NZ621790B2

NZ621790B2 - Cardiovascular therapeutics

Info

Publication number: NZ621790B2
Application number: NZ621790A
Authority: NZ
Inventors: Christopher John Charles; Christopher Joseph Pemberton; Arthur Mark Richards; Maithri Siriwardena
Original assignee: Otago Innovation Limited
Priority date: 2011-08-18
Filing date: 2012-08-17
Publication date: 2018-01-04

Abstract

Disclosed is the use of a pharmaceutical composition comprising a therapeutically effective amount of a Type-B natriuretic signal peptide (BNP-SP) fragment agent and a pharmaceutically acceptable carrier in the manufacture of a medicament for preventing and/or treating a cardiovascular disorder, wherein the Type-B natriuretic signal peptide fragment is an amino acid sequence as defined by a sequence or formula as disclosed in the specification. rein the Type-B natriuretic signal peptide fragment is an amino acid sequence as defined by a sequence or formula as disclosed in the specification.

Description

OTAPC CARDIOVASCULAR THERAPEUTICS FlELD The inventions relate to phamiaceuticals. compositions and s useful for treating. preventing and ameliorating the effects of cardiovascular diseases, disorders and conditions. as well as articles and kits comprising such compounds and compositions.

BACKGROUND The following includes infomiation that may be useful in understanding the present invention. It is not an admission that any of the infomtation, publications or documents specifically or implicitly referenced herein is prior art. or essential, to the presently described or claimed inventions. All publications and patents mentioned herein are hereby incorporated herein by reference in their entirety.

Heart disease. including ischernic heart disease. myocardial tions and other acute coronary syndromes. as well as heart failure, is a major health problem throughout the world.

It is tood. for example. that rnyocardiai infarctions are a signiﬁmnt source of mortality among those individuals with heart disease. Myocardial infarction (MI) or acute dial infarction (AMI). commonly known as a heart attack. is the interruption of blood supply to a part of the heart. causing heart cells to die. This is most commonly due to occlusion (blockage) of a coronary artery following the rupture of a vulnerable sclerotic plaque. which is an unstable collection of lipids and white blood cells (especially macrophwes) in the wall of an . The resulting ischemia and oxygen shortage. if left untreated for a sufficient period of time. can cause damage or death (infarction) of heart muscle . i.e., the myocardium. Classical symptoms of acute myocardial infarction include sudden chest pain (typically radiating to the left arm or left side of the neck). shortness of breath. nausea. vomiting. palpitations. sweating. and anxiety. Approximately one quarter of all myocardial infarctions. however. are 'silent.‘ i.e., without chest pain or other symptoms. Immediate treatment for suspected acute myocardial tion es oxygen. n. and subiingual nitroglycerin. Most cases of ST elevation Ml (STEMI. also sometimes referred to as transmural myocardial tion. or Q-wave dial infarction) are treated with olysis or aneous coronary intervention (PCI). NSTEMI (non-ST elevation Ml, also sometimes ed to as nontransmural myocardial infarction. or non-Q-wave dial infarction) is managed with medication, although PCI is often performed during hospital admission. Heart attacks are the leading cause of death for both men and women worldwide.

Heart failure (HF). often called congestive heart failure (CHF), is a cliniml syndrome characterized by systemic perfusion inadequate to meet the body‘s metabolic demands as a result of impaired c pump on. i.e., it is generally defined as the inability of the heart to supply sufficient blood flow to meet the needs of the body. Heart failure is a common. costly. disabling, and potentially deadly condition. McMurray JJ, Pfefier MA (2005) 0TA-1 101-PC 'Heart failure‘. Lancet 365 (9474): 9. In developed countries. around 2% of adults suffer from heart e. but in those over the age of 65. this increases to 640%. Id. Currently. it is estimated that more than 5 million ans are afflicted with heart failure. approximately 2% of the population. American Heart Association. Heart Disease and Stroke Statistics - 2008 Update. Dallas: American Heart Association. 2008. ircahaigumalsorg.

Both the human suffering and the al burden associated with HF are substantial. Patients with heart failure account for about 1 trillion hospital admissions annually. and another 2 million patients have heart failure as a secondary diagnosis. One third of these patients are readmitted within 90 days for recurrent decompensation.

Common causes of heart failure include myocardial infarction and other forms of ischemic heart disease. hypertension. valvular heart disease, and cerdiomyopathy. McMurray JJ, Pfeffer MA (2005) 'Heart failure'. Lancet 365 (9474): 1877-89.

Heart failure may be further subdivided into systolic or diatolic heart failure. In systolic heart failure. there is reduced wrdiac contractility, whereas in diastolic heart e there is ed cardiac relaxation and abnormal ventricular ﬁlling. The most common cause of heart failure is left cular (LV) systolic ction (about 60% of patients). In this mtegory, most cases are a result of end-stage coronary artery disease, either with a history of dial infarction or with a cally underperfused. yet viable. myocardium. In many patients. both processes are present simultaneously. Other common muses of LV systolic ction include idiopathic dilated cardiomyopathy. valvular heart disease. hypertensive heart disease. toxin-induced cardiomyopathics (9.9., doxorubicin. herceptin. alcohol). and congenital heart disease. Heart failure can also develop as a result of right ventricular infarction. pulmonary ension, chronic severe tricuspid regurgitation. or arrhythmogenic right ventricular dysplasia. A less-common cause of heart failure is utput failure caused by thyrotoxicosis, arteriovenous ﬁstulae. Paget's disease, pregnancy. or severe chronic anemia. Diastolic LV dysfunction (impaired relaxation) usually is related to chronic hypertension or ischemic heart e. Other causes include restrictive. inﬁttrative. and hypertrophic cardiomyopathies. Inadequate ﬁlling of the right ventricle can result from dial constriction or cardiac tamponade. ts at high risk for developing heart failure are those with hypertension. coronary artery e. diabetes mellitus. family history of cardiomyopathy. use of cardiotoxins, and obesity. Heart failure is a common me. especially in older adults. Although more patients survive acute myocardial infarction because of reperfusion therapy. most have at least some residual LV systolic dysfunction. which can lead to heart failure. Currently. heart failure has no cure. While treatments such as medicines and lifestyle changes can help people live longer and more active lives. researchers continue to look for new ways to treat heart failure and its complications.

Chest pain is a nonspeciﬁc symptom that can have cardiac muses. and the term angina is typically reserved for pain syndromes arising from presumed myocardial ischemia. The term unstable angina was ﬁrst used to signify the intermediate state between myocardial infarction and the more chronic state of stable angina. The old term. preinfarction angina, conveys the clinical intent of intervening to attenuate the risk of myocardial infarction or OTAPC death. Patients with this condition have also been categorized according to their presentation, diagnostic test results. or course over time; these categories include new-onset angina, accelerating angina. rest angina. early postinfarct angina. and early postrevascularization angina. Unstable angina is considered to be an acute coronary syndrome in which there is no release of the enzymes and biomarkers of myocardial necrosis. Although the etiology and definition of unstable angina can be broad. interplay between disrupted atherosclerotic plaque and id thrombi is present in many cases of unstable angina. with consequent hemodynamic deﬁcit or microembclization. This is distinct from stable angina. in which the typical underlying cause is a fixed ry stenosis with compromised blood flow and slow. progressive plaque growth that allows for the occasional development of collateral flow.

"Acute Coronary Syndrome' (ACS) has been applied to a group of ry disorders that result from ic insult to the heart. ACS includes patients who have or are at high risk of developing an Ml. ts with ACS present to the physician with conditions that span a continuum that includes unstable angina. STEMl.

NSTEMl and transmural (Q-wave) Ml. ACS also include cardiac ischemia. and is believed to result largely from thrombus deposition and growth within one or more coronary arteries. resulting in a partial or complete ion of the artery, and ntly involves rupture of the plaque, resulting in an ischemic injury. ACS may also be precipitated by a coronary vasospasm or increased myocardial demand. For . see. e.g., Davies. Clin. Cardiol.

(Supp. I): 12 17 (1997). The seriousness of ACS is ined by the morbidity and mortality that follow the ischemic insult. For example. workers have estimated that within four to six weeks of presentation with ACS. the risk of death or a subsequent MI is 844%. and the rate of death. Ml. or refractory ia is 15-25%. Theroux and Fuster. Circulation 97:1195 1206 (1998). Given that the total number of deaths in the US. from acute MI is about 600,000. the search within the art for information that relates to the therapeutic management of ACS has understandably been extensive.

B-type natriuretic peptide (BNP or BNP-32) is a 32-aminc acid neurohorrnone that is synthesized in ventricular myocardium and released into the ation in response to cular dilation and pressure overload.

The plasma concentration of BNP is elevated among CHR patients. and increases in proportion to the degree of left ventricular ction and the severity of CHF symptoms. For review, see. e.g., Wiese et al.. Circulation 102: 3074 9 (2000); Yasue et al.. Circulation 90: 195 203 (1994); Yoshimura et al.. tion 87: 464 9 (1993); Stein and Levin. Am. Heart J. 135: 914 23 ; and Omland et al.. Heart 76: 232 7 (1996). The precursor to BNP is synthesized as a 134-amino acid precursor molecule referred to as ‘pre pro BNP.” Miich is cleaved into a signal peptide comprising amino acids 1-26 and a 108-amino acid molecule consisting of amino acids 27-134, referred to as 'pro BNP.’ Pro BNP is proteolytically processed into a no acid N-terrninal peptide (amino acids 1-76), referred to as ‘NT pro BNP' and the 32-amino acid mature hormone, ed to as BNP or BNP 32 (amino acids 77-108). It has been reported that NT pro-BNP, BNP-32. and the pre pro BNP can circulate in human . See, e.g., Tateyama et al.. Biochem. Biophys. Res. Commun. 185: 760 7 (1992); Hunt at al.. Biochem. Biophys. Res. . 214: 1175 83 (1995).

OTA—1101-PC In August 2001, hBNP (native peptide) was approved by the FDA under the trade name Natrecor (nesiritide) for the treatment of acute congestive heart failure. or was the first drug approved for the treatment of CHF in over twelve years. It is administered by intravenous continuous infusion over a period of 48 hours in patients with acute decompensated or advanced CHF who have dyspnea at rest or with minimal ty. As the drug is expensive and requires hospitalization. Natrecor is only used for the most acute cases. Additionally, the therapeutic ness of BNP is limited by endopeptidase degradation. as well as natriuretic peptide clearance receptor ) mediated internalization. which causes these proteins to have a fairly short half-life in vivo. For example. the plasma half life of BNP is estimated to be approximately 20 s (Potter 9! at. Endocrine Reviews 27(1):42-72 (2006)), and previous therapeutic administration of these peptides has been limited to time consuming enous infusion. lly in a al or other l care facility.

There remains a need in the art for new therapeutics useful in ng patients having or at risk for developing cardiovascular diseases. disorders and conditions. including ischemic heart disease. acute coronary mes and heart failure. There is a particular need for new therapeutics that span the entire spectrum of cardiovascular diseases. disorders and conditions associated with ischemia and/or oxidative stress. Such therapeutics are described and claimed herein. based on surprising discoveries indicating. for example, that signal peptide fragments of BNP are novel cardioprotective and therapeutic agents.

BRIEF 80mm! The inventions described and d herein have many attributes and embodiments including. but not limited to. those set forth or described or referenced in this Brief Summary. It is not intended to be clusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this Brief Summary. which is included for purposes of illustration only and not restriction.

In one aspect, the inventions provided herein include compounds. The compounds are useful for the treatment of cardiovascular disorders. in another aspect, the ions include compositions comprising or cOnsisting essentially of one or more of those compounds.

Compounds of the invention. which in a non-limiting preferred embodiment are isolated or substantially pure. include the following peptides: LHLAFLGGRS (SEQ.ID.N021). LHLAFLGGR (SEQ.lD.NO:2).

LHLAFLGG (SEQ.ID.NO:3), LHLAFLG (SEQ.ID.N0:4). LHLAFL (SEQ.ID.N015) and LHLAF D.N026). LHLA (SEQ.ID.NO:7). LHL (SEQ.ID.N028). LH D.N029). in the above peptides shown as SEQ.ID.NO:1-9. any one or more of the Leucines (L) can be substituted with lsoleucine (l). with D-Ieucine or D-isoieucine, or with tert-leucine. cine, L-allo-isoleucine, D-allo-isoleucine. D-tert-leucine and D-norleucine. and/or the histidine can be substituted with any non-naturally occurring amino acid that has or is prepared to have a side chain terminating with an imidazole ring all of which are SEQ.ID.NO:1-9 analogs.

OTAPC nds of the invention. which in a non-limiting preferred embodiment are isolated or substantially pure. also include peptides according to the ing Formula I: LHX1X2X3X4X5X5X7Xa wherein X1 is Norleucine. lle. Val, Met. Ala. Phe or Gly; X2 is Val. Leu, lie or Gly; )9 is Leu, Val. lle. Ala. Tyr or Gly; X4 is Norleucine. lle. Val. Met. Ala. Phe or Gly; X5 is Pro. Ala, Arg or Ser; X5 is Pro. Ala. Arg or Ser; X1 is Arg, Gln. Asn or Gly; and X3 is Thr or Gly.

Compounds of the invention, which in a non-limiting red embodiment are ed or substantially pure. also include peptides according to the following Formula II: LHXixth4X5XsX7 wherein X1 is Norleucine. lle, Val. Met. Ala, Phe or Gly; X2 is Val. Leu. lle or Gly; X3 is Leu. Val, lle. Ala. Tyr or Gly; X4 is Norleucine. lle, Val. Met. Ala. Phe or Gly; X5 is Pro. Ala, Arg or Ser: X5 is Pro, Ala. Arg or Ser; and X7 is Arg. Gin, Asn or Gly; provided that where X1 is Norleucine. Ile. Val. Met, Ala. Phe or Gly. X: can also be Ala, X3 can also be Phe. X4 can also be Leu. Xscan also be Gly. Xscan also be Gly. and X1 can also be Arg; where X; is Val. Leu or lle or Gly. X1 can also be Leu. X; can also be Phe. X4 can also be Leu. Xscan also be Gly. Xs can also be Gly. and X7 can also be Arg; where X1 is Leu. Val, lle. Ala. Tyr or Gly. X1 can also be Leu. X2 can also be Ala. X1 can also be Leu. Xscan also be Gly. Xscan also be Gly. and X1 can also be Arg: where X4 is Norleucine. lle. Val. Met. Ala. Phe or Gly. X1 can also be Leu. X2 can also be Ala. X1 can also be Phe. )gcan also be Gly. Xscan also be Gly. and X1 can also be Arg; where X1 is Pro. Ala. Arg or Ser. X1 can also be Leu, X2 can also be Ala. X: can also be Phe. X4 can also be Leu, Xscan also be Gly. and X1 can also be Arg; where Xsis Pro. Ala. Arg or Ser. X1 can also be Leu. X2 can also be Ala. X; can also be Phe. X4 can also be Leu. Xscan also be Gly. and wan also be Arg; where Xyis Lys, Gln, Asn or Gly. X1 can also be Leu. X2 can also be Ala, X2 can also be Phe. X4 can also be Leu. Xscan also be Gly. and Xecan also be Gly.

Compounds of the invention. which in a non-limiting preferred embodiment are isolated or substantially pure. also e peptides according to the following Formula lll: L H X1 X2 X2X4 X1 X5 wherein X1 is Norleucine. lle. Val. Met. Ala. Phe or Gly; X2 is Val. Leu. lie or Gly; X1 is Leu. Val, lle. Ala. Tyr or Gly; X4 is Norleucine. lle. Val. Met. Ala. Phe or Gly; X5 is Pro. Ala. Arg or Ser; and X5 is Pro. Ala, Arg or Ser: provided where X1 is Norleucine. lie. Val, Met. Ala. Phe or Gly. X2 can also be Ala. X3 can also be Phe. X4 can also be Leu. X5 can also be Gly. X; can also be Gly. and X; can also be Arg; 0TAPC where X2 is Val, Leu or lie or Gly, X1 can also be Leu. X; can also be Phe. X4 can also be Leu. Xscan also be Gly. Xewn also be Gly. and X7 can also be Arg: where X; is Leu, Val. Ile, Ala, Tyr or Gly, X. can also be Leu, X2 can also be Ala, X4 can also be Leu. kmn also be Gly. Xecan also be Gly. and X1 can also be Arg; where X4 is Norleucine, lle, Val. Met, Ala, Phe or Gly. Xi can also be Leu. X2 can also be Ala. X; can also be Phe, Xscan also be Gly. Xs can also be Gly. and X] can also be Arg; where X5 is Pro. Ala, Arg or Ser, Xi can also be Leu. X2 can also be Ala, X; can also be Phe. X4 can also be Leu. Xscan also be Gly, and X7 can also be Arg; where X5 is Pro. Ala. Arg or Ser, X. can also be Leu. X2 can also be Ala. X; can also be Phe, X4 can also be Leu. Xscan also be Gly. and Xyoan also be Arg.

Compounds of the invention, which in a non-limiting preferred embodiment are isolated or substantially pure, also e peptides according to the following Formula W: l. H X1 X2 X3X4 is X5 wherein X1 is Norleucine. Ile, Val. Met, Ala, Phe or Gly; X2 is Val. Leu. He or Gly; X3 is Leu, Val. Ile. Ala, Tyr or Gly; X4 is Norleucine, Ile, Val. Met. Ala. Phe or Gly; and X5 is Pro, Ala, Arg or Ser; provided that where X1 is Norleucine. lie. Val, Met. Ala. Phe or Gly. X2 can also be Ala. X; can also be Phe, X4 can also be Leu. Xscan also be Gly. Xscan also be Gly. and X7 can also be Arg; where X2 is Val. Leu or lie or Gly. X: can also be Leu, X; can also be Phe, X4 can also be Leu. )Qcan also be Gly. Xscan also be Gly. and X7 can also be Arg: where k is Leu, Val, Ile. Ala. Tyr or Gly, Xi can also be Leu, X2 can also be Ala, X4 can also be Leu. Xscan also be Gly, Xscan also be Gly, and X1 can also be Arg; where X4 is cine. lle. Val. Met, Ala. Phe or Gly. X1 can also be Leu. X2 can also be Ala. X2 can also be Phe. Xscan also be Gly, Xscan also be Gly. and X7 can also be Arg; Miere Xsis Pro, Ala. Arg or Ser. X1 can also be Leu. X2 can also be Ala, X; can also be Phe, X4 can also be Leu. Xscan also be Gly. and X; can also be Arg. nds of the invention, which in a non-limiting preferred embodiment are isolated or substantially pure. also include peptides according to the following Formula V: L H X1 X2 X3X4 wherein X! is Norleucine, Ile. Val. Met. Ala. Phe or Gly; X2 is Val, Leu. lie or Gly; X2 is Leu, Val, lie, Ala, Tyr or Gly; and X4 is Norleucine, Ile. Val, Met, Ala. Phe or Gly; provided that where Xi is Norleucine. lle. Val. Met. Ala. Phe or Gly, X2 can also be Ala. X3 can also be Phe, X4 can also be Leu. Xscan also be Gly. Xscan also be Gly. and X7 can also be Arg; where X2 is Val, Leu or lie or Gly. Xi can also be Leu, X; can also be Phe, X4 can also be Leu. Xscan also be Gly. Xscan also be Gly. and X1 can also be Arg; OTA-l101-PC where X1 is Leu, Val. lle. Ala. Tyr or Gly. X1 can also be Leu. X2 can also be Ala. X4 can also be Leu. Xscan also be Gly. Xscan also be Gly. and X7 can also be Arg; where X1 is Norleucine, lle, Val. Met. Ala, Phe or Gly. X1 can also be Leu. X2 can also be Ala, X; can also be Phe. Xscan also be Gly. Xscan also be Gly. and X1 can also be Arg.

Compounds of the invention, which in a non-limiting preferred embodiment are isolated or substantially pure, also include peptides according to the following Formula VI: L H X1 X2 X1 wherein X1 is Norleucine, Ile. Val, Met, Ala. Phe or Gly; X2 is Val. Leu. lie or Gly; and )Qi is Leu, Val. lle. Ala.

Tyr or Gly; provided that where X1 is Norleucine, Ile. Val. Met. Ala. Phe or Gly. X2 can also be Ala. and X1 can also be Phe; Where X2 is Val, Leu or lle or Gly. X1 can also be Leu. and X; can also be Phe.; and where X; is Leu, Val. lle, Ala. Tyr or Gly. X1 can also be Leo. and X2 can also be Ala.

Compounds of the invention. which in a non-limiting preferred embodiment are isolated or substantially pure. also include es according to the following Formula VII: L H X1 X2 wherein X1 is Norleucine, lle. Val. Met, Ala. Phe or Sly; and X2 is Val. Leu, He or Gly; provided that where X1 is Norleucine, lle. Val. Met, Ala, Phe or Gly. X2 can also be Ala; where X2 is Val. Leu or lie or Gly. X1 can also be Leo; and provided that the peptide is not LHAL or LHGG. nds of the ion, which in a non-limiting preferred embodiment are isolated or substantially pure. also include peptides according to the following Formula VIII: L H Xi n X1 is cine.

Included in the scope of the ion are active analogs and vative variants of these nds, including truncations thereof. preferably C-tenninal truncations. Additionally, for example. in the peptides shown in Formulae l-Vlll, any one or more of the Leucines (L) can be substituted with lsoleucine (l). with D-leucine or D-isoleucine, or with ten-leucine. norleucine. L-allo-isoleucine. D-alloisoleucine, D-tert-leucine and D-norleucine. and/or the histidine can be substituted with any non-naturally occurring amino acid that has or is prepared to have a side chain terminating with an imidazole ring, all of which are further analogs thereof.

In one non-limiting embodiment. one or more of the amino acids of the peptides within the scope of the invention, including .NOS:1-9 and ces within Formulae I-VIII. may be in the L- or D- configuration.

In other embodiments. one or more of the amino acids of the peptides within the scope of the invention are naturally- occuring non-genetically coded amino acids. In still other embodiments, one or more of the amino acids of the peptides within the scope of the invention are amino acid analogs or synthetic amino acids. 0TAPC In another non-limiting embodiment. the N-terminal Leucine (or lsoleucine D-leucine, D-isoleucine. tert-leucine. norieucine. L-allo-isoleucirie. D-alloisoleucine. D-tert-leucine or eucine) of the peptides within the scope of the invention. ing SEQ.ID.NOS:1-9 and sequences within Formulae I-Vlll. may be modiﬁed to contain a formyl group, a group comprising a fonnyl group. an ester of a carboxylic acid (preferably an aldehyde ester. 9.9.. a yethyl group. a carboxymethyl group. etc). or a group comprising a an ester of a carboxylic acid.

Modiﬁcations with formyl. carboxyethyl. and carboxymethyl groups are presently preferred.

In another embodiment, one or more the amino acids in compounds within the scope of the invention. including SEQ.ID.NOS:1-9 and sequences within Formulae l-Vlll. are substituted for another amino acid from a similar amino acid class or subclass. based primarily upon the chemical and physical ties of the amino acid side chain. For example. one or more hydrophilic or polar amino acids can be tutred for another hilic or polar amino acid. Likewise, one or more hydrophobic or nonpolar amino acids can be tuted for another hydrophobic or nonpolar amino acid. In making such substitutions. polar amino acids can be r subdivided into amino acids having acidic, basic or hydrophilic side chains and nonpolar amino acids can be further subdivided amino acids having aromatic or hydrophobic side chains. Nonpolar amino acids may be further subdivided to include. among others. aliphatic amino acids.

Also within the scope of the invention are nds of the invention that have been modiﬁed to improve their biophannaceutical properties. In certain embodiments. the compounds of the invention are modiﬁed. for example. to provide increased stability. increased resistance to proteolytic inactivation. decreased to nonexistent genicity. increased circulatory lives, including modiﬁed serum half-lives and modiﬁed therapeutic ives, and low toxicity. Modiﬁed forms of compounds of the invention include g forms. representative examples of which are described elsewhere herein. Methods by which the nds of the invention can be modiﬁed also include. for example. by PEGylation. by chemical derivitization. and by fusion or conjugation with es or .

Modiﬁded compounds include modiﬁed Type-B natriuretic signal peptide fragment agents. including. for example. modiﬁed BNPspl17-26) (SEQ ID N021). and modiﬁed analogs. variants (e.g.. conservative variants) and truncations thereof. Other embodiments include peptides selected from SEQ.lD.NOS:2 to 9 that have been modiﬁed. and peptides according to Formula I. Formula II. Formula lll. a IV. Formula V. Formula Vl. Formula Vll and/or Formula Vlll that has been modiﬁed. and active analogs. variants (e.g.. conservative variants) and truncations thereof that have been modiﬁed.

Other embodiments include peptidiomimetics of compounds of the invention.

The present inventions also include phan'naceutical compositions comprising or consisting essentially of a Type-B natriuretic signal peptide fragment agent and a phannaceuticalty acceptable carrier. in one embodiment, the pharmaceutical composition comprises or ts ially of BNPsp(17-26) (SEQ ID N011). In r embodiment. the pharrnaceutiml composition comprises or consists ially of a sequence selected from SEQ.ID.NOS:2 to 9. In another embodiment. the pharmaceutical composition ses or consists essentially of a OTAPC sequence selected from Formula I. Formula II. Formula lll. Formula IV. Formula V. Formula VI. Formula Vil and/or Formula Vlll. Included in the scope of the ion are pharmaceutical compositions including one or more active analogs and vative variants of these compounds. including truncations thereof. preferably C-temiinal truncations. In one ment. the inventions include pharmaceutical compositions comprising or consisting essentially of a Type-B natriuretic signal peptide fragment or a therapeutically active analog or variant or tmncation In another embodiment. the inventions include ceutical compositions comprising or consisting essentially of compounds of the invention. including analogs. variants. truncations. etc. that have been modified to improve their rrrraceutical properties. In certain embodiments, the compounds of the invention are modiﬁed. for example, to provide increased stability. increased resistance to proteolytic inactivation. decreased to nonexistent immunogenicity. increased half-lives or circulatory lives. and low toxicity. Methods by which the compounds of the invention can be ed e, for example. by PEGylation. by chemical derivitization. and by fusion or conjugation with peptides or lipids.

