WO2019237157A1 - Cardiac treatment - Google Patents

Abstract

The invention relates to a method of improving cardiac function in an individual having sub optimal cardiac function comprising administering to the individual a therapeutically effective amount of PDGF-AB or a functional derivative thereof, thereby improving cardiac function in the individual.

Description

Cardiac treatment

Field of the invention

The invention relates to methods for the treatment of a cardiovascular event.

Background of the invention

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art. Myocardial infarction (Ml) is one of the leading causes of mortality worldwide. Ml induces necrosis of cardiomyocytes that are unable to significantly proliferate and replace damaged tissue which leads to the development of collagenous scar, arrhythmias and heart failure.

Despite significant progress in coronary artery reperfusion and medical therapy for Ml there are no available therapies that directly impact cardiac scar in its early stages of formation. Such a therapy would lead to significant improvement in cardiac function after an initial cardiovascular event such as Ml. There thus remains a need to identify new treatments that restore cardiac function in patients suffering cardiovascular events such as Ml. WO 2004/045531 discusses methods of promoting vascular health, and for protecting cardiac tissue in a patient from damage during myocardial infarction by administration of a combination of VEGF, angiopoietin-2 and PDGF-AB by intra- myocardial injection prior to or at the time of ligation. WO 2004/045531 shows that administration of PDGF-AB alone by intramyocardial injection conferred a benefit only when the administration occurred 24-48 hrs prior to ligation. WO 2004/045531 does not discuss improvement to tissue arising from administration of PDGF-AB at the time of myocardial infarction. WO 2008/157733 discusses a controlled release formulation of a PDGF protein. The formulation is said to useful for any condition in which PDGF is known to have application. Administration of PDGF to infarcted tissue was not a known application of PDGF, and such administration is not discussed in WO 2008/157733. US 2015/0250822 discusses loading agents into platelets which are then incorporated into sites of active angiogenesis or blood vessel injury. There is no discussion of treatment of myocardial infarction nor administration of PDGF-AB to infarcted myocardium.

US 8795652 discusses a treatment of infarcted tissue which utilises cells, and additionally, supply scaffolding or growth factors. According to US 8795652, thinning of the infarct region is addressed by replacing dead cells with viable cells. There is no discussion of utilising PDGF-AB in the absence of cell therapy.

Summary of the invention

The invention is directed to a method of improving cardiac function in an individual having sub optimal cardiac function comprising or consisting of, administering to the individual a therapeutically effective amount of PDGF-AB or a functional derivative thereof, thereby improving cardiac function in the individual.

In an embodiment, the sub optimal cardiac function is caused by, or associated with a cardiovascular event that causes cardiac tissue ischemia or infarction. The cardiovascular event may be selected from the group consisting of: unstable angina, coronary artery spasm, coronary artery embolism, coronary artery dissection or coronary artery spasm. In a preferred embodiment the infarction is acute ST elevation myocardial infarction from coronary artery occlusion.

In an embodiment, PDGF-AB or a functional derivative thereof may be

administered after the cardiovascular event or after the cardiac tissue infarction. In certain embodiments, the PDGF-AB or a functional derivative thereof is not

administered prior to the cardiovascular event or is not administered prior to cardiac tissue infarction. In an embodiment, the administration of PDGF-AB prevents a further increase in sub optimisation of cardiac function. Preferably, the administration of PDGF-AB restores cardiac function to normal cardiac function with respect to the individual profile.

In an embodiment, the administration of PDGF-AB leads to an improvement in left ventricular ejection fraction, thereby leading to an improvement in cardiac function.

In an embodiment, the administration of PDGF-AB leads to an improvement in stroke volume, thereby leading to an improvement in cardiac function.

In an embodiment, the administration of PDGF-AB leads to an improvement in left ventricular end systolic volume, thereby leading to an improvement in cardiac function.

In an embodiment, the administration of PDGF-AB leads to an improvement in anisotropy or alignment of collagen fibre in myocardial scar tissue, thereby leading to an improvement in cardiac function.

In an embodiment, the administration of PDGF-AB leads to minimisation of likelihood of sudden cardiac death.

In an embodiment, the administration of PDGF-AB leads to improvement in left ventricular ejection fraction and delta left ventricular ejection fraction.

In an embodiment, the administration of PDGF-AB leads to a reduction in ventricular tachycardia or arrhythmia, thereby leading to an improvement in cardiac function and/or a reduced risk of life threatening cardiac events.

In an embodiment, the administration of PDGF-AB leads to improvement in scar anisotropy, organisation and mechanotransduction without reduced left ventricular compliance.

In an embodiment, the improvement in cardiac function is assessed by

electrocardiography (ECG) or magnetic resonance imaging (MRI).

In an embodiment of the invention, there is provided use of a therapeutically effective amount of PDGF-AB or a functional derivative thereof in the preparation of a medicament for improving cardiac function in an individual having sub optimal cardiac function, thereby improving cardiac function in the individual.

In an embodiment of the invention, there is provided use of a therapeutically effective amount of PDGF-AB or a functional derivative thereof for improving cardiac function in an individual having sub optimal cardiac function, thereby improving cardiac function in the individual.

In an embodiment of the invention, there is provided a method of remodelling left ventricular cardiac tissue associated with infarction or ischemia in an individual comprising, or consisting of administration of PDGF-AB to an individual having ischemic or infarcted left ventricular cardiac tissue, thereby remodelling the left ventricular cardiac tissue.

In an embodiment of the invention, there is provided a method of improving survival in an individual who has suffered a cardiovascular event comprising, or consisting of administering to the individual a therapeutically effective amount of PDGF- AB or a functional derivative thereof, thereby improving survival in the individual.

In an embodiment, survival is disease - free survival. In another embodiment, survival may be progression - free survival. In another embodiment, the improvement in survival is associated with an improvement in cardiac function.

In an embodiment, PDGF-AB is a recombinant protein.

In an embodiment, PDGF-AB is administered from less than 1 minute to 120 hours after a cardiovascular event, or 30 minutes to 72 hours after tissue infarction. In an embodiment, PDGF-AB is administered over a period of 1 second to 120 hours or up to 240 hours.

In an embodiment, PDGF-AB may be administered one or more times. In an embodiment, PDGF-AB is administered once.

In an embodiment, PDGF-AB is administered by intravenous infusion, intravenous injection, intra-arterial injection or osmotic minipump. In an embodiment, PDGF-AB is administered together with a further compound selected from the group consisting of: an anti-inflammatory, an anti-fibrotic, a stimulator of cardiomyocyte dedifferentiation, proliferation or migration, or a regulator of extracellular matrix deposition or composition.

The invention further relates to a method for increasing anisotropy or alignment of collagen fibre in myocardial scar tissue of an individual comprising administering a therapeutically effective amount of PDGF-AB or a functional derivative thereof to an individual requiring treatment, wherein the PDGF-AB is administered to infarcted myocardial tissue, preferably to infarcted myocardial tissue that includes scar tissue or precursor tissue thereof. Preferably, PDGF-AB is administered over a period of 1 second to 240 hours. Preferably, PDGF-AB is administered by intravenous (IV) injection, intra-cardiac (trans-epicardial or trans-endocardial) injection, intra-arterial injection or by osmotic minipump.

In an embodiment, PDGF-AB may be administered over a period of 1 second and up to 10 days. In another embodiment, PDGF-AB may be administered over a period of 1 second to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more.

In this embodiment, the administration of PDGF-AB may lead to an improvement in one or more of left ventricular ejection fraction, an improvement in stroke volume, an improvement in left ventricular end systolic volume, a reduction in ventricular tachycardia or arrhythmia, thus leading to an improvement in cardiac function. Preferably, the method decreases the likelihood of lethal arrhythmia arising in absence of said treatment in the individual. In one embodiment, the improvement in cardiac function is assessed by electrophysiology study, electrocardiography (ECG) or magnetic resonance imaging (MRI).

In an embodiment, any method described herein does not comprise administering vascular endothelial growth factor (VEGF) or angiopoietin-2 (Ang-2). In other words, the method may comprise administering PDGF-AB or a functional derivative thereof, but does not comprise administering VEGF or Ang-2 to the individual.

In another embodiment, any method described herein does not comprise administering PDGF-AB in the form of a fibrin sealant or surgical glue. In other words, the method may comprise administering PDGF-AB or a functional derivative thereof but not in the form of a fibrin sealant or surgical glue to the individual.

In another embodiment, any method described herein does not comprise administering platelets. In other words, the method may comprise administering PDGF- AB or a functional derivative thereof but does not comprise administering platelets to the individual.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1. Kaplan-Meier estimates of the probability of survival according to study group. Treatment with rh-PDGF-AB post Ml is associated with significantly improved survival at 1 month in pigs.

Figure 2. Representative cine cardiac magnetic resonance imaging. Pigs treated with PDGF-AB show improvement in cardiac function, less thinning and dilation of the injured left ventricular myocardium when compared to the control group.

Figure 3. (a) Pigs treated with PDGF-AB show improvement in left ventricular ejection fraction (LVEF) when compared to the placebo treated group over a treatment period of 28 days (b) Pigs treated with PDGF-AB also show improvement in absolute LVEF when compared to the placebo treated group over a treatment period of 28 days. ALVEF represents the absolute difference between LVEF at 1 month and 2 days after acute myocardial infarction.

