WO2017055248A1 - Methods and pharmaceutical compositions for the treatment of heart failure - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of heart failure. In particular, the present invention relates to a method of treating heart failure in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a RCN-3 polypeptide or nucleic acid molecule encoding thereof.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT

OF HEART FAILURE

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of heart failure.

BACKGROUND OF THE INVENTION:

The financial burden of major cardiovascular (CV) diseases to public health is heavy.

Moreover, this cost will increase due to aging of the population. Alarming remarks may be made when considering congestive heart failure (HF) as a consequence of myocardial infarction (MI) and atherosclerosis, ventricular hypertrophy as a result of essential hypertension, and chronic renal disease that derives from diabetic nephropathy and hypertension. Despite optimal therapy, patients with HF experience clinically meaningful disease progression (1). HF is now considered as a complex disorder in which neuro- hormonal, inflammatory and immunity systems are involved (2). During the development of HF, cardiac fibrosis occurs. Cardiac fibrosis is defined by the changes in heart geometry, structural organization and gene expression (3). Major progresses are needed to improve HF prevention and treatment: 1) ensure accurate and early diagnosis of heart dysfunction in patients, before cardiac failure becomes clinically overt; 2) optimize the prevention and treatment of HF; 3) target the right treatment to the right patients.

Inappropriate mineralocorticoid signaling has been shown to play an important role in the progression of CV disease (4). Adosterone is a main regulator of renal sodium reabsorption with an overall effect on volemia and blood pressure. Adosterone binds to the mineralocorticoid receptor (MR), a transcription factor of the nuclear receptor family present in the kidney and also in non-epithelial cells. New extra-renal pathophysiological effects of this hormone have been characterized, extending its actions to the CV system (5). MR is over- expressed in the myocardium of the failing heart (6). Inappropriate MR activation has been shown to promote cardiac fibrosis in experimental models (5). The RALES, EPHESUS and more recently the EMPHASIS-HF clinical trials demonstrated that the addition of MR antagonists (MRA) to standard care markedly reduced the overall and cardiovascular mortality in patients with HF (7, 8, 9). The beneficial effects were associated with a reduction of cardiac fibrosis (10). These conclusions are reinforced at the cellular level in cardiac fibroblasts and vascular smooth muscle cells (VSMCs) where Adosterone increases collagen synthesis via MR (Rewieved in Reference 5). Taken together, the published data indicate the potential contribution of Adosterone to cardiac fibrosis, acting as a collagen regulator. However, the precise mechanisms responsible for Adosterone-induced collagen synthesis remain to be determined.

Several studies in recent years have contributed to outline the complex interactions between the renin-angiotensin-aldosterone system receptors and other molecules such as cytokines, on the one hand, and the cardiac hypertrophy phenotype, on the other. Cardiotrophin- 1 (CT-1) is a cytokine produced by cardiomyocytes and fibroblasts that induces cardiac hypertrophy and interstitial fibrosis both in vitro and in vivo (1 1). Thus, CT- 1 -treated rats presented increased myocardial interstitial and perivascular fibrosis, leading to increased cardiac stiffness and myocardial dysfunction (11). Moreover, plasma levels of CT-1 are increased in hypertension and associated with left ventricular hypertrophy and fibrosis in hypertensive patients (12). Importantly, we have recently identified CT-1 as one of the key molecules involved in Adosterone-induced cardiomyocyte hypertrophy and interstitial fibrosis in vitro (13) and in vivo (14). By using a CT-1 knockout mouse model we have described that CT-1 is a necessary factor for the accumulation of fibrous tissue and the development of left ventricular hypertrophy induced by Adosterone (14). Thus, CT-l-null mice were resistant to Adosterone-induced interstitial and perivascular myocardial fibrosis (14). In addition, a significant association has been found between abnormally high CT-1 and abnormally high Adosterone in HF patients, suggesting that the MR pathways might be involved in CT-1 overproduction in HF (12).

Galectin-3 (Gal-3), also known as Mac-2, carbohydrate-binding protein [CBP]-35, εΒΡ, RL-29, HL-29, L-34, or lipopolysaccharide-binding protein [LBP]) is a 29-35 kDa protein. Gal-3 is involved in numerous physiological and pathological processes some of which, inflammation and fibrosis, are pivotal contributing to pathophysiological mechanisms in the development and progression of HF. Indeed, Gal-3 has been demonstrated to be robustly expressed in the failing heart and linked to myocardial fibrosis and remodeling (15). We have recently demonstrated that Gal-3 is a direct Adosterone-target gene in cultured vascular cells (16). In the Adosterone/salt mouse model, Gal-3 invalidation blunts cardiac fibrosis (17). Therefore Gal-3 may be a biomarker of MR activation, as well as a biotarget involved in the remodeling occurring in HF.

Taken together, Adosterone/CT-1 /Gal-3 is recognized as promoter of cardiac fibrosis. However the underlying mechanisms are not well defined. Identifying such mechanisms may be useful to decider novel target pathway in cardiac fibrosis modulated in HF and 2) identifying novel therapeutic target to counteract fibrosis mediated by Adosterone/CT-l/Gal-3 or other neurohormonal factors.

RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand calcium-binding proteins localized to the secretory pathway (18). Therefore, RCN can be classified into the EF-hand calcium-binding protein superfamily, which includes calmodulin, troponin C, and myosin light chain. The function of this protein remains unknown. However, its localization in the lumen of the endoplasmic reticulum suggests a role in protein synthesis, modification, and intracellular transport.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of heart failure. In particular, the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:

Using an unbiased approach based on proteomic analysis, the inventors have identified RCN-3 as a protein downregulated by Adosterone, CT-1 and Gal-3. Recombinant RCN-3 exerts antifibrotic effects in adult cardiac fibroblasts, diminishing the synthesis and the secretion of collagen type I. Downregulation of RCN-3 could be a new mechanism by which Adosterone, CT-1 and Gal-3 induce cardiac fibrosis and RCN-3 treatment could exert beneficial effects reducing hypertrophy in cardiomyocytes.