The ions include a pharmaceutical composition comprising one or more pharmaceutically acceptable Type-B natriuretic signal peptide agents for the treatment of a cardiovascular disorder. 9.9.. an acute coronary syndrome. heart failure. ischemic heart disease, etc. and related cardiovascular diseases. disorders and conditions characterized at least in party by ischemia and/or oxidative stress. and related disorders and conditions.

Certain preferred Type-B natriuretic signal peptide agents are identiﬁed herein as .NOS:1 to 9. BNPsp(17- 26) (SEQ ID NO:1) is most preferred. Other Type-B retic signal peptide agents are within Formula I. Formula II.

Formula Ill. Formula IV. Formula V. Formula VI. Fon'nuia VII and Formula Vlll. Other embodiments include active analogs. variants (6.9.. conservative variants) and tions of the foregoing. and a pharrnaceutically acceptable carrier. Thus, the inventions include pharmaceutical compositions in a form le for. or adapted to. treatment of a subject for a cardiovascular disease. disorder or condition. In one embodiment. the cardioiovascular disease. disorder or condition is associated with ischemia and/or oxidative stress. In certain ments. the cardiovascular disease, disorder or condition is an acute coronary syndrome. The acute coronary syndrome may. for e, be selected from the group consisting of ST-segment elevation myocardial infarction. non-ST-segment elevation dial infarction and unstable . In other embodiments. the cardiovascular disease. disorder or condition is ischemic heart disease. In other embodiments, the vascular disease. disorder or condition is heart e (any form). For example. the heart failure may be systolic or diastolic heart failure. The heart failure may result from left ventricular systolic dysfunction. The heart failure may also be a result of right ventricular infarction. pulmonary hypertension. c severe tricuspid regurgitation. or arrhythmogenic right ventricular dysplasia. The heart failure may also be a result of diastolic LV dysfunction. In another embodiment the cardioiovascular disease. disorder or condition is ischemic heart disease.

OTAPC in one aspect. the ion includes pharmceutical compositions useful for preventing and/or treating a vascular disorder in a subject, 9.9.. an acute ry syndrome. heart failure. ischemic heart e. etc.. and related cardiovascular diseases. ers and conditions involving ischemia and/or ive stress. and related disorders and conditions. including parenteral delivery forms and formulations. as well as other fomis of delivery including fomis for delivery by infusion. injection and instillation. and delayed. slow, extended or controlled release compositions. s and matrices. comprising or consisting essentially of therapeutically effective s of a Type-B natriuretic signal peptide nt agent alone or in combination with another cardiovascular therapeutic agent(s). and a pharrnaceutically acceptable carrier. In certain preferred embodiments. the pharmaceutical compositions are formulated for intravenous administration. ing by infusion or as a bolus.

Other formulations for other routes of administration are also within the scope of the invention. including. for example. formulations for nasal. pulmonary. buccal. rectal. transderrnal and oral delivery.

In another aspect. the compositions of the invention comprise about 0.01 to about 100 milligrams. about 100 to about 500 rams. or about 500 to about 1000 milligrams or more of a compound of the invention. for example. a Type-B natriuretic signal peptide fragment or Type-B natriuretic signal peptide fragment analog. including one or more of SEQ.ID.NOS:1-9 and peptides according to any of Formulae l to Vlll. Other doses are bed herein and include doses ranging from at least about 100 nanograms. including. for example at least about 200 nanograms. 600 nanograms. 2000 nanograms. 6000 nanograms and at least about 10.000 nanograms or rrrore.

Dose concentrations include concentrations of at least about 0.1 moles per liter. including, for example, at least about 0.3, 1.0. 3.0 and 10.0 nMoles/L. Dose concentrations also include concentrations of 0.1 /L. 0.3 nMoles/L. 1.0 nMoles/L. 3.0 nMoles/L and 10.0 nMoles/L These dose concentrations are equivalent to 0.1, 0.3. 1. 3. 11 rig/L and administrable weight doses of 0.4. 1.0. 4.0. 10 and 39 micrograms/kg (pg/kg). Also within the invention are other doses ranging from 0.1 to 5.0 rig/kg and 0.1 to 10.0 pg/kg. Additionally. doses of about 0.4. 1.0. 4.0. 10 and 39 pg/kg are within the invention. Doses of at least about 0.4. 1.0. 4.0. 10 and 39 rig/kg are also within the invention.

These compositions and amounts may be provided as single or muliple closes.

The inventions also e methods of treatment of a subject having or at risk for developing a cardiovascular disease. disorder or condition. comprising administering to the subject a therapeutically effective amount of one or more of the nds or pharmaceutical compositions described . In one non-limiting embodiment. the cardiovascular disease. disorder or condition is associated with ischemia and/or oxidative stress. In one embodiment. the cardiovascular e. disorder or condition is an acute coronary me. e.g.. ST-segment elevation myocardial infarction. non-ST-segment elevation myocardial infarction or unstable angina. In another embodiment. the cardiovascular disease. disorder or condition is heart failure. in other embodiments. the cardiovascular disease. er or condition is ischemic heart disease. In another embodiment. the cardiovascular disease. disorder or condition is stable angina.

OTAPC The inventions include methods of treating a subject having or at risk for developing a cardiovascular e. disorder or ion. sing a therapeutically effective amount of a Type~B natriuretic signal peptide fragment agent and a pharrnaceutically acceptable r. In one embodiment. the Type-B natriuretic signal peptide fragment agent in the pharmaceutical composition is BNPsp(17-26) (SEQ ID NO:1). In another embodiment. the Type-B natriuretic signal peptide fragment in the pharmaceutical composition comprises or consists essentially of a sequence ed from SEQ.ID.NOS:2 to 9. in another embodiment. the Type-B natriuretic signal peptide fragment agent in the pharmaceutical composition comprises or consists essentially of a sequence selected from a I. Formula II. Formula Ill. Formula IV. Formula V, Formula VI. Formula VII or Formula VIII. Type-B natriuretic signal peptide nt agents also include active analogs. variants. truncations. and modified forms of the Type-B natriuretic signal peptide fragment agents bed herein.

In another aspect. the inventions include methods of treating and/or preventing a cardiovascular disease, disorder or condition that is associated with ischemia and/or oxidative stress in a subject by increasing Type-B natriuretic signal peptide fragment activity in the subject. This may be accomplished. for example. by administering to the subject a composition comprising a therapeutically effective amount of a Type-B retic signal peptide fragment agent. 9.9.. a Type-B natriuretic signal peptide fragment or a Type-B natriuretic signal peptide fragment, including a BNPsp fragment comprising or consisting essentially of a sequence selected from .NOS:1-9. or a peptide comprising or consisting essentially of a peptide according to any of Formulae I to VIII. or an analog. t. truncation or modification thereof. In certain embodiments. about 0.01 to about 100. 500 or 1000 nanograms or milligrams or more (9.9.. at least about 100 nanograms or milligrams. at least about 500 nanograms or milligrams. or at least about 1000 nanograms or milligrams) of a BNPsp nt or Type-B natriuretic signal peptide fragment , 9.9.. a BNPsp fragment comprising or consisting essentially of a sequence selected from SEQ.ID.NOS:1-9. or a peptide sing or consisting essentially of a peptide according to any of Formulae l to VIII. is administered per day in single or divided doses or by continuous infusion. for example.

In another aspect. the inventions e s of ng a t ing from chest pain of any cause, including acute coronary syndrome. sing administering to the patient a therapeutically effective amount of a Type-B natriuretic signal peptide fragment agent. wherein the t is not suffering from a Q-wave MI or STEMI. In a certain embodiment of this method. the patient is suffering from unstable angina. In another embodiment of this method. the patient is suffering from non-Q-wave cardiac necrosis. In still another embodiment of this method. the patient has a blood troponin I level of no more than 0.4 nglml. In yet another embodiment of this method. the patient has a blood troponin T level of no more than 0.1 ng/ml. In yet another embodiment of this method. the patient does not have elevated blood ne kinase. In still another embodiment of this method. the patient does not have ST-segment elevation. In yet another embodiment of this method. the patient does not exhibit a ogical Q-wave. In another embodiment of this method. the patient exhibits one or more of the following OTAPC symptoms: chest pain greater than 15 minutes in duration. chest pain at rest, or chest pain following minimal exertion that is poorly responsive to sublingual nitrates.

In one embodiment. the Type-B natriuretic signal e fragment agent is administered in a single dose. In another embodiment. the Type-B natriuretic signal peptide fragment agent is administered in more than one dose. In yet another embodiment. the Type-B natriuretic signal peptide fragment agent is administered continuously over a period of time. for example a predetermined period of time. In still another embodiment. glucose or a potassium salt. or a combination thereof. is co-administered with the Type-B natriuretic signal peptide fragment agent In another . the ions e methods for treatment of a patient. comprising administering to the individual a therapeutically effective amount of a Type-B retic signal peptide nt agent. wherein the administration is after the onset of one or more of the following symptoms: chest pain lasting longer than 15 minutes. chest pain at rest. chest pain following minimal exertion. nausea. shortness of breath. palpitations. or dizziness. In other embodiments. the patient has not suffered a Q-wave MI or STEMI prior to the onset of the symptom or symptoms; patient is suffering from unstable angina: the patient is suffering from non-Q- wave cardiac necrosis; the patient has a blood troponin I level of no more than 0.4 nglml; the patient has a blood troponin T level of no more than 0.1 nglml; the patient does not have elevated blood creatine kinase myocardial isoenzyme; the t does not have ST-segment elevation; the patient does not exhibit a pathological Q-wave; the administration occurs between the time of onset of the one or more symptoms. and the time the patient s a 0- wave Ml or STEMI. In another embodiment. the method further comprises the step of continuing the stration of a Type-B natriuretic signal peptide fragment agent during the time that the patient suffers a Qwave MI or STEMI.

In yet another embodiment. the method further ses the step of continuing the administration of a Type-B natriuretic signal peptide fragment agent after the Iirne the patient suffers a Q-wave Ml or STEMI. In other embodiments of this method. the patient has ic heart disease. or is at risk for developing ischemic heart disease. In still another ment of the method, the patient has one or more of the following cardiac abnormalities: congestive heart failure. worsening heart murmur due to mitral regurgitation. or evidence of cardiac conduction disturbances. In other embodiments. the patient has a normal ECG. In another embodiment of this method. the patient has stable angina. In other embodiments of the . the Type-B natriuretic signal peptide nt agent is administered in a single dose. or is stered in more than one dose. or is administered continuously. In an additional embodiment of this method. glucose or a potassium salt. or a combination thereof. is co-administered with the Type-B natriuretic signal peptide fragment agent.

The inventions also include methods for treating a t suffering from stable angina. comprising administration of a Type-B natriuretic signal peptide fragment agent. In a further ment. the administration is continuous over a period of time. including a predetermined period of time.

OTA~1101-PC The inventions also provide a method for performing angioplasty on a patient in need thereof. comprising administering a Type-B natriuretic signal peptide fragment agent to the patient during the angioplasty procedure. In a further embodiment. the method comprises or further comprises administering a Type-B retic signal peptide fragment agent to the t prior to the angioplasty procedure. In a further ment. the method ses or further ses administering a Type-B natriuretic signal peptide fragment agent to the t following the angioplasty procedure. In other embodiments, a Type-B natriuretic signal peptide fragment agent is administerd to the patient before, . and/or after the angioplasty ure. in any ation.

The inventions also include methods for treatment of a patient with ischemic heart disease. or is at risk for developing ischemic heart disease. including patients who exhibit one or more of the following symptoms: nausea, shortness of breath. palpitations. or dizziness. and further wherein the patient does not t chest pain. comprising administering to the patient a therapeutically effective amount of a Type-B natriuretic signal peptide nt agent. wherein the patient is not suffering a Q-wave Ml or STEMI. In another embodiment of this method. the patient has a normal ECG.

Also provided are methods for increasing the time during which thrombolytic therapy will be effective following the ﬁrst symptom of cardiac distress, comprising administering a therapeutically ive amount of a Type-B natriuretic signal peptide fragment agent after the onset of one or more of the following symptoms: chest pain g longer than 15 minutes. chest pain at rest. chest pain following minimal exertion. nausea, shortness of breath, palpitations. or dizziness.

In another aspect. the d subject is a mammal. preferably a human. Other mammals include domestic and farm animals. and zoo, sports. or pet animals. such as dogs. horses. and cats.

The inventions also include articles of manufacture comprising package al ning one or more of the compounds or pharmaceutical compositions described herein. Then inventions also include articles of manufacture comprising e material containing one or more of the compounds or pharmaceutical compositions bed herein. together with instructions for use in or on a subject in order to prevent and/or treat a cardioiovascular disease. disorder or condition. In one embodiment. the cardioiovascular disease. disorder or ion referred to in the instructions is associated with ischemia and/or oxidative stress. In another embodiment the cardioiovascular disease. disorder or condition referred to in the instructions is ischemic heart disease. In one embodiment, the cardioiovascular disease. disorder or condition referred to in the instructions is an acute coronary syndrome, 9.9.. unstable angina. STEMI. and/or NSTEMI. In another embodiment the iovascular disease. disorder or condition referred to in the instructions is heart failure (any form). The instructions may be electronic and/or associated with a website.

The inventions also e methods of preparing a medicament for preventing or treating one or more of the cardioiovascular disease, disorder or conditions referenced herein. including. 9.9.. an acute coronary syndrome. heart failure. etc. comprising bringing together a therapeutically effective amount of a compound OTAPC referenced herein, 9.9.. a Type-B natriuretic signal peptide fragment or a Type-B natriuretic signal peptide fragment analog or variant, and a ceutically acceptable carrier. in one embodiment the Type-B natriuretic signal peptide fragment comprises a sequence selected from SEQ.lD.NOS:1 to 9. In another embodiment the Type-B natriuretic signal peptide fragment analog is a compound selected from one or more of Formulae I-VIII. In one embodiment the medicament is formulated for parenteral administration.

Compositions and methods of the invention for the prevention and/or treatment of a cardiovascular disorder. 9.9.. an acute coronary me, heart failure. ic heart disease. etc. and related cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. also comprise administration of a Type-B nab‘iuretic signal peptide fragment agent in series or in combination with (e.g.. in physical combination. provided as a combined preparation) one or more other cardiovascular treatment agents. Such other vascular treatment agents e nitrates. B-blockers. calcium channel blockers (particularly for stable or unstable angina. but also for heart e in the case of B-blockers). diuretic agents. vasodilator agents. positive inotropes. ACE inhibitors and aldosterone antagonists. e.g. spironolactone (particularly for heart failure). blood thinning therapeutics (e.g.. aspirin. heparins. warfarins) and lycerin (particularly for Ml).

Compositions and methods of the invention for the prevention and/or treatment of a cardiovascular disorder. 9.9.. an acute coronary syndrome. heart failure. ischemic heart e. etc. and related vascular diseases. disorders and ions involving ischemia and/or oxidative . may also comprise administration of a Type-B natriuretic signal peptide fragment agent in series or in combination with (9.9.. in physical combination. provided as a combined preparation) one or more anti-thrombolytic therapies (e.g.. streptokinase inhibitors. anti- platelet thereapetuics. such as. for e. clcpidogrel).

Compositions and methods of the invention for the prevention and/or treatment of a cardiovascular disorder. 9.9.. an acute coronary me. heart failure. ischemic heart disease. etc.. and d cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. may also comprise administration of a Type-B natriuretic signal e nt agent in series or in ation with (9.9., in physical combination. provided as a combined preparation) a Type-B natriuretic peptide. including for example nesiritide. a recombinant form of Type-B natriuretic peptide. in certain methods and itions (including pharmaceutical compositions. forrnuiations. articles of manufacture and kits) of the invention for the prevention and/or treatment of a cardiovascular disorder. 9.9.. an acute coronary syndrome. heart failure. ischemic heart disease. etc. and related cardiovascular es. disorders and conditions involving ischemia and/or oxidative stress. sub-therapeutically effective amounts of a Type-B retic signal peptide fragment agent. and one or more other cardiovascular treatment agents are used or provided for ed administration (separately or jointly as a combined preparation) to provide a combined action that is eutically effective.

OTAPC Thus. it will be understood that compositions and methods of the invention for the treatment of a cardiovascular disorder, 6.9.. an acute coronary syndrome. heart e. ic heart disease. etc. and related cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. that employ a Type-B natriuretic signal peptide fragment agent. ing active analogs thereof. and r cardiovascular eutic agent are disclosed. A Type-B retic signal peptide fragment agent may be selected. for example. from the group consisting of BNPsp(l7-26) (SEQ ID N021). BNPsp(17-25) (SEQ ID NO:2). BNPsp(17-24) (SEQ ID N023).

BNPsp(l7-23) (SEQ ID N024). BNPsp(17-22) (SEQ ID N025). BNPsp(i7-21) (SEQ ID N026). BNPsp(1'I-20) (SEQ.ID.N0:7). BNPsp(17-19) D.NO:8). and BNPsp(17-18) (SEQ.ID.NO:9). and active analogs thereof. In another embodiment. a Type-B natriuretic signal peptide agent may be selected from the group consisting of a ce ing any one of Formula I. Formula II. Formula III. Formula lV. Formula V. Formula VI. a VII and Formula VIII. and active analogs thereof. Optionally. a cardiovascular agent is selected. for e. from the group comprising or wnsisting essentially of nitrates. B-blockers. calcium channel blockers. diuretic agents. vasodilator agents. positive inotropes. ACE inhibitors. aldosterone antagonists. nitroglycerin. blood thinning agents. anti-thrombolytic agents. and TypeB natn'uretic peptides. ent of a subject as ed herein with one or more compounds or pharmaceutical compositions as described herein may comprise their aneous, separate. sequential or sustained administration.

Pharmaceutical compositions useful for ting and/or treating a cardiovascular disorder. 9.9.. an acute coronary syndrome. heart failure. ischemic heart disease. etc.. and related cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. are also provided in the form of a combined preparation. for example, as an admixture of two or more Type-B natriuretic signal peptide fragment agents.

The term "a combined preparation' includes not only physical ations of compounds. but compounds provided as a 'kit of parts' in the sense that the combination partners as defined above can be dosed ndently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b). i.e. simultaneously. separately or sequentially. The parts of the kit can then. for example. be administered simultaneously or logically staggered. that is at diﬂerent time points and with equal or different time intervals for any part of the kit of parts.

In one embodiment. the inventions e a kit comprising one or more doses of a Type-B natriuretic signal peptide fragment agent. the kit comprising one or more of a syringe. a 'pen' injector that delivers a metered dose. a needle-less injector. a liquid formulation. 3 Iyophilized powder and a sterile liquid for reconstitution. a dry-powder r. a buccal , and a sublingual tablet.

In one embodiment a combined preparation is administered. wherein two or more separate compositions are administered to a subject. wherein the first composition comprises a therapeutically effective amount of a Type~B natriuretic signal peptide fragment agent and the second composition comprises a therapeutically effective amount of another cardiovascular therapeutic agent. In another embodiment a third OTAPC composition is administered comprising a Type-B natriuretic signal peptide fragment agent or another cardiovascular therapeutic agent.

Thus. pharmaceutical compositions useful for preventing and/or ng a cardiovascular disorder. e.g.. an acute coronary syndrome. heart failure, ischemic heart disease. etc.. and related cardiovascular diseases. disorders and conditions invotving ischemia and/or oxidative stress. are provided for combined. simultaneous, separate sequential or ned administration. In one embodiment, a composition sing or consisting essentially of a Type-B natriuretic signal peptide fragment agent is administered at or about the same time as another vascular therapeutic agent(s). In one embodiment. a composition sing a Type-B natriuretic signal peptide fragment agent is stered within at least about thirty minutes of another vascular therapeutic agent(s). In one embodiment. a composition comprising a Type-B natriuretic signal peptide nt agent is stered within at least about one hour of another cardiovascular therapeutic agent(s). In one embodiment. a composition sing a Type-B natriuretic signal peptide fragment agent is administered within at least about 2-12 or 12 to 24 hours of r cardiovascular therapeutic agent(s). In one embodiment. a composMon comprising a Type-B natriuretic signal peptide fragment agent is administered within at least about 24-48 hours of another vascular therapeutic agent(s). In another embodiment the Type-B natriuretic signal peptide fragment agent and another cardiovascular therapeutic agent(s) are administered within about 1-8 hours of each other. within about one day of each other. or within about one week of each other.

In another aspect. the invention includes methods for administering a therapeutically effective amount of a Type-B natriuretic signal peptide fragment agent. alone or in combination with another cardiovascular therapeutic agent. formulated in a delayed release preparation. a slow release preparation. an extended release preparation, a controlled release preparation. and/or in a repeat action preparation to a subiect having or at risk for ping a cardiovascular disorder. e.g.. an acute coronary syndrome, heart failure. ischemic heart disease. etc. and d cardiovascular diseases, disorders and conditions involving ischemia and/or oxidative stress. or a related disorder or ion.

In certain other aspects. the ion also relates to methods of using such compositions to treat subjects suffering from or at risk for a cardiovascular disorder. e.g.. an acute coronary syndrome. heart failure. ischemic heart disease. etc. and related cardiovascular diseases. disorders and conditions ing ischemia and/or oxidative stress. and related disorders and conditions.

In other aspects. the inventions include methods and compositions for preventing and/or treating a subject having or suspected of having or posed to. or at risk for, any diseases. disorders and/or conditions characterized in whole or in part by angina.

According to one aspect. the present invention is directed to methods of halting or decreasing or providing relief from the ms of a cardiovascular disorder. e.g.. an acute ry syndrome. heart failure.

OTA-i 101-PC ischemic bean disease. etc. and related cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. in another . the invention provides a method of ting and/or treating a cardiovascular disorder. 9.9.. an acute coronary syndrome. heart e. ischemic heart disease. etc.. and related cardiovascular diseases. disorders and conditions ing ischemia and/or oxidative stress. comprising administering to a t in need thereof a ition comprising therapeutically effective amounts of a Type-B natriuretic signal e fragment agent agent. alone or together or in combination with another vascular eutic agent. wherein said first agent is selected from the group consisting of BNPsp(17-26) (SEQ |D N011). BNPsp(17-25) (SEQ lD N0:2).

BNPsp(17-24) (SEQ ID N0:3). BNPsp(17-23) (SEQ lD NO:4), BNPsp(17-22) (SEQ ID NO:5). BNPsp(17-21) (SEQ 10 N06). BNPsp(17-20) (SEQ.lD.NO:7). BNPsp(t7-19) (SEQ.lD.NO:8). and BNPsp(17-18) (SEQ.lD.NO:9) and sequences according any one of Formula I, Formula II. Formula Ill. Formula IV. Formula V. Formula VI. Formula VII and Formula VIII. and active s f. wherein the second cardiovascular agent is selected from the group comprising or consisting essentially of es. ers. calcium channel blockers. diuretic agents. vasodilator agents. positive inotropes. ACE inhibitors. aldostercne antagonists. nitroglycerin. blood thinning agents. anti- thrombolytic agents. and TypeB natriuretic peptides.

Methods of the invention include the sequential or simultaneous administration a first and second agents as bed herein. either or both of which are provided in amounts or doses that are less that those used when the agent or agents are administered alone. i.e.. when they are not administered in combination. Such lesser amounts of agents administered are typically from about one-twentieth to about one-tenth the amount or amounts of the agent when administered alone. and may be about one-eighth the amount. about one-sixth the amount. about one-ﬁfth the amount. about one-fourth the amount. about one-third the amount. and about one-half the amount when administered alone.

In another aspect, the invention includes an article of cture comprising a vessel containing a therapeutically effective amount of a Type-B natriuretic signal peptide nt agent(s). such as. for example.

BNPsp(17-26) (SEQ ID NO:1). BNPsp(17-25) (SEQ ID NO:2). BNPsp(17-24) (SEQ ID N023). 17-23) (SEQ ID N024). BNPsp(17-22) (SEQ ID NO:5). BNPsp(17-21) (SEQ lD NO:6). 17-20) (SEQ.lD.NO:7). BNPsp(17- 19) (SEQ.lD.N018). and BNPsp(17-18) (SEQ.lD.N029) and compounds selected from any one of Formula I. Formula II. Formula III. Formula IV. Formula V. Formula Vl. Formula VII. and Formula VIII. and active analogs thereof. together or in physical combination with a second cardiovascular agent. such as one or more nitrates. [3-biockers. calcium channel blockers. diuretic agents. vasodilator agents. ve inotropes. ACE inhibitors. aldostercne antagonists, nitroglycerin. blood thinning agents, anti~thrombolytic agents. and/or Type-B natriuretic peptides. and instructions for use. including use for the treatment of a subject as described herein.

The invention includes an article of cture comprising packaging material containing one or more dosage forms as described herein. n the packaging material has a label that indicates that the dosage 0TA—1101-PC form can be used fora subject having or suspected of having or predisposed to any of the diseases. disorders and/or conditions described or referenced herein. including acute coronary sydromes. ischemic heart disease. angina and heart failure.

The invention includes method of preparing a medicament for preventing and/or treating a cardiovascular disorder. e.g.. an acute coronary syndrome. heart failure. ischemic heart disease. etc. and related cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. sing bringing er and an amount of a Type-B retic signal peptide fragment agent and a pharmaceutically acceptable carrier together with one or more other cardiovascular agents useful for preventing and/or treating a cardiovascular disorder. 9.9.. an acute coronary syndrome. heart failure. ischemic heart disease. etc.. and related cardiovascular es. disorders and conditions involving ischemia and/or ive stress.

The invention includes methods for the use of a therapeutically effective amount of a Type-B natriuretic signal peptide nt s) in the manufacture of a dosage form useful for preventing and/or treating a cardiovascular disorder. e.g.. an acute coronary syndrome. heart failure. ischemic heart disease. etc. and related cardiovascular es. ers and conditions involving ischemia and/or oxidative stress. and related disorders and conditions. Such dosage fomrs include, for example. oral delivery toms and ations. well as other forms of delivery including forms for delivery by infusion. injection and instillation. and compositions and devices including slow-release. extended release. and delayed release compositions. depots and matrices. for example. Such dosage forms include those for the ent of a subject as disclosed herein.

In certain other aspect. the invention provides a package comprising a Type-B natriuretic signal peptide fragment agent(s) together with instructions for use. alone or in combination with one or more other cardiovascular therapeutic agents for preventing and/or treating a cardiovascular disorder. e.g.. an acute coronary syndrome. heart failure. ischemic heart e. etc. and related vascular diseases. disorders and conditions involving ischemia and/or oxidative stress. and related disorders and ions.

