The present invention relates to the use of interleukin-4 (IL-4) in the treatment of myocardial infarction (Ml).

Myocardial infarction is a leading cause of death and disability, affecting over 2.7 million patients in the UK. This number is likely to soar with the aging population and current therapies have only limited effects in treating heart failure. Due to the recent progress in the diagnosis and treatment, the early mortality of acute Ml has been markedly reduced. This success, however, has resulted in an increase in the number of patients suffering post-MI heart failure. Existing medical therapies have limited efficacy for heart failure and have failed to aid recovery of cardiac performance, thus heart transplantation is the only radical treatment but is problematic because of donor-shortage. Therefore, the development of new therapies for the treatment of post-MI heart failure is a high priority in the UK.

Due to the insufficient regenerative capacity of the heart, significant loss of cardiomyocytes after Ml leads to scar formation. This process depends on a tightly orchestrated inflammatory response (Gajarsa JJ, et al. Heart Fail Rev. 201 1 ;16:13-21 ; Frangogiannis N. Circ Res. 2012;1 10:159-73). Cardiac cell death by Ml induces inflammatory signals that recruit neutrophils within 24 hours, and monocytes/macrophages thereafter. These leukocytes degrade extracellular matrices (ECM) and macromolecules released by injured cells, and aid clearance of dead cells. After ~5 days monocytes/macrophages initiate resolution of inflammation and promote angiogenesis to assure blood supply in the scar, with parallel ECM synthesis via stimulating fibroblasts, forming supportive scars. However, inflammation and ischaemia often persists and induces adverse molecular and cellular changes (remodelling) throughout the heart, particularly in the border area (margin of the lesion). Macrophages thus represent a potential therapeutic target for attenuating adverse remodelling and enhancing myocardial repair after Ml. In general, macrophages can be functionally polarized into at least two subgroups; classically-activated M1 and alternatively-activated M2 phenotypes (Gordon S, et al. Immunity. 2010;32:593-604; Mosser DM, et al. Nat Rev Immunol. 2008;8:958-69). Stimulation with IFN-γ or TNFa (T helper 1 cell-type activation) drives macrophages into the pro-inflammatory M1 phenotype, which plays a central role in host defence against bacterial/viral infections and in sterile inflammation.

In contrast, exposure to T helper 2 cytokines, IL-4 and/or IL-13, induces anti-inflammatory M2 macrophages, which promote repression of inflammation and enhance wound-healing. M2 macrophages display increased phagocytic activity and synthesis anti-inflammatory IL-10. The importance of the M1/M2 polarization balance in the repair of damaged organs has been reported, however, the M1/M2 classification is almost certainly an oversimplification, and it is increasingly recognized that the macrophage acquire a wide spectrum of phenotypes with distinct properties.

Previous attempts to treat heart failure by targeting M2 macrophages (indirectly), including transfusion of ex vivo programmed M2-like macrophages (Circulation. 2006;1 14:194-100) or apoptotic leukocytes (Lancet. 2008; 371 :228-36), and injection of phosphatidylserine-presenting liposomes (Proc Natl Acad Sci USA. 201 1 ;108:1827-32) have not been very successful in the clinical arena. Our novel data obtained in this present and follow-up experimentations could inform advanced therapeutic strategies (new drug, small molecules, microRNAs) for heart failure by targeting heart-resident M2-like macrophages.

IL-4 is a cytokine principally involved in the stimulation of activated B-cell and T-cell proliferation. Overproduction of IL-4 is associated with allergies. IL-4 has been reported to suppress matrix metalloproteinases by immunomodulation and to improve cardiac function in murine myocarditis (Li et al. Eur. J. Pharm.2007;554:60-68 and PhD thesis of S. Rutschow (201 1) at j t o://vvww diss.iu- beriin de/diss/receive/FUDISS thesis 000C00022 30?ianf.i- n). However, myocarditis is an inflammatory/immune system disease which does not cause ischaemia/hypoxia or significant amounts of cardiac cell death. IL-4 in extravascular tissues promotes alternative activation of macrophages into M2 cells and inhibits activation of macrophages into M1 cells. However, systemic IL-4 injection may cause adverse side effects, e.g. provoking type I allergic diseases including asthma. Another concern with respect to its use in patients post-MI is that IL-4 might exaggerate atherosclerosis development (even though pharmacological delivery of IL-4 can rather suppress atherosclerosis (Kleemann R, et al. Cardiovasc Res. 2008;79:360-76)). Also, the half-life of IL-4 may be too short to effectively exert the biological activity when injected in the free form (Jenkins SJ, et al. Science. 201 1 ;332:1284-8).

There is therefore a need for a treatment of myocardial infarction which addresses the problem of post-MI heart failure. The present invention uses treatment with IL-4 which may activate cardiac resident macrophages more directly. The invention therefore offers a superior outcome in the treatment of Ml.

According to a first aspect of the invention, there is provided interleukin-4 (IL-4) for use in the treatment of myocardial infarction (Ml).

According to a second aspect of the invention there is provided a composition comprising IL-4 for use in the treatment of Ml. The composition may be prepared as an injectable formulation.

Suitably, the IL-4 molecule is human IL-4, including isoforms thereof. Conventional IL-4 is encoded by four exons, whereas the alternatively spliced isoform is encoded by exons 1 , 3 and 4 (IL-452). A reference sequence for IL-4 is NCBI Reference Sequence: NP_000580.1 with the sequence:

1 mgltsqllpp lffllacagn fvhghkcdit lqeiiktlns lteqktlcte ltvtdifaas

61 kntteketfc raatvlrqfy shhekdtrcl gataqqfhrh kqlirflkrl drnlwglagl

121 nscpvkeanq stlenflerl ktimrekysk ess

In the above reference sequence, residues 1 to 24 represent the signal peptide for IL-4 and the mature IL-4 protein corresponds to residues 25 to 153. References to IL-4 therefore include isoforms, derivatives, analogues, fragments and variants which have IL-4 activity. Glycosylated forms and non-glycosylated forms are also included. IL-4 activity includes the property of binding to the lnterleukin-4 (IL-4) receptor (IL-4R) which exists in soluble form or as a cell-surface membrane bound protein. The term "protein" in this text means, in general terms, a plurality of amino acid residues joined together by peptide bonds. It is used interchangeably and means the same as peptide, oligopeptide, oligomer or polypeptide, and includes glycoproteins and derivatives thereof. The term "protein" is also intended to include isoforms, fragments, analogues, variants and derivatives of a protein wherein the fragment, analogue or derivative retains essentially the same biological activity or function as a reference protein, i.e. references herein to IL-4 include references to other isoforms, fragments, analogues, variants and derivatives of IL-4 that have IL-4 activity.

