibrinolytic therapy. Without being bound by any mechanism of action, activation of plasminogen to plasmin can take place by enzymatic cleavage (e.g., by tissue-type plasminogen activator or uPA) or by a conformational change. Plasminogen activators include, but are not limited to, urokinase (uPA), tissue- type plasminogen activator (tPA), streptokinase, single chain urokinase (scuPA), two chain uPA (tcuPA), rt-PA, alteplase, reteplase, lanoteplase, TNK-rt-PA, anisoylated plasminogen streptokinase complex, anistreplase, and derivatives thereof (see, for example, U.S. Patent No. 7,271,143 indicated by reference as if fully set forth herein). "Heart failure" includes, but is not limited to congestive heart failure, compensated heart failure, decompensated heart failure, and the like.

According to some embodiments, the subject is a mammal. According to a certain embodiment, the mammal is a human.

Determination of a therapeutically effective amount of the peptide to be administered is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

The precise amount of the peptide to be administered to a particular subject, preferably a mammal, more preferably a human being, in the method of treatment of the present invention will depend on a number of factors, for example the specific peptide administered; its mode of administration; the particular clinical condition being treated and/or its severity; and/or the age, body mass and/or past clinical history of the patient to be treated, and always lies within the sound discretion of the person administering and/or supervising the treatment, for example a medical practitioner such as nurse and/or physician. Nevertheless, a suitable daily dose of the peptide for administration to a mammal is generally from about 0.01 mg/day per kg of the mammal's body mass to about 80 mg/kg/day, more usually 0.2-40 mg/kg/day given in a single dose and/or in divided doses at one or more times during the day. The pharmaceutical composition can contain from about 0.1% to about 99% by weight of the peptide and is generally prepared in unit dose form, a unit dose of a peptide generally being from about 0.1 mg to about 500 mg.

Dosage amount and administration intervals may be adjusted individually to provide sufficient plasma levels of the peptide to induce an anti-hypertensive effect.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks, or until cure is effected or diminution of the disease state is achieved.

The peptides of the present invention can be administered alone or in conjunction with other therapeutic agents.

"Therapeutic agent" includes any therapeutic agent that can be used to treat or prevent the diseases described herein. Therapeutic agents include, for example, aldosterone antagonists, alpha-adrenergic receptor antagonists, angiotensin II antagonists, angiotensin-converting enzyme (ACE) inhibitors, anti-hyperlipidemic compounds, vasodilator compounds, β-adrenergic antagonists, calcium channel blockers, diuretics, endothelin antagonists, phosphodiesterase inhibitors, potassium channel blockers, proton pump inhibitors, renin inhibitors, selective cyclooxygenase-2 (COX-2) inhibitors, and the like. The following examples are presented to provide a more complete understanding of the invention. The specific techniques, conditions, materials, proportions and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention. EXAMPLE 1

The hypotensive effect of THR-18 in dogs

A cardiovascular safety study was conducted in telemetered beagle dogs. The study was designed to examine the effects of the 18-mer peptide of SEQ ID NO:l designated THR-18 on the blood pressure (systolic, diastolic, mean), heart rate, ECG and clinical signs after escalating single intravenous slow bolus doses of 0 (vehicle), 5, 10 and 20 mg/kg (3 males and 3 females/dose group).

Treatment with THR-18 at a low dose of (5 mg/kg) resulted in a slight but not statistically significant decrease of the blood pressure parameters in females (15% in systemic blood pressure), 30 min after the treatment.

Treatment with THR-18 at a mid dose (10 mg/kg) or at a high dose (20 mg/kg) caused a moderate decrease of the blood pressure (the systolic, diastolic and average blood pressure) in both sexes to a similar degree at 30 min as a minimum value. The decrease was slightly stronger in the females (35%-49% in SP) than in the males (25%- 42% in SP). At approximately 180 min, the blood pressure parameters returned to the baseline in both genders.

EXAMPLE 2

The hypotensive effect of THR-18 in humans A phase I escalating dose study was conducted in healthy volunteers by the

Clinical Pharmacology Unit, Division of Medicine, Hadassa Medical Center, Jerusalem.

Subjects (6 subjects/group) were exposed to escalating doses of 0.25, 0.5, 0.75 and 1 mg/kg of THR-18, administered by intravenous infusion during 15 min. No significant effects were seen in the 0.25 and 0.5 mg/kg treated groups. However, a moderate lowering of blood pressure was seen in two subjects in the 0.75 mg/kg group, and a further drop of about 20 mm Hg was evident in 5 out of the 6 subjects in the 1 mg/kg group. The effect in this group was maximal at 40 min post infusion. EXAMPLE 3

The hypotensive effect of THR-18 in spontaneously hypertensive rats

Male rats (three months old) having spontaneous hypertension were fed ad libitum a commercial rodent diet and housed under standard laboratory conditions in a climate controlled environment at a temperatures of 20-24°C and relative humidity of 30-70% with a 12 hours light and 12 hours dark cycle.

The rats were anesthetized by subcutaneous administration of ketamine:xylazine during the surgical procedure to expose the right jugular vein, left carotid artery and trachea. A heparinized polyethylene cannula was inserted into the carotid artery for monitoring the pulsatile blood pressure (BP) via a pressure transducer connected to a Biopac system TSD 104 A.