In other aspects. the inventions provide for use of one or more of the compounds and compositions bed herein in the manufacture of a medicament. In other aspects. the ions provide for use of one or more of the compounds and compositions described herein in the manufacture of a ment for use in the ent of one or more of the diseases. disorders and conditions described herein. In other aspects. the inventions provide for use of one or more of the compounds. compositions and medicaments described and claimed herein in the treatment of a subject for one or more of the diseases. disorders and conditions described herein.

These and other s of the present inventions. which are not limited to or by the information in this Brief Summary. are provided below.

BRIEF DESCRIPTION OF FIGURES OTA-1101‘PC This application ns at least one ﬁgure executed in color. Copies of this application with color drawing(s) will be provided upon request and payment of the necessary fee. A brief summary of each of the ﬁgures is provided below.

Figure 1 demonstrates the beneficial effects of human 17-26) administration in an isolated rat heart model of ischemia reperfusion injury. Figure 1(A) shows that administration of 0.3nMol and 1nMol human BNPsp(17-26) either before (pre) or after (IDR) a 40 minute period of ia improves the contractile function of the left ventricle as assessed by developed pressure. These effects were most pronounced at 0.3nMol pre and 1nMot lDR. Figure 1(8) documents vascular reactivity, as assessed by perfusion pressure in the same hearts as in Figure 1A. Perfusion pressures during the reperfusion phase after ischemia are beneﬁcially reduced by pre or lDR treatment with BNPsp(17-26). Figure 1(C) shows signiﬁcant reductions in troponin I release (a biomarker of cardiac cell necrosis) during reperfusion resulting from lDR administration of human BNPsp(17-26). Figure 1(D) shows itant improvements in reperfusion myoglobin levels in the same s described in Figure 1C.

Figure 2 demonstrates in vivo nce and lack of haemodynamic effects to human BNPsp(17- 26) administration in normal. healthy sheep. Figure 2(A) shows the lack of response in cardiac output in sheep 1 when given constant infusion of human BNPsp(17-26) at tong/kglmin and 100kg/nglmin. compared with control on (saline). Such a se is indicative of a well tolerated agent. Figure 2(B) shows the same lack of administered. response of cardiac output in sheep 2, when the same doses of BNPsp(17-26) as in Figure 2A were Figure 3 shows normalized contractile function (developed pressure) in isolated hearts preconditioned with synthetic human BNPsp(17-26) and control buffer. Doses and group size are as shown.

Figure 4 shows normaﬁzed vascular function (perfusion pressure) in isolated hearts preconditioned with tic human BNPsp(17-26) and control buffer. Doses and group size are as shown.

Figure 5 shows the cumulative e of troponin 1 (AUG) in hearts preconditioned with synthetic human BNPsp(17-26) and control buffer. Doses and group size are as per Figures 3 and 4. = P<0.01 vs. control.

Figure 6 shows the ped pressures in isolated hearts given BNPsp(17-26) during reperfusion after ischemia. Doses and sample size are as shovm.

Figure 7 shows the ion pressure (upper panel) and cumulative troponin release (tower panel) in isolated hearts given BNPsp917-26) during reperfusion after ischemia.

Figure 8 shows Hematoxylin and Eosin (HE) staining demonstrating a r degree of myocyte oel swelling and myotibritlar derangement in l hearts compared with BNPsp(17-26) treated hearts.

Figure 9 shows capsase-3 staining of slides of left ventricular free wall yocytes. Caspase-3 activity is indicated by the brown colouration. Colouration was virtually absent from hearts infused with inmol/L BNPsp(17- 26) at reperfusion and ly reduced in hearts preconditioned with 03an BNPsp(17-26). compared with control.

Figure 10 shows marked reduction in TUNEL ve cells from hearts infused with BNPsp(17-26).

TUNEL ve nuclei (red-brown colouration) was markedly reduced in all hearts infused with BNPsp(17-26).

OTA'1101-PC (0082] Figure 11 shows that the infusion of human BNPsp(17-26) into 4 normal sheep at 100 and 1000ug/kg.min had no effect upon venous pressure. heart rate. mean arterial pressure or cardiac output. Similar results were found for hormones and renal indices.

Figure 12 shows the cumulative troponin l release in sheep undergoing cardiac ischemia and receiving human BNPsp(17-26). Treated sheep had significantly lower cumulative troponin I release (P<0.01) compared with control.

Figure 13 shows the elution profile of proteolytically cleaved human BNPsp(18-26) that has been passed through either an ia isolated rat heart or in vivo sheep under cardiac coronary ligation. The elution position (fraction 34) is four fractions earlier than synthetic human BNPsp( 17-26). indicated by the downward arrow.

Figure 14 shows the developed pressures in ed ed rat hearts ing 03an of altered BNPsp sequences (n=3 for each group).

DETAILED Descrurrnou Practice of the present inventions may include or employ various conventional techniques of molecular biology (including recombinant ques), iology. cell biology. biochemistry. nucleic acid chemistry. and immunology. which are within the skill of the art. Such techniques are explained fully in the literature. and include but are not limited to. by my of example only. Molecular Cloning: A Laboratory Manual. second edition r et al.. 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel. 2001). jointly and individually referred to herein as 'Sambrook'; Oligcnucleotide Synthesis (M. J. Gait. ed.. 1984): Animal Cell Culture (R. I. ey. ed.. 1987); Handbook of Experimental Immunology (D. M. Weir & C. C. Blackwell, eds); Gene Transfer Vectors for Mammalian Cells (J. M. Miller 8 M. P. Calos. eds. 1987); t Protocols in Molecular Biology (F. M. Ausubel et al.. eds. 1987, including supplements h 2001); PCR: The Polymerase Chain Reaction, (Mullis et al.. eds. 1994); Current Protocols in lmmunology (J. E. Coligan et al.. eds. 1991); The Immunoassay ok (D. Wild. ed.. Stockton Press NY. 1994); Bioccnjugate Techniques (Greg T. Herrnanson. ed.. Academic Press. 1996); Methods of immunological Analysis (R. Masseyeff. W. H. Albert. and N. A. Staines. eds, im; VCH Verlags gesellschaﬂ mbH. 1993). Harlow and Lane (1988) Antibodies. A tory Manual, Cold Spring Harbor ations. New York. and Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY (jointly and dually referred to herein as Harlow and Lane). Beaucage el al. eds. Current Protocols in Nucleic Acid Chemistry John Wiley 8 Sons. Inc.. New York, 2000); and Agrawal. ed.. Protocols for Oligonucleotides and Analogs. Synthesis and Properties Humana Press Inc.

New . 1993) It is to be understood that the inventions are not limited to the particular ology. protocols. constructs. and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only. and is not intended to limit the scope of the OTAPC present invention. which will be limited only by the appended claims. As used herein and in the appended claims. the singular forms ‘a.' ‘an.' and “the“ include plural nce unless the context clearly indicates otherwise. Thus, for example. reference to a 'Type-B natriuretic signal peptide fragment' is a reference to one or more such peptides and includes equivalents thereof now known or later developed. Unless defined ise, all technical and scientiﬁc terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the inventions belong. Although any methods. devices. and materials similar or equivalent to those described herein an be used in the practiw or g of the invention. the preferred methods. devices and materials are now described.

It is intended that reference to a range of s disclosed herein (for example 1 to 12) also incorporates reference to all related numbers within that range (for example. 1. 1.1. 2, 3. 3.9.4. 5. 6. 6.5. 7. 8. 9.5. 10, 11 and 12) and also any range of rational numbers within that range (for example 210 8. 1.5 to 5.5 and 3.1 to 4.7) and. therefore, all sub ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is speciﬁcally intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be ered to be expressly stated in this application in a similar manner. The following temis have the ing meanings when used herein.

Amino acids used in compounds provided herein (e.g. peptides and proteins) can be genetically encoded amino acids. naturally occurring non-genetically d amino acids. or synthetic amino acids. Both Lo and tiomers of any of the above can be utilized in the compounds. The following abbreviations may be used herein for the following genetically encoded amino acids (and residues f): alanine (Ala. A); arginine (Arg. R); asparagine (Asn, N); aspartic acid (Asp. D); cyteine (Cys. C): glycine (Gly. G); glutamic acid (Glu. E); glutamine (Gin.

Q); ine (His. H); isoleucine (lie. I): leucine (Leu. L): lysine (Lys. K); methionine (Met. M); phenylalanine (Phe, F); proline (Pro. P); serine (Ser. S); threonine (Thr. T); tryptophan (Trp. W); tyrosine (Tyr. Y); and valine (Val. V).

Certain commonly tered amino acids that are not genetically encoded and that can be present in active compounds of the invention include. but are not limited to. B—alanine (b-Ala) and other omega-amino acids such as opropionic acid (Dap), 2.3diaminopropionic acid (Dpr. Z). 4-aminobutyric acid and so forth: a- aminoisobutyric acid (Aib): eaminohexanoic acid (Aha); 8-aminovaleric acid (Ava); rnethylglycine (MeGly); ornithine (Om); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine ); N-methylisoleucine (Melle): phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle. J): 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2- fiuorophenylalanine (Phe(2-F)); 3-lluorophenyialanine (Phe(3-F)): 4-ﬂuorophenylalanine (Phe(4-F)); penicillarnine (Pen); 1,2.3,4-tetrahydroisoquinoline-S-carboxylic acid (Tic); beta.thienylalanine (Thi): nine ide (MSO); homoarginine (hArg); N-acetyl lysine (Act.ys); 2.3-diaminobutyric acid (Dab): 2.3-diaminobutyric acid (Dbu): p- aminophenylalanine (Phe(pNH2)); N-methyl valine (MeVal); homocysteine ; 3-benzothiazoIyl-alanine (thAla. B); and homoserine (hSer). Additional amino acid analogs contemplated include phosphoserine. othreonine. phosphotyrosine. hydroxyproline. gamma-carboxyglutamate. hippuric acid, droindole-Z- carboxylic acid. statine. a-methyl-alanine, para-benzoyiphenylalanine. propargylglycine. and sarcosine. Peptides 0TAPC that are encompassed within the scope of the invention can have any of the foregoing amino acids in the L- or D- conﬁguration. or any other amino acid described herein or known in the art. whether currentiy or in the future. whilst ing a biological activity.

Amino acids that are substitutable for each other generally reside within similar ciasses or subclasses. As known to one of skill in the art. amino acids can be placed into different s depending primarily upon the chemical and physical properties of the amino acid side chain. For example. some amino acids are generally considered to be hydrophilic or polar amino acids and others are considered to be hydrophobic or nonpoiar amino acids. Polar amino acids include amino acids having acidic. basic or hydrophilic side chains and nonpoiar amino acids include amino acids having aromatic or hydrophobic side chains. Nonpoiar amino acids may be further subdivided to include. among others. aliphatic amino acids. The ions of the s of amino acids as used herein are as follows: 'Nonpolar Amino Acid' refers to an amino acid having a side chain that is uncharged at physiological pH. that is not polar and that is generally repelled by aqueous on. Exampies of genetically encoded hydrophobic amino acids include Ala. lie. Leu. Met. Trp. Tyr and Val. Examples of non-genetically encoded nonpoiar amino acids include t-BuA. Cha and Me.

"Aromatic Amino Acid' refers to a nonpoiar amino acid having a side chain containing at least one ring having a conjugated n-eiectron system (aromatic group). The aromatic group may be further substituted with tuent groups such as alkyi. aikenyt, atkynyi. hydroxyl. suifonyt, nitro and amino groups. as well as others. es of cally encoded aromatic amino acids include phenylalanine. tyrosine and tryptophan. Commonly tered non-genetically encoded aromatic amino acids include phenyiglycine, 2-naphthylalanine. [5 thienylalanine. 3-benzothiazotyl-alanine. 1.2.3.4-tetrahydroisoquinolinecarboxylic acid. 4-chtorophenyiatanine. 2-tiuorophenylaianine. 3-tiuorophenylalanine and 4-tiuorophenylalanine.

"Aliphatic Amino Acid' refers to a nonpoiar amino acid having a saturated or unsaturated straight chain. branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids include Ala. Leu. Val and lie. Examples of non-encoded aliphatic amino acids include Nie.

'Polar Amino Acid' refers to a hydrophilic amino acid having a side chain that is charged or uncharged at physiological pH and that has a bond in Miich the pair of electrons shared in common by two atoms is held more ctosety by one of the atoms. Polar amino acids are generally hydrophilic, meaning that they have an amino acid having a side chain that is attracted by aqueous solution. Examples of cally encoded polar amino acids include asparagine. ne. ine, lysine and . Examples of non-genetically encoded polar amino acids include citrulline. homocysteine. N-acetyl lysine and methionine suifoxide.

”Acidic Amino Acid' refers to a hydrophilic amino acid having a side chain pK value of less than 7.

Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. es of genetically encoded acidic amino acids include ic acid (aspartate) and ic acid (glutamate).

OTAPC 'Basic Amino Acid' refers to a hydrophilic amino acid having a side chain pK value of greater than 7. Basic amino acids typically have positively charged side chains at logical pH due to association vn'th hydronium ion. Examples of genetically encoded basic amino acids include arginine. lysine and histidine. Examples of non-genetically encoded basic amino acids include omithine. 2.3diaminopropionic acid. 2.4-diaminobutyric acid and hornoarginine. ‘lonizable Amino Acid' refers to an amino acid that can be charged at a physiological pH. Such ionizable amino acids include acidic and basic amino acids. for example. p—aspartic acid. D-glutamic acid. D—histidine.

D-arginine. D-lysine. o-hydroxylysine. D-omithine. L-aspartic acid. L-glutamic acid. L-histidine. L-arginine. L-lysine, L- hydroxylysine or L-omithine.

As will be appreciated by those having skill in the art. the above classifications are not absolute.

Several amino acids exhibit more than one characteristic ty, and can therefore be ed in more than one category. For example. tyrosine has both a nonpolar aromatic ring and a polar hydroxyl group. Thus. tyrosine has several characteristics that could be bed as nonpolar. aromatic and polar. However. the nonpolar ring is dominant and so tyrosine is lly considered to be nonpolar. rly. in addition to being able to form disulﬁde linkages, cysteine also has nonpolar character. Thus. while not strictly classiﬁed as a hydrophobic or nonpolar amino acid, in many instances cysteine can be used to confer hobicity or nonpolarity to a peptide.

In some embodiments. polar amino acids contemplated by the present invention include. for example. arginine. asparagine. aspartic acid. cysteine. glutamic acid. glutamine, histidine. homocysteine. lysine. hydroxylysine. omithine, serine, threonine. and structurally related amino acids. in one embodiment the polar amino is an ionizable amino acid such as arginine. aspartic acid. glutamic acid. histidine. hydroxylysine. lysine, or ne. [00100) Examples of polar or nonpolar amino acid residues that can be utilized include. for example. alanine. valine. leucine. methionine. isoleucine. phenylalanine. tryptophan. tyrosine and the like.

As used herein. a 'cardiovascular er' is any cardiovascular disease. er or condition that involves or may be characterized at least in part by oxidative stress and/or ischemia.

During physiological processes molecules undergo chemical s involving reducing and oxidizing reactions. A molecule with an unpaired electron can combine with a molecule capable of donating an electron. The donation of an on is temied as ion s the g of an electron is called reduction.

Reduction and oxidation can render the d molecule unstable and make it free to react with other molecules to cause damage to cellular and sub-cellular components such as membranes. proteins and DNA. As used herein. ‘oxidative stress' refers to excessive production of reactive oxidant species (ROS) resulting in oxidative stresslnitrosative stress. a process that is an important mediator of cell damage. Important aspects of redox imbalance that triggers the activity of a number of signaling pathways ing ription factors activity. a s that is ubiquitous in vascular disease related to ischemialrepertusion injury, for example. Reactive oxidant species can originate from a variety of sources such as nitric oxide (NO) synthase (NOS). ne oxidases 0TA-1101~PC (X0). the cyclooxygenases. nicotinamide adenine dinucleotide phOSphate )H) oxidase isofonns and metalcatalyzed reactions. These include free ls such as superoxide anion (02‘). yi radical (HO). lipid ls (R00) and nitric oxide (NO). Other reactive oxygen species, for example. hydrogen peroxide . nitrite (ONOO“) and lorous acid (HOCl). although are not free ls but have oxidizing effects that contribute to ive stress.

“Ischemia“ is a condition that occurs when blood flow and oxygen are diminished in a particular part of the body. Cardiac ischemia is the name for this condition when the heart is the body part targeted. Ischemic heart disease is a term that covers heart issues caused by narrowing of the arteries. With arteries narrowed. less blood and oxygen are able to reach the heart . This is also referred to as coronary artery disease and coronary heart disease and may ultimately lead to heart attack. Ischemia often causes chest pain or discomfort known as angina pectoris. People with angina also may have undiagnosed episodes of silent ischemia.

Cardiovascular disorders e. for example, heart failure (including congestive heart es and other forms of heart failure noted anyvirhere herein) and acute coronary syndromes (including Q-wave Ml, STEMI. non-Gwave MI. NSTEMI and unstable angina) and ischemic heart disease. Cardiovascular disorders also include diseases. disorders and conditions involving the heart or blood vessels in which Type-B natriuretic peptide is elevated within a ally relevant timefrarne. Cardiovascular disorders also include diseases. disorders and conditions involving the heart or blood vessels in which one or more of cardiac troponin I. cardiac troponin T. creatine kinase-MB, Type-A and/or Type-B natriuretic peptide signal peptides or signal peptide fragments. uric acid. C- reactive protein and/or osteoprotegerin islare present in increased levels in clinically relevant timefrarnes. Other vascular disorders include non-Q-wave cardiac necrosis.

As used herein. a patient suffering from “unstable angina' denotes a patient who has one or more of the following symptoms and signs: (1) ST segment depression. as measured by ECG; (2) slightly elevated troponin T levels, of no more than 0.1 ng/ml; or (3) ly elevated troponin I levels, of no more than 0.4 ng/ml. In contrast to Q-wave Ml. CK-MB and LDH ievels are typically not elevated during unstable angina. Also in contrast to Q—wave Ml. a patient with le angina typically has no ST segment elevation nor any pathological Q-wave. Finally. unstable angina can be diagnosed solely on the basis of chest pain. typically chest pain lasting longer than 15 minutes. chest pain at rest. or chest pain following minimal exertion and that is pooriy responsive to sublingual nitrates.

Alternatively. even in the absence of chest pain. a patient can be sed with le angina if previously diagnosed with ischemic heart e or is considered to be at strong risk for developing ischemic heart disease, and who presents with nausea. shortness of breath. palpitations. or dizziness. miore. the skilled artisan will understand that the diagnosis of unstable angina is one of medical nt.

As used herein. "ischemic heart disease' denotes disease of cardiac tissue that results from a decreased oxygen supply to the cardiac tissue that is due to reduced coronary artery blood ﬂow. Typically, this reduced blood ﬂow results from the partial or complete obstruction of blood vessels that service the heart. A 0TAPC diagnosis of ischemic heart disease can be based on the presence of chronic. stable angina. elicited by exercise (also known as 'exertional angina") that is relieved by sublingual nitrates. A diagnosis of ischemic heart disease also can be based on an ECG reading that is consistent with ischemic heart disease. such as one exhibiting ST segment deviations and/or T wave ions.

As used herein. 'Type-B natriuretic signal e fragment agent' in one aspect refers to a fragment of a TypeB natriuretic signal peptide from any species. including mun'ne. bovine. ovine. e, , avian, and preferably human, in native ce or in a cally engineered form. and from any source. whether natural. tic. or recombinantly produced, having one or more of the biologic or eutic activities described . The term ‘Type«B natriuretic signal peptide fragment agent' also includes pharmaceutically acceptable salts and prodrugs. and prodrugs of the salts. polymorphs. hydrates. solvates. biologically-active fragments. biologically active variants and stereoisorners of the naturally-occurring any Type-B retic signal peptide fragment, as well as agonist and mimetic variants of any naturally-occurring Type-B natriuretic signal peptide nt and active analogs (e.g.. peptides containing. for example. speciﬁc deletions or other modiﬁcations that maintain biological activity) and polypeptide fusions thereof. Fusions comprising additional amino acids at the amino terminus. carboxyl terminus. or both. are encompassed by the term 'Type-B natriuretic signal peptide fragment agent." Fusions comprising onal amino acids at the carboxyl terminus of a Type-B natriuretic signal peptide fragment. or other Type-B natriuretic signal peptide fragment agent (including. for e, variants and analogs of a Type-B natriuretic signal peptide fragment). are preferred. Exemplary Type-B natriuretic signal peptide fragment agents include 17- 26) (SEQ ID N021), BNPsp(17-25) (SEQ ID N022), BNPsp(17-24) (SEQ ID N03), BNPsp(17-23) (SEQ ID N024), BNPsp(17-22) (SEQ ID N0:5), BNPsp(17-2‘I) (SEQ ID NO:6), BNPsp(17-20) (SEQ.ID.NO:7). t7-19) (SEQ.ID.NO:8). and BNPsp(17-18) (SEQ.ID.NO:9). Other Type-B natriuretic signal peptide fragment agents e peptides according any of Formula I. Formula II. Formula III. Formula IV. Formula V. Formula VI. Formula VII and Formula VIII.

The art is familiar with modification of peptides. for example. by polymer conjugation or glycosylation. The term ”Type-B natriuretic signal peptide fragment agent' includes modiﬁed peptides including peptides conjugated to a polymer such as PEG. and may be comprised of one or more additional derivitizations of ne, lysine. or other residues. In addition. the Type-B natriuretic signal peptide fragment agent may comprise a linker or polymer. wherein the amino acid to which the linker or polymer is conjugated may be a non-natural amino acid according to the present invention. or may be conjugated to a naturally encoded amino acid utilizing techniques known in the art such as coupling to lysine or cysteine.

Substitutions. ons. modiﬁcations or additions of amino acids described herein in reference to compounds of the invention. for e. SEQ ID NO: 1. 2. 3. 4. 5, 6. 7. 8. 9 or other es as deﬁned. for example. in Formula I. Formula II. a III. Formula IV. Formula V. Formula VI. Formula VII and Formula VIII. are OTAPC intended to also refer to substitutions. deletions. modiﬁcations or additions in corresponding positions in fusions. variants, fragments. conjugations. etc.

The term 'Type~B natriuretic signal peptide fragment agent' also encompasses homodirners. heterodimers. homomultimers. and heteromultimers that are linked. including but not limited to those linked directly via non-naturally encoded amino acid side chains, either to the same or different non-naturally encoded amino acid side chains. to naturally-encoded amino acid side . or indirectly via a linker. Exemplary s include small organic compounds. water e polymers of a variety of lengths such as poly(ethylene glycol) or polydextran or polypeptides of various lengths.

] The term ‘Iinker' is used herein to refer to groups or bonds that normally are formed as the result of a chemical reaction and typically are covalent es. Hydrolytically stable linkages means that the es are substantially stable in water and do not react with water at useful Ph values. including but not limited to. under physiological conditions for an extended period of time. perhaps even indefinitely. Hydrolytically le or degradable linkages mean that the linkages are degradable in water or in aqueous solutions. including for example. blood. Enzymatically unstable or able linkages mean that the linkage can be degraded by one or more enzymes. As understood in the art. PEG and related polymers may include able linkages in the r backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. For example. ester linkages ionned by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent. Other hydrolytically degradable linkages include. but are not limited to. carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrozone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the on product of an aldehyde and an alcohol; orthoester linkages that are the on product of a formate and an alcohol; peptide es formed by an amine group. including but not limited to. at an end of a polymer such as PEG. and a carboxyl group of a e; and oligonucleotide linkages formed by a phosphoramidite group. including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.

The term 'active.‘ “biologically active" or gically active agent' when used herein means any substance which can affect any physical or biochemical ties of a biological . pathway. molecule. or interaction relating to an organism. including but not limited to. viruses, bacteria. bacteriophage. transposons. prions, insects, fungi. plants. animals. and humans. ln particular, as used herein. biologically active molecules include, but are not limited to. any substance ed for cure. mitigation. treatment, or prevention of cardiovascular disorder in humans or other animals, or to otherwise enhance physical or mental well-being of humans or s.

As used herein. the term “water soluble polymer” refers to any polymer that is soluble in aqueous solvents. e of water soluble polymers to a Type-B natriuretic signal peptide fragment agent. 9.9.. Type-B OTAPC natriuretic signal peptide fragments. can result in changes including. but not limited to. increased or modulated senim half-life. or increased or ted therapeutic half-life relative to the unmodified form. modulated immunogenicity. modulated physical association characteristics such as aggregation and multimer formation. altered receptor binding. and altered receptor dimerization or multimerization. The water soluble polymer may or may not have its own biological activity, and may be utilized as a linker for attaching Type-B natriuretic signal peptide fragment agents. e.g.. TypecB natriuretic signal peptide fragments. to other substances. including but not limited to one or more Type-B natriuretic signal peptide fragment agents. e.g.. TypeB natriuretic signal e fragments. or one or more biologically active molecules. Suitable polymers include. but are not limited to, polyethylene glycol. polyethylene glycol propionaldehyde. mono C1-C10 alkoxy or aryloxy derivatives thereof (described in US. Pat. No. 5.252.714), monomelhoxy-polyethylene glycol. polyvinyl pyrrolidone. polyvinyl alcohol. polyamino acids. divinylether maleic anhydride. N-(2-Hydroxypropyl)~methacrylamide. dextran. n derivatives including dextran sulfate. polypropylene glycol. polypropylene oxide/ethylene oxide copolymer. polyoxyethylated polyol. heparin, heparin fragments. ccharides. oligosaocharides. glycans. cellulose and cellulose tives, including but not d to methylcellulose and carboxymethyl cellulose. starch and starch derivatives. polypeptides. polyalkylene glycol and derivatives thereof. oopolymers of polyalkylene glycols and derivatives thereof. polyvinyl ethyl ethers. and alpha-beta- poly[(2-hydroxyethyl)-DL-aspartamide. and the like. or mixtures thereof. es of such water soluble polymers include. but are not limited to. polyethylene glycol and serum albumin.

