WO2009097893A1 - Methods for the treatment of cardiac disease associated to myocardial fibrosis using an inhibitor of pcp - Google Patents

Abstract

The invention relates to methods for the treatment of cardiac diseases associated with fibrosis, in particular, chronic cardiac failure or hypertensive heart disease by the use of compounds which are capable of decreasing the activity of the Procollagen C-Proteinase or PCP. The invention also relates to methods for the developing a personalized therapy for the treatment of a patient suffering from a cardiac disease associated to myocardial fibrosis comprising determine in a sample from said patient a parameter indicative of the PCP activity and if the value of said parameter is higher than a predefined threshold value, then the patient is candidate to the treatment with a therapeutically effective amount of a PCP inhibitory agent, in particular, with torasemide.

Description

METHODS FOR THE TREATMENT OF CARDIAC DISEASE ASSOCIATED TO MYOCARDIAL FIBROSIS USING AN INHIBITOR OF PCP

FIELD OF THE INVENTION

The invention relates to the methods for the treatment of myocardial diseases associated with increased fibrosis using PCP inhibitors. The invention also relates to a method for providing a personalized therapy of diseases related to an increased fibrosis which involving the determination of a parameter associated with PCP activity.

BACKGROUND ART

Maintained increase of blood pressure is associated to a significant increase in cardiovascular morbidity and mortality in hypertensive patients. This effect is due to the fact that arterial hypertension (AHT) may damage the structure and function of arteries, heart, brain and kidneys. In particular, patients suffering from AHT show a high risk of developing structural and functional heart alterations which are known as the hypertensive heart disease (HHT). Left ventricle hypertrophy is the most typical hallmark of HHT, although there exist other microscopic changes in said conditions leading to the so-called myocardium remodeling. Hypertrophy and cardiomyocyte apoptosis, myocardial fibrosis and hypertrophy of the intramyocardial artery and arterioles are hallmarks of the myocardial remodeling taking place in HHT.

Much effort has been devoted to the identification of strategies for the treatment of HHT. In particular, Kagitani et al. (J. Hypertens., 2004, 22:1007-1015) have reported the use of the anti-inflammatory compound tranilast for the treatment of HHT. Rasoul et al. (J Hypertens 2004; 22:593-603) have described that the stem cell proliferation inhibitor JV-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is capable of decreasing myocardial inflammation and fibrosis in a rat model of hypertension. These compounds are probably acting by inhibiting the activity of TGF-β.

WO07146981 describes 4-heterocyclyl 2-arylamino-aminopirimidines derivatives which are able to reduce cardiac failure. These compounds act by the activity of protein kinase C. WO07129646 describes different macro lides which are suitable for the treatment of chronic cardiac failure. These compounds are believes to exert their therapeutic properties by inhibition of the matrix metalloprotease -9.

JP2007204416 describes the use of compositions comprising isoleucine, valine and leucine for the treatment of chronic cardiac failure.

US2007072825 describes the use of N-bencilpiridine -modified porfϊrinic compounds for the treatment of cardiovascular diseases, including chronic cardiac failure.

US2006111361 describes a method for reverting left ventricle remodeling occurring during chronic cardiac failure by the administration of ranolazine or of a combination of ranolazine and enalapril or of ranolazine and metoprolol tartrate.

WO07072564 describes a method for the treatment and prevention of chronic cardiac failure by the use of voglibose o acarbose, two compounds which exert their therapeutic effects due to their antihyperglycemic effect caused by an inhibition of the glycosidase α activity.

WO07077122, WO07006688 and WO07039438 describe methods for the treatment of chronic cardiac failure based on the use of antagonists of the vasopressin Via receptor, in particular, compounds carrying a indol-3-carbonyl-spiro group

WO05087233 describes a method for the treatment of chronic cardiac failure based on the administration of perhexiline, a known antianginal agent capable of modifying cardiac metabolism from a free fatty acid-based metabolism to the more efficient glucose-based metabolism.

WO04060489 describes a method for the treatment of chronic cardiac failure based on the administration of xanthine oxidase inhibitor, which results in an increase of the high-energy phosphate available to the cardiac muscle US2006094715 describes a method for the treatment and prevention of chronic cardiac failure based on the administration of 5-HT₄ receptor antagonists.

US2004082641 describes a method for the treatment and prevention of early cardiovascular diseases, including chronic cardiac failure, based on the administration of inhibitors or glycogen phosphorylase.

WO04105786 describes a method for the treatment of chronic and acute cardiac failure un based in the administration of adiponectine, isolated or in combination with an agent such as pentoxifylline, which is capable of regulating the production of tumor necrosis factor α.

Ogata et al. (J Am Coll Cardiol 2004; 43:1481-1488) have described that Fenofibrate, an agonist of the peroxisomal proliferator alpha receptor, is capable of reducing fibrosis in the heart of rats showing an overload in arterial pressure. This effect is thought to be due to the increased in the repression of pro-inflammatory (e.g. NF -KB) (Schiffrin et al., Hypertension 2003; 42:664-668).

Meiners et al. (Circulation 2004; 44:471-477) have described that the inhibitor of the ubiquitin-proteasome system MG 132 is capable of suppressing the expression of fibrillar collagen in isolated fibroblasts and to reduce myocardial fibrosis in SHRs.

Although the mechanisms underlying said effects are unknown, it has been suggested that this are caused by the complex cross-talk among different transcription factors involved in collagen biosynthesis, which includes some targets of peroxisomal degradation such as NF-κB (Ghish, A.K., Exp. Biol. Med. 2002; 227:301-314).

Hasegawa et al., (J MoI Cell Cardiol 2003; 35:953-960), Bezerra, et al., (Clin Sci 2005; 108:349-355) and Porter et al., (Cardiovasc Res 2004; 61 :745-755.) have described that statins are capable of decreasing cardiac fibrosis in hypertensive rats. Preliminary results indicate that this anti-fϊbrotic effect is the result of a direct effect in signals causing fibroblast activation and growth (Porter et al., Cardiovasc Res 2004; 61 :745- 755). Swaney, et al. (Proc Natl Acad Sci USA 2005; 102:437-442) have described that activation and over-expression of adenylate cyclase results in the inhibition of myofibroblasts in adults rats as well as collagen synthesis, thus providing a basis for the developing of therapeutic strategies directed to increasing AMPc levels in cardiac fibroblasts.

Thus, all these results open new strategies for the treatment of myocardial fibrosis, although a more extensive characterization of the underlying mechanisms is needed before these therapies can be applied. Thus, there remains a need in the art for alternative therapeutic approaches for the treatment of HHT and chronic cardiac failure.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method for the treatment or prevention of a cardiac disease associated to myocardial fibrosis in a subject comprising the administration of a therapeutically effective amount of an agent which is capable of inhibiting PCP.

In a second aspect, the invention relates to a method for developing a personalized therapy for the treatment of a patient suffering from a cardiac disease associated to myocardial fibrosis comprising:

(a) determine in a sample from said patient a parameter indicative of the

PCP activity and (b) if the value of said parameter is higher than a predefined threshold value, then the patient is candidate to the treatment with a therapeutically effective amount of a PCP inhibitory agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Schematic representation of the steps involved in the synthesis and processing of collagen type I molecules. DNA = deoxyribonucleic acid, PCP = procollagen type I carboxy-terminal proteinase; PCPE = procollagen type I carboxy-terminal proteinase enhancer; PICP = carboxy- terminal propeptide of procollagen type I, that is released as result of the action of PCP on its substrate and secreted into the blood stream.

Figure 2: Western blots and autoradiograms of PCP.

Representative Western blot autoradiograms of myocardial PCP from 1 patient with chronic heart failure at baseline and 8 months after randomization to furosemide (left), and chronic heart failure patient at baseline and 8 months after randomization to torasemide (right). Abbreviations as in figure 1.

Figure 3: Western blots and autoradiograms of PCPE.

Representative Western blot autoradiograms of myocardial PCPE from 1 patient with chronic heart failure at baseline and 8 months after randomization to furosemide (left), and chronic heart failure patient at baseline and 8 months after randomization to torasemide (right). Abbreviations as in figure 1.

Figure 4: PCP and PCPE expression.

Histological sections of myocardial biopsy specimens from 1 patient with chronic heart failure. (A, C) immunostaining for PCP and PCPE, respectively. These molecules were identified in brown and located mostly in areas of interstitial and perivascular fibrosis

(arrows). Positive staining also was seen in cardiomyocytes (arrowheads). (B, D)

Negative controls for the correspondent primary antibody omission. Magnification

XlOO. Abbreviations as in Figure 1.

Figure 5 : Association between aldosterone and PCP activation.

Positive correlation (y=0.004x + 1.907) between plasma aldosterone and activation of myocardial PCP (as assessed by the ratio of PCP active form to PCP zymogen) in all patients with chronic heart failure. Abbreviations as in Figure 1.