Figure 4. (a) Pigs treated with PDGF-AB show improvement in stroke volume when compared to the placebo and sham treated groups over a treatment period of 28 days. (b) Pigs treated with PDGF-AB also show improvement in absolute stroke volume when compared to the placebo treated group over a treatment period of 28 days.

Figure 5. Pigs treated with PDGF-AB show improvement in indexed left ventricular end systolic volume when compared to the placebo treated groups over a treatment period of 28 days.

Figure 6. Pigs treated with PDGF-AB show improvement in (a) absolute left ventricular end systolic volume but not (b) absolute left ventricular end diastolic volume, when compared to the placebo treated groups over a treatment period of 28 days.

Figure 7. Pigs treated with PDGF-AB and induced with cardiac arrhythmia show less risk of sudden cardiac death when compared to the control group.

Figure 8. rhPDGF-AB improves myocardial contractility and energetics with little effect on ventricular compliance. Improved systolic function in rhPDGF-AB treated animals versus vehicle shown by an increase in (A) maximal rate of pressure change during systole (dP/dt max) at steady-state. (B) Representative pressure-volume (PV) recordings with corresponding end-systolic pressure volume relationships (ESPVR) during IVC occlusion showing a steeper slope in the rhPDGF-AB-group compared to vehicle, increased slope of the regression curve for C) ESPVR and (D) preload recruitable stroke work index (PRSW), demonstrating improved myocardial load- independent contractility. PV diastolic function was significantly different as assessed by decrease in (E) dP/dt min, but no difference was observed in (F) the slope of the regression curve for EDPVR or (G) tau, the isovolumic relaxation time. n=10 in rhPDGF- AB group; n=6 in vehicle. All data are presented as mean ± SEM. ^*p<0.05 (unpaired t- test); ^**p<0.005 (unpaired t-test); ns=non-significant.

Figure 9. rhPDGF-AB promotes vasculogenesis and arteriolar collateralization after Ml. (A) Representative images show von Willebrand factor (vWF) immunostaining with enhanced capillary density in the peri-infarct zone in the rhPDGF-AB-treated animals compared to Ml vehicle but (B) no difference in arteriolar density (a-SMA+). Scale bar, 50pm. n=10 in rhPDGF-AB group; n=6 in vehicle group. (C) Pig heart tissue sections (5 mm thick) cleared using CUBIC R1 a protocol. (D) Representative images of a-SMA- stained vessels using 3D light sheet microscopy show increased arteriolar density and branching points with smaller arteriolar diameter in rhPDGF-AB-treated animals compared to vehicle (E-G). n=3/group. All data are presented as mean ± SEM. ^*p<0.05 (unpaired t-test); ^**p<0.005 (unpaired t-test); ns=non-significant.

Figure 10. rhPDGF-AB promotes collagen fiber alignment and wound healing without reduction of overall scar size. (A) Representative cine cMR 4 chamber LGE views (scar = white; viable myocardium = black), (B) macroscopic hearts (scar = white; viable myocardium = brown); scale bar, 10mm and (C) collagen quantification by Gomori trichrome staining demonstrates scar size in vehicle and rhPDGF-AB-treated groups at day 28 are similar (scar = purple; viable myocardium = pink); scale bar, 100pm. n=4 vehicle and n=6 rhPDGF-AB. (D) Representative peri-infarct images using SHG 2- photon and polarized light imaging show organized collagen fiber alignment in the rhPDGF-AB group; scale bar 50pm (E) Representative distribution of collagen fiber alignment assessed by SHG 2-photon imaging. (F) Representative images of picrosirius red-stained infarct sections viewed with polarized light (Type I collagen = orange; Type III collagen = green); scale bar, 75pm. (G) Atomic force microscopy showing no difference in stiffness within infarct and border zones; n=5/group. n=5/group for infarct zone; n=5 vehicle and n=7 rhPDGF-AB for border zone. (H) Endogenous tension generation in mesenchymal cell microtissues show increased tissue condensation rate and tension generation after rhPDGF-AB treatment. n=15-18/condition. cMR = cardiac magnetic resonance; LGE = late gadolinium enhancement; IZ = infarct zone; BZ = border zone; RZ = remote zone; SHG = second harmonic generation. LGE and collagen data are presented as mean ± SEM. AFM and organoid data are presented as mean and 95% confidence intervals. ^**p<0.005 (unpaired t-test); ^***p<0.0005 (unpaired t-test); ns=non-significant.

Figure 11. rhPDGF-AB reduces arrhythmogenicity. (A) Compared to vehicle, rhPDGF- AB-treated animals had significantly decreased inducibility of ventricular tachycardia (VT) with a reduced number of extrastimuli required to induce VT in the positive animals, and (B) reduced myofiber heterogeneity within the scar core and scar border regions. Heterogeneity map: representative images of myocardial scar tissue heterogeneity (color blue to red indicates increased myofiber clustering) in vehicle (top) and rhPDGF-AB-treated animals (bottom). Heterogeneity index, where small values indicates multiple small clusters of myocytes separated by small distances thus facilitating an increased propensity for cell-cell coupling and large values indicating the opposite, where decreased coupling may facilitate electrophysiological conduction slowing or blockade. (C) Gomori trichrome stain of infarcted myocardium in vehicle shows “islands” of preserved myocardium (red) surrounded by scar tissue (blue) compared to a more homogenous distribution of scar in the rhPDGF-AB-treated group. A schematic representation of a potential VT circuit is depicted (white dashed line). Scale bar, 1 mm. Scar heterogeneity data are presented as mean ± SEM.^*p<0.05 (unpaired t-test).

Figure 12. (A-B) The administration of PDGF-AB to human cardiac organoids expressing PDGFR-alpha leads to the upregulation of tenascin-C, ITGB1 and alpha- SMA.

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the patient features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. All of the patents and publications referred to herein are incorporated by reference in their entirety.

For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa. The inventors have found that when PDGF-AB is administered to an individual after the occurrence of a cardiovascular event, PDGF-AB has the ability to restore cardiac function. In particular, the inventors have found that PDGF-AB has the ability to decrease mortality, improve cardiac function including left ventricular ejection fraction, improve stroke volume, improve left ventricular end systolic volume, improve LV remodelling and minimise ventricular remodelling.

The inventors finding herein is surprising because it has previously been shown that PDGF-AB has been ineffective as a treatment regime. This is demonstrated in US 7,504,379 and WO 03/070083 . Importantly, whilst it has been reported that PDGF-AB can reduce infarct size when given as a pretreatment prior to the induction of Ml (Edelberg et al (2002) Circulation 105:508-613), pretreatment with PDGF-AB has no clinical correlate. Further, the same study has reported that when PDGF-AB is given at ligation, PDGF-AB confers no beneficial effect in preserving heart function.

Furthermore, the inventors study is the first to address the treatment of cardiac arrhythmias which is an enormous clinical problem following myocardial infarction. The inventors’ clinically relevant large animal data show a benefit on individual survival which is associated with decreased heart rhythm abnormalities. The inventor’s finding thus provides for a new therapeutic approach to the treatment of cardiovascular events such as myocardial infarction. Platelet-derived growth factor (PDGF)

Naturally occurring, platelet-derived growth factor ("PDGF") is a disulfide-bonded dimer having two polypeptide chains, namely the "A" and "B" chains, with the A chain being approximately 60% homologous to the B chain.

Naturally occurring PDGF is found in three dimeric forms, namely PDGF-AB heterodimer, PDGF-BB homodimer, or PDGF-AA homodimer. PDGF-AB has been identified as the predominate naturally occurring form. Each monomeric subunit of the biologically active dimer, irrespective of whether it is an A chain monomer or a B chain monomer, contains eight cysteine residues. Some of these cysteine residues form interchain disulfide bonds that hold the dimer together. As used herein, the term PDGF means any PDGF polypeptide or protein, including PDGF A, PDGF B, PDGF AB, PDGF BB, and PDGF AA.

There different isoforms of PDGF act through two different receptors, the alpha (PDGFRA) and beta (PDGFRB) forms. The receptor for PDGF, PDGFR is classified as a receptor tyrosine kinase (RTK), a type of cell surface receptor. The alpha type binds to PDGF-AA, PDGF-BB and PDGF-AB, whereas the beta type PDGFR binds with high affinity to PDGF-BB and PDGF-AB. PDGF binds to the PDGFR ligand binding pocket located within the second and third immunoglobulin domains. Upon activation by PDGF, these receptors dimerise, and are "switched on" by auto-phosphorylation of several sites on their cytosolic domains, which serve to mediate binding of cofactors and subsequently activate signal transduction.