As used herein. the term ‘polyalkylene glycol' or 'poly(alkene glycol)‘ refers to polyethylene glycol (polylethylene glycol», polypropylene glycol. polybutylene glycol. and derivatives thereof. The term 'polyalkylene glycol' and/or ‘polyethylene glycol' asses both linear and branched polymers and average molecular weights of between 0.1 kDa and 100 kDa or more. Other exemplary embodiments are listed. for example, in commercial supplier catalogs. such as Shearwater Corporation's catalog thylene Glycol and Derivatives for ical Applications" (2001).

As used herein. the term "modified serum half-life' means an increased circulating half-life of a modified Type-B natriuretic signal peptide fragment agents, e.g.. a Type-B natriuretic signal peptide nt, relative to its non-modified form. Serum half-life is measured by taking blood s at various time points after administration of a Type-B natriuretic signal e fragment agent, e.g.. a Type-B natriuretic signal peptide fragment. and determining the tration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum ife. Increased serum half-life bly has at least about two-fold. but a smaller increase may be , for example where it enables a satisfactory dosing n or avoids a toxic effect. in some embodiments. the increase is at least about three-fold. at least about ﬁve-fold. or at least about ten-fold or more.

] The term ied therapeutic half-life' as used herein means an increase in the half-life of the therapeutically ive amount of a modiﬁed Type-B natriuretic signal peptide fragment agent. e.g.. a Type-B 01-PC retic signal peptide fragment. relative to its non-modiﬁed form. Therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properﬁes of the molecule at various time points after administration. sed therapeutic half-life desirably enables a ular beneﬁcial dosing regimen. a particular beneﬁcial total dose. or avoids an undesired effect. In some embodiments. the increased therapeutic half-life results from increased potency. increased or decreased binding of the modiﬁed molecule to its target. sed or decreased breakdown of the molecule by enzymes such as proteases. or an se or decrease in another parameter or mechanism of action of the non-modiﬁed molecule.

The term “isolated." when applied to a peptide. denotes that the peptide is free of at least some of the cellular or other biological components with which it is ated in the natural state. or that the peptide has been concentrated to a level greater than the concentration of its in vivo or in vitro production. It can be in a homogeneous or substantially homogenous state. Isolated substances can be in either a dry or semi-dry state, or in on. including but not limited to. an aqueous solution. It can be a component of a pharmaceutical composition that comprises additional phannaceutically acceptable carriers and/or excipients. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacryfamide gel electrophoresis or high perfomiance liquid tography. for e.

] By “substantially pure' is meant a degree of purity of total Type-B natriuretic signal peptide agent. 6.9.. BNPsp(17-26) (SEQ ID NO:1). to total protein Where there is at least 70% Type-B natriuretic signal peptide agent. more preferably at least 80%. and even more preferably increasing to at least 90%. 95% or 99%. A ularly preferred purity is at least 95%. By “essentially pure' is meant that the composition is at least 90% or more pure for the desired Type-B natriuretic signal peptide agent. A peptide which is the predominant species present in a preparation is also substantially puriﬁed.

The term 'effective amount' as used herein refers to that amount of the Type-B natriuretic signal peptide fragment agent being administered that will relieve to some extent one or more of the symptoms of the disease. condition or disorder being treated. itions containing the Type-B natriuretic signal peptide fragment agents described herein can be administered for prophylactic. ing. and/or therapeutic ents.

As used herein, ‘subject' refers to any mammal. including humans. domestic and farm animals. and 200. sports. or pet animals. such as dogs. horses. cats. sheep. pigs. cows. etc. The preferred mammal herein is a human. including adults. children. and the elderly. Preferred sports animals are horses and dogs. Preferred pet s are dogs and cats.

As used herein. nting" means ting in whole or in part. rating or controlling. reducing. lessening. or decreasing. or retarding or halting.

As used herein. a ‘therapeutically effective amount" in reference to the nds or compositions of the instant invention refers to the amount sufficient to induce a desired biological. pharmaceutical. or therapeutic result. That result can be alleviation of one or more of the signs. symptoms. or causes of a disease or disorder or OTA—1101-PC condition. or any other desired alteration of a ical . In the present invention. the result will involve the treatment, tion and/or ion of one or more of symptoms of a cardiovascular disorder, including, for example. an acute coronary syndrome. a heart failure, an ischemic heart e. and angina and any cardiovascular er, disease, or condition that involves ischemia and/or oxidative .

As used herein, the terms 'treating' and 'treatment' refer to both therapeutic treatment and prophylactic or preventative measures.

'Analogs' or ‘peptide analogs' refer to the compounds with properties analogous to those of the template peptide and may be non-peptide drugs. “Peptidomimetics" (also known as “mimetic es"). which include peptidebased compounds, also include such non-peptide based compounds such as peptide analogs.

Peptidomimetics that are urally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, omimetics are stnicturally identical or similar to a paradigm polypeptide (i.e.. a polypeptide that has a biological or pharmacological function or activity). but can also have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of, for example. -CH2NH-, -CH2$-, -CH2-CH2-, - CH=CH- (cis and trans). -. -CH(OH)CH2-. and -CH2$O-. The mimetic can be either entirely composed of natural amino acids, or non-natural analogues of amino acids. or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also comprise any amount of l amino acid conservative substitutions as long as such substitutions also do not ntially alter mimetic activity in general. the term ‘peptide' refers to any polymer of two or more individual amino acids (Mtether or not naturally occurring) linked via e bonds, as occur when the carboxyl carbon atom of the carboxylic acid bound to the amino group bonded to the alpha-carbon clone amino acid (or amino acid residue) becomes covalently nitrogen atom of the amino group bonded to the alpha-carbon of an adjacent amino acid. These peptide bond linkages, and the atoms comprising them (i.e.. alphacarbon atoms, carboxyl carbon atoms (and their substituent oxygen atoms), and amino nitrogen atoms (and their tuent hydrogen atoms» form the 'polypeptide of the protein. in addition. as used herein, the terms ‘polypeptide' and “peptide" may be used interchangeably.

Similarly, protein fragments. analogs. derivatives, and variants are may be ed to herein as “peptides" or "peptide agents.'. The term ‘fragment' of a peptide refers to a polypeptide sing fewer than all of the amino acid residues of the peptide.

As used herein, taneously“ is used to mean that the one or more agents of the invention are administered concurrently. whereas the term “in ation" is used to mean they are administered, if not simultaneously or in physical combination, then 'sequentially' within a timeframe that they both are available to act therapeutically. Thus. administration 'sequentially' may permit one agent to be administered within minutes (for example, 1. 2. 3, 4. 5. 10. 15. 20, 25. 30) minutes or a matter of hours. days. weeks or months after the other provided that both the Type-B natriuretic signal peptide fragment agent and another cardiovascular therapeutic OTA-1 101-PC agent, for example. are concurrentty present in effective amounts. The time delay n administration or administrations of the ents will vary depending on the exact nature of the components. the interaction therebetween. and their respective half-lives.

WEB Natriuretlc Signal e Fragment Agnts Type-B natriuretic signal peptide fragment agents of the invention described herein are capable of modulating one or more of the symtoms of a cardiovascular disorder. Preferably. the vascular disorder is an acute coronary syndrome. but others are intended as described herein.

Compounds of the invention, Mrich in a non-limiting preferred embodiment are isolated or substantially pure. include the following peptides: Compounds of the invention. which in a non-limiting preferred embodiment are ed or substantially pure, also include peptides according to the following Formula I: LHX1X2X3X4X5X5X7Xa wherein Xi is cine. lle, Val. Met. Ala. Phe or Gly; X2 is Val. Leu. lie or Gly; X3 is Leu. Val. lle. Ala, Tyr or Gly; X4 is Norleucine. Ile. Val. Met. Ala. Phe or Gly; )Q is Pro, Ala. Arg or Ser; X5 is Pro. Ala. Arg or Ser; X7 is Arg.

Gln. Asn or Gly; and X3 is Thr or Gly. nds of the invention. which in a non-limiting red embodiment are isolated or substantially pure. also include peptides awarding to the following Formula ll: LHX1X2X3X4X5X6X7 wherein X1 is Norleucine. lle. Val. Met. Ala. Phe or Gly; X2 is Val. Leu. lie or Gly; X3 is Leu. Val. lle. Ala. Tyr or Gly; X4 is Norleucine. lie. Val. Met. Ala. Phe or Gly; X5 is Pro. Ala. Arg or Ser; X5 is Pro. Ala. Arg or Ser. and X7 is Arg. Gin. Asn or Gly; provided that 0TAPC where X1 is Norieucine, Ile, Val. Met. Ala. Phe or Gly. X2 can also be Ala. X1 can also be Phe. X4 can also be Leu. Xscan also be Gly. Xacan also be Gly. and X7 can also be Arg; where X2 is Val. Leu or Ile or Gly. X1 can also be Leu. X; can also be Phe. X4 can also be Leu. Xscan also be Gly. Xscan also be Gly. and X1 can also be No: where X1 is Leu. Val. lle. Ala. Tyr or Gly. X1 can also be Leu. X2 can also be Ala. X4 can also be Leu. Xscan also be Gly. Xacan also be Gly. and X7 can also be Arg; where X4 is Norleucine. Ile, Val, Met. Ala. Phe or Gly. X1 can also be Leu, X2 (an also be Ala. X; can also be Phe, Xscan also be Gly. Xecan also be Gly. and chan also be Arg; where kis Pro. Ala. Arg or Ser, X1 can also be Leu. X2 can also be Ala. )Q can also be Phe, X4 can also be Leu. Xsmn also be Gly. and X1 can also be Arg; where Xsis Pro. Ala. Arg or Ser. X1 can also be Leu. X2 can also be Ala. X1 an also be Phe. X4 can also be Leu. kcan also be Gly. and X1 can also be Arg; where X2 is Lys. Gln, Asn or Gly. X1 can also be Leu. X2 can also be Ala. X3 can also be Phe, X4 can also be Leu, Xscan also be Gly. and Xacan also be Gly.

Compounds of the ion. which in a non-limiting preferred embodiment are isolated or substantially pure. also e peptides according to the following Formula Ill: L H X1 X2 X3X4 X5 X5 wherein X1 is Norleucine. Ile. Val. Met, Ala. Phe or Gly; X2 is Val. Leu. Ile or Gly; X1 is Leu. Val. Ile. Ala. Tyr or Gly: X4 is Norleucine. Ile. Val. Met. Ala. Phe or Gly; X1 is Pro. Ala. Arg or Ser; and X1, is Pro, Ala. Arg or Ser; provided that where X1 is Norieucine. Ile. Val, Met. Ala, Phe or Gly. X2 can also be Ala, X1 can also be Phe. X4 can also be Leu. X5 can also be Gly. Xscan also be Gly. and X; can also be Arg; Miere X2 is Val. Leu or lie or Gly. X1 can also be Leu. X1 can also be Phe. X4 can also be Leu. Xscan also be Gly. X5 can also be Gly. and X1 can also be Arg; where X; is Leu. Val. Ile. Ala. Tyr or Gly. X1 can also be Leu. X2 man also be Ala. X4 can also be Leu, Xscan also be Gly. Xscan also be Gly. and chan also be Arg; where X4 is Norleucine. Ile, Val. Met. Ala. Phe or Gly. X1 can also be Leu, X2 can also be Ala. X2 can also be Phe. Xscan also be Gly. Xacan also be Gly. and X1can also be Arg; where Xsis Pro. Ala. Arg or Ser. X1 can also be Leu. X2 can also be Ala. X1 can also be Phe. X4 can also be Leu. Xscan also be Gly. and Xycan also be Arg; where X; is Pro. Ala. Arg or Ser. X1 can also be Leu. X2 can also be Ala, X; can also be Phe. X4 can also be Leu. Xscan also be Gly. and X7 can also be Arg.

Compounds of the invention. which in a non-limiting preferred ment are isolated or substantially pure. also include peptides according to the following Formula IV: OTA-11011PC L H X1 X2 X1X4 is X1 wherein X1 is Norleucine. lle. Val. Met. Ala. Phe or Gly; X2 is Val. Leu. lie or Gly: X3 is Leu. Val. lie. Ala, Tyr or Gly: X4 is Norieucine. Ile. Val. Met. Ala. Phe or Gly: and X5 is Pro. Ala. Arg or Ser; provided that where X1 is Norleucine. lle. Val. Met. Ala, Phe or Gly. X2 can also be Ala. X1 can also be Phe. X4 can also be Leu. X5 can also be Gly. Xscan also be Gly. and X1 can also be Arg; where X2 is Val. Leu or lie or Gly, X1 can also be Leu. X1 can also be Phe. X4 can also be Leu. Xscan also be Gly. Xacan also be Gly. and X; can also be Arg; where )Q is Leu. Val, lle. Ala. Tyr or Gly. X1 can also be Leu. X2 can also be Ala. X4 can also be Leu. Xscan also be Gly. Xscan also be Gly, and chan also be Arg; where X4 is Norleucine. lle. Val. Met. Ala. Phe or Gly. X1 can also be Leu. X2 an also be Ala, X3 can also be Phe. Xscan also be Gly. Xscan also be Gly. and X1 can also be Arg; where Xsis Pro. Ala, Arg or Ser. X1 can also be Leu, X2 can also be Ala. X1 can also be Phe, X4 can also be Leu. Xscan also be Gly, and X1 can also be Arg.

Compounds of the invention. which in a non-limiting preferred embodiment are isolated or substantially pure. also include peptides according to the following Formula V: L H X1 X2 X1 X4 wherein X1 is cine. lle. Val. Met. Ala. Phe or Gly: X2 is Val. Leu. lie or Gly: )Q is Leu. Val. lie. Ala, Tyr or Gly: and X4 is Norleucine. lie. Val. Met. Ala. Phe or Gly: ed that where X1 is Norleucine, lle, Val. Met. Ala, Phe or Gly. X2 can also be Ala. X1 can also be Phe. X4 can also be Leu. Xscan also be Gly, Xscan also be Gly. and X1 can also be Arg: where X2 is Val. Leu or lie or Gly. X1 can also be Leu, X1 can also be Phe. X4 can also be Leu. Xscan also be Gly. Xacan also be Gly. and X1 wn also be Arg; where )9 is Leu. Val. lle. Ala. Tyr or Gly. X1 can also be Leu. X2 can also be Ala. X4 can also be Leu. Xscan also be Gly. Xecan also be Gly, and X1 can also be Arg; where X1 is Norleucine, lie. Val. Met. Ala. Phe or Gly, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe. Xscan also be Gly. Xscan also be Gly. and X1 can also be Arg.

] Compounds of the invention. which in a non-limiting red embodiment are isolated or substantially pure. also include peptides awarding to the follovw'ng Formula VI: L H X1 X2 X3 wherein X1 is Norleucine, Ile. Val, Met. Ala. Phe or Gly: X2 is Val. Leu. lie or Gly: and X1 is Leu. Val. lle. Ala.

Tyr or Gly: provided that where X1 is Norleucine. lle. Val. Met. Ala, Phe or Gly. X2 can also be Ala. and X3 can also be Phe; Where X2 is Val, Leu or lie or Gly, X1 can also be Leu. and X1 can also be Phe; and where )Q is Leu, Val. lle, Ala. Tyr or Gly, X1 can also be Leu. and X2 can also be Ala.

OTA-1101~PC Compounds of the invention. which in a non-limiting preferred embodiment are isolated or substantially pure, also include peptides according to the following Formula VII: L H X1 X2 wherein X1 is Norteucine. lie. Val. Met. Ala. Phe or Gly; and X2 is Val. Leu. lie or Gly; ed that where X is Norleucine. Ile. Val. Met. Ala. Phe or Gly, X2 can also be Ala.; and where X; is Vat, Leu or lie or Gly. X: can also be Leu.

Compounds of the invention. which in a non-limiting preferred embodiment are isolated or substantially pure. also include peptides awarding to the following Formula VIII: L H X1 n X1 is Norleucine. lte. Val. Met. Ala. Phe or Gly.

Included in the scope of the invention are biologically and/or therapeutically active analogs and conservative variants of these compounds. including truncations thereof, preferably C-terminal truncations. For example. in the above peptides shown as SEQ.ID.NO:1-9 and in Formulae l-VIll. any one or more of the Leucines (L) can be substituted with lsoleucine (l). with D-leucine or D-isoleucine. or with ten-leucine. norteucine. L~allo- isoleucine. isoleucine. -leucine and D-norleucine. and/or the ine can be substituted with any non- naturally occurring amino acid that has or is prepared to have a side chain terminating with an imidazole ring.

In one non-ﬁmiting embodiment. one or more of the amino acids of the peptides within the scope of the invention. including SEQ.ID.NOS:1-9 and sequences within Formulae l-Vlll. may be in the L- or D conﬁguration.

In other embodiments. one or more of the amino acids of the peptides within the scope of the ion are naturally- occuring non-genetically coded amino acids. In still other ments. one or more of the amino acids of the peptides within the scope of the ion are amino acid analogs or synthetic amino acids. in another non-limiting embodiment. the N-terrninal Leucine (or lsoleucine D-leucine. D-isoleucine. ten-leucine, norteucine. L-allo-isoleucine. D-allo-isoleucine. D~tert~leucine or D—norleucine) of the peptides within the scope of the invention. including SEQ.ID.NOS:1-9 and sequences within ae I-Vllt. may be may be modified to contain a fonnyl group. a group comprising a formyl group. an ester of a carboxylic acid (preferably an aldehyde ester. 6.9.. a carboxyethyl group. a carboxymethyl group. etc). or a group sing a an ester of a carboxylic acid.

Modiﬁcations with fonnyl. carboxyethyl. and carboxymethyl groups are presently red.

In another embodiment. one or more the amino acids in compounds within the scope of the invention. including SEQ.ID.NOS:1-9 and sequences within ae l-Vlll. are tuted for another amino acid from a r amino acid class or subclass. based primarily upon the al and physical properties of the amino acid side chain. For example. one or more hydrophilic or polar amino acids can be tuted for another hydrophilic or polar amino acid. Likewise. one or more hydrophobic or nonpolar amino acids can be substituted for another hydrophobic or nonpolar amino acid. In making such substitutions. polar amino acids can be r subdivided into amino acids having acidic. basic or hydrophilic side chains and nonpolar amino acids can be further subdivided OTAPC amino acids having aromatic or hydrophobic side chains. Nonpolar amino acids may be further subdivided to include. among others. aliphatic amino acids.

Also within the scope of the invention are compounds of the invention that have been modiﬁed to improve their biopharrnaceutical properties. ln certain embodiments. the compounds of the invention are d. for example. to provide increased stability. sed resistance to proteolytic inactivation. decreased to nonexistent immunogenicity. increased atory lives. ing modiﬁed serum half-lives and modiﬁed therapeutic half-lives. and low toxicity. Methods by which the compounds of the invention can be modiﬁed include. for example. by PEGylation. by chemical tization, and by fusion or conjugation with peptides or lipids. Modiﬁded compounds include ed Type-B natn‘uretic signal peptide fragment . including. for example. modiﬁed BNPsp(17-26) (SEQ ID N021), and modiﬁed analogs. variants (e.g., conservative variants) and tmncations thereof. Other embodiments include peptides selected from SEQ.ID.NOS:2 to 9 that have been modiﬁed. and peptides according to Formula I, Formula II. Formula lll. Formula IV. Forrnuia V. a VI. a VII and/or Formula Vlll that has been modiﬁed, and active s. ts (e.g., conservative variants) and truncations thereof that have been modiﬁed.

This invention envisions prodrug toms of the therapeutic peptides of the invention. A ‘prodrug' is a modiﬁed form of a therapeutic peptide that includes a reversible chemical ation that can reliably d to convert the prodrug to the parent peptide through either an enzymatic or ymatic catalytic reaction under physiological conditions following delivery to a patient. Such modiﬁcations can e chemical ity, alter aqueous solubility. extend biological ife. broaden therapeutic indices. improve pharmacodynamics, and/or improve bioavailability. for example. while preserving the pharmacological properties of the parent therapeutic peptide. Such modiﬁcations can also allow the parent peptide to be released after it reaches the biological compartment where it can exert the desired . A 'prodrug' is a compound that may include one or more specialized non-toxic protective groups used in a transient manner to alter or to eliminate certain limiting properties in the parent peptide, which protective group(s) can be removed by enzymatic or chemical cleavage. Any suitable protective group(s) can be employed to generate a peptide prodnig of the invention. Such specialized modiﬁcations include inclusion of one or more amino acid residues at either or both the amino- and/or carboxy- terrninus of the parent peptide. ge sites that allow for the efﬁcient in vivo removal of additional N- or C- terrninal amino acids or amino acid sequences are preferably included in such prodrug molecules. ations other than the addition of one or more N- and/or C-terminal amino acid residues are also envisioned. For example. diketopiperazine and diketomorpholine (DKP and DMP) strategies for prodrug conversion may be used (see. e.g.. Application of Peptide-Badsed Prodrug try in Drug Development, Springer. Ed. De. Amab (2012)). where prodrugs slowly convert to the parent drug at physiological conditions driven by the compounds' inherent chemical instability. without the need of any enzymatic cleavage. To improve stability. parent es of the invention can be protected against exopeptidase-mediated hydrolysis by bioreversibly masking N- and/or Gterminal amino acids.

OTA~1101~PC Examples of prodrugs of the invention are those wherein the parent peptide includes one or more additional amino acid residues appended to the N- and/or C-terminus of the parent e. The compounds of the invention also e prodmg forms of the agents of the invention. For example. prodrug forms include those having one to 16 amino acid residues appended to the N-terminus of, for example, the peptide BNPsp(17-26) (SEQ ID N0:1). Examples of such prodrug forms e those that have one or more of the amino acids listed in Table 1 linked to the parent peptide via a suitable bond. Representative prodrug embodiments include residues 1-16, 2-16, 346. 4-16, 5-16. 6-16, 7-16, 8-16, 9-16. 10-16. 11-16. 12-16, 13-16. 14-16, and 15-16 linked to the N-terminus of, for example. BNPsp(17-26). or any of the other peptides from SEQ.|D.NOS:2 to 9. and peptides according to Formula 1, Formula II. Formula lll, Formula IV. Formula V. Formula Vl, Formula Vll and/or Formula VIII, and active analogs and variants (e.g.. conservative variants) of the foregoing.

BNPs - Amino Acid Residue Position Ammo Acrd at Resrdue Posrtion ———_ ——Q orCorL _— T or K or M or A ___-_ —_—3EE— —__-_ —r——m- —_—L __—-— __—L _—1 L __—F —17 —L _2——L _£—__ ___am— .- S (or F) Further es of a prong according to the invention include those wherein an amino group of parent peptide is acylated. alkylated. phosphorylated. eioosanoylated, alanylated. pentytaminocarbonylated. (5- methyI-Z-oxo-i,3-dioxolenyl)rnethoxycarbonylated, tetrahydrofuranylated. pyrrolidylmethylated. yloxymethylated or tert-butylated, and the like; a compound n a hydroxy group of the parent peptide is OTAPC acylated. alkyiated. phosphorylated. acetylated. palmitoylated. propanoylated. pivaioylated, succinylated, lated. alanylaled or dimethyiaminornethylcarbonylated. and the like; and a compound wherein a carboxy group of the parent peptide is esteritied or amidated (e.g., ethyl esterilied. phenyl esteriﬁed. carboxymethyl esteritied. dimethyiaminomethyi esteritied. pivaioyioxymethyl tied. ethoxycarbonyloxyethyi esteritied. phthaiidyl esterified. (5-methyl~2-exo-l .3dioxolew4-yl)methyl esteriﬁed, cyciohexyloxycarbonylethyl esteriiied or methylamidated, and the like) and the like.

Other prodrugs fomis are also envisioned, including those ning chemical modiﬁcations to one or more amino acids residues that are not positioned at the N- or C-tenninus of the parent peptide. As those in the art will appreciate, any suitable chemical modification that can be removed under physiological conditions to yield a pharmaceutically active form of a compound of the invention can be ed.

Other embodiments include peptidiomimetics of compounds of the invention.

A presently preferred Type-B natriuretic signal peptide fragment agent is 17-26) (SEQ ID N021). ] illustrations of cardioprotective activities of Type-B natriuretic signal peptide fragment agents are provided in the below Examples. e 1 shows the ability of Type-B natriuretic signal peptide fragment agents to improve cardiac contractility by stration of BNPsp(l7-26) (SEQ ID N021) before and aﬂer 45 minutes of global ischemia in isolated rat heart preparations. In the in vivo sheep experiments of Example 2. it is shown that cardiac contractile function and troponin release. diagnostic markers of myocardial damage. are improved by stration of a Type-B natriuretic signal e fragment agent, BNPsp(17-26) (SEQ lD N011). Example 3 describes experiments to assess BNPsp fragment peptides of various lengths and their bioactivity as observed in Example 1 and referred to in Example 2.

Synthesis of Type-B natriuretic signal peptide iragrnent agents. as well as modifed Type-B natriuretic signal peptide fragment agents. is carried out using methods known in the art. Compunds of the inventions that are peptides. such as SEQ.lD.NOS:1-9. can be made by solid-state chemical peptide synthesis.

Other nds. such as fusion peptides. can also be made by conventional recombinant techniques using standard procedures described in. for example. ok & Maniaitis. The peptides and other compounds of the invention may be chemically modified. This may enhance their resistance to ases and other enzymes. restrict clearance by the kidney. etc. Methods of ing such modified compounds are known in the art.

] The precise ce of the Type-B natriuretic signal peptide fragment agent used will depend upon its ability to ameliorate on or more of the symtoms or effects of a wrdiovascular disorder. Means for determining such effects are ed in Examples 1 and 2. Other means for assessing the utility of a Type-B natriuretic signal peptide fragment agent for treatment or prevention of a cardiovascular disorder include in vitro cell culture experiments using c myocyte and non-myocyte cell lines, as well as in vivo and ex vivo ments with models of cardiac congenital disease and toxicity.

OTA—1101-PC Suitable Type-B natriuretic signal peptide fragment agents for the ation of the pharmaceutical compositions of the invention include LHLAFLGGRS (SEQ.|D.N0:1). LHLAFLGGR (SEQ.|D.N022).