Figure 6: Association between PICP and CVF. Positive correlation (y=1.338x + 18.73) between the reduction in serum and the reduction in myocardial collagen volume fraction (CVF) in patients with chronic heart failure treated with torasemide. Abbreviations as in figure 1.

Figure 7: Associations among PCP activation and CVF and PICP.

(A) Positive correlation (y=0.012 + 27.48) between the reduction in PCP activation (as assessed by the ratio as assessed by the ratio of PCP active form to PCP zymogen) and the reduction in myocardial CVF in patients with chronic heart failure treated with torasemide. (B) positive correlation (y=0.483x + 9.979) between the reduction in PCP activation (as assessed by the ratio as assessed by the ratio of PCP active form to PCP zymogen) and the reduction in serum PICP in patients with chronic HF treated with torasemide. Abbreviations as in figure 6

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have found out that, surprisingly, torasemide is capable of interfering with the myocardial PCP/PCPE system and that said effect may be one of the factors contributing to the therapeutic effect on myocardial diseases. These results have led to the conclusion that compounds capable of interfering with the collagen fiber formation by inhibiting PCP might be generally applicable to the treatment and prevention of cardiac diseases associated with undesired myocardial fibrosis.

Thus, in a first aspect, the invention relates to a method for the treatment or prevention cardiac disease associated to myocardial fibrosis in a subject comprising the administration of a therapeutically effective amount of an agent which is capable of inhibiting PCP.

In the context of the present invention, PCP is understood as the type I procollagen carboxyterminal proteinase, a zinc protease secreted by fibroblasts in response to TGFβ activation. PCP belongs to the family of astacin-related proteases and is capable of cleaving the C-terminal peptide of type I, II and III collagens. Additionally, PCP is capable of activating lysyl oxidase, an enzyme essential for the cross-linking of the collagen fibers which stabiles the structure of collagen. Thus, PCP plays a double role in collagen formation since it converts procollagen precursor molecules into the units that can associate to form collagen fibers and promoter the cross-linking among the collagen fibers.

As used herein, "PCP inhibitor" is understood as a compound capable of inhibiting the capacity of PCP of activating type I, II and YYY collagen. Preferably, the PCP inhibitors useful in the context of the present invention show IC50 values within the range of about 0.001 μM to about 100 μM. Preferably, the inhibitors have IC50 values within the range of about 0.01 μM to about 10.0 μM, more preferably, between about 0.1 μM and 1 μM. Methods for determining the capacity of a compound of acting as a PCP inhibitor are widely available to the skilled person. These methods are usually based in determining the capacity of said compound in inhibiting the proteolytic activity of PCP on a peptide whose sequence resembles the target site of PCP on the type I procollagen peptide chain, releasing thereby a detectable fragment. Assays adequate for the determination of the PCP inhibitory activity include:

- Assays based on the detection of fluorescence emitted by fluorescent compounds released from fluorogenic peptides whose sequence is based in the sequence of the type I procollagen alpha2 such as DABCYL - Asp - Phe - Tyr - Arg - Ala - Asp - Gen - Pro - Arg - Asp (EDANS) - NH₂ (Beekman et al, 1996.,

FEBS Lett., 390:221-225), Dnp-Pro-β-cyclohexyl-Ala-Gly-CysCMeVHis-Ala- LyS(N-Me-AIa)-NH₂ (Bickett et al., Anal.Biochem, 212, 58-64, 1993) and DACMCys-Pro-Tyr-Gly-Asp-Glu-Pro-nLeu-Lys-FITC-OH (US2002169133).

- Assays based in the autoradiographic detection of the collagen formed from ¹⁴C labeled procollagen after separating both compounds by SDS-polyacrylamide gel electrophoresis (described en WO9705865).

- Assays based in the detection of the C peptide by immunological jeans (e.g. ELISA) using specific antibodies (described in WO9705865).

- Assays based in the capacity of a compound to promote release into the medium of a procollagen from a culture of a cell line which secretes collagen into the medium. These methods usually involved detection of procollagen in the conditioned media of a cell culture in the presence of the candidate compound (described in WO9705865). - Animal models of fϊbrotic deposits such as injury models in rats (Schilling et al., 1959, Surgery, 46:702-710), the estradiol-induced uterine expansion model (Mandell et al., 1982, J.Biol.Chem., 257:5268-5273), the induced angiogenesis model (Matrigel) (Passaniti et al., 1992, Lab. Invest. 67:519-528), hepatic fibrosis models (Tsukamoto et al., 1990, Seminar in Liver Disease 10:56-65; Kock-Weser, 1952, Lab. Invest. 1 :324-331; Marrione, 1949, Am. J. Pathol, 25:273-285; Tarns, 1957, Am. J. Pathol. 33:13-27; Wahl et al., 1986, J. Exp. Med. 163:884-902), pulmonary fibrosis models (Kelly et al., 1980, J. Lab. Clin. Med. 96: 954-964), arterial restenosis models (Jackson, 1994, Trends of Cardiovascular Medicine 4: 122- 130; Clowes et al., 1983, Lab. Invest. 9:327- 333), kidney fibrosis models (Yamarnoto et al., 1987, Kidney International 32:514-525), tendon reparation models (Franklin et al., 1986, J. Lab. Clin. Med. 108:103- 108), tumour growth models (Kiohs, et al., 1985, JNCL 75:353-359), trabeculectomy models (Lahery et al., 1989, Journal of Ocular Pharmacology 5:155-179) and abdominal adhesion model (Williams et al., 1992, J. Surg. Res. 2:65-70).

PCP inhibitors adequate in the context of the present invention include:

Phosphinate peptide analogues

These compounds, described in WO0307980, show a general structure of the formula

wherein Rl is hydrogen or a methyl group.

Sulfonamide hydroxamates

These compounds, described in US2003199520, show a general structure of the formula:

wherein Z is -OH, -NHOH, or OR¹²wherein R¹² is alkyl;

Rl is alkyl, haloalkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)-C(O)-X where X is alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, amino, monosubstituted amino, disubstituted amino, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl, heterocyclylalkyl, hydroxy, alkoxy, cycloalkoxy, cycloalkylalkoxy, heteroalkyloxy, aralkyloxy, or heteroaralkyloxy), or -C(=NR')NHSO,R" (where R' is hydrogen or alkyl, and R" is alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or heterocyclylalkyl); R is -CH(R²)Arl or -CH(R^CH=CHAr¹ where R² is hydrogen or alkyl; and ArI is aryl or heteroaryl; Ar² is either:

(i) a phenyl ring of formula:

wherein:

R³ and R⁷ are, independently of each other, hydrogen, alkyl, alkylthio, or halo; R⁴ and R⁶ are, independently of each other, hydrogen, alkyl, or halo; R⁵ is alkyl, haloalkyl, heterocyclyl, alkylthio, arylthio, aralkylthio, heteroarylthio, heteroaralkylthio, cycloalkylthio, cycloalkylalkylthio, alkoxy, aryloxy, aralkoxy, heteroaryloxy, heteroaralkyloxy, cycloalkoxy, cycloalkylalkoxy, alkyloxycarbonyl, hydroxy, halo, cyano, carboxy, nitro, amino, monoalkylamino, dialkylamino, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, heteroarylsulfonyl, heteroaralkylsulfonyl, cycloalkylsulfonyl, cycloalkylalkylsulfonyl, or -Y-(alkylene)-C(O)-Z [where Y is a bond, -NR^a-, -O-, or -S(O)_n- (where n is 0 to 2), R^a is hydrogen or alkyl, and Z is alkoxy, hydroxy, amino, monosubstituted amino, or disubstituted amino]; or R⁵ together with R4 forms -0-(CR⁸R⁹),- where n is 2 or 3 and each R⁸ and R⁹ are, independently of each other, hydrogen or alkyl; or the carbon atoms to which R⁵ and R⁴ are attached are fused to the C2-C3 carbons of a benzofurane ring; provided that at least two of R³, R⁴, R⁶, and R⁷ are not hydrogen at the same time; or (ii) a naphthyl ring of formula:

wherein:

R¹⁰ is hydrogen, alkyl, alkoxy, or halo; and

R¹¹ is hydrogen, alkyl, haloalkyl, alkylthio, alkoxy, alkyloxycarbonyl, aryloxy, hydroxy, halo, cyano, carboxy, nitro, amino, monoalkylamino, dialkylamino or alkylsulfonyl provided that both R¹⁰ and R¹¹ are not hydrogen at the same time. -heterocvclylpropanohidroxamic acid derivatives These compounds, described in US6645993, show a general structure of the formula:

wherein:

X is

alkylene or

alkenylene, each of which is unsubstituted or substituted by one or more fluorine atoms;

R is aryl,

cycloalkyl or

cycloalkenyl unsubstited or substituted by one or more fluorine atoms W is N,

X^I es hydrogen or

alkyl,

Y¹ is

alkyl, unsubstituted or substituted by aryl, or by one or more halogen atoms, with the proviso that when Y¹ is methyl, X¹ is not H; or Y¹ is aryl; or

Y¹ is a mono or bicyclic non-aromatic carbocyclic or heterocyclic moiety containing up to 10 ring atoms and which can include up to 3 ring heteroatoms, independently selected from N, O and S, which ring moiety is unsubstituted or substitued by one or more substituents independently selected from halogen, alkoxy and alkyl

unsubstituted or substituted by one or more halogen; aryl is a mono or bicyclic aromatic carbocyclic or heterocyclic moiety containing up to 10 ring atoms, and which can include up to 3 ring heteroatoms, independently selected from N, O and S, which ring moiety is unsubstituted or substituted by one or more substitutents, independently selected from halogen,

alkoxy and Ci-C₄ alkyl unsubstituted or substituted by halogen.