Accordingly, a first object of the present invention relates to a method of treating heart failure in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a RCN-3 polypeptide or nucleic acid molecule encoding thereof.

The term "heart failure" (HF) as used herein embraces congestive heart failure and/or chronic heart failure. Functional classification of heart failure is generally done by the New York Heart Association Functional Classification (Criteria Committee, New York Heart Association. Diseases of the heart and blood vessels. Nomenclature and criteria for diagnosis, 6th ed. Boston: Little, Brown and co, 1964;1 14). This classification stages the severity of heart failure into 4 classes (I-IV). The classes (I-IV) are:

Class I: no limitation is experienced in any activities; there are no symptoms from ordinary activities. Class II: slight, mild limitation of activity; the patient is comfortable at rest or with mild exertion.

Class III: marked limitation of any activity; the patient is comfortable only at rest. Class IV: any physical activity brings on discomfort and symptoms occur at rest.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

The method of the present invention is particularly suitable for reducing cardiac fibrosis and/or cardiac hypertrophy. As used herein, the term "RCN-3" has its general meaning in the art and refers to the reticulocalbin 3 protein. An exemplary amino acid sequence of RCN-3 is represented by SEQ ID NO: l . SEQ ID NO : 1 (RCN-3_homo sapiens)

MMWRPSVLLL LLLLRHGAQG KPSPDAGPHG QGRVHQAAPL SDAPHDDAHG NFQYDHEAFL GREVAKEFDQ LTPEESQARL GRIVDRMDRA GDGDGWVSLA ELRAWIAHTQ QRHIRDSVSA AWDTYDTDRD GRVGWEELRN ATYGHYAPGE EFHDVEDAET YKKMLARDER RFRVADQDGD SMATREELTA FLHPEEFPHM RDIVIAETLE DLDRNKDGYV

QVEEYIADLY SAEPGEEEPA WVQTERQQFR DFRDLNKDGH LDGSEVGHWV LPPAQDQPLV EANHLLHESD TDKDGRLSKA EILGNWNMFV GSQATNYGED LTRHHDEL Accordingly the term "RCN-3 polypeptide" has its general meaning in the art and includes naturally occurring RCN-3 and conservative function variants and modified forms thereof.

In some embodiments, the RCN-3 polypeptide comprises an amino acid sequence having at least 90% of identity with SEQ ID NO : 1.

According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981 ; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73 :237-244, 1988; Higgins and Sharp, CABIOS, 5 : 151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al, Comp. Appls Biosci., 8:155-165, 1992; and Pearson et al, Meth. Mol. BioL, 24:307-31, 1994). Altschul et al, Nat. Genet., 6: 119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11 , and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet, 3:266-272, 1993; Madden et al. Meth. EnzymoL, 266: 131-141, 1996; Altschul et al, Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.

In some embodiments, it is contemplated that the RCN-3 polypeptides of the invention used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

In some embodiments, the RCN-3 polypeptide of the invention is fused a Fc domain of an immunoglobulin. Suitable immunoglobins are IgG, IgM, IgA, IgD, and IgE. IgG and IgA are preferred IgGs are most preferred, e.g. an IgGl . Said Fc domain may be a complete Fc domain or a function-conservative variant thereof. The RCN-3 polypeptide of the invention may be linked to the Fc domain by a linker. The linker may consist of about 1 to 100, preferably 1 to 10 amino acid residues.

According to the invention, the polypeptide of the invention may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art. The polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as described in Stewart and Young; Tarn et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides. A variety of expression vector/host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al, 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al, 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful in recombinant protem productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC 12, K562 and 293 cells. As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA or RNA molecule. However, the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8- hydroxy-N6-methyladenosine, aziridmylcytosine, pseudoisocytosine, 5-

(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine,

1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,

2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

In some embodiments, the nucleic acid molecule of the present invention is included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. Typically, the vector is a viral vector which is an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking IT sequences. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species. Typically, the nucleic acid molecule or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters".

By a "therapeutically effective amount" is meant a sufficient amount of the RCN-3 polypeptide or the nucleic acid molecule encoding thereof for treating heart failure at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. According to the invention, the RCN-3 polypeptide or the nucleic acid molecule

(inserted or not into a vector) of the present invention is administered to the subject in the form of a pharmaceutical composition. Typically, the RCN-3 polypeptide or the nucleic acid molecule (inserted or not into a vector) of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral- route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The RCN-3 polypeptide or the nucleic acid molecule (inserted or not into a vector) of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

A further object of the present invention relates to a method for assessing mineralocorticoid receptor activation in the cardiac tissue of a subject comprising the steps of i) determining the expression level of RCN-3 in a sample obtained from the subject, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding of the mineralocorticoid receptor activation in the cardiac tissueof the subject when the expression level determined at step i) is lower than the predetermined reference value.

As used herein, the term "mineralocorticoid receptor" or "MR" has its general meaning in the art and refers to the nuclear receptor subfamily 3, group C, member 2, (NR3C2) that is a receptor with high affinity for mineralocorticoids. The mineralocorticoid receptor is also called aldosterone receptor.

In some embodiments, the sample is a blood sample. As use herein the term "blood sample" refers to a whole blood, serum, or plasma sample. Determination of the expression level of RCN-3 can be performed by a variety of techniques. In some embodiments, the methods of the invention comprise contacting the sample with a binding partner capable of selectively interacting with the RCN-3 protein present in the blood sample. The binding partner may be an antibody that may be polyclonal or monoclonal, typically monoclonal. In some embodiments, the binding partner may be an aptamer.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally and the EBV-hybridoma technique. Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti- CN-3, single chain antibodies. Antibodies useful in practicing the present invention also include anti-RCN-3 fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to RCN3. For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., Ml 3. Briefly, spleen cells of a suitable host, e. g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

In some embodiments, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.

