WO2007003594A1 - Screening methods for inhibitors of the metalloprotease meprin - Google Patents

Abstract

Compounds that regulate the zinc metalloprotease meprin can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including cardiovascular and renal disorders. The invention provides assays for the identification of such compounds.

Description

Solvay Pharmaceuticals GmbH 30173 Hannover

SCREENING METHODS FOR INHIBITORS OF THE METALLPROTEASE MEPRIN

Description

FIELD OF INVENTION

The present invention relates to the use of compounds inhibiting or modulating the activity of the meprin protein for the treatment and/or prevention of cardiovascular disorders. In addition, methods for identifying inhibitors, ligands or modulators of the BNP degrading activity of meprin are disclosed in the present invention.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference herein and are not admitted to be prior art.

Meprin A and Meprin B

Meprin A (EC number 3.4.24.18), also termed Endopeptidase-2, and Meprin B (EC number 3.4.24.63) are highly regulated, secreted, and cell-surface zinc metalloendopeptidases that are abundantly expressed in the kidney and intestine. They are members of the "astacin family" and "metzincin superfamily". Meprin A and B form homo- and hetero-oligomeric complexes consisting of evolutionarily related alpha and/or beta subunits that are approximately 50% identical at the amino acid level. The subunits derived from a common multidomain ancestor protein but have evolved to have markedly different substrate and peptide bond specificities, structural properties, chromosomal locations, and membrane associations.

The alpha and beta subunits are encoded on two genes: the alpha gene is on human chromosome 6 (mouse 17) near the histocompatibility complex; the beta subunit on chromosome 18 in both the mouse and human genomes. While both meprin subunits are expressed in embryonic kidney proximal tubule cells and intestinal epithelial cells, the subunits are expressed differentially postnatally, and one or the other subunit appears to be up-regulated in cancer cells. For example, meprin alpha is expressed and secreted in colon cancer cells, while meprin beta is expressed in breast cancer cells. The different enzymatic properties, expression profiles, and localization of the subunits indicate different functions for meprin alpha and beta. The meprin subunits can be expressed separately, or coordinately, and consequently they form homo- and hetero-oligomeric where the basic unit is a disulfide-linked dimer. The meprin alpha subunit has a propensity to form large homo-oligomeric complexes containing 12-100 sub- units, while meprin beta alone forms only homodimers. Both subunits are synthesized as membrane-spanning type 1 proteins in the endoplasmic reticulum with small cytoplasmic tails, whereas the bulk of the protein is extracellular. However, while the meprin beta subunit remains membrane- bound through the secretory pathway and at the plasma membrane, the meprin alpha subunit is proteolytically processed during biosynthesis and thus loses its transmembrane domain. Accord- ingly, meprin alpha exists at the membrane only when it associates with meprin beta, which exists as a type I protein at the plasma membrane. Membrane bound forms of meprins are dimeric (homodimeric beta = Meprin B) or tetrameric (hetero-oligomeric 2* alpha + 2* beta or 3* alpha + 1 * beta complexes = Meprin A). The secreted form of meprin alpha (also called Meprin A) containing 10-100 subunits consists of noncovalently associated dimers forms the largest proteases known to exist [reviewed in Bond et al, 2005].

Both meprin subunits cleave a variety of peptides and proteins and have a preference for peptides larger than six amino acids, indicating extended substrate binding sites. They are capable of cleaving a wide range of substrates, from bioactive peptides, such as gastrin, bradykinin, angio- tensin, cholecystokinin and parathyroid hormone, cytokines such as osteopontin and monocyte chemotactic peptide-1 , as well as extracellular matrix proteins, such as gelatin, collagen IV, fi- bronectin and casein. The peptide bond specificities of Meprins A and B differ substantially, indicating different functions; however, they can both hydrolyze extracellular matrix proteins. The best peptide substrates identified for Meprin A were gastrin-releasing peptide, cholecystokinin, gluca- gon, substance P, and valosin. The first three of the latter peptides are also substrates for Meprin B; the last two are not. Gastrin is by far the best substrate identified for Meprin B, and it is not cleaved by mouse Meprin A. Osteopontin is a further substrate for Meprin B. In contrast, Meprin A, but not B, cleaves cytokines such as monocyte chemoattractant protein- 1 , and this could be important in immune responses. In general, the meprin beta subunit shows a preference for hydrolysis of peptide bonds containing acidic amino acid residues, whereas meprin alpha prefers to cleave bonds flanked by small or hydrophobic residues [Bertenshaw et al, 2001].

It is known that the cleaving activity of human Meprin is inhibited by hydroxamic acid derivatives such as batimastat, galardin and Pro-Leu-Gly-hydroxamate, by TAPI-O (tumour necrosis factor alpha protease inhibitor-O) and TAPI-2, and by thiol-based compounds such as captopril [Kruse et al, 2004]. For determination of meprin activity, Kruse et al used N-benzoyl-L-tyrosyl-p- aminobenzoic acid as substrate. The most effective inhibitor of human meprin alpha described was the naturally occurring hydroxamate actinonin

Actinonin as inhibitor of Meprin A was also described by WoIz [1994] who analyzed the substrate specificity and kinetic data of Meprin A in comparison to the metalloprotease astacin from crayfish. Both, neither Kruse et al (2004) or Bertenshaw et al (2001) nor WoIz (1994) have pre- sented any evidence that meprin can also cleave BNP and that meprin can be used in a screening assay to identify potential therapeutic targets for cardiovascular diseases. Bertenshaw was not successful in identifying BNP as Meprin A substrate despite the profound search for novel meprin A substrates.

Meprins are known to play a critical role in development and have been implicated in cancer metastasis, inflammatory bowel disease, and kidney diseases. The US patent application US 2004/033582 discloses - based on the knowledge that Meprin degrades bradykinin, a vasodila- tatory peptide - the use of single nucleotide polymorphisms (SNP) of the human meprin gene in a method for identifying an individual at risk of developing a disorder upon administration of a phar- maceutically acceptable amount of an angiotensin-converting enzyme (ACE) inhibitor and/or vasopeptidase inhibitor. Furthermore, US 2004/033582 discloses that certain SNPs might be associated with the incidence of cardiovascular disorders.

The term "meprin" in the context of the present invention usually refers to both forms of me- prin, i.e. Meprin A and / or Meprin B.

Actinonin and derivatives thereof

European patent application EP 0 474 553 describes the use of novel actinonin derivatives as enzyme inhibitors; particularly, these compounds have strong inhibitory activities against en- kephalinase and angiotensin-converting enzymes, and are therefore useful for therapeutically treating hypertension, cardiac failure and the like. It is also stated that actinonin and derivatives thereof already known have levels of inhibitory activity against angiotensin-converting enzymes that are too low to utilize the compounds in practice.

International patent application WO 02/53169 discloses the use of Actinonin as inhibitor of amino peptidase N (APN; E.C. 3.4.11.2.; CD 13) in combination with inhibitors of Dipeptidyl Peptidase IV (DPP-IV, E.C. 3.4.14.5.; CD 26) for the prevention and therapy of cerebral damages caused by an ischemia, preferably of ischemic or hemorrhagic stroke, after a cranial/cerebral trauma, after a heart standstill, after a cardiac infarction or as a consequence of heart surgical operations (e. g. bypass surgeries).

B-Type Natriuretic Peptide (= Brain Natriuretic peptide)

Natriuretic peptides (NP) like atrial (ANP), B-type or brain (BNP) and C-type natriuretic peptide (CNP) are cyclic peptide hormones with relevance to cardiovascular, endocrine and renal homeostasis. In all three peptides an intact 17 amino acid disulfide-linked loop is the essential structural feature for their biological activity.

NPs are quickly cleared from circulation by (a) binding to a clearance receptor with the subsequent internalization and cleavage by cytoplasmic enzymes and (b) degradation by extracellular peptidases. The neutral endopeptidase (NEP), a membrane bound type-ll metallopeptidase of the M13 family, is generally regarded to be the main enzyme for NP-degradation, despite the fact that the degradation of ANP, BNP and CNP seems to be achieved on quite different catabolic routes.

However, recent analyses have shown that the hypothesis that NEP is the main or even single enzyme responsible for the degradation of NPs cannot be further uphold: It was shown by using studies with purified NEP from mouse tissue and with membrane preparations of wildtype and NEP-knockout mice that, in contrast to ANP and CNP, BNP is not cleaved by NEP and that there must be a NP-degrading activity independent from NEP [Walther et al, 2004A and 2004B]. A recently developed model of the active site of NEP tries to explain why BNP is not efficiently cleaved by NEP, since the two large tails of the BNP(I -32) might hinder the correct orientation of the cyclic BNP in the NEP cavity, and therefore, the potential cleavage site cannot be oriented towards the catalytic site [Pankow et al, 2004 and 2005].

The new NP-degading enzyme activity might present a further interesting target for the treatment of cardiovascular disorders.

Dipeptidylpeptidase IV

The exopeptidase dipeptidyl-peptidase IV (DPP-IV; EC 3.4.14.5), a cell-surface protease that occurs in a soluble form in plasma, cleaves many bioactive peptides of medical importance. It preferentially cleaves dipeptides from the amino terminus of peptides with a proline or alanine in the second position. The substrate specificity of DPP-IV is not that strict, however; cleavage after a penultimate Ser, GIy, Thr, VaI, and Leu also has been observed. Moreover, vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide, both of which have a serine in this position, are known DPP-IV substrates. In a recent paper it was shown that DPP-IV is responsible for the N-terminal cleavage of human BNP(I -32) to deliver hBNP(3-32) [Brandt et al, 2006]. However, it was also shown that loss of the amino-terminal dipeptide does not change the resistance of human BNP to human NEP-mediated degradation.

Furthermore, international patent application WO 2005/049022 discloses that DPP-IV inhibitors may be useful for the prevention, delay of progression or treatment of cardiovascular diseases or damages, renal diseases or damages, Heart Failure, or Heart Failure associated diseases. Preferred cardiovascular diseases or damages are selected from cardiac hypertrophy, cardiac remodeling after myocardial infarction, pulmonary congestion and cardiac fibrosis in dilated or in hypertrophic cardiomyopathy, cardiomyopathy such as dilated cardiomyopathy or hypertrophic cardiomyopathy or diabetic cardiomyopathy, left or right ventricular hypertrophy, diabetic myopathy, stroke prevention in congestive heart failure, hypertrophic medial thickening in arteries and/or in large vessels, mesenteric vasculature hypertrophy, and artherosclerosis, whereby preferred renal diseases or damages are selected from renal hyperfiltration such as after portal renal ablation, proteinuria in chronic renal disease, renal arteriopathy as a consequence of hypertension, nephrosclerosis, hypertensive nephrosclerosis and mesanglial hypertrophy.

US application US 2006/046978 discloses novel compounds and compositions containing the compounds which inhibit dipeptidyl peptidase (especially DPP-IV) and neprilysin (NEP, neutral endopeptidase) as well as dipeptidyl peptidase (especially DPP-IV) and angiotensin converting enzyme (ACE) and/or dipeptidyl peptidase (especially DPP-IV) and vasopeptidases (especially ACE and NEP). These compounds and pharmaceutical compositions thereof are useful for the treatment as well as the prevention of type 2 diabetes mellitus.

Cardiovascular Diseasese - Congestive Heart Failure

Congestive heart failure (CHF) is a clinical syndrome caused by heart disease, characterised by breath lessness and abnormal sodium and water retention, and resulting in oedema. This occurs when the heart is unable to generate a cardiac output sufficient to meet the demands of the body without marked increase of diastolic pressure. It is a consequence of a cardiac disease which impairs ventricular systolic or diastolic function, or both. It is not a single disease but the end stage of many different forms of heart diseases, the most common of which are the coronary artery diseases, hypertension and diabetes. Heart failure is manifested by symptoms of poor tissue perfusion (e.g. fatigue, poor exercise tolerance) or congestion of vascular beds (e.g. dyspnoea, pulmonary edema, and peripheral edema) or both. Treatment of heart failure is generally directed towards its underlying causes.

The prevalence of symptomatic heart failure in the general population in Europe is estimated to be about 0.4-2 %. As the prevalence rises rapidly with age, the increasing life expectancy is expected to have a major impact on the incidence of heart failure in the near future. The asymptomatic form of left-ventricular systolic dysfunction is estimated to be as common as symptomatic congestive heart failure.