Other derivatives of the 3-heterocyclylpropanehydroxamic acid have been described in US2003/0069291 and show the general structure:

wherein:

X is alkylene or

C C alkenylene, each of which is unsubstituted or substituted by one or more fluorine atoms; R is aryl cycloalkenyl or cycloalkyl unsubstituted or substituted by one or

more fluorine atoms; wherein aryl comprises phenyl unsubstituted or substituted by one or more substituents independently selected from

alkyl) and C(O)p

alkyl) groups; wherein p=0, 1 or 2; [HET] is a divalent heterocyclic moiety selected from:

wherein Z is H or C^1"4 alkyl;

Y is a mono- or bicyclic unsaturated ring system containing from 5 to 10 ring atoms, of which up to 4 of said ring atoms are hetero-atoms independently selected from N, O and S, and said ring system is unsubstituted or substituted by one or more substituents independently selected from =0, C_1-4 alkyl, C_1-4 alkoxy, NR¹R², SO₂NR¹R², CO₂R¹, CO NR¹R², CH₂CO₂R¹, NR¹CO₂R², NR¹SO₂R²N, or het¹;

wherein R¹ and R² are each independently selected from H and Ci_4 alkyl unsubstituted or substituted by Ci_4 alkoxy; wherein het¹ is a N-linked 4- to 6-membered mono- or bicyclic heterocycle unsubstituted or containing 1 or 2 further hetero ring atoms independently selected from N and O, which heterocycle is unsubstituted or substituted by one or more substituents independently selected from =0, C_1-4 alkyl, C_1-4 alkoxy, NR¹R², SO₂NR¹R², CO₂R¹, CO NR¹R², CH₂CO₂R¹, NR¹CO₂R², NR¹SO₂R²N or het²; wherein het² is a N-linked 4- to 6-membered mono- or bicyclic heterocycle unsubstituted or containing 1 or 2 further hetero ring atoms independently selected from N and O.

Hydroxamate dipeptide derivatives

These compounds, described by Ovens et al (J.Peptide ScL, 2000, 6:489-495), are selected from the group of

and

Ox(adi)azolili hydroxaminc acid derivatives

These compounds, described in WO0147901, have a general structure of the formula:

wherein:

X is alkylene or alkenylene, each of which is optionally substituted by one

or more fluorine atoms;

R is aryl or

C C cycloalkyl optionally substituted by one or more fluorine atoms; W is N or CZ; Y and Z are each independently H, C₁-C₄ alkyl (optionally substituted by one or more substituents independently selected from halogen, S(O)pR⁶, OR⁵, CONR¹R², o CO₂R⁷ and aryl),

alkanoyl optionally substituted by one or more halogen,

alkoxycarbonyl optionally substituted by one or more halogen or CONR¹R², R¹ and R² are each independently selected from H cycloalkyl, alkyl

(optionally substituted by C₃-C₈ cycloalkyl, aryl, CO₂H, CO₂R⁵, and/or NR³R⁴), or R¹ and R² can be taken together with the nitrogen to which they are attached to represent a 4- to 6-membered heterocyclic ring optionally containing one or two further hetero atoms in the ring independently selected from N, O and S, which heterocyclic ring is optionally benzo- or pyrido-fused, and which heterocyclic ring is optionally substituted by

alkyl

⁵ aryl and/or NR³R⁴;

R³ and R⁴ are each independently selected from H, C

alkyl or

alkoxycarbonyl optionally substituted by one or more halogen, or R³ and R⁴ can be taken together with the nitrogen atom to which they are attached to represent a morpholine, piperidine, azetidine or piperazine (optionally N-substituted by

Ci-C₄ alkyl)

R⁵ is alkyl optionally substituted by CO₂R⁷, CONR³R⁴, o R⁵ is aryl;

R⁶ is

alkyl optionally substituted by one or more halogen, or aryl; R⁷ is H or R⁶; p is 0, 1 or 2,

"Aryl" is a mono- or bicyclic aromatic carbocyclic or heterocyclic system comprising from 5 to 10 ring atoms, including up to 3 hetero-atoms selected from N, O and S, where, if there is a N atom in the ring, it can be present as the N-oxide, which ring system is optionally substituted by up to 3 substituents independently selected from halogen, Ci-C₄ alkyl optionally substituted by one or more halogen, Ci-C₄ alkoxy optionally substituted by one or more halogen, phenyl, pyridyl, CO₂H,

CONR³R⁴, CO₂(Ci-C₄ alquilo), NR³R⁴, OH y OC(O)(Ci-C₄ alquilo).

Peptide analogs

These inhibitors have been described in WO9705865 and in US20020169133 and comprise the following families of compounds:

(i) Family A, formed by compounds of the general formula

o

wherein:

Ri is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo substitituted aralkyl, heteroaralkyl, biaryl, biarylalky, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalAyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(tio, sulfmyl or sulfonyl)-alkyl;

R₂ is selected from the group consisting of H, lower alkyl; R₃ is selected fϊom the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo substitituted aralkyl, heteroaralkyl, biaryl, biaryialkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl; R₄ is selected from the group consisting of aryl, heteroaryl, alkyl, aralkyl, heteroaralkyl, alkylamino, arylalkylamino;

X is selected from the group consisting of SO₂, C=O;

Y is selected from the group consisting of OH, HOHN (hydroxylamine), H₂N, alkylamino; Z s a direct bond; methylene, oxygen, sulfur, amino; and n is 0 or 1.

(ii) Family B formed by compounds having the follosing general formula:

wherein

Ri is selected from the group consisting of H, lower alkyl, mono- or poly-haloalhyl, carboxyalkyl, aryi, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoallcyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfϊnyl or sulfony l)-alkyl;

R₂, is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfony l)-alkyl;

R₃ is selected fϊom the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalk~l, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialLylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalAylalkyl, heterocycloalkylalkyl , alkyl-(thio, sulfϊnyl or sulfony l)-alkyl;

R₄, is selected from the group consisting of H, lower alkyl; and

R₅ is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylarnino)alkyl, alkyl-(thio, sufϊnyl or sulfonyl)alkyl, alkoy alky lacy lalkyl.

(iii) Family C formed by compounds having the following structural formula:

wherein Ri is selected from the group consisting of H, lower alkyl, mono- or poly-haloalhyl, carboxyalkyl, aryi, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoallcyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfϊnyl or sulfony l)-alkyl;

R₂, is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfony l)-alkyl;

R₃ is selected fϊom the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalk~l, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialLylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalAylalkyl, heterocycloalkylalkyl , alkyl-(thio, sulfmyl or sulfony l)-alkyl;

R₄, is selected from the group consisting of H, lower alkyl; and

R₅ is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylarnino)alkyl, alkyl-(thio, sufϊnyl or sulfonyl)alkyl, alkoy alky lacy lalkyl.

and (iv) Family D, formed by compounds having the following general formula:

wherein

Ri is selected from the group consisting of H, lower alkyl, mono- or poly-haloalhyl, carboxyalkyl, aryi, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoallcyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfϊnyl or sulfony l)-alkyl;

R₂, is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfϊnyl or sulfony l)-alkyl;

R₃ is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalk~l, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalAylalkyl, heterocycloalkylalkyl , alkyl-(thio, sulfmyl or sulfony l)-alkyl;

R₄, is selected from the group consisting of H, lower alkyl; and

R₅ is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylamino)alkyl, alkyl-(thio, sufϊnyl or sulfonyl)alkyl, alkoyalkylacylalkyl.