The A polypeptide of human PDGF can be any mammalian PDGF A polypeptide including, for example, human, mouse, rat, rabbit, goat, bovine, horse, sheep and any other mammalian PDGF A polypeptide. The B polypeptide of human PDGF can be any mammalian PDGF B polypeptide including, for example, human, mouse, rat, rabbit, goat, bovine, horse, sheep and any other mammalian PDGF B polypeptide. The following sequence is one example of an amino acid sequence of a human PDGF-AB recombinant protein:

Alpha chain: SIEEAVPAVC KTRTVIYEIP RSQVDPTSAN FLIWPPCVEV KRCTGCCNTS SVKCQPSRVH HRSVKVAKVE YVRKKPKLKE VQVRLEEHLE CACATTSLNP DYREEDTGRP RESGKKRKRK RLKPT (SEQ ID NO:1 )

Beta chain: SLGSLTIAEP AMIAECKTRT EVFEISRRLI DRTNANFLVW

PPCVEVQRCS GCCNNRNVQC RPTQVQLRPV QVRKIGIVRK KPIFKKATVT LGDHLACKCE TVAAARPVT (SEQ ID NO:2). The following sequence is one example of an amino acid sequence of a human

PDGF A polypeptide:

1 MRTWACLLLL GCGYLAHALA EEAEIPRELI ERLARSQIHS

41 IRDLQRLLEI DSVGAEDALE TNLRAHGSHT VKHVPEKRPV

81 PIRRKRSIEE AIPAVCKTRT VIYEIPRSQV DPTSANFLIW 121 PPCVEVKRCT GCCNTSSVKC QPSRVHHRSV KVAKVEYVRK 161 KPKLKEVQVR LEEHLECACA TSNLNPDHRE EETGRRRESG

201 KKRK (SEQ ID NO:3)

The following sequence is one example of a human sequence for the PDGF B polypeptide:

1 MNRCWALFLS LCCYLRLVSA EGDPIPEELY EMLSDHSIRS 41 FDDLQRLLHG DPGEEDGAEL DLNMTRSHSG GELESLARGR 82 RSLGSLTIAE PAMIAECKTR TEVFEISRRL IDRTNANFLV 121 WPPCVEVQRC SGCCNNRNVQ CRPTQVQLRP VQVRKIEIVR

161 KKPIFKKATV TLEDHLACKC ETVAAARPVT RSPGGSQEQR 201 AKTPQTRVTI RTVRVRRPPK GKHRKFKHTH DKTALKETLG 241 A (SEQ ID NO:4)

Other sequences for PDGF-AB can readily be obtained by one of skill in the art, for example, in from the publicly available GenBank database. A skilled person would understand that these sequences can be modified to generate functional derivative so long as the polypeptide retains its ability to bind and activate its cognate receptor, PDGFR.

The invention provides PDGF-AB polypeptides that are useful in treating cardiac conditions. Polypeptides of the invention include a polymeric form of amino acids of any length, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, and depsipeptides, and polypeptides having modified, cyclic, bicyclic, depsicyclic, or depsibicyclic peptide backbones. They include single chain proteins as well as multimers. They also include conjugated proteins, fusion proteins, including, but not limited to, glutathione S-transferase (GST) fusion proteins, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, fusion proteins with or without N- terminal methionine residues, pegylated proteins, and immunologically tagged, or his- tagged proteins. Also included in the polypeptides of the invention are variations of naturally occurring proteins, where such variations are homologous or substantially similar to the naturally occurring protein, as well as corresponding homologs from different species. Variants of polypeptide sequences include insertions, additions, deletions, or substitutions compared with the subject polypeptides. The polypeptides of the invention also include peptide aptamers.

It is to be understood that the PDGF-AB polypeptides according to the invention include biologically active fragments and analogs of therapeutic polypeptides specifically identified, such as the growth factors and proteins mentioned above. Thus, for example, a reference to PDGF-AB encompasses not only the full-length PDGF-AB, but also functional derivatives, biologically active fragments and analogs of PDGF-AB. A functional derivative, biologically active fragment or analog is capable of treating ischemic cardiac injury or other cardiac conditions. Analogs of a particular therapeutic polypeptide can differ from the therapeutic polypeptide by amino acid sequence differences, or by modifications (e.g., post-translational modifications), which do not affect sequence, or by both. Analogs of the invention will generally exhibit at least 80%, at least 85%, at least 90%, or at least 99% amino acid identity with all or part of the amino acid sequence of a therapeutic polypeptide. Methods for assaying the capacity of biologically active fragments and analogs to treat ischemic cardiac injury or other cardiac conditions are known in the art.

Protein engineering may be employed to improve or alter the characteristics of the therapeutic polypeptides of the invention. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins" including single or multiple amino acid substitutions, deletions, additions, or fusion proteins. Such modified polypeptides can show desirable properties, such as enhanced activity or increased stability.

In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. For many proteins, including the extracellular domain of a membrane associated protein or the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function.

However, even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature from of the protein generally will be retained when less than the majority of the residues of the complete or mature protein are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete protein retains its required activity can be determined by routine methods known in the art.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequences of the molecules. Similarly, many examples of biologically functional C-terminal deletion muteins are known.

However, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature form of the protein generally will be retained when less than the majority of the residues of the complete or mature protein are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete protein retains such biological activity can be determined by routine methods known in the art.

In addition to terminal deletion forms of the protein discussed above, it also will be recognized by one of ordinary skill in the art that some amino acid sequences of the therapeutic polypeptides of the invention can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, a skilled person would understand how to determine which domains are critical areas to retain function of the protein such as binding of PDGF-AB to its cognate receptor. Such mutants include deletions, insertions, inversions, repeats, and type substitutions, selected according to general rules known in the art, so as have little effect on activity. For example, guidance on how to make phenotypically silent amino acid substitutions is provided in Bowie et al., (1990) Science 247:1306-1310, wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.

The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections, or screens, to identify sequences that maintain functionality. These studies report that proteins are surprisingly tolerant of amino acid substitutions. It is known that certain amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.

Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg, and replacements between the aromatic residues Phe and Tyr. Thus, a fragment, derivative, or analog of a polypeptide may be (i) one in which one or more of the amino acid residues are substituted with a conserved or nonconserved amino acid residue; such a substituted amino acid residue may or may not be one encoded by the genetic code; (ii) one in which one or more of the amino acid residues includes a substituent group; (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 above form of the polypeptide, such as an IgG Fe fusion region peptide, a leader or secretory sequence, a sequence employed to purify the above form of the polypeptide, or a proprotein sequence. Such fragments, derivatives, and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. Thus, the therapeutic polypeptides of the invention may include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human manipulation. As indicated, these changes may be of a minor nature, such as conservative amino acid substitutions, that do not significantly affect the folding or activity of the protein.

Conservative amino acid substitutions include the aromatic substitutions Phe, Trp, and Tyr; the hydrophobic substitutions Leu, Iso, and Val; the polar substitutions Glu and Asp; the basic substitutions Arg, Lys, and His; the acidic substitutions Asp and Glu; and the small amino acid substations Ala, Ser, Thr, Met, and Gly.

Amino acids essential for the functions of the therapeutic polypeptides of the invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. The latter procedure introduces single alanine mutations. The resulting mutant molecules are then tested for biological activity such as receptor binding, or in vitro or in vitro proliferative activity.

Further, substitutions of charged amino acids with other charged or neutral amino acids which may produce proteins with highly desirable improved characteristics, such as less aggregation. Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical formulations, because, for example, aggregates can be immunogenic.

Replacing amino acids can also change the selectivity of the binding of a ligand to cell surface receptors. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labelling.

Administration route

PDGF-AB or a functional derivative thereof can be administered to the individual or patient through various means, e.g., intravenously, intracardially, and intraperitoneally, and in a variety of formulations, e.g., with or without material that slowly releases the therapeutic agent, with or without matrix material that serves as scaffold, and with or without certain kinds of stem cells including cardiac stem cells. Various materials can be used as matrix material, including, but not limited to, collagen, nanofiber, and alginate. In some embodiments, the therapeutic agent can be administered with or without use of devices such as catheters, and with or without monitoring, e.g., via echocardiography. The therapeutic agent can be used to treat patients, including, but not limited to, patients with pathological conditions including, but not limited to, heart failure, myocardial infarction, coronary artery disease, and cardiomyopathy.

The invention also provides PDGF-AB nucleic acids or fragments thereof comprising a sequence of DNA or RNA, including one having an open reading frame that encodes the therapeutic polypeptide and is capable, under appropriate conditions, of being expressed as one of the therapeutic polypeptides of the instant invention. The term ‘nucleic acid’ also encompasses genomic DNA, cDNA, mRNA, splice variants, antisense RNA, RNAi, DNA comprising one or more single-nucleotide polymorphisms (SNPs), and vectors comprising the subject nucleic acid sequences. Also encompassed in this term are nucleic acids that are homologous or substantially similar or identical to the nucleic acids encoding the therapeutic proteins. Thus, the subject invention provides genes encoding a subject protein, and homologs thereof.

Polynucleotides or nucleic acids of the invention refer to polymeric forms of nucleotides of any length. The polynucleotides can contain deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives. For example, nucleic acids can be naturally occurring DNA or RNA, or can be synthetic analogs, as known in the art. Polynucleotides of the invention also encompass genomic DNA, genes, gene fragments, exons, introns, regulatory sequences, or regulatory elements, such as promoters, enhancers, initiation and termination regions, other control regions, expression regulatory factors, and expression controls; DNA comprising one or more single-nucleotide polymorphisms (SNPs), allelic variants, isolated DNA of any sequence, and cDNA; mRNA, tRNA, rRNA, ribozymes, splice variants, antisense RNA, antisense conjugates, RNAi, and isolated RNA of any sequence; recombinant polynucleotides, heterologous polynucleotides, branched polynucleotides, labelled polynucleotides, hybrid DNA/RNA, polynucleotide constructs, vectors comprising the subject nucleic acids, nucleic acid probes, primers, and primer pairs. Polynucleotides of the invention encompass modified nucleic acid molecules, with alterations in the backbone, sugars, or heterocyclic bases, such as methylated nucleic acid molecules, peptide nucleic acids, and nucleic acid molecule analogs, which may be suitable as, for example, probes if they demonstrate superior stability and/or binding affinity under assay conditions. They also encompass single-stranded, double- stranded, and triple helical molecules that are either DNA, RNA, or hybrid DNA/RNA and that may encode a full-length gene or a biologically active fragment thereof.

