WO2020070089A1 - Chromogranin a and uses thereof - Google Patents

Abstract

The present invention refers to Chromogranin A (CgA) or a fragment thereof for use in the treatment and/or prevention of cardiotoxicity induced by an anthracycline and to relative pharmaceutical composition. The invention also relates to a method to assess the risk of developing cardiotoxicity induced by an anthracycline.

Description

Chromogranin A and uses thereof

TECHNICAL FIELD

The present invention refers to Chromogranin A (CgA) or a fragment thereof for use in the treatment and/or prevention of cardiotoxicity induced by an anthracycline and to relative pharmaceutical composition. The invention also relates to a method to assess the risk of developing cardiotoxicity induced by an anthracycline.

BACKGROUND ART

The therapeutic application of anthracy clines, a class of drugs widely used in cancer chemotherapy, is strongly limited by their cardiotoxic effects (Gianni et al, 2008; Ych & Bickford, 2009). Indeed, anthracyclines have been associated with the development of cardiomyopathy, left ventricular dysfunction and/or heart failure, depending on cumulative dose, administration schedule, and concomitant use of other cardiotoxic therapies (Yeh & Bickford, 2009). Cardiotoxicity may appear as congestive heart failure, or asymptomatic left ventricular ejection fraction drops, hypertension, arrhythmias, QT prolongation and myocardial ischemia (Yeh & Bickford, 2009). Cardiotoxic reactions to anthracyclines may be acute, i.e. during drug administration, subacute, or chronic (Yeh & Bickford, 2009). Among anthracyclines, doxorubicin (Doxo) is widely used for the treatment of various malignancies, such as breast cancer, bladder cancer, Kaposi’s sarcoma, acute lymphocytic leukemia, lymphoma and others (Minotti et al, 2004). This drug can exert anti-tumor effects by inhibiting topoisomerase and blocking DNA resealing during cell replication (Zhang et al, 2012). Doxo-induced cardiotoxicity may occur acutely after 2-3 days of treatment, or chronically within 1 year, causing dilated cardiomyopathy (Legha et al, 1982; Wojnowski et al, 2005). Moreover, some studies have shown that patients receiving Doxo have an increased risk of myocardial infarction (MI) persisting up to 25 years after Doxo treatment (Swerdlow et a/,2007).

Doxo-dependent cardiotoxicity involves various signaling pathways, including free radical generation, peroxynitrite formation, calcium overloading, mitochondrial dysfunction, alteration in b-adrenergic receptor signaling, and activation of matrix metalloproteinase (Minotti et al, 2004; Singal et al, 2000). Doxo can also induce in the human heart a dramatic alteration of the redox balance, possibly via an iron-mediated increase in reactive oxygen species (ROS) (Ichikawa et al, 2014).

Over the past decade, several adjuvant therapies have been proposed to decrease the cardiotoxic effects of Doxo. However, despite various therapeutic interventions, the deterioration of the cardiac functions is often accompanied by high mortality rates (Singal et al, 2000). Dexrazoxane is the only Food and Drug Administration-approved cardioprotective agent for anthracycline- induced cardiotoxicity (Zhang et al, 2016). This drug reduces cardiotoxicity possibly by removal of iron from Doxo by its EDTA-like hydrolysis product and by decreasing the levels of hydroxyl- free radicals. However, only 26% of patients treated with dexrazoxane tolerate higher cumulative doses of Doxo, compared with 5% of patients treated with placebo (Swain & Vici, 2004).

Chromogranin A (CgA) is a 439-residue long protein located in secretory vesicles of many neuroendocrine cells, neurons, granulocytes and cardiomyocytes (Helle et al, 2007). Upon cell stimulation CgA is exocytotically released in the blood to reach, in healthy subjects, 0.5-1 nM concentrations (Ceconi et al, 2002; Tota et al, 2014). Increased levels of circulating CgA have been observed in a variety of diseases, including cancer, sepsis, and various inflammatory disorders (Massironi et al, 2016; Hsu et al, 2015). For example, marked increase of CgA plasma levels have been detected in patients with neuroendocrine tumors, reaching up to 1000-fold the normal values in certain patients with carcinoid tumors (Syversen et al, 1994).

Clinical studies have suggested that CgA is also increased in cardiovascular pathologies (Angelone et al, 2012). High plasma CgA levels have been found in patients with hypertension, chronic and acute heart failure, myocardial infarction, decompensated and hypertrophic heart and acute coronary syndromes, with important prognostic implications (Ceconi et al, 2002; Goetze et al, 2014; Pieroni et al, 2007). In patients with dilated and hypertrophic cardiomyopathy, tissue expression of CgA co-localizes with B-type natriuretic peptide (BNP) in ventricular cardiomyocytes, while plasma CgA strongly correlates with circulating BNP or with C-terminal endothelin-l precursor fragment (CT-proET-l) (Goetze et al, 2014).

Proteolytic cleavage of CgA can lead to the production of various bioactive peptides, including, but not limited to, CgAi_₇₆(vasostatin-l), CgA352-372(catestatin), and CgA_{4i i-}436(serpinin), all characterized by cardiovascular activity (Helle et al, 2007; Tota et al, 2014). Ex vivo studies have shown that vasostatin-l and catestatin can induce cardiodepressive effects on isolated and perfused rat hearts through an anti-beta-adrenergic-nitric oxide (NO)-cGMP signaling (Cerra et al, 2006; Angelone et al, 2008), whereas serpinin acts like a beta 1 -adrenergic agonist through adenylate-cAMP-independent NO signaling (Tota et al, 2012). These peptides can also exert ex vivo cardioprotective effects against ischemia/reperfusion (I/R): in this setting vasostatin-l acts as a pre-conditioning inducer (Cappello et al, 2007), catestatin as a post-conditioning agent (Penna et al, 2010) and serpinin as a pre and post-conditioning agent (Pasqua et al, 2015). Notably, the full-length CgA, when added to perfused rat hearts, can reduce myocardial contractility and relaxation, and induces coronary dilation, by activating the endothelial NO-dependent pathway. Recent studies have also shown that hemodynamic and excitatory stimulation of rat hearts induce intra-cardiac CgA proteolytic processing to generate low molecular-weight fragments containing the vasostatin-l sequence (Pasqua et al, 2013).

There is still the need for novel pharmacological cardioprotective molecules to contrast toxicity induced by anthracy cline compounds.

SUMMARY OF THE INVENTION

In the present invention the effects of systemic administration of low-dose CgA or fragments thereof on anthracycline-induced inflammation, heart damage, and I/R injury in a rat model, as well as the effect of CgA on the anti-cancer activity of this drug in various murine models of solid tumors were investigated. It was found that that CgA or fragments thereof can indeed exert cardioprotective effects against anthracycline-induced cardiotoxicity, by reducing inflammation and by activating anti-apoptotic and pro-survival cascades, without impairing antitumor activity. Furthermore, the inventors show that intracardiac expression of CgA and its plasma levels are markedly decreased by anthracy dines. In particular such effects are observed when CgA is administered at physiological concentrations. Preferably the anthracycline is doxorubicin.

The inventors have surprisingly found that anthracycline-induced cardiotoxicity in a rat model can be prevented by administration of low-dose chromogranin A (CgA), a cardio-regulatory protein normally released in the blood by the neuroendocrine system and by the heart itself. Mechanistic studies, performed in vivo and ex vivo with isolated rat hearts, showed that exogenous CgA could prevent several anthracycline-induced adverse events in the heart, such inflammation, oxidative stress, apoptosis, fibrosis, and ischemic injury. On the other hand, anthracycline, such as Doxo, reduced intra-cardiac expression and release of CgA in the blood, suggesting that this anti-cancer drug can impair the production of an important endogenous cardioprotective agent. Finally, the inventors observed that CgA does not impair the anticancer activity of anthracycline in murine models of melanoma, fibrosarcoma, lymphoma and lung cancer. These findings suggest that changes in the endogenous levels of CgA in cancer patients may impact on anthracycline- induced cardiotoxicity and that administration of exogenous CgA might represent a novel approach to prevent adverse events without impairing anti-tumor effects.

The present invention demonstrates in a rat model that anthracycline such as doxorubicin, a cardiotoxic anticancer drug, can reduce the plasma levels of chromogranin A (CgA), an endogenous cardioprotective protein, and that systemic administration of low-dose exogenous CgA and/or its fragments can restore cardioprotection. These findings point to a novel cardioprotective role for circulating CgA and suggest that monitoring plasma levels of CgA before and after chemotherapy in cancer patients provide important prognostic information regarding drug-related cardiotoxicity. Furthermore, the inventors’ results suggest that administration of exogenous CgA and/or its fragments to patients might represent a novel pharmacological strategy to limit cardiac damage typically associated with anthracycline therapy particularly in patients with reduced plasma levels of this protein.

The present results demonstrate, on one hand, that systemic administration of CgA to rats can prevent anthracycline-induced cardiotoxicity, and, on the other hand, that administration of anthracycline to rats can reduce the endogenous CgA in the blood, i.e. of a potential protective factor.

The present invention provides chromogranin A (CgA) or a fragment thereof for use in the treatment and/or prevention of a cardiotoxicity induced by an anthracycline.

Preferably, the fragment is selected from a fragment comprising or consisting of: Catestatin, Cts (CgA_352-372), Vasostatinl, VS1 (CgAi_-76), Serpinin, Serp (CgA₄n-₄₃₆), CgAi-372, CgAi-373 and CgA_352-373. Preferably the fragment comprises VS I (CgA 1-₇₀) or Cts (CgA_352-372). More preferably the fragment consists of Catestatin, Cts (CgA_352-372), Vasostatinl, VS I (CgA _{1 -70}), Serpinin, Serp (CgA₄n-₄₃₆), CgAi-372, CgAi-373 or CgA352-373. Indicated fragments refer to fragments of SEQ ID No. 1.

In a preferred embodiment the cardiotoxicity induced by an anthracycline is acute, subacute or chronic.

Preferably the cardiotoxicity induced by an anthracycline is selected from the group consisting of: inflammation, cardiac fibrosis, ischemia/reperfusion injury, cardiomyocyte apoptosis, cardiomyopathy, left ventricular dysfunction and/or heart failure.

Preferably the anthracycline is selected from the group consisting of: doxorubicin, epirubicine, daunorubicine, idarubicine, nemorubicin, pixantrone, sabarubicine, valrubicine.

More preferably CgA is used at a physiological dose, preferably at a dose of between 3 pg/kg/day and 30 pg/kg/day.

Still preferably chromogranin A (CgA) or a fragment thereof is administered prior to or simultaneously with the anthracycline.

In a preferred embodiment chromogranin A (CgA) or a fragment thereof is administered in a subject with low levels of CgA and/or a fragment therefor, preferably the fragment CgAi-₃₇₂ or the fragment CgAi-₃₇₃.