(v) Family E, formed by compounds having the following general formula

wherein

R¹ and R⁴ are, independently of each other, hydrogen or alkyl; R² is

(i) cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heterocyclyl or heterocycloalkyl; or (ii) -(alkylene)-B-X where B is -0-, -NR⁸-, -S(O)_n,- (where n is 0, 1 or 2), - C=O, -CONR⁸-, -NR⁸CO₂-, NR⁸SO₂- o -C(=NR⁸)NR⁸SO₂- (where R⁸ is H or alkyl), and X is cycloalkyl, cycloalkylalkyl, aryl, aralkyl heteroaryl or heteroaralkyl; or (iii) -(alkylene)-B-X where B is -NR⁸-C0- (where R⁸ is H or alkyl), and X is cycloalkyl, cycloalkylalkyl, aryl, aralkyl heteroaryl or heteroaralkyl; or

(iv) and R² and R³ form an alkylene or heteroalkylene chain: R³ is hydrogen or alkyl R⁶ is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl or heteroaralkyl;, R⁵ is

(i) hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heterocycloalkyl, heteroalkyl, or - (alkylene)-C(O)-X¹ where X¹ is alkyl, hydroxy, alkoxy, aryl, aralkyl, aryloxy, aralkyloxy, heteroaryl, heteroaryloxy, heteroaralkyloxy or NRR" (where R and R" are independently H or alkyl, or R and R" form an alkylene chain); or

(ii) R⁵ and R⁴ form an alkylene chain; or (iii) R⁵ and R⁶ form an alkylene chain; n is 0 ό 1

A is -C(=O)-CH(R⁹)-(CH₂)_m-NR¹⁰- wherein m is an integer from 0-5 inclusive;

R⁹ is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, hetergcycloalkyl, heteroalkyl, or -(alkylene)-C(O)-X¹ where X¹ is alkyl, hydroxy, alkoxy, aryl, aralkyl, aryloxy, aralkyloxy, heteroaryl, heteroaryloxy, heteroaralkyloxy or NRR" (where R and R" are independently H or alkyl, or R and R" form an alkylene chain) R¹⁰ is hydrogen, alkyl, aralkyl or heteroaralkyl; Z is Y-B wherein

Y is alkylene or a bond; and B is -CO-, -C(O)O-, -CONR⁸-, -SO₂-, o -SO₂NR⁸- (wherein R⁸ is hydrogen or alkyl), alkylene (optionally substituted by hydroxy, alkoxy, amino, monoalkylamino or dialkylamino) or a bond;

R⁷ is cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl or heteroaralkyl; provided that when n=0 and Z is SO₂, then R² does not contain an imidazole group

The application relates to methods of treatment using the compounds as defined above, alone or in combination of one or more as well as methods of treatment using their salts, solvates, hydrates or prodrugs.

Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example include those mentioned by Berge et al, in J. Pharm. Sci., 66, 1-19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, pamoate, carasylate, and p- toluenesulphonate salts.

Pharmaceutically acceptable base addition salts are well known to those skilled in the art, and for example include those mentioned in the art cited above, and can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium and zinc salts, as well as alkaline organic salts such as diethanolamine, ethanolamine, triethanolamine, glucamine and basic amino acid salts.

Certain of the PCP inhibitory agents suitable for the methods of the invention may exist in one or more zwitterionic forms. Certain of the compounds may exist in one or more tautomeric forms. Certain of the compounds, their salts, solvates, prodrugs, etc. may exist in one or more polymorphic forms. It is to be understood that the methods of the invention contemplate the use of all such zwitterions, tautomers and polymorphs of the compounds mentioned above. Certain of the PCP inhibitory compounds suitable for use in the methods of the invention can be used in crystalline form or as solvates (e. g. hydrates) and it is intended that both forms be within the scope of the present invention. The term "solvate" defines a complex of variable stoichiometry formed by a solute (in this invention, a compound of the invention) and a solvent. Such solvents should not interfere with the biological activity of the solute. Methods of solvation are generally known within the art.

Moreover, the compounds for use in the methods of the invention, their salts, hydrates, prodrugs etc. can exhibit isotopic variation, e.g. forms with enriched ²H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O and the like may be prepared, for example by suitable variation of the synthetic methods described herein using methods and reagents known in the art or routine modification thereof. All such isotopic variants are included in the scope of the invention.

Prodrug moieties are well-known to those skilled in the art (see for example the article by H Feres, in Drugs of Today, vol 19, no.9 (1983) pp.499-538, especially section Al), and for example include those specifically mentioned in A.A. Sinkula's article in Annual Reports in Medicinal Chemistry, vol 10, chapter 31, pp.306-326, herein incorporated by reference, and the references therein. Specific prodrug moieties which may be specifically mentioned are aliphatic-aromatic, carbonate, phosphate and carboxylic esters, carbamates, peptides, glycoside, acetals and ketals, tetrahydropyranyl and silyl ethers. Such prodrug moieties can be cleaved in situ, e.g. are hydrolysable in physiological conditions, to give compounds listed above.

Certain of the compounds suitable for the methods of the invention may exist as geometric isomers. The compounds of the formula (I) may possess one or more asymmetric centers, apart from the specified centers in formula (I), and so exist in two or more stereoisomeric forms. The present invention includes all the individual stereoisomers and geometric isomers of the compounds and mixtures thereof.

The compounds listed above or their salts or solvates are preferably in pharmaceutically acceptable or substantially pure form. By pharmaceutically acceptable form is meant, inter alia, having a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels. Purity levels for the drug substance are preferably above 50%, more preferably above 70%, most preferably above 90%. In a preferred embodiment, the purity is above 95% of the compound or of its salts, solvates or prodrugs.

In general, the compounds used in the methods of the invention will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compounds are outweighed by the therapeutically beneficial effects. The actual amount of the compound i.e., the active ingredient, will depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors. The drug can be administered more than once a day, preferably once or twice a day. Therapeutically effective amounts of compounds used in the methods of the invention may range from approximately 0.05-35 mg per kilogram body weight of the recipient per day; preferably about 0.3-20 mg/kg/day. Thus, for administration to a 70 kg person, the dosage range would most preferably be about 21 mg to 1.4 g per day.

The choice of the optimal route of administration of the pharmaceutical compositions will be influenced by several factors including the physico-chemical properties of the active molecules within the compositions, the urgency of the clinical situation and the relationship of the plasma concentrations of the active molecules to the desired therapeutic effect. For instance, if necessary, the PCP inhibitors may be prepared with carriers that will protect them against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can inter alia be used, such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, polyorthoesters, and polylactic acid. Furthermore, it may be necessary to coat the PCP inhibitors with, or coadminister the agonistic binding molecules with, a material or compound that prevents the inactivation of the PCP inhibitors. For example, the PCP inhibitors may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.

The routes of administration of the PCP inhibitors can be divided into two main categories, oral and parenteral administration. These two categories include, but are not limited to, bolus, buccal, epidermal, epidural, inhalation, intra- abdominal, intra-arterial, intra-articular, intrabronchial, intracapsular, intracardiac, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebronventricular, intracolic, intracervical, intradermal, intragastric, intrahepatic, intramedullary, intramuscular, intramyocardial, intranasal, intra-ocular intra-orbital, intra-osteal, intrapelvic, intrapericardiac, intraperitoneal, intraplaque, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasternal, intrasynovial, intrathecal, intrathoracic, intratumoral, intra-uterine, intravenous, intraventricular, intravesical, rectal, spinal, subarachnoid, subcapsular, subcutaneous, subcuticular, sublingual, topical, transdermal, and transmucosal, transtracheal, vaginal administration. The preferred manner of administration is systemic using a convenient daily dosage regimen which can be adjusted according to the degree of affliction.

Intranasal delivery is typically accomplished with dry powder formulations, liquid solutions or suspensions suitable for nebulization or with aerosol propellants suitable for use in a metered dose inhaler. Alternatively, drug substance may be associated with microspheres made of materials such as gelatin, dextran, collagen or albumin The microspheres are conveniently delivered in freeze dried form with a nasal insufflator device or a pressurized aerosol cannister.

Penetration enhancers such as amphiphilic steroids may also be used as additives to increase the systemic absorption of the drug into the tissue. Effective administration may also be accomplished by pulmonary or respiratory delivery since polypeptides are readily absorbed through the cellular lining of the alveolar region of the mammalian lung. Advantageously, such administration frequently does not require the use of penetration enhancers as additives. Devices and methods for pulmonary delivery deep into the lung are described in U.S. Pat. No. 5,780,014, issued JuI. 14, 1998 and U.S. Pat. No. 5,814,607, issued Sep. 29, 1998. Oral dosage forms can be formulated inter alia as tablets, troches, lozenges, aqueous or oily suspensions, dispersable powders or granules, emulsions, hard capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, liquids, gels, or slurries. These formulations can contain pharmaceutically excipients including, but not limited to, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid ; binding agents such as starch, gelatin or acacia; lubricating agents such as calcium stearate, glyceryl behenate, hydrogenated vegetable oils, magnesium stearate, mineral oil, polyethylene glycol, sodium stearyl, fumarate, stearic acid, talc, zinc stearate; preservatives such asn-propyl-p-hydroxybenzoate ; colouring, flavouring or sweetening agents such as sucrose, saccharine, glycerol, propylene glycol or sorbitol; vegetable oils such as arachis oil, olive oil, sesame oil or coconut oil; mineral oils such as liquid parafϊn; wetting agents such as benzalkonium chloride, docusate sodium, lecithin, poloxamer, sodium lauryl sulfate, sorbitan esters; and thickening agents such as agar, alginic acid, beeswax, carboxymethyl cellulose calcium, carageenan, dextrin or gelatin.