Polynucleotides of the invention include single nucleotide polymorphisms. Single nucleotide polymorphisms (SNPs) occur frequently in eukaryotic genomes. The nucleotide sequence determined from one individual of a species may differ from other allelic forms present within the population. The present invention encompasses such SNPs. The subject polynucleotides include those that encode variants of the polypeptides described in the instant specification. Thus, in some embodiments, a subject polynucleotide encodes variant polypeptides that include insertions, deletions, or substitutions compared with the polypeptides described herein. Conservative amino acid substitutions include serine/threonine, valine/leucine/isoleucine, asparagine/histidine/glutamine, glutamic acid/aspartic acid, etc.

Nucleic acids encoding the proteins and polypeptides of the subject invention may be cDNA or genomic DNA or a fragment thereof. The term "gene" shall be intended to mean the open reading frame encoding specific proteins and polypeptides of the subject invention, and intrans, as well as adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 20 kb beyond the coding region, but possibly further in either direction. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome. The subject polynucleotides are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a sequence or fragment thereof of the subject genes, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant," i.e,. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome. The invention provides plasmids, i.e., small, independently replicating pieces of extrachromosomal cytoplasmic DNA that can be transferred from one organism to another, comprising the therapeutic polynucleotides of the invention. Plasmids can become incorporated into the genome of a host or can remain independent. Artificially constructed plasmids are commonly used as cloning vectors. The invention also provides vectors, i.e., plasmids that can be used to transfer DNA sequences from one organism to another.

Expression vectors can be used to express the therapeutic gene products of the invention and typically comprise restriction sites to provide for the insertion of nucleic acid sequences encoding heterologous protein or RNA molecules.

The subject genes and gene fragments are useful in therapy to treat ischemic cardiac injury and other cardiac conditions. Expression vectors may be used to introduce the gene into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the subject gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g., plasmid; retrovirus, e.g., lentivirus; adenovirus; adenoassociated virus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.

An adenoviral vector preparation can be administered in combination with a vasoactive agent to enhance gene delivery. The vector can be delivered into a blood vessel such as an artery or into a tissue that is preinfused and/or co-infused with a vasoactive agent. Vasoactive agent, as used herein, refers to a natural or synthetic substance that induces increased vascular permeability and/or enhances transfer of macromolecules such as gene delivery vectors from blood vessels, e.g. across capillary endothelia. By making the vascular system more permeable to macromolecules or otherwise more amenable to the transfer of macromolecules into the capillary bed perfused by an artery, vasoactive agents can enhance delivery of these vectors to the targeted sites and thus effectively enhance overall expression of the transgene in the target tissue. Vasoactive agents that can be used in the instant invention include histamine; histamine derivatives and agonists, such as those that interact with histamine H receptors, which include, for example, 2-methylhistamine, 2-pyridylethylamine, betahistine, and thiazolylethylamine; vascular endothelial growth factors (VEGFs) and VEGF agonists (as described herein and in the cited references); and nitric oxide donors, such as sodium nitroprusside (SNP).

Cardiovascular event

Any interruption of blood flow to the heart that leads to injury or infarction of the heart is known as a cardiovascular event. Causes of a cardiovascular event may include arrhythmias, heart valve disease, hypertension, left ventricular hypertension, diabetes, cardiomyopathy (enlarged heart), certain types of cancers (malignancies), and carotid or coronary artery disease. Cardiovascular events can precede conditions such as acute myocardial infarction (AMI) or heart attack, and congestive heart failure. Typically, symptoms of a cardiovascular event include chest pain, tiredness, coughing, shortness of breath, odema, sweating, palpitations, and nausea.

Cardiac ischemia

Cardiac ischemia arises when the blood flow inside a coronary artery is restricted. The restricted blood flow is most commonly caused by plaque build-up on the inner walls or lining of the artery. Unable to obtain optimal amounts of oxygen and nutrients because of the reduced blood flow, cardiomyocytes function at sub-optimal levels and may die. The heart eventually is not able to pump blood efficiently. Episodes of cardiac ischemia can cause abnormal heart rhythms (arrhythmias), which can lead to either fainting or cardiac arrest and sudden cardiac death. Weakening of the heart muscle (cardiomyopathy) may also result. When a blood clot completely obstructs blood flow through an artery already narrowed by plaque, a heart attack may occur.

Ischemic cardiac injury is sustained by the myocardium as a result of cardiac ischemia. At the cellular level, ischemic cardiac injury is characterized by a central region of cellular necrosis, surrounded by a penumbra or "volume at risk" (VAR) where cells typically undergo a delayed death. A substantial portion of cardiomyocyte loss after myocardial infarction and reperfusion has been shown to arise from apoptosis within this region. In addition, further injury occurs as a result of recruiting inflammatory cells into the infarcted region. The inflammatory cells release chemotactic and cytotoxic cytokines and other inflammatory molecules, thus expanding the volume of injury. These forms of cell death and injury eventually may lead to heart failure.

Changes in gene expression after ischemia have been observed. Myocardial ischemia can induce transcription of the apoptosis regulator BAX gene, the early growth response factor Egr-1 and Egr-3 genes, and genes associated with cardiac muscle development such as those encoding a-myosin heavy chain (a-MHC) and fetal myosin alkali light chain (MLC).

Acute myocardial infarction

In some embodiments, methods are provided for treating an individual who has suffered an acute myocardial infarction (AMI) comprising administering to the individual a therapeutically effective dose of PDGF-AB or a functional derivative. In some embodiments, a method is provided for treating a condition caused by adverse ventricular remodelling, wherein the ventricular remodelling is caused by an AMI. In some embodiments, the ventricular remodelling is fibrosis. Thus, in some embodiments a method is provided for reducing adverse ventricular remodelling (e.g., ventricular fibrosis) in an individual who has suffered an AMI. The ventricular remodelling (e.g., ventricular fibrosis) is reduced relative to an amount of ventricular remodelling (e.g., an amount of ventricular fibrosis) in the absence of administration of PDGF-AB or a functional derivative (e.g., in comparison to an individual who was not administered the therapeutic).

Acute myocardial infarction (AMI) refers to infarction (damage or death) of heart tissue due to an acute, immediate blockage of one or more of the coronary arteries. Coronary arterial occlusion (blockage) due to thrombosis is the cause of most cases of AMI. This blockage restricts the blood supply to the muscle walls of the heart and is often accompanied by symptoms such as chest pain, heavy pressure in the chest, nausea, and shortness of breath, or shooting pain in the left arm. In an acute Ml, severe restriction of blood flow in the coronary conduit vessels leads to reduced oxygen delivery to the myocardium and a subsequent cascade of inflammatory reactions resulting in death (infarction) of myocardial tissue. Rapid restoration of blood flow to jeopardized myocardium can limit necrosis and reduce mortality. AMI leads to rapid death of myocytes and vascular structures in the supplied region of the ventricle. The loss of myocytes, arterioles, and capillaries in the infarcted area is irreversible, resulting with time in the formation of scarred tissue. AMI may be divided into ST elevation myocardial infarction (STEMI), diagnosed by elevation of the ST segment of the electrocardiogram, and non-ST elevation myocardial infarction (non-STEMI), diagnosed by absence of such electrocardiographic changes. STEMI may be treated with thrombolysis or percutaneous coronary

intervention (PCI). Non-STEMI may be managed with medication, although PCI is often performed during hospital admission.

Patients with AMI can be diagnosed by characteristically elevated levels of troponin, creatine kinase and myoglobin. Troponin levels are now considered the criterion standard in defining and diagnosing Ml. Cardiac troponin levels (troponin-T and troponin-l) have a greater sensitivity and specificity than myocardial muscle creatine kinase (CK-MB) levels in detecting Ml. Serum levels typically increase within 3-12 hours from the onset of chest pain, peak at 24-48 hours, and return to baseline over 5-14 days.

Creatine kinase comprises 3 isoenzymes, including creatine kinase with muscle subunits (CK-MM), which is found mainly in skeletal muscle; creatine kinase with brain subunits (CK-BB), predominantly found in the brain; and myocardial muscle creatine kinase (CK-MB), which is found mainly in the heart. Serial measurements of CK-MB isoenzyme levels were previously the standard criterion for diagnosis of Ml. CK-MB levels typically increase within 3-12 hours of onset of chest pain, reach peak values within 24 hours, and return to baseline after 48-72 hours. Levels peak earlier (wash out) if reperfusion occurs.