Preferably the chromogranin A (CgA) or a fragment thereof does not prevent the anti-tumor activity of the anthracycline. The present invention provides a pharmaceutical composition comprising chromogranin A (CgA) or a fragment thereof and pharmaceutically acceptable carriers for use in the treatment and/or prevention of a cardiotoxicity induced by an anthracy cline.

Preferably the pharmaceutical composition further comprises an anthracycline, preferably the anthracycline is selected from the group consisting of: doxorubicin, epirubicine, daunorubicine, idarubicine, nemorubicin, pixantrone, sabarubicine, valrubicine.

The present invention also provides a method to assess the risk of developing cardiotoxicity induced by an anthracycline in a subject, comprising the step of measuring the level of Chromogranin A and/or a fragment thereof in a biological sample obtained from the subject. Preferably the biological sample is selected from the group consisting of: blood, plasma, urine, saliva, serum. Still preferably the subject is affected by cancer.

In the present invention a fragment of CgA may be any fragment that inhibits the cardiotoxicity induced by an anthracycline. The fragment may comprise the vasostatin-l (CgAl-76) and/or catestatin domains (CgA352-372) such as CgAl-373, CgAl-372, CgA352-373, CgA352-372, CgAl-76.

In the present invention CgA or the fragments thereof may be modified by removing 1 or more aminoacid residues from their N-terminus, and/or by coupling with compounds that increase their plasma half-life and bioavailability (e.g. polyethylene glycol or nanoparticles) and/or by introducing chemical modifications that reduce their susceptibility to proteolytic degradation.

The cardiotoxicity induced by an anthracycline may be a cardiac injury induced by anthracycline therapy that is detected as left ventricular ejection fraction (LVEF) changes (Vejpongsa and Yeh 2014). In particular, cardiotoxicity is defined as an LVEF decline of >5% to <55% with heart failure symptoms or an asymptomatic decrease of LVEF >10% to <55% (Curigliano et al., 2012). The LVEF changes anthracycline-induced are measured by echocardiography, multigated acquisition scans, or magnetic resonance imaging (de Geus-Oei et al., 2011; Sawaya et al., 2011; Wassmuth et al; 2001). Factors that influence LVEF in patients exposed to anthracycline - containing regimens include fluid overload, sepsis, ischemic heart disease, or other chemotherapy drugs (Vejpongsa and Yeh 2014).

Acute, early-onset chronic progressive, and late-onset chronic progressive cardiotoxicity may be defined according to Grenier and Lipshultz (1998) and to Lipshultz et al. (2008) in which the cardiotoxicity induced by anthracycline therapy is categorized into acute, early-onset chronic progressive, and late-onset chronic progressive. Acute cardiotoxicity occurs in <1% of patients immediately after infusion of the anthracycline and manifests as an acute, transient decline in myocardial contractility, which is usually reversible (Wouters et al., 2005; Yeh and Bickford; 2009). The early-onset chronic progressive form occurs in 1.6% to 2.1% of patients, during therapy or within the first year after treatment (Wouters et al., 2005; Yeh and Bickford; 2009). Late-onset chronic progressive anthracycline-induced cardiotoxicity occurs at least 1 year after completion of therapy in 1.6% to 5% of patients (Wouters et al., 2005; Yeh and Bickford; 2009). In the present invention, the doses of CgAi-439 or the fragment CgAi-372 or the fragment CgAi-373 are between 3 and 30 pg/kg/day (0.07-6 nmol/kg/day), the doses of CgA352-373 are between 0.05- 10 nmol/kg/day, and the doses of the other fragments are between 6 and 100 nmol/kg/day.

A subject with low levels of CgA comprises a subject with plasma levels lower than 1 nM, preferably lower than 0.5 nM, or a subject with the fragment CgAi-372 or the fragment CgAi-373 lower than 0.5 nM, preferably lower than 0.2 nM.

The present invention also provides a method to assess the risk of developing cardiotoxicity induced by an anthracycline in a subject, comprising the step of measuring the level of Chromogranin A and/or of a fragment thereof in a biological sample obtained from the subject and comparing said measured level to a proper control. Any known method in the art (e.g. immune assay, radio-immuno assay, ELISA, fluoro-immunoassay, chemiluminescent-immunoassays, gold- and metal-based immunoassay, lanthanide-based immunoassay, HPLC-based assays) may be used to measure the level of Chromogranin A, in particular as described below or using a reference standard of known quantity of recombinant or synthetic CgA or a fragment thereof. Proper control may be the level of Chromogranin A and/or of a fragment thereof in a biological sample of a healthy subject or a non-cancer subject.

In the present invention CgA or fragment thereof may include other amino acid residues. In one embodiment, CgA or fragment thereof includes additional amino acids at the amino-ter inal end, the carboxy-terminal end, or both the amino- and carboxy-terminal ends.

Further, at least one amino acid of CgA or a fragment thereof may be substituted with a conservative substitution. A conservative substitution for an amino acid in a polypeptide described herein may be selected from other members of the class to which the amino acid belongs. For example, it is known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and Gin for Asn to maintain a free -NH2. The overall structural similarity for CgA in different vertebrates is around 40%, but the most highly conserved regions occur at the N and C -termini, which show much higher levels of structural similarity.

Guidance on how to modify the amino acid sequences of a protein disclosed herein may be provided by comparing the amino acid sequences of a human CgA and a mouse CgA in a protein alignment. Identical amino acids strongly conserved amino acids and weakly conserved amino acids can be identified. The skilled person can predict which alterations to an amino acid sequence are likely to modify the biological activity of CgA or a fragment thereof, as well as which alterations are unlikely to modify biological activity.

Thus, as used herein, in one embodiment CgA or a fragment thereof includes those having, having at least, or having no greater than, 1 , 2 or 3 amino acid substitutions, for instance a conservative substitution, compared to a reference amino acid sequence, such as SEQ ID NO:l. In one embodiment, CgA or a fragment thereof includes those with at least 50%, at least 55%, at least

60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least

87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least

94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to a reference amino acid sequence, such as SEQ ID NO:l.

CgA or a fragment thereof has biological activity. Whether a polypeptide has biological activity may be determined by in vitro or in vivo assays. In one embodiment, biological activity refers to the ability to exert cardioprotective effects against anthracycline-induced cardiotoxicity.

As discussed above, CgA or a fragment thereof may include other amino acid residues. In one embodiment, the additional amino acids are heterologous amino acids. As used herein, "heterologous amino acids" refers to amino acids that are not normally or naturally found flanking the sequence depicted at, for instance, SEQ ID NO:l in a natural CgA protein. The number of heterologous amino acids at the amino-terminal end, the carboxy-terminal end, or both the amino- and carboxy-terminal ends may be, may be at least, or may be no greater than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 amino acids, and so on. Such a polypeptide that includes, for instance, SEQ ID NO:l or fragment thereof and heterologous amino acids may be referred to as a fusion polypeptide.

In one embodiment, the additional amino acid sequence may be useful for purification of the fusion polypeptide by affinity chromatography. Various methods are available for the addition of such affinity purification moieties to proteins. Representative examples include, for instance, polyhistidine-tag (His-tag) and maltose-binding protein (see, for instance, Hopp et al. (U.S. Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137), Sgarlato (U.S. Pat. No. 5,935,824), and Sharma (U.S. Pat. No. 5,594,115)). In one embodiment, the additional amino acid sequence may be a carrier polypeptide. The carrier polypeptide may be used to increase the iminunogenicity of the fusion polypeptide to increase production of antibodies that specifically bind to a polypeptide of the invention. The invention is not limited by the types of carrier polypeptides that may be used to create fusion polypeptides. Examples of carrier polypeptides include, but are not limited to, keyhole limpet hemacyanin, bovine serum albumin, ovalbumin, mouse serum albumin, rabbit serum albumin, and the like. In another embodiment, the additional amino acid sequence may be a fluorescent polypeptide (e.g., green, yellow, blue, or red fluorescent proteins) or other amino acid sequences that can be detected in a cell, for instance, a cultured cell, or a tissue sample that has been removed from an animal. If a polypeptide described herein includes an additional amino acid sequence not normally or naturally associated with the polypeptide, the additional amino acids are not considered when percent structural similarity to a reference amino acid sequence is determined.

Polypeptides described herein can be produced using recombinant DNA techniques, such as an expression vector present in a cell. Such methods are routine and known in the art. The polypeptides may also be synthesized in vitro, e.g., by solid phase peptide synthetic methods. The solid phase peptide synthetic methods are routine and known in the art. A polypeptide produced using recombinant techniques or by solid phase peptide synthetic methods can be further purified by routine methods, such as fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on an arrion- exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, or ligand affinity. Such methods may also be used to isolate CgA polypeptide or a fragment thereof from a cell. Also provided are polynucleotides. In one embodiment, a polynucleotide encodes a polypeptide described herein. Also included are the complements of such polynucleotide sequences. A polynucleotide encoding a polypeptide having biological activity is referred to herein as CgA polynucleotide or a fragment thereof.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxynucleotides, peptide nucleic acids, or a combination thereof, and includes both single-stranded molecules and double-stranded duplexes. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. In one embodiment, a polynucleotide is isolated. An "isolated" polynucleotide is one that has been removed from a cell. For instance, an isolated polynucleotide is a polynucleotide that has been removed from a cell and many of the polypeptides, nucleic acids, and other cellular material of its natural environment are no longer present. A "purified" polynucleotide is one that is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components of a cell. Polynucleotides that are produced by recombinant, enzymatic, or chemical techniques are considered to be isolated and purified by definition, since they were never present in a cell.

Given the amino acid sequence of any one of CgA polypeptide or a fragment thereof described herein, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods. The class of nucleotide sequences encoding a selected polypeptide sequence is large but finite, and the nucleotide sequence of each member of the class may be readily determined by one skilled in the art by reference to the standard genetic code, wherein different nucleotide triplets (codons) are known to encode the same amino acid.

A CgA polynucleotide or a fragment thereof described herein may include heterologous nucleotides flanking the coding region encoding the CgA-derived polypeptide. As used herein, the terms "coding region" and "coding sequence" are used interchangeably and refer to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A "regulatory sequence" is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term "operably linked" refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence. As used herein, "heterologous nucleotides" refers to a nucleotide sequence that is not normally or naturally found flanking an open reading frame in a cell encoding CgA polypeptide or a fragment thereof. Nucleotides normally or naturally found flanking nucleotides encoding CgA polypeptide or a fragment thereof include those present in exon VII of the CgA gene. Typically, heterologous nucleotides may be at the 5' end of the coding region, at the 3' end of the coding region, or the combination thereof. Examples of heterologous nucleotides include, but are not limited to, a regulatory sequence. The number of heterologous nucleotides may be, for instance, at least 10, at least 100, or at least 1000.