The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, mi l l, Rel86, Rel88.

The aforementioned assays generally involve the binding of the binding partner (ie. Antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

The concentration of RCN-3 may be measured by using standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said RCN-3. A biological sample containing or suspected of containing said RCN-3 is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art. Measuring the concentration of the RCN-3 protein (with or without immunoassay- based methods) may also include separation of the compounds: centrifugation based on the compound's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the compound's affinity for the particular solid-phase that is used. Once separated, said RCN-3 may be identified based on the known "separation profile" e. g., retention time, for that compound and measured using standard techniques.

Alternatively, the separated compounds may be detected and measured by, for example, a mass spectrometer.

A reference value can be relative to a number or value derived from population studies, including without limitation, such subjects having similar body mass index, total cholesterol levels, LDL/HDL levels, systolic or diastolic blood pressure, subjects of the same or similar age range, subjects in the same or similar ethnic group, and subjects having the same severity of heart failure. Such predetermined reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of metabolic syndrome. In some embodiments, the predetermined reference values are derived from the level of RCN-3 in a control sample derived from one or more subjects who were not subjected to heart failure. Furthermore, retrospective measurement of the level of RCN-3 in properly banked historical subject samples may be used in establishing these predetermined reference values. Typically, the levels of RCN-3 in a subject having mineralocorticoid receptor activation in the cardiac tissue is deemed to be lower than the reference value obtained from the general population or from healthy subjects. The method for assessing mmeralocorticoid receptor activation in the cardiac tissue according to the invention may find in various applications, In particular, the method of the present invention is particularly suitable for the treatment of subjects suffering from heart failure. Even more particularly, mmeralocorticoid receptor antagonists have been suggested as beneficial for the treatment of heart failure. However, up to now, it was not possible to discriminate subjects that could benefit from such a treatment. Administration of a MR antagonist in a subject may be accompanied with serious adverse side effects such as hyperkalemia and therefore it is highly desirable to clearly identify subjects suffering from heart failure that could benefit of a treatment with a MR antagonist.

In some embodiments the method of the present invention for assessing assessing mmeralocorticoid receptor activation in the cardiac tissue further comprises determining the expression level of Galectin-3 and/or cardiotrophin-1.

In one further aspect, the present invention relates to a method of treating heart failure comprising the steps of i) determining the expression level of RCN-3 in a sample obtained from the subject, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) administering the subject with a therapeutically effective amount of a mmeralocorticoid receptor antagonist when the expression level determined at step i) is lower than the predetermined reference value.

As used herein the term "MR antagonist" has its general meaning in the art. The MR antagonistic of a compound may be determined using various methods as described in J, Souque A, Wurtz JM, Moras D, Rafestin-Oblin ME. Mol Endocrinol. 2000 Aug; 14(8): 1210- 21; Fagart J, Seguin C, Pinon GM, Rafestin-Oblin ME. Mol Pharmacol. 2005 May;67(5):1714-22 or Hellal-Levy C, Fagart J, Souque A, Wurtz JM, Moras D, Rafestin- Oblin ME. Mol Endocrinol. 2000 Aug; 14(8): 1210-21. Typically, the transfection of the human mmeralocorticoid receptor in COS cells together with a luciferase-expressing reporter gene allows to measure its transactivation activity in the presence of a candidate compound. In the context of the present invention, mmeralocorticoid receptor antagonists are typically selective for the mmeralocorticoid receptor as compared with the related receptors such as androgen receptor, estrogen receptors, glucocorticoid receptor, progesterone receptor, thyroid hormone receptors, peroxisome proliferator-activated receptors, retinoic acid receptor, farnesoid x receptor, pregnane x receptor, liver X receptor, vitamin D receptor, retinoid x receptor and the constitutive androstane receptor. By "selective" it is meant that the affinity of the antagonist for the mineralocorticoid receptor is at least 10-fold, typically 25-fold, more typically 100-fold, still typically 500-fold higher than the affinity for the related receptors. MR antagonists constitute a class of pharmacological compounds that are well known by the skilled artisan.

For example, the mineralocorticoid receptor antagonists according to the invention generally are spirolactone-type steroidal compounds. The term "spirolactone-type" is intended to characterize a structure comprising a lactone moiety attached to a steroid nucleus, typically at the steroid "D" ring, through a spiro bond configuration. A subclass of spirolactone-type mineralocorticoid receptor antagonist compounds consists of epoxy-steroidal mineralocorticoid receptor antagonist compounds such as eplerenone. Another subclass of spirolactone-type antagonist compounds consists of non-epoxy-steroidal mineralocorticoid receptor antagonist compounds such as spironolactone.

The epoxy-steroidal mineralocorticoid receptor antagonist compounds used in the method of the present invention generally have a steroidal nucleus substituted with an epoxy- type moiety. The term "epoxy-type" moiety is intended to embrace any moiety characterized in having an oxygen atom as a bridge between two carbon atoms.