Sensitivity is approximately 95%, with high specificity. However, sensitivity and specificity are not as high as for troponin levels. Urine myoglobin levels rise within 1 -4 hours from the onset of chest pain in AMI. Although myoglobin levels are highly sensitive but not specific. The electrocardiogram (ECG) is an important tool in the initial evaluation and triage of patients in whom an Ml is suspected. It is confirmatory of the diagnosis in approximately 80% of cases. An ECG is obtained immediately if Ml is considered or suspected. In patients with inferior Ml, a right-sided ECG is recorded to rule out right ventricular (RV) infarct. Convex ST-segment elevation with upright or inverted T waves is generally indicative of Ml in the appropriate clinical setting. ST depression and T wave changes may also indicate evolution of Ml (non-ST-elevated Ml). Progression of Ml can be evaluated by performing ECGs serially, e.g. daily serial ECGs for the first 2-3 days and additionally as needed.

Imaging studies can be helpful for diagnosis of Ml, particularly if the diagnosis is questionable. An echocardiogram can identify regional wall motion abnormalities indicating tissue damage or death. An echocardiogram can also define the extent of the infarction and assess overall left ventricle (LV) and right ventricle (RV) function. In addition, an echocardiogram can identify complications, such as acute mitral regurgitation (MR), LV rupture, or pericardial effusion.

Myocardial perfusion imaging (MPI) utilizes an intravenously administered radiopharmaceutical to depict the distribution of blood flow in the myocardium. The radiopharmaceutical distribution in the heart is imaged using a gamma camera. Perfusion abnormalities, or defects, are assessed and quantified as to location, extent and intensity. Myocardial perfusion imaging can identify areas of reduced myocardial blood flow associated with infarct. Cardiac catheterization defines the patient's coronary anatomy and the extent of the blockage(s) via cardiac angiography.

AMI may be distinguished from chronic myocardial infarction using any appropriate method known in the art. In some embodiments, the presence of myocardial edema involving a disruption of the energy-regulated ionic transport mechanisms across the cell membrane after the Ml is indicative of AMI. The relatively large extracellular matrix of the developed scar allows gadolinium-based contrast media to accumulate, resulting in delayed enhancement (DE). T2-weighted cardiovascular magnetic resonance (T2-weighted CMR) is the cardiac magnetic resonance imaging (cMRI) modality that sensitively detects infarct-associated myocardial edema and may be used to differentiate acute from chronic Ml. In certain embodiments, a combination of DE and T2-weighted CMR is used to differentiate acute from chronic Ml.

Congestive Heart Failure (CHF)

In some embodiments, methods are provided wherein the incidence of congestive heart failure (CHF) or complications of CHF are reduced when PDGF-AB or a functional derivative is administered to the patient. The incidence of CHF or complications of CHF are reduced relative to the incidence of CHF or complications of CHF in the absence of administration of the therapeutic (e.g., in comparison to a patient who was not administered the therapeutic). The incidence of CHF may be reduced by at least 10% when a therapeutic having an anti-fibrotic effect is administered to a patient in comparison to a patient who was not administered the therapeutic. In further embodiments, the incidence of CHF may be reduced by at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or more when a therapeutic is administered to a patient in comparison to a patient who was not administered the therapeutic.

CHF may be a complication of AMI and results from a decline in the pumping capacity of the heart. CHF can also result from cardiac malformations, such as valve disease, or other disorders that damage cardiac tissue, e.g. cardiac myopathy. Due to the activation of one or more compensatory mechanisms, the damaging changes caused by CHF can be present and ongoing even while the patient remains asymptomatic. In fact, the compensatory mechanisms which maintain normal cardiovascular function during the early phases of CHF may actually contribute to progression of the disease, for example by exerting deleterious effects on the heart and circulation.

Myocardial remodelling is a complex process which accompanies the transition from asymptomatic to symptomatic heart failure, and may be described as a series of adaptive changes within the myocardium. Components of myocardial remodelling may include fibrosis, alterations in myocyte biology, loss of myocytes by necrosis or apoptosis, alterations in the extracellular matrix and alterations in left ventricular chamber geometry.

The diagnosis of congestive heart failure is most often a clinical one that is based on knowledge of the patient's pertinent medical history, a careful physical examination, and selected laboratory tests. Symptoms include dyspnoea (shortness of breath) which worsens upon lying supine, fluid retention and swelling in the lungs and extremities, e.g. with pulmonary rales or oedema in the legs.

Congestive heart failure is strongly suggested by the presence of cardiomegaly (enlarged heart) or pulmonary vascular congestion on chest X-ray. Electrocardiogram (ECG) may show anterior Q waves or left bundle branch block on the electrocardiogram. The echocardiogram is the diagnostic standard for identifying congestive heart failure. The patient may undergo two-dimensional echocardiography with Doppler flow studies. Radionuclide angiography or contrast cineangiography may be helpful if the echocardiogram is equivocal.

Preservation of Viable Cardiac Tissue and Reduction of Infarct Size

In some embodiments, methods are provided wherein the cardiac tissue is preserved from necrosis when PDGF-AB is administered to a patient suffering an AMI, in comparison to the amount of viable cardiac tissue in the absence of administration of the therapeutic (e.g., in comparison to a patient who was not administered a therapeutic). The amount of cardiac tissue preserved from necrosis can be increased at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 90%, or at least 95%. The increase in viable cardiac tissue can be determined by MRI or computerized tomography (CT) scan.

Methods are also provided herein to control or reduce myocardial infarct size. In other words, PDGF-AB or the functional derivative is effective at reducing infarct size in a patient that suffers a cardiovascular event, such as myocardial infarction.

To“control,” "improve" or "restore" as used herein means to reduce, reduce the incidence of, or prevent the progression of a cardiac related condition. In some cases, methods are provided wherein the infarct size of a patient is reduced when a therapeutic is administered to said patient, in comparison to the infarct size of a patient in the absence of administration of the therapeutic (e.g., in comparison to a patient who was not administered a therapeutic). The infarct size can be reduced at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 90%, or at least 95%. The reduction in infarct size can be determined by MRI and/or by voltage/conduction mapping.

In some embodiments, methods are provided wherein the cardiac function is preserved when PDGF-AB or a functional derivative is administered to a patient suffering an AMI, in comparison to the cardiac function of a patient suffering an AMI in the absence of administration of the therapeutic (e.g., in comparison to a patient who was not administered a therapeutic). Preservation of cardiac function can be determined by measuring ejection fraction using echocardiography, wherein the ejection fraction can be improved by at least 1 %, at least 3%, at least 5%, at least 7%, at least 10%, at least 12%, or at least 15%. Preservation of cardiac tissue can also be determined by measuring ejection fraction using MRI, wherein the ejection fraction can be improved by at least 1 %, at least 3%, at least 5%, at least 7%, at least 10%, at least 12%, or at least 15%, and/or the infarct size can be decreased by at least 1 %, at least 3%, at least 5%, at least 7%, at least 10%, at least 12% or at least 15%. Other methods of determining cardiac function are known in the art and include but are not limited to nuclear imaging, functional capacity, exercise capacity, New York Heart Association (NYHA) functional classification system, and myocardial oxygen consumption (MVO2). A skilled person would understand that an assessment of stroke volume and left ventricular end systolic volume are reflective of cardiac function.

Reduction in the Incidence of Ventricular Tachycardia

Methods are provided herein wherein the incidence of ventricular tachycardia in a patient is reduced when PDGF-AB or a functional derivative is administered to said patient, in comparison to the incidence of ventricular tachycardia in a patient who was not administered the therapeutic. The incidence of ventricular tachycardia can be reduced at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 90%, or at least 95%. The reduction in incidence of tachycardia can be determined by electrophysiology study or ambulatory electrocardiogram (Holter monitor or event monitor)..

Ventricular Fibrillation

In some embodiments, methods are provided for treating or preventing ventricular fibrillation in a patient in need thereof, comprising administering to the patient PDGF-AB or a functional derivative thereof. In some embodiments, the amount or degree of ventricular fibrillation is reduced relative to the amount or degree of ventricular fibrillation in the absence of administration of the therapeutic.

Ventricular fibrillation (VF) is a condition in which the heart's electrical activity becomes disordered. When this happens, the heart's ventricles contract in a rapid, unsynchronized way. The ventricles "quiver" rather than beat, causing the heart to pump little or no blood.

VF is life threatening and requires prompt treatment. Without medical treatment, collapse and sudden cardiac death can occur. Ventricular fibrillation (VF) may occur spontaneously with unpredictable timing and requires specialized tests to acquire an accurate diagnosis.

VF may be diagnosed using an electrocardiogram (ECG or EKG), e.g. a Holter Monitor. A Holter monitor is a small, portable machine that records the patient's ECG and is typically worn for 24 hours. This monitor may detect arrhythmias that might not show up on a resting electrocardiogram, which only records a heartbeat for a few seconds at rest.

VF may also be diagnosed using an event monitor -- this is a small monitor about the size of a pager that the patient can have for up to a month. Since the arrhythmia may occur at unpredictable times, this monitor records the abnormal rhythm when the patient signals that he or she is experiencing symptoms. An exercise stress or treadmill test also may be used to diagnose VF, by recording the electrical activity of the patient's heart during exercise, which differs from the heart's electrical activity at rest.