The fragment, analogue or derivative of the protein as defined in this text, may be at least 6, preferably 10 or 20, or up to 50 or 100 amino acids long. The fragment, derivative or analogue of the protein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably, a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence which is employed for purification of the polypeptide. Such fragments, derivatives and analogues are deemed to be within the scope of those skilled in the art from the teachings herein. Particularly preferred are variants, analogues, derivatives and fragments having the amino acid sequence of the protein in which several e.g. 5 to 10, or 1 to 5, or 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein of the present invention. Also especially preferred in this regard are conservative substitutions. An example of a variant of IL-4 used in the present invention is a protein as defined above, apart from the substitution of one or more amino acids with one or more other amino acids. The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance.

Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). Substitutions of this nature are often referred to as "conservative" or "semi- conservative" amino acid substitutions.

Amino acid deletions or insertions may also be made relative to the amino acid sequence for the IL-4 protein referred to above. Thus, for example, amino acids which do not have a substantial effect on the activity of the IL-4 polypeptide, or at least which do not eliminate such activity, may be deleted. Such deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced - for example, dosage levels can be reduced.

Amino acid insertions relative to the sequence of the IL-4 protein above can also be made. Such amino acid changes relative to the sequence for IL-4 can be made using any suitable technique e.g. by using site-directed mutagenesis. It should be appreciated that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non- naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present.

An IL-4 protein for use in the present invention may have additional N-terminal and/or C-terminal amino acid sequences. Such sequences can be provided for various reasons, for example, glycosylation.

"Identity" as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness (homology) between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

According to a third aspect of the invention the IL-4 may be provided in the form of pharmaceutical composition for use in the treatment of Ml. The composition may delivered systemically or locally. The pharmaceutical composition may be administered in any effective, convenient manner effective for treating Ml including, for instance, administration by intravenous, subcutaneous, intramuscular, oral, transdermal and intraperitoneal routes among others. The composition may be administered as a bolus or as part of a series of repeated administrations or a continuous infusion. In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. In one embodiment, the pharmaceutical composition is suitable for injection. Such injectable formulations may be administered by any convenient route as above, including, for example intracoronary injection or intramyocardial injection. A formulation suitable for injection may be prepared as an aqueous or substantially aqueous formulation. The formulation may comprise such additional salts, preservatives and stabilisers and/or excipients or adjuvants as required. The dosage forms of the invention may be provided as anhydrous powders ready for extemporaneous formulation in a suitable aqueous medium. Intracoronary injection may be made using a catheter. Intramyocardial injection may be made using a needle-syringe (during open-heart surgery) or a catheter, for example a cardiac catheter.

It may be generally preferred to formulate such dosage forms as a buffered aqueous formulation. Suitable buffer solutions may include, but are not limited to amino acids (for example histidine), salts of inorganic acids and alkali metals or alkaline earth metals, (for example sodium salts, magnesium salts, potassium salts, lithium salts or calcium salts - exemplified as sodium chloride, sodium phosphate). Other components such as detergents or emulsifiers (for example, Tween 80 ^® or any other form of Tween ^® ) may be present and stabilisers (for example benzamidine or a benzamidine derivative). Excipients such as sugars, (for example sucrose) may also be present. Suitable values for pH are physiological pH, e.g. pH 6.8 to 7.4. Liquid dosage forms may be prepared ready for use in such administration vehicles.

The composition may also be formulated for slow release/delayed release using a suitable biomaterial. Such compositions may be injected into the myocardium, or sprayed or placed onto the heart surface of the exposed heart during surgery. Alternatively, such compositions formulated for slow release/delayed release may be for intrapericardial or intramyocardial injection. Such intrapericardial injection or intramyocardial injection may be by percutaneous catheter.

The IL-4 may be administered either before an Ml has occurred or subsequently. The invention includes pre-treatment of a subject prior to Ml or treatment post-MI.

In one embodiment of the invention, IL-4 is co-administered with an anti-IL-4 neutralizing monoclonal antibody, or a fragment thereof as defined herein. Without wishing to be bound by theory, it is believed that the co-administration may extend the half-life of the IL-4 in vivo.

The compositions for use according to the invention may also comprise another (i.e. one or more other) pharmaceutically active substances in addition to the interleukin-4 (IL-4) which is defined as a first pharmaceutically active substance. The second pharmaceutically active substance may be a pro-angiogenic drug, an anti-inflammatory drug, a cytokine, a calcium-channel blocker (a calcium antagonist), an angiotensin-converting-enzyme (ACE) inhibitor (or ACEI), or a growth factor. The second pharmaceutically active substance may be present in the composition in addition to an anti- IL-4 neutralizing monoclonal antibody as described above.

The pro-angiogenic drug may be selected from the group consisting of VEGF, HIF1 a and bFGF.

The anti-inflammatory drug may be a steroid drug. The steroid may be a corticosteroid drug and can be a drug selected from the group consisting of: hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, prednisone, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, halcinonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-valerate, halometasone, alclometasone diproprionate, betamethasone valerate, betamethasone diproprionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-proprionate, fluocortolone caproate, fluocortolone pivalate, fluprednidene acetate, flunisolide, fluticasone furoate, fluticasone proprionate, beclomethasone diproprionate, hydrocortisone-17-butyrate, hydrocortisone-17- aceponate, hydrocortisone-17-buteprate, ciclesonide and prednicarbate.