The pharmaceutical compositions of the present invention can also be formulated for parenteral administration. Formulations for parenteral administration can be inter alia in the form of aqueous or non-aqueous isotonic sterile non-toxic injection or infusion solutions or suspensions. Preferred parenteral administration routes include intravenous, intraperitoneal, epidural, intramuscular and intratumoral injection or infusion. The solutions or suspensions may comprise agents that are non-toxic to recipients at the dosages and concentrations employed such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic sodium chloride solution, oils such as synthetic mono-or diglycerides or fatty acids such as oleic acid, local anaesthetic agents, preservatives, buffers, viscosity or solubility increasing agents, water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like, oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like, and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Lastly, compounds may be systemically administered by transdermal delivery, which typically involves placing the drug on the surface of the skin and allowing it to permeate through the skin. Transdermal delivery devices employ a structure such as an adhesive patch or the like that serves as a reservoir for the drug and brings the drug into diffusive contact with the skin. In one general type, the structure is a three dimensionally stable matrix known as a monolithic matrix. Such matrices are described in more detail in U.S. Pat. Nos. 5,804,214, 5,149,538 and 4,956,171 which describe matrices made of polymers and copolymers of acrylic latexes, acrylic esters, methacrylic esters and vinyl acetates.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

The choice of formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills or capsules are preferred) and the bioavailability of the drug substance. Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a cross-linked matrix of macromolecules. U.S. Pat. No. 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability.

The compositions are comprised of in general, a compound of formula (I) in combination with at least one pharmaceutically acceptable excipient. Acceptable excipients are non-toxic, aide administration, and do not adversely affect the therapeutic benefit of the compound of formula (I). Such excipient may be any solid, liquid, semisolid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.

Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols.

Compressed gases may be used to disperse a compound of this invention in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc.

Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).

The amount of the compound in a formulation can vary within the full range employed by those skilled in the art. Typically, the formulation will contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt % of a compound of formula I based on the total formulation, with the balance being one or more suitable pharmaceutical excipients. Preferably, the compound is present at a level of about 1-80 wt %. The diseases that can be treated using the method of the invention include any disorder wherein there is an abnormal increase in the amount of collagen fibres in the myocardium. Preferably, Said disease is selected from the group of chronic cardiac failure and HHT.

The authors of the present invention have also shown that measuring the activity of the parameters associated to myocardial fibrosis in a subject suffering from a cardiac disease associated to myocardial fibrosis provides a clear indication as to whether said subject is candidate for treatment with a PCP inhibitor. Thus, in a further aspect, the invention relates to a method for providing a personalized therapy for the treatment of a patient suffering from a cardiac disease associated to myocardial fibrosis comprising:

(i) determining in a sample from said patient a parameter indicative of the PCP activity and (ii) if the value of said parameter is higher than a predefined threshold value, then the patient is candidate to the treatment with a therapeutically effective amount of a PCP inhibitory agent.

Parameters indicative of PCP activity are known to the skilled person. By way of example, said parameters can be selected from the group of:

(i) PICP levels,

(ii) PCP activity,

(iii) PCP levels, (iv) ratio of active PCP and PCP zymogen,

(v) PCPE levels and

(vi) a combination of one or more of (i) to (v).

PICP or carboxy terminal propeptide of type I procollagen corresponds to the peptide which is released from the procollagen type I alpha subunit upon cleavage of this molecule by PCP (see figure 1). Since PP activity is necessary for the generation of PICP, the levels of this peptide in a sample from a patient will show a positive correlation with the activity of PCP in said subject. Thus, increased PICP will be indicative of an increase fibrosis and hence, that the patient might benefit from a therapy aimed at inhibiting PCP activity. Techniques suitable for detecting PICP in a sample include any technique capable of quantitative determination of a protein in a sample. Usually, these assays are immunoassays using anti-PICP specific monoclonal or polyclonal antibodies. Adequate techniques include RIA, ELISA, Western blotting, immunofluorescence, immunoturbidimetry, nepehelometry, turbidimetry, latex- amplified nepehelometry. The RIA methods described by Melkko et al., (Clin.Chem., 1990, 36:1328-1332) and in GB2245568 are particularly preferred.

PCP activity in a sample of the patient can also be used as a parameter indicative on whether the patient will benefit from the treatment with a PCP inhibitor. Methods for determining PCP activity have been explained in detail above.

PCP levels and the ratio of mature PCP to PCP zymogen are also indicative of the activity of said protein since PCP is synthesized as a zymogen which requires proteolytic cleavage for activation. Thus, by detecting the level of PCP using techniques which allow distinguishing it from the inactive zymogen) or by measuring the ratio of

PCP to PCP zymogen, it is possible to conclude whether the patient can be a candidate for the treatment with a PCP inhibitor. The skilled person will appreciate that the determination of the levels of PCP must be carried out using techniques which allow the specific detection of said protein while avoiding the simultaneous detection of the inactive zymogen. Preferably, the determination of PCP and the PCP zymogen in a biological sample is carried out after fractionating the proteins of said sample based on their size followed by immunodetection with an antibody capable of binding to both PCP and to the zymogen. By detecting both proteins on the same experiment, it is possible to calculate the expression ratios.

In another aspect, the parameter indicative of PCP activity is the amount of the PCP enhancer (PCPE), a glycoprotein that binds PICP (Kessler, E. and Adar,R. 1989, Eur.J.Biochem., 186:115-121) and that stimulates PCP activity (Adar, R. et al., 1986, Collagen Relat. Res. 6:267-277). The concentration of PCPE is an indication of the PCP overall activity and thus, by measuring the amount of PCPE activity, it is possible to determine the activity of PCP. PCPE is measured using any of the methods known in the art for quantitative determination of a protein in a sample. Preferably, the levels of PCPE are determined by immunoassays using anti-PCPE specific monoclonal or polyclonal antibodies. Adequate techniques include RIA, ELISA, Western blotting, immunofluorescence, immunoturbidimetry, nepehelometry, turbidimetry and the like. Preferably, PCPE is determined by Western blot as shown in fig. 3.

The skilled person will also appreciate that the different parameters indicative of PCP activity outlined above need not be taken in consideration in isolated manner but that it is possible to determine at least two, preferably three, more preferably four and even more preferably five different parameters from said sample and compare all of them to the reference threshold values in order to determine the PCP activity of the sample.

The determination of the parameters indicative of PCP activity is carried out in any biological sample that contains sufficient amount of the proteins so that their presence can be detected either by measuring expression levels or by determining the activity. The different parameters indicative of PCP activity can be determined in myocardial biopsies. However, it is preferred to determine the parameters on samples which do not require invasive methods for their preparation. In a preferred embodiment, the parameter indicative of PCP activity is the PICP levels and said PCIP levels are determined in serum or plasma.

The skilled person will appreciate that the determination of the expression level of a given protein can only be informative when considered in respect to reference values. Typically, reference values are those obtained from similar samples from patients which do not show any signs of myocardial fibrosis. Thus, in another aspect, the invention relates to the methods of determining parameters associated to myocardial fibrosis using wherein said values are calculated in relation to a threshold value wherein said threshold value is the value of the parameter indicative of PCP activity in a patient which does not suffer of cardiac disease associated to myocardial fibrosis.

In a preferred embodiment, the PCP inhibitor which is selected based on the value of the parameter indicative of PCP activity is torasemide. Torasemide is a pyridine- sulfonylurea type loop diuretic having the following formula:

In another embodiment, the cardiac disease associated to myocardial fibrosis is selected from the group of chronic cardiac failure and HHT.

The invention is described hereinafter with the following examples which are to be construed as illustrative and in no way limitative of the scope of the invention.

EXAMPLES

Methods

Patients and study protocols. This was an individually randomized, open-label, parallel-group pilot study. AU subjects gave written, informed consent before participating in the study. The investigation conformed to the principles outlined in the Declaration of Helsinki. The study protocol was approved by the Institutional Ethics Committee of the Donostia University Hospital.

The study population consisted of 22 white patients. All patients were required to have a previous diagnosis of chronic HF by the presence of a least 1 major and 2 minor criteria of the Framingham study (Ho et al, 1993, J.Am.Coll.CardioL, 22 Suppl A:6A-13A) during the last 6 months. Whereas 80% of the patients enrolled had hypertensive heart disease, the remaining 20% showed ischemic heart disease. None of the patients had suffered from previous myocardial infarction. A depressed ejection fraction (<0.40) was observed in 55% of patients. Although all patients were clinically stable, they were randomly assigned to receive either torasemide or furosemide in accordance with recent data (Faris et al, 2002, Int.J.CardioL, 82:149-158). After randomization, 11 patients were assigned to torasemide 10 to 20 mg daily (torasemide ! group) and 11 patients to furosemide 20 to 40 mg daily (furosemide group) for 8 months. The doses were used in accordance with current guidelines for patients with chronic HF (Hunt et al., 2005, J.Am.Coll. Cardiol., 46:el-82). Existing recommended salt intake restriction (4 g/day) and concomitant HF medications (i.e., an angiotensin-converting enzyme inhibitor, or an angiotensin-receptor antagonist, and a beta-adrenergic blocker) were continued during the study (Hunt et al., 2005, J.Am.Coll.CardioL, 46:el-82). None of the patients were treated with aldosterone antagonists.