The term "steroidal," as used in the phrase "epoxy-steroidal," denotes a nucleus provided by a cyclopenteno-phenanthrene moiety, having the conventional "A", "B", "C", and "D" rings. The epoxy-type moiety may be attached to the cyclopentenophenanthrene nucleus at any attachable or substitutable positions, that is, fused to one of the rings of the steroidal nucleus or the moiety may be substituted on a ring member of the ring system. The phrase "epoxy-steroidal" is intended to embrace a steroidal nucleus having one or a plurality of epoxy-type moieties attached thereto. Epoxy-steroidal mmeralocorticoid receptor antagonists suitable for use in the present methods include a family of compounds having an epoxy moiety fused to the "C" ring of the steroidal nucleus. Examples include 20-spiroxane compounds characterized by the presence of a 9a, 11 a-substituted epoxy moiety, such as: Pregn-4-ene-7,21-dicarboxylic acid, 9,1 l-epoxy-17-hydroxy-3-oxo-,y- lactone, methyl ester, (7α,11α,17β)

Pregn-4-ene-7,21-dicarboxylic acid, 9,ll-epoxy-17-hydroxy-3-oxo- ,dimethyl ester, (7 ,11α,17β)

- 3' H-cyclopropa[6,7]pregna-4,6-diene-21- carboxylic acid,9,l 1-epoxy- 6,7-dihydro- 17-hydroxy-3-oxo-, y -lactone, (6 β ,7 β ,11 ,17 j3 )

Pregn-4-ene-7,21-dicarboxylic acid,9,ll- epoxy-17-hydroxy-3-oxo-,7-(l- methylethyl) ester,monopotassium salt, (7α,11 ,17β)

Pregn-4-ene-7,21-dicarboxylic acid,9,ll- epoxy-17-hydroxy-3-oxo-,7- methylethyl) ester,monopotassium salt, (7a, 11α,17β)

3' H-cyclopropa[6,7]pregna-l,4,6-triene- 21 -carboxylic acid,9,l 1-epoxy- 6,7-dihydro- 17-hydroxy-3-oxo-, y -lactone(6 β ,7 β ,11 ) 3⁷ H-cyclopropa[6,7]pregna-4,6-diene-21- carboxylic acid, 9,11-epoxy- 6,7-dihydro-17- hydroxy-3-οχο-, methyl ester, (6 β ,7 β ,11 ,17 β ) - 3' H-cyclopropa[6,7]pregna-4,6-diene-21- carboxylic acid, 9,11-epoxy-

6,7-dihydro-17- hydroxy-3-οχο-, monopotassium salt, (63,73,lla,173) 3' H-cyclopropa[6,7]pregna-l,4,6-triene-21- carboxylic acid, 9,11-epoxy- 6,7-dihydro-17- hydroxy-3-οχο-, y -lactone(6 j3 , 7 /3,11a, 17/3)

Pregn-4-ene-7,21-dicarboxylic acid,9,ll- epoxy-17-hydroxy-3-oxo-,y- lactone,ethyl ester, (7a, 11 ,17β)

Pregn-4-ene-7,21-dicarboxylic acid,9,ll- epoxy-17-hydroxy-3-oxo-,Y- lactone,l- methylethyl ester (7α,11 ,17β)

A particular benefit of using epoxy-steroidal mineralocorticoid receptor antagonists, as exemplified by eplerenone, is the high selectivity of this group of mineralocorticoid receptor antagonists for the mineralocorticoid receptor. The superior selectivity of eplerenone results in a reduction in side effects that can be caused by mineralocorticoid receptor antagonists that exhibit non-selective binding to related receptors, such as androgen or progesterone receptors. These epoxy steroids may be prepared by procedures described in Grob et al., U.S.

Pat. No. 4,559,332. Additional processes for the preparation of 9, 11-epoxy steroidal compounds and their salts are disclosed in Ng et al, WO97/21720 and Ng et al, W098/25948.

Of particular interest is the compound eplerenone ((Pregn-4-ene-7,21-dicarboxylic acid, 9,l l-epoxy-17-hydroxy-3-oxo-,Y-lactone, methyl ester, (7 ,11α,17β)) (CAS No. 107724-20-9), also known as epoxymexrenone. Eplerenone is a mmeralocorticoid receptor antagonist and has a higher selectivity for mmeralocorticoid receptors than does, for example, spironolactone. Selection of eplerenone as the mmeralocorticoid receptor antagonist in the present method would be beneficial to reduce certain side-effects such as gynecomastia that occur with use of mmeralocorticoid receptor antagonists having less specificity.

Non-epoxy-steroidal mmeralocorticoid receptor antagonists suitable for use in the present methods include a family defined by Formula I:

(I)

Wherein:

- R is lower alk l of up to 5 carbon atoms, and

Lower alkyl residues include branched and unbranched groups, for example, methyl, ethyl and n-propyl. Specific compounds of interest within Formula I are the following:

7 -acetylthio-3-oxo-4, 15-androstadiene-[17( β -Y )-spiro- 5' Jperhydrofuran-2' -one;

3-0X0-7 -propionylthio-4,15-androstadiene-[17(( β -Y )-spiro- 5' ]perhydrofuran-2^/ -one;

6 β ,7 j3 -methylene-3-oxo4,15-androstadiene-[17(( j3 -l' ^s ires' ]perhydrofuran-2^/ -one;

15 a ,16 a -methylene-3-oxo-4,7 -propionylthio-4-androstene[ 17( β - V )-spiro-5 ]perhydrofuran-2 -one;

- 6 β ,7 β ,15 a ,16 a -dimethylene-3-oxo-4-androstene[17( β -l' )-spiro-

5' J-perhydrofuran-2' -one;

7 a -acetylthio-1 β , 16 β -Methylene-3-oxo-4-androstene-[17( /3 - )- spiro-5^; ]perhydrofuran-2⁷ -one;

15 β ,16 β -methylene-3 -oxo-7 β -propionylthio-4-androstene-[ 17( β -\' )- spiro-5^; Jperhydrofuran^⁷ -one; and

6 β ,7 j3 ,15 β ,16 β -dimethylene-3-oxo-4-androstene-[17( β -\' )-spiro- 5' lperhydrofuran-2' -one.

Methods to make compounds of Formula I are described in U.S. Pat. No. 4, 129,564 to Wiechart et al. issued on 12 Dec. 1978.