Another method of diagnosing ventricular tachycardia (VT) and predicting future VT or VF is through an electrophysiological study. In an electrophysiological (EP) study, physicians insert special electrode catheters - long, flexible wires - into veins and guide them into the heart. These catheters sense electrical impulses and also may be used to stimulate different areas of the heart. Physicians can then locate the sites that are causing arrhythmias. The EP study allows physicians to examine an arrhythmia under controlled conditions and acquire more accurate, detailed information than with any other diagnostic test. The EP study with programmed electrical stimulation has been proven to predict future VT/VF and sudden cardiac death.

VF can be monitored and measured by any one or more of the parameters described. In some embodiments, the incidence of VF can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 90%, or at least 95%, compared to incidence of VF in a patient who was not administered the therapeutic.

Sudden Cardiac Death

Sudden cardiac death (also called sudden arrest) is death resulting from an abrupt loss of heart function (cardiac arrest). The patient may or may not have diagnosed heart disease. The time and mode of death are unexpected. It occurs within minutes after symptoms appear. The most common underlying reason for patients to die suddenly from cardiac arrest is AMI due to coronary heart disease. Other types of arrhythmia can also cause cardiac arrest.

Most of the cardiac arrests that lead to sudden death occur when the electrical impulses in the diseased heart become rapid (ventricular tachycardia) or chaotic (ventricular fibrillation) or both. This irregular heart rhythm (arrhythmia) causes the heart to suddenly stop beating. Some cardiac arrests are due to extreme slowing of the heart, bradycardia. If a cardiac arrest was due to ventricular tachycardia or ventricular fibrillation, survivors are at higher risk for another arrest, especially if they have underlying heart disease.

Therefore, in some cases, methods are provided wherein the incidence of sudden cardiac death is reduced when PDGF-AB or a functional derivative thereof is administered to said patient, in comparison to the incidence of cardiac death in a patient who was not administered a therapeutic. The incidence of sudden cardiac death can be reduced at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 90%, or at least 95%.

Arrhythmia

Methods of the invention are contemplated to control arrhythmia by administering PDGF-AB or a functional derivative thereof. In some embodiments, a method is provided to reduce the incidence or risk of arrhythmia. The incidence or risk can be reduced at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 90%, or at least 95%.

An arrhythmia is an abnormal heart rhythm. In an arrhythmia the heartbeats may be too slow, too rapid, too irregular, or too early. There are many types of arrhythmias, including premature atrial contractions (early extra beats that originate in the atria (upper chambers of the heart), premature ventricular contractions (PVCs) (skipped heartbeat), atrial fibrillation (an irregular heart rhythm that causes the atria, the upper chambers of the heart to contract abnormally), atrial flutter (an arrhythmia caused by one or more rapid circuits in the atrium), paroxysmal supraventricular tachycardia (PSVT) (a rapid heart rate, usually with a regular rhythm, originating from above the ventricles), accessory pathway tachycardias (a rapid heart rate due to an extra abnormal pathway or connection between the atria and the ventricles), AV nodal reentrant tachycardia (a rapid heart rate due to more than one pathway through the AV node), ventricular tachycardia (VT) (a rapid heart rhythm originating from the lower chambers (or ventricles) of the heart), ventricular fibrillation (an erratic, disorganized firing of impulses from the ventricles), bradyarrhythmias (slow heart rhythms, which may arise from disease in the heart's electrical conduction system), and/or long QT syndrome (the QT interval is the area on the electrocardiogram (ECG) that represents the time it takes for the heart muscle to contract and then recover, or for the electrical impulse to fire impulses and then recharge). When the QT interval is longer than normal, it increases the risk for "torsade de pointes," a life-threatening form of ventricular tachycardia.

Symptoms of arrhythmia include chest pain, fainting, fast or slow heartbeat (palpitations), light-headedness, dizziness, paleness, shortness of breath, skipping beats, changes in the pattern of the pulse, and sweating. Arrythmias may be diagnosed by those of skill in the art using such methods as electrocardiogram, Holter monitor, event monitor, stress test, echocardiogram, cardiac catheterization, electrophysiology study (EPS), and head-up tilt table test.

The amount of a therapeutic effective to control arrhythmia may be an amount effective to reduce ventricular remodelling, e.g. in an animal model or during clinical trial.

Ventricular remodelling refers to the changes in size, shape, and function of the heart after injury to the left ventricle. The injury is typically due to AMI. In some embodiments, the ventricular remodelling is due to ventricular fibrosis caused by an AMI. The remodelling process is characterized by progressive expansion of the initial infarct area and dilation of the left ventricular lumen, with cardiomyocyte replacement by fibrous tissue deposition in the ventricular wall. Another integral component of the remodelling process is the development of neoangiogenesis within the myocardial infarct scar, a process requiring activation of latent collagenase and other proteinases.

In some embodiments, the therapeutically effective amount of PDGF-AB or the functional derivative may have an anti-fibrotic effect that reduces tissue remodelling or fibrosis. In some embodiments, the therapeutic having an anti-fibrotic effect reduces the activity of transforming growth factor-beta (TGF-b) signalling pathway. The extent of fibrosis and the effect of PDGF-AB can be assessed by routine means in the art including measurement of protein and/or gene expression levels of type I collagen, smooth muscle actin (aSMA) histological staining (ie Masson trichrome staining). Survival assessment

In one embodiment there is provided a method for improving the survival rate in a patient having suffering a cardiovascular event including administering to the patient PDGF-AB or a functional derivative thereof, as described above to the patient. The administration of this compound may improve, modify or influence various types of survival rate depending on the cardiovascular event and progression. In one embodiment the method improves disease - free survival (i.e. the period after curative treatment (i.e. where the disease is eliminated) when no disease can be detected) in the patient. In one embodiment the method improves the progression-free survival (i.e. the period after treatment when disease remains stable (i.e. does not progress)) in the patient.

The effectiveness of PDGF-AB or a functional derivative thereof may be assessed for propensity of improved survival by a survival analysis using the Kaplan- Meier method (as described in Example 1 herein and shown in Figure 1 ). The Kaplan- Meier method estimates the survival function from life-time data. The method can be used to measure the fraction of patients living for a certain amount of time after treatment. A plot of the Kaplan-Meier method of the survival function is a series of horizontal steps of declining magnitude which, when a large enough sample is taken, approaches the true survival function for that population. The value of the survival function between successive distinct sampled observations ("clicks") is assumed to be constant.

An important advantage of the Kaplan-Meier curve is that the method can take into account "censored" data- losses from the sample before the final outcome is observed (for instance, if a patient withdraws from a study). On the plot, small vertical tick-marks indicate losses, where patient data has been censored. When no truncation or censoring occurs, the Kaplan-Meier curve is equivalent to the empirical distribution.

In statistics, the log-rank test (also known as the Mantel-Cox test) is a hypothesis test to compare the survival distributions of two groups of patients. It is a nonparametric test and appropriate to use when the data are right censored. It is widely used in clinical trials to establish the efficacy of new drugs compared to a control group when the measurement is the time to event. The log-rank test statistic compares estimates of the hazard functions of the two groups at each observed event time. It is constructed by computing the observed and expected number of events in one of the groups at each observed event time and then adding these to obtain an overall summary across all time points where there is an event. The log-rank statistic can be derived as the score test for the Cox proportional hazards model comparing two groups. It is therefore asymptotically equivalent to the likelihood ratio test statistic based from that model.

Administration and dosage

In some embodiments, the PDGF-AB or a functional derivative thereof includes derivatives that are modified to enhance suitability for administration, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition. For example, but not by way of limitation, derivatives include compositions that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more nonclassical amino acids.

In still other embodiments, the PDGF-AB or a functional derivative thereof may be modified to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.

The PDGF-AB or a functional derivative thereof can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Pharmaceutically acceptable salts include, by way of non-limiting example, may include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotlnate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, triftuoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, a-hydroxybutyrate, butyne-1 ,4- dicarboxylate, hexyne-1 ,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-brornobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2- hydroxyethylsulfonate, methylsulfonate, naphthiene-1 -sulfonate, naphthalene-2- sulfonate, naphthiene-1 ,5-sulfonate, xylenesulfonate, and tartarate salts.

The term "pharmaceutically acceptable salt" also refers to a salt of the compositions of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethyiamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-0H- lower alkyiamines, such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert- butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2- hydroxyethyl)amine; N-methyl-D-giucamine; and amino acids such as arginine, lysine, and the like.

Further, PDGF-AB or a functional derivative thereof can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.

Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.

In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, ftour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

In one embodiment, PDGF-AB or a functional derivative thereof can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.

Where necessary, PDGF-AB or a functional derivative thereof also includes a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art.

The therapies outlined herein can be co-delivered in a single delivery vehicle or deliver/ device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection. The formulations comprising PDGF-AB or a functional derivative thereof may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).

In one embodiment, PDGF-AB or a functional derivative thereof is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.

Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transderrmal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In some embodiments, the administering is effected orally or by parenteral injection. The mode of administration can be left to the discretion of the practitioner, and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.

In certain embodiments, PDGF-AB or a functional derivative thereof may be administered orally or by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.

In specific embodiments, it may be desirable to administer locally to the area in need of treatment. In one embodiment, PDGF-AB or a functional derivative thereof is formulated in accordance with routine procedures as a composition adapted for oral administration to humans. Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time.