LHLAFLGG (SEQ.|D.N023). LHLAFLG D.NO:4). LHLAFL (SEQ.|D.NO:5). LHLAF (SEQ.|D.NO:6), LHLA (SEQ.|D.N0:7). LHL D.N0:8). and LH (SEQ.|D.N029). Other suitable Type-B retic signal peptide fragment agents for the preparation of the pharmaceutical compositions of the invention include peptides within Formulae i-Vlll. Other suitable TypeB natriuretic signal peptide fragment agents for the preparation of the pharmaceutical compositions are described herein. and include. for example. analogs, ion. ions and modiﬁcations (including fusions) of the foregoing compounds.

Type-B natriuretic signal peptide fragment agent activity can be selected in terms of their sequence and desired activity by any convenient. and conventional. approach including, for example. as bed in the Examples below.

Homology and gues of Type-B natriuretic signal peptide fragment agents. for example.

Type-B retic signal peptide fragments. are discussed herein. Such a Type-B natriuretic signal peptide fragment typically has at least about 70% homology. preferably at least about 80%. at least about 90%. at least about 95%, at least about 97% or at least about 99% homology with the relevant sequence. ] gy may be ated based on any method in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings).

The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for e as described in Aitschul S. F. (1993) J Mol Evoi 36: 290-300; Altschul. S, F et al (1990) J Mol Biol 215: 403-10.

Software for performing BLAST es is publicly available through the National Center for Biotechnology lnfonnation (http://www.ncbi.nim.nih.govl). This algorithm involves first identifying high scoring sequence pair (HSPs) by fying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned vn'th a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et at, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as tar as the tive alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below. due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.

The BLAST algorithm ters W. T and X ine the sensitivity and speed of the alignment.

The BLAST program uses as defaults a word length (W). the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) ents (B) of 50. expectation (E) of 10, M=5, N=4, and a comparison of both strands.

OTAPC The BLAST algorithm performs a statistical analysis of the similarity n two sequences; see e.g.. Karlin and Altschul (1993) Proc. Natl. Acad. 80‘. USA 90: 5873-5787. One measure of similarity provided by the BLAST thm is the smallest sum probability (P(N)). which provides an indication of the probability by which a match between two amino acid sequences would occur by . For example. a sequence is considered similar to another sequence ii the st sum probability in comparison of the ﬁrst sequence to a second sequence is less than about 1, preferably less than about 0.1. more preferably less than about 0.01. and most preferably less than about 0.001.

The homologous sequence typically differs from the relevant sequence by at least about (or by no more than about) 2. 5. 10. 15, 20 more ons (which may be substitutions. deletions or insertions). These mutations may be measured across any of the regions mentioned above in on to calculating homology.

Cardiovascular Theragutlc Agents Compositions and methods of the invention for the tion and/or treatment of a cardiovascular disorder. e.g.. an acute coronary syndrome. heart failure. ic heart disease. etc. and related cardiovascular diseases. ers and conditions involving ischemia and/or oxidative stress. also comprise administration of a Type-B natriuretic signal peptide fragment agent in series or in combination with (e.g.. in physical combination. provided as a combined preparation) one or more other cardiovascular treatment agents. Cardiovascular therapeutic agents include nitrates. B—blockers. calcium channel blockers (particularly for stable or unstable angina. but also for heart failure in the case of B-blockers). diuretic agents. vasodilator . positive inotropes. ACE inhibitors and aldosterone antagonists. e.g. spironolactone cularly for heart failure). blood thinning therapeutics (e.g.. aspirin. ns. warfarins) and nitroglycerin cularty for Ml).

Compositions and s of the invention for the tion and/or treatment of a vascular disorder. e.g.. an acute coronary syndrome. heart failure. ischemic heart disease. etc.. and related cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. may also comprise administration of a Type-B natriuretic signal peptide fragment agent in series or in combination with (e.g.. in physical ation. provided as a combined preparation) one or more anti-thrombolytic therapies (e.g.. streptokinase inhibitors. anti- plateiet thereapetuics. such as. for example. clopidogrel).

Compositions and methods of the invention for the prevention and/or treatment of a cardiovascular disorder. e.g.. an acute coronary syndrome. heart failure. ischemic heart disease. etc., and related cardiovascular es. disorders and conditions involving ischemia and/or oxidative stress. may also comprise administration of a Type-B natriuretic signal peptide fragment agent in series or in ation with (e.g.. in physical combination. ed as a combined preparation) a Type-B natriuretic peptide. including for example nesiritide. a recombinant form of Type-B natriuretic peptide.

In certain methods and compositions (including pharmaceutical compositions. ations. articles of manufacture and kits) of the invention for the tion and/or treatment of a cardiovascular disorder. e.g.. an 0TA—1101-PC acute coronary syndrome. heart failure. ischemic heart e. etc.. and related cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. sub-therapeutically effective amounts of a Type-B natriuretic signal peptide fragment agent. and one or more other cardiovascular treatment agents are used or provided for combined administration (separately or jointly as a combined preparation) to provide a combined action that is therapeutically effective.

Thus. it will be understood that compositions and methods of the invention for the treatment of a cardiovascular disorder. 9.9.. an acute coronary me. heart failure. ischemic heart disease. etc. and related cardiovascular diseases. disorders and conditions involving ischemia and/or oxidative stress. that employ a TypeB natriuretic signal peptide fragment agent and r cardiovascular therapeutic agent are disclosed. A Type-B natriuretic signal peptide fragment agent may be ed. for e, from the group ting of BNPsp(17-26) (SEQ ID N021). BNPsp(17-25) (SEQ ID NO:2). BNPsp(17-24) (SEQ ID N023). BNPsp(17-23) (SEQ ID NO:4), BNPsp(17-22) (SEQ ID NO:5), BNPsp(17-21) (SEQ ID N0:6), BNPsp(17-20) (SEQ.ID.NO:7). BNPsp(17-19) (SEQ.ID.NO:8). and BNPsp(17-18) D.NO:9). In another embodiment. a Type-B natriuretic signal peptide fragment agent may be ed from the group consisting of a sequence according any one of Formula I. Formula II.

Formula lll. Formula IV, Formula V. Formula VI. Formula VII and Formula Vlll. Optionally. a cardiovascular agent is selected. for example. from the group comprising or consisting essentially of nitrates. B-blockers. calcium channel blockers. ic agents. vasodilator agents. positive inotropes. ACE inhibitors. aldosterone antagonists. lycerin. blood thinning agents. anti-thrombolytic agents. and Type-B retic peptides.

Treatment of a subject as provided herein with one or more compounds or ceutical itions as described herein may comprise their acute or sustained administration and. in the case of ations. their simultaneous. separate. or sequential administration. as further described herein.

The agents of the invention of the may be administered to a subject in need of treatment. such as a subject with any of the diseases. disorders or conditions mentioned herein. The condition of the subject can thus be improved. The agents may be used in the manufacture of a medicament to treat any of the diseases. disorders or condtions mentioned herein. Thus. in accordance with the ion. there are provided formulations by which cardiovascular disorders can be d.

A therapeutically effective amount of each of the combination rs (9.9.. a Type-B natriuretic signal peptide fragment agent and another cardiovascular therapeutic agent) may be administered simultaneously. separately or sequentially and in any order. The agents may be administered separately or as a ﬁxed combination.

When not stered as a ﬁxed combination. preferred methods e the tial administration of a TypeB natriuretic signal peptide fragment agent and another cardiovascular therapeutic agent. either or both of which are provided in amounts or doses that are less that those used when the agent or agents are administered alone. i.e.. when they are not administered in combination, either physically or in the course of treatment. Such lesser s of agents administered are typically from about one-twentieth to about one-tenth the amount or amounts of the agent OTAPC when administered alone. and may be about ghth the . about one-sixth the amount. about one-ﬁfth the amount, about one-fourth the amount. about one-third the , and about one-half the amount when administered alone. Preferably. the agents are stered sequentially within at least about one-half hour of each other. The agents may also be administered within about one hour of each other. within about one day to about one week of each other. or as otherwise deemed appropriate.

The agents of the invention of the may be administered to a subject in need of treatment. such as a subject with an acute coronary syndrome or any of the diseases or conditions mentioned . The condition of the subject can thus be improved. The compounds may thus be used in the treatment of the subject's body by therapy.

They may be used in the manufacture of a medicament to treat any of the conditions mentioned herein. Thus, in accordance with the invention. there are provided forrnulaticns by which cardiotherapy and cardioprotection can be speciﬁcally evoked.

Dosage Forms and Formulations and Administration The compounds of the invention may be present in an isolated or substantially or essentially pure form. It will be understood that the product may be mixed with carriers or diluents which will not interfere with the intended purpose of the product and still be regarded as isolated or substantially pure. A product of the ion may also be in a substantially or essentialy puriﬁed form, preferably comprising or consisting essentially of about 80%. 85%. or 90%. 9.9. at least about 95%. at least about 98% or at least about 99% of the compound or dry mass of the preparation.

Depending on the intended route of administration. the pharmaceutical products. pharmaceutical compositions. combined preparations and medicaments of the invention may. for example. take the form of solutions. suspensions. instillations. sustained release formulations. or powders, and typically contain about DJ %-95% of active ingredient(s). preferably about 0.2%-70%. Other suitable ations include injection- and infusion-based formulations. Other useful formulations include sustained e preparations. including. for example, controlled. slow or delayed release preparations. s of the invention include controlled or other doses. dosage forms. formulations, compositions and/or devices containing one or more Type-B natriuretic signal e fragment agents. wherein the Type-B natriuretic signal peptide fragment agents are. for example. one or more Type-B natriuretic signal peptide fragments. The present invention includes. for e. doses and dosage forms for at least oral administration. transdermal delivery. topical application. itory delivery. ucosal delivery. ion (including subcutaneous administration. subderrnal stration. uscular administration. depot administration. and intravenous administration, including delivery via bolus. slow intravenous injection, and intravenous drip). on devices (including implantable infusion devices. both active and passive). administration by tion or insutflation. buccal administration and sublingual stration. it will be appreciated that any of the dosage forms. compositions. formulations or devices described herein particularly for intravenous administration may be utilized.

OTAPC where applicable or desirable. in a dosage form. composition. formulation or device for administration by any of the other routes herein contemplated or commonly employed. For example, a dose or doses could be given parenterally using a dosage form suitable for parenteral administration which may incorporate features or compositions described in respect of dosage forms suitable for oral administration. or be delivered in an sustained dosage form. such as a modiﬁed release. extended release. delayed e. slow release or repeat action dosage form.

Preferably the Type-B natriuretic signal peptide fragment agents of the invention are combined with a aceutically acceptable carrier or diluent to produce a ceutical composition. Suitable carriers and diluents e isotonic saline solutions. for example phosphate-buffered saline. Suitable diluents and excipients also include. for example. water. saline. dextrose. glycerol. or the like. and combinations thereof. In addition. if desired substances such as wetting or emulsifying agents. stabilizing or pH buffering agents may also be present.

] The term ’pharmaceutically acceptable carrier' refers to any useful carriers. excipients. or stabilizers which are nontoxic to the cell or mammal being exposed o at the dosages and trations employed. and include pharmaceutical carriers that do not induce the production of antibodies harmful to the individual ing the composition. Suitable can'iers can be large. slowly metabolized macromolecules such as proteins. polysaccharides. polylactic acids. polyglycolic acids. polymeric amino acids. and amino acid copolymers.

Often the physiologically acceptable carrier is an s pH buffered solution. Other examples of physiologically acceptable carriers include buffers such as phosphate. citrate. and other organic acids; antioxidants including ascorbic acid; low lar weight (less than about 10 residues) polypeptide; proteins. such as serum albumin. n, or immunoglobulins; hydrophilic rs such as polyvinylpyrrolidone; amino acids such as glycine. glutamine, asparagine. arginine or lysine; monosaccharides. disaccharides. and other ydrates including glucose. mannose. or dextrins; chelating agents such as EDTA; sugar ls such as mannitol or sorbitol; salt- fon'ning counterions such as sodium; and/or nonionic surfactants such as Tween. polyethylene glycol (PEG). and Pluronics.

Pharmaceutimlly ameptable salts can also be t. e.g.. mineral acid salts such as hydrochlorides. hydrobromides. phosphates. sulfates. and the like; and the salts of organic acids such as acetates. propionates. tes. benzoates. and the like.

Suitable carrier materials include any carrier or vehicle commonly used as a base for . lotions. gels. emulsions. or paints for topical administration. Examples include emulsifying agents. inert carriers including hydrocarbon bases. fying bases. non-toxic solvents or water-soluble bases. Particularly suitable es e pluronics. HPMC. CMC and other cellulose-based ingredients. n. hard parafﬁn. liquid parafﬁn. soft yellow parafﬁn or soft white parafﬁn. white beeswax. yellow beeswax. cetostearyl alcohol, cetyl alcohol. icones. emulsifying waxes, isopropyl myristate. microcrystalline wax. oleyl alcohol and stearyl alcohol.

An auxiliary agent such as casein. gelatin, albumin. glue. sodium alginate. carboxymethylcellulose. methylcellulose. hydroxyethylcellulose or polyvinyl alcohol may also be included in the formulation of the invention.

OTAPC The dosage fomts. formulations. devices and/or compositions of the invention may be formulated to optimize bioavailability and to maintain plasma concentrations within the therapeutic range. including for extended s. Sustained delivery preparations. e.g.. controlled delivery preparations. also optimize the drug concentration at the site of action and minimize periods of under and over medication, for example.

The dosage forms. devices and/or compositions useful in the invention may be ed for periodic stration. including once daily administration. for low dose controlled and/or low dose long-lasting in vivo release of a Type-B natriuretic signal peptide fragment agent.

Examples of dosage forms suitable for oral administration include, but are not limited to tablets. capsules. es. or like forms. or any liquid forms such as syrups. aqueous solutions. emulsions and the like. capable of providing a therapeutically effective amount of a Type-B natriuretic signal peptide fragment agent.

Examples of dosage forms suitable for ennal administration include. but are not limited to. transdermal patches, ermal bandages. and the like. es of dosage forms suitable for topical administration of the compounds and formulations useful in the invention are any . stick. spray. nt. paste. cream. gel. etc. whether applied directly to the skin or via an intermed.

Examples of dosage forms suitable for suppository administration of the compounds and formulations useful in the invention e any solid dosage form inserted into a bodily orifice particularly those inserted rectally. vaginally and urethrally.

Examples of dosage forms suitable for transmucosal delivery of the compounds and formulations useful in the invention e depositories ons for enemas. pessaries. tampons. creams. gels. pastes. foams. nebulised solutions. powders and similar formulations containing in addition to the active ingredients such carriers as are known in the art to be appropriate.

Examples of dosage of fomis suitable for injection of the nds and formulations useful in the invention include delivery via bolus such as single or multiple strations by intravenous injection. subcutaneous, subdennal. and uscular administration or oral administration.

Examples of dosage forms suitable for depot administration of the nds and formulations useful in the invention include pellets or small cylinders of active agent or solid forms wherein the active agent is entrapped in a matrix of biodegradable polymers. mlcroemulsions, liposomes or is microencapsulated.

Examples of infusion devices for compounds and formulations useful in the invention include infusion pumps containing one or more Type-B natriuretic signal peptide fragment agents and/or pre-complexed Type-B natriuretic signal peptide fragment agents. at a desired amount for a desired number of doses or steady state administration. and include implantable drug pumps.

Examples of implantable infusion devices for compounds and formulations useful in the ion include any solid form in which the active agent is encapsulated within or dispersed throughout a biodegradable polymer or synthetic. polymer such as silicone. ne . silastic or similar polymer.

OTAPC Examples of dosage fomts suitable for inhalation or insufflation of compounds and formulations useful in the invention include compositions comprising solutions and/or suspensions in pharmaceutically acceptable. aqueous, or organic solvents. or mixture thereof and/or powders.

Examples of dosage fomis suitable for buccal administration of the compounds and formulations useful in the invention include lozenges. tablets and the like. compositions comprising solutions and/or suspensions in pharmaceutimlly acceptable. aqueous. or organic solvents. or mixtures thereof and/or powders.

Examples of dosage fomis suitable for sublingual administration of the compounds and formulations useful in the invention include lozenges. tablets and the like. compositions sing solutions and/or suspensions in pharmaceutically able. aqueous. or organic solvents. or mixtures thereof and/or powders.

Examples of controlled drug formulations for delivery of the compounds and formulations useful in the ion are found in. for example. Sweetman. SC. (Ed). Martindale. The Complete Drug Reference. 33rd Edition. Pharmaceutical Press. Chicago. 2002. 2483 pp.; Aulton. M. E. (Ed) Pharmaceutics. The Science of Dosage Form Design. Churchill Livingstone. Edinburgh. 2000. 734 pp.; and. Ansel. H. C.. Allen. L. V. and Popovich. N. G. Pharmaceutical Dosage Forms and Drug Delivery Systems. 7th Ed.. cott 1999. 676 pp.

Excipients employed in the manufacture of drug ry systems are described in s publications known to those skilled in the art including, for example. Kibbe, E. H. Handbook of ceutical Excipients. 3rd Ed..

American ceutical Association. Washington. 2000. 665 pp. The USP also provides examples of modiﬁed- release oral dosage forms. including those formulated as tablets or capsules. See. for example. The United States Pharrnacopeia ional Formulary 18, The United States Pharmacopeial Convention, Inc. Rockville MD. 1995 (hereinafter ‘the USP'). which also describes specific tests to ine the drug release capabilities of extended- release and delayed-release tablets and es. Further guidance oonceming the analysis of extended release dosage fomts has been provided by the FDA. See Guidance for Industry. ed release oral dosage forms: development. evaluation. and application of in vifrolin vivo correlations. Rockville. MD: Center for Drug Evaluation and Research. Food and Drug Administration (1997).

Further examples of dosage forms useful in the methods of the ion include. but are not limited to. ed-release (MR) dosage forms ing delayed-release (DR) forms: prolonged-action (PA) forms; controlled-release (CR) forms: extended-release (ER) forms; timed-release (TR) fomis: and cting (LA) forms.

For the most part, these terms are used to describe orally administered dosage forms. however these terms may be applicable to any of the dosage forms. formulations. compositions and/or devices described . These formulations effect delayed total drug release for some time after drug stration. and/or drug release in small aliquots ittently after administration, and/or drug release slowly at a controlled rate governed by the delivery system, and/or drug release at a constant rate that does not vary. and/or drug release for a significantly longer period than usual formulations.

OTA—1101-PC Modiﬁed-release dosage forms of the invention include dosage forms having drug release features based on time. course. and/or location which are ed to accomplish eutic or convenience ives not offered by conventional or immediate-release forms. See. for example. Bogner. R.H. U.S. Pharmacist 22 (Suppl.):3- 12 (1997); Scale-up of oral extended-release drug delivery systems: part I. an overview. Pharmaceutical Manufacturing 2:23-27 (1985). Extended-release dosage toms of the invention include. for example. as deﬁned by The United States Food and Drug Administration (FDA). a dosage form that allows a ion in dosing frequency to that presented by a conventional dosage form, 9.9.. a solution or an immediate-release dosage form. See, for example. . RH. (1997) supra. Repeat action dosage toms of the ion include, for example. forms that contain two single doses of medication. one for immediate release and the second for delayed release. Bi-Iayered tablets. for example. may be prepared with one layer of drug for immediate release with the second layer designed to release dnig later as either a second dose or in an extended-release manner. Targeted-release dosage toms of the invention include. for example. formulations that facilitate drug release and which are directed towards isolating or concentrating a drug in a body region. tissue. or site for absorption or for drug action.

Also useful in the invention are coated beads. es or microspheres containing one or more Type-B natriuretic signal peptide fragment agents and/or pre-complexed Type-B natriuretic signal e fragment agents. which may be used to achieve modified release of one or more Type-B natriuretic signal peptide fragment agents and/or pre-complexed Type-B natriuretic signal peptide fragment agents by incorporation of the drug into coated beads. granules. or microspheres. In such systems. the Type-B natriuretic signal peptide fragment agent and/or pre-complexed Type-B natriuretic signal peptide fragment agent is distributed onto beads. pellets, granules or other particulate systems. See Ansel. H.C.. Allen. L.V. and Popovich, N.G.. Pharmaceutical Dosage Forms and Drug Delivery Systems. 7th Ed. Lippinoott 1999. p. 232.

Methods for cture of microspheres suitable for dmg delivery have been described. See. for example. Arshady, R. Polymer Eng Sci 6-1758 (1989); see also. Arshady. R.. Polymer Eng Sci 30:905-914 (1990); see also: y R.. Polymer Eng Sci 30:915-924 (1990). Various coating s are commercially available. E.g.. Aquacoat“l [FMC ation. Philadelphia] and lease“ [Coleman]; Aquacoat aqueous polymeric dispersion. Philadelphia: FMC Corporation. 1991; Surerelease aqueous lled e coating system.

West Point. PA: Colorcon. 1990; Butler. J.. et al.. Pharm Tech 22:122-138 (1998); Yazici. E.. et al., ceut Dev Technol1:175-183 (1996).

Variation in the ess of the coats and in the type of coating materials used s the rate at which the body ﬂuids are capable of penetrating the coating to dissolve the Type-B natriuretic signal peptide fragment agent. Generally. the thicker the coat, the more resistant to penetration and the more delayed will be Type-B natriuretic signal peptide fragment agent e and dissolution. See Madan. P. L. US. Pharmacist 15:39-50 (1990). This provides the different desired sustained or extended release rates and the targeting of the coated beads to the desired segments of the gastrointestinal tract. Examples of ﬁlm-forming polymers which can be used in water- 0TAPC insoluble release-slowing intermediate layerts) (to be applied to a pellet. spheroid or tablet core) include ethylcellulose. polyvinyl acetate, Eudragit® RS. Eudragit® RL. etc. (Each of Eudragit® RS and Eudragit® RL is an ammonia methacrylate copolymer. The release rate can be controlled not only by incorporating therein suitable water-soluble pore s. such as lactose, ol. sorbitol, etc. but also by the thickness of the coating layer applied. Multi-tablets may be formulated which include small spheroid-shaped ssed mini-tablets that may have a diameter of between 3 to 4 mm and can be placed in a n capsule shell to provide the desired pattern of Type-B natriuretic signal peptide fragment agent release. Each capsule may contain 8-10 minitablets. some uncoated for immediate release and others coated for extended release of the Type-B natriuretic signal peptide fragment agent.

] A number of methods may be employed to generate modified-release dosage foms of one or more TypeB natriuretic signal e fragment agents suitable for oral administration to humans and other mammals.

Two basic mechanisms available to achieve modiﬁed release drug delivery include altered dissolution or diffusion of drugs and excipients. Within this context. for example, four processes may be ed. either simultaneously or consecutively. These are as follows: (i) hydration of the device (9.9.. swelling of the ); (ii) diffusion of water into the device; (iii) controlled or delayed ution of the drug; and (iv) controlled or delayed diffusion of dissolved or solubilized drug out of the device.

In order to formulate a range of dosage values. cell culture assays and animal s can be used.

The dosage of such compounds ably lies within the dose that is therapeutically ive for at least 50% of the population. and that exhibits little or no toxicity at this level.

The effective dosage of each of the Type-B natriuretic signal peptide fragment agents employed in the methods and compositions of the invention may vary depending on a number of factors including the particular Type-B natriuretic signal peptide fragment agent or agents ed, the cardiovascular therapeutic ccmbinational partner if present. the mode of administration, the frequency of administration. the condition being treated. the severity of the condition being treated. the route of administration, the needs of a patient sub-population to be treated or the needs of the dual patient which different needs can be due to age. sex. body weight. relevant condition speciﬁc to the patient.

A suitable dose may be from about 0.001 to about 1 or to about 10 mglkg body weight such as about 0.01 to about 0.5 mg/kg body weight. A suitable dose may however be from about 0.001 to about 0.1 mg/kg body weight such as about 0.01 to about 0.05 mg/kg body weight. Doses from about 1 to 100. 0. 200-300. 300-400. and 0 miligrams are riate. as are doses of about 500-750 micrograms and about 750-1000 micrograms. Other useful doses include from about 300 to about 1000 picomoles per dose, and about 0.0510 about 0.2 nanomoles per dose. Still other doses are within the following claims.

For example. in certain embodiments. the Type-B natriuretic signal peptide fragment agent composition may be administered at about 0.01 nanomolar (mM) or 0.05 nM to about 200 nM final concentration.

OTAPC Preferably. the Type-B natn'uretic signal peptide fragment agent composition is administered at about 0.1 nM to about 150 nM ﬁnal concentration. more preferably. the Type-B natriuretic signal peptide fragment agent composition is applied at about 1 nM to about 100 nM ﬁnal concentration, and more preferably, the Type-B natriuretic signal peptide fragment agent composition is administered at about 10-20 nM to about 100-150 nM ﬁnal concentration. Additionally.

Type-B natriuretic signal peptide fragment agent dose amounts include. for example, about 0.1-1. 1-2, 2-3, 3-4, or 4- milligrams (mg). from about 5 to about 10 mg, from about 10 to about 15 mg. from about 15 to about 20 mg. from about 20 to about 30 mg. from about 30 to about 40 mg, from about 40 to about 50 mg. from about 50 to about 75 mg. from about 75 to about 100 mg. from about 100 mg to about 250 mg. and from 250 mg to about 500 mg. Dose amounts from 500 to about 1000 rams or more or also provided. as noted above. Other doses include doses ranging from at least about 100 nanograms. including. for example at least about 200 nanograms. 600 nanograms. 2000 nanograms. 6000 nanograrns and at least about 10.000 ams or more. Dose concentrations include concentrations of at least about 0.1 moles per liter, including. for example. at least about 0.3. 1.0. 3.0 and 10.0 nMoles/L. Dose concentrations also include trations of 0.1 nMoles/L, 0.3 nMoles/L. 1.0 /L. 3.0 nMoles/L and 10.0 nMoles/L. These dose concentrations are equivalent to 0.1. 0.3, 1. 3, 11 pg”. and administrable weight doses of 0.4. 1.0. 4.0, 10 and 39 micrograms/kg (pglkg). Also within the invention are other dam ranging from 0.1 to 5.0 pig/kg and 0.1 to 10.0 pglkg. Additionally. doses of about 0.4, 1.0. 4.0. 10 and 39 jug/kg are within the invention. Doses of at least about 0.4, 1.0, 4.0, 10 and 39 pg/kg are also within the invention.