A number of studies, including endomyocardial biopsy, were performed in each patient at enrollment (baseline) and 8 months after randomization. A group of 12 normotensive subjects (7 men and 5 women, mean age 56 years, range 39 to 72 years) were used as control subjects for histomorpho logic and biochemical studies. They were subjects with clinically presumed coronary artery disease who were found to lack the disease at a coronary angiography.

Echocardiographic assessment. Two-dimensional echocardiographic imaging, targeted M-mode recordings, and Doppler ultrasound measurements were obtained in each patient as recommended (Sahn et al., 1978, Circulation, 58:1072-1083). Left ventricular mass was measured, and left ventricular mass index was calculated by dividing left ventricular mass by body surface area. The following pulsed Doppler measurements were obtained: maximum early transmittal velocity in diastole, maximum late transmittal velocity in diastole, the deceleration time of the early mitral filling wave, and isovolumic relaxation time. Ejection fraction was calculated according to

Quinones et al. (Quinones et al, 1978, Chest, 74:59-65).

Biochemical determinations. Venous blood samples were drawn at 09:00 h in an upright position. Plasma aldosterone was measured by radioimmunoassay using a commercial kit. Serum PICP was determined by radioimmunoassay according to a method previously described (Querejeta et al., Circulation, 2000, 101 :1729-1735). Histomorphologic and immunohistochemical studies.

Three transvenous endomyocardial biopsies were taken from the middle area of the interventricular septum with a bioptome Cordis 96 cm (7-F) under fluoroscopic guidance after angiographic examination. The CVF was determined by quantitative morphometry with an automated image analysis system in sections stained with collagen- specific picrosirius red, as previously reported (Diez et al, 1995, Circulation, 91 :1450-1456). Immunohistochemical analysis for PCP and PCPE was performed on formalin-fixed and paraffin-embedded sections. Immunohistochemical staining was performed by the avidin peroxidase-labeled dextran polymer method. Positive staining was visualized with DAB Plus (Boehringer Mannheim Corp., Indianapolis, Indiana), and tissues were counterstained with Harris hematoxylin (Sigma, St. Louis, Missouri). A mouse monoclonal antibody against PCP (dilution 1 :100; R8cD Systems, Abingdom, England) and PCPE (dilution 1 : 100; R8cD Systems) was used as the primary antibody.

Western blot studies. A 5-pg sample of total protein obtained from transvenous endomyocardial biopsies was processed for Western blot as recently described (Lopez et al., 2006, J.Am.Coll.CardioL, 48:89-96). Specific rabbit polyclonal antibodies against PCP (R&D Systems, specificity 95%), PCPE (R&D Systems; specificity 100%), MMP- 1 (Oncogene, Cambridge, Massachusetts; specificity 100%) and TIMP-I (Chemicon, Hofheim, Germany, specificity 95%) were incubated at dilutions of 1 :500, 1 :100, 1 :2,000, and 1 :200, respectively. Bands were detected by peroxidase-conjugated secondary antibodies (Amersham Biosciences, Barcelona, Spain) and visualized with the ECL-Plus chemiluminescence system (Arnersham Biosciences). Autoradiograms were analyzed using an automatic densitometer (Molecular Imager FX, Bio-Rad, Barcelona, Spain). The blots were also probed with a monoclonal beta-actin antibody (Sigma) as a control for loading. Data are expressed as arbitrary densitometric units relative to beta-actin expression.

Reverse transcriptase-polymerase chain reaction (RTPCR) study. The mRNA levels of the al chain of procollagen type I were analyzed by real-time quantitative RT- PCR as recently described (Goikoetxea et al., 2006, Cardiovasc. Res., 69:899-907). Reverse transcription was performed with 200 pg of total RNA by using Superscript 111 reverse transcriptase (Invitrogen, Eugene, Oregon). Real-time PCR was performed with an ABI PRISM 7000 Sequence Detection System according to the manufacturer's recommendations (Applied Biosystems, Madrid, Spain) by using specific TaqMan MGB fluorescent probes for human mRNA of the αl chain of procollagen type I (HsOO 176329), and a specific TaqMan MGB fluorescent probe for human constitutive 18s ribosomal RNA as endogenous control. Data are expressed as arbitrary units relative to 18s ribosomal RNA.

Statistical analysis. Differences in parameters between normotensive controls and the whole group of HF patients at baseline and between the 2 groups of HF patients at baseline and after treatment (absolute values and deltas) were tested using a Student t test for unpaired data once normality was shown (Shapiro-Wilks test); otherwise, a nonparametric test (Mann- Whitney U test) was used. Differences in parameters before and after treatment within each group of patients were tested by a Student t test for paired data once normality was shown (Shapiro-Wilks test); otherwise, a nonparametric test (Wilcoxon test) was used. The correlation between continuously distributed variables was tested by univariate regression analysis and bivariate association (Spearman coefficient). Data are expressed as mean value + SEM. A value of p < 0.05 was considered statistically significant.

Results

Baseline characteristics. Baseline clinical and echocardiographic characteristics of the 2 groups of patients are presented in the Table 1.

Table 1. Effects of treatment on Clinical Parameters assessed in the 2 groups of patients with chronic failure

No significant differences were observed between the groups in the parameters tested. As shown in Figure 2, 2 PCP bands of 112 and 96 kDa corresponding to zymogen and active form, respectively, were identified in myocardial samples from all patients. In addition, the 55 -kDa 111 -length and the 36-kDaproteolytic fragment of PCPE were also detected in all samples (Fig. 3). Figure 4 shows that although PCP and PCPE were mostly expressed in fibroblasts and areas of interstitial and panvascular fibrosis, these molecules also were present in cardiomyocytes. Myocardial parameters evaluated in the normotensive group and the whole group of HF patients are presented in Table 2.

Table 2. Clinical and Cardiac Parameters assessed in normotensive subjects and in the whole group of patients with chronic cardiac failure.

As expected, values related to collagen expression and deposition, as well as PICP values, were higher in patients than in normotensive subjects. In addition, PCP zymogen was decreased and PCP activation, assessed as the ratio between its active form and its zymogen, and 36-kDa PCPE were increased in HF patients compared with normotensive subjects. No significant differences were found between the 2 groups of patients in the baseline values of parameters assessing myocardial fibrosis and collagen type I synthesis and degradation (Table 3). The PCP activation did tend to be higher in the torasemide group (2.59 ± 0.21) than in the furosemide group (2.14 ± 0.16), but the difference did not reach statistical significance. A positive correlation (r =0.687, p < 0.001) was found between plasma aldosterone and PCP activation in all patients (Fig. 5). Effects of treatment. GENERAL ASPECTS. Eight months after randomization, patients in the torasemide group (n = 11) and the furosemide group (n = 11) received mean daily dosages of 10.9 ± 0.6 mg and 34.5 ± 3.8 mg of these agents, respectively. Baseline medications other than loop diuretics were maintained unchanged during the treatment period in the 2 groups of patients. No adverse effects occurred during the study in either group. The frequency of complications (including hospitalizations and exacerbations of HF) was similar in the 2 groups (data not shown).

CLINICAL AND ECHOCARDIOGRAPHY DATA. As shown in Table 1, the changes in body weight and blood pressure were similar in the 2 groups of patients. The values of left ventricular end-diastolic volume showed a nonsignificant trend toward a decrease in torasemide-treated but not in furosemide-treated patients (Table 1). The values of left ventricular ejection fraction showed a non- significant trend toward an increase in torasemide-treated but not in furosemide-treated patients (Table 1). The number of patients showing improvement of at least 1 grade in New York Heart

Association functional class was greater (p < 0.01) in the torasemide group than in the furosemide group.

MYOCARDIAL FIBROSIS. Although CVF decreased (p < 0.01) in the torasemide- treated patients, it remained unchanged in furosemide-treated patients (Table 3). In addition, CVF was lower (p < 0.01) in torasemide-treated patients than in furosemide- treated patients. Furthermore, the delta for this parameter was also different between the 2 groups (torasemide-treated patients -43.20 ± 6.40%, furosemide-treated patients -4.11 + 7.30%, p < 0.05).

Table 3. Effects of treatment on parameters related to myocardial fibrosis and collagen type I synthesis and degradation assessed in the 2 groups of patients with chronic heart failure.