A polynucleotide described herein can be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide of the invention employs standard ligation teclmiques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, transposon vectors, and artificial chromosome vectors. Examples of viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors. A vector may be replication-proficient or replication- deficient. A vector may result in integration into a cell’s genomic DNA. Typically, a vector is capable of replication in a host cell, for instance a mammalian and/or a bacterial cell, such as E. coli.

Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, use in gene transfer into cells of the gastrointestinal tract, and the like. Suitable host cells for cloning or expressing the vectors herein are prokaryotic or eukaryotic cells. Suitable eukaryotic cells include mammalian cells, such as murine cells and human cells. Suitable prokaryotic cells include eubacteria, such as gram negative organisms, for example, E. coli.

An expression vector optionally includes regulatory sequences operably linked to the polynucleotide of the present invention. An example of a regulatory sequence is a promoter. A promoter may be functional in a host cell used, for instance, in the construction and/or characterization of CgA polynucleotide or a fragment thereof, and/or may be functional in the ultimate recipient of the vector. A promoter may be inducible, repressible, or constitutive, and examples of each type are known in the art. A polynucleotide of the present invention may also include a transcription terminator. Suitable transcription terminators are known in the art. Polynucleotides described herein can be produced in vitro or in vivo. For instance, methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for in vitro synthesis are known. Methods for in vitro synthesis also include, for instance, in vitro transcription using a circular or linear expression vector in a cell free system. Expression vectors can also be used to produce a polynucleotide of the present invention in a cell, and the polynucleotide may then be isolated from the cell.

Also provided are compositions including one or more polypeptides or polynucleotides described herein. Such compositions typically include a pharmaceutically acceptable carrier. As used herein "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Additional compounds can also be incorporated into the compositions.

A composition may be prepared by methods known in the art of pharmacy. In general, a composition can be formulated to be compatible with its intended route of administration. A formulation may be solid or liquid. Administration may be systemic or local. In some aspects local administration may have advantages for site-specific, targeted disease management. Local therapies may provide high, clinically effective concentrations directly to the treatment site, with less likelihood of causing systemic side effects.

Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular), enteral (e.g., oral or rectal), and topical (e.g., epicutaneous, inhalational, transmucosal) administration. Appropriate dosage forms for enteral administration of the compound of the present invention may include tablets, capsules or liquids. Appropriate dosage forms for parenteral administration may include intravenous administration. Appropriate dosage forms for topical administration may include nasal sprays, metered dose inhalers, dry-powder inhalers or by nebulization.

Solutions or suspensions can include the following components: a sterile diluent such as water for administration, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; electrolytes, such as sodium ion, chloride ion, potassium ion, calcium ion, and magnesium ion, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A composition can be enclosed in, for instance, ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Compositions can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline. A composition is typically sterile and, when suitable for injectable use, should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compound (e.g., a polypeptide or polynucleotide described herein) in the required amount in an appropriate solvent with one or a combination of ingredients such as those enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a dispersion medium and other ingredient such as from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation that may be used include vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

For enteral administration, a composition may be delivered by, for instance, nasogastric tube, enema, colonoscopy, or orally. Oral compositions may include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier. Pharmaceutically compatible binding agents can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the active compounds may be delivered in the form of an aerosol spray, a nebulizer, or an inhaler, such as a nasal spray, metered dose inhaler, or dry- powder inhaler.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds may be formulated into ointments, salves, gels, or creams as generally known in the art. An example of transdermal administration includes iontophoretic delivery to the dermis or to other relevant tissues. The active compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

The active compounds may be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthocstcrs, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. Delivery reagents such as lipids, cationic lipids, phospholipids, liposomes, and microencapsulation may also be used.

In one embodiment, an active compound may be associated with a targeting group. As used herein, a "targeting group" refers to a chemical species that interacts, either directly or indirectly, with the surface of a cell, for instance with a molecule present on the surface of a cell, e.g., a receptor. The interaction can be, for instance, an ionic bond, a hydrogen bond, a Van der Waals force, or a combination thereof Examples of targeting groups include, for instance, saccharides, polypeptides (including hormones), polynucleotides, fatty acids, and catecholamines. Another example of a targeting group is an antibody. The interaction between the targeting group and a molecule present on the surface of a cell, e.g., a receptor, may result in the uptake of the targeting group and associated active compound. Cells that may be targeted include, but are not limited to, cell of the gastrointestinal tract such as macrophages, CD4+ and CD8+ T cells, NKT cells, mast cells, intraepithelial T cells, T regulatory cells, granulocyte (neutrophils, basophils, eosinophils), B cells, and dendritic cells.

When a polynucleotide is introduced into cells using a suitable technique, the polynucleotide may be delivered into the cells by, for example, transfection or transduction procedures. Transfection and transduction refer to the acquisition by a cell of new genetic material by incorporation of added polynucleotides. Transfection can occur by physical or chemical methods. Many transfection techniques are known to those of ordinary skill in the art including, without limitation, calcium phosphate DNA co- precipitation, DEAE-dextrin DNA transfection, electroporation, naked plasmid adsorption, cationic liposome-mediated transfection (commonly known as lipofection), use of glycoconjugatcs and polyplexes, targeting serpin-enzyme complex receptors, and polyefhyleneimine. Transduction refers to the process of transferring nucleic acid into a cell using a DNA or R A virus.

A polynucleotide described herein may be used in combination with other agents assisting the cellular uptake of polynucleotides, or assisting the release of polynucleotides from endosomes or intracellular compartments into the cytoplasm or cell nuclei by, for instance, conjugation of those to the polynucleotide. The agents may be, but are not limited to, peptides, especially cell penetrating peptides, protein transduction domains, and/or dsRNA-binding domains which enhance the cellular uptake of polynucleotides (Dowdy et ah, US Published Patent Application 2009/0093026, Eguchi et ah, 2009, Nature Biotechnology 27:567-571, Lindsay et ah, 2002, Curr. Opin. Pharmacol., 2:587- 594, Wadia and Dowdy, 2002, Curr. Opin. Biotechnol. 13:52-56. Gait, 2003, Cell. Mol. Life Sci., 60: 1-10). The conjugations can be performed at an internal position in the oligonucleotide and/or at a terminal position, such as the 5'-end and/or the 3'-end.

Toxicity and therapeutic efficacy of such active compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the ED50(the dose therapeutically effective in 50% of the population).

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such active compounds lies preferably within a range of concentrations that include the ED50with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For an active compound used in the methods of the invention, it may be possible to estimate the therapeutically effective dose initially from cell culture assays. A dose may be formulated in animal models to achieve a concentration range that includes the IC50(i.e., the concentration of the test compound which achieves a half-maximal inhibition of signs and/or symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

The compositions can be administered one or more times per day to one or more times per week, including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with an effective amount of a polynucleotide or a polypeptide can include a single treatment or can include a series of treatments.

As used herein, an "effective amount" relates to a sufficient amount of CgA or a fragment thereof to provide the desired effect. For instance, in one embodiment an "effective amount" is an amount effective to exert cardioprotective effects against anthracycline-induced cardiotoxicity, by reducing inflammation and by activating anti-apoptotic and pro-survival cascades, without impairing antitumor activity.

The present invention will be illustrated by means of non-limiting examples in reference to the following figures.

Fig. 1. Schematic diagram of the experimental protocol used in rats.

Fig. 2. Effect of CgA on the systemic inflammation and cardiac fibrosis induced by Doxo in rats. Plasma levels of interleukin- 1b (IL- 1 b) (n=7 rats/group) (A), tumor necrosis factor-a (TNF- a) (n=7 rats/group) (B), reactive oxygen species (ROS) (n=4 rats/group) (C), lactate dehydrogenase (LDH) (n=3-6 rats/group) (D) and total plasma proteins (n=5-l2 rats/group) (E) in Control, Doxo, Doxo+CgA and CgA groups. (E) Western blot analysis of non-perfused heart tissues (n=3 hearts/group) with anti-connective tissue growth factor (CTGF) antibodies. Histograms represent the ratio of densitometric analysis of protein/loading control: p<0.05 (*), p<0.0l (**), p<0.00l (***), by One-Way ANOVA/Newman-Keuls Multiple Comparison Test. Fig. 3. Effect of Doxo and CgA, alone and in combination, on I/R injury of the rat heart.

(A) dLVP and (B) LVEDP variations. Data are expressed as changes of dLVP and LVEDP values (mmHg) from the stabilization to the end of the l20-min of reperfusion with respect to the baseline values for Control, Doxo, Doxo+CgA and CgA groups (n=7-l0 hearts/group). Grey boxes indicate ischemic administration (Bonferroni Multiple Comparison test), dLVP=29.3% of total variation between groups (p<0.00l); LVEDP=39.86% of total variation between groups (p <0.001). Inset graph shows the dLVP and LVEDP at the end of reperfusion (One-way ANOVA/Newman-Keuls Multiple Comparison Test, *=p <0.05; **=p<0.01 ). (C) Infarct size (h=7-10 hearts/group). The amount of necrotic tissue measured after 30-min global ischemia and l20-min reperfusion is expressed as percent of the LV mass (% IS/LV). p<0.00l (***), by One- way ANOVA/Newman-Keuls Multiple Comparison Test.

Fig. 4. Effect of Doxo and CgA, alone and in combination, on protective signaling cascades.

Western blot analysis of (A) Akt, (B) Erkl/2, (C) GSK3a/p, (D) STAT-3 and (E) AMPKa phosphorylation in heart tissues from SHAM, Control, Doxo, Doxo+CgA and CgA groups (n=3 hearts/ group) after I/R. Histograms represent the ratio of densitometric analysis of protein/loading control: p<0.05 (*), p<0.0l (**), p<0.00l (***), by One-Way ANOVA/Newman-Keuls Multiple Comparison Test.

Fig. 5. Effect of Doxo and CgA, alone and in combination, on markers of tissue inflammation. Detection of (A) Cox-2 and iNOS, (B) NLRP3, (C) AOX-l, and (D) XO (by western blotting) and (E) ROS production (by ELISA) in heart tissues from SHAM, Control, Doxo, Doxo+CgA and CgA groups (n=3 hearts/group). Histograms represent the ratio of densitometric analysis of protein/loading control: p<0.05 (*), p<0.0l (**), p<0.00l (***), by One- Way ANOVA/Newman-Keuls Multiple Comparison Test.

Fig. 6. Effect of Doxo and CgA on cardiomyocytes apoptosis in rats. Immunohistochemical localization of ARC in ventricular cardiomyocytes (A). Western blotting analysis of Bcl-2 (B), Bax (C) and active caspase 3 (D) expression in heart tissues of SHAM, Control, Doxo, Doxo+CgA and CgA groups (n=3 hearts/group) after I/R. Histograms represent the ratio of densitometric analysis of protein/loading control: p<0.05 (*), p<0.0l (**), p<0.00l (***), by One-Way ANOVA/Newman-Keuls Multiple Comparison Test. (E) TUNEL assay of rat heart sections (representative images). TUNEL-positive nuclei of cardiomyocytes are indicated by white arrows; “Negative” (without TdT); nuclei stained with propidium iodide (red).