Another family of non-epoxy-steroidal compounds of interest is defined by Formula

wherein Rl is Cl-3-alkyl or Cl-3 acyl and R2 is H or Cl-3-alkyl.

Specific compounds of interest within Formula II are the following:

1 a-acetylthio- 15β, 16p-methylene-7a-methylthio-3-oxo- 17 -pregn-4-ene- 21,17-carbolactone; and

15p,16p-methylene-l ,7 -dimethylthio-3-oxo-17a-pregn-4-ene-21 , 17- carbolactone.

Methods to make the compounds of Formula II are described in U.S. Pat. No. 4,789,668 to Nickisch et al. which issued 6 Dec. 1988.

Yet another family of non-epoxy-steroidal compounds of interest is defined by a structure of Formula III:

wherein R is lower alkyl, examples of which include lower alkyl groups of methyl, ethyl, propyl and butyl. Specific compounds of interest include:

3 β,21 -dihydroxy- 17 -pregna-5 , 15 -diene- 17-carboxylic acid γ-lactone;

3 β,21 -dihydroxy- 17 -pregna-5, 15 -diene- 17-carboxylic acid γ-lactone 3- acetate; 3 P,21-dihydroxy-17a-pregn-5-ene-l 7-carboxylic acid γ-lactone;

3 P,21-dihydroxy-17a-pregn-5-ene-l 7-carboxylic acid γ-lactone 3-acetate;

21-hydroxy-3-oxo-17a-pregn-4-ene-17-carboxylic acid γ-lactone;

2 l-hydroxy-3-oxo- 17a-pregna-4,6-diene- 17-carboxylic acid γ- lactone;

21 -hydroxy-3-oxo- 17a-pregna- 1 ,4-diene- 17-carboxylic acid γ- lactone;

7a-acylthio-21 -hydroxy-3 -oxo- 17a-pregn-4-ene- 17-carboxylic acid γ- lactone; and

7a-acetylthio-21 -hydroxy-3 -oxo- 17 -pregn-4-ene- 17-carboxylic acid γ- lactone.

Methods to make the compounds of Formula III are described in U.S. Pat. No. 3,257,390 to Patchett which issued 21 Jun. 1966.

Still another family of non-epoxy-steroidal compounds of interest is represented by Formula IV:

wherein E' is selected from the group consisting of ethylene, vinylene and (lower alkanoyl)thioethylene radicals, Έ" is selected from the group consisting of ethylene, vinylene, (lower alkanoyl)thioethylene and (lower alkanoyl)thiopropylene radicals; R is a methyl radical except when E' and Έ" are ethylene and (lower alkanoyl) thioethylene radicals, respectively, in which case R is selected from the group consisting of hydrogen and methyl radicals; and the selection of E' and Έ" is such that at least one (lower alkanoyl)thio radical is present.

One family of non-epoxy-steroidal compounds within Formula IV is represented by Formula V: lower

Another compound of Formula V is l-acetylthio-17a-(2-carboxyethyl)-17 -hydroxy- androst-4-en-3-one lactone.

Another family of non-epoxy-steroidal compounds within Formula IV is represented by Formula VI:

Exemplary compounds within Formula VI include the following:

7a-acetylthio- 17a-(2-carboxyethyl)- 17P-hydroxy-androst-4-en-3 -one lactone;

7P-acetylthio- 17a-(2-carboxyethyl)- 17 -hydroxy-androst-4-en-3 -one lactone;

la,7 -diacetylthio-17a-(2-carboxyethyl)-17P-hydroxy-androsta-4,6-dien-3- one lactone;

7a-acetylthio- 17ae-(2-carboxyethyl)- 17β-hydro y-androsta-l ,4-dien-3-one lactone;

7a-acetylthio- 17a-(2-carboxyethyl)-l 7P-hydroxy- 19-norandrost-4-en-3- one lactone; and

7a-acetylthio-17 -(2-carboxyethyl)-17P-hydroxy-6a-methylandrost-4-en- 3-one lactone. In Formulae IV- VI, the term "alkyl" is intended to embrace linear and branched alkyl radicals containing one to about eight carbons. The term "(lower alkanoyl)thio" embraces

O

radicals of the formula lower alkyl ° Of particular interest is the compound spironolactone (17-hydroxy-7ct-mercapto-3- oxo-17a-pregn-4-ene-21-carboxylic acid γ- lactone acetate) having the following structure:

Methods to make compounds of Formulae IV-VI are described in U.S. Pat. No.

3,013,012 to Cella et al. which issued 12 Dec. 1961. Spironolactone is sold by G. D. Searle & Co., Skokie, 111., under the trademark "ALDACTONE", in tablet dosage form at doses of 25 mg, 0 mg and 100 mg per tablet. Another family of steroidal mineralocorticoid receptor antagonists is exemplified by drospirenone, (6R-(6 a , 1 a , 8 β , 9 a , 10 β , 13 β , 14 a , 15 a , 16 a , 17 β ))-1, 3^; , 4' , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 21-hexadecahydro-lO, 13-dimethylspiro [17H- dicyclopropa(6,7: 15,16)cyclopenta(a)phenanthrene-17,2 (5' H)-furan)-3,5' (2H)-dione, CAS registration number 67392-87-4. Methods to make and use drospirenone are described in patent GB 1550568 1979, priority DE 2652761 1976.

Crystalline forms that are easily handled, reproducible in form, easily prepared, stable, and which are non-hygroscopic have been identified for the mineralocorticoid receptor antagonist eplerenone. These include Form H, Form L, various crystalline solvates and amorphous eplerenone. These forms, methods to make these forms, and use of these forms in preparing compositions and medicaments, are disclosed in Barton et al, WO 01/41535 and Barton et al., WO 01/42272 both incorporated herein in their entirety.