The usefulness of measurement of single peptides derived from atrial natriuretic peptide prohormone (proANP) and brain natriuretic peptide pro-hormone (proBNP) in the diagnosis of heart failure was demonstrated. Cardiac impairment is associated with elevated circulating levels of ANP, BNP, N-terminal fragment of proANP (NT-proANP) and N-terminal fragment of proBNP (NT- proBNP). High plasma concentrations correlate with poor prognosis after myocardial infarction and heart failure.

Relationship NP and Cardiovascular diseases

NPs - especially BNP - become more important for diagnosis but also for the treatment of cardiovascular diseases, especially chronic heart failure (see e.g. international patent application WO 2005/003764, and Rademaker et al [2005]). BNP is regarded as having cardioprotective properties, since it reduces blood volume over the short term by sequestering plasma and over the longer term by promoting renal salt and water excretion and by antagonizing the renin-angiotensin- aldosterone system at many levels [reviewd by Woods et al., 2004]. Even recombinantly produced BNP is used for the treatment of CHF [Schreiner & Protter, 2002]. Therefore, the inhibition of the metabolism of BNP is an attractive option for increasing BNP-levels, and thereby increasing the cardioprotective potential of endogenous BNP.

PROBLEM OF THE INVENTION

As outlined above, the commonly accepted model of the natriuretic peptide system with NEP as the central degrading peptidase has to be partly revised. In particular, NEP does not seem to represent the initial degrading enzyme of BNP, but instead another - so far unknown peptidase - seems to be responsible for BNP degradation. This NEP-independent BNP degradation provides an effective means for achieving a beneficial BNP increase in cardiovascular pathology by inhibiting the assumed novel NP-degrading peptidase(s). Accordingly, it was the object of the present invention to identify the protein responsible for initial BNP degradation, and to demonstrate that inhibitors or modulators of said enzyme target are useful for therapeutic applications where in- creased BNP levels are desired.

SUMMARY OF INVENTION The aim of the present invention was to identify the enzymatic activity responsible for the initial degradation of BNP(I -32). For further biochemical characterization, a broad spectrum of peptidase inhibitors was tested and the inhibition pattern was compared to known kidney peptidases. The NP-degrading activity was completely inhibited by EDTA, partially by chymostatin, but not by phosphoramidon and was identified as Meprin A (EC 3.4.24.18). The compound Actinonin of the following formula (I),

the most effective inhibitor for Meprin A known today [Kruse et al., 2004], totally blocked the mouse BNP degradation in mice kidney membranes. Both kidney membrane and purified Meprin A cleave within the N-terminal tail of mouse BNP(I -32) at position His6-lle7. It was shown that the resulting peptide mouse BNP(7-32) (also termed BNP-26) is more accessible for degradation by NEP, since the large N-terminal tails hindering the binding of the BNP(I -32) to NEP are now partially removed. In addition, it was shown that also human BNP(I -32) is cleaved by meprin A of mice kidney membranes within the N-terminal tail of human BNP(I -32) at position Gly7-Ser8.

Since BNP becomes more important for diagnosis but also for the treatment of chronic heart failure, the inhibition of the metabolism of BNP is an attractive option for increasing BNP-levels. With Meprin A the present invention identified a new important target for pharmacological intervention, since the inhibition of Meprin A directly interferes with the degradation of BNP. Accordingly, the inhibition of Meprin A is a method for stopping the degradation of BNP and therefore giving rise to sustained or even elevated BNP levels which have e.g. cardioprotective effects.

In a first aspect, the present invention therefore pertains to a) a method of treating a human patient having need of maintaining or increasing BNP levels by administering a therapeutically effective amount of a compound modulating or inhibiting meprin activity; b) a method of treating a disease or disorder associated with decreased or insufficient levels of BNP in mammals and humans comprising administering to a subject in need thereof a therapeutically effective amount of a compound modulating or inhibiting meprin activity or downregulating the meprin expression; c) a method of treating or preventing cardiovascular disorders or diseases and/or renal diseases or disorders in mammals and humans comprising administering to a subject in need thereof a therapeutically effective amount of a compound modulating or inhibiting meprin activity; or d) the use of compound inhibiting, modulating or decreasing meprin activity for the manufacture of a medicament for the prophylaxis and/or treatment of cardiovascular disorders or diseases and/or renal diseases or disorders.

In one embodiment, the compound modulating or inhibiting meprin activity or downregulating meprin expression is administered or manufactured together with a therapeutically effective amount of an inhibitor of dipeptidyl peptidase IV (DPP-IV). Alternatively, the compound modulating or inhibiting meprin activity or downregulating meprin expression itself is also an inhibitor of dipeptidyl peptidase IV (DPP-IV) or decreases DPP-IV activity (dual DPP-IV/Meprin inhibitor).

In one embodiment, the preferred meprin of the invention is Meprin A.

In a further embodiment, the cardiovascular disorder or disease is selected from the group consisting of atherosclerosis, peripheral vascular disease, cerebral vascular disease, cardiac ischemia; ischemic heart disease, acute coronary syndrome, stable and unstable angina, acute myocardial infarction, post myocardial infarction, acute and chronic heart failure, including congestive heart failure; peripheral occlusive disease; ischemic stroke; hypertension, including essential hypertension and secondary forms of hypertension such as renal hypertension and/or pulmonary hypertension.

In a further embodiment of the present invention, the renal disorder or disease is selected from acute renal failure and chronic renal failure.

Additionally, the present invention relates to the administration of compound modulating or inhibiting meprin activity in combination with a therapeutically effective amount of a NEP inhibitor. The compound modulating or inhibiting meprin activity or downregulating meprin expression may be administered or manufactured together with a therapeutically effective amount of an inhibitor of NEP. Alternatively, the compound modulating or inhibiting meprin activity or downregulating meprin expression itself is also an inhibitor of NEP or decreases NEP activity (dual NEP/Meprin inhibitor).

In a further aspect, the present invention relates to an in vitro screening method to identify therapeutic agents useful in the treatment of cardiovascular and/or renal disorders or diseases in a mammal.

In one embodiment, the method screens for a test compound which specifically binds to a meprin metalloprotease, the method comprising the steps of: a) providing a test compound; b) contacting the test compound with the meprin metalloprotease for a sufficient time and under suitable conditions for binding; and c) detecting binding of the meprin metalloprotease to the test compound, thereby identifying the test compound which specifically binds the meprin metalloprotease.

In a further aspect, the method screens for agents which modulate the activity of a meprin metalloprotease, said method comprising the steps of : a) providing a test compound; b) contacting the test compound with the meprin metalloprotease; and c) assaying a biological activity of the meprin metalloprotease, wherein a test compound which increases said biological activity is identified as a potential therapeutic agent for increasing the activity of the meprin metalloprotease, and wherein a test compound which decreases said biological activity is identified as a potential therapeutic agent for decreasing the activity of the meprin metalloprotease.

In another aspect, the method screens for therapeutic agents useful for the treatment of a cardiovascular disorder or disease and/or renal disease or disorder, which inhibit or decrease the activity of a meprin metalloprotease, said method comprising the steps of: a) providing a test compound; b) contacting the test compound with the meprin metalloprotease; c) assaying a biological activity of the meprin metalloprotease in presence and absence and/or at different concentrations of said test compound, and d) optionally assaying said biological activity in the presence of a compound known to be a regulator of the meprin metalloprotease. wherein a test compound which decreases or inhibits said biological activity is identified as a potential therapeutic agent for inhibiting or decreasing meprin activity.

In a preferred embodiment, the screening methods are performed in a way that the biological activity of the meprin metalloprotease is assayed by measuring the influence of the test compound on the activity of the meprin metalloprotease in the presence of a suitable substrate for the meprin metalloprotease. In particular, the substrate is BNP. Furthermore, the biological activity of the meprin metalloprotease is preferably assayed by measuring the degree of BNP degradation. In another embodiment, the compound known to be a regulator of the meprin metalloprotease is actinonin.

Therefore, in an additional aspect, the present invention relates to a method of screening for agents which inhibit or decrease the activity of a meprin metalloprotease, comprising the steps of: a) providing a test compound; b) contacting the test compound with said meprin metalloprotease in the presence of BNP, c) measuring the degree of BNP degradation, and d) comparing the degree of BNP degradation by the meprin metal loprotease in presence and absence of the test compound, wherein a test compound which induces a lower degree of BNP degradation is identified as a potential therapeutic agent for inhibiting or decreasing the activity of a meprin metalloprote- ase.

In one embodiment of the invention, the meprin metalloprotease used in all screening methods is meprin A, preferably Meprin A of human, mouse or rat origin. In the context of the present invention, the meprin metalloprotease used in all screening methods is a meprin A having at least 90%, 92%, 94%, 96%, 98%, 99% or 100% identity to an amino acid sequence as displayed in SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3.

In a further embodiment of the present invention, the BNP used in all screening methods is mouse BNP or human BNP, preferably mouse BNP(I -32) or human BNP(I -32). In the context of the present invention, the BNP used in all screening methods is a BNP having the amino acid sequence as displayed in SEQ ID NO: 4 or SEQ ID NO: 6.

In a further embodiment of the present invention, the meprin metalloprotease used in all screening methods comprises at least one meprin fusion protein, i.e. the oligomeric complex Meprin A or Meprin B comprises at least one meprin alpha and/or meprin beta subunit which was genetically modified to be a meprin fusion protein.

The individual steps of the screening methods according to the present invention might be characterized by one or more of the following details: The step b) of contacting the test compound with the meprin metalloprotease is performed in or at the surface of a cell, whereby the cell preferably is present in an in vitro system. Alternatively, the step b) of contacting is performed in a cell-free system. The meprin metalloprotease used in the assay might be provided i) as isolated protein, ii) in the form of a membrane preparation bearing said meprin protein, or iii) in the form of an intact cell or cell extracts comprising said meprin protein. In a further embodiment, the meprin metalloprote- ase and/or the test compound and/or the optionally present BNP is coupled to a detectable label. Optionally, the meprin metalloprotease may be attached to a solid support.

In a further embodiment of the present invention, the screening methods as displayed above are further characterized in that a test compound already being identified as a potential therapeutic agent for inhibiting or decreasing meprin activity is further tested for its ability to inhibit or decrease the activity of a dipeptidyl peptidase IV, comprising the steps of a) contacting the test compound with said dipeptidyl peptidase IV, b) assaying a biological activity of said dipeptidyl peptidase IV in the presence and absence and/or at different concentrations of said test compound, wherein a test compound which decreases or inhibits said biological activity is identified as a potential therapeutic agent for inhibiting or decreasing meprin and dipeptidyl peptidase IV activity.

In a further embodiment of the present invention, the screening methods as displayed above are further characterized in that a test compound already being identified as a potential therapeutic agent for inhibiting or decreasing meprin activity is further tested for its ability to inhibit or decrease the activity of a NEP, comprising the steps of a) contacting the test compound with said NEP, b) assaying a biological activity of said NEP in the presence and absence and/or at different concentrations of said test compound, wherein a test compound which decreases or inhibits said biological activity is identified as a potential therapeutic agent for inhibiting or decreasing meprin and NEP activity.

In a further aspect, the invention related to a method for identifying compounds which are useful for the treatment of a cardiovascular disease or disorders, wherein the screening method comprises the step of determining whether the compound inhibits or decreases meprin activity as set out above. Preferably, the step of determining whether the compound inhibits or decreases meprin activity is carried out by measuring the degree of BNP degradation by the meprin metallo- protease.

In an additional aspect, the present invention relates to a compound which inhibits or decreases meprin activity, whereby said compound is identified by a method of the invention as described above. Preferably, said compound is used for the treatment and/or prophylaxis of cardio- vascular or renal disorders or diseases. Accordingly, the present invention also relates to a method of treating or preventing cardiovascular disorders or diseases in mammals and humans comprising administering to a subject in need thereof a therapeutically effective amount of a compound, whereby said compound is identified by a method of the invention as described above. Preferably, the compound is a small molecule, an RNA molecule, an antisense oligonucleotide, a polypeptide, an antibody, a small interfering RNA molecule (siRNA), or a ribozyme.