Alternatively, the ant-inflammatory drug may be an antibody for a pro-inflammatory cytokine such as TNF-a, or IL1 -β, i.e. an anti-TNFa antibody, or an anti-IL-1 β antibody, or a fragment thereof.

An "antibody fragment" as referred to herein means any portion of a full length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody and Fd fragments. The term "single chain variable fragment" or "scFv" refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked. One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites.

Suitable cytokines and growth factors may be selected from the group consisting of IL-10, TGF-β, IGF-I and SDF-I, or combinations thereof.

The calcium-channel blocker (CCB) drug may be a dihydropyridine. The dihydropyridine may be selected from the group consisting of amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, isradipine, efonidipine, felodipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine. Alternatively, the calcium-channel blocker may be a non-dihydropyridine compound such as a phenylalkylamine selected from the group consisting of verapamil, gallopamil, fendiline, or a benzothiazepene compound, for example diltiazem.

The angiotensin-converting-enzyme (ACE) inhibitor (or ACEI) may be selected from the group consisting of captopril, zofenopril, enlapril, ramipiril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, cilazpril, or fosinopril.

The administration of IL-4 and pharmaceutically active substance may be simultaneous, separate or sequential. According to a fourth aspect of the invention there is provided a composition comprising IL-4 or isoforms, derivatives, analogues, fragments and variants thereof and a pharmaceutically active substance as defined above as a combined preparation for simultaneous, separate or sequential use in the treatment of myocardial infarction.

It is also envisaged that an anti-IL-4 neutralizing monoclonal antibody as described above may be also administered as part of this use in accordance with the invention. Consequently, the invention also provides a composition comprising IL-4 or isoforms, derivatives, analogues, fragments and variants thereof and an anti-IL-4 neutralizing monoclonal antibody as a combined preparation for simultaneous, separate or sequential use in the treatment of myocardial infarction.

It is also possible that the use in accordance with this aspect of the invention may comprise IL-4 or isoforms, derivatives, analogues, fragments and variants thereof, an anti-IL-4 neutralizing monoclonal antibody and a pharmaceutically active substance as defined above as a combined preparation for simultaneous, separate or sequential use in the treatment of myocardial infarction.

It is also envisaged that the IL-4 may be delivered by gene therapy by expression of a suitable nucleic acid construct encoding IL-4, or by cell therapy by administration of cells expressing IL-4. The cells which express IL-4 may be cells which have been genetically manipulated to express IL- 4, as well as cells which naturally have a high level of IL-4 expression.

In accordance with the present invention, the IL-4 can be delivered to a patient by means of a nucleic acid encoding a gene for IL-4. Such a nucleic acid construct may be therefore used therapeutically in a method of the invention by way of gene therapy.

Administration of the nucleic acid construct may be directed to the target site by physical methods. Examples of these include topical administration of the "naked" nucleic acid in the form of a vector in an appropriate vehicle, for example, in solution in a pharmaceutically acceptable excipient, such as phosphate buffered saline, or administration of a vector by physical method such as particle bombardment according to methods known in the art.

Other physical methods for administering the nucleic acid construct directly to the recipient include ultrasound, electrical stimulation, electroporation and microseeding. Further methods of administration include oral administration or administration through inhalation. Particularly preferred is the microseeding mode of delivery which is a system for delivering genetic material into cells in situ in a patient. This method is described in US Patent No. 5697901 . The nucleic acid construct may also be administered by means of delivery vectors. These include viral delivery vectors, such as adenovirus, retrovirus or lentivirus delivery vectors known in the art. Other non-viral delivery vectors include lipid delivery vectors, including liposome delivery vectors known in the art. As used herein the term "gene therapy" refers to the introduction of genes by recombinant genetic engineering of body cells (somatic gene therapy) for the benefit of the patient. Furthermore, gene therapy can be divided into ex vivo and in vivo techniques. Ex vivo gene therapy relates to the removal of body cells from a patient, treatment of the removed cells with a vector i.e., a recombinant vector, and subsequent return of the treated cells to the patient. In vivo gene therapy relates to the direct administration of the recombinant gene vector by, for example, intravenous or intravascular means. Preferably the method of gene therapy is carried out ex vivo.

Preferably in gene therapy, the expression vector is administered such that it is expressed in the subject to be treated. Thus for human gene therapy, the promoter is preferably a human promoter from a human gene, or from a gene which is typically expressed in humans, such as the promoter from human CMV.

The gene therapy may comprise the manipulation of somatic cells of human or non-human mammals. Alternatively, the gene therapy may involve the manipulation of the germ line cells of a non-human mammal. The present invention therefore also provides a method for providing a human with IL-4 for use in the treatment of myocardial infarction by introducing human cells into a human, the human cells having been treated in vitro to insert therein a nucleic acid construct encoding IL-4 wherein the construct is caused to express IL-4. An ex vivo somatic gene therapy method comprises manipulating the cells removed from a patient with the nucleic acid construct encoding IL-4 in an appropriate vector As used herein, the term "manipulated cells" covers cells transfected with a recombinant vector.

The nucleic acid construct encoding IL-4 preferably includes a promoter or other regulatory sequence which controls expression of the nucleic acid. Promoters and other regulatory sequences which control expression of a nucleic acid have been identified and are known in the art. The person skilled in the art will note that it may not be necessary to utilise the whole promoter or other regulatory sequence. Only the minimum essential regulatory element may be required and, in fact, such elements can be used to construct chimeric sequences or other promoters. The essential requirement is, of course, to retain the tissue and/or temporal specificity. The promoter may be any suitable known promoter, for example, the human cytomegalovirus (CMV) promoter, the CMV immediate early promoter, the HSV thymidine kinase, the early and late SV40 promoters or the promoters of retroviral LTRs, such as those of the Rous Sarcoma virus (RSV) and metallothionine promoters such as the mouse metallothionine-l promoter. The promoter may comprise the minimum comprised for promoter activity (such as a TATA element without enhancer elements) for example, the minimum sequence of the CMV promoter. Preferably, the promoter is contiguous to the nucleic acid sequence encoding IL-4.