Fig. 7. Effect of Doxo on intracardiac CgA expression in rats. Western blot analysis of human recombinant CgA (hCgA) and of tissue extracts of not-perfused heart (NP) from Control, Doxo, Doxo+CgA and CgA groups (n=3 hearts/group). Histograms represent the densitometric analysis of the ~60 kDa, ~45 kDa and ~25 kDa bands: bars represent the area of each band expressed as a percentage of the total area of the same band in the four groups p <0.05 (*), p<0.0l (**), by One- Way ANOVA/Newman-Keuls Multiple Comparison Test.

Fig. 8. Effect of Doxo on the circulating levels of CgA in rats. Plasma levels of Control, Doxo, Doxo+CgA and CgA treated rats (n=4-l3 for each group) were detected by two ELISA based on the use of mAh 5A8 in the capture step and rabbit antiserum against full-length CgA (A, assay- 1) or a rabbit antiserum against CgA410-439 (B, assay-2) p <0.05 (*), p<0.0l (**), by unpaired two-tailed /-test. The levels of CgA in adrenal gland extracts are also shown (C). Fig. 9. Anti-tumor activity of Doxo in the absence and presence of CgA in tumor-bearing mice. Mice bearing subcutaneous tumors (B16 melanoma, RMA lymphoma, WEHI-164 fibrosarcoma, or LLC Lewis lung carcinoma) were treated as indicated. CgA: 1.5 pg (i.p., all models); Doxo: 220 pg (i.p., B16F1 melanoma); 130 pg (i.p., RMA lymphoma); 150 pg (day 6) and 200 pg (day 15) (i.v., WEHI-164 fibrosarcoma); 80 pg (day 6) and 100 pg (day 9) (i.p., Lewis lung carcinoma) p <0.05 (*), p<0.0l (**), by unpaired two-tailed t-test. Arrows, time of treatment; tumor volume (six mice per group, mean +/- SE). The area under the curve for each mouse was calculated using GraphPad Prism. Differences between calculated areas were evaluated by Mann-Whitney tests (6 animals per group; **, P < 0.01; *, P < 0.05).

Fig. 10. (A) Representative immunoblots of CgA levels (~70 kDa) in plasma samples from rats of Control, Doxo, Doxo+CgA and CgA groups (n=3 plasma samples/group), as detected with mAb5A8. (B) Negative control using only goat anti-mouse without primary antibody (mAb 5A8), showing that only the ~70 kDa band was specific.

Fig. 11. (A) dLVP and (B) LVEDP variations. Data are expressed as dLVP and LVEDP values (mmHg) at the end of the l20-min of reperfusion for Doxo (h=10), Doxo+CgA 1-372 (n=3), Doxo+CgA 1-373 (n=3), Doxo+CgA 352-373 (n=2), Doxo+Cts (n=4), Doxo+rhVSl (n=4) and Doxo+Serp (n=3). (C) Infarct size. The amount of necrotic tissue measured after 30-min global ischemia and l20-min reperfusion is expressed as percent of the left ventricle mass (LV) (% IS/LV). (D) Levels of total plasma proteins for Doxo (n=l 1), Doxo+CgA 1-372 (n=3), Doxo+CgA 1-373 (n=3), Doxo+CgA 352-373 (n=3), Doxo+Cts (n=5), Doxo+rhVSl (n=5) and Doxo+Serp (n=4). For comparison, the inventors reported here Control group. *p<0.05, **p < 0.01, ***p < 0.001 by One-way ANOVA/Newman-Keuls Multiple Comparison Test.

Fig. 12. Body weight measurements in rats chronically daily treated for one week with (A) Saline (Control) (n=6), (B) Doxo (n=9), (C) Doxo+CgA (n=6), (D) Doxo+Cts (n=5), (E) Doxo+rhVSl (n=5), (F) Doxo+Serp (n=4), (G) CgA (n=6), (H) Doxo+CgAi-372, (I) Doxo+CgA 1-373 and (L) Doxo+CgA 352-373. Values of body weight (g) were registered at the start and at the end of the treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by t-test.

Fig. 13. Levels of total CgA, CgAl-439, 1-76 and troponin T in a lymphoma patient treated with CHOP (6 cycles), a chemotherapy regimen that inludes doxorubicine. Assays were performed in plasma samples collected before each cycle of treatment (thus three weeks after each treatment) and 6 months after the last cycle. CgA and its fragments and troponin T levels were detected by ELISA, including an ELISA for full-length CgAi-439, an ELISA for CgAi-76 and an ELISA unable to discriminate between full-length CgA and fragments (total-CgA-ELISA). Fig. 14. Levels of C Ai-372/3 and troponin T in 15 patients with breast cancer treated with FEC (4 cycles), a chemotherapy regimen that includes the antracycline epirubicin. The correlation between CgAi -372/3 levels before the I^st and 2^nd cycles and troponin T after the 4^th cycle is shown. Correlation coefficients (r and r²) and p values are also shown.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Faboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Faboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, NY.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Filley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

Materials and Methods

Animals. Male Wistar rats (~300 g body weight) (Harlan Laboratories, Udine, Italy), identically housed under controlled lighting and temperature conditions, fed a standard diet and water ad libitum. All protocols were conducted in accordance with the Declaration of Helsinki, the Italian law (DL.26/2014), the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011) and the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific. The project was approved by the Italian Ministry of Health, Rome and by the ethics review board.

Drugs.

Doxorubicin (Doxo) was provided from Sigma Aldrich (cat. Number 25316-40-9, Milan, Italy) Full-length recombinant CgAi_₄39(CgA) was prepared by expression of the human CgA cDNA in E. coli cells and purified as previously described (Crippa et al, 2013).

CgAl-439 (SEQ ID No. 1)

LPVNSPMNKGDTEVMKCIVEVISDTLSKPSPMPVSQE (37) CFETLRGDERILS ILRHQNLLKELQDLALQGAKERAH (74)

QQKKHSGFEDELSEVLENQSSQAELKEAVEEPSSKDV (111)

MEKREDSKEAEKSGEATDGARPQALPEPMQESKAEGN (148)

NQAPGEEEEEEEEATNTHPPASLPSQKYPGPQAEGDS (185)

EGLSQGLVDREKGLSAEPGWQAKREEEEEEEEEAEAG (222)

EEAVPEEEGPTVVLNPHPSLGYKEIRKGESRSEALAV (259)

DGAGKPGAEEAQDPEGKGEQEHSQQKEEEEEMAVVPQ (296)

GLFRGGKSGELEQEEERLSKEWEDSKRWSKMDQLAKE (333)

LTAEKRLEGQEEEEDNRDSSMKLSFRARAYGFRGPGP (370)

QLRRGWRPSSREDSLEAGLPLQVRGYPEEKKEEEGSA (407)

NRRPEDQELESLSAIEAELEKVAHQLQALRRG (439)

In vivo experiments in rats

Animal treatments. Wistar rats were divided in 4 groups and treated daily for 1 week (i.p.), as follows: a) group I (Control): 3 mL/kg/day of saline (16 rats); b) group II (Doxo): 3 mg/kg/day of Doxo (cumulative dose, 21 mg/kg (16 rats); c) group III (Doxo+CgA) (13 rats); 3 mg/kg/day of Doxo plus 10.71 pg/kg/day of CgA (13 rats); d) group IV (CgA): 10.71 pg/kg/day of CgA (13 rats). This dose of CgA (about 0.2 nanomoles/kg/day, which is very low) generates physiologically relevant plasma levels of CgA (Cumis et al, 2016).

Animals were sacrificed after 7 days to evaluate: i) plasma levels of inflammatory cytokines, LDH, ROS, and CgA; ii) heart performance by Langendorff perfusion technique; iii) intracellular cardiac signalling. To these aims, 3 hearts/group were directly used for western blotting assays, whereas 10 hearts/group were subjected to Langendorff perfusion and I/R protocols (see below). In addition, 3 hearts from the“Control” group were perfused with Krebs-Henseleit (KH) buffer (see below) for 190 min (named“SHAM”). Finally, 3 hearts from each Langendorff perfused groups were used for immunohistochemical and intracellular signalling studies (apex of hearts). After sacrifice, blood samples were collected from the abdominal aorta with heparinized syringes. Plasma was then separated by centrifugation at 3000 x g (l5min, 4°C) and stored at -80°C until analysis. Samples of coronary effluent, during reperfusion, were withdrawn with a catheter inserted into the right ventricle via the pulmonary artery.

Enzyme-Linked Immunosorbent Assays (ELISAs). Detection of ROS, TNF-a and IL-l b in plasma samples was performed by ELISA using commercial kits (ROS, Sunred Biological Technology, Shanghai, China; TNF-a and I L- 1 b, Thermo Scientific, Rockford, USA). The cardiac production of ROS was evaluated as follows: the left ventricle of hearts was homogenized using Ultra-Turrax® in Phosphate-Buffered Saline (137 mM NaCl, 2.7 mM KC1, 10 mM Na₂HP0₄, 1.8 mM KH₂P0₄; pH 7.4) plus a mixture of protease inhibitors (1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride, and 200 mmol/L sodium orthovanadate) and centrifuged at 15000 x g for 20 min (4°C). The supernatants were then tested by ELISA.

The plasma CgA levels were detected using two sandwich ELISAs based on the use of mAh 5 A8 (cross reactive with human, mouse and rat CgA) in the capture step and rabbit antisera against murine full-length CgA (assay- 1) or against the CgA_4io ₄39 region (assay 2). The assays were performed as described previously (Crippa et al, 2013). Assay validation studies showed that the assay- 1 can detect CgA as well as fragments lacking the C-terminal region, whereas the assay -2 specifically detects CgA. The ratio between the results of assay-2 and -1 is an index of C-terminal fragmentation. Recombinant full-length murine CgA was used to calibrate the assays. Assay- 1 and -2 analytical recovery of rat CgA from plasma samples spiked with Doxo was >90% when Doxo concentrations were lower than 100 lig/mL.

Lactate dehydrogenase (LDH) determination. LDH enzymatic activity in blood and in coronary effluent from isolated Langendorff heart perfusion were measured spectrophotometrically at 340 nm by monitoring NADH consumption during the reduction of pyruvate to lactate as described (Penna et al, 2006). Data (IU/L) were expressed as cumulative values for the entire reperfusion period.