Furthermore, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound which inhibits or decreases meprin activity in combination with a pharmaceutically acceptable carrier. Preferably, the said compound is identified by a method of the invention as described above. In a further embodiment, said pharmaceutical composition may comprise one or more additional pharmaceutical agents. These additional pharmaceutical agent is preferably an cardiovascular-active agent, which may be selected from the group consisting of beta-blockers, calcium channel blockers, diuretics, renin inhibitors, ACE inhibi- tors, AT-1 receptor antagonists, ET receptor antagonists, NEP inhibitors, SEP inhibitors, ECE inhibitors, DPP-IV inhibitors and nitrates.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A shows the degradation of mBNP(1-32) in kidney membrane preparations from wild- type mice

Fig. 1 B shows the accumulation of mBNP7-32 in Candoxatrilat-treated kidney membrane preparations from wildtype mice

Fig. 2 shows a screening for different peptidase inhibitors and their effect on inhibiting the degradation of mBNP(1-32) by kidney membrane preparations of NEP-deficient mice (mean values with SD, n = 5).

Fig. 3A shows the HPLC elution profile of the degradation of mouse BNP(1-32) by pure Me- prin A.

Fig. 3B shows the HPLC elution profile of the degradation of mouse BNP(I -32) by kidney membrane preparations of NEP-knockout mice.

Fig. 4 shows the degradation of mouse BNP(I -32) by kidney membrane preparations from wildtype mice in the presence and absence of peptide inhibitors specific for NEP (Candoxatrilat) and Meprin A (Actinonin), respectively.

Fig. 5A shows the degradation of different NP substrates by recombinant murine NEP.

Fig. 5B shows the degradation of different NP substrates by isolated Meprin A.

Fig. 6 shows the degradation of human BNP(I -32) by kidney membrane preparations from wildtype mice in the presence and absence of peptide inhibitors specific for NEP (Candoxatrilat), Meprin A (Actinonin) and Dipeptidyl Peptidase IV (Diprotin), respectively.

Fig. 7 shows the occurrence of different cleavage products of human BNP(I -32) after degra- dation by kidney membrane preparations from wildtype mice in the presence and absence of peptide inhibitors specific for Meprin A (Actinonin, abbreviated as A - A1 corresponds to 1 μM

Actinonin, A2 corresponds to 10 μM Actinonin) and Dipeptidyl Peptidase IV (Diprotin, abbreviated as D), respectively. BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 amino acid sequence of mature human Meprin A

SEQ ID NO: 2 amino acid sequence of mature murine Meprin A SEQ ID NO: 3 amino acid sequence of mature rat Meprin A

SEQ ID NO: 4 amino acid sequence of human BNP(I -32)

SEQ ID NO: 5 amino acid sequence of murine BNP(I -45)

SEQ ID NO: 6 amino acid sequence of murine BNP(I -32)

SEQ ID NO: 7 amino acid sequence of porcine BNP(I -32) SEQ ID NO: 8 amino acid sequence of rat BNP(I -45)

SEQ ID NO: 9 amino acid sequence of rat BNP(I -32)

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Acronyms

ACE angiotensin-converting enzyme

ANP atrial natriuretic peptide

AT-1 angiotensin type 1 BNP B-type or brain natriuretic peptide

CHF congestive heart failure

CNP C-type natriuretic peptide

DPP-IV Dipeptidyl Peptidase IV

ECE endothelin converting enzyme ET endothelin

NEP neutral endopeptidase

NP natriuretic peptide

NT-proANP N-terminal fragment of proANP

NT-proBNP N-terminal fragment of proBNP proANP atrial natriuretic peptide prohormone proBNP brain natriuretic peptide prohormone

SEP soluble endopeptidase

SNP single nucleotide polymorphisms

RSA rat serum albumin HPLC high performance liquid chromatography hBNP human BNP mBNP murine BNP rBNP rat BNP Definitions:

The term "meprin metalloprotease" or simply "meprin" in the context of the present invention always refers to both forms of meprin, i.e. Meprin A and / or Meprin B.

The terms "meprin activity" or "biological activity of a meprin metalloprotease" refer to any biological activity which can be measured using meprin. In particular, the activity is measured in the form of proteolytic cleavage of a suitable substrate. Preferably, the substrate is a peptide known to be a natural substrate of meprin, in particular of Meprin A, or a peptide containing an optimized cleavage site for meprin [e.g. as depicted by Bertenshaw et al., 2001]. The substrate mostly preferred in the context of the present invention is BNP or a derivative thereof, still susceptible to hydrolysis by meprin.

"Meprin A" also termed "Endopeptidase-2" is a known enzyme, the enzymatic properties of which are summarized under the EC number 3.4.24.18. In the context of the present invention, a preferred substrate for Meprin A is BNP. Meprin A is a hetero-oligomeric complex comprising at least one meprin alpha polypeptide and at least on meprin beta polypeptide OR a homo-oligomeric complex comprising at least 10 meprin alpha polypeptides.

"Meprin B" is a known enzyme, the enzymatic properties of which are summarized under with the EC number 3.4.24.18. Meprin B is a protein homo-oligomeric complex comprising at least two meprin beta polypeptides.

The term "meprin alpha" refers to the alpha subunit of Meprin A. The meprin alpha used in the context of the present invention is preferably mammalian meprin alpha, more preferably human, mouse, rat or porcine meprin alpha. The amino acid and coding nucleotide (mRNA) sequences of the murine meprin alpha precursor are accessible in public databases by the accession numbers P28825 and M74897. respectively. The corresponding human amino acid and nucleotide sequences are accessible in public databases by the accession numbers Q16819 and M82962. whereas the rat sequences can be found at accession numbers Q64230 and S43408. respectively. The term "meprin alpha" may also comprise meprin variants, in particular naturally occurring or synthetically modified variants, of meprin alpha, as long as the derivatives - in form of their corresponding homo- or hetero-oligomeric complexes - still show their known enzymatic activity profile; i.e. in particular they still should be able to cleave BNP.

In particular, human Meprin A comprises the mature amino acid sequence as displayed in SEQ ID NOM (1 -letter-code)

NGLRDPNTRWTFPIPYILADNLGLNAKGAILYAFEMFRLKSCVDFKPYEGESSYIIFQQFDGC WSEVGDQHVGQNISIGQGCAYKAIIEHEILHALGFYHEQSRTDRDDYVNIWWDQILSGYQHNFDTY DDSLITDLNTPYDYESLMHYQPFSFNKNASVPTITAKIPEFNSIIGQRLDFSAIDLERLNRMYNCTTT HTLLDHCTFEKANICGMIQGTRDDTDWAHQDSAQAGEVDHTLLGQCTGAGYFMQFSTSSGSAEE AALLESRILYPKRKQQCLQFFYKMTGSPSDRL WWVRRDDSTGNVRKLVKVQTFQGDDDHNWKI AHWLKEEQKFRYLFQGTKGDPQNSTGGIYLDDITLTETPCPTGVWTVRNFSQVLENTSKGDKLQ SPRFYNSEGYGFGVTLYPNSRESSGYLRLAFHVCSGENDAILEWPVENRQVIITILDQEPDVRNRM SSSMVFTTSKSHTSPAINDTVIWDRPSRVGTYHTDCNCFRSIDLGWSGFISHQMLKRRSFLKNDDL IIFVDFEDITHLSQTEVPSKGKRLSPQGLILQGQEQQVSEEGSGKAMLEEALPVSLSQGQPSRQKR SVENTGPLEDHNWPQYFRDPCDPNPCQNDGICVNVKGMASCRCISGHAFFYTGERCQSAEVHG SVLGMVI GGTAGVI FLTFSI I Al LSQRPRK

In particular, murine Meprin A comprises the mature amino acid sequence as displayed in SEQ ID NO: 2 (1 -letter-code)

NAMRDPSSRWKLPIPYILADNLELNAKGAILHAFEMFRLKSCVDFKPYEGESSYIIFQKLSGC WSMIGDQQVGQNISIGEGCDFKATIEHEILHALGFFHEQSRTDRDDWNIWWDQIITDYEHNFNTY DDNTITDLNTPYDYESLMHYGPFSFNKNESIPTITTKIPEFNTIIGQLPDFSAIDLIRLNRMYNCTATHT LLDHCDFEKTNVCGMIQGTRDDADWAHGDSSQPEQVDHTLVGQCKGAGYFMFFNTSLGARGEA ALLESRILYPKRKQQCLQFFYKMTGSPADRFEVWVRRDDNAGKVRQLAKIQTFQGDSDHNWKIAH VTLNEEKKFRYVFLGTKGDPGNSSGGIYLDDITLTETPCPAGVWTIRNISQILENTVKGDKLVSPRF YNSEGYGVGVTLYPNGRITSNSGLLGLTFHLYSGDNDAILEWPVENRQAIMTILDQEADTRNRMSL TLMFTTSKNQTSSAINGSVIWDRPSKVGVYDKDCDCFRSLDWGWGQAISHQLLKRRNFLKGDSLII FVDFKDLTHLNRTEVPASARSTMPRGLLLQGQESPALGESSRKAMLEESLPSSLGQRHPSRQKR SVENTGPMEDHNWPQYFRDPCDPNPCQNEGTCVNVKGMASCRCVSGHAFFYAGERCQAMHVH GSLLGLLIGCIAGLIFLTFVTFSTTNGKLRQ

In particular, Meprin A derived from rat comprises the mature amino acid sequence as displayed in SEQ ID NO: 3 (1 -letter-code)

NALRDPSSRWKPPIPYILADNLDLNAKGAILNAFEMFRLKSCVDFKPYEGESSYIIFQQFSGC WSMVGDQHVGQNISIGEGCDYKAIIEHEILHALGFFHEQSRTDRDDYVNIWWNEIMTDYEHNFNTY DDKTITDLNTPYDYESLMHYGPFSFNKNETIPTITTKIPEFNAIIGQRLDFSATDLTRLNRMYNCTRT HTLLDHCAFEKTNICGMIQGTRDDADWVHEDSSQPGQVDHTLVGRCKAAGYFMYFNTSSGVTGE VALLESRILYPKRKQQCLQFFYKMTGSPSDRLLIWVRRDDNTGNVRQLAKIQTFQGDSDHNWKIA HVTLNEEKKFRYVFQGTKGDPGNSDGGIYLDDITLTETPCPTGVWTIRNISQVLENTVKGDRLVSP RFYNSEGYGFGVTLYPNGRITSNSGYLGLAFHLYSGDNDVILEWPVENEQAIMTILDQEPDARNRM SLSLMFTTSKYQTSSAINGSVIWDRPTKVGVYDKDCDCFRSIDWGWGQAISHQMLMRRNFLKDDT LIIFVDFKDLTHLRQTEVPISSRSVIPRGLLLQGQEPLALGDSRIAMMEESLPRRLDQRQPSRPKRS VENTGPMEDHNWPQYFRDPCDPNPCQNEGTCVNVKGMASCRCVSGHAFFYTGERCQAMHVH GSLLGLLIGCITALIFLTFITFSNTYQKLRQ The term "meprin beta" refers to the beta subunit of Meprin A and/or Meprin B. The meprin beta used in the context of the present invention is preferably mammalian meprin beta, more preferably human, mouse, rat or porcine meprin beta. The amino acid and nucleotide sequences of the murine meprin beta precursor are accessible in public databases by the accession numbers Q61847 and L15193 (GenBank), respectively. The corresponding human amino acid and nucleotide sequences are accessible in public databases by the accession numbers Q 16820 and X81333. whereas the rat sequences can be found at accession numbers P28826 and M88601. respectively. The term "meprin beta" may also comprise meprin variants, n particular naturally occurring or synthetically modified variants, of meprin beta, as long as the derivatives - in form of their corre- sponding homo- or hetero-oligomeric complexes - still show their known enzymatic activity profile; i.e. in particular they still should be able to cleave BNP.

A "meprin subunit", within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of (i) polypeptides having the sequence of known meprin alpha or beta subunits as defined above, (ii) polypeptides comprising the sequence of known meprin alpha or beta subunits, and (iii) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i) or (ii), wherein said polypeptide has meprin activity.

"Active" or "biologically active", with respect to a meprin metalloprotease, refers to those forms, fragments, domains or variants of a meprin alpha and/or beta polypeptide which retain the biological metabolic or physiologic and/or immunogenic and antigenic activity of the known meprin alpha and beta polypeptides, respectively, as defined above, including similar activities or improved activities or these activities with decrease undesirable side effects.

"Naturally occurring meprin (alpha or beta)" refers to a meprin alpha or beta polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.

"Chimeric" or "Fusion" molecules may be constructed by introducing all or part of the nucleotide sequence of the meprin subunits into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following Meprin characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.