As stated herein, the nucleic acid construct may be in the form of a vector. Vectors frequently include one or more expression markers which enable selection of cells transfected (or transformed) with them, and preferably, to enable a selection of cells containing vectors incorporating heterologous DNA. A suitable start and stop signal will generally be present.

As described herein administration may also take place via transformed host cells. Such cells include cells harvested from the subject, into which the nucleic acid construct is transferred by gene transfer methods known in the art. Followed by the growth of the transformed cells in culture and grafting to the subject.

One such method relates to a cell comprising the nucleic acid construct encoding IL-4. The cell may be termed a "host" cell, which is useful for the manipulation of the nucleic acid, including cloning. Alternatively, the cell may be a cell in which to obtain expression of the nucleic acid. Representative examples of appropriate host cells for expression of the nucleic acid construct include mesenchymal stem cells. However, it is anticipated that the host cell may also be an autologous cell derived from the patient to be treated, or from an allogenic donor or source of allogenic donor cells, such as a cell line. In the case of an allogenic donor cell, the recipient subject may receive additional immunosuppressant drug therapy to alleviate or reduce the possibility of rejection of the cells administered.

Introduction of an expression vector into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic - lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook et al, Molecular Cloning, a Laboratory Manual, Second Edition, Coldspring Harbor Laboratory Press, Coldspring Harbor, N.Y. (1989).

Endogenous levels of IL-4 in a patient may also be increased by modulation of other molecules in the IL-4 signalling pathway, for example other IL-4 receptor agonists such as IL-13.

As used herein, the term "treatment" includes any regime that can benefit a human or a non-human animal. The treatment of "non-human animals" extends to the treatment of domestic animals, including horses and companion animals (e.g. cats and dogs) and farm/agricultural animals including members of the ovine, caprine, porcine, bovine and equine families.

According to the present invention, the treatment of myocardial infarction includes prophylactic (preventive) treatment for patients at risk of the condition as well as the therapeutic treatment for patients after a myocardial infarction has occurred. Treatment according to the present invention before myocardial infarction improves post-infarction survival and cardiac function with enhanced myocardial repair. Treatment after myocardial infarction according to the present invention improves cardiac function and attenuates heart dilatation.

For administration to mammals, and particularly humans, it is expected that the daily dosage of the active agent will be from 5 to 20 μg/kg body weight per day, typically around ^g/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual which will be dependent on factors including the age, weight, sex and response of the individual. The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention

The term "myocardial infarction or (Ml)" includes both acute myocardial infarction (AMI) and postinfarction ischaemic heart failure. Myocardial infarction is more commonly referred to as a heart attack in which the flow of blood to a part of the heart stops which results in damage to the heart muscle. Ml is most frequently the result of coronary heart disease. Risk factors for Ml include various medical conditions or diseases as well as certain genetic associations.

According to the present invention, the treatment of myocardial infarction includes treatment of patients undergoing coronary artery bypass grafting (CABG) post-myocardial infarction, percutaneous coronary intervention (PCI), stem cell therapy of the heart, volume reduction surgery, cardiac resynchronisation therapy or in therapy using a ventricular assist device (artificial heart).

The invention therefore also extends to the use of IL-4 or isoforms, derivatives, analogues, fragments and variants thereof in the manufacture of a medicament for the treatment of myocardial infarction.

According to a fifth aspect of the invention, there is provided a method of treatment of myocardial infarction, comprising the step of administering a composition comprising IL-4 or isoforms, derivatives, analogues, fragments and variants thereof to a subject in need thereof. The method may further comprise the step of administering one or more other pharmaceutically active substances as defined above. Such methods also extend to methods by which IL-4 is delivered by means of gene therapy or cell therapy as described above. It is also envisaged that an anti-IL-4 neutralizing monoclonal antibody as described above may be administered as part of this method in accordance with the invention. References to a "subject" in the present disclosure includes a patient under the supervision of a medical practitioner.

According to a sixth aspect of the invention, there is provided a kit comprising a lyophilized preparation of interleukin-4 (IL-4) or isoforms, derivatives, analogues, fragments and variants thereof and a sterile aqueous medium for use in the treatment of myocardial infarction (Ml), optionally with instructions for use. The kit may further comprise one or more other pharmaceutically active substances as defined above. The kit may further also comprise an anti- IL-4 neutralizing monoclonal antibody as well as or instead of the other pharmaceutically active substances.

According to a seventh aspect of the invention, there is provided a drug eluting implantable medical device coated with a drug eluting polymer comprising interleukin-4 (IL-4) for use in the treatment of myocardial infarction (Ml), wherein the device is an endoprosthesis, stent, vena cava filter, embolic protection filter, valve, occlusive device, trocar, or aneurysm treatment device.

IL-4 can also be administered through the implantation of a device coated with a drug eluting polymer comprising IL-4. The device can be placed in a coronary artery where the IL-4 can be released by the drug eluting polymer. The use of such devices may be part of a therapy in which the subject to be treated can receive a second pharmaceutically active substance and/or an anti-IL- 4 neutralizing monoclonal antibody which may be administered separately or as part of the drug eluting polymer.

Such a drug eluting implantable medical device may be an endoprosthesis, stent, vena cava filter, embolic protection filter, or similar which are coated with a suitably drug release polymer comprising IL-4. The device is thereby configured with a controlled drug delivery profile that allows for enhanced drug delivery into the lumen tissue adjacent to the implantable medical device and that inhibits drug delivery into the systemic blood circulation. Suitably, the device will be self- expanding. Alternatively it may be expanded once inserted into its final position. The preferential drug delivery into the lumen tissue can be facilitated by a hydrophobic component being included in a coating on the medical device (e.g., stent or vena cava filter) that is in contact with the lumen tissue. The hydrophobic components of the tissue cooperate with the hydrophobic component of the coating so as to facilitate preferential diffusion of a hydrophilic drug into the tissue over into systemic blood. Similarly, the drug can be hydrophilic or amphipathic by having both lipophilic and hydrophilic portions. The polymer can include a hydrophilic component for the hydrophilic drug, and hydrophilic and/or hydrophobic components for the lipophilic, hydrophilic, or amphipathic drugs.