Ex vivo experiments

Heart Perfusion. At the end of the treatments described above, rats were anesthetized with ethyl carbamate (2 g/kg body weight, i.p) and sacrificed. Then, hearts were rapidly excised, immediately placed in ice-cold perfusion buffer, cannulated via the aorta and perfused in the Langendorff apparatus at a constant flow-rate of 12 mL/min (37°C), as previously described (Pasqua et al, 2015). Perfusion was performed by using a KH buffer containing 4.7 mM KC1, 113 mM NaCl, 25 mM NaHCOs, 1.8 mM CaCl₂, 1.2 mM MgS0₄, 1.2 mM KH₂P0₄, 1.1 mM mannitol, 11 mM glucose, 5 mM Na-pyruvate (Sigma Aldrich, Saint Louis, Missouri, USA) (pH 7.4; 37°C; 95% 0₂ and 5% C0₂). A water-filled latex balloon was connected to a pressure transducer (BLPR; WRI, Inc., Sarasota, FL, USA) and inserted into the left ventricle through the mitral valve, to record cardiac mechanical parameters. Another pressure transducer was located above the aorta to measure coronary pressure (CP). The developed left ventricular pressure (dLYP) and the left ventricular end diastolic pressure (LVEDP) were measured to evaluate inotropism (Pasqua et al, 2013). The endurance of the preparations was stable up to 190 min. The performance variables were measured every 10 min. Parameters were recorded by using the PowerLab data acquisition system (AD Instruments, Oxford-UK) as previously reported (Pasqua et al, 2013).

Ischemia/Reperfusion (I/R) protocols. After chronic treatment, 7 hearts from each group described above (Control, Doxo, Doxo+CgA, and CgA) were subjected to 30 min of global, no- flow ischemia, followed by 120 min of reperfusion (I/R).

At the end of treatments, cardiac parameters were analysed by Langendorff technique. Cardiac performance before and after ischemia was evaluated by analyzing LVP recovery, as an index of contractile activity, and LVEDP as an index of contracture, defined as an increase in LVEDP of 4 mmHg above the baseline level (Pasqua et al, 2015).

Infarct size (IS). To measure infarct areas hearts were rapidly removed from the perfusion apparatus at the end of reperfusion. The left ventricles were dissected transversely into 2-3 mm slices. After 20 min of incubation at 37°C in 0.1% nitro blue tetrazolium in phosphate buffer (59.8mM NaH₂P0₄, 484.9mM Na₂HP0_4, pH:7.4), unstained necrotic tissues were carefully separated from stained viable tissues by an independent observer who was not aware of the nature of the intervention. The weights of the necrotic and non-necrotic tissues were then determined, and the necrotic mass was expressed as a percentage of total left ventricular mass, including septum (Pasqua et al, 2015).

A comprehensive diagram showing the experimental protocol for both in-vivo and ex-vivo studies is detailed in Fig.1.

Immunoblotting analysis. After chronic treatments and perfusion (I/R protocol) apex of cardiac ventricles (n=3 for each group) were homogenized in ice-cold RIPA lysis buffer (Sigma-Aldrich) containing a mixture of protease inhibitors (1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride, and 200 mmol/L sodium ortho vanadate). Then homogenates were centrifuged at 15000 x g for 20 min at 4°C for debris removal. Protein concentration was determined using a Bradford reagent according to the manufacturer instructions (Sigma-Aldrich, Missouri, USA). Equal amounts of proteins (30 pg) were loaded on 8% SDS-PAGE gels (NLRP3, iNOS, Cox-2, XO and AOX-l), or 10% SDS-PAGE gels (CTGF, p-AMPKa, AMPKa, p-Akt, Akt, p-Erkl/2, Erkl/2, p-GSK-3 a/b, GSK-3a/p, p-STAT3 and STAT3), or 12% SDS-PAGE gels (Bcl-2 Bax, and active caspase 3), subjected to electrophoresis and transferred to polyvinyl difluoride membranes. The membranes were blocked with not fat dried milk and incubated overnight at 4°C with different antibodies including monoclonal rabbit antibodies against GSK- 3a/b, GAPDH (Santa Cruz Biotechnology, California USA), p-AMPKa and NLRP3 (Cell Signaling Technology, Danvers, USA), or polyclonal rabbit antibodies against p-Akt, Akt, Erkl/2, iNOS, AOX-l (Santa Cruz Biotechnology, California USA), AMPKa (Cell Signaling Technology, Danvers, USA), p-GSK-3a/p and active caspase 3 (Sigma Aldrich, Missouri, USA), or monoclonal mouse antibodies against p-Erkl/2, Bax, Bcl-2 (Santa Cruz Biotechnology, California USA), p-STAT3 and STAT3 (Cell Signaling Technology, Danvers, USA) or polyclonal goat antibodies against XO, CTGF and Cox-2 (Santa Cruz Biotechnology, California USA), diluted 1 : 1000 in Tris-buffered saline containing 0.1% Tween 20 and 5% not fat dry milk (TBSTM). Antibodies against Akt, ERK1/2, AMPKa, GSK-3a/p, GAPDH, or STAT-3 were used as loading controls. Anti-rabbit, anti-goat and anti-mouse peroxidase-linked secondary antibodies (Santa Cruz Biotechnology, California USA) were diluted 1 :2000 in TBSTM. Immunodetection was performed using the ECL PLUS enhanced chemiluminescence kit. Autoradiographs were obtained by membrane exposure to X-ray films (Amersham, UK). Immunoblots were digitalized; densitometric analyses of the bands areas and the pixel intensity represented by 256 Gray values (0=white; 256=black) and the background was subtracted. The analyses were carried out using NIH IMAGE 1.6 (National Institutes of Health, Bethesda, USA).

CgA intracardiac expression. CgA expression was evaluated by western blotting using a mouse monoclonal antibody (5A8) (Ratti et al, 2000) directed against the vasostatin-l sequence of CgA (CgA -76). Perfused and not perfused hearts homogenates (3 different hearts/group) were separated on 10% SDS-PAGE gels (30 pg of total proteins) and immunodetected with mAb 5A8 as described above.

Histological analysis. After I/R, hearts (n=3 for each group) were removed from the perfusion apparatus and flushed with PBS, pH 7.4. The hearts were blocked in diastole with an excess of KC1 (0.5g/L) and fixed with 2:2: 1 v/v methanol:acetone:water (MAW fixative). The samples were dehydrated with graded ethanol, embedded in paraplast (Sherwood, St. Louis, MO, USA), and serially sectioned (thickness, 8pm). Sections were placed onto Superfrost Plus slides (Menzel- Glaser, Germany). Hearts sections, after deparaffinization and rehydratation, were analysed by immunohistochemistry (HRP/DAB detection kit; Abeam, Cambridge, MA, USA). Briefly, the sections were pretreated with H2O2, to remove endogenous peroxidase activity, incubated for 1 h with Protein Block and overnight with rabbit polyclonal anti-ARC (apoptosis repressor with card domain) antibodies (1 :100) at 4 °C. The slides were washed with PBS, then incubated with biotinylated goat anti-rabbit-IgG (immunoglobulin G), and finally with streptavidin-peroxidase complex. The signal was visualized using 3,3’-diaminobenzidine (DAB) as chromogen. “Negative” control was obtained by omitting primary anti-ARC. The sections were observed using a ZEISS AXIOSKOP microscope and the images were digitalized by Axiocam 105 color, ZEISS.

Apoptosis detection. TUNEL staining (in situ Cell Death Detection Kit, POD, Roche Diagnostics-Germany) was performed, as previously described (Amelio et al, 2013). Briefly, rehydrated sections were incubated with proteinase K (20pg/mL; 37°C; 20min), washed, rinsed and incubated with TUNEL mixture (fluorescein conjugated nucleotides and Terminal deoxynucleotidyl transferase (TdT) (37°C, 60min). TdT was omitted in“Negative” control. Sections were observed using a ZEISS AXIOSKOP fluorescence microscope and images were digitalized by Axiocam 105 color, ZEISS.

In vivo studies in tumor-bearing mice. Studies in murine models were approved by the Ethical Committee of the San Raffaele Scientific Institute, and performed according to the prescribed guidelines. B ALB/c and C57BL/6/N female mice (6-7 weeks old, from Charles River Laboratories, Calco, Italy) were challenged with s.c. injection in the left flank of 2xl0⁵ B16-F1 melanoma cells (C57BL/6/N), RMA cells (C57BL/6/N), or 1.5x106 WEHI-164 cells (BALB/c) or Lewis Lung Carcinoma (BALB/c). Mice were treated with or without recombinant CgA in 0.9% sodium chloride containing 100 pg/ml endotoxin-free human serum albumin, and with or without Doxo. Drug doses and treatment schedules for each experiment are reported in figures and figure legends. Tumor growth was monitored daily by measuring the tumor size with calipers. Statistics. All data were expressed as mean±SEM. One-way ANOVA, non-parametric Newman- Keuls Multiple Comparison Test (for post-ANOVA comparisons) was used for western blot and ELISA analysis. Mann-Whitney test was used for antitumoral activity. *p=<0.05, **p=<0.0l, ***p=<o 001 were considered statistically significant. Two-way ANOVA, non-parametric Bonferroni's multiple comparison test (for post-ANOVA comparisons) was used for the time course of hemodynamic analysis. The statistical analysis was carried out using Graphpad Prism5.

EXAMPLES

Example 1: CgA protects against Doxo-induced cardiotoxicity in a rat model

The effect of exogenous CgA on Doxo-induced cardiotoxicity was evaluated in rats (n=l3- l6/group). The experimental design, drug doses and administration time are described in Fig.l .

Exogenous CgA reduces the Doxo-dependent systemic impairment

To assess the effect of CgA on Doxo-induced systemic inflammation (Elsherbiny et al, 2016) and cardiac damage (Holmgren et al, 2015) the inventors analysed, first, the levels of IL-l b, TNF-a, ROS and LDH in plasma samples obtained from rats treated with Control, Doxo, Doxo+CgA or CgA. Doxo-treated rats, but not those treated with Doxo+CgA, showed increased plasma levels of IL-l b, TNF-a, ROS, and LDH (Fig. 2A-D) compared to the controls. These data suggest that CgA could markedly reduce the Doxo-induced systemic inflammation.

Doxo treatment was also associated with a significant reduction of total plasma protein levels. This reduction was not observed in rats treated with Doxo+CgA or CgA alone (Fig. 2E).

Exogenous CgA prevents Doxo-induced heart fibrosis

The presence of fibrosis in the rat hearts from each group was analysed. To this aim the inventors investigated the expression of connective tissue growth factor (CTGF), a pro-fibrotic marker, in heart tissue extracts by western blotting using a specific antibody. Rat hearts from the Doxo group, but not from the Doxo+CgA group, showed increased expression of CTGF, compared to the controls (Fig.2F), indicating that CgA prevents the fibrotic response caused by Doxo in the heart.

CgA limits I/R injury in rat hearts

The effects of Doxo and CgA on I/R injury were then investigated using the isolated and perfused Langendorff hearts. The basal cardiac parameters of all groups after 40 min of stabilization are reported in Table 1.