A meprin fusion protein comprises two polypeptide segments fused together by means of a peptide bond. A fusion protein is encoded by two, often unrelated, fused genes or fragments thereof. The first polypeptide segment comprises at least 50 contiguous amino acids of a meprin subunit, preferably a meprin alpha subunit, or of a biologically active variant thereof, such as those described above. The first polypeptide segment also can comprise full-length meprin subunit. The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include, but are not limited to β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags may be used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus(HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located adjacent to the meprin subunit.

A "(meprin) variant" refers to a (meprin) polynucleotide or (meprin) polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleo- tide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Synthetically modified variant" refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.

"Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonin with a serine.

"Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systemati- cally making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.

A "signal sequence" or "leader sequence" can be used, when desired, to direct the polypep- tide through a membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.

"BNP" refers to B-type (or brain) natriuretic peptide, in particular to the active BNP derived from the precursor protein. The BNP used in the context of the present invention is preferably mammalian BNP, more preferably human, mouse, rat or porcine BNP, and corresponds to the C- terminal part of the individual precursor proteins (the amino acid sequences of which are accessible in public databases, e.g. by the accession numbers P 16860 for the human precursor and P40753 for the mouse precursor protein).

The human BNP(1-32) has the amino acid sequence as displayed in SEQ ID NO: 4 (3 letter code): Ser Pro Lys Met VaI GIn GIy Ser GIy Cys Phe GIy Arg Lys Met Asp Arg Ne Ser Ser Ser Ser GIy Leu GIy Cys Lys VaI Leu Arg Arg His; and has the CAS-Number 114471-18-0.

The murine BNP(1-45) has the amino acid sequence as displayed in SEQ ID NO: 5 (3 letter code): Ser GIn GIy Ser Thr Leu Arg VaI GIn GIn Arg Pro GIn Asn Ser Lys VaI Thr His Ne Ser Ser Cys Phe GIy His Lys Ne Asp Arg Ne GIy Ser VaI Ser Arg Leu GIy Cys Asn Ala Leu Lys Leu Leu

The murine BNP(1-32) fragment has the amino acid sequence as displayed in SEQ ID NO: 6 (3 letter code): Asn Ser Lys VaI Thr His Ne Ser Ser Cys Phe GIy His Lys Ne Asp Arg Ne GIy Ser VaI Ser Arg Leu GIy Cys Asn Ala Leu Lys Leu Leu

The porcine BNP(1-32) has the amino acid sequence as displayed in SEQ ID NO: 7 (3 letter code): Ser Pro Lys Thr Met Arg Asp Ser GIy Cys Phe GIy Arg Arg Leu Asp Arg Ne GIy Ser Leu Ser GIy Leu GIy Cys Asn VaI Leu Arg Arg Tyr

The rat BNP(1-45) has the amino acid sequence as displayed in SEQ ID NO: 8 (3 letter code): Ser GIn Asp Ser Ala Phe Arg Ne GIn GIu Arg Leu Arg Asn Ser Lys Met Ala His Ser Ser Ser Cys Phe GIy GIn Lys Ne Asp Arg Ne GIy Ala VaI Ser Arg Leu GIy Cys Asp GIy Leu Arg Leu Phe

The rat BNP(1-32) fragment has the amino acid sequence as displayed in SEQ ID NO: 9 (3 letter code): Asn Ser Lys Met Ala His Ser Ser Ser Cys Phe GIy GIn Lys Ne Asp Arg Ne GIy Ala VaI Ser Arg Leu GIy Cys Asp GIy Leu Arg Leu Phe "Inhibitor" is any substance which retards or prevents a chemical or physiological reaction or response. Accordingly, the term "inhibit", refers to a decrease or inhibition of a chemical or physiological reaction or response, in particular a decrease or inhibition of the biological activity of meprin. Common inhibitors include but are not limited to antagonists, antisense molecules, and antibodies. Preferably, a meprin inhibitor in the context of the present invention acts by reducing the ability or velocity of meprin to cleave peptidic substrates, in particular BNP, e.g. by blocking the active site of meprin, by irreversible binding and inactivating meprin, by diminishing the concentration of meprin etc.

The term "modulate", as it appears herein, refers to a change in the activity of meprin metal- loprotease. Accordingly, the term "modulator" refers to a compound that modulates the activity of a meprin metalloprotease. For example, modulation may cause an increase or a decrease in enzymatic activity, binding characteristics, or any other biological, functional, or immunological properties of meprin.

A "label" can be used to marker a compound, target or polypeptide; suitable labels which can be employed include, but are not limited to, fluorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g. biotin-avidin).

The term "Dipeptidyl peptidase IV" or simply "DPP-IV" in the context of the present invention encompasses DPP-IV from different species, but preferably refers to mammalian, in particular human DPP-IV. "Dipeptidyl peptidase IV" is a known enzyme, the enzymatic properties of which are summarized under the EC number EC 3.4.14.5.

The term "DPP-IV inhibitor" is intended to indicate a molecule that exhibits inhibition of the enzymatic activity of DPP-IV and functionally related enzymes, such as from 1-100% inhibition, and specially preserves the action of substrate molecules, including but not limited to brain natriuretic peptide, glucagon-like peptide-1 , gastric inhibitory polypeptide, peptide histidine methionine, substance P, neuropeptide Y, and other molecules typically containing alanine or proline residues in the second amino terminal position. Treatment with DPP-IV inhibitors prolongs the duration of action of peptide substrates and increases levels of their intact, undegraded forms leading to a spectrum of biological activities relevant to the disclosed invention. For the purpose of the invention, chemical compounds are tested for their ability to inhibit the enzyme activity of purified CD26/DPP-IV. Briefly, the activity of CD26/DPP-IV can be measured in vitro by its ability to cleave the synthetic substrate Gly-Pro-p-nitroanilide (Gly-Pro-pNA). Cleavage of Gly-Pro-pNA by DPP-IV liberates the product p-nitroanilide (pNA), whose rate of appearance is directly proportional to the enzyme activity. Inhibition of the enzyme activity by specific enzyme inhibitors slows down the generation of pNA. Stronger interaction between an inhibitor and the enzyme results in a slower rate of generation of pNA. Thus, the degree of inhibition of the rate of accumulation of pNA is a direct measure of the strength of enzyme inhibition. The accumulation of pNA is measured with a spectrophotometer. The inhibition constant, Ki, for each compound is determined by incubating fixed amounts of enzyme with several different concentrations of inhibitor and substrate.

In the present context "a DPP-IV inhibitor" is also intended to comprise active metabolites and prodrugs thereof, such as active metabolites and prodrugs of DPP-IV inhibitors. A "DPP-IV metabolite" is an active derivative of a DPP-IV inhibitor produced when the DPP-IV inhibitor is metabolised. A "DPP-IV prodrug" is a compound that is either metabolised to a DPP-IV inhibitor or is metabolised to the same metabolite (s) as a DPP-IV inhibitor.

DPP-IV inhibitors are known in the art. In particular, DPP-IV inhibitors are those as summarized in international patent application WO 2005/049022, preferably as displayed from page 4, bottom to page 11 , bottom of said patent specification, and as summarized in US patent application US 2006/0046978, the content of which is hereby incorporated into the present application by reference to these publications.

The term "Neutral Endopeptidase", "Neprilysin" or simply "NEP" in the context of the present invention encompasses NEP from different species, but preferably refers to mammalian, in particular human NEP. "Neutral Endopeptidase" is a known enzyme, the enzymatic properties of which are summarized under the EC number 3.4.24.11.

Treatment methods

In one embodiment, the present invention provides methods of treating abnormal conditions such as, for instance, cardiovascular and/or renal dysfunctions, disorders or diseases, hereinabove generally referred to as "the diseases" in the context of the present invention. The cardiovascular and/or renal diseases, which are explained in more detail below, may be treated by maintaining or increasing BNP levels, whereby the BNP levels are measured in the plasma and/or the tissue, preferably cardiac tissue.

Since the present invention provides for the first time the link between the degradation of cardioprotective BNP and the metalloprotease meprin, in particular Meprin A, the treatment methods of the present invention preferably aim to inhibit or at least reduce or modulate the activity of meprin.

If the activity of the meprin has to be down regulated, several approaches are available. One approach comprises administering to a subject in need thereof an inhibitor compound of meprin A, as hereinabove described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function of the meprin polypeptide, such as, for example, by blocking the binding of substrates, enzymes, etc., and thereby protecting the further cleavage of BNP. In another approach, soluble forms of the polypeptides still capable of binding the substrate, enzymes, etc. in competition with endogenous polypeptide may be administered. Typical examples of such competitors include fragments of the meprin polypeptides.

In still another approach, expression of the gene encoding endogenous meprin alpha or beta polypeptides can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered. Alternatively, oligonucleotides which form triple helices ("triplexes") with the gene can be supplied. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo. Synthetic antisense or triplex oligonucleotides may comprise modified bases or modified backbones. Examples of the latter include methylphosphonate, phosphorothioate or peptide nucleic acid backbones. Such backbones are incorporated in the antisense or triplex oligonucleotide in order to provide protection from degradation by nucleases and are well known in the art. Antisense and triplex molecules synthesized with these or other modified backbones also form part of the present invention as potential inhibitors or antagonists of meprin subunit polypeptides.

In addition, expression of the meprin polypeptides may be prevented by using ribozymes specific to the meprin alpha and/or beta mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic. Synthetic ribozymes can be designed to specifically cleave meprin mRNAs at selected positions thereby preventing translation of the meprin mRNAs into functional polypeptide. Ribozymes may be synthesized with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribosymes may be synthesized with non-natural backbones to provide protection from ribonuclease degradation, for example, 2'-O- methyl RNA, and may contain modified bases.

Cardiovascular Disorders

Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low- output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (Ml) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by arteriosclerosis. Ml prophylaxis (primary and secondary prevention) is included as well as the acute treatment of Ml and the prevention of complications. Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen. This group of diseases includes stable angina, unstable angina and asymptomatic ischemia.

Arrhythmias include all forms of atrial and ventricular tachy-arrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias.

Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others. The genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.

Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.

Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel. The atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion.

Although the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.

The formation of the atherosclerotic lesion can be considered to occur in five overlapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition. Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance of the lesion is unclear. Thus, a need exists for therapeutic methods and agents to treat cardiovascular pathologies, in particular congestive heart failure and atherosclerosis and other conditions related to coronary artery disease.

Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis.

Renal disorders

Kidney disorders may lead to hypertension or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney. Excessive urination may lead to hypotension.

Meprin Subunit Polypeptides

The present invention relates to the use of meprin metalloprotease, in particular Meprin A and/or Meprin B, preferably Meprin A, in screening assays. As described above, Meprin A and Meprin B are oligomeric protein complexes which comprise at least one meprin alpha and/or meprin beta subunit. In particular, the present invention uses mouse, rat and/or human meprin polypeptides (or enzymes), and also to the corresponding meprin polypeptide fragments comprising a substantial portion of said entire meprin polypeptide.

Thus, in a first aspect, the meprin subunit polypeptides used in the present invention include isolated polypeptides, in particular isolated mammalian (mouse, human, pork and/or rat) species meprin polypeptides, comprising an amino acid sequence which has at least 70% identity, preferably at least 80% and in particular at least 85 % identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to one of the amino acid sequences of naturally occurring meprin alpha or beta subunits from rat, mouse, human or pork, as identified above by the accession numbers in public databases, over the entire length of the respective amino acid sequences. Such polypeptides include those comprising one of the amino acid sequences selected from said group of meprin sequences.

In a second aspect, the meprin subunit polypeptides used in the present invention include isolated polypeptides, in particular isolated mammalian (mouse, human, pork and/or rat) species meprin polypeptides, having an amino acid sequence of at least 70% identity, preferably at least 80% and in particular at least 85 % identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to one of the amino acid sequences of naturally occurring meprin alpha or beta subunits from rat, mouse, human or pork, as identified above by the accession numbers in public databases, over the entire length of the respective amino acid sequences. Such polypeptides include the meprin subunit polypeptide defined by the accession numbers given above.