The drug eluting polymer may be polyurethane (PUR), polyglycolide (PGA), polyactide (PLA), cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, polyorganophosphazene (POP), poly ethylene (PE), poly ethylene glycol (PEG), poly-l-lactic acid (PLLA) or po lyg lycol ic/lactic acid (PGLA).

In accordance with the present invention, a drug eluting endoprosthesis can be provided for improved drug delivery within a body lumen of a human or other animal. Examples of drug eluting endoprostheses can include stents, filters, grafts, valves, occlusive devices, trocars, aneurysm treatment devices, or the like. In the context of the present invention, the drug eluting endoprosthesis can be configured for a variety of intralumenal applications, including vascular, arterial and coronary.

Drug eluting implantable medical devices include a vena cava filter that can be implanted in the vena cava to elute drug over time. The device becomes a systemic drug release device instead of a device to treat an area of stenosis. The drug release device could replace daily pills for individuals in need of the therapy provided by the drug, such as in heart transplant patients.

Generally, an endoprosthesis of the present invention can include at least a first set of interconnected strut elements that cooperatively define an annular element. A strut element can be more generally described as an endoprosthetic element, wherein all well- known endoprosthetic elements can be referred to here as a "strut element" for simplicity. Usually, each strut element can be defined by a cross-sectional profile as having a width and a thickness, and including a first end and a second end bounding a length. The stent element can be substantially linear, arced, rounded, squared, combinations thereof, or other configurations. The strut element can include a bumper, crossbar, connector, interconnector, intersection, elbow, foot, ankle, toe, heel, medial segment, lateral segment, coupling, sleeve, combinations thereof, or the like, as described in more detail below. The strut element can have improved structural integrity by including crack- inhibiting features.

Usually, the annular elements can include a plurality of circumferentially-adjacent crossbars that are interconnected end-to-end by an elbow connection, intersection, or a foot extension. As such, at least one annular element or endoprosthesis can include an elbow, intersection, or a foot extension (foot) extending between at least one pair of circumferentially-adjacent crossbars. The elbow or foot can thus define an apex between the pair of circumferentially-adjacent crossbars of the annular element or endoprosthesis. Also, an intersection can have a shape similar to a crossbar or interlinked crossbars so as to provide a junction between two coupled pairs of circumferentially-adjacent crossbars. The elbow can be configured in any shape that connects adjacent ends of circumferentially-adjacent crossbars, and can be described as having a U-shape, V-shape, L-shape, X-shape, Y-shape, H-shape, K-shape, or the like. The elbow and/or intersection can be configured in any shape that connects longitudinal and circumferentially adjacent crossbars, and can be described as having a cross shape, X-shape, Y-shape, H-shape, K- shape, or the like. The foot can have a foot shape having a first foot portion extending circumferentially from an end of one of the adjacent strut members and a second foot portion extending circumferentially from a corresponding end of the other of the circumferentially-adjacent strut members. In combination, the first and second foot portions generally define an ankle portion connected to a toe portion through a medial segment and the toe portion connected to a heel portion through a lateral segment.

As described herein, an endoprosthesis, in one configuration, can include two or more interconnected annular elements. Each annular element can generally define a ring- like structure extending circumferentially about a longitudinal or central axis. The cross- sectional profile of each annular element can be at least arcuate, circular, helical, or spiral, although alternative cross- sectional profiles, such as oval, oblong, rectilinear or the like, can be used. The different annular elements can be defined as having the same characterization or different characterizations. The first and second annular elements generally define a tubular structure. For example, each annular element can define a continuous closed ring such that the longitudinally-aligned annular elements form a closed tubular structure having a central longitudinal axis. Alternatively, each annular element can define an open ring shape such that a rolled sheet, open tubular, or "C-shape" type structure is defined by the annular elements. That is, the annular element is not required to be closed. Furthermore, each annular element can define substantially a 360-degree turn of a helical pattern or spiral, such that the end of one annular element or endoprosthesis can be joined with the corresponding end of a longitudinally-adjacent annular element or endoprosthesis to define a continuous helical pattern along the length of the endoprosthesis.

In one embodiment, the endoprosthesis is a stent, but it will be understood that the benefits and features of the present invention are also applicable to other types of endoprosthesis or other medical devices known to those skilled in the art. The present invention uses IL-4 treatment which may activate cardiac resident macrophages more directly, hence it may offer a superior outcome. In some embodiments, the present invention also provides effective therapy for treatment of patients by intraperitoneal injection before the onset of Ml, as well as treatment with IL-4 after the onset of Ml. In the scenario of therapeutic delivery of IL-4 in post-MI heart failure, the main target for treatment is the border area that suffers from adverse remodeling with persistent inflammation and ischaemia.

It is envisaged that for some embodiments the present invention may find greatest use in procedures the heart is exposed during treatment post-MI, for example for patients undergoing coronary artery bypass grafting (CABG).

Preferred features of the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be described by way of reference to the following Examples and drawings in which:

FIGURE 1 shows IL-4 administration (prior to Ml) enhanced prognosis and cardiac function post-MI. (A) Post-MI survival rate of the mice was significantly improved in the IL-4 group (n=31) compared with the PBS group (n=29). *P<0.05 versus the PBS group; Log-rank test. (B) Cardiac rupture rate over 28 days post-MI was reduced in the IL-4 group (n=31) compared with the PBS group (n=29). *P<0.05 versus the PBS group; χ ² test. (C) Echocardiographic analysis showed that the IL-4 group displayed improved cardiac function and attenuated ventricular dilatation compared to the PBS group. n=12 in each point of each group. *P<0.05 versus the PBS group in corresponding time point; Repeated measures ANOVA. (D) Cardiac catheterization assessments detected improved hemodynamics and enhanced cardiac performance at Day 28 post-MI in the IL-4 group (n=10) compared to the PBS group (n=10). *P<0.05 versus the PBS group; Student's t- test. LVEDP, left ventricular end-diastolic pressure, Dev. Pressure, developed pressure.