Mineralocorticoid receptor antagonists according to the invention may also be nonsteroidal. For example, classes of non-steroidal MR antagonists have just begun to emerge over the past few years (Meyers, Marvin Jl ; Hu, Xiao Expert Opinion on Therapeutic Patents, Volume 17, Number 1, January 2007 , pp. 17-23(7) and Piotrowski DW. Mineralocorticoid Receptor Antagonists for the Treatment of Hypertension and Diabetic NephropathyJ. Med. Chem. 2012, 55, 7957-7966). For instance, dihydropyrymidines have been shown to display MR antagonism (Activation of Mineralocorticoid Receptors by Exogenous Glucocorticoids and the Development of Cardiovascular Inflammatory Responses in Adrenalectomized Rats. Young MJ, Morgan J, Brolin K, Fuller PJ, Funder JW. Endocrinology. 2010 Apr 21). Furthermore, Arhancet el al. disclose other class of non-steroidal MR antagonists (Arhancet GB, Woodard SS, Dietz JD, Garland DJ, Wagner GM, Iyanar , Collins JT, Blinn JR, Numann RE, Hu X, Huang HC. Stereochemical Requirements for the Mineralocorticoid Receptor Antagonist Activity of Dihydropyridines. J Med Chem. 2010 Apr 21). Other exemplary non-steroidal mmeralocorticoid receptor antagonists include but are not limited to those described in US 20090163472 WO2004052847, WO 2008053300 WO2008104306, WO2007025604, WO201264631 , WO2008126831, WO2012008435, WO2010104721 , WO200985584, WO200978934, WO2008118319, WO200917190, WO200789034, WO2012022121, WO2012022120, WO2011 141848 and WO200777961 that are hereby incorporated by reference into the present disclosure.

In some embodiments, the mineralocorticoid receptor antagonist is selected from the group consisting of:

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1. Adosterone, CT-1, Gal-3 decreased RCN-3 expression in human adult cardiomyocytes. All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of 4 assays, in arbitrary units normalized to β-actin. *p<0.05 vs Control.

Figure 2. Recombinant RCN-3 decreased collagen type I and collagen type III expressions in human adult fibroblasts. All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of 4 assays, in arbitrary units normalized to β-actin. *p<0.05 vs Control

Figure 3. Recombinant RCN-3 decreased collagen type I secretion in human adult fibroblasts without modifying MMP-1 activity. All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of 4 assays, in arbitrary units normalized by stain free. *p<0.05 vs Control

Figure 4. Recombinant RCN-3 decreased alpha-smooth muscle in human adult cardiomyocytes. All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of 4 assays, in arbitrary units normalized to β-actin. *p<0.05 vs Control

Figure 5. Aldosterone decreases RCN-3 protein levels through mineralocorticoid receptor activation in human adult cardiac fibroblasts. All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of 4 assays, in arbitrary units normalized β-actin. *p<0.05 vs control; $p<0.05 Aldosterone.

Figure 6. RCN-3 exerts anti-fibrotic effects in human adult cardiac fibroblasts.

All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of 4 assays, in arbitrary units normalized by stain free gel. *p<0.05 vs scramble activation; $p<0.05 vs scramble KO.

Figure 7. Recombinant RCN-3 increases the activation of Akt, and decreases STAT3 and ERKl/2 activation in human adult fibroblasts. All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of 4 assays, in arbitrary units normalized by stain free gel. *p<0.05 vs Control.

Figure 8. Aldosterone decreases RCN-3 protein levels in the heart through mineralocorticoid receptor activation in vivo in rats. All conditions were performed at least in triplicate. Histogram bars represent the mean ±SEM of each group of animals (Control, n=8; Aldo, n=8; Spiro, n=8), in arbitrary units by stain free gel. *p<0.05; **p<0.01 vs Control group. Figure 9. Cardiac RCN-3 mRNA levels are increased after 8 days and 8 weeks post-Myocardial Infarction (MI) in mice. All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of each group of animals (Sham, n=7; MI; n=7), in arbitrary units normalized to 18s. **p<0.01 vs Sham group. Figure 10. Cardiac RCN-3 mRNA levels are increased after 3 days and 3 weeks

Angiotensin II (Ang II) treatment in mice. All conditions were performed at least by triplicate. Histogram bars represent the mean ±SEM of each group of animals (Control, n=7; Ang II; n=7), in arbitrary units normalized to 18s. *p<0.05; **p<0.01 vs Control group.

EXAMPLE 1: Methods

Human cardiac fibroblasts (from Promocell) were treated with Aldosterone 10 ⁸M (A9477, Sigma-Aldrich), recombinant human CT-1 10^"9M (612-CD, R&D Systems) and recombinant human Gal-3 10^"8M (1154-GA, R&D Systems) for 24 hours. Independent experiments were performed to compare cellular proteomes derived from three biological replicates for each experimental condition (Aldosterone-, CT-1-, and Gal-3-treated cells), against unstimulated cells. Cellular pellets were resuspended in lysis buffer containing 7 M urea, 2 M thiourea, 4% (v/v) CHAPS, 50 mM DTT. Homogenates were spined down at 14,000 x rpm for 1 h at 15°C. A shotgun comparative proteomic analysis of total cell extracts using iTRAQ (isobaric Tags for Relative and Absolute Quantitation) was performed.