The meprin polypeptides used in the present invention are members of the metalloprotease family of polypeptides. The meprin polypeptides are of particular interest in the present invention, since for the first time the link to BNP degradation and therefore, the link to the direct treatment of cardiovascular diseases was established. Accordingly, the meprin polypeptides, and the assembled meprin metalloproteases, are of particular interest for identifying modulators or inhibitors of these polypeptides, for providing diagnostic assays for detecting diseases associated with inappropriate BNP levels, and for treating conditions associated with BNP imbalance with compounds identified to be modulators or inhibitors of meprin, preferably Meprin A. Hence, the meprin polypeptides may be used for designing or screening for selective modulators or inhibitors, and thus can lead to the development of new drugs. The properties of the meprin polypeptides, in particular of the assembled Meprin A and/or Meprin B enzymes, preferably the Meprin A enzymes, are hereinafter referred to as "meprin activity" or "meprin metalloprotease activity" or "biological activity of meprin". Also included amongst these activities are antigenic and immunogenic activities of said meprin enzymes and polypeptides. Preferably, an enzyme or polypeptide used in the context of the present invention exhibits at least one biological activity of meprin, preferably of Meprin A.

Preferably, a meprin subunit polypeptide comprises or represents an amino acid sequence as displayed in SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3.

The meprin subunit polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. The meprin used in the context of the present invention may be purified meprin from natural sources (isolated from rat or pork kidney membranes) or recombinantly produced meprin. For recombinant production, it is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production (see also the paragraphs referring to fusion proteins).

For optimal use in screening assays, the present invention also includes variants of the aforementioned meprin polypeptides these are polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, VaI, Leu and lie; among Ser and Thr, among the acidic residues Asp and GIu; among Asn and GIn; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5- 10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.

The present invention furthermore pertains to the use of fragments of the meprin alpha and/or beta polypeptides, in particular to meprin polypeptide fragments comprising a substantial portion of the entire meprin (alpha or beta) polypeptide. A fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned meprin polypeptides. As with meprin polypeptides, fragments may be "free- standing", or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region.

Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of meprin polypeptides, except for deletion of a continuous series of residues that includes the amine terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amine terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are these that mediate enzyme activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.

With regard to the variant of the invention pertaining to polypeptide fragments comprising a substantial portion of the entire meprin alpha or beta subunits as defined above, the term "substantial" has the meaning that the fragment of the individual meprin polypeptide has in particular a size of at least about 50 amino acids, preferably a size of at least about 100 amino acids, more preferably a size of at least about 200 amino acids, most preferably a size of at least about 300 amino acids. In this context "about" includes the particularly recited sizes larger or smaller by several, 5, 4, 3, 2 or 1 amino acid. The meprin polypeptide fragments according to the invention preferably show at least to some extent at least one of the properties which are characteristic for the meprin polypeptides themselves.

Meprin enzymes and polypeptides used in the context of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

Expression of Meprin Polypeptides used in Screening Assays

Recombinant meprin subunit polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Said expression systems comprise a polynucleotide or polynucleotides encoding meprin, and the host cells are genetically engineered with such expression systems; and the meprin polypeptides are produced by recombinant techniques. Cell-free translation systems can also be employed to produce said meprin subunits using RNAs derived from the corresponding meprin encoding DNA constructs.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides encoding meprin subunits. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold

Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.. Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals, i.e. derived from a different species.

If a meprin subunit polypeptide is to be expressed for use in screening assays, it is generally possible that the polypeptide be produced at the surface of the cell or alternatively in a soluble protein form. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellular^, the cells must first be lysed before the polypeptide is recovered. If the polypeptide is bound at the surface of the cell (membrane bound polypeptide), usually membrane fractions are prepared in order to accumulate the membrane bound polypeptide.

The meprin subunit polypeptides can be recovered and purified from from any cell that expresses the polypeptide, including host cells that have been transfected with meprin metalloprotease expression constructs (recombinant cell cultures). A purified meprin metalloprotease polypeptide is separated from other compounds that normally associate with the meprin metalloprotease polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (HPLC) is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and or purification. A preparation of purified meprin metalloprotease polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.

Fusion proteins

In a further aspect, the present invention relates to the use of genetically engineered soluble fusion proteins comprising a meprin polypeptide, or a fragment thereof, as already defined above, in the screening methods for identifying meprin activity inhibiting or modulating compounds.

Screening Assays

It is an essential aspect of the present invention, to provide screening assays to identify compounds which modulate or inhibit the function of the meprin polypeptide, preferably the Meprin A metalloprotease. In particular, it was the aim of the present invention to provide screening assays to identify compounds which modulate or inhibit the function of meprin to degrade the B-type natriuretic peptide BNP, which cannot be cleaved by the neutral endopeptidase NEP, to a shortened BNP fragment, which can be further degraded by NEP.

Candidate or test compounds or agents which bind to meprin and/or have a modulatory or inhibitory effect on the activity or the expression of meprin are identified either in assays that employ cells which express meprin (cell-based assays) or in assays with isolated meprin (cell-free assays). The various assays can employ a variety of variants of meprin (e.g., full-length meprin, a biologically active fragment of meprin, or a fusion protein which includes all or a portion of meprin). Moreover, meprin can be derived from any suitable mammalian species (e. g., human meprin, rat meprin or murine meprin).

The screening method may simply measure the influence of a candidate compound on the activity of the meprin polypeptide, or on cells or membranes bearing the polypeptide. Alternatively, the screening method may involve competition with a competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the activity of the polypeptide or to the cells or membranes bearing the polypeptide. Inhibition of polypeptide activity is generally assayed in the presence of a known substrate (e.g. naturally occurring BNP or synthetically derived derivatives thereof), and the effect of the candidate compound is observed by altered activity, e.g. by testing whether the candidate compound results in inhibition or stimulation of the meprin activity.

For example, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a meprin metalloprotease as defined in the present invention, and a suitable substrate to form a mixture, measuring meprin activity in the mixture, and comparing the meprin activity of the mixture to a standard without candidate compound. Further possible variations of the screening methods of the invention were already given above.

The assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known meprin ligand, e.g. Actinonin, to meprin. The assay can also be an activity assay entailing direct or indirect measurement of the activity of meprin. The assay can also be an expression assay entailing direct or indirect measurement of the expression of meprin mRNA or meprin protein. The various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms of diseases related to meprin.

The present invention includes biochemical, cell free assays that allow the identification of inhibitors and activators of proteases suitable as lead structures for pharmacological drug development. Such assays involve contacting a form of meprin or a meprin polypeptide (e.g., full- length meprin, a biologically active fragment of meprin, or a fusion protein comprising all or a portion of meprin) with a test compound and determining the ability of the test compound to act as an inhibitor (preferably) or an activator of the enzymatic activity of meprin.

The activity of meprin molecules of the present invention can be measured using a variety of assays that measure meprine activity. For example, meprine enzyme activity can be assessed by a standard in vitro zinc/metal lo-protease assay. Those of skill in the art are aware of a variety of substrates suitable for in vitro assays. In addition, protease assay kits are available from commercial sources, such as Calbiochem (San Diego, Calif.). However, the preferred substrate in the context of the present invention is BNP.

Solution in vitro assays can be used to identify a meprin modulator or inhibitor. Solid phase systems can also be used to identify a modulator or inhibitor of a meprin polypeptide. For example, a meprin polypeptide or meprin fusion protein can be immobilized onto the surface of a receptor chip of a commercially available biosensor instrument (BIACORE™, Biacore AB; Uppsala, Sweden). The use of this instrument is disclosed, for example, by [Karlsson, (1991), and Cunningham and Wells, (1993)].

In brief, a meprin polypeptide or fusion protein is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within a flow cell. A test sample is then passed through the cell. If a meprin modulator or inhibitor is present in the sample, it will bind to the immobilized polypeptide or fusion protein, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination on-and off-rates, from which binding affinity can be calculated, and assessment of the stoichiometry of binding, as well as the kinetic effects of meprin mutation. This system can also be used to examine antibody-antigen interactions, and the interactions of other complement/anti-complement pairs.

In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of meprin. Such assays can employ full-length meprin, a biologically active fragment of meprin, or a fusion protein which includes all or a portion of meprin.

As described in greater detail below, the test compound can be obtained by any suitable means, e.g., from conventional compound libraries.

Determining the ability of the test compound to modulate the activity of meprin can be accomplished, for example, by determining the ability of meprin to bind to or interact with a target molecule, e.g. preferably BNP in the context of the present invention. The target molecule can be a molecule with which meprin binds or interacts with in nature (i.e. the cleavage of BNP by meprin). The target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal, or can induce a further signal. The target meprin molecule can be, for example, a second intracellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with meprin. Determining the ability of meprin to bind to or interact with a target molecule can be accomplished by one of the methods described here within for determining direct binding. In one embodiment, determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3, etc. ), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e. g. , a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e. g. , luciferase), or detecting a cellular response.

In various embodiments of the above assay methods of the present invention, it may be desirable to immobilize meprin (or a meprin fragment or even the target molecule) to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to meprin, or interaction of meprin with a target molecule (i.e.BNP) in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro- centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or meprin, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of meprin can be determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either meprin or its target molecule (preferably BNP) can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, III.), and immobilized in the wells of streptavidin-coated plates (Pierce Chemical). Alternatively, antibodies reactive with meprin or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with meprin or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with meprin or target molecule.

Functional Assays

Test compounds can be tested for the ability to increase or decrease the biological activity of a meprin polypeptide. The meprin activity can be measured, for example, using methods described above. Meprin activity can be measured after contacting either a purified meprin or an intact cell with a test compound. A test compound which decreases meprin activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing meprin activity. A test compound which increases meprin activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing meprin activity.

High Throughput Screening

Test compounds can be screened for the ability to bind to meprin metalloprotease polypeptides or to affect meprin metalloprotease activity or meprin metalloprotease gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 5001. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format. Alternatively, "free format assays" or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al., Proc. Natl. Acad. Sci. U. S. A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, and then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors. Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10,1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change. Yet another example is described by Salmon et al., Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar. Another high throughput screening method is described in Beutel et al., U. S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

Binding Assay

For binding assays, the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the meprin metalloprotease, preferably the Meprin A enzyme, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules. In binding assays, either the test compound or the meprin metalloprotease can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the meprin metalloprotease can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. Alternatively, binding of a test compound to a meprin metalloprotease can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a meprin metalloprotease. A microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Determining the ability of a test compound to bind to a meprin metalloprotease polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

Gene expression

In another embodiment, test compounds are identified which increase or decrease meprin gene expression. As used herein, the term "correlates with expression of a polynucleotide" indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding meprin, by northern analysis or real-time PCR is indicative of the presence of nucleic acids encoding meprin in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding meprin. The term "microarray", as used herein, refers to an array of distinct polynucleotides or oligonucleotides arrayed on a substrate, such as paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support. A meprin polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of meprin polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a regulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of meprin mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of meprin polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immuno-histochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into meprin.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses meprin polynucleotide can be used in a cell-based assay system. The meprin polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line can be used.

Computer Aided Drug Design

Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate meprin expression or activity.

Having identified such a compound or composition (i.e. the known Meprin A inhibitor Actinonin), the active sites or regions are identified. Such sites might typically be the enzymatic active site, allosteric sites, regulator binding sites, or ligand binding sites. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand (in particular BNP). In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found. Next, the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined. If an incomplete or insufficiently accurate structure is determined, the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy. Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods. Finally, having determined the structure of the active site, either experimentally, by modeling, or by a combination, candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential meprin modulating compounds.

Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand. The composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.

A preferred method of computer aided drug design in order to provide a method for the structure-based design of a modulator or inhibitor of the meprin enzyme, which modulates or inhibits the degradation of BNP comprises the following steps: (a) determining in the first instance the three-dimensional structure of the meprin, preferably determining in the first instance the three- dimensional structure of the meprin with the bound BNP ligand; (b) deducing the three-dimensional structure for the likely reactive or binding site(s) of a potential modulator or inhibitor (e.g. Actinonin, which is a known strong inhibitor of Meprin A); (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and (d) testing whether the candidate compounds are indeed modulators or inhibitors. It will be further appreciated that this will normally be an iterative process.

BNP as substrate

In a preferred embodiment, BNP is used as a substrate for assaying meprin activity in the screening assays of the present invention. The BNP used is preferably the physiologically active C- terminal part of the native BNP precursor. In particular, mammalian BNP, more preferably human, mouse, rat or porcine BNP, and the corresponding C-terminal parts of the individual precursor proteins (the amino acid sequences of which are accessible in public databases, e.g. by the accession numbers P 16860 for the human precursor and P40753 for the mouse precursor protein) are used. The present invention preferably uses a peptide comprising the C-terminal 50, 45, 40, 39, 38, 37, 36, 35, 34, 33 or 32 contiguous amino acids of the precursor proteins. Preferably, the BNP(I- 32) or BNP(I -45) peptides as defined above are used in the context of the present invention. In particular, the BNP peptides as displayed in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 are used.