Table 1 Basal cardiac parameters after stabilization.

*p < 0.05, ***p< 0.001 : Doxo vs Saline, and ^§p < 0.05, ^§§p< 0.01, ^§§§p< 0.001 : Doxo+CgA vs Doxo, CgA vs Doxo (one-way ANOVA/Newman-Keuls Multiple Comparison Test)

The inventors observed that the hearts from the Doxo group had higher coronary pressure (CP) and lower heart rate (HR) compared to Control.

To assess the effect of Doxo and CgA on the systolic and diastolic function in the post-ischemic phase the inventors examined the dLVP (i.e. the inotropic activity) and the LVDEP value (i.e. the contracture state) before and after ischemia. In the Control and Doxo groups the dLVP at the end of reperfusion was significantly lower than before ischemia (Fig.3A). In contrast, in Doxo+CgA and CgA groups dLVP returned to the pre-ischemic value in the recovery phase. Furthermore, in both Doxo and Control groups, the LVEDP significantly increased during reperfusion, while in the Doxo+CgA and CgA groups it was unchanged (Fig.3B). These findings indicate that CgA induced systolic recovery after ischemia damage.

The effect of Doxo and CgA on ischemia-induced infarct size (IS) (expressed as a percentage of left ventricular mass) was also investigated. The IS was similar in Control and Doxo groups (73.6±3% and 74.8±7% of IS/LV, respectively) treated hearts. Notably, in Doxo+CgA and CgA groups the IS was significantly smaller (45.5+3% and 40.5+3%, respectively) (Fig.3C). Accordingly, CgA reduced significantly in the coronary effluent the LDH levels, a specific marker of ischemic damage, compared to Doxo and Control groups. These results imply that CgA can protect the heart from ischemic injury either alone or in combination with Doxo.

CgA activates protective signaling cascades and switches-ojf the up-regulation of tissue inflammatory components in the heart after I/R

Activation of intrinsic pro-survival signaling cascades, such as the reperfusion injury salvage kinase (RISK) pathway and the recently described survivor activating factor enhancement (SAFE) pathway, can mediate cardioprotection from I/R (Hausenloy et al, 2005). Thus, the activation of these signaling pathways in the hearts of rats treated with or without Doxo and CgA was evaluated by western blotting analysis of various components of these patterns after I/R. Untreated hearts (only perfused for 190 min without ischemic manoeuvre, SHAM group) were used as control, while hearts from the Control group received only I/R manoeuvre without pharmacological conditioning. The phosphorylation of Akt and Erkl/2, key component of the RISK pathway, was lower in the Doxo group compared with the SHAM group. CgA treatment prevented the Doxo- induced inhibition of Akt and Erkl/2 phosphorylation (Fig.4A and B) and increased the expression of GSK-3a/p, another important component of the RISK pathway (Fig.4C). Of note, in the CgA group, the expression of these RISK components was similar to that observed in the SHAM group. The phosphorylation of STAT3, a specific component of the SAFE pathway, showed a similar trend, being reduced in Control and Doxo groups, and increased in the Doxo+CgA group. Furthermore, the expression in the CgA group was similar to that of SHAM animals (Fig.4D).

The expression of phosphorylated AMPKalpha, a cell energy stress biosensor (Kim et al, 2016), was increased in Doxo+CgA and CgA groups compared to the Doxo group (Fig.4E).

Similarly, the expression of iNOS and COX2, specific markers of tissue inflammation (Kelleni et al, 2015), was increased in the Doxo group, but not in the Doxo+CgA and CgA groups (Fig.5 A and B). Additionally, NFPR3, a specific intracellular complex responsible for the activation of pro-inflammatory cytokines (Abderrazak et al, 2015), was up-regulated in the Doxo group, compared to Control, but not in the Doxo+CgA and CgA groups (Fig.5C).

CgA reduces the production of free radicals induced by I/R in the heart of rats treated with Doxo The free radical generating properties of anthracyclines and I/R in cardiomyocytes are well documented (Octavia et al, 2012). To assess the redox balance in the heart of rats the inventors analyzed a) the expression of specific markers involved in the production of free radicals, such as xanthine oxidase (XO) (Yee & Pritsos, 1997) and aldehyde oxidase 1 (AOX-l) (Kundu et al, 2012), and b) the intracardiac ROS production (Octavia et al, 2012). Western blot analysis of heart extracts showed significant reduction of AOX-l and XO in Doxo+CgA compared to Doxo groups (Fig.5D and E). Similar levels of XO and AOX-l were observed in hearts perfused with KH buffer alone (SHAM) or buffer containing 4nM CgA without ischemic manoeuvre.

Intracardiac ROS concentrations, by ELISA, increased in Control rats and even more in the Doxo group. A significant reduction of intracardiac ROS was observed in the CgA+Doxo and CgA groups (Fig.5E).

CgA protects the heart by turning off apoptosis

The effect of Doxo and CgA on cardiomyocytes apoptosis was also investigated. Immunohistochemical analysis of heart tissue sections showed that ARC (apoptosis repressor with caspase recruitment domain), a protein capable of counteracting apoptosis in cardiac cells (Lu et al, 2013), was strongly increased in Doxo+CgA and CgA groups compared to Doxo and Control groups (Fig.6A). Western blotting analysis of heart homogenates of Bcl-2, an anti-apoptotic protein and Bax, a pro-apoptotic protein (Gustafsson & Gottlieb, 2007), confirmed the cardioprotective effect induced by CgA: indeed, Bcl-2 and Bax and Caspase3 were increased and decreased, respectively, in Doxo+CgA and CgA groups, compared to SHAM, Control, and Doxo groups (Fig.6B,C and D). Accordingly, TUNEL assay evidenced a lower number of apoptotic myocytes (identified by fluorescent nuclei) in Doxo+CgA and CgA groups compared to Doxo and Control (Fig.6E).

Example 2: Doxorubicin treatment reduces the intracardiac expression of CgA in rats

CgA is produced by the heart and physical and chemical stimulations promote its intracardiac proteolytic processing (Glattard et al, 2006; Pasqua et al, 2013). To evaluate whether Doxo affects intracardiac CgA production and fragmentation, the inventors performed western blotting analyses of homogenates of not-perfused (NP) hearts with mAb 5A8, a cross-reactive antibody against an epitope located in the N-terminal region of human/rat CgA (Ratti et al, 2000). Western blot analysis of heart tissue extracts from the Control group showed a major band of ~60 kDa and other bands with lower mass (45 and 25 kDa), possibly corresponding to fragmented CgA (Fig. 7). The intensity of all these bands decreased in the Doxo group (Fig. 7), suggesting that this drug could reduce the expression of CgA in the rat hearts or cause proteolytic processing to fragments undetectable with the inventors’ antibody. Administration of exogenous CgA to rats did not rescue tissue expression of endogenous CgA, suggesting that the protective mechanism of exogenous CgA was not mediated by an increased expression of endogenous CgA in the heart. Of note, reduction of band intensity was observed also in the CgA group, suggesting that exogenous CgA could somehow reduce intra-cardiac CgA expression or promote its degradation.

Example 3: Doxorubicin causes a reduction of endogenous circulating CgA in rats

The effect of Doxo on the circulating levels of endogenous CgA was then investigated using ELISA. CgA plasma levels of rats treated with Doxo were lower than controls, as measured with an assay capable of detecting full-length rat CgA plus fragment lacking the C-terminal region (assay-l) or with an assay capable of detecting full-length CgA (assay-2) (Fig. 8A and B). This suggests that Doxo can reduce the circulating levels of endogenous CgA. The observed reduction was unlikely related to full-length CgA degradation, as the ratio between the results of assay-2 and assay-l was not significantly affected. Noteworthy, exogenous CgA could not inhibit the Doxo-induced reduction of endogenous CgA (Fig. 8A and B). The reduction of the circulating CgA after Doxo treatment was confirmed by WB analysis of plasma samples: also in this case a marked reduction of a 70 kDa band was observed in Doxo-treated rats (see Fig. 10). These and the above results suggest that Doxo can reduce CgA expression in the heart as well as its circulating levels. No reduction of CgA expression was observed in adrenal gland extracts (Fig. 8C), suggesting that the effect of Doxo on the circulating CgA levels was not related to a lower expression in this organ.

Example 4: CgA does not prevent the anti-tumor activity of Doxo in murine models

The effect of CgA on the anti-cancer activity of Doxo was then investigated in various murine models of solid tumors. To this aim mice bearing subcutaneous tumors, including melanomas (B16F10), fibrosarcomas (WEHI-164), lymphomas (RMA), or Lewis Lung Carcinomas (LLC), were treated with Doxo alone or in combination with CgA. In all these models Doxo significantly delayed tumor growth either when injected alone or in combination with CgA (Lig. 9), suggesting that CgA does not abrogate the anti-tumor activity of Doxo. Also CgA alone could delay tumor growth in some models. The present results show that CgA can prevent anthracycline-induced cardiotoxicity in a rat model. In particular, the results show that systemic administration of low-dose recombinant CgA (10.71 pg/kg/day, i.p.) can protect rats from anthracycline-induced inflammation, cardiac fibrosis and damage, and from I/R heart injury. Preferably anthracycline is doxorubicin.

To evaluate the protective activity of CgA the inventors have measured a series of markers of anthracycline-induced inflammation and cardiotoxicity in rat plasma, and assessed heart vulnerability to I/R injury by ex vivo experiments with explanted hearts. Considering that anthracycline such as Doxo can cause the release of pro-inflammatory cytokines in the blood of patients, such as IL-l b and TNF-a (Sauter et al, 2011), and the release of ROS and LDH, two markers of cytotoxic and cardiotoxic responses (Holmgren et al, 2015), the inventors investigated, first, the effect of anthracycline and CgA on the plasma levels of these markers. Remarkably, exogenous CgA could prevent the Doxo-induced release of all these markers in the blood of rats, pointing to a protective effect against the pro-inflammatory and cytotoxic effects of Doxo. Interestingly, CgA could also attenuate the reduction of plasma protein levels consequent to Doxo administration. Given that anthracycline, such as Doxo binds plasma proteins (Chassany et al, 1996) and that lower levels of plasma proteins may result in an increase of free anthracycline and in a change of its tissue distribution (Saleem et al, 2016), this effect of CgA might represent an important protective mechanism. Overall, these results support the ability of CgA to revert several systemic toxic effects caused by anthracycline. CgA could also prevent the anthracycline -induced expression of connective tissue growth factor (CTGF), a fibrosis marker increased in heart failure (Szabo et al, 2014), suggesting that CgA could prevent anthracycline-induced heart fibrosis.