PARAMETERS RELATED TO COLLAGEN TYPE I SYNTHESIS AND DEGRADATION. As shown in Table 3, the expression of al(l) mRNA decreased (p < 0.05) to a similar extent in the 2 groups of treated patients. Whereas administration of torasemide was associated with a reduction (p < 0.01) in serum PICP, this parameter did not change in the furosemide group (Table 3). In addition, serum PICP measured 8 months after randomization was lower (p < 0.01) in the torasemide group than in the furosemide group. The delta for this parameter was different between the 2 groups (torasemide-treated patients -19.30 ± 3.30%, furosemide-treated patients -4.12 ± 6.40%, p < 0.05). A positive correlation was found between changes in serum PICP and changes in CVF (r = 0.692, p < 0.01) in torasemide-treated patients (Fig. 6). No significant changes in either MMP-I or TIMP-I were observed in the 2 groups with treatment (Table 3).

PCP AND PCPE EXPRESSION. The expression of both PCP zymogen and PCP active form increased (p < 0.05) in the furosemide group, but remained unchanged in the torasemide group (Table 3, Fig. 2). Whereas PCP activation remained unchanged in furosemide-treated patients (1.96 ± 0.16), it decreased (p < 0.05) in torasemide-treated patients (2.14 + 0.19). The expression of full-length PCPE did not change with treatment in either group of patients (Table 3, Fig. 3). The 36-kDa PCPE fragment decreased (p < 0.05) in torasemide-treated patients and remained unchanged in furosemide-treated patients (Table 3, Fig. 3). Changes in PCP activation were positively correlated with both changes in CVF (r = 0.876, p < 0.001) (Fig. 7A) and changes in serum PICP (r = 0.897, p < 0.001) (Fig. 7B) in torasemide -treated patients. In addition, a positive correlation (r = 0.588, p < 0.05) was found between final values of plasma aldosterone and the inhibition of PCP activation in torasemide -treated patients. These correlations were not found in furosemide-treated patients. No other correlations were found among the remaining parameters tested in this study.

Claims

1. Method for the treatment or prevention of a cardiac disease associated to myocardial fibrosis in a subject comprising the administration of a therapeutically effective amount of an agent which is capable of inhibiting PCP.

2. Method according of claim 1 wherein the PCP inhibitory agent is selected from the group of:

(a) a compound having the general formula

wherein Rl is hydrogen or a methyl group.

(b) A compound having a general structure of the formula:

wherein

Z is -OH, -NHOH, or OR¹²wherein R¹² is alkyl; Rl is alkyl, haloalkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heterocyclylalkyl, cycloalkylalkyl, -(alkylene)- C(O)-X where X is alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, amino, monosubstituted amino, disubstituted amino, aryl, aralkyl, heteroaryl, heteroaralkyl, heterocyclyl, heterocyclylalkyl, hydroxy, alkoxy, cycloalkoxy, cycloalkylalkoxy, heteroalkyloxy, aralkyloxy, or heteroaralkyloxy), or - C(=NR')NHSO,R" (where R is hydrogen or alkyl, and R" is alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or heterocyclylalkyl); R is -CH(R²)Arl or -CH(R^CH=CHAr¹ where R² is hydrogen or alkyl; and ArI is aryl or heteroaryl; Ar² is either: (i) a phenyl ring of formula:

wherein:

R³ and R⁷ are, independently of each other, hydrogen, alkyl, alkylthio, or halo;

R⁴ and R⁶ are, independently of each other, hydrogen, alkyl, or halo;

R⁵ is alkyl, haloalkyl, heterocyclyl, alkylthio, arylthio, aralkylthio, heteroarylthio, heteroaralkylthio, cycloalkylthio, cycloalkylalkylthio, alkoxy, aryloxy, aralkoxy, heteroaryloxy, heteroaralkyloxy, cycloalkoxy, cycloalkylalkoxy, alkyloxycarbonyl, hydroxy, halo, cyano, carboxy, nitro, amino, monoalkylamino, dialkylamino, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, heteroarylsulfonyl, heteroaralkylsulfonyl, cycloalkylsulfonyl, cycloalkylalkylsulfonyl, or -Y-(alkylene)-C(O)-Z [where Y is a bond, -NR^a-, -O-, or -S(O)_n- (where n is 0 to 2), R^a is hydrogen or alkyl, and Z is alkoxy, hydroxy, amino, monosubstituted amino, or disubstituted amino]; or R⁵ together with R4 forms -0-(CR⁸R⁹),- where n is 2 or 3 and each R⁸ and R⁹ are, independently of each other, hydrogen or alkyl; or the carbon atoms to which R⁵ and R⁴ are attached are fused to the C2-C3 carbons of a benzofuran ring; provided that at least two of R³, R⁴, R⁶, and R⁷ are not hydrogen at the same time; or (ii) a naphthyl ring of formula:

wherein:

R¹⁰ is hydrogen, alkyl, alkoxy, or halo; and R¹¹ is hydrogen, alkyl, haloalkyl, alkylthio, alkoxy, alkyloxycarbonyl, aryloxy, hydroxy, halo, cyano, carboxy, nitro, amino, monoalkylamino, dialkylamino or alkylsulfonyl provided that both R¹⁰ and R¹¹ are not hydrogen at the same time.

(C) a compound having a general structure of the formula:

wherein:

X is alkylene or alkenylene, each of which is unsubstituted or substituted

by one or more fluorine atoms;

R is aryl, cycloalkyl or cycloalkenyl unsubstited or substituted by one or

more fluorine atoms W is N,

X¹ es hydrogen or Ci -C₄ alkyl,

Y¹ is Ci-C₄ alkyl, unsubstituted or substituted by aryl, or by one or more halogen atoms, with the proviso that when Y¹ is methyl, X¹ is not H; or Y¹ is aryl; or

Y¹ is a mono or bicyclic non-aromatic carbocyclic or heterocyclic moiety containing up to 10 ring atoms and which can include up to 3 ring heteroatoms, independently selected from N, O and S, which ring moiety is unsubstituted or substitued by one or more substituents independently selected from halogen,

alkoxy and alkyl

unsubstituted or substituted by one or more halogen; aryl is a mono or bicyclic aromatic carbocyclic or heterocyclic moiety containing up to 10 ring atoms, and which can include up to 3 ring heteroatoms, independently selected from N, O and S, which ring moiety is unsubstituted or substituted by one or more substitutents, independently selected from hologen,

alkoxy and

alkyl unsubstituted or substituted by halogen

(d) a compound having a general formula

wherein:

X is alkylene or kenylene, each of which is unsubstituted or substituted

by one or more fluorine atoms;

R is aryl, cycloalkenyl or ycloalkyl unsubstituted or substituted by one or

more fluorine atoms; wherein aryl comprises phenyl unsubstituted or substituted by one or more substituents independently selected from

alkyl) groups; wherein p=0, 1 or 2;

[HET] is a divalent heterocyclic moiety selected from:

wherein Z is H or C^1"4 alkyl;

Y is a mono- or bicyclic unsaturated ring system containing from 5 to 10 ring atoms, of which up to 4 of said ring atoms are hetero-atoms independently selected from N, O and S, and said ring system is unsubstituted or substituted by one or more substituents independently selected from =0, Ci_4 alkyl, Ci_₄ alkoxy, NR¹R², SO₂NR¹R², CO₂R¹, CO NR¹R², CH₂CO₂R¹, NR¹CO₂R², NR¹SO₂R²N, or het¹;

wherein R¹ and R² are each independently selected from H and Ci_₄ alkyl unsubstituted or substituted by Ci_₄ alkoxy; wherein het¹ is a N-linked 4- to 6-membered mono- or bicyclic heterocycle unsubstituted or containing 1 or 2 further hetero ring atoms independently selected from N and O, which heterocycle is unsubstituted or substituted by one or more substituents independently selected from =0, Ci_4 alkyl, Ci_₄ alkoxy, NR¹R², SO₂NR¹R², CO₂R¹, CO NR¹R², CH₂CO₂R¹, NR¹CO₂R², NR¹SO₂R²N or het²; wherein het² is a N-linked 4- to 6-membered mono- or bicyclic heterocycle unsubstituted or containing 1 or 2 further hetero ring atoms independently selected from N and O.