However, also derivatives of the BNP fragments defined above may be used as substrates in the screening methods of the present invention as long as the BNP derivatives are still cleaved by meprin A. Preferably, one of the resulting subfragments should represent a preferred substrate for the NEP enzyme, in order to reflect the natural situation of the two step BNP degradation.

Accordingly, also BNP peptides may be used , which comprise or have an amino acid sequence which has at least 70% identity, preferably at least 80% and in particular at least 85 % identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to one of the amino acid sequences of naturally occurring BNP peptides from rat, mouse, human or pork, as identified above, and defined by the accession numbers in public databases, over the entire length of the respective amino acid sequences. Such polypeptides include those having or comprising one of the amino acid sequences selected from said group of BNP sequences.

For optimal use in screening assays, the present invention also includes variants of the aforementioned BNP peptides. These BNP variants vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, VaI, Leu and lie; among Ser and Thr, among the acidic residues Asp and GIu; among Asn and GIn; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination. BNP peptides can be obtained, for example, by purification from natural sources, by recombinant expression of BNP encoding polynucleotides in the appropriate host systems, or by direct chemical synthesis.

Labeling of proteins, substrates or compounds

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various assays. Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Compounds

A test compound preferably binds to a meprin metalloprotease. More preferably, a test compound decreases meprin metalloprotease activity by at least about 10, preferably about 50, more preferably about 75, 80, 90, 95 or 100% relative to the absence of the test compound.

Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such stimulators or inhibitors so- identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof.

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. Libraries of compounds can be presented in solution or on beads, chips, bacteria or spores, plasmids, or phage.

Examples of potential polypeptide inhibitors may include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc., or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Administration forms (Pharmaceutical Formulations)

The method of the invention is primarily intended for treatment in a mammal, preferably in humans and other primates, of cardiovascular diseases or disorders and/or renal disease or disorders, wherein the disease or disorder preferably requires maintaining or increasing the BNP level, preferably by administration of a compound modulating or inhibiting meprin activity or by downregulating the meprin expression.

The compounds may be administered orally, dermally, parenterally, by injection, by pulmonal or nasal delivery, or sublingually, rectally or vaginally in dosage unit formulations. The term "administered by injection" includes intravenous, intraarticular, intramuscular (e.g. by depot injection where the active compounds are released slowly into the blood from the depot and carried from there to the target organs), intraperitoneal, intradermal, subcutaneous, and intrathecal injections, as well as use of infusion techniques. Dermal administration may include topical application or transdermal administration. One or more compounds may be present in association with one or more non-toxic pharmaceutically acceptable auxiliaries such as excipients, adjuvants (e.g. buffers), carriers, inert solid diluents, suspensing agents, preservatives, fillers, stabilizers, antioxidants, food additives, bioavailability enhancers, coating materials, granulating and disintegrating agents, binding agents etc., and, if desired, other active ingredients.

The pharmaceutical composition may be formulated for example as immediate release, sustained release, pulsatile release, two or more step release, depot or other kind of release formulations.

The manufacture of the pharmaceutical compositions according to the invention may be performed according to methods known in the art and will be explained in further detail below. Commonly known and used pharmaceutically acceptable auxiliaries as well as further suitable diluents, flavorings, sweetening agents, coloring agents etc. may be used, depending on the intended mode of administration as well as particular characteristics of the active compound to be used, such as solubility, bioavailability etc. Suitable auxiliaries and further ingredients may be such as recommended for pharmacy, cosmetics and related fields and which preferably are listed in the European Pharmacopoeia, FDA approved or cited in the "GRAS" list (FDA List of food additives that are 'generally recognized as safe' (GRAS)).

One mode of application of the compounds of general formula (I) or of pharmaceutical compositions comprising one or more of said compounds is oral application, e. g., by tablets, pills, dragees, hard and soft gel capsules, granules, pellets, aqueous, lipid, oily or other solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, elixiers, solid emulsions, solid dispersions or dispersible powders. For the preparation of pharmaceutical compositions for oral administration, the compounds suitable for the purposes of the present invention as defined above can be admixed with commonly known and used adjuvants and excipients such as for example, gum arabic, talcum, starch, sugars (such as, e. g., mannitose, methyl cellulose, lactose), gelatin, surface-active agents, magnesium stearate, aqueous or nonaqueous solvents, paraffin derivatives, cross-linking agents, dispersants, emulsifiers, lubricants, conserving agents, flavoring agents (e. g., ethereal oils), solubility enhancers (e. g., benzyl benzoate or benzyl alcohol) or bioavailability enhancers (e.g. Gelucire™). In the pharmaceutical composition, the active ingredients may also be dispersed in a microparticle, e. g. a nanoparticulate, composition.

For parenteral administration, the active agents can be dissolved or suspended in a physiologically acceptable diluent, such as, e. g., water, buffer, oils with or without solubilizers, surface-active agents, dispersants or emulsifiers. As oils for example and without limitation, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil may be used. More generally spoken, for parenteral administration the active agent can be in the form of an aqueous, lipid, oily or other kind of solution or suspension or even administered in the form of liposomes or nano- suspensions.

Transdermal application can be accomplished by suitable patches, as generally known in the art, specifically designed for the transdermal delivery of active agents, optionally in the presence of specific permeability enhancers. Furthermore, also emulsions, ointments, pastes, creams or gels may be used for transdermal delivery.

Another mode of application is by implantation of a depot implant comprising an inert carrier material, such as biologically degradable polymers or synthetic silicones such as e. g. silicone rubber. Such implants are designed to release the active agent in a controlled manner over an extended period of time (e. g., 3 to 5 years).

It will be appreciated by those skilled in the art that the particular method of administration will depend on a variety of factors, all of which are considered routinely when administering therapeutics. It will also be understood, however, that the actual dosages of the agents of this invention for any given patient will depend upon a variety of factors, including, but not limited to the activity of the specific compound employed, the particular composition formulated, the mode of administration, time of administration, route of administration and the particular site, host, and disease being treated, and furthermore the age of the patient, the body weight of the patient, the general health of the patient, the gender of the patient, the diet of the patient, rate of excretion, drug combinations, and the severity of the condition undergoing therapy. It will be further appreciated by one skilled in the art that the optimal course of treatment, i.e., the mode of treatment and the daily number of doses of a compound of Formula I or a pharmaceutically acceptable salt thereof given for a defined number of days, can be ascertained by those skilled in the art using conventional treatment tests. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using conventional dosage-determination tests in view of the experimental data for a given compound. For oral administration, an exemplary daily dose generally employed will be from about 0.01 μg/kg to about 100 mg/kg of total body weight, whereby courses of treatment may be repeated at appropriate time intervals. Administration of pro-drugs may be dosed at weight levels that are chemically equivalent to the weight levels of the fully active compounds. The daily dosage for parenteral administration will generally be from about 0.01 μg/kg to about 100 mg/kg of total body weight. A daily rectal dosage regimen will generally be from about 0.01 μg/kg to about 200 mg/kg of total body weight. The daily topical dosage regimen will generally be from about 0.1 μg to about 100 mg administered between one to four times daily. The transdermal concentration will generally be that required to maintain a daily dose of from 0.01 μg/kg to 100 mg/kg of total body weight.

EXAMPLES

It was recently shown by using studies with purified NEP from mouse tissue and with membrane preparations of wildtype and NEP-knockout mice that, in contrast to ANP and CNP, BNP(I- 32) is not cleaved by NEP and that there must be a NP-degrading activity independent from NEP [Walther et al, 2004A and 2004B]. The assays described below were performed according to the protocols given within Walther et al. [2004A], as well as the animals and materials used, unless explicitly stated otherwise.

The analyses of BNP and degradation fragments were performed by high performance liquid chromatography (HPLC) with UV-detection (Shimadzu LC10-AT, Kyoto, Japan) on a 100 C18 nucleosil column, a 40 μl injection volume and a flow rate of 1 ml/min. The BNP fragments were eluted using a linear gradient of A) 1 % Trifluoroacetic acid (TFA) in H₂O (80 - 65%) and B) Acetoni- trile (20-35%) for the first 25 minutes (0-25 min), and a second gradient A) TFA (0.1 % in H₂O) (80%) and B) Acetonitril (20%) for the following 10 minutes (25-35 min). The peptides were UV- detected at 216 nm and 220 nm. A linear correlation between peak areas and natriuretic peptide concentration was confirmed before analysis. Therefore, the value "area under the curve" (peak area) of the peak for the BNP(I -32) and for the smaller degradation fragments for each individual HPLC chromatogram were used as a measure for peptide concentration.

I. Identification of the initial degradation product of BNP(1-32)

In order to characterize the NEP independent degradation of BNP(I -32), kidney membrane preparations of wildtype mice were incubated with BNP(I -32) in the presence of known inhibitors of NEP and aminopeptidase. The time course of BNP degradation and the generation of BNP cleavage products were monitored by HPLC.

Performance: mBNP(1-32) with a final concentration of 10 μM was incubated with 80 μl solubilized mice kidney membrane (0.4 mg/ml protein) in a final volume of 600 μl Tris-Puffer sup- plemented with 0.1% RSA (Tris/RSA) in the presence of the aminopeptidase inhibitor Bestatin (100 μM final concentration). Identical samples were incubated in the presence of additional candoxatri- lat (0.1 mM final concentration) for NEP-independent degradation. Samples of 100 μl were repeatedly taken during an incubation time of 120 min and stopped by addition of 50 μl 0.35 M perchloric acid. The degradation of mBNP(1-32) and the formation of a smaller BNP fragment was monitored by analyzing all samples by HPLC. The peak area for the BNP(I -32) and for the smaller, newly appearing BNP fragment of each individual HPLC chromatogram was plotted against the time course of incubation. Results: The results are depicted in Fig 1. Fig. 1A shows that the BNP(1-32) is quickly degraded by the murine kidney membranes; however, the degradation is NEP independent, since the addition of the NEP inhibitor candoxatrilat does not influence BNP(1-32) degradation. Fig. 1 B shows that an initial BNP degradation product - which was identified to correspond to mBNP(7-32) by mass spectroscopic analysis - is formed; this mBNP(7-32) accumulates in presence of the NEP-inhibitor candoxatrilat, but is further degraded in the absence of candoxatrilat. Accordingly, the NEP enzyme is involved in the further degradation of the BNP(7-32) fragment. This confirms the results of the BNP-NEP degradation studies, and supports the model of the NEP catalytic site that the mBNP(7-32) carrying a shortened N-terminal tail now better fits into the NEP cave and can be more easily cleaved. This means, that NEP in spite of its inability to degrade BNP, is involved in BNP inactivation.

II. Identification of the enzymatic activity responsible for the initial degradation of BNPd -32)

In order to identify the enzymatic activity potentially involved in degradation of BNP(I -32), the influence of a broad spectrum of typical peptidase inhibitors on mBNP degradation (degradation of mouse BNP by kidney membrane preparations of NEP-deficient mice) was tested and the inhibition pattern was compared to known kidney peptidases.

Performance: 5 samples each containing mouse BNP(I -32) at a final concentration of 10 μM were incubated with 5 μl solubilized kidney membrane of NEP-deficient mice (5.4 mg protein/ml) in a final volume of 100 μl Tris/RSA Puffer in the presence of effective concentrations of different peptidase inhibitors. Samples were stopped after 30 min by addition of 50 μl 0.35 M perchloric acid. The degradation of mBNP(1-32) was monitored by analyzing all samples by HPLC. The peak area for the BNP(I -32) of the HPLC chromatograms was determined for each peptidase inhibitor.

For the peptidase inhibitors, a commercially available set (Genotech, Cat#786-207) was used. All inhibitors were present in IOOfold concentration. From each preparation, 2 μl were used in a sample volume of 100 μl. The individual concentrations are as follows:

The peptidase inhibitor Lisinopril was used in a final concentration of 0.02 mM.

Results: The results are depicted in Fig 2 (given are the mean values with SD, n=5): The BNP degrading activity was strongest inhibited by EDTA. Minor effects were found by high concentrations of chymostatin, but not by phosphoramidon. Consequently, the prospected peptidase should be a metalloprotease.