Still other dosage levels n about 1 nanogram (ng)/kg and about 1 mglkg body weight per day of each of the agents described herein. In certain embodiments, the dosage of each of the subject compounds will lly be in the range of about 1 ng to about 1 microgram per kg body weight, about 1 ng to about 0.1 microgram per kg body weight. about 1 ng to about 10 ng per kg body weight. about 10 ng to about 0.1 microgram per kg body weight. about 0.1 microgram to about 1 microgram per kg body weight, about 20 ng to about 100 ng per kg body weight. about 0.001 mg to about 0.01 mg per kg body weight. about 0.01 mg to about 0.1 mg per kg body weight. or about 0.1 mg to about 1 mg per kg body weight. In n embodiments. the dosage of each of the subject compounds will generally be in the range of about 0.001 mg to about 0.01 mglkg body weight. about 0.01 mg to about 0.1 mglkg body weight. about 0.1 mg to about 1 mglkg body . if more than one Type-B natriuretic signal peptide fragment agent is used. the dosage of each Type-B natriuretic signal e fragment agent need not be in the same range as the other.

Conveniently, if infused. the Type-B natriuretic signal peptide fragment agent is administered for at least about 0.5 to 1 hour. at least about 1-2 hours. at least about 2-4 hours. at least about 4-6 hours, at least about 6 8 hours. at least about 8-10 hours. at least about 12 hours. or at least about 24 hours.

As noted herein. the doses of a Type-B natriuretic signal peptide fragment. peptide or peptidomimetic. for example. administered in ation, or other cardiovascular therapeutic agents stered in ation with either or both, can be adjusted down from the doses administered when given alone. 01-PC The combined use of several agents may reduce the required dosage for any individual agent because the onset and duration of effect of the different agents may be complementary. In a preferred ment. the combined use of two or more Type~B natriuretic signal peptide fragment and/or cardiovascular therapeutic agents has an additive, synergistic or super-additive effect.

In some cases. the combination of a Type-B natriuretic signal peptide fragment agent and a cardiovascular therapeutic agent. or other agents administered in combination with either or both. have an additive effect. In other cases, the combination can have greater-than-additive . Such an effect is referred to herein as a 'supra-additive' effect. and may be due to synergistic or potentiated interaction.

In another preferred embodiment, the ed use of a Type~B natriuretic signal peptide fragment agent and another cardiovascular therapeutic agent. reduces the frequency in which said agent is administered compared to the frequency when said agent is administered alone. Thus. these combinations allow the use of lower and/or fewer doses of each agent than previously required to achieve desired therapeutic goals.

Doses may be administered in single or d applications. The doses may be administered once. or application may be repeated. Typically. administration can be by infusion in on to or instead of multiple single adminstrations.

One or more Type-B natriuretic signal peptide fragment agents and another cardiovascular therapeutic agent. if desired. may be stered by the same or different routes. The s agents of the invention can be administered separately at different times during the course of therapy. or concurrently in divided or single combination forms.

In one aspect of the invention a Type-B retic signal peptide fragment agent is administered in one composition and another cardiovascular therapeutic agent is administered in a second composition. In one ment the first ition sing a Type-B natriuretic signal peptide fragment peptide agent is administered before the second composition comprising another cardiovascular therapeutic agent. In one embodiment the ﬁrst composition sing a Type-B natriuretic signal peptide fragment peptide agent is administered after the second composition comprising another cardiovascular therapeutic agent. In one preferred embodiment the ﬁrst composition comprising a Type-B natriuretic signal peptide nt peptide agent is administered before and after the second composition sing another cardiovascular therapeutic agent. In one embodiment the second composition sing another cardiovascular therapeutic agent is administered before and after the ﬁrst composition comprising a Type-B natriuretic signal peptide fragment peptides agent. In one embodiment the ﬁrst composition comprising a Type-B natriuretic signal peptide fragment peptide agent is administered about the same time as the second composition comprising another cardiovascular therapeutic agent.

] The delivery of a formulation comprising a Type-B natriuretic signal peptide fragment agent, alone or together with another cardiovascular therapeutic agent. over a period of time. in some instances for about 1-2 OTAPC hours, about 2-4 hours, about 4-6 hours, about 68. or about 24 hours or longer, may also be awomplished using slow release or depot formulations, for example, as well as transdennal formulations and devices.

Strategies to improve the oral bioavailability of proteins have ranged from changing their physicochemical properties by modiﬁcation of their lipophilicity and enzyme susceptibility. to adding novel functionality using transport-carrier les that are recognized by endogenous transport-carrier systems in the gastrointestinal tract and/or to their inclusion in specially adapted drug carrier systems. ed polymeric-based systems have attracted erable attention in the controlled e in targeting particular organs/tissues, and in their ability to deliver proteins and peptides. They can effectively deliver the proteins to a target site and thus se the therapeutic beneﬁt. while minimizing side effects. Protein association with polymer-based carriers. Such as polymeric microparticles. nanoparticles. ets or patches is a useful approach to improve oral protein bioavailability. Polymer-based carriers can protect proteins from the gastrointestinal environment and allow the modulation of physicochemical and protein release properties and consequently the biological behavior. Also, from the perspective of improving oral absorption, the major effect of carriers is to se epithelial membrane permeability. y leading to higher bioavailability.

Dosage forms of the compounds and formulations of the invention, extended Type-B natriuretic signal peptide fragment agent action may be achieved by ing the rate at which the Type-B natriuretic signal peptide fragment agent is released from the dosage form and/or by slowing the transit time of the dosage form through the gastrointestinal tract (see Bogner. R.H.. US cist 22 (Suppl.):3-12 (1997)). The rate of dnig release from solid dosage forms may be modiﬁed by the technologies described below which. in general. are based on the following: 1) modifying drug dissolution by controlling access of biologic fluids to the drug through the use of barrier gs; 2) controlling drug diffusion rates from dosage forms; and 3) chemically ng or interacting between the drug substance or its pharmaceutical barrier and site-speciﬁc ical ﬂuids. s by which these objectives are achieved are also provided herein. In one approach. employing digestion as the release mechanism. the Type-B natriuretic signal peptide fragment agent is either coated or entrapped in a substance that is slowly digested or dispersed into the intestinal tract. The rate of availability of the Type-B retic signal peptide fragment agent is a function of the rate of digestion of the dispersible material. Therefore. the e rate, and thus the effectiveness of the Type-B natriuretic signal e fragment agent varies from subject to subject depending upon the ability of the subject to digest the material.

A r form of slow release dosage form of the compounds and formulations of the invention is any suitable osmotic system where semi-permeable membranes of for e cellulose acetate. cellulose acetate butyrate. cellulose acetate propionate. is used to control the release of Type-B natriuretic signal peptide fragrnenl agent. These can be coated with aqueous dispersions of c lacquers without changing release rate. An e of such an osmotic system is an osmotic pump device, such as the Orosm device developed by Alza Inc.

(USA). 01-PC Other s useful in the methods of the invention utilize monolithic matrices including. for example. slowly eroding or hydrophilic polymer matrices. in which one or more TypeB natriuretic signal peptide fragment agents are compressed or embedded.

Monolithic matrix devices comprising compounds and ations useful in the invention include those formed using. for example. TypeB retic signal e fragment agents sed in a soluble . which become increasingly ble as the matrix dissolves or swells; examples include hydrophilic colloid matrices. such as hydroxypropyloellulose (BP) or hydroxypropyl cellulose (USP); hydroxypropyl rnethylcellulose (HPMC; BP.

USP); rnethylcellulose (MC; BP. USP); calcium carboxymethylcellulose (Calcium CMC; BP. USP); acrylic acid r or carboxy polyrnethylene pol) or Carbomer (BP. USP); or linear glycuronan rs such as c acid (BP. USP). for example those formulated into microparticles from alginic acid (alginate)-gelatin hydrocolloid coacervate systems, or those in which liposomes have been encapsulated by coatings of alginic acid with poly-L- lysine membranes. Type-B natriuretic signal peptide fragment agent e occurs as the polymer swells. forming a matrix layer that controls the diffusion of aqueous ﬂuid into the core and thus the rate of diffusion of Type-B natriuretic signal peptide fragment agent from the system.

In such systems. the rate of Type-B natriuretic signal peptide fragment agent release depends upon the tortuous nature of the channels within the gel. and the viscosity of the entrapped ﬂuid. such that different release kinetics can be achieved. for example. zero-order, or first~order combined with pulsatile release. Where such gels are not cross-linked. there is a weaker. rmanent association between the polymer chains, which relies on ary bonding. With such devices. high loading of the Type-B natriuretic signal peptide fragment agent is achievable. and effective blending is frequent. Devices may contain 20 - 80% of Type-B natriuretic signal peptide fragment agent (wlw). along with gel modiﬁers that can enhance Type-B natriuretic signal peptide fragment agent diffusion; examples of such modifiers include sugars that can enhance the rate of hydration. ions that can influence the content of cross-links. and pH buffers that affect the level of polymer ionization. Hydrophilic matrix devices may also contain one or more pH buffers. surfactants. counter-ions, ants such as magnesium stearate (BP. USP) and a glidant such as colloidal silicon dioxide (USP; colloidal anhydrous silica. BP) in addition to Type-B retic signal peptide fragment agent and hydrophilic matrix.

Monolithic matrix devices comprising compounds and formulations useful in the invention also include those d using. for example. Type-B natriuretic signal peptide fragment agent particles are dissolved in an insoluble matrix. from which Type-B natriuretic signal peptide fragment agent becomes available as solvent enters the matrix. often through channels. and ves the Type-B natriuretic signal peptide fragment agent particles.

Examples include s formed with a lipid matrix, or insoluble r , including preparations formed from Camauba wax (BP; USP); mediurn-chain ceride such as fractionated coconut oil (SP) or triglycerida saturata media (PhEur); or cellulose ethyl ether or ethylcellulose (BP. USP). Lipid matrices are simple and easy to manufacture. and incorporate the following blend of powdered components: lipids (20-40% hydrophobic solids WM) OTAPC which remain intact during the release process; Type-B natriuretic signal peptide fragment agent . e.g., channeling agent. such as sodium chloride or sugars, which leaches from the formulation, forming aqueous micro-channels (capillaries) through which solvent enters. and through which TypeB natn‘uretic signal peptide fragment agent is released. in this system. the Type-B natriuretic signal peptide fragment agent is embedded in an inert insoluble polymer and is released by leaching of aqueous ﬂuid. which diffuses into the core of the device through capillaries formed between particles. and from which the TypeB natriuretic signal e fragment agent diffuses out of the device. The rate of release is controlled by the degree of compression. particle size. and the nature and ve content (w/w) of excipients. An example of such a device is that of s Gradumet (Martindale 33rd Ed, 1360.3).

A further example of a suitable insoluble matrix is an inert plastic matrix. By this method. Type-B natriuretic signal peptide nt agents are granulated with an inert c material such as polyethylene. polyvinyl acetate. or polymethacrylate, and the granulated mixture is then compressed into tablets. Once ingested. the Type-B natriuretic signal peptide fragment agent is slowly ed from the inert plastic matrix by diffusion. See. for example.

Bodmeier. R. & Paeratakul. 0.. J Pharm Sci 79:32-26 (1990); Laghoueg. N.. ef al.. Int J Pharm 502133-139 (1989): Buckton. G., et aI.. Int J Pharm 74:153-158 (1991). The ssion of the tablet creates the matrix or plastic form that retains its shape during the leaching of the Type-B natriuretic signal peptide fragment agent and through its passage through the gastrointestinal tract. An immediate-release portion of Type-B natriuretic signal peptide fragment agent may be compressed onto the surface of the . The inert tablet , expended of Type-B natriuretic signal peptide fragment agent. is excreted with the feces. An example of a suwessful dosage form of this type is Gradumet (Abbott; see, for example. radumet. dale 33rd Ed. p. ).

Further examples of monolithic matrix devices useful in the methods of the invention include compositions and formulations of the invention incorporated in pendent attachments to a polymer matrix. See. for example. Scholsky. KM. and Fitch, R.M.. J Controlled Release 3:87-108 (1986). In these devices. Type-B natriuretic signal peptide fragment agents may be attached by means of an ester linkage to poly(acrylate) ester latex particles prepared by s emulsion polymerization. Still further examples of monolithic matrix s of the ion orate dosage forms in which the Type-B natriuretic signal peptide nt agent is bound to a biocompatible polymer by a labile chemical bond. e.g., polyanhydrides prepared from a substituted anhydride (itself ed by reacting an acid chloride with the drug: ryloyl chloride and the sodium salt of methoxy benzoic acid) have been used to form a matrix with a second polymer (Eudragit RL) which releases drug on hydrolysis in gastric ﬂuid.

See Chafi. N.. et al.. Int J Pharm 67:265-274 (1992).

Modiﬁed release fomis of one or more Type-B natriuretic signal peptide fragment agents may also be prepared by rnicroencapsulation. Microencapsulation is a process by which solids. liquids, or even gasses may be encapsulated into microscopic size particles h the formation of thin coatings of 'wall' material around the nce being encapsulated such as disclosed in US. Patent Nos. 418; 3.391.416 and 3.155.590. Gelatin (BP. USP) is commonly employed as a wall-forming material in microencapsulated preparations. but synthetic 0TAPC polymers such as polyvinyl alcohol (USP). ethylcellulose (BP. USP). polyvinyl chloride. and other materials may also be used. See, for example, Zentner. GM. et al.. J lled Release 22217-229 ; Files. AL. et at. J Pharm Sci 59:610-613 (1970); Samuelov. Y.. et al. J Pharm Sci 68325329 (1979). Different rates of Type-B natriuretic signal peptide fragment agent release may be obtained by changing the core-to-wall ratio. the polymer used for the coating. or the method of microencapsulation. See. for e: Yazici, E.. Oner. et al..Pharmaceut Dev Technol: 1:175—183 (1996).

Other useful ches include those in which the Type-B natriuretic signal peptide fragment agent is incorporated into polymeric colloidal particles or microencapsulates (micrcparticles, microspheres or nanoparticles) in the form or reservoir and matrix devices. See: Douglas. S. J.. et at. C.R. C. Crit Rev Therap Drug Carrier Syst 261 (1987); Oppenheim. R.C.. Int J Pharm 8:217-234 (1981 ); Higuchi, T.. J Pharm Sci 52:1145- 1149 (1963).

] Formulations of drugs suitable for transdennal delivery are known to those d in the art. and are described in references such as Ansel et at. (supra). Methods known to enhance the delivery of drugs by the percutaneous route include al skin penetration enhancers, which increase skin permeability by reversibly damaging or otherwise altering the physicochemical nature of the stratum comeum to decrease its resistance to drug diffusion. See Shah. V.. Peck. C.C.. and Williams. R.L.. Skin penetration enhancement: clinical pharmacological and regulatory considerations. In: Walters. K.A. and ft, J. (Eds) Pharmaceutical skin penetration enhancement.

New York: Dekker. (1993). Skin ation ers suitable for forrnulaticn with Type-B natriuretic signal peptide fragment agents in transdermal drug delivery systems may be chosen from the ing list: acetone. laurocapram. dimethylacetamide, ylfonnamide. dimethylsulphoxide. ethanol. oleic acid. polyethylene glycol. propylene glycol and sodium Iauryl sulphate. Further skin penetration enhancers may be found in publications known to those skilled in the art. See. for example, Osborne. D.W.. & Henke. J.J.. Pharm Tech 2125066 (1997); Roll. 0.. ‘Pharm Tech 12:130-139 (1988). In addition to chemical means. there are physical methods that enhance transdennal drug delivery and penetration of the compounds and formulations of the invention. These include ionlophoresis and sonophoresis. Formulations suitable for administration by ionlophoresis or soncphoresis may be in the form of gels. creams. or lotions.

Transdermal delivery. methods or formulations of the invention, may utilize, among others. monolithic delivery systems. drug-impregnated adhesive delivery systems (9.9.. the Latitude” drug-in-adhesive system from 3M). active transport devices and membrane-controlled systems. Transdermal delivery dosage forms of the invention include those which tute the Type-B retic signal peptide fragment agent. for the diclofenic or other pharmaceutically acceptable salt thereof referred to in the transdennal delivery systems disclosed in. by way of e. US. Patent Nos. 6,193,996. and 6.262.121.

Other dosage forms include variants of the oral dosage fomrs adapted for itory or other parenteral use. When rectally administered in the form of suppositories. for example. these compositions may be OTAPC prepared by mixing one or more compounds and formulations of the invention with a suitable non-irritating excipient. such as cocoa butter. synthetic glyceride esters or polyethylene glycols. which are solid at ordinary temperatures. but liquify and/or dissolve in the rectal cavity to release the Type-B retic signal peptide fragment agent.

Suppositories are generally solid dosage forms ed for insertion into body oriﬁces including rectal, vaginal and occasionally urethrally and can be long acting or slow release. Suppositories e a base that can include. but is not limited to. materials such as alginic acid. which will prolong the release of the pharmaceutically acceptable active ingredient over several hours (5-7).

Transmucosal administration of the compounds and formulations useful in the invention may utilize any mucosa! membrane but commonly es the nasal. buccal, vaginal and rectal s. Formulations suitable for nasal administration of the compounds and formulations of the invention may be administered in a liquid form. for example, nasal spray, nasal drops. or by aerosol administration by nebulizer. including aqueous or city solutions of the Type-B signal e fragment agent. Formulations for nasal administration. wherein the carrier is a solid. include a coarse powder having a particle size, for example. of less than about 100 microns. preferably less. most preferably one or two times per day than about 50 microns. which is administered in the manner in which snuff is taken. i.e.. by rapid tion through the nasal passage from a container of the powder held close up to the nose.

Compositions in solution may be nebulized by the use of inert gases and such nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be ed to a facemask. tent or intermittent Type- B natriuretic signal e fragment agents may be administered orally or nasally from devices that deliver the formulation in an appropriate . Formulations may be ed as aqueous ons for example in saline. solutions ing benzyl alcohol or other suitable preservatives. absorption ers to enhance bio-availability. fluorocarbons. and/or other solubilising or dispersing agents known in the art.

Compositions may be prepared according to conventional s by dissolving or ding an amount of a Type-B natriuretic signal peptide fragment agent(s) (s) ingredient in a diluent. The amount of Type-B natriuretic signal peptide fragment agent is from between 0.1 mg to 1000 mg per ml of diluent. In some embodiments. dosage forms of 100 mg and 200 mg of a Type-B natriuretic signal peptide fragment agent are provided. By way of example only. the amount of Type-B natriuretic signal peptide fragment agent may range from about 1 mg to about 750 mg or more (for example. about 1 mg. about 10 mg. about 25 mg. about 50 mg. about 100 mg. about 150 mg. about 200 mg. about 250 mg. about 400 mg. about 500 mg. about 600 mg. about 750 mg. about 800 mg. about 1000 mg. and about 1200 mg). Other doses include doses ranging from at least about 100 nanograms. including, for example at least about 200 nanograms. 600 nanograms. 2000 nanograms. 6000 nanograms and at least about 10.000 ams or more. Dose concentrations include concentrations of at least about 0.1 moles per liter. including. for example. at least about 0.3. 1.0. 3.0 and 10.0 nMoles/L. Dose concentrations also include concentrations of 0.1 nMoles/L. 0.3 nMoles/L. 1.0 nMoles/L. 3.0 nMoles/L and 10.0 nMoles/L. These dose concentrations are equivalent to 0.1. 0.3. 1. 3. 11 ngL and adminlstrable weight doses of 0.4. 1.0. 4.0. 10 and OTAPC 39 ramshrg (pg/kg). Also within the invention are other doses ranging from 0.1 to 5.0 [191th and 0.1 to 10.0 pglkg. Additionally, doses of about 0.4. 1.0. 4.0. 10 and 39 pglkg are within the invention. Doses of at least about 0.4, 1.0. 4.0. 10 and 39 pglkg are also within the invention. Other amounts within these ranges may also be used and are specifically contemplated though each number in n is not expressly set out.

Type-B natriuretic signal e fmgment agents can be provided and administered in forms suitable for once-aday dosing. An acetate. phosphate, citrate or glutamate buffer may be added allowing a pH of the ﬁnal composition to be from about 5.0 to about 9.5; optionally a carbohydrate or dric alcohol toniciﬁer and. a preservative selected from the group consisting of m~cresol. benzyl alcohol. methyl. ethyl. propyl and butyl parabens and phenol may also be added. Water for injection. tonicifying agents such as sodium chloride. as well as other excipients. may also be present. if desired. For parenteral administration. formulations are isotonic or substantially isotonic to avoid inﬂation and pain at the site of stration.

The terms . buffer solution and buffered solution. when used with reference to hydrogen-ion concentration or pH. refer to the ability of a . particularly an aqueous solution. to resist a change of pH on adding acid or alkali. or on dilution with a solvent. Characteristic of buffered solutions, which undergo small changes of pH on on of acid or base. is the presence either of a weak acid and a salt of the weak acid. or a weak base and a salt of the weak base. An example of the fomier system is acetic acid and sodium acetate. The change of pH is slight as long as the amount of hydroxyl ion added does not exceed the capacity of the buffer system to neutralize Maintaining the pH of the formulation in the range of approximately 5.0 to about 9.5 can enhance the stability of the parenteral formulation of the t invention. Other pH ranges. for example. include. about 5.5 to about 9.0. or about 6.0 to about 8.5, or about 6.5 to about 8.0. or. preferably, about 7.0 to about 7.5.

The buffer used may be selected from any of the ing. for example. an acetate buffer. a phosphate buffer or glutamate buffer. the most preferred buffer being a ate buffer. Carriers or excipients can also be used to facilitate administration of the compositions and ations of the invention. Examples of carriers and excipients include calcium carbonate. calcium phosphate. various sugars such as lactose. glucose. or sucrose. or types of starch. cellulose tives. gelatin, polyethylene glycols and physiologically compatible solvents. A stabilizer may be included. but will generally not be needed. If included. however. an example of a stabilizer useful in the practice of the invention is a carbohydrate or a polyhydric alcohol. The polyhydric alcohols include such compounds as sorbitol. mannitol. glycerol. l. and polypropylene/ethylene glycol copolymer, as well as various polyethylene glycols (PEG) of molecular weight 200. 400, 1450. 3350. 4000. 6000. and 8000). The carbohydrates include. for example. mannose, . trehalose. maltose. inositol. lactose. galactose. arabinose. or lactose. lsotonicity agents, or agents to maintain isotonicity. may also be used or included.

The United States Pharrnacopeia (USP) states that anti-microbial agents in bacteriostatic or fungistatic concentrations must be added to preparations contained in multiple dose containers. They must be OTAPC present in adequate concentration at the time of use to prevent the multiplication of microorganisms inadvertently uced into the ation while withdrawing a portion of the contents with a hypodermic needle and syringe. or using other invasive means for delivery. such as pen ors. Antimicrobial agents should be ted to ensure compatibility with all other components of the formula. and their activity should be evaluated in the total formula to ensure that a particular agent that is effective in one ation is not ineffective in another. It is not uncommon to find that a particular agent will be effective in one formulation but not effective in another formulation. While the vative for use in the practice of the invention can range from 0.005 to 1.0% (w/v), the red range for each preservative. alone or in combination with others. is: benzyl alcohol (OJ-1.0%). or m-cresol (OJ-0.6%). or phenol (OJ-0.8%) or combination of methyl (ODS-0.25%) and ethyl or propyl or butyl %-0.03%) parabens. The ns are lower alkyl esters of para-hydroxybenzoic acid. A detailed ption of each preservative is set forth in 'Remington's Pharmaceutical Sciences' as well as Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1. 1992. Avis et al. For these purposes. the Type-B natriuretic signal peptide fragment agent may be administered parenterally (including subcutaneous injections. intravenous. intramuscular. intradennal injection or infusion techniques) or by inhalation spray in dosage unit formulations ning conventional non-toxic phannaceutically acceptable carriers. adjuvants and vehicles.

If d. the eral formulation may be thickened with a thickening agent such as a methylcellulose. The ation may be prepared in an emulsified form. either water in oil or oil in water. Any of a wide variety of pharmaceutically aweptable emulsifying agents may be employed including. for example. acacia . a non-ionic surfactant or an ionic surfactant. It may also be desirable to add suitable dispersing or suspending agents to the pharmaceutical ation. These may include. for example. aqueous suspensions such as synthetic and natural gums. e.g., tragacanth. acacia. alginate. dextran. sodium carboxymethylcellulose. lcellulose. polyvinyl-pyrrolidone or gelatin.

It is possible that other ingredients may be present in a parenteral pharmaceutical formulation useful the invention. Such additional ingredients may include wetting agents. oils (9.9.. a ble oil such as sesame. peanut or olive). analgesic agents. emulsiﬁers. antioxidants. bulking agents. tonicity modiﬁers. metal ions. oleaginous vehicles. proteins (9.9.. human serum albumin. gelatin or proteins) and a zwitterion (9.9.. an amino acid such as e. taurine. arginine. e. lysine and histidine). Such additional ingredients. of course. should not adversely affect the overall stability of the pharmaceutical formulation of the present invention. Regarding pharmaceutical formulations. see also. Pharmaceutical Dosage Forms: Parenteral Medications. Vol. 1. 2nd ed., Avis ef al.. Eds. Marcel Dekker. New York, NY. 1992.

Suitable routes of parenteral administration include intramuscular. intravenous. subcutaneous. intraperitoneal. subdennal, intraderrnal. intraarticular. intrathecal and the like. Mucosal delivery is also permissible.

The dose and dosage regimen will depend upon the weight and health of the subject.

OTAPC ] In addition to the above means of achieving extended drug action. the rate and duration of Type-B natriuretic signal peptide nt agent delivery may be controlled by. for example by using mechanically controlled drug infusion pumps.

The Type-B retic signal peptide fragment agent(s) can be administered in the form of a depot injection that may be formulated in such a manner as to permit a sustained release of the Type-B natriuretic signal peptide fragment agent. The Type-B natriuretic signal e fragment agent can be ssed into pellets or small cylinders and implanted subcutaneously or intramuscularly. The pellets or cylinders may additionally be coated with a suitable biodegradable r chosen so as to provide a desired release proﬁle. The Type-B natriuretic signal peptide fragment agent may aitematively be micropeileted. The Type-B natriuretic signal peptide fragment agent micropellets using bioacceptable polymers can be designed to allow release rates to be manipulated to e a desired release . Alternatively. injectable depot tomis can be made by forming microencapsulated matrices of the Type-B natriuretic signal peptide fragment agent in biodegradable polymers such as polylactide-polyglyoolide.