Selectively permeable membranes surrounding an osmotically active driving pharmaceutically active compounds described herein are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade. Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystaliine cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, etc., and mixtures thereof.

Dosage forms suitable for parenteral administration (e.g. intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

Cardiac catheterization is an example of a method of local delivery to the heart that involves the passage of a catheter (typically, a thin flexible tube) into the right or left side of the heart. Generally this procedure is performed to obtain diagnostic information about the heart or its blood vessels or to provide therapeutic interventions in certain types of heart conditions, such as in balloon angioplasty. Cardiac catheterization can be used to determine pressure and blood flow in the heart's chambers, collect blood samples from the heart, and examine the arteries of the heart with an X-ray technique called fluoroscopy. It can also be done on infants and children to examine or treat congenital heart defects. The technique has not been used in methods for local delivery of therapeutic agents to the myocardium for treating cardiac conditions, as disclosed in the instant invention.

The therapeutic agent may be delivered by introducing a catheter into either a vein or an artery, which is then advanced into a heart chamber and ultimately to an affected area in the myocardium, for example, areas that have sustained ischemic cardiac injury. In one embodiment of the invention, a catheter can be inserted into a femoral vein and then advanced from the femoral vein into the right atrium, and from the right atrium into the myocardium of the affected area; or from the right atrium to the right ventricle and into the myocardium of the affected area. In another embodiment, a catheter can be introduced into a femoral artery and advanced from the femoral artery into the aorta and left ventricle and then into the myocardium of the affected area; or :from the left ventricle to the left atrium into the myocardium of the affected area.

Cardiac catheterization has been described in detail elsewhere, for example, Bairn & Grossman (2000) Grossman's Cardiac Catheterization, Angiography, and

Intervention. 6th ed., Lippincott, Williams, & Wilkins.

A variety of catheters and delivery routes can be used to achieve intracoronary delivery, as is known in the art (see, for example, Textbook of interventional Cardiology (1994) E.J. Topol, ed., 2nd ed., W.B. Saunders Co.; Vascular Surgery (1989). Direct intracoronary (or graft vessel) injection can be performed using standard percutaneous catheter based methods under fluoroscopic guidance. Any variety of coronary catheter, or a Stack perfusion catheter, for example, can be used in the present invention. A variety of general purpose catheters and modified catheters can also be used in the instant invention. They are available commercially, for example, from Advanced Cardiovascular Systems (ACS), Target Therapeutics, Boston Scientific and Cordis. Where delivery to the myocardium is achieved by injection directly into a coronary artery, a number of approaches can be used to introduce a catheter into the coronary artery, as is known in the art. For example, a catheter can be conveniently introduced into a femoral artery and threaded retrograde through the iliac artery and abdominal aorta and into a coronary artery.

Alternatively, a catheter can be first introduced into a brachial or carotid artery and threaded retrograde to a coronary artery. The capillary bed of the myocardium can also be reached by retrograde perfusion, for example, from a catheter placed in the coronary sinus. Such a catheter can include a proximal balloon to prevent or reduce anterograde flow as a means of facilitating retrograde perfusion. A therapeutic composition of the invention can be adapted to be delivered to the cardiac area by catheter.

The therapeutic agent can be administered locally at the time of cardiac surgery, while treating a cardiac event, or while performing a diagnostic procedure. The therapeutic agent can also be delivered in anticipation of events that can result in ischemic cardiac injury or other cardiac conditions. In this regard, the therapeutic agent serves to prevent ischemic cardiac injury or other cardiac conditions. For example, the therapeutic agent can be delivered a plurality of days prior to non-cardiac surgery, complex percutaneous revascularization, or complex cardiac surgery. The therapeutic agent can also be delivered to donor hearts prior to cardiac transplantation to prevent any ischemic cardiac injury or other cardiac conditions that may arise during the entire transplantation process (explantation, transport, implantation). The therapeutic agent can also be useful in providing myocardial protection to patients with diffuse, nonrevascularizable coronary artery disease. For these patients, a life-long regimen of the therapeutic agent may be needed.

Therapeutic compositions have also been delivered to the heart by direct injection into the cardiac muscle (myocardium). Direct injection may be performed during open heart surgery. Surgical visualization of the heart facilitates accurate implantation into the myocardium. Direct injection may also be performed without surgical access to the heart by injecting the therapeutic composition through the chest wall, guided by the use of an imaging procedure. Any known imaging technique which provides information in real time is suitable for use with the methods disclosed herein of injecting therapeutic compositions of the invention into the myocardium. For example, echocardiography and other real-time imaging techniques can be used to guide direct injection.

In an embodiment, the therapeutic agent is delivered to the heart by direct intracoronary injection using standard percutaneous catheter-based methods under fluoroscopic guidance. The injection can be made substantially (such as at least 1 cm) into the lumen of the coronary arteries or one or more saphenous veins or internal mammary artery grafts or other conduits delivering blood to the myocardium. Any coronary artery can be injected. Any suitable variety of coronary catheter, or a Stack perfusion catheter, can be used in accordance with the present invention.

In some embodiments, the instant invention employs a catheter suitable for injecting therapeutic agents into specific parts of the heart, for example, the VAR region, presumptive VAR region, pericardial space, myocardium, or pericardium. Magnetic resonance (MR) may be used to precisely guide delivery of therapeutic agents to defined locations within the infarct or elsewhere in the heart. A catheter as described by Karmarkar et al., Magnetic Resonance in Medicine (2004) 51 :1 163-1 172 or by U.S. Patent No. 6,304,769, can be used. The components of such a catheter can be arranged to form a loopless RF antenna receiver coil that enables tracking by magnetic resonance imaging (MRI). Different types of RF receiver antennas (for example, loop, loopless, opposed solenoid, etc.) can be used to enable active tracking. Myocardial delayed-enhancement (MDE) imaging can identify the infarcted myocardium, and real time MRI can be used to guide catheterization. The distal end of the catheter can be seen under MRI with a bright signal at the distal tip of the catheter. Using MRI tracking, the catheter can be steered into position and the needle advanced to inject the therapeutic agent intramyocardially or into the pericardial space or into any other desired location in the heart.

Using the catheterization delivery methods of the instant invention, the therapeutic agent can be delivered to specific areas of the heart. The therapeutic agent can be delivered to the injury site, the VAR region, or presumptive VAR region. In other embodiments, the therapeutic agent is delivered to the pericardial space. The pericardial space may potentially serve as a convenient, safe, and effective drug delivery reservoir that might be used to administer therapeutic agents to the heart. The pericardial space can be accessed by transthoracic devices (for example, needles or catheters) or by a transventricular approach using a catheter. The pericardial space can also be accessed transvenously via the right auricle. A therapeutic composition of the invention can also be adapted to be delivered to the cardiac area by direct injection.

The dosage of PDGF-AB or a functional derivative thereof as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject's general health, and the administering physician's discretion. Any agent described herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional therapeutic agent, to a subject in need thereof. In various embodiments any agent described herein is administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 1 1 hours 35 to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.

In various embodiments any agent described herein is administered less than 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after a cardiovascular event or a myocardial infarction.

The amount of PDGF-AB or a functional derivative thereof that is admixed with the carrier materials to produce a single dosage can vary depending upon the subject being treated and the particular mode of administration. In vitro or in vivo assays can be employed to help identify optimal 5 dosage ranges.

In general, the doses that are useful are known to those in the art. For example, doses may be determined with reference Physicians' Desk Reference. 66th Edition. PDR Network; 2012 Edition (December 27, 2011 ). In some embodiments, the present invention allows a patient to receive doses that exceed those determined with reference Physicians' Desk Reference or doses that are below the approved label amount.

The dosage of PDGF-AB or a functional derivative thereof can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic} information about a particular subject may affect dosage used. Furthermore, the exact patient dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.

The dosage regimen utilizing PDGF-AB or a functional derivative thereof can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the patient; and the specific compound of the invention employed. These pharmaceutical actives can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, these actives can be administered continuously rather than intermittently throughout the dosage regimen.

The therapeutics disclosed herein can be dosed at a total amount of about 0.2 to about 2400 mg per day. The dosage can be divided into two or three doses over the day or given in a single daily dose. Specific amounts of the total daily amount of the therapeutic contemplated for the disclosed methods include about 0.2mg, 0.5mg, 5mg, 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 267 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 534 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1068 mg, about 1 100 mg, about 1 150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1335 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1869 mg, about 1900 mg, about 1950 mg, about 2000 mg, about 2050 mg, about 2100 mg, about 2136 mg, about 2150 mg, about 2200 mg, about 2250 mg, about 2300 mg, about 2350 mg, and about 2400 mg.

In some embodiments, the patient or individual is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In some embodiments, the subject and/or animal is a transgenic animal comprising a fluorescent cell.

In some embodiments, the patient or individual is a human. In some embodiments, the human is a paediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. In other embodiments, the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from 25 about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from 30 about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old. In other embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the nonhuman animal is a livestock animal. The invention provides kits that can simplify the administration of any agent described herein. An exemplary kit of the invention comprises any composition described herein in unit dosage form. In one embodiment, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anaesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein. In one embodiment, the kit comprises a PDGF-AB or a functional derivative thereof.