Further investigations with a variety of commercially available metal lopeptidase and specific inhibitors showed that the enzyme Meprin A is involved in the initial degradation of mBNP in the mouse kidney [data not shown].

III. Confirmation that Meprin A is the enzyme responsible for the initial degradation of BNP(1-32)

In order to confirm that Meprin A is involved in the initial degradation of BNP, the degradation pattern of mBNP treated with commercially available Meprin A was compared to the degradation pattern of mBNP incubated with kidney membrane preparations from NEP knockout mice by HPLC analysis.

Performance:

A) A sample containing mouse BNP(I -32) at a final concentration of 10 μM was incubated with 0.2 ng/μl Meprin A (Cat. No. 445001 from Calbiochem, derived from rat) at 37°C in a final volume of 600 μl Tris/RSA puffer in the presence of the aminopeptidase inhibitor bestatin (100 μM). Samples of 100 μl were taken after 20 and 60 min and stopped by addition of 50 μl 0.35 M perchloric acid.

B) A sample containing mouse BNP(I -32) at a final concentration of 10 μM was incubated with 5 μl solubilized kidney membrane of NEP-deficient mice (5.4 mg protein/ml) at 37°C in a final volume of 100 μl Tris/RSA puffer in the presence of the aminopeptidase inhibitor bestatin (100 μM) and stopped after 20 min by addition of 50 μl 0.35 M perchloric acid.

The degradation of mBNP(1-32) and the formation of a smaller BNP fragment was monitored by analyzing all samples by HPLC. The signals detected are plotted against the time course of elution for each individual probe.

Results: The results are depicted in Fig 3A and Fig 3B: Fig. 3A shows the HPLC elution profile of the degradation of mouse BNP by pure Meprin A. The degradation leads to one major prod- uct (UV-peak of the HPLC separation) at 28.3 min. Fig. 3B shows the HPLC elution profile of the degradation of mouse BNP by kidney membrane preparations of NEP-knockout mice. An identical peak as in Fig. 3A appears. The product belonging to the UV-peak of the HPLC separation at 28.3 min could again be identified as mouse BNP 7-32 by mass spectroscopy. Therefore, it could be confirmed that Meprin A is involved in the initial cleavage of mBNP in the kidney and that it cleaves the mBNP(1-32) between the His6-lle7 amino acids, thereby deliberating a smaller BNP(7-32) fragment of 26 amino acids.

The same experiment as described under A was also performed in the presence of 0.1 mM Actinonin. The BNP(I -32) fragment was not degraded and the resulting HPLC chromatogram corresponds to the chromatogram obtained for the BNP sample at 0 min [data not shown].

IV. Degradation of mBNP by kidney membrane preparations of wildtvpe mice

In order to further characterize the BNP degradation, mouse BNP was incubated with kidney membrane preparations from wildtype mice in the presence and absence of known inhibitors of NEP and Meprin A, namely Candoxatrilat (Pfizer) and Actinonin (CAS No. 13434-13-4; obtained from Sigma, Cat.-No. A6671). respectively.

Performance: 3 samples of mouse BNP(I -32) at a final concentration of 10 μM were incu- bated with 5 μl solubilized kidney membrane of wildtype mice (5.4 mg protein/ml) at 37°C in a final volume of 100 μl Tris/RSA puffer in the presence of the aminopeptidase inhibitor bestatin (100 μM) and optionally additionally with Candoxatrilat (final concentration 0.1 mM) and/or Actinonin (final concentration 1 μM), and stopped after 20 min by addition of 50 μl 0,35 M perchloric acid. The degradation of mBNP(1-32) was monitored by analyzing all samples by HPLC. The peak area for the BNP(I -32) of the HPLC chromatogram was determined for each sample. The initial value for the mBNP(1-32) at 0 min without incubation was taken as reference value and set to 100%; the % values for the mBNP(1-32) still present in the samples after 20 min of incubation were calculated in comparison to the reference value. The data given are mean values from 3 samples. Results: The results are depicted in Fig 4 (given are the mean values with SD, n=3): The incubation of murine BNP(I -32) with kidney membrane preparations from wildtype mice (i.e. NEP+/+) results in fast degradation of the mBNP. Whereas candoxatrilat (C) treatment of the samples in order to inhibit the NEP enzyme does not significantly decrease the degradation of the mBNP(1-32), the Meprin A inhibitor actinonin (A) blocks degradation of mouse-type BNP(I -32) up to 90 %.

V. Comparison of the enzymatic degradation of different NPs by isolated NEP and Meprin A

In order to analyze the substrate specifity of NEP and Meprin A, different types of natriuretic peptides (NP) were incubated with isolated NEP and isolated Meprin A, respectively.

Performance:

A: Different NPs (mANP, mBNP(1-32); mBNP(7-32); hBNP, CNP) at a final concentration of 10 μM each were incubated with 0.27 ng/μl recombinant mouse NEP (rmNEP, R&D Laboratories) in a final volume of 900 μl Tris-buffer supplemented with 0.1% RSA (Tris/RSA). Samples of 100 μl were taken after 0, 20 and 60 min (for mBNP; hBNP) or after 0, 10, 20 min (for mBNP(7-32), CNP, mANP) and stopped by addition of 0.35 M perchloric acid. In the presence of Candoxatrilat (NEP inhibitor), the peptides were not degraded (data not shown).

B: Different NPs (mANP, mBNP(1-32); mBNP(7-32); hBNP(1-32), CNP) at a final concentration of 10 μM each were incubated with 0.2 ng/μl Meprin A (Cat. No. 445001 from Calbiochem, derived from rat) at 37°C in a final volume of 600 μl Tris/RSA in presence of the aminopeptidase inhibitor bestatin (100 μM). Samples were repeatedly taken during an incubation time of 60 min and stopped by addition of 0.35 M perchloric acid. In the presence of additional actinonin (1 μM; Meprin A inhibitor), the peptides were not degraded (data not shown). The degradation of the NPs was monitored by analyzing all samples by HPLC as described above; however, different elution gradients were used for the NPs: The murine BNP(I -32) and mBNP(7-32) fragments were eluted as described above. The murine ANP(1-28) was eluted using a linear gradient of A) 0.1 % Trifluoroacetic acid (TFA) in H₂O (80 - 68%) and B) Acetonitrile (20-32%) for the first 25 minutes (0- 25 min), and a second linear gradient A) TFA (0.1% in H₂O) (80%) and B) Acetonitril (20%) for the following 10 minutes (25-35 min). The human BNP(1-32) was eluted using a linear gradient of A) 0.1% Trifluoroacetic acid (TFA) in H₂O (80 - 73%) and B) Acetonitrile (20-27%) for the first 15 minutes (0-15 min), and a second linear gradient A) TFA (0.1 % in H₂O) (80%) and B) Acetonitril (20%) for the following 10 minutes (15-25 min). The CNP(I -22) was eluted using a linear gradient of A) 0.1 % Trifluoroacetic acid (TFA) in H₂O (75 - 68%) and B) Acetonitrile (25 - 32%) for the first 15 minutes (0-15 min), and a second linear gradient A) TFA (0.1% in H₂O) (75%) and B) Acetonitril (25%) for the following 10 minutes (15-25 min). The peptides were UV-detected at 216 nm and 220 nm. A linear correlation between peak areas and natriuretic peptide concentration was confirmed before analysis. The peak area for the corresponding NP fragments of each individual HPLC chro- matogram was plotted against the time course of incubation.

Results: The results are depicted in Fig 5A and 5B (given are the mean values of 3 samples each): Comparison of the degradation of NPs by NEP (Fig. 5A) and Meprin A (Fig. 5B) results in opposite substrate preferences for both peptidases: The best substrate for NEP is CNP-28, which lacks the C-terminal tail. On the other hand, CNP is not a substrate for Meprin A. Moreover, Meprin

A cleaves the NEP-resistant human BNP(I -32). In addition, it could be confirmed that the initial degradation product resulting from the cleavage with Meprin A, the mBNP 7-32 fragment, is cleaved more rapidly by NEP than mBNP1-32.

Vl. Degradation of hBNP by kidney membrane preparations of wildtvpe mice

In order to further characterize the BNP degradation, human BNP (hBNP) was incubated with kidney membrane preparations from wildtype mice in the presence and absence of known inhibitors of NEP and Meprin A, namely Candoxatrilat (Pfizer) and Actinonin (CAS No. 13434-13-4: obtained from Sigma, Cat-No A6671), respectively. Furthermore, an inhibitor of the enzyme dipep- tidyl peptidase IV was used for this assay, namely Diprotin (obtained from Sigma, Cat-No J9759), since DDP-IV was recently discovered to be also known to be responsible for degradation of hu- man BNP [Brandt et al, 2006]. The cleavage of hBNP with DDP-IV results in hBNP(3-32).

Performance: 4 samples of human BNP(I -32) at a final concentration of 10 μM were incubated with 5 μl solubilized kidney membrane of wildtype mice (5.4 mg protein/ml) at 37°C in a final volume of 100 μl Tris/RSA puffer in the presence of the aminopeptidase inhibitor bestatin (100 μM) or additionally with Candoxatrilat (final concentration 0.1 mM) and/or Actinonin (final concentration 1 μM) and/or Diprotin (final concentration 0.1 mM), and stopped after 20 min by addition of 50 μl 0.35 M perchloric acid. The degradation of hBNP(1-32) was monitored by analyzing all samples by HPLC. The peak area for the BNP(I -32) of the HPLC chromatogram was determined for each sample. The initial value for the hBNP(1-32) at 0 min without incubation was taken as reference value and set to 100%; the % values for the hBNP(1-32) still present in the samples after 20 min of incubation were calculated in comparison to the reference value. The data given are mean values from 4 samples.

Results: The results are depicted in Fig 6 (given are the mean values with SD, n=4): The in- cubation of human BNP(I -32) with kidney membrane preparations from wildtype mice (i.e. NEP+/+) results in fast degradation of the hBNP. Candoxatrilat (C) treatment of the samples in order to inhibit the NEP enzyme does not decrease the degradation of the hBNP(1-32) at all. However, the Meprin A inhibitor actinonin (A) alone was also not able to significantly block the degradation of hBNP(1-32). Also the combined inhibition with Actinonin and Candoxatrilat had no significant effect, as well as the addition of the dipeptidyl peptidase IV inhibitor Diprotin alone. Only the combined inhibition of meprin A and dipeptidyl peptidase IV by addition of Actinonin and Diprotin induced a significant reduction of hBNP(1-32) degradation. The reduced effect of Diprotin can be partly explained by the fact that Diprotin itself is a substrate for the enzymes of kidney membrane and is rapidly degraded itself [Rahfeld et al, 1991]

VII. Analysis of cleavage products after degradation of hBNP by kidney membrane preparations of wildtvpe mice

The assay was performed according to Example VII. The degradation of the hBNP was monitored by analyzing all samples by HPLC as described herewithin, thereby differentiating between the cleavage products.

The degradation of hBNP(1-32) and the formation of smaller hBNP fragments were moni- tared by analyzing all samples by HPLC. The peak height for the BNP(1-32) and for the smaller, newly appearing BNP fragment of each individual HPLC chromatogram was determined, and was taken as a qualitative measure for peptide concentration.

Results: The results are depicted in Fig 7 (given are the mean values with SD, n=3): The in- cubation of human BNP(I -32) with kidney membrane preparations from wildtype mice (i.e.

NEP+/+) results in fast degradation of the hBNP, whereby detectable BNP fragments were hBNP(8-32) and hBNP(8-30) resulting from Meprin A cleavage at the N-terminus, whereas the two

C-terminal amino acids are degraded by another enzymatic activity. Diprotin (D) treatment did not alter the resulting BNP fragment pattern. Actinonin (A) treatment of the samples in order to inhibit the meprin A enzyme does not decrease the degradation of the hBNP(1-32), but instead of a hBNP(8-32) or hBNP(8-30) fragment now a hBNP(3-32) fragment was detected at a high level.

This fragment seems not be further degraded. The combined inhibition with Diprotin and Actinonin

(D + A) induced a significant reduction of any kind of hBNP(1-32) degradation.

CITED LITERATURE

• Bertenshaw GP, Turk BE, Hubbard SJ, Matters GL, Bylander JE, Crisman JM, Cantley LC, Bond JS. (2001) "Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity." J Biol Chem. 2001 Apr 20;276(16): 13248-55.