Peptide labeling. iTRAQ labeling of each sample was performed according to the manufacturer's protocol (ABSciex). Briefly, a total of 80 μg of protein from each treated and untreated cell sample was reduced with 50 mM tris (2-carboxyethyl) phosphine (TCEP) at 60 °C for 1 h, and cysteine residues were alkylated with 200 mM methylmethanethiosulfonate (MMTS) at room temperature for 15 min. Protein enzymatic cleavage was carried out with trypsin (Promega; 1 :20, w/w) at 37 °C for 16 h. Each tryptic digest was labeled according to the manufacturer's instructions with one isobaric amine-reactive tags. After lh incubation, each set of labeled samples were independently pooled and evaporated until <40 μΐ in a vacuum centrifuge. To increase proteome coverage, the peptide pool was injected to an Ettan LC system with a X-Terra RP18 precolumn (2.1 x 20mm) and a high pH stable X-Terra RP18 column (C 18; 2.1 mm x 150mm; 3.5μηι) (Waters) at a flow rate of 40 μΐ min. Peptides were eluted with a mobile phase B of 5-65% linear gradient over 35 min (A, 5 mM ammonium bicarbonate in water at pH 9.8; B, 5 mM ammonium bicarbonate in acetonitrile at pH 9.8). 8 fractions were collected, evaporated under vacuum and reconstituted into 20 μΐ of 2% acetonitrile, 0.1% formic acid, 98% MilliQ-H20 prior to mass spectrometric analysis. Peptides mixtures were separated by reverse phase chromatography using an Eksigent nanoLC ultra 2D pump fitted with a 75 μηι ID column (Eksigent 0.075 x 150). Samples were first loaded for desalting and concentration into a 0.5 cm length 300 μηι ID precolumn packed with the same chemistry as the separating column. Mobile phases were 100% water 0.1% formic acid (FA) (buffer A) and 100% Acetonitrile 0.1% FA (buffer B). Column gradient was developed in a 70 min two step gradient from 2% B to 30% B in 60 min and 30%B to 40% B in 10 min. Column was equilibrated in 95% B for 5 min and 2% B for 15 min. During all process, precolumn was in line with column and flow maintained all along the gradient at 300 nl min. Eluting peptides from the column were analyzed using an AB Sciex 5600 TripleTOF system. Information data acquisition was acquired upon a survey scan performed in a mass range from 350 m/z up to 1250 m/z in a scan time of 250 ms. Top 25 peaks were selected for fragmentation. Minimum accumulation time for MS/MS was set to 75 ms giving a total cycle time of 2.1 s. Product ions were scanned in a mass range from 100 m z up to 1700 m/z and excluded for further fragmentation during 15 s. For relative quantification and protein identification, data files were processed using ProteinPilot 4.5 software from AB Sciex which uses the algorithm Paragon (Shilov IV, Seymour SL, Patel AA, et al. Mol Cell Proteomics. 2007) for database search and Progroup for data grouping and searched against Uniprot human database. False discovery rate was performed using a non-lineal fitting method and displayed results were those reporting a 1 % Global False Discovery Rate (FDR) or better. The search parameters allowed for cysteine modification by MMTS. Reporter ion intensities were bias corrected for the overlapping isotope contributions from the iTRAQ tags according to the certificate of analysis provided by the reagent manufacturer (ABsciex). The peptide and protein selection criteria for relative quantitation were performed as follows. Only peptides unique for a given protein were considered for relative quantitation, excluding those common to other isoforms or proteins of the same family. Proteins were identified on the basis of having at least one peptide with an ion score above 99% confidence. Among the identified peptides, some of them were excluded from the quantitative analysis for one of the following reasons: (i) The peaks corresponding to the iTRAQ labels were not detected; (ii) the peptides were identified with low identification confidence (<1.0%); (iii) the sum of the signal-to-noise ratio for all of the peak pairs was <6 for the peptide ratios. The protein sequence coverage (95% conf.) was estimated for specific proteins by the percentage of matching amino acids from the identified peptides having confidence greater than or equal to 95% divided by the total number of amino acids in the sequence. Several quantitative estimates provided for each protein by ProteinPilot were utilized: the fold change ratios of differential expression between labelled protein extracts; the p-value, representing the probability that the observed ratio is different than 1 by chance. A decoy database search strategy was also used to estimate the false discovery rate (FDR), defined as the percentage of decoy proteins identified against the total protein identification. The FDR was calculated by searching the spectra against the decoy database generated from the target database. The results were then exported into Excel for manual data interpretation. Although relative quantification and statistical analysis were provided by the ProteinPilot software, an additional 1.3 -fold change cutoff for all iTRAQ ratios (ratio <0.77 or >1.3) was selected to classify proteins as up- or down-regulated. Proteins with iTRAQ ratios below the low range (0.77) were considered to be underexpressed, whereas those above the high range (1.3) were considered to be overexpressed.

Results

More than 30 proteins were differentially expressed when human adult cardiac fibroblasts were treated with Aldosterone.

More than 89 proteins were differentially expressed when human adult cardiac fibroblasts were treated with CT-1.

More than 15 proteins were differentially expressed when human adult cardiac fibroblasts were treated with Gal-3. Among all these proteins, we have selected Reticulocalbin (RCN) proteins family that is down-regulated by either Aldosterone, CT-1 or Gal-3. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand calcium-binding proteins localized to the secretory pathway (18). Therefore, RCN can be classified into the EF-hand calcium-binding protein superfamily, which includes calmodulin, troponin C, and myosin light chain. The function of this protein remains unknown. However, its localization in the lumen of the endoplasmic reticulum suggests a role in protein synthesis, modification, and intracellular transport. EXAMPLE 2:

Methods

Human cardiac fibroblasts (from Promocell) were treated with Aldosterone (10^"8M; Sigma) in presence or absence of Spironolactone (10^"5M; Sigma) for 24 hours.

Human cardiac fibroblasts (Promocell) were treated with recombinant human RCN-3 (abl23203, Abeam) for 24 hours. The effects of recombinant RCN-3 on extracellular matrix components were analyzed by western blot (cell lysates).