FIGURE 2 shows IL-4 administration (prior to Ml) reduced infarct size and increased thickness of infarcted wall. Picrosirius red staining of Day 28 post-MI heart samples showed that the infarct size was markedly reduced (A), with thicker ventricular walls of the infarct area (B), in the IL-4 group compared to the PBS group. Scale bars =1 mm. n=6 in each group, *P<0.05 versus the PBS group; Student's t-test. FIGURE 3 shows IL-4 administration (prior to Ml) enhanced formation of fibrotic connective tissue in the infarct area. (A) In the picrosirius red staining samples above (refer to Figure 2), quantity of collagen fraction was calculated by using NIH image-analysis software (ImageJ). Results are presented as collagen volume fraction (%) in the graph. (B) Total RNA was extracted from isolated heart tissue using the Gene Jet PCR purification Kit

(Thermo Scientific) and quantified with a Nano-Drop 8000 spectrophotometer (Thermo Scientific). cDNA was synthesized by using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems) from 25ng and 150ng of total RNAs from M2-macrophages and heart tissues, respectively. Quantitative PCR for Col1 a1 and Col3a1 was performed by Rotor-Gene 6000 (Qiagen) with SYBR Green I master mix (Roche) in following conditions:

95 C for 10 min followed by 50 cycles at 95 C for 15 sec, 64 C for 30 sec and 72 C for 30 sec. Gene expression levels were normalized by Hprt.

FIGURE 4 shows IL-4 administration (prior to Ml) increased activation of and reduced apoptosis of fibroblasts post-MI. (A) Thy1 ⁺ cardiac fibroblasts were increased in number and activation (transformation to Thy1 ⁺ aSMA ⁺ myofibroblasts) in the infarct area at Day 7 post-MI in the IL-4 group compared to the PBS group. Scale bars=50 μηι. aSMA ratio = percentages of aSMA ⁺ myofibroblasts in Thy1 ⁺ fibroblasts. n= in each group, *P<0.05 versus the PBS group; Student's t-test. (B) Double immunofluorescent staining for Thy1 and cleaved caspase 3 (c-caspase 3) showed that apoptosis of cardiac fibroblasts in the infarct area at Day 7 post-MI was attenuated in the IL-4 group compared to the PBS group. Scale bars=50 μηι, n=6 in each group, *P<0.05 versus the PBS group; Student's t-test.

FIGURE 5 shows IL-4 administration (prior to Ml) improved the capillary formation post-MI. Immunohistolabelling for isolectin B4 was performed to assess microvascular formation after IL-4 treatment using a primary antibody (biotinylated Griffonia simplicifolia lectin I- isolectin B4; Vector L-1 104; 1 :100 dilution). Staining protocols were the same as above.

FIGURE 6 shows IL-4 administration (prior to Ml) amplified post-MI augmentation of cardiac M2-like macrophages in the damaged myocardium. Immunohistolabelling for

CD206 was performed to assess M2-like cardiac macrophages after IL-4 treatment using a primary antibody (AlexaFlour488-conjugated rat anti-CD206, BioLegend 14709, 1 :100 dilution). Staining protocols were the same as above. Stained sections were mounted with DAKO Fluorescence Mounting Medium and images were acquired with All-in-one microscope (Keyence).

FIGURE 7 shows IL-4 injection post-MI enhanced cardiac function and attenuated cardiac dilatation. (A) Echocardiographic analysis showed that the IL-4 group displayed improved cardiac function and attenuated ventricular dilatation compared to the PBS group. n=7 in each group. *P<0.05, **P<0.01 versus the PBS group; Student's t-test. (B) Cardiac catheterization assessments detected improved hemodynamics and enhanced cardiac performance at Day 28 post-MI in the IL-4 group (n=6) compared to the PBS group (n=6). **P<0.01 , ***P<0.001 ; Student's t-test. FIGURE 8 shows IL-4 administration post-MI amplified augmentation of cardiac M2-like macrophages. Staining protocols were the same as above. Stained sections were mounted with DAKO Fluorescence Mounting Medium and images were acquired with All-in-one microscope (Keyence). FIGURE 9 shows IL-4 administration post Ml enhanced formation of fibrotic connective tissue in the infarct area. Scale bars=100 μηι. n=4 in each group, Student's t-test.

FIGURE 10 shows IL-4 administration post-MI increased cardiac fibroblasts in the infarct areas. Scale bars=50 μηι. n=4 in each group, Student's t-test.

FIGURE 1 1 shows IL-4 administration post-MI improved the capillary formation in the border areas. Scale bars=50 μηι. n=4 different hearts were studied in each point, Student's t-test. The Examples are present for the purposes of illustration only and are not to be construed as being limitations to the invention. Examples 1 to 6 show that IL-4 administration before myocardial infarction improves post-infarction survival and cardiac function with enhanced myocardial repair. Examples 7 to 1 1 show that IL-4 administration post myocardial infarction improves cardiac function and attenuates heart dilatation.

Example 1 : IL-4 administration (prior to Ml) enhanced prognosis and cardiac function post- Ml

C57BL/6 mice (6-8 weeks of age) were purchased from Charles River UK. Mice were maintained specific pathogen free in our animal facility on 12-hour light-dark cycle, with free access to food and water. Mice were randomly assigned to groups, and where possible, in vitro studies as well as in vivo procedures and assessments were all carried out in a blinded manner.

5μg recombinant IL-4 (PeproTech, 214-14) was dissolved in 100μΙ Phosphate Buffered Saline (PBS) and injected, together with 25μg anti-IL-4 neutralizing monoclonal antibody [BD Biosciences, 554387]) which extends the half-life of IL-4 in vivo, into the peritoneal cavity of mice twice (2 days before and immediately before induction of myocardial infarction [Ml]). Equivalent volume of PBS was similarly injected as a control.