• Bond JS, Matters GL, Banerjee S, Dusheck RE. (2005) "Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer." FEBS Lett.:

579(15):3317-22. • Brandt I, Lambeir AM, Ketelslegers JM, Vanderheyden M, Scharpe S, De Meester I. (2006) "Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form." Clin Chem. 52(1 ):82-7, Epub 2005 Oct 27.

• EP 0 474 553 • Kruse MN, Becker C, Lottaz D, Kohler D, Yiallouros I, Krell HW, Sterchi EE, Stacker W. (2004) Human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors. Biochem J. 378(Pt 2):383-9.

• Pankow K, Becker M, Krause G, Diehl A, Walther T, Siems EW (2004) "Sequential differences in the natriuretic peptides influence their catabolism by neutral endopeptidase (EC 3.4.24.11 )" Naun.-Schmied. Arch, of Pharmacology 369, R32 126 Suppl.

• Pankow K, Sun X, Krause G, Diehl A, Walther T, Siems WE (2005) "Neue Enzyme fϋr den Abbau natriuretischer Peptide?" Poster Presentation, 46. Frϋhjahrstagung der DGPT vom 15. - 17. Marz 2005 in Mainz, Germany

• Rademaker MT, Richards AM (2005) Cardiac natriuretic peptides for cardiac health. Clin Sci (Lond). 108(1 ):23-36. Review.

• Rahfeld J, Schierhorn M, Hartrodt B, Neubert K, Heins J. (1991 ) "Are diprotin A (lle-Pro-lle) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV?" Biochim Biophys Acta. 1076(2):314-6.

• Schreiner GF & Protter AA (2002) "B-type natriuretic peptide for the treatment of congestive heart failure." Curr Opin Pharmacol. 2002 Apr;2(2): 142-7.

• US 2004/033582 US 2006/046978

• Walther T, Stepan H, Pankow K, Becker M, Schultheiss HP, Siems WE (2004A) "Biochemical Analysis of Neutral Endopeptidase Activity Reveals Independent Catabolism of Atrial and Brain Natriuretic Peptide." Biol. Chem. 385, 179-84.

• Walther T, Stepan H, Pankow K, Gembardt F, Faber R, Schultheiss HP, Siems WE (2004B) "Relation of atrial (ANP) and brain natriuretic peptide (BNP) to their N-terminal fragments in fetal circulation: evidence for enhanced neutral endopeptidase (NEP) activity and resistance of BNP to NEP in the fetus." Br. J. Obstet. Gyn., 111 , 452-5. • WO 2002/053169

• WO 2005/049022

• WO 2005/003764

• Woods RL. (2004) Cardioprotective functions of atrial natriuretic peptide and B-type natriuretic peptide: a brief review. Clin Exp Pharmacol Physiol. 31(11):791-4. Review. • WoIz (1994) "A kinetic comparison of the homologous proteases astacin and meprin A." Arch Biochem Biophys. 310(1):144-51.

Claims

Claims

1. A method of treating a human patient having need of maintaining or increasing BNP levels by administering a therapeutically effective amount of a compound modulating or inhibiting meprin activity.

2. The method according to claim 1, wherein the therapeutically effective amount of a compound modulating or inhibiting meprin activity is administered in combination with a therapeutically effective amount of an inhibitor of dipeptidyl peptidase IV.

3. A method of treating a disease or disorder associated with decreased or insufficient levels of BNP in mammals and humans comprising administering to a subject in need thereof a therapeutically effective amount of a compound modulating or inhibiting meprin activity or downregulating meprin expression.

4. The method according to claim 3, wherein the therapeutically effective amount of a compound modulating or inhibiting meprin activity or downregulating meprin expression is administered in combination with a therapeutically effective amount of an inhibitor of dipeptidyl peptidase IV.

5. A method of treating or preventing cardiovascular and/or renal disorders or diseases in mammals and humans comprising administering to a subject in need thereof a therapeutically effective amount of a compound modulating or inhibiting meprin activity.

6. The method according to claim 5, wherein the therapeutically effective amount of a compound modulating or inhibiting meprin activity is administered in combination with a therapeutically effective amount of an inhibitor of dipeptidyl peptidase IV.

7. A method according to any of claims 1 to 6, wherein the meprin is Meprin A.

8. A method according to claim 5, wherein the cardiovascular disorder or disease is selected from the group consisting of atherosclerosis, peripheral vascular disease, cerebral vascular disease, cardiac ischemia; ischemic heart disease, acute coronary syndrome, stable and unstable angina, acute myocardial infarction, post myocardial infarction, acute and chronic heart failure, including congestive heart failure; peripheral occlusive disease; ischemic stroke; hypertension, including essential hypertension and secondary forms of hypertension such as renal hypertension and/or pulmonary hypertension.

9. A method according to claim 5, wherein the renal disorder or disease is selected from acute renal failure and chronic renal failure.

10. A method according to claim 5 or 8 further comprising administering to said subject a thera- peutically effective amount of a NEP inhibitor.

11. An in vitro screening method to identify therapeutic agents useful in the treatment of cardiovascular and/or renal disorders or diseases in a mammal, wherein

(i) the method screens for therapeutic agents, which specifically bind to a meprin metalloprote- ase, said method comprising the steps of a) providing a test compound; b) contacting the test compound with said meprin metalloprotease for a sufficient time and under suitable conditions for binding; and c) detecting binding of the meprin metalloprotease to the test compound, thereby identifying a therapeutic agent which specifically binds the meprin metalloprotease; and/or

(ii) the method screens for therapeutic agents, which modulate the activity of a meprin metalloprotease, said method comprising the steps of a) providing a test compound; b) contacting the test compound with said meprin metalloprotease; and c) assaying a biological activity of said meprin metalloprotease, wherein a test compound which increases said biological activity is identified as a potential therapeutic agent for increasing the activity of the meprin metalloprotease, and wherein a test compound which decreases said biological activity is identified as a potential therapeutic agent for decreasing the meprin activity; and/or (iii) the method screens for therapeutic agents, which inhibit or decrease the activity of a meprin metalloprotease, said method comprising the steps of : a) providing a test compound; b) contacting the test compound with the meprin metalloprotease; c) assaying a biological activity of the meprin metalloprotease in presence and absence and/or at different concentrations of said test compound, d) optionally assaying said biological activity in the presence of a compound known to be a regulator of the meprin metalloprotease, wherein a test compound which decreases or inhibits said biological activity is identified as a potential therapeutic agent for inhibiting or decreasing meprin activity.

12. A method according to claim 11 , wherein the biological activity of the meprin metalloprotease is assayed by measuring the influence of the test compound on the activity of the meprin metalloprotease in the presence of a suitable substrate for the meprin metalloprotease.

13. A method according to claim 12, wherein the substrate is BNP.

14. A method according to claim 13, wherein the biological activity of the meprin metalloprotease is assayed by measuring the degree of BNP degradation.

15. A method according to claim 11 , wherein the compound known to be a regulator of the meprin metalloprotease is actinonin.

16. A method according to claim 11 , wherein the method screens for therapeutic agents which inhibit or decrease the activity of a meprin metalloprotease, comprising the steps of a) providing a test compound; b) contacting the test compound with said meprin metalloprotease in the presence of BNP, c) measuring the degree of BNP degradation, and d) comparing the degree of BNP degradation by said meprin metalloprotease in presence and absence and/or at different concentrations of said test compound, wherein a test compound which induces a lower degree of BNP degradation is identified as a potential therapeutic agent for inhibiting or decreasing meprin activity.

17. A method according to any of the claims 11 to 16, wherein the meprin metalloprotease is meprin A.

18. A method according to claim 17, wherein the meprin A is of human, mouse or rat origin.

19. A method according to claim 17, wherein the meprin A has at least 90% identity to an amino acid sequence as displayed in SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3.

20. A method according to claim 13, 14 or 16, wherein the BNP used is mouse BNP or human BNP, preferably mouse BNP(I -32) or human BNP(I -32).

21. A method according to claim 20, wherein the BNP has the amino acid sequence as displayed in SEQ ID NO: 4 or SEQ ID NO: 6.

22. A method according to any of claims 11 to 21 , wherein the meprin metalloprotease comprises at least one meprin fusion protein.

23. A method according to any of claims 11 to 21 , wherein the step of contacting is in or at the surface of a cell.

24. A method according to any of claims 11 to 21 , wherein the step of contacting is in a cell- free system.

25. A method according to any of claims 11 to 21 , wherein the meprin metalloprotease is pro- vided i) as isolated protein, ii) in the form of a membrane preparation bearing said meprin protein, or iii) in the form of an intact cell or cell extracts comprising said meprin protein.

26. A method according to any of claims 11 to 21 , wherein the meprin metalloprotease is coupled to a detectable label.

27. A method according to any of claims 11 to 21 , wherein the test compound is coupled to a detectable label.

28. A method according claim 13, 14, 16, 20 or 21 , wherein the BNP is coupled to a detectable label.

29. A method according to any of claims 11 to 21 , wherein the meprin metalloprotease is attached to a solid support.

30. A method according to any of claims 11 to 29, wherein a test compound being identified as a potential therapeutic agent for inhibiting or decreasing meprin activity is further tested for its ability to inhibit or decrease the activity of a dipeptidyl peptidase IV, comprising the steps of a) contacting the test compound with said dipeptidyl peptidase IV, b) assaying a biological activity of said dipeptidyl peptidase IV in the presence and absence and/or at different concentrations of said test compound, wherein a test compound which decreases or inhibits said biological activity is identified as a potential therapeutic agent for inhibiting or decreasing meprin and dipeptidyl peptidase IV activity.

31. A compound for use in therapy that modulates, inhibits or decreases the activity of a meprin metalloprotease, wherein said compound is identified by any of the methods of claims 11 to 30.

32. A compound according to claim 31 for the treatment and/or prophylaxis of cardiovascular or renal disorders or diseases.

33. A method of treating or preventing cardiovascular disorders or diseases in mammals and humans comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to claim 31.

34. Use of a compound inhibiting, modulating or decreasing meprin activity for the manufacture of a medicament for the prophylaxis and/or treatment of cardiovascular disorders or diseases and/or renal diseases or disorders.

35. Use of a compound according to claim 34, wherein the compound further inhibits or decreases dipeptidyl peptidase IV activity.

36. Use according to Claim 34, wherein the cardiovascular disorder or disease is selected from the group consisting of atherosclerosis, peripheral vascular disease, cerebral vascular disease, cardiac ischemia; ischemic heart disease, acute coronary syndrome, stable and unstable angina, acute myocardial infarction, post myocardial infarction, acute and chronic heart failure, including congestive heart failure; peripheral occlusive disease; ischemic stroke; hypertension, including essential hypertension and secondary forms of hypertension such as renal hypertension and/or pulmonary hypertension.

37. Use according to claim 34, wherein the renal disorder or disease is selected from acute renal failure and chronic renal failure.

38. Use according to any of the claims 34 to 37, wherein the compound is identified by the method according to any of the claims 11 to 30.

39. Use according to claim 38, wherein said compound is a) a small molecule, b) an RNA molecule, c) an antisense oligonucleotide, d) a polypeptide, e) an antibody, f) a small interfering RNA molecule (siRNA), or g) a ribozyme.

40. Use according to any of the claims 34 to 39, wherein the medicament further comprises one or more additional pharmaceutical agents.

41. Use according to claim 40, wherein said additional pharmaceutical agent is an cardiovascular-active agent selected from the group consisting of beta-blockers, calcium channel blockers, diuretics, renin inhibitors, ACE inhibitors, AT-1 receptor antagonists, ET receptor antagonists, NEP inhibitors, SEP inhibitors, ECE inhibitors, dipeptidyl peptidase IV inhibitors and nitrates.

42. A pharmaceutical composition comprising a therapeutically effective amount of a compound which inhibits or decreases meprin activity in combination with a pharmaceutically acceptable carrier.

43. A pharmaceutical composition according to claim 42, wherein the compound is identified by the method according to any of the claims 11 to 30.

44. A pharmaceutical composition according to one of the claims 42 or 43 further comprising one or more additional pharmaceutical agents.

45. The pharmaceutical composition of claim 44, wherein said additional pharmaceutical agent is an cardiovascular-active agent selected from the group consisting of beta-blockers, calcium channel blockers, diuretics, renin inhibitors, ACE inhibitors, AT-1 receptor antagonists, ET receptor antagonists, NEP inhibitors, SEP inhibitors, ECE inhibitors, dipeptidyl peptidase IV inhibitors and nitrates.