Adult human cardiomyocytes (Promocell) were treated with recombinant human RCN-3 (ab 123203, Abeam) for 24 hours

Human cardiac fibroblasts (from Promocell) were transfected with RCN-3 CRISPR Activation Plasmid (sc-410060-ACT, Santa Cruz Biotechnology) which consists of reticulocalbin-3-specific 20 nt guide RNA sequences derived from the GeC O (v2) library. Supernatant of the cells and cellular pellets were collected after 24 hours of transfection.

Human cardiac fibroblasts (from Promocell) were transfected with RCN-3 CRISPR/Cas9 Knock-out (KO) Plasmid (sc-410060, Santa Cruz Biotechnology). Supernatant of the cells and cellular pellets were collected after 24 hours of transfection.

Human cardiac fibroblasts (from Promocell) were treated recombinant human Gal-3 10^"8M (11 4-GA, R&D Systems) for different times (5, 10, 15, 30 and 60 minutes) and compared against unstimulated cells.

Male Wistar rats (250 g; Harlan Iberica) were treated for 3 weeks with vehicle (sunflower oil, subcutaneous injection, n=10), Aldo-salt (1 mg/kg/day, subcutaneous injection, n=10 and 1% NaCl as drinking water) and Aldo-salt plus Spironolactone (Spiro) (200 mg/kg/day, subcutaneous injection, n=10).

Left coronary artery ligations were performed in 8-week-old male mice and littermate controls under anesthesia (xylazine [3.6 mg/kg IP] plus 2% isoflurane). Analgesia was induced using ουρΓεηο ηίηε (0.05 mg/kg SC just after induction of anesthesia and 6, 12, 24, and 48 hours post coronary artery ligation). The snare was not tied for sham-operated mice.

8-week-old mice were treated with Ang II (1.44 mg/kg day; dissolved in phosphate- buffered saline Sigma) administered with Alzet osmotic mini-pumps for 2 weeks.

Results

We have validated that Aldosterone, CT-1 and Gal-3 decreased RCN-3 expression in human adult cardiac fibroblasts in a new set of experiments. Human cardiac fibroblasts (from Promocell) were treated with Aldosterone 10^"8M, recombinant human CT-1 10^"9M and recombinant human Gal-3 10^"8M for 24 hours. The results are shown in Figure 1.

We show that recombinant RCN-3 decreased collagen type I and collagen type III expressions in human adult fibroblasts (Figure 2).

Conditioned media from human cardiac fibroblasts treated with RCN-3 was analysed to measure collagen and metalloproteinases secretions and we show that recombinant RCN-3 decreased collagen type I secretion in human adult fibroblasts without modifying MMP-1 activity (Figure 3).

Adult human cardiomyocytes from Promocell were treated with recombinant human RCN-3 for 24 hours, and cardiomyocyte hypertrophy was evaluated and we show that recombinant RCN-3 decreased alpha-smooth muscle in human adult cardiomyocytes (Figure 4).

Aldosterone decreased RCN-3 protein levels in human adult cardiac fibroblasts. This effect was prevented by Spironolactone (10^"6M; Sigma), showing that Aldosterone decreases RCN-3 through mineralocorticoid receptor activation (Figure 5).

RCN-3 CRISPR Activation Plasmid induced an overexpression of RCN-3 and decreased the secretion of collagen I protein levels in the supernatant of the cells measured by ELISA after 24 hours of stimulation in human cardiac fibroblasts without modifications on collagen III, a-SMA and fibronectin intracellular protein content. RCN-3 CRISPR Cas9 Knock-out (KO) Plasmid decreased RCN3 protein levels without modifications in the secretion of collagen I measured in the supernatant of the cells and collagen III, a-SMA and fibronectin intracellular protein levels after 24 hours in human adult cardiac fibroblasts (Figure 6).

In human cardiac fibroblasts, recombinant RCN-3 (0.1 g/ml; Abeam) increased Akt phosphorylation after 15 and 30 minutes of stimulation and decreased ERK 1/2 phosphorylation after 60 min of stimulation. In addition, recombinant RCN-3 decreased STAT3 phosphorylation in all the times studied (5-60 minutes) (Figure 7).

In vivo treatment of adult male Wistar rats with Aldo-salt (1 mg/kg/day and 1% NaCl in the drinking water) decreased RCN-3 cardiac protein levels. This effect was prevented with 3 weeks administration of spironolactone (200 mg/kg/day; Sigma), a mineralocorticoid receptor antagonist (Figure 8).

Cardiac RCN-3 mRNA levels were increased in 8-week-old mice at 8 days or 8 weeks after left coronary artery ligation (Figure 9) as well as in mice treated with Ang II for 2 weeks (Figure 10).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of treating heart failure in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a RCN-3 polypeptide or nucleic acid molecule encoding thereof.

2. The method of claim 1 wherein the RCN-3 polypeptide comprises an amino acid sequence having at least 90% of identity with SEQ ID NO: 1.

3. The method of claim 1 wherein the RCN-3 polypeptide is fused to a Fc domain of an immunoglobulin.

4. The method of claim 1 wherein the nucleic acid molecule is included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

5. A method for assessing mmeralocorticoid receptor activation in the cardiac tissue of a subject comprising the steps of i) determining the expression level of RCN-3 in a sample obtained from the subject, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding of the mmeralocorticoid receptor activation in the cardiac tissue of the subject when the expression level determined at step i) is lower than the predetermined reference value.

6. The method of claim 5 wherein the sample is a blood sample.

7. The method of 5 which comprises determining the expression level of Galectin-3 and/or cardiotrophin- 1.

8. A method of treating heart failure comprising the steps of i) determining the expression level of RCN-3 in a sample obtained from the subject, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) administering the subject with a therapeutically effective amount of a mmeralocorticoid receptor antagonist when the expression level determined at step i) is lower than the predetermined reference value.