Ml was induced by ligating the left coronary artery under 1 .0% isoflurane anesthesia and mechanical ventilation. Successful generation of Ml was confirmed by changes in color and motion of left ventricular walls. The chest was closed, and when the mice regained consciousness they were returned to the cage.

At chosen time (before Ml, and day 7 and 28 after Ml), transthoracic echocardiography was performed by using Vevo-770 (VisualSonics) with a 30 MHz high-frequency transducer under 1 .0% isoflurane inhalation. Left ventricular (LV) ejection fraction was calculated with Simpson's method from 2-dimensional tracing. LV end-diastolic and end-systolic dimensions were measured with M- mode. LV end-diastolic and end-systolic areas were measured with B-mode. Data were collected from 3 different measurements in a blinded manner. In addition, hemodynamic parameters were measured at day 28 post-MI by using cardiac catheterization. Briefly, under general anesthesia using isoflurane and mechanical ventilation, a catheter (SPR-839NR; Millar Instruments) was inserted into the LV cavity through the LV free wall. Intra-LV pressure signals were measured (MPVS-300; Millar Instruments) and digitally recorded with a data acquisition system (PowerLab 8/30; ADInstruments). Data were collected from at least 5 different measurements in a blinded manner. Results are shown in Figure 1 .

Example 2: IL-4 administration (prior to Ml) reduced infarct size and increased thickness of infarcted wall

At day 28 after Ml, the mouse was euthanized, and heart was flushed with ice-cold PBS and perfused with ice-cold 4% paraformaldehyde in PBS. Cardiac tissues were embedded in O.C.T. compound (VWR International) and frozen in isopentane chilled in liquid nitrogen. 8μηι frozen sections were produced and incubated in 1 .5% phosphomolybic acid for 60 min, next in 0.1 % Picrosirius red for 15 min, and then in 0.5% acetic acid solution for 3 min. After dehydration through increasing concentrations of ethanol to xylene, sections were mounted using DPX mounting medium (VWR International). Infarct size (the ratio of scar length to total left ventricular circumference) and wall thickness were measured at five independent regions in the infarct area. Results are shown in Figure 2.

Example 3: IL-4 administration (prior to Ml) enhanced formation of fibrotic connective tissue in the infarct area

An increased amount of and improved organisation (alignment) of collagen fibrils in the infarct area was observed in the IL-4 group compared to the PBS group. In contrast, extracellular collagen deposition in the remote and border areas was unchanged or attenuated in the IL-4 group. Scale bars=100 μηι. n=6 in each point in each group, *P<0.05 versus the intact (no Ml) heart in each group, ^† P<0.05 versus the WT group in the corresponding time and area; Repeated measures ANOVA.

It should be noted that "physiological" fibrotic tissue formation in the infarct areas is beneficial to support and prevent the fragile infarcted myocardium post-MI from rapture and dilatation. This was enhanced by IL-4 treatment. In contrast, extracellular collagen deposition in the remote and border areas represents adverse (unwanted) ventricular remodeling. This was not increased by IL-4 treatment, resolving a concern of excessive, "pathological" myocardial fibrosis by this treatment.

Quantitative RT-PCR showed that collagen genes (Col1a1 and Col3a1) were up-regulated in the heart post-MI in the IL-4 group, compared to the PBS group, at Day 7 but not Day 28 post-MI. n=6 in each point in each group, *P<0.05 versus the corresponding time point of the PBS group; Repeated measures ANOVA. Results are shown in Figure 3.

This production of more solid and supportive scar tissue in the fragile infarct areas played a role in prevention of post-MI rapture of the heart, attenuation of cardiac dilatation, and thereby improvement of cardiac function.

Example 4: IL-4 administration (prior to Ml) increased activation of and reduced apoptosis of fibroblasts post-MI

To investigate the changes in fibroblasts, frozen tissue sections (δμηι), were incubated in PBS containing 0.1 % Triton X for 5 min at the room temperature, and non-specific antibody-binding sites were pre-blocked with blocking buffer (PBS plus 5% goat serum or PBS plus 5% bovine serum albumin). Then, primary antibody (rat anti-Thyl monoclonal antibody [eBiosciences 14-0901 ; 1 :100 dilution], rabbit anti-creaved caspase 3 polyclonal antibody [Cell Signaling 9661 ; 1 :200 dilution], or rabbit anti-aSMA polyclonal antibody [Abeam ab5694; 1 :100 dilution]) was applied overnight at 4 °C. For visualization, sections were incubated with fluorophore-conjugated secondary antibodies and 4',6-diamidino-2-phenylindole (DAPI) in blocking buffer for 1 hour at the room temperature. Stained sections were mounted with DAKO Fluorescence Mounting Medium and images were acquired with All-in-one microscope (Keyence). Results are shown in Figure 4.

Example 5: IL-4 administration (prior to Ml) improved the capillary formation post-MI

Immunohistolabelling for isolectin B4 was performed to assess microvascular formation after IL-4 treatment using a primary antibody (biotinylated Griffonia simplicifolia lectin l-isolectin B4; Vector L- 1 104; 1 :100 dilution). Staining protocols were the same as above.

As a result, it was observed that the density of capillary vessels was improved at Day 7 and 28 post-MI in the IL-4 group (IL-4 treatment) compared to the PBS group (PBS injection). Graphs show the averaged capillary density in each area in each point. Scale bars=50 μηι. n=6 different hearts were studied in each point, *P<0.05 versus the intact (no Ml) heart in each group, ^† P<0.05 versus the PBS group in the corresponding time and area; Repeated measures ANOVA. Results are shown in Figure 5.