The results of ex vivo experiments show that systemic administration of CgA to rats can protect the hearts from subsequent I/R damage. These experiments were performed using the isolated and Langendorff perfused hearts explanted from rats after pharmacological treatment. This model is widely used to study I/R pathophysiology and disease states and represents a versatile tool to analyze the effects of pharmacological agents on heart physiology and vulnerability to I/R injury, such as that induced by anthracycline, for instance Doxo (Bell et al, 2011). Remarkably, it appears that CgA can protect the heart of rats treated with Doxo, as judged from the reduction of fibrosis, inflammation and infarct size in rats that received Doxo plus CgA compared to Doxo alone (from -75% to -45%). This protective effect is corroborated by the improved post-ischemic systolic recovery and reduction of contracture. The same preconditioning-like protective effect was observed also with hearts explanted from rats treated with CgA alone. The results of mechanistic studies suggest that CgA could protect the heart against I/R injury by activating the protective RISK and SAFE pathways and by switching-off the up-regulation of various tissue inflammatory components. In particular, the characterization of heart homogenates from rats treated with or without anthracycline, and then exposed to I/R, showed low expression of components of the RISK and SAFE pro-survival cascades, such as phosphorylated Akt, ERK1/2, GSK-3a/p, and STAT3. Notably, CgA enhanced the expression of these components to levels similar to those found in the hearts of rats treated with CgA alone. CgA also increased the expression of phosphorylated AMPK, a kinase known to regulate redox homeostasis, to preserve the mitochondrial function and to mediate cardioprotection by hormones or other exogenous substances (Carling et al, 2012). Regarding myocardial inflammation after I/R, which is known to contribute to heart dysfunction (Ha et al, 2011), the results show that anthracycline increased the expression of iNOS and COX-2, two inflammatory mediators involved in Doxo cardiotoxicity (Valdez et al, 2011), and of NLPR3, an intracellular component of the inflammasome responsible for the activation of pro-inflammatory cytokines (Abderrazak et al, 2015). The levels of these components were reduced by co-treatment with CgA. These observations, altogether, suggest that CgA can protect the myocardium from anthracycline -induced injury by activating pro-survival cascades and by reducing systemic and tissue inflammation. This view is further supported by the results of other mechanistic studies showing that CgA can also reduce the production of free radicals and activate anti-apoptotic cascades in the heart of rats treated with anthracycline such as Doxo. It has been previously demonstrated that increased production of intra-cardiac ROS, e.g. mitochondrial superoxide radical anion (0₂ ) and hydrogen peroxide (H2O2), are responsible for Doxo-induced cardiomyocyte apoptosis and death (Gianni et al, 2008), and that this occurs also during I/R (Valdez et al, 2011). Indeed, Doxo can induce xanthine oxidase (XO), an enzyme responsible for superoxide ion production and free radical-mediated myocardial damage (Yee & Pritsos, 1997). Furthermore, Doxo can also induce aldehyde oxidase 1 (AOX-l), another enzyme that contributes to myocardial oxidative stress by catalyzing aldehyde oxidation and H2O2 production (Kundu et al, 2012). Both XO and AOX-l are strongly involved in ischemia-elicited cell damages in the heart (Yee & Pritsos, 1997; Kundu et al, 2012). Interestingly, CgA significantly reduced intra-cardiac expression of both enzymes and decreased Doxo-induced production of ROS. CgA could also change the expression of ARC (Lu et al, 2013) and Bcl-2, two apoptosis inhibitors, and of BAX and caspase 3, two apoptosis promoters (Gustafsson & Gottlieb, 2007): compared to the Doxo group, the hearts of rats exposed to Doxo plus CgA showed an enhanced expression of ARC and Bcl-2 and a reduction of BAX and caspase 3. Considering that Doxo increases oxidative stress-dependent apoptotic signaling in cardiomyocytes (Holmgren et al, 2015), these observations contribute to support that CgA can prevent Doxo-induced cardiotoxicity also by activating anti-apoptotic mechanisms. Accordingly, TUNEL assays showed a decrease of apoptotic nuclei in cardiomyocytes of animals treated with CgA. These together with the above results indicate that CgA protects the myocardium from Doxo-induced injury by activating pro-survival cascades, by switching-off the up-regulation of tissue inflammatory targets, by reducing free radical production, and by engaging anti-apoptotic pathways.

The fact that the protective activity of CgA, either in presence or absence of anthracycline such as Doxo, was achieved with a relatively low-dose seems to be a physiologically relevant finding (10.71 pg/kg/day, i.p.). This dose can generate 2-4 nM peak plasma levels, i.e. within the range of 2-5 nM levels of endogenous CgA observed in untreated rats. Conceivably, endogenous CgA, at concentrations close to physiological values, may have a significant role in cardioprotection. Other cardioprotective agents described in literature, e.g. 17 b-estradiol, testosterone, and catestatin, can exert significant protective effects only at high non-physiological concentrations (Fraser et al, 1999; Borst et al, 2010; Wang et al, 2016). Thus, changes in the pathophysiological levels of circulating CgA in patients might result in different Doxo-induced cardiotoxic effects.

In this regard, the observation that anthracycline administration to rats is associated with a reduction of CgA plasma levels and heart tissue expression of this protein may represent another important finding of the inventors’ study. Considering that no reduction of CgA expression was observed in the adrenal gland, a tissue specific mechanism can be underlined. Based on this and the above data, it seems that the anthracycline-induced decrease of circulating full-length CgA may correspond to the loss of an important physiologic cardioprotective agent. Moreover, exposure of rat hearts to stress challenges, like adrenergic and ET-l stimulation, promotes CgA proteolytic processing into short fragments endowed of different cardioregulatory functions (Pasqua et al, 2013; Tota et al, 2014). Thus, given that both circulating and tissue CgA may have cardioregulatory functions, the marked reduction of CgA in plasma and heart tissue observed in anthracycline-treated rats indicates that this drug alter important physiological cardioregulatory factors, thereby representing a novel additional mechanism of anthracycline-related cardiotoxicity.

Finally, the present results of studies performed in tumor bearing mice, showing that the anti tumor activity of anthracycline is not inhibited by CgA, indicate that the anti-tumor and cardiotoxic effects of anthracycline are independent events, and that only the latter are affected by CgA. Thus, administration of exogenous CgA represents a novel approach to prevent adverse cardio-toxic events of anthracycline without impairing its anti-tumor effects.

In conclusion, the results, overall, indicate that anthracycline, such as Doxo, can reduce the endogenous levels of CgA in the blood of rats, i.e. of a potential protective factor, and that exogenous administration of CgA can prevent anthracycline-induced cardiotoxicity. These findings, together with the observation that exogenous CgA does not impair the anti-tumor activity of anthracycline in various murine models. Then, detection of circulating full-length CgA before, during, and after therapy with anthracycline can predict cardiotoxicity, and administration of exogenous CgA is useful as a support therapy in anthracycline-treated patients, to limit or prevent cardiotoxicity, preferably in subjects having low CgA plasma levels.

Example 5: Effects of chromogranin A-derived peptides (Vasostatin, Catestatin, Serpinin, CgA 1-372, CgA 1-373 and CgA 352-373) on Doxorubicin-induced cardiotoxicity

The effects of CgA-derived peptides [Catestatin, Cts (CgA 352-372), human recombinant Vasostatinl, rhVSl (CgA i_7₆), Serpinin, Serp (CgA 41 1-436,), CgA 1-372, CgA 1-373 and CgA 352-373] were investigated on Doxo-induced cardiotoxicity by applying ischemia/reperfusion (I/R) protocols.

Materials and Methods

Animals. Male Wistar rats (~ 450 g body weight) (Harlan Laboratories, Udine, Italy), identically housed under controlled lighting and temperature conditions, fed a standard diet and water ad libitum. All protocols were conducted in accordance with the Declaration of Helsinki, the Italian law (DL.26/2014), the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011) and the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific. The project was approved by the Italian Ministry of Health, Rome and by the ethics review board.

Drugs. Doxorubicin (Doxo) was provided from Sigma Aldrich (Milan, Italy). Catestatin, Cts (CgA352-372), human recombinant Vasostatinl, rhVSl (CgAi-7₆), Serpinin, Serp (CgA4ii-436), CgA 1-372, CgA 1-373 and CgA 352-373 were produced by recombinant and chemical synthesis according to standard procedures.

Animal treatments. Wistar rats were divided in 7 groups and treated daily for 1 week (i.p.), as follows:

a) group I (Doxo): 3 mg/kg/day of Doxo (cumulative dose, 21 mg/kg); b) group II (Doxo+Cts): 3 mg/kg/day of Doxo plus 33 nmol/kg/day of Cts

c) group III (Doxo+rhVSl): 3 mg/kg/day of Doxo plus 33 nmol/kg/day of rhVSl

d) group IV (Doxo+Serp): 3 mg/kg/day of Doxo plus 33 nmol/kg/day of Serp

e) group V (Doxo+CgA 1-372): 3 mg/kg/day of Doxo plus 0.2 nmol/kg/day of CgA 1-372 f) group VI (Doxo+ CgA 1-373): 3 mg/kg/day of Doxo plus 0.2 nmol/kg/day of CgA 1-373 g) group VII (Doxo+CgA 352-373): 3 mg/kg/day of Doxo plus 0.2 nmol/kg/day of CgA 352-373

Doses of the CgA-derived peptides Cts, rhVSl and Serp correspond to the first concentration that elicited significant effects on ex vivo cardiac performance in rat (Cerra et al., 2006; Angelone et al., 2008; Tota et al., 2012), while the dose of the CgA-derived peptides CgA 1-372, CgA 1-373 and CgA 352-373 is the same than full length CgA.

Animals were sacrificed after 7 days to evaluate the heart performance by Langendorff perfusion technique; After sacrifice, blood samples were collected from the abdominal aorta with heparinized syringes. Plasma was then separated by centrifugation at 3000 x g (l5min, 4°C) for total plasma proteins quantification that was determined using a Bradford reagent according to the manufacturer instructions (Sigma- Aldrich, Missouri, USA).

Ex vivo experiments

Heart Perfusion (as above)

Ischemia/Reperfusion (I/R) protocols. After chronic treatment, the hearts from each group described above (Doxo, Doxo+Cts, Doxo+rhVSl, Doxo+Serp, Doxo+CgA 1-372, Doxo+CgA 1-373 and Doxo+CgA 352-373) were subjected to 30 min of global, no-flow ischemia, followed by 120 min of reperfusion (I/R).

At the end of treatments, cardiac parameters were analysed by Langendorff technique. Cardiac performance before and after ischemia was evaluated by analyzing LVP recovery, as an index of contractile activity, and LVEDP as an index of contracture, defined as an increase in LVEDP of 4 mmHg above the baseline level (Pasqua et al, 2015).