The 18-mer peptide of SEQ ID NO: l (designated THR-18) was prepared as a trifluoroacetic acid salt and administered to the rats at a dose of 2.5 mg/kg or 10 mg/kg in a volume of 10 ml/kg (6 rats in each group). The carrier was 5% mannitol in water for injection. A control group received the carrier only. The peptide or carrier were administered by a bolus intravenous administration of 10% of the dose volume during two minutes and the rest of the dose was administered by infusion (dosing) during one hour. The rats weight on average 300 gr.

The results of the blood pressure as monitored during the bolus injection, infusion and thereafter are presented in FIG. 1. The initial blood pressure (BP) of the rats was 100 ± 5 mmHg (Mean ± S.D.). Administration of the carrier or 2.5 mg/kg THR-18 caused a rapid increase of 5-10% of the initial blood pressure (FIG. 1). This small increase was not statistically significant (p>0.05; according to paired t-test), and it may be related to the effect of anesthesia on the SHR rats.

The blood pressure of the rats receiving the high dose of THR-18 (10 mg/kg) did not increase after bolus injection and infusion, and one hour later, a decrease in the BP of

5-10% was observed. At 2 hours after peptide infusion, an additional decrease in BP of about 10% was observed, and this was statistically different from the control treated group (p<0.05). At three hours post-infusion the decrease in BP was about 15% of the initial value, and was statistically different (p<0.05) from the BP of the THR-18 2.5 mg/kg group.

Thus, a dose of 10 mg/kg of THR-18, delivered during 1 hour infusion, resulted in statistically significant decrease in BP of hypertensive rats.

It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described herein above. Rather the scope of the invention is defined by the claims that follow.

Claims

1. A method for treating hypertension comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of an isolated peptide of the amino acid sequence set forth in SEQ ID NO:2:

R₁-Arg-Met-Ala-Pro-Xi-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X4-Val-Val-Arg-R₂ or a fragment thereof comprising the amino acid sequence X₁-X₂-Ile-Ile-Met-X₃ as set forth in SEQ ID NO:3,

wherein R] is selected from the group consisting of a hydrogen, acetyl, alkyl, and an amino blocking group; Xj is selected from the group consisting of

Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, and a carboxyl blocking group; the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

2. The method according to claim 1, wherein X] is Glu, X₂ is Glu, and X₃ is Asp.

3. The method according to claim 1, wherein Ri is acetyl and R₂ is amide.

4. The method according to claim 1, wherein the peptide is of the amino acid sequence selected from the group consisting of SEQ ID NOs:l and 4 to 10.

5. The method according to claim 4, wherein the peptide is of the amino acid sequence as set forth in SEQ ID NO: 1.

6. The method according to claim 4, wherein the peptide is of the amino acid sequence as set forth in SEQ ID NO:7.

7. A method for treating a hypertension-related cardiovascular disease comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of an isolated peptide of the amino acid sequence set forth in SEQ ID NO:2:

Ri-Arg-Met-Ala-Pro-Xi-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X4-Val-Val-Arg-R₂ or a fragment thereof comprising the amino acid sequence X₁-X₂-Ile-Ile-Met-X₃ as set forth in SEQ ID NO:3,

wherein Rj is selected from the group consisting of a hydrogen, acetyl, alkyl, and an amino blocking group; Xi is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, and a carboxyl blocking group; the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, and wherein the cardiovascular disease is not a thrombus-related cardiovascular disease.

8. The method according to claim 7, wherein Xi is Glu, X₂ is Glu, and X₃ is Asp.

9. The method according to claim 7, wherein Rj is acetyl and R₂ is amide.

10. The method according to claim 7, wherein the peptide is of the amino acid sequence selected from the group consisting of SEQ ID NOs:l, 4 to 10.

1 1. The method according to claim 10, wherein the peptide is of the amino acid sequence as set forth in SEQ ID NO:l .

12. The method according to claim 10, wherein the peptide is of the amino acid sequence as set forth in SEQ ID NO: 7.

13. The method according to claim 7, wherein the cardiovascular disease is selected from the group consisting of reduced blood vessel patency, diastolic dysfunction, a coronary artery disease, atherosclerosis, atherogenesis, angina, aneurysm, ischemic heart disease, myocardial infarction, peripheral vascular disease, and restenosis.

14. The method according to claim 7, wherein the cardiovascular disease is myocardial infarction.

15. The method according to claim 7, wherein the cardiovascular disease is reduced blood vessel patency.

16. The method according to claims 1 and 7, wherein the pharmaceutical composition is administered by intravenous, subcutaneous, intramuscular, intraperitoneal, oral, topical, intradermal, transdermal, intranasal, epidural, ophthalmic, vaginal or rectal administration route.

17. The method according to claims 1 and 7, wherein the pharmaceutical composition is administered intravenously.

18. The method according to claim 17, wherein the pharmaceutical composition is administered by bolus injection.

19. The method according to claims 1 and 7, wherein the pharmaceutical composition is formulated in a form selected from the group consisting of a solution, suspension, emulsion, tablet, gel, powder, cream, depot, and a sustained-release formulation.