This increased microvasculature by IL-4 treatment is thought to enhance local perfusion and improve the viability and functionality of fibroblasts/myofibroblasts, which enhances formation of connective tissues, in addition to improving perfusion of surviving cardiomyocytes. Example 6: IL-4 administration (prior to Ml) amplified post-MI augmentation of cardiac M2- like macrophages in the damaged myocardium

Immunohistolabelling for CD206 was performed to assess M2-like cardiac macrophages after IL-4 treatment using a primary antibody (AlexaFlour488-conjugated rat anti-CD206, BioLegend 14709, 1 :100 dilution). Staining protocols were the same as above. Stained sections were mounted with DAKO Fluorescence Mounting Medium and images were acquired with All-in-one microscope (Keyence). Results in Figure 6 showed that post-MI augmentation of CD206 ⁺ M2-like macrophages seen in the border and infarct areas of the PBS group (injection of PBS; n=6 in each point) was further amplified in the IL-4 group (IL-4 treatment; n=6 in each point). Scale bars=50 μηι. *P<0.05 versus the intact (no Ml) heart in each group, ^† P<0.05 versus the PBS group in the corresponding time and area; Repeated measures ANOVA.

Example 7: IL-4 injection post-MI enhanced cardiac function and attenuated cardiac dilatation

Ml was induced by ligating the left coronary artery in C57BL/6 mice (6-8 weeks of age; from Charles River UK) under 1 .0% isoflurane anesthesia and mechanical ventilation. Successful generation of Ml was confirmed by changes in color and motion of left ventricular walls. The chest was closed, and subsequently, IL-4 ^g recombinant IL-4 [PeproTech, 214-14] dissolved in 100μΙ Phosphate Buffered Saline (PBS) was injected into the peritoneal cavity of mice, together with 25μg anti-IL-4 neutralizing monoclonal antibody [BD Biosciences, 554387]), which is known to enhance half-life of IL-4 in vivo. Equivalent volume of PBS was similarly injected as a control. The mice were returned to the cage. Results are shown in Figure 7. At day 28 after Ml, transthoracic echocardiography and cardiac catheterization was performed as above (see Figure 1).

Example 8: IL-4 administration post-MI amplified augmentation of cardiac M2-like macrophages

At day 7 after Ml, the mouse was euthanized, and heart was flushed with ice-cold PBS and perfused with ice-cold 4% paraformaldehyde in PBS. Cardiac tissues were embedded in O.C.T. compound (VWR International) and frozen in isopentane chilled in liquid nitrogen. Immunohistolabelling for CD206 was performed to assess M2-like cardiac macrophages using a primary antibody (AlexaFlour488-conjugated rat anti-CD206, BioLegend 14709, 1 :100 dilution). Staining protocols were the same as above. Stained sections were mounted with DAKO Fluorescence Mounting Medium and images were acquired with All-in-one microscope (Keyence).

Results in Figure 8 showed that post-MI augmentation of CD206 ⁺ M2-like macrophages seen the PBS group (injection of PBS; n=5) was further amplified in the IL-4 group throughout the heart (all infarct, border and remote areas) (IL-4 treatment; n=5). Scale bars=50 μηι. *P<0.05, **P<0.01 ; Student's t-test.

Example 9: IL-4 administration post Ml enhanced formation of fibrotic connective tissue in the infarct area

At day 28 after Ml, frozen sections were produced as above and incubated in 1 .5% phosphomolybic acid for 60 min, next in 0.1 % Picrosirius red for 15 min, and then in 0.5% acetic acid solution for 3 min. After dehydration through increasing concentrations of ethanol to xylene, sections were mounted using DPX mounting medium (VWR International). Infarct size (the ratio of scar length to total left ventricular circumference) and wall thickness were measured at five independent regions in the infarct area. Quantity of collagen fraction was calculated by using NIH image-analysis software (ImageJ). Results are shown in Figure 9.

It was shown that formation of fibrotic tissues in the infarct area (quantity and organization) was augmented in the IL-4 group compared to the PBS group. This production of more solid and more supportive scar tissue played a role in attenuation of cardiac dilatation and improvement of cardiac function. Scale bars=100 μηι. n=4 in each group, Student's t-test.

Example 10: IL-4 administration post-MI increased cardiac fibroblasts in the infarct areas To investigate the changes in cardiac fibroblasts at day 7 post-MI, frozen tissue sections (δμηι), were incubated in PBS containing 0.1 % Triton X for 5 min at the room temperature, and nonspecific antibody-binding sites were pre-blocked with blocking buffer (PBS plus 5% goat serum or PBS plus 5% bovine serum albumin). Then, primary antibodies (rat anti-Thyl monoclonal; eBiosciences 14-0901 , 1 :100 dilution) were applied. After rinsing 3 times for 15 minutes in PBS, sections were incubated with fluorophore-conjugated secondary antibodies and 4',6-diamidino-2- phenylindole (DAPI) in blocking buffer for 1 hour at the room temperature. Stained sections were mounted with DAKO Fluorescence Mounting Medium and images were acquired with All-in-one microscope (Keyence). Results are shown in Figure 10. It was shown that Thy1 ⁺ fibroblasts in the infarct area in the IL-4-treated mice displayed increased occurrence at Day 7 post-MI. This contributed to the production of more solid fibrotic scar in the fragile infarct areas by IL-4 treatment. Scale bars=50 μηι. n=4 in each group, Student's t-test.

Example 11 : IL-4 administration post-MI improved the capillary formation in the border areas

Immunohistolabelling for isolectin B4 was performed to assess microvascular formation after IL-4 treatment using a primary antibody (biotinylated Griffonia simplicifolia lectin l-isolectin B4; Vector L- 1 104; 1 :100 dilution). Staining protocols were the same as above. Stained sections were mounted with DAKO Fluorescence Mounting Medium and images were acquired with All-in-one microscope (Keyence). Results are shown in Figure 1 1 . Results demonstrated that the density of capillary vessels was improved in the border areas surrounding the infarct at Day 7 post-MI in the IL-4 group (IL-4 treatment) compared to the PBS group (PBS injection). Scale bars=50 μηι. n=4 different hearts were studied in each point, Student's t-test.