Depending on the ratio of Type-B natriuretic signal peptide fragment agent to polymer. and the nature of the particular polymer employed. the rate of Type-B natriuretic signal peptide fragment agent release can be controlled.

Depot injectable formulations can also be prepared by ping the Type-B natriuretic signal e fragment agent in liposomes. examples of which include unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids. such as terol. stearyl amine or phosphatidylcholines. Depot injectable formulations can also be prepared by entrapping the Type-8 natriuretic signal peptide fragment agent in microemulsions that are compatible with body tissue. By way of e. reference is made to US. Patent Nos. 6410.041 and 6.362.190.

Implantable infusion devices may employ inert material such as biodegradable rs listed above or synthetic silicones. for example. cylastic. silicone rubber or other polymers manufactured by the Dow- Corning Corporation. The polymer may be loaded with Type-8 natriuretic signal peptide fragment agent and any excipients. Implantable infusion devices may also comprise a coating of. or a portion of, a medical device wherein the coating comprises the polymer loaded with Type-B natriuretic signal peptide fragment agent and any excipient.

Such an implantable on device may be prepared as disclosed in US. Patent No. 6.309.380 by coating the device with an in vivo biocompatible and biodegradable or bioabsorbable or bioerodibleerodible liquid or gel on containing a polymer with the solution comprising a d dosage amount of Type-B natriuretic signal peptide fragment agent and any excipients. The solution is converted to a ﬁlm adhering to the medical device thereby forming the implantable Type-B natriuretic signal e fragment agent-deliverable l device. An implantable infusion device may also be prepared by the in situ formation of a Type-B natriuretic signal peptide fragment agent ning solid matrix as disclosed in US. Patent No. 789. Implantable infusion devices may be passive or active. as known in the art.

OTAPC Also useful in methods of the invention are microemulsions. i.e.. such as ﬂuid and stable homogeneous solutions composed of a hydrophilic phase. a Iipophilic phase. at least one surfactant (SA) and at least one cosurfactant . Examples of le surfactants include mono.. di- and triglycerides and polyethylene glycol (PEG) mono- and diesters. A cosurfactant. also sometimes known as 'co-surface-active agentm.’ is a al compound having hydrophobic character. intended to cause the mutual solubilization of the aqueous and oily phases in a microemulsion. es of le co-surfactants include ethyl diglycol, lauric esters of propylene glycol. oleic esters of polyglycerol. and related compounds.

Type-B natriuretic signal peptide fragment agents may also be delivered using various polymers to enhance bioavailability by increasing on to mucosal surfaces. by sing the rate of degradation by hydrolysis or enzymatic degradation of the Type-B natriuretic signal peptide fragment agent, and by increasing the surface area of the Type-B retic signal e fragment agent relative to the size of the particle. Suitable polymers can be natural or synthetic. and can be biodegradable or non-biodegradable. Delivery of low molecular weight active . such as for e Type-B natriuretic signal peptide fragment agents . may occur by either diffusion or degredation of the polymeric system. Representative natural polymers include proteins such as zein. modiﬁed zein. casein. gelatin, gluten. serum n. and collagen. polysaccharides such as cellulose. dextrans, and polyhyaluronic acid. Synthetic polymers are generally preferred due to the better characterization of degradation and release proﬁles. Representative synthetic polymers include polyphosphazenes. poly(vinyl alcohols). polyamides. polycarbonates. polyacrylates. polyalkylenes. polyacryiamides. polyalkylene glycols. kylene oxides. polyalkylene terephthalates. polyvinyl ethers. nyl esters. polyvinyl halides. polyvinylpyrrolidone. polyglyoolides. polysiloxanes. ethanes and mers thereof. Examples of le rylates include poly(methyl rylate). thyl methacrylate). poly(butyl methacrylate). poly(isobutyl methacrylate). poly(hexyl methacrylate). poly(isodecyl methacrylate). poly(lauryl methacrylate). poly(phenyl rylate). poly(methyl acrylate). poly(isopropyl acrylate). poly(isobutyl acrylate) and poly(octadecyl acrylate). Synthetically modiﬁed natural polymers include cellulose derivatives such as alkyl celluloses. hydroxyalkyl celluloses. cellulose ethers. cellulose esters, and nitrocelluloses. Examples of suitable cellulose derivatives include methyl cellulose, ethyl cellulose. ypropyl ose. hydroxypropyl methyl cellulose. hydroxybutyl methyl cellulose. cellulose acetate. cellulose propionate. cellulose acetate butyrate. cellulose acetate phthalate. carboxymethyl cellulose. cellulose tate and cellulose sulfate sodium salt. Each of the polymers described above can be obtained from commercial sources such as Sigma Chemical Co.. St. Louis. Mo., Polyscienoes. Warrenton. Pa. Aldrich Chemical 00.. Milwaukee. Wis.. Fluka.

Ronkonkoma. N.Y.. and . Richmond. Calif. or can be synthesized from monomers obtained from these suppliers using standard techniques.

The polymers described above can be separately characterized as biodegradable. non- biodegradable. and bioadhesive polymers. Representative synthetic degradable polymers include polyhydroxy acids such as polylactides. polyglyoolides and copolymers thereof. poly(ethylene terephthalate). poly(butic acid). 0TAPC poly(valeric acid). poly(lactide-co-mprolactone). polyanhydrides. polyorthoesters and blends and mers thereof.

Representative natural biodegradable polymers include polysaccharides such as alginate, dextran, cellulose, collagen. and chemical derivatives thereof (substitutions. additions of chemiwl groups. for example. alkyl, alkylene. hydroxylations. oxidaticns, and other cations routinely made by those skilled in the art), and proteins such as albumin. zein and oopolymers and blends thereof. alone or in combination with synthetic polymers. Examples of non-biodegradable polymers include ne vinyl acetate. poly(meth)acrylic acid. polyamides. polyethylene. polypropylene. polystyrene. polyvinyl chloride. nylphenol, and copolymers and mixtures thereof. Hydrophilic polymers and hydrogels tend to have bioadhesive ties. Hydrophilic polymers that contain carboxylic groups (9.9.. poly[acrylic acidl) tend to exhibit the best bioadhesive properties. Polymers with the highest trations of carboxylic groups are preferred when bioadhesiveness on soft tissues is desired. Various cellulose derivatives, such as sodium alginate. carboxymethylcellulose, hydroxyrnethylcellulose and rnethylcellulose also have bioadhesive properties. Some of these bioadhesive materials are water-soluble. while others are hydrogels. Polymers such as hydroxypropylmethylcellulose acetate succinate (HPMCAS). cellulose acetate trimellitate (CAT). cellulose e phthalate (CAP). hydroxypropylcellulose acetate phthalate (HPCAP), hydroxypropylmethylcellulose acetate phthalate (HPMCAP). and methylcellulose acetate phthalate (MCAP) may be utilized to enhance the bioavailability of Type-B natriuretic signal peptide nt agents with which they are complexed. Rapidly bioerodible polymers such as poly(lactide-co-glycolide), polyanhydrides, and poiyorthoesters. whose carboxylic groups are exposed on the external e as their smooth surface erodes, can also be used for bioadhesive Type-B natriuretic signal peptide fragment agent ry systems. In addition. rs containing labile bonds. such as polyanhydrides and polyesters, are well known for their hydrolytic reactivity. Their hydrolytic degradation rates can generally be altered by simple changes in the r backbone. Upon degradation, these materials also expose carboxylic groups on their external surface. and can also be used as B retic signal e fragment agent delivery systems.

Other agents that may e ilability or absorption of one or more Type-B natriuretic signal peptide fragment agents can act by facilitating or inhibiting transport across the inal mucosa. For example, agents that increase blood flow. such as vasodilators. may increase the rate of absorption of orally administered Type-B natriuretic signal peptide fragment agent by increasing the blood ﬂow to the gastrointestinal tract. Vasodilators constitute another class of agents that may e the bioavailability of Type-B natriuretic signal peptide fragment .

Other mechanisms of enhancing bioavailability of the compositions and formulations useful in the invention include the inhibition of reverse active ort mechanisms. For example. it is now thought that one of the active transport mechanisms t in the intestinal epithelial cells is p-glycoprotein transport mechanism which facilitates the reverse transport of substances. which have diffused or have been orted inside the epithelial cell. back into the lumen of the intestine. Inhibition of this p-glycoprotein mediated active transport system will cause less drug to be transported back into the lumen and will thus increase the net drug transport across the gut epithelium and 0TAPC will increase the amount of dnig tely available in the blood. s p-glycoprotein tors are well known and appreciated in the art. These include. water soluble vitamin E; polyethylene glycol; poloxamers including Pluronic F-68; Polyethylene oxide; yethylene castor oil derivatives including Cremophor EL and Crernophor RH 40; Chrysin, (+)-Taxifolin; Naringenin; Diosmin; tin: and the like.

Thus. while the delivery period will be dependent upon both the condition and the agent and the therapeutic effect which is desired, continuous or slow-release delivery for about 0.5-1 hour, about 1-2 hours. about 2-4 hours. about 4-6 hours, about 6-8, or about 24 hours or longer is provided. In accordance with the present invention. this is achieved by inclusion of a Type-B natriuretic signal peptide nt agent, alone or toether with another cardiovascular therapeutic agent, in a formulation together with a pharmaceutically acceptable carrier or vehicle. particularly in the form of a formulation for continuous or slow-release administration.

As noted. the one or more agents of the invention may be administered before, during. immediately following a procedure in or on a subject. for e an angioplasty procedure or other physical intervention, such as stenting. They are preferably administered. for example. before and/or during a procedure or within about 24, about 12. about 10. about 9. about 8, about 7, about 6, about 5, about 4, about 3, about 2 hours or within about 60, about 45. about 30, about 15, about 10, about 5, about 4, about 3, about 2. about 1 minute following a procedure. for example.

The routes of administration and s described herein are intended only as a guide since a skilled physician will er the optimum route of administration and dosage for any particular patient and condition.

Any of the methods of ng a subject having or at risk fora cardiovascular disorder may utilize the administration of any of the doses, dosage forms. formulations. and/or compositions herein described. ceutical Compositions The present invention is directed to pharmaceutical compositions and their methods of use for preventing and/or treating a cardiovascular er wherein the composition comprises a therapeutically effective amount of a Type-8 natriuretic signal peptide fragment agent. alone or together with another cardiovascular therapeutic agent.

Accordingly, in one aspect, the ion provides compositions for use in ting and/or treating a cardiovascular disorder, which comprises or consists essentially of at least one Type—B natriuretic signal peptide fragment agent, alone or together with another cardiovascular therapeutic agent. In a red embodiment. the composition further comprises a pharmaceutically acceptable carrier or vehicle. in one preferred form, the composition contains one or more Type-B natriuretic signal peptide fragment e agents. Most preferably. the agent is BNPsp(17-26) (SEQ.ID.NO:1).

Kits, Medicamants and Articles of Manufacture 01-PC A Type-B retic signal peptide fragment agent may also be used in the manufacture of the ment for preventing and/or treating a cardiovascular er and related disorders and conditions.

In one aspect. the invention es a kit for preventing and/or treating a cardiovascular disorder comprising one or more compositions or formulations described. For example, the invention includes a kit comprising a composition comprising a therapeutically effective amount of a Type-B natriuretic signal peptide fragment agent. alone or in combination with one or more cardiovascular therapeutic . For example. the kit may include a composition sing an effective amount of a Type-B retic signal peptide fragment agent and or more of the following: nitrates. B-blockers, calcium channel blockers (particularly for stable or unstable angina. but also for heart failure in the case of B—blockers); diuretic agents, lator agents. positive inotropes, ACE inhibitors and aldoslerone antagonists. e.g. spironolactone (particularly for heart failure); blood thinning eutics (9.9.. aspirin. heparins. warfarins) and nitroglycerin (particularly for Mt). Kits may also include compositions comprising or consisting essentially of a Type-B natriuretic signal peptide fragment agent in alone or in ation with (9.9.. in physical combination. provided as a combined preparation) one or more anti-thrombolytic therapies (e.g.. streptokinase inhibitors, anti-platelet thereapetuics. such as. for example, clopidogrel). Kits may also include a Type- B natriuretic signal peptide fragment agent alone or in combination with (9.9.. in physical combination. provided as a combined preparation) a Type-B natriuretic peptide. including for example tide. and/or a inant form of TypeB natriuretic peptide.

Articles of cture are also provided comprising a vessel containing a composition or formulation of the invention (in any dose or dose form or device) as described herein and instructions for use for the treatment of a subject. For example, in another aspect. the invention includes an e of manufacture comprising a vessel containing a therapeutically effective amount of a Type-B natriuretic signal peptide nt agent. alone or in combination with one or more other cardiovascular therapeutic agents.

The compositions and formulations of the invention may be used for preventing and/or treating a wrdiovascular disorder and related disorders and conditions.

The inventions also include methods of treatment of a t having or at risk for developing a cardiovascular disease. disorder or condition. comprising administering to the subject a therapeutically effective amount of one or more of the compounds or ceutical compositions described herein. In one non-limiting embodiment. the cardiovascular disease, disorder or condition is associated with ischemia and/or oxidative stress. In one embodiment. the cardiovascular disease. disorder or condition is an acute coronary syndrome. e.g., ST-segment elevation myocardial infarction. non-ST-segment elevation dial tion or unstable angina. in another embodiment. the vascular disease. disorder or condition is heart failure. In other embodiments. the cardiovascular disease. disorder or condition is ischemic heart disease. In another embodiment. the cardiovascular disease. disorder or condition is stable angina.

OTAPC The inventions include methods of treating a subject having or at risk for developing a cardiovascular disease. disorder or condition, comprising a therapeutically effective amount of a Type-B natriuretic signal peptide fragment agent and a phannaceutically acceptable carrier. In one embodiment. the Type-B natriuretic signal peptide fragment agent in the pharmaceutical composition is BNPsp(17-26) (SEQ ID N0:1). In another embodiment. the Type-B natriuretic signal e fragment in the ceutical composition comprises or consists ially of a sequence selected from SEQ.ID.NOS:2 to 9. In another embodiment. the Type-B natriuretic signal peptide fragment agent in the pharmaceutical ition comprises or consists essentially of a sequence selected from Fomiula I. Formula II, Formula lll. Formula IV. Formula V. Formula Vl. Formula VII andlor Formula VIII. Type-B natriuretic signal e fragment agents also include active analogs, variants, truncations. and modified forms of the Type-B natriuretic signal e fragment agents described herein.

In another aspect. the inventions include methods of treating and/or preventing a cardiovascular disease. disorder or condition that is associated with ischemia and/or oxidative stress in a subject by increasing Type-B natriuretic signal e fragment activity in the subject. This may be accomplished. for example. by administering to the subject a ition comprising a therapeutically effective amount of a Type-B natriuretic signal peptide fragment agent. e.g.. a Type-B retic signal peptide fragment or a Type-B retic signal peptide fragment. including a BNPsp fragment comprising or ting essentially of a sequence selected from SEO.ID.NOS:1~9. or a peptide comprising or consisting essentially of a peptide according to any of Formulae l to Vlll, or an analog, variant. tion or modiﬁcation thereof. In certain embodiments. doses ibed above are utilized. In other embodiments. about 0.01 to about 100. 500 or 1000 milligrams or more (9.9.. at least about 100 milligrams. at least about 500 milligrams. or at least about 1000 ams) of a BNPsp fragment or Type-B natriuretic signal peptide fragrnenl analog, 9.9., a BNPsp fragment comprising or consisting essentially of a sequence selected from SEQ.ID.NOS:1-9. or a peptide comprising or consisting essentially of a peptide awarding to any of Formulae I to VIII. is administered per day in single or d doses or by continuous infusion. for example.

In another aspect. the inventions include methods of treating a patient suffering from acute coronary syndrome, comprising administering to the patient a eutically effective amount of a Type-B natriuretic signal peptide fragment agent. wherein the patient is not suffering from a Q-wave MI or STEMI. In a certain embodiment of this . the patient is suffering from unstable angina. In another embodiment of this method. the patient is ing from non-Q-wave cardiac necrosis. In still another embodiment of this method. the patient has a blood in I level of no more than 0.4 nglml. In yet another embodiment of this method. the patient has a blood troponin T level of no more titan 0.1 nglml. In yet another embodiment of this method. the t does not have elevated blood creatine kinase, In still another embodiment of this method. the patient does not have ST-segment elevation. in yet r embodiment of this method. the patient does not exhibit a pathological Gwave. In another embodiment of this . the patient exhibits one or more of the following ms: chest rain greater than 15 0TA—1101aPC minutes in duration. chest pain at rest. or chest pain following minimal exertion that is poorly responsive to sublingual nitrates.

In one embodiment. the Type-B natriuretic signal peptide fragment agent is administered in a single dose. In another embodiment, the Type-B natriuretic signal peptide fragment agent is administered in more than one dose. In yet r embodiment. the Type-B natriuretic signal peptide fragment agent is administered uously over a period of time. for example a predetermined period of time. In still another embodiment. glucose or a potassium salt. or a combination thereof. is co-administered with the Type-B natriuretic signal peptide fragment agent In another aspect. the inventions e methods for treatment of a t, comprising stering to the dual a therapeutically effective amount of a Type-B natriuretic signal peptide fragment agent. wherein the administration is after the onset of one or more of the following symptoms: chest pain lasting longer than 15 minutes, chest pain at rest. chest pain following minimal exertion, . shortness of breath. palpitations. or dizziness. In other embodiments. the patient has not ed a Q-wave MI or STEMI prior to the onset of the m or symptoms; t is suffering from unstable angina; the patient is ing from non-Q- wave cardiac necrosis; the patient has a blood troponin I level of no more than 0.4 ng/ml; the patient has a blood in T level of no more than 0.1 ng/ml ; the patient does not have elevated blood creatine kinase myocardial isoenzyme; the patient does not have ST-segrnent elevation; the patient does not exhibit a pathological Q-wave; the stration occurs between the time of onset of the one or more symptoms, and the time the patient suffers a Q- wave MI or STEMI. In another ment. the method further comprises the step of continuing the administration of a Type-B natriuretic signal peptide nt agent during the time that the patient suffers a Q-wave MI or STEMI.

In yet another embodiment. the method further comprises the step of continuing the administration of a Type-B natriuretic signal peptide fragment agent after the time the patient s a Q-wave Ml or STEMI. In other embodiments of this method. the t has ischemic heart disease. or is at risk for developing ischemic heart disease. In still another embodiment of the method. the patient has one or more of the following cardiac abnormalities: congestive heart failure, worsening heart murmur due to mitral regurgitation. or evidence of cardiac conduction disturbances. In other embodiments. the patient has a normal ECG. In another embodiment of this method. the patient has stable angina. In other embodiments of the method. the Type-B natriuretic signal e fragment agent is administered in a single dose. or is administered in more than one dose. or is administered continuously. In an additional embodiment of this method, glucose or a p0tassium salt. or a combination thereof, is inistered with the Type-B natriuretic signal peptide fragment agent.

The inventions also include methods for treating a patient suffering from stable angina. comprising administration of a Type-B natriuretic signal peptide fragment agent. In a funher embodiment, the administration is continuous over a period of time. including a predetermined period of time.

OTAPC The ions also provide a method for performing angioplasty on a patient in need thereof. comprising administering a Type-B natriuretic signal peptide nt agent to the patient during the angioplasty procedure. in a r embodiment. the method comprises or further ses stering a Type-B natriuretic signal peptide nt agent to the patient prior to the angioplasty procedure. In a further embodiment. the method comprises or further ses administering a Type-B natriuretic signal peptide fragment agent to the patient following the angioplasty procedure. In other embodiments. a Type-B natriuretic signal peptide fragment agent is administerd to the patient before. during, and/or after the lasty procedure. in any ation.

The inventions also include methods for treatment of a patient with ischemic heart disease, or is at risk for developing ischemic heart disease. including patients who exhibit one or more of the following symptoms: nausea, shortness of breath. palpitations. or dizziness. and further wherein the patient does not exhibit chest pain. comprising administering to the t a therapeutically effective amount of a Type-B retic signal peptide fragment agent. n the t is not suffering a Q-wave Ml or STEMl. In another embodiment of this method. the patient has a normal ECG.

Also provided are methods for increasing the time during which thrombolytic therapy will be effective following the first symptom of cardiac distress. comprising administering a therapeutically effective amount of a Type-B natriuretic signal peptide fragment agent after the onset of one or more of the following symptoms: chest pain lasting longer than 15 minutes. chest pain at rest. chest pain following minimal exertion. nausea, shortness of breath. palpitations, or dizziness. in another aspect. the treated subject is a , preferably a human. Other mtammals include domestic and farm animals. and 200. sports. or pet animals. such as dogs. horses, and cats. in one aspect the invention is directed to sustained administration of a Type-B retic signal peptide fragment agent and. optionally. antoher cardiovascular therapeutic agent. In one ment. the agent(s) are administered for at least about 0.5 hours. about 1- 24 hours. at least about 2. hours. at least about 3 hours. at least about 4 hours. at least about 5 hours. at least about 6 hours, at least about 7 hours. at least about 8 hours. at least about 9 hours. at least about 10 hours, at least about 11 hours. at least about 12 hours or at least about 24 hours.

Any of the methods of treating a subject having or ted of having or predisposed to a disease. disorder. and/or condition referenced or described herein may utilize the administration of any of the doses. dosage forms, formulations. compositions and/or devices herein described.

A better understanding of the invention will be gained by reference to the following non-limiting experimental section which is illustrative and is not intended to limit the ion or the claims in any way. The data support the use of the compounds and compositions described herein for treatment of cardiovascular diseases. disorders and conditions. as described.

EXAMPLES 01-PC Data show that BNPsp(17-26) is rapidly cleared from the circulation. However. it has been unexpectedly and surprisingly discovered that compounds. such as 17-26) for example. can act as a protective/therapeutic agent in, by way of example, mental cardiac ischemia and infarction.

Animal models may be used to test the efﬁcacy of the administration of compounds of the invention to an dual with a cardiovascular disorder. such as unstable angina. for example. a disorder within the ACS spectrum. whether or not they have yet suffered an actual infarction. Rat models and sheep models have been found to be particularly well suited for this purpose. In rats, BNPsp(17-26) administered during the last 3 minutes of a 40 min ischemia period and then throughout a 2-hour reperlusion period signiﬁcantly reduced infarct size (-30%). and the rats also had signiﬁcantly improved hemodynamics. ln sheep. administration of BNPsp(17-26) significantly d the stunning period. during usion after a period of subcritical ischemia. s showing cardioprotective properties of compounds of the invention, such as BNPsp(17~ 26) and other BNPsp fragments for example, are provided. The Examples include experiments showing cardioprotection in an in vitro isolated rat heart ischemia model, and in an in viva sheep model of myocardial infarction.

Exmpm1 Rat Heart lschemia Model Isolated rat heart. Male Sprague-Dawley rats weighing 250 g to 350 9 were anesthetized by sodium pentobarbitone (50 mg/kg i.p.) and sacrificed by decapitation. The ed. Langendorff perfused rat heart set up was prepared as previously described. Pemberton et at. Ghrelin induces vasoconstriction in the rat coronary ature without altering c peptide production. Am J. Physiol (Heart and Circ. Physiology) 2004 287: H1522- H1529; Piuhola et at. Direct Cardiac actions of erythropoietin (EPO): effects on c contractility. BNP ion and ischemia-reperlusion . Clinical Science 2008 114: 293-304.

Left ventricular end diastolic pressure (LVEDP). developed pressure (DP) and the maximal and minimal derivatives of the left ventricular pressure (+dP/dtm. and -dP/dt..... respectively) were measured with a liquid-tilled balloon in the left cle. Perfusion pressure was monitored with a side arm cannula above the aortic root. A constant flow rate of 12 mUmin was maintained with a peristaltic pump (Gilson ls. model MP-2). The animal ethics committee of the Christchurch School of Medicine. University of Otago approved the study protocol.

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US al Institutes of Health (NIH publication no. 85-23. revised 1996). ia-reperfusion protocol. The preparations for ischemia-reperlusion experiments were paced with a stimulator (Digitimer Ltd.. England) using a bipolar electrode placed on the right atrium (15 V. 1 ms. 300 bpm).

The temperature in the moisturized chamber where the heart was positioned was monitored to remain between 35- 37°C throughout the experiments. In this set of experiments. the cardioprotective effects of increasing doses of BNPsp(17-26) were ted (0.1 . 0.3. 1.0. 3.0 and 10.0 nMoles/L). These doses are equivalent to about 0.1 , 0.3.

OTAPC 1.0. 3.0, and 10-11 pg/L and administrable weight doses of about 400. 1000. 4000. 10.000 and 39.000 nglkg or about 0.4. 1.0, 4.0. 10 and 39 micrograms / kg. Doses were compared under two ent strategies: (1) a preconditioning effect prior to 45 minutes of global ia (“PRE'). and (2) a direct. 'real time" effect given at the initiation of 120 minutes reperfusion ('lDR'). The ents were given. respectively. for 30 minutes either prior to ischemia or starting at the time of reperfusion. During the repertusion, 35 minutes after reinitiating the coronary flow, the LVEDP was temporarily set to 5 mmHg by adjusting intraventricutar balloon volume to obtain contractile parameters with comparable end-diastolic pressure.

Measurement of perfusate cTnl and bin. Cardiac in I ('cTnl') levels in isolated heart pertusate were measured on a hospital laboratory high throughput er (Abbott Architect. Canterbury Health tories. Christchurch Hospital. New Zealand). using a late generation cTnl assay. Myoglobin was measured using a Chemilumineswnt Microparticle Immunoassay (Canterbury Health Labs. Christchurch. New Zealand) on an Abbot ect i2000 analyser.

Tissue is also analysed for markers of apoptosis. namely TUNEL staining and caspase 3 determination. Trypan blue exclusion (0.4% trypan in PBS) is performed to provide an estimate of necrotic cells.

Three regions with in the infarct tenitory are analyzed. The number is expressed as a tage of necrotic cells out of 250 cells.

TUNEL staining. DNA fragmentation [terminal deoxynucleotidyltransferase-mediated UTP end- ng (TUNEL) assay] was detected from formalin ﬁxed sections of LV free wall using a kit from Chemioon lntemational according to the manufacturer's protocol. as usly reported. Piuhola et al., Direct Cardiac actions of erythropoietin (EPO): effects on cardiac oontraclility. BNP secretion and ischemia-reperfusion injury. Clinical Science 2008 114: 293-304. From each heart a cross n at mid ventricle level was used for ng and all the TUNEL positive cells were counted. Sections were counterstained with DAPI to determine the total number of cells.