Infarct size (IS). To measure infarct areas hearts were rapidly removed from the perfusion apparatus at the end of reperfusion. The left ventricles were dissected transversely into 2-3 mm slices. After 20 min of incubation at 37°C in 0.1% nitro blue tetrazolium in phosphate buffer (59.8 mM NaH₂P0₄, 484.9 mM Na₂HP0_4, pH:7.4), unstained necrotic tissues were carefully separated from stained viable tissues by an independent observer who was not aware of the nature of the intervention. The weights of the necrotic and non-necrotic tissues were then determined, and the necrotic mass was expressed as a percentage of total left ventricular mass, including septum (Pasqua et al, 2015).

Statistics. All data were expressed as mean±SEM. One-way ANOVA, non-parametric Newman- Keuls Multiple Comparation Test (for post-ANOVA comparisons) was used for the analysis. *p=<0.05, **r=<0.01, ***p=<0.00l were considered statistically significant. Two-way ANOVA, non-parametric Bonferroni's multiple comparison test (for post-ANOVA comparisons) was used for the time course of hemodynamic analysis. The statistical analysis was carried out using Graphpad Prism5.

Results

The basal cardiac parameters of all groups after stabilization are reported in Table 2.

Table 2. Basal cardiac parameters after stabilization.

*p < 0.05: Doxo vs Saline, Doxo+rhVS 1 v.s Saline, Doxo+Serp vs Saline, Doxo+CgAi-372 vs Saline (one-way ANOVA/Newman-Keuls Multiple Comparison Test)

The inventors observed that the hearts from the Doxo, Doxo+rhVS 1, Doxo+Serp and Doxo+CgAi -372 groups showed a significant higher coronary pressure (CP) compared to Control. The effects of Doxo and CgA-derived peptides on the systolic and diastolic functions in the post- ischemic phase were evaluated through the developed Left Ventricular Pressure [dLVP (as index of systolic activity)] and the Left Ventricular EndoDiastolic Pressure [LVDEP values (as index of diastolic activity)] values.

In the Control and Doxo groups the dLVP, at the end of reperfusion, was significantly lower than before ischemia (Pig. 11 A). In contrast, in Doxo+CgAi-373, Doxo+CgA352-373, Doxo+Cts, Doxo+rhVS 1 and Doxo+Serp groups the dLVP was higher, although only in Doxo+Cts the increase was significant compared to Doxo and Control groups (Pig. 11 A). Regarding the diastolic function, at the end of reperfusion, LVEDP appeared reduced in Doxo+CgAi-373, Doxo+CgA352- 373 , Doxo+Cts, Doxo+rhVSl and Doxo+Serp even though not in a significant manner (Pig. 11B). At the end of I/R protocols, the amount of necrotic tissue (IS) was assessed. IS was expressed as percent of the left ventricle mass (%IS/LV); this analysis revealed that IS was similar in Control and Doxo groups (Fig. 11C). Notably, in Doxo+CgAi-373, Doxo+CgA352-373, Doxo+Cts and Doxo+rhVSl groups the IS was significantly smaller compared to the Doxo (Fig. 11C). Fig. 11D shows that Doxo+CgAi-372, Doxo+CgAi-373, Doxo+CgA352-373, Cts, rhVSl or Serp could not significantly reduce the total plasma protein levels induced by Doxo.

Fig.12 shows the animal weight before and after treatment. Neither CgA, nor Doxo+CgAi-372, Doxo+CgAi-373, Cts, rhVSl or Serp could inhibit the loss of body weight induced by Doxo.

These hemodynamic findings and infarct size analyses suggest that, among tested peptides, CgAi_ _373, CgA_352-373, Cts and rhVSl exert protection in the presence of Doxo treatment. Despite the basal systolic and diastolic functions in the hearts treated with the CgA-derived peptides was improved compared with Doxo alone hearts, the post-ischemic recovery of the peptides was not notable. Altogether, data indicate that the full length CgA is the most active agent in protecting the heart from ischemic susceptibility in a rat model of Doxo-dependent cardiotoxicity. Noteworthy, the two CgA-derived peptides, CgAi-_373, CgA_352-373, exert protection at the same dose of the full length CgA (0.2 nanomoles/kg/day) only on infarct size, while their action on systolic recovery was not remarkable compared to full length CgA. The cardioprotection obtained with the other CgA- derived peptides, Cts and rhVSl, is achieved at higher dose (33 nanomoles/kg/day of Cts and rhVSl) in respect to full length CgA.

Example 6: Detection of CgA in cancer patients

Materials and Methods

Detection of CgA fragments and troponin T in lymphoma and breast cancer patients was approved by the Ethical Committe of the San Raffaele Hospital, Milan, and all patients signed an informed consent form on the trial. CgA fragments and troponin T levels were measured, by ELISA, in plasma samples obtained from patients before each cycle of treatment and stored at -80°C until analysis. The ELISA for total CgA, CgAi-_372/3 and CgAi-₇₆ have been performed as described previously (M. Bianco, Cancer Research 2016, Apr 1; 76(7): 1781-91). Troponin T was measured with a commercial ELISA kit.

Results

The inventors monitored the levels of CgA and its fragments, by ELISA, in a lymphoma patient treated with CHOP (cyclophosphamide, doxorubicin (hydroxydaunomycin), vincristine (Oncovin ®), prednisolone) (6 cycles), a chemotherapy regimen that includes the antracycline doxorubicin. A progressive increase of troponin T levels, a marker of cardiotoxicity, was observed in plasma samples collected before each cycle of treatment (thus three weeks after each treatment) (Fig. 13), suggesting that myocardial damage occurred during treatment. An ELISA assay unable to discriminate between full-length CgA and fragments (total-CgA-ELISA) showed modest increase of total-CgA levels after treatment (presumably as a consequence of the administration of a proton pump inhibitor, a gastro-protective drug known to induce the release of CgA fragments in the blood from the gastric mucosa), which returned close to baseline 6 months after the first cycle. Interestingly, ELISA assays specific for the full-length CgA and the CgAl-76 N-terminal fragment (vasostatin-l, VS-l), showed that these molecules progressively decreased upon treatment (Fig. 13, lower panels), suggesting that prolonged treatment with chemotherapy decreases the levels of these cardioprotective factors.

A similar study was performed in 15 patients with breast cancer treated with FEC (fluorouracil (5FU), epirubicin and cyclophosphamide) (4 cycles), a chemotherapy regimen that includes the antracycline epirubicin. Also in these patients we observed a) a progressive increase of troponin T levels, and b) a progressive increase of total-CgA after the 2-3 cycles (not shown). No changes in full-length CgA were observed in this case, likely because the treatment was limited to 4 cycles with a lower dose of antracycline. However, correlation studies showed that the plasma levels of troponin T after the 4th cycle correlated in a negative manner with the plasma levels of the fragment CgAi-372 _and/or the fragment CgAi-373 (tested with a specific ELISA) before treatment (r=0.018, r=-0.58) or before the second cycle (P= 0.0014, r=-0.75) (Fig. 14). These data suggest that the initial plasma levels of this fragment can predict a cardiotoxic response to this chemotherapeutic regimen observed 3 months later. No significant prediction was observed with basal full-length CgA or total-CgA. Interestingly, the fragment CgAi-372 or the fragment CgAi-373 contain the vasostatin-l and the catestatin sequences, i.e. sequences that are endowed with cardioprotective activity in the rat model. This notion and the observation that patients having low levels of troponin T after the 4th cycle of treatment had relatively high basal levels of the fragment CgA -372 and/or the fragment CgAi-373 before treatment suggest that this fragment contributed to cardioprotection in patients. Administration of CgAi-373, or ofCgAi-₃₇₂, or CgA_352-372 (catestatin), or CgA352-373 or alternatively CgAi-76 (vasostatin-l) to patients having low levels of CgAi-372/3 represent a therapeutic strategy to prevent cardiotoxicity induced by antracy dines.

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Claims

1. Chromogranin A (CgA) or a fragment thereof for use in the treatment and/or prevention of a cardiotoxicity induced by an anthracy cline.

2. Chromogranin A (CgA) or a fragment thereof for use according to claim 1 wherein the fragment is selected from a fragment comprising or consisting of: Catestatin, Cts (CgA₃₅2- 372), Vasostatinl, VS1 (CgAi_-76), Serpinin, Serp (CgA_4i 1-430), CgA 1-372, CgA 1-373 and CgA

352 373

3. Chromogranin A (CgA) or a fragment thereof for use according to claim 1 or 2 wherein the cardiotoxicity induced by an anthracycline is acute or chronic.

4. Chromogranin A (CgA) or a fragment thereof for use according to any one of previous claim wherein the cardiotoxicity induced by an anthracycline is selected from the group consisting of: inflammation, cardiac fibrosis, ischemia/reperfusion injury, cardiomyocyte apoptosis, cardiomyopathy, left ventricular dysfunction and/or heart failure.

5. Chromogranin A (CgA) or a fragment thereof for use according to any one of previous claim wherein the anthracycline is selected from the group consisting of: doxorubicin, epirubicine, daunorubicine, idarubicine, nemorubicin, pixantrone, sabarubicine, valrubicine.

6. Chromogranin A (CgA) or a fragment thereof for use according to any one of previous claim wherein CgA is used at a physiological dose, preferably at a dose of between 3 Lig/kg/day and 30 pg/kg/day.

7. Chromogranin A (CgA) or a fragment thereof for use according to any one of previous claim wherein chromogranin A (CgA) or a fragment thereof is administered prior to or simultaneously with the anthracycline.

8. Chromogranin A (CgA) or a fragment thereof for use according to any one of previous claim being administered in a subject with low levels of CgA and/or of the fragment CgAi_ 372 and/or the fragment CgAi-373.

9. Chromogranin A (CgA) or a fragment thereof for use according to any one of previous claim wherein said chromogranin A (CgA) or a fragment thereof does not prevent the anti tumor activity of the anthracycline.

10. A pharmaceutical composition comprising chromogranin A (CgA) or a fragment thereof, and pharmaceutically acceptable carriers for use in the treatment and/or prevention of a cardiotoxicity induced by an anthracycline.

11. The pharmaceutical composition according to claim 10 further comprising an anthracycline.

12. A method to assess the risk of developing cardiotoxicity induced by an anthracycline in a subject, comprising the step of measuring the level of Chromogranin A and/or of a fragment thereof in a biological sample obtained from the subject and comparing said measured level to a proper control

13. The method according to claim 12 wherein the Chromogranin A (CgA) fragment is selected from a fragment comprising or consisting of: Catestatin, Cts (CgA352-372), Vasostatinl, VS1 (CgAi_-76), Serpinin, Serp (CgA₄n-₄36), CgA 1-372, CgA 1-373 and CgA 352- 373.

14. The method according to claim 12 or 13 wherein the biological sample is selected from the group consisting of: blood, plasma, urine, saliva, serum.

15. The method according to claim 12 -14 wherein the subject if affected by cancer.