(e) a compound selected from the group of

and

(f) a compound having the formula

wherein:

X is Ci-C₆ alkylene or C₂-C₆ alkenylene, each of which is optionally substituted by one or more fluorine atoms;

R is aryl or C₃-Cs cycloalkyl optionally substituted by one or more fluorine atoms; W is N or CZ; Y and Z are each independently H, C₁-C₄ alkyl (optionally substituted by one or more substituents independently selected from halogen, S(O)pR⁶, OR⁵, CONR¹R², o CO₂R⁷ and aryl),

alkanoyl optionally substituted by one or more halogen,

alkoxycarbonyl optionally substituted by one or more halogen or CONR¹R², R¹ and R² are each independently selected from H, C₃-C₈ cycloalkyl, alkyl

(optionally substituted by C₃-C₈ cycloalkyl, aryl, CO₂H, CO₂R⁵, and/or NR³R⁴), or R¹ and R² can be taken together with the nitrogen to which they are attached to represent a 4- to 6-membered heterocyclic ring optionally containing one or two further hetero atoms in the ring independently selected from N, O and S, which heterocyclic ring is optionally benzo- or pyrido-fused, and which heterocyclic ring is optionally substituted by C₁-C₄ alkyl, CO₂H, CO₂R⁵, aryl and/or NR³R⁴;

R³ and R⁴ are each independently selected from H,

alkyl or

lkoxycarbonyl optionally substituted by one or more halogen, or R³ and R⁴ can be taken together with the nitrogen atom to which they are attached to represent a morpholine, piperidine, azetidine or piperazine (optionally N-substituted by

Ci-C₄ alkyl)

R⁵ is Ci-C₄ alkyl optionally substituted by CO₂R⁷, CONR³R⁴, o R⁵ is aryl;

R⁶ is Ci-C₄ alkyl optionally substituted by one or more halogen, or aryl; R⁷ is H or R⁶; p is 0, 1 or 2,

"Aryl" is a mono- or bicyclic aromatic carbocyclic or heterocyclic system comprising from 5 to 10 ring atoms, including up to 3 hetero-atoms selected from N, O and S, where, if there is a N atom in the ring, it can be present as the N-oxide, which ring system is optionally substituted by up to 3 substituents independently selected from halogen, Ci-C₄ alkyl optionally substituted by one or more halogen, Ci-C₄ alkoxy optionally substituted by one or more halogen, phenyl, pyridyl, CO₂H,

CONR³R⁴, CO₂(Ci-C₄ alquilo), NR³R⁴, OH y OC(O)(Ci-C₄ alquilo).

(g) a compound having the general formula

or

wherein:

Ri is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo substitituted aralkyl, heteroaralkyl, biaryl, biarylalky, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalAyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(tio, sulfϊnyl or sulfonyl)-alkyl;

R₂ is selected from the group consisting of H, lower alkyl;

R₃ is selected fϊom the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, halo substitituted aralkyl, heteroaralkyl, biaryl, biaryialkyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfonyl)-alkyl;

R₄ is selected from the group consisting of aryl, heteroaryl, alkyl, aralkyl, heteroaralkyl, alkylamino, arylalkylamino;

X is selected from the group consisting of SO₂, C=O;

Y is selected from the group consisting of OH, HOHN (hydroxylamine), H₂N, alkylamino;

Z s a direct bond; methylene, oxygen, sulfur, amino; and n is 0 or 1.

CO a compound having the general formula

wherein

Ri is selected from the group consisting of H, lower alkyl, mono- or poly-haloalhyl, carboxyalkyl, aryi, heteroaryl, aralkyl, heteroaralkyl, biaryl, biaryialkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoallcyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfϊnyl or sulfony l)-alkyl;

R₂, is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfϊnyl or sulfony l)-alkyl;

R₃ is selected fϊom the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalk~l, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialLylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalAylalkyl, heterocycloalkylalkyl , alkyl-(thio, sulfmyl or sulfony l)-alkyl;

R₄, is selected from the group consisting of H, lower alkyl; and

R₅ is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylarnino)alkyl, alkyl-(thio, sufϊnyl or sulfonyl)alkyl, alkoy alky lacy lalkyl.

(i) a compound having the general formula

wherein

Ri is selected from the group consisting of H, lower alkyl, mono- or poly-haloalhyl, carboxyalkyl, aryi, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoallcyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfony l)-alkyl; R₂, is selected from the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfϊnyl or sulfony l)-alkyl;

R₃ is selected fϊom the group consisting of H, lower alkyl, mono- or poly-haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalk~l, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialLylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalAylalkyl, heterocycloalkylalkyl , alkyl-(thio, sulfmyl or sulfony l)-alkyl;

R₄, is selected from the group consisting of H, lower alkyl; and

R₅ is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylarnino)alkyl, alkyl-(thio, sufϊnyl or sulfonyl)alkyl, alkoy alky lacy lalkyl.

and (j) a compound having the general formula

wherein

Ri is selected from the group consisting of H, lower alkyl, mono- or poly- haloalhyl, carboxyalkyl, aryi, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoallcyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfmyl or sulfony l)-alkyl; R₂, is selected from the group consisting of H, lower alkyl, mono- or poly- haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalkyl, hydroxyl, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialkylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyl-(thio, sulfinyl or sulfony l)-alkyl;

R₃ is selected from the group consisting of H, lower alkyl, mono- or poly- haloalkyl, carboxyalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, biaryl, biarylalk~l, hydroxyalkyl, alkyoxyalkyl, acyloxyalkyl, mercaptoalkyl, (amino, mono- or dialLylamino)alkyl, acylaminoalkyl, cycloalkyl, heterocycloalkyl, cycloalAylalkyl, heterocycloalkylalkyl , alkyl-(thio, sulfinyl or sulfony l)-alkyl;

R₄, is selected from the group consisting of H, lower alkyl; and

R₅ is selected from the group consisting of H, lower alkyl, carboxyalkyl, (mono- or dialkylarnino)alkyl, alkyl-(thio, sufinyl or sulfonyl)alkyl, alkoy alky lacy lalkyl.

a compound having the general formula

wherein

R¹ and R⁴ are, independently of each other, hydrogen or alkyl; R² is

(i) cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heterociclo or heterocycloalkyl; or

(ii) -(alkylene)-B-X where B is -0-, -NR⁸-, -S(O)_n,- (where n is 0, 1 or 2), - C=O, -CONR⁸-, -NR⁸CO₂-, NR⁸SO₂- o -C(=NR⁸)NR⁸SO₂- (where R⁸ is H or alkyl), and X is cycloalkyl, cycloalkylalkyl, aryl, aralkyl heteroaryl or heteroaralkyl; or (iii) -(alkylene)-B-X where B is -NR⁸-CO- (where R⁸ is H or alkyl), and X is cycloalkyl, cycloalkylalkyl, aryl, aralkyl heteroaryl or heteroaralkyl; or

(iv) and R² and R³ form an alkylene or heteroalkylene chain: R³ is hydrogen or alkyl

R⁶ is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl or heteroaralkyl;,

R⁵ is

(i) hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, hetergcycloalkyl, heteroalkyl, or -(alkylene)-C(O)-X¹ where X¹ is alkyl, hydroxy, alkoxy, aryl, aralkyl, aryloxy, aralkyloxy, heteroaryl, heteroaryloxy, heteroaralkyloxy or NRR" (where R and R" are independently H or alkyl, or R and R" form an alkylene chain); or (ii) R⁵ and R⁴ form an alkylene chain; or

(iii) R⁵ and R⁶ form an alkylene chain; n is 0 ό 1

A is -C(=O)-CH(R⁹)-(CH₂)_m-NR¹⁰- wherein m is an integer from 0-5 inclusive; R⁹ is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, hetergcycloalkyl, heteroalkyl, or - (alkylene)-C(O)-X¹ where X¹ is alkyl, hydroxy, alkoxy, aryl, aralkyl, aryloxy, aralkyloxy, heteroaryl, heteroaryloxy, heteroaralkyloxy or NR'R" (where R and R" are independently H or alkyl, or R and R" form an alkylene chain) R¹⁰ is hydrogen, alkyl, aralkyl or heteroaralkyl;

Z is Y-B wherein Y is alkylene or a bond; and

B is -CO-, -C(O)O-, -CONR⁸-, -SO₂-, o -SO₂NR⁸- (wherein R⁸ es hydrogen or alkyl), alkylene (optionally substituted by hydroxy, alkoxy, amino, monoalkylamino or dialkylamino) or a bond;

R⁷ is cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl or heteroaralkyl; provided that when n=O and Z is SO₂, then R² does not contain an imidazole group

3. Method according to any one of claims 1 or 2 wherein the cardiac disease is chronic cardiac failure or HHT.

4. Method for developing a personalized therapy for the treatment of a patient suffering from a cardiac disease associated to myocardial fibrosis comprising: a) determine in a sample from said patient a parameter indicative of the PCP activity and b) if the value of said parameter is higher than a predefined threshold value, then the patient is candidate to the treatment with a therapeutically effective amount of a PCP inhibitory agent.

5. Method according to claim 4 wherein the parameter indicative of PCP activity is selected from the group of i) PICP levels, ii) PCP activity, iii) PCP levels iv) ratio of active PCP and PCP zymogen, v) PCPE levels and vi) a combination of one or more of (i) to (v).

6. Method according to claims 4 or 5 wherein said threshold value is the value of the parameter indicative of PCP activity in a patient which does not suffer of cardiac disease associated to myocardial fibrosis.

7. Method according to any of claims 4 to 6 wherein the PCIP level are determined in serum or plasma.

8. Method according to any one of claims 4 to 7 wherein the PCP inhibitor is torasemide.

9. Method according to any one of claims 4 to 8 wherein the cardiac disease associated to myocardial fibrosis is selected from the group of chronic cardiac failure and HHT.