Immunohistochemical detection of cleaved caspase-3. Caspase3 is one of the terminal effectors of the apoptotic e. It exists in cells as an inactive 32 kDa protein. and in apoptotic cells it is cleaved to 20/17 kDa active form. An immunohistochemical que for detection of cleaved caspase-3 was used. Briefly. formalin- tixed sections were deparatiinized, rehydrated and incubated in 1% H20; for 30 min to quench endogenous peroxidase. Following antigen retrieval with heat. the sections were incubated overnight at 4°C with a onal rabbit antibody recognizing the cleaved form of human caspase-3 (Cell Signaling Technology. Beverly. MA). Primary antibody binding was detected with peroxidase labelled polymer conjugated to goat abbit immunoglobulins (DAKO Corporation, Carpinteria. CA) and diaminobenzldine solution (DAKO) used as the substrate. The tissues were y counterstained with haematoxylin. PBS replaced the primary antibody as ve control for these experiments. The mean number of caspase-3 positive cells per 7 randomly selected 40x objective ﬁelds was counted in each sample.

OTAPC Isolation of mitochondrial and cytosolic proteins. Cardiac LV free walls were homogenized in a buffer containing 250 mM sucrose. 10 mM Tris. 1 mM EDTA. protease inhibitors and phosphatase inhibitors. The lysate was fuged for 5 min at 1000 g to pellet the unbroken cells and the nuclei. The supernatant was further centrifuged 20 min at 13,000 g to pellet the mitochondria. The pellet was resuspended in homogenization buffer and r washed twice with the same buffer. Finally. the mitochondrial pellet was resuspended in solubilization buffer consisting of 150 mM NaCI. 20 mM Tris, 10 mM EDTA. 1% NP-40. se inhibitors and phosphatase inhibitors.

After 30-minute incubation on ice the lysate was centrifuged 10 min at 9 to pellet the unsoluble material. The supernatant was further centrifuged 60 min at 100,000 g to separate the cytosolic fraction (the supernatant).

Assessth ofBNPsp(f 7-26) activity in ed perfused rat heart. Mass spectrometry was used to document oxidative stress reaction product addition to unmodiﬁed BNPsp(17-26) in isolated rat heart perfusate samples. Two samples were analysed: the ﬁrst was 10nmoVL unmodiﬁed 17-26) in isolated heart perfusate that had not passed through an ischemic heart: the second was 10nmoVL BNPsp(17-26) that had passed through a rat heart that had undergone no ﬂow ischemia for 45 minutes. BNPsp(17-26) was added at the time of reperfusion and sample was collected for 3 minutes after flow initiation. ate sample were extracted on solid phase cartridges rton et al.. n induces vasoconstriction in the rat coronary vasculature without altering cardiac e tion. Am J. Physiol (Heart and Circ. Physiology) 2004 287: H1522-H1529) and further puriﬁed by xclusion high performance liquid chromatography (SE-HPLC) using a isocratic gradient of 60% itrilelO.1% triflouroacetic acid (TFA). reactive BNPsp(17~26) was quantitated by immunoassay (Piuhola et al.. Direct Cardiac actions of erythropoietin (EPO): s on cardiac contractility, BNP ion and ischemia-reperfusion injury. Clinical Science 2008 114: 293-304) and then structurally assessed by matrix assisted laser desorptionironization time of ﬂight mass spectroscopy (MALDl-TOF MS). All MS spectra were acquired in positive-ion mode with 800-1000 laser pulses per sample spot. A maximum of six precursor ions of each sample spot were selected for MS/MS collision-induced fragmentation (CID) analysis. Structural modifications to BNPsp(17-26) were analysed by LC-MS3 LTQ-Orbitrale. mass spectrometry (Thermo Scientiﬁc. San Jose. CA). g peptides were monitored by a full mass scan using the linear ion trap in a mass range from mlz 400-1400. The predicted mlz value of the doubly charged peptide was ed as the exclusive precursor mass triggering subsequent scan events.

Statistical analysis. Results are presented as mean :l: standard error of the mean (SEM). Multiple group comparisons were made by one-way or repeated-measures ANOVA as appropriate followed by the post hoc test for least significant differences. For the comparison between two groups. Student's t test was used.

Significance was assumed at P<0.05. All the Statistical analyses were performed Mth SPSS on 17).

Results ] Isolated rat heart preparations infusion of BNPsp(17-26) either for 30 minutes prior to (pre). or for minutes immediately after (IDR), 45 minutes of ischemia resulted in significant improvements in cardiac OTAPC contractility (developed pressure, Figure 1. Panel A) and in vascular tone (perfusion pressure, Figure 1. Panel B). compared with control infusion that ed vehicle buffer alone. Thus. control developed pressures ed to only ~75% of pre-ischemic values, whereas pre-ischemia infusion with 0.3nrnol/L or post-ischemia infusion of 1nmol/L BNPsp(17-26) returned ped pressures to between 110~120% pre-ischemia values (P<0.01). An element of dose response was observed and there was a trend for pre-infusion of 03an BNPsp(17-26) to have positive inotropic effect prior to ischemia. Likewise, vascular tone during the post~ischemic reperfusion phase was well preserved with pre-ischemia infusion of l/L BNPsp(17-26) (P<0.01) and with post-ischemia use of 0.3 and 1nmollL BNPsp(17-26) (P<0.05. Figure 1. Panel B).

In agreement with the haemodynamic data. cardiac bicmarker analysis revealed marked and signiﬁcant reductions in both Tnl and myoglobin release during the reperfusion phase after ischemia. when BNPsp(17-26) was given either pre- or post-ischemia. Exemplar results. from post-ischemia reperfusion (lDR). are shown in Figure 1. panels C and 0. Thus. both 0.3 and 1nmollL BNPsp(17-26) resulted in ~20% the Tnl release of control infusion (Panel C. P<0.01) and ~60°lo the l myoglobin release (Panel 0, P<0.05). Given that Tnl and myoglobin release have both been correlated with size of cardiac infarct and subsequent prognosis (mortality. adverse events). these substantial BNPsp(17-26) inspired reductions have meaningful clinical utﬂity.

Further analysis using reduced sequence variants of BNPsp reveals therapeutic and cardioprotective effects.

Taken together, these results support a favourable clinical utility for BNPsp signal peptide fragment agents in the areas of cardiotherapy and cardioprotection (before and after ischemic episodes of any cause).

These data support the concept that human BNPsp( 17-26), and shorter yl terminal ted versions . as well as N-terminal addition peptides variants thereof. are powerful. clinically useful cardiotherapeutic and cardioprotective agents. Accordingly. the clinical potential for use of these peptide sequences is strong in acute cardiac coronary syndromes and other diseases. disorders and conditions noted herein. Other ian and lower rate forms of BNPsp ces, variants. derivatives. and analogs will also s such therapeutic and protective properties.

EXAMPLE 2 Sheep Model ] Data show that BNPsp(17-26) is rapidly d from the circulation. However. it has been unexpectedly and surprisingly ered that unmodified 17-26) can act as novel protective/therapeutic agent in experimental cardiac ischemia and infarction. as indicated herein. This e demonstrates that the compounds are safe. on of BNPsp(17-26) into two , healthy sheep (achieving circulating levels found to be favourably bioactive in ed rat hearts) resulted in no detectable changes to haernodynamics. renal function or OTAPC circulating biornarkers (cardiac output is an exemplar shown in Figure 2). This is a favourable proﬁle in normal EXAMPLE 3 Rat Heart lschemia Model Ex vivo Isolated perfused rat heart model of cardiac ischemic injury heart. In this Example. more than 100 male Sprague-Dawley rats were used, all male. The heart was removed the heart under global anaesthesia and placed in an it in our experimental rig setup as described in Example 1. The hearts were is perfused with a standard. well used buffer system containing glucose to provide energy and calcium to ensure the intrinsic beating activity of the heart is ved. After equilibration, we perform two types of experiments were performed. First. we infuse the hearts were infused with either vehicle control (buffer itself) or human BNPsp(17-26) for 30 minutes prior to 40 minutes of global ischemia (refened to : this is known as preconditioning). Second. we infuse the hearts were infused with vehicle or human BNPsp(t7-26) after ischemia (referred to : this is known as repertusion treatment). which and more closely mimics the real clinical situation (ie.given that doctors can only invoke Tx AFTERtherapy after a heart attack has occurred). End points of interest are improvements in cardiac contractility after ischemia, reduction in cardiac troponin release. improvements in post-ischemia coronary blood pressure. reduction in infarct size. At the end of the experiment. left ventricular free wall s were biopsied for subsequent ination of markers of apoptosis (TUNEL staining. caspase 3) and Western Blot of ERKl/2. Pl3K. Akt and .4 TUNEL staining was done on samples of the left ventricular free wall that were fixed in 10% fomaldehyde overnight and then stored in parafﬁn. Prior to staining the sections were rehydrated with saline buffer and endogenous dase activity blocked by incubation with 0.3% H202. TUNEL staining was perfomied as per the manufacturer’s protocol (Chemicon International). The mean number of TUNEL positive cells were counted and ed as a ratio of the entire cell count per ten randomly selected 400x obiective ﬁelds in each sample.

] Caspase 3 staining was med on separate slides prepared as for TUNEL staining. Prior to staining slides were rehydrated and incubated with 1% (v/v)H202. The hearts were incubated for hours at 4°C with a polyclonal rabbit dy directed towards the activated form of Caspase 3 (Cell Signalling Technology). Primary antibody binding was detected with perxidase labelled polymer conjugated with goat anti rabbit lgG (DAKO). The slides were then lightly rstained with hematoxylin. The slides were photographed at X400 magniﬁcation. isolated rat heart data Human BNPsp(17-26) reduced the damage caused to heart tissue by a period of ischemia. When hearts receiving vehicle o global ischemia for 40 minutes they red to about 70% of their pre-ischemia ctile function (developed pressure). This is true for tire-conditioned and repertusion ent hearts. In contrast, hearts preconditioned or treated at reperfusion with BNPsp(17-26) recover to slightly over 100% of their pre-ischemia contractile function (Figure 3). Thus, when considering l versus 0.3nmollL BNPsp(17-26). there was a significant se in contractility during infusion with 0.3nmollL concentration % versus control, OTAPC P=0.003). More importantly, during the usion phase after ischemia. there were statistically signiﬁcant improvements in developed pressure in the BNPsp(17-26) treated hearts (0.1-0.3nmol/L +21% versus control.

P=0.007). Analysis showed signiﬁcant differences between control and 0.1 and 03an with both these trations achieving improved contractility across all time points analyzed.

] Concomitant with the improvements in developed re, 17-26) induces improvements in coronary vascular tone, such that there is reduced post-ischemia ry nstriction (Figure 5). During the reperfusion stage, there were signiﬁcant changes in perfusion pressures between groups. Compared with control values, repeated measures ANOVA with post-hoc analysis identified signiﬁcant reductions (-25 to -50%. P=0.008) in reperfusion vascular pressures at the doses of 3nmol/L BNPsp(17-26). These s began immediately post reperfusion and continued until the and of the sampling .

] Following this. reperfusion-only were conducted ments in isolated rat hearts. In rat hearts receiving 0.3-1.0 nmoI/L BNPsp(17-26) at reperfusion. cardiac contractile function was cantly improved compared with control values (Figure 6). During the reperfusion stage improved contractility was observed in the study hearts administered 0.3 and 1 nmol/L BNPsp(17-26) (+7% and +26% versus control, P=0.003, respectively).

Corresponding to this, reperfusion perfusion pressures and c troponin release were also improved in hearts receiving BNPsp(17-26) (Figure 5). During reperfusion. hearts administered 0.3 and 1nmol/L BNPsp(17-26) had lower mean perfusion pressures (-10%, P<0.05) compared with control.

In accordance with this positive ynamlc proﬁle. the release of troponin I from the ischemic myocardium was signiﬁcantly d in hearts receiving BNPsp(17-26). Thus. compared with control, there was a 50% reduction in tive Troponin I release in hearts administered 03an (P<0.05) and a 66% ion (P<0.01) in hearts receiving inmoIlL 17-26).

We also investigated the effect of BNPsp(17-26) upon markers of cellular apoptosis and necrosis.

Figure 8 displays the cellular preservation effects of BNPsp(17-26) as determined by HE staining. as indicated by improved ity and less disruption in in form in BNPsp(17-26) treated hearts.

Staining for caspase-3 activity is shown in Figure 9. There was a signiﬁcant reduction in caspase-3 positive cells (indicated by brown colouration) in hearts treated with BNPsp(17-26).

Staining with TUNEL ed less brown coloured nuclei in BNPsp(17-26) treated hearts, which indicates a greater degree of DNA integrity and less cellular fragmentation. See Figure 10.

EXAMPLE 4 In vivo myocardial infarction sheep models (healthy and) Infusion of human BNPsp(17-26) during in vivo cardiac ischemia in sheep will also result in beneﬁcial effects upon cardiac contractile function, signiﬁcant reductions in release of biomarkers (troponin I, myoglobin) of necrosis and signiﬁcant reductions in ventricular wall stress abnormalities that accompany remodelling after ischemia. 0TAPC ] In this set of experiments, four normal healthy sheep were infused with human BNPsp(17-26) at 1001000uglkgmin-1 to document any effects upon normal blood pressure. heart rate or renal on. This ment was carried out to ine achieved circulating levels of BNPsp(17-26) in response to the dose given and to document any signiﬁcant effects upon haemodynamic, renal or hormonal indices.

Experimental myocardial infarction was performed on 8 sham operated and 7 experimental sheep.

Each sheep was surgically ed under anaesthesia with jugular and carotid access catheters, ECG electrodes and 3 Swan Ganz catheter to measure cardiac output. All 15 sheep undenrvent 90 min ischaemia of the 2nd diagonal of the LAD coronary artery by means of a releasable snare. 30 min prior to the start of ia. each sheep received (depending on their group) either saline or 500nglkglmin BNPsp(17-26) for 120 min. Thus. this study was a preconditioning and during design. Serial ynamic recordings and venous blood ng were taken preanaesthetic and then at -10. occlusion (O). 0+30. 0+60. 0+90. and then at 120. 150. 240. and 360 min and 5, 24 and 48 hours. Serial echocardiography (basal. mid and apical regions in the short-axis plane) was performed pre. during and 30 min post occlusion.

Infusion of human BNPsp(17-26) at 100-1000ug/kgmin in 4 normal sheep had no icant effects upon haemodynamic. renal or hormonal indices (Figure 11). The clearance of BNPsp(17-26) from the circulation in sheep was very fast. being in the order of minutes. This ts a plasma half-life of less than 1minute. or a very rapid proteolytic cleavage to a non-immunoreactive form.

Following this positive safely/tolerance proﬁle. we then stered 500nglkg.min tic human BNPsp(17-26) to 7 sheep undergoing cardiac ischemia induced by coronary ligation. lmportantly. when compared with l saline infusions. BNPsp(17-26) signiﬁcantly reduced cumulative cardiac troponin I (P<0.01) release post-ischemia (Figure 12).

EXAMPLE 5 Analysis of BNPsp(17-26) metabolites fanned in viva during ischemia In this Example. the degradation of human BNPsp(17-26) into metabolites was assessed. Two methods were used. in a ﬁrst mental. an ex vivo set up was used wherein 1nM 17-26) was infused,at the time of reperlusion after 40min ischemia into an isolated rat heart. The system was set to recirculate the BNPsp(17-26) containing buffer so the peptide was exposed to ischemic tissue for more than one pass through the heart A 10ml sample of perfusate was collected after 20 minutes of recirculation, extracted on a Sep Pak C18 cartridge and puriﬁed by immunoafﬁnity purification and reverse phase HPLC. This d material was then subjected to tandem MSIMS for precise identiﬁcation. The second experimental was in vivo. wherein 3ml of peripheral plasma from sheep receiving 500ng/kg.min BNPsp(17-26) during cardiac ischemia was puriﬁed as for the ex vivo isolated rat heart perfusate and analysed on tandem MSIMS.

These experiments assessed the degradation during ischemia of human BNPsp(17-26) to metabolites when passed h an isolated rat heart preparation or whole animal (sheep). In both setups.

OTAPC synthetic human BNPsp(17-26) was degraded to a smaller form. namely BNPsp(i8-26). resulting from proteolytic cleavage of the amino terminal leucine. This is shown in Figure 13. A single sharp peak resolved on RP-HPLC and was conﬁrmed as human 18-26) by tandem MS/MS. This indicates that the amino terminal end is most susceptible to initial degradation.

Effect of modifying the amino acid sequence of BNPsp(17-26) ] This set of experiments assessed modiﬁed BNPsp(17-26) peptides. This experiment repeated the preconditioning work outlined in the isolated rat heart model Examples. but with C-terminal ablated and an N-terminal extended version of BNPsp(17-26). Hearts were ditioned with 30 minute doses of 03an BNPsp(16—26) and BNPsp(17-24) prior to 40 minutes of global ischemia and 90 minutes reperfusion. c contractile and perfusion pressure indices were recorded.

] Modiﬁcation of the BNPsp(17-26) sequence in these initial ments. either through N-terminal addition or C-terminal ablation, and the effects upon responses observed in isolated hearts are shown in Figure 14. lmpcrtantly, the addition of phenylalanine (F) at position 16 to the N-terminus (thus creating BNPsp(16-26)) gave the same haemodynamic protective proﬁle as BNPsp(17-26). A modiﬁed peptide iM'th two C-terminal amino acids removed (i.e.. 17-24) also had a protective effect.

All patents, publications. scientiﬁc articles. web sites. and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains. and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its ty. Applicants reserve the right to physically incorporate into this speciﬁcation any and all materials and information from any such s, ations. scientiﬁc articles, web sites, electronically available information. and other referenced materials or documents.

The speciﬁc methods and compositions described herein are representative of preferred ments and are exemplary and not intended as limitations on the scope of the ion. Other objects. aspects. and embodiments will occur to those skilled in the art upon consideration of this cation. and are encompassed within the spirit of the invention as deﬁned by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modiﬁcations may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or ts. or limitation or limitations. which is not cally disclosed herein as essential. Thus, for example. in each instance herein. in embodiments or examples of the present ion. any of the terms ising". “consisting essentially of'. and ‘ccnsisting of" may be replaced with either of 0TA-1101PC the other two terms in the speciﬁcation. Also. the terms ising', 'including'. containing'. etc. are to be read expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the . it is also that as used herein and in the appended claims. the singular forms 'a,’ 'an.’ and 'the’ include plural nce unless the context clearly es ise. Under no circumstances may the patent be reted to be "de to the speciﬁc examples or embodiments or methods speciﬁcally disclosed herein. Under or any other no circumstances may the patent be interpreted to be limited by any statement made by any Examiner official or employee of the Patent and Trademark Ofﬁce unless such statement is speciﬁcally and t ication or ation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms of description and not of limitation. and there is no intent in the use of such terms and expressions to exclude any equivalent of the es shown and described or portions thereof. but it is recognized that various modiﬁcations are possible within the scope of the invention as claimed. Thus, it will be understood that gh the present invention has been speciﬁcally disclosed by preferred embodiments and optional features. modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modiﬁcations and variations are considered to be within the scope of this invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any t matter from the genus. regardless of whether or not the excised material is speciﬁcally recited herein.

Other embodiments are within the follovn'ng claims. In addition. where features or aspects of the invention are described in terms of Markush groups. those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or up of members of the Markush group.

We

Claims

claim:

1. Use of a pharmaceutical ition comprising a therapeutically effective amount of a Type-B natriuretic signal peptide (BNPsp) nt agent and a pharmaceutically acceptable carrier in the manufacture of a medicament for preventing and/or treating a cardiovascular disorder in a subject, wherein the Type-B natriuretic signal peptide fragment is an amino acid sequence as defined in any one of SEQ ID NOs: 1-9 or Formulae I-VIII; wherein the peptide of Formula I is defined by: LH X1 X2 X3 X4 X5 X6 X7 X8 (Formula I) wherein X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; X2 is Val, Leu or Ile or Gly; X3 is Leu, Val, Ile, Ala, Tyr or Gly ; X4 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; X5 is Pro, Ala, Arg or Ser; X6 is Pro, Ala, Arg or Ser; X7 is Gln, Asn or Gly; and X8 is Ser, Thr or Gly; and the peptide of Formula II is defined by: L H X1 X2 X3 X4 X5 X6 X7 (Formula II) n X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; X2 is Val, Leu, Ile or Gly; X3 is Leu, Val, Ile, Ala, Tyr or Gly; X4 is, Norleucine, Ile, Val, Met, Ala, Phe or Gly; X5 is Pro, Ala, Arg or Ser; X6 is Pro, Ala, Arg or Ser; and X7 is Arg, Gln, Asn or Gly; provided that where X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly, X6 can also be Gly, and X7 can also be Arg; where X2 is Val, Leu or Ile or Gly, X1 can also be Leu, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly, X6 can also be Gly, and X7 can also be Arg; where X3 is Leu, Val, Ile, Ala, Tyr or Gly, X1 can also be Leu, X2 can also be Ala, X4 can also be Leu, X5 can also be Gly, X6 can also be Gly, and X7 can also be Arg; where X4 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X5 can also be Gly, X6 can also be Gly, and X7 can also be Arg; where X5 is Pro, Ala, Arg or Ser, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu, X6 can also be Gly, and X7 can also be Arg; where X6 is Pro, Ala, Arg or Ser, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly, and X7 can also be Arg; where X7 is Arg, Gln, Asn or Gly, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly, and X6 can also be Gly; and the e of Formula III is defined by: L H X1 X2 X3 X4 X5 X6 (Formula III) wherein X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; X2 is Val, Leu, Ile or Gly; X3 is Leu, Val, Ile, Ala, Tyr or Gly; X4 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; X5 is Pro, Ala, Arg or Ser; and X6 is Pro, Ala, Arg or Ser; provided that where X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly, X6 can also be Gly; where X2 is Val, Leu or Ile or Gly, X1 can also be Leu, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly, X6 can also be Gly; where X3 is Leu, Val, Ile, Ala, Tyr or Gly, X1 can also be Leu, X2 can also be Ala, X4 can also be Leu, X5 can also be Gly, X6 can also be Gly; where X4 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X5 can also be Gly, X6 can also be Gly; where X5 is Pro, Ala, Arg or Ser, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu, X6 can also be Gly; where X6 is Pro, Ala, Arg or Ser, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly; and the peptide of Formula IV is defined by: L H X1 X2 X3 X4X5 la IV) wherein X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; X2 is Val, Leu, Ile or Gly; X3 is Leu, Val, Ile, Ala, Tyr or Gly; X4 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; and X5 is Pro, Ala, Arg or Ser; provided that where X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly; where X2 is Val, Leu or Ile or Gly, X1 can also be Leu, X3 can also be Phe, X4 can also be Leu, X5 can also be Gly; where X3 is Leu, Val, Ile, Ala, Tyr or Gly, X1 can also be Leu, X2 can also be Ala, X4 can also be Leu, X5 can also be Gly; where X4 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X5 can also be Gly; where X5 is Pro, Ala, Arg or Ser, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu; and the peptide of Formula V is defined by: L H X1 X2 X3 X4 (Formula V) wherein X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; X2 is Val, Leu, Ile or Gly; X3 is Leu, Val, Ile, Ala, Tyr or Gly; and X4 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; provided that where X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X2 can also be Ala, X3 can also be Phe, X4 can also be Leu; where X2 is Val, Leu or Ile or Gly, X1 can also be Leu, X3 can also be Phe, X4 can also be Leu; where X3 is Leu, Val, Ile, Ala, Tyr or Gly, X1 can also be Leu, X2 can also be Ala, X4 can also be Leu; where X4 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X1 can also be Leu, X2 can also be Ala, X3 can also be Phe; and the peptide of Formula VI is d by: L H X1 X2 X3 (Formula VI) wherein X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; X2 is Val, Leu, Ile or Gly; and X3 is Leu, Val, Ile, Ala, Tyr or Gly; provided that where X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X2 can also be Ala, and X3 can also be Phe; where X2 is Val, Leu or Ile or Gly, X1 can also be Leu, and X3 can also be Phe; and where X3 is Leu, Val, Ile, Ala, Tyr or Gly, X1 can also be Leu, and X2 can also be Ala; and the peptide of Formula VII is defined by: L H X1 X2 n X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly; and X2 is Val, Leu, Ile or Gly; provided that where X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly, X2 can also be Ala,; and where X2 is Val, Leu or Ile or Gly, X1 can also be Leu; and the peptide of Formula VIII is defined by: L H X1 wherein X1 is Norleucine, Ile, Val, Met, Ala, Phe or Gly.

2. Use according to claim 1, wherein said Type-B natriuretic signal peptide fragment agent is BNPsp defined by residues 17-26 (SEQ ID NO:1).

3. Use according to claim 1 or claim 2, wherein said Type-B natriuretic signal peptide fragment agent comprises a ce selected from SEQ.ID.NOS:2 to 9.

4. Use according to any one of claims 1 to 3, wherein said composition is suitable for parenteral administration.

5. Use ing to any one of claims 1 to 4, wherein said parenteral administration is infusion.

6. Use according to any one of claims 1 to 5, wherein said composition is suitable for slow, delayed or controlled release administration.

7. Use according to any one of claims 1 to 6, wherein said cardiovascular disorder is an acute coronary syndrome.

8. Use according to claim 7, wherein the acute coronary syndrome is selected from the group consisting of ST-segment elevation myocardial infarction, non–ST-segment elevation myocardial infarction and le angina.

9. Use according to any one of claims 1 to 7, wherein said cardiovascular disorder is a heart failure or an ischemic heart disease.

10. Use according to any one of claims 1 to 9, wherein the ment comprises about 0.01 to about 100 milligrams of said Type-B natriuretic signal peptide nt.

11. Use according to any one of claims 1 to 10, wherein the subject is a mammal.

12. Use according to claim 11, n the mammal is a human.

13. A use according to any one of claims 1 to 12, substantially as hereinbefore described and with or without reference to any one or more of the Examples and/or