An "effective amount," when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a cardiovascular event, e.g. myocardial infarction. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the patient features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Examples

Example 1

PDGF-AB restores cardiac function in a porcine model of ischaemia reperfusion (l/R) injury

This study was conducted to determine the role of PDGF-AB in a model of ischaemia reperfusion (l/R) injury (myocardial infarction) in a large animal model which more closely resembles the heart of humans. For this purpose, the inventors have translated this therapy into the clinically-relevant porcine model of ischaemia reperfusion (l/R) injury. Methods

All procedures in this study were approved by the Royal North Shore Hospital Animal Ethics Committee. Briefly, Landrace swine (25-30kg) underwent experimental Ml followed by intravenous rhPDGF-AB (n=12) or placebo (n=1 1 ) for 7 days. Cardiac magnetic resonance imaging (CMR) and micromanometer conductance catheter pressure volume (PV) loop analyses were conducted at days 2 and 1 month to assess structural and functional changes. Animals were euthanased at 1 month. The

experimental model is represented schematically in Table 1. For all procedures, anesthesia was induced with intramuscular ketamine and propofol, and maintained with 0.5-2.5% inhaled isoflurane.

Table 1

Table 1 : Study Outline and randomization. rh-PDGF-AB = recombinant human Platelet- Derived Growth Factor AB; LVEF = left ventricular ejection fraction; cMRI = cardiac Magnetic Resonance Imaging; 2D = 2 dimensional.

Myocardial Infarction

Ml was induced in female Landrace mini-pigs by endoluminal balloon occlusion of the left anterior descending coronary artery and distal to the first major diagonal for 90 minutes, followed by complete reperfusion and recovery. Animals were then randomized to intravenous mini-pump infusion of rhPDGF-AB (65pg/kg) or placebo (n=1 1 /group) for 7 days. All interventions/analyses were blinded to randomization.

Preparation and Delivery of rhPDGF-AB rhPDGF-AB was commercially synthesized by Peprotech (Cat# 120-28, Lot# 1 1 1 1 S396, Peprotech, London, UK). rhPDGF-AB / placebo was delivered at a fixed infusion rate of 65mcg/kg for 7 days via a 2mL1 ALZET (Durect, CA, USA) osmotic mini pump inserted into the right external jugular vein. rhPDGF-AB was detected in the plasma on Day 2 of treated animals using a PDGF AB Fluman Elisa kit (Abeam).

Cardiac MRI

CMR imaging was performed using a 3 Tesla Philips MRI scanner (Ingenia, Philips, Netherlands). After appropriate planning, a short axis cine stack was acquired using a balanced turbo field echo sequence (bTFE) with the following parameters: time to echo (TE) 1.5ms, repetition time (TR) 3.0ms, flip angle 45°, 8mm slice thickness, 0.08mm overlap, 30 phases per cardiac cycle with 125% sampling. Endocardial contours were drawn in a semi-automated fashion using cvi42 version 5.2.2 (Circle Cardiovascular Imaging Inc, Calgary, Canada) according to Society of Cardiac Magnetic Resonance guidelines. Papillary muscles were excluded from the left ventricular volume.

Electrophysiology Study Protocol

Programmed electrical stimulation (PES) was performed at 1 month to identify animals at risk of sudden cardiac death (SCD) post-MI. PES was performed at the right ventricular apex at twice diastolic threshold using programmed stimulation. A drive train (S1 S1 ) of 8 beats at 400ms was followed by up to 4 extrastimuli delivered one at a time. Initial extrastimulus was delivered at a coupling interval of 300ms then decremented by 10ms until ventricular refractoriness. There was no set lower limit for the shortest permissible extrastimulus-coupling interval. Sustained monomorphic VT ventricular tachycardia (VT) was defined as cycle length (CL) >200ms lasting 10sec (or resulting in hemodynamic instability) induced by <4 extrastimuli was considered a positive result.

Assessment of Vascular

and Collagen Heterogeneity

Paraffin-embedded biopsies will be sectioned into 4-pm slices. Subsequently, the slides are stained for determination of vascular density, collagen density, and tissue heterogeneity. Each tissue section is to be digitally scanned (Nanozoomer, Hamamatsu, Japan) at 20x objective magnification and imported into Image J analysis software.

Arterioles and capillaries are expressed as number per millimeter squared. A validated algorithm is used to calculate the heterogeneity index of viable (surviving) myocardium of each histological specimen excised from remote infarct (normal myocardium), proximity scar border (normal myocardium), scar border (scarred myocardium) and dense scar regions.

Micromanometer conductance catheterization

Haemodynamic measurements were performed at endpoint (day 28) before euthanasia. The right femoral artery was cannulated and a 5-Fr micromanometer conductance catheter (Ventri-cath-510S, Millar, Houston, Texas, USA) that was advanced across the aortic valve and into the LV. Baseline end-systolic and end- diastolic pressures and volumes were recorded. Transient LV preload reduction was achieved by inflation of an 18-20 mm balloon catheter (CRE Pulmonary balloon, Boston Scientific) in the IVC. Load-independent measures of cardiac function including slopes of ESPVR, PRSW and EDPVR were obtained during inferior vena cava occlusion. Ves and V_w were calculated as volume-axis intercepts of the ESPVR and PRSW relationships respectively. The time constant of fall in isovolumic LV pressure (tau) was studied in isovolumic beats after ejection.

Gross-examination and histopathological analysis

On day 28, hearts were arrested with 75-150mg/kg potassium chloride and excised for subsequent analysis. For histological studies, the hearts and tissue samples from the lungs, liver, spleen, kidneys and ovaries were harvested and fixed in 10% neutral buffered-formalin followed by paraffin embedding. For AFM, hearts were quickly snap frozen with liquid nitrogen and stored at -80°C until process.

2-dimensional vascular density

Neo-angiogenic effects were assessed by immunohistochemical staining to determine capillary density. Briefly, paraffin embedded sections were deparaffinized, rehydrated and antigen retrieved in 10mM sodium citrate buffer with 0.05% Tween-20. Sections were blocked with 5% goat serum in PBS/Tween-20 0.05% (blocking buffer) for 1 hr. Primary antibodies for von Willebrand Factor (vWF, 1 :500, Dako, #A0082) and alpha-smooth muscle actin (a-SMA, 1 :500, Dako, #M0851 ) were then applied to the sections and incubated for 1 hr at room temperature. Following this, sections were washed and stained with appropriate secondary antibodies - goat anti-mouse AlexaFluor-488 (1 :500, Life Technologies, A1 1029) or goat anti-rabbit AlexaFluor-594 (1 :500, Life technologies, A1 1037). After counterstained with DAPI (1 pg/ml), sections were mounted and examined. Each tissue section was digitally scanned (Nanozoomer, Hamamatsu, Japan) at 20x objective magnification and imported into Image J analysis software. Arteriolar and capillary density were represented as a frequency per millimeter squared. Capillaries were defined as vessels stained with vWF; > 4 DAPI positive cells in the endothelium and arterioles were considered to be structures containing vWF co localized with a-SMA.

3-dimensional vascular density

Light sheet microscopy was utilized to employ a 3D method of assessing vasculogenesis. Tissue samples (10 mm) from the infarct zones were optically cleared using the CUBIC (Clear, Unobstructed Brain / Body Imaging Cocktails and Computational analysis) R1 a method. Briefly, tissue sections were fixed overnight in 4% paraformaldehyde at 4^QC and immersed in CUBIC Reagent 1 A at 37^QC for 3 days, with fresh Reagent 1 A replaced daily. Samples were then washed in PBS and blocked in PBS-containing 0.5% Triton-X100-10% and 10% goat serum overnight at 4^QC., and then incubated in with anti-a-SMA, diluted 1 :250 in blocking buffer at 4^QC for 5 days with gentle rocking. Tissue was washed 3x1 hour in PBS and incubated in AlexaFluor488 secondary antibody (1 :500) for 2 days, washed in PBS 1 hour, and incubated with DAPI (10mM) for 1 hour. Samples were then transferred to CUBIC Reagent 2 at 37^QC for 24 hours. Samples were mounted on metal pins and imaged using the light sheet microscope (Z.1 , Zeiss) whilst immersed in CUBIC Reagent 2 and imported into IMARIS 9.2.0 for analysis. A total of 10 images per pig were analyzed for arteriolar density, branching points and diameter, using IMARIS quantification. Initially, background noise was reduced through use of the volume blend tool and applied equally across all samples. Following this, the filament trace mode was utilized using a manual trace and autopath (no loop) setting. The draw tool was set to analyze the cone with an adaptive diameter setting. Correction parameters were enabled to account for vessel diameter and vessel centre discrepancies by checking both“automatic centre” and“automatic diameter”. Full traces were conducted for each image ensuring that all vessels were accurately traced across the x, y and z axes. Excel data generated by the program allowed a range of comparisons and statistical analyses. In this case, we analyzed mean diameter, branching points and vessel numbers. Histological analysis

To quantify fibrosis, myocardial sections were stained with Gomori trichrome for collagen, digitally scanned (Nanozoomer, Hamamatsu, Japan) at 20x objective magnification and imported into an in-house histology analysis software. The software was able to differentiate between viable myocardium and collagen within the scar, based on a threshold algorithm utilizing the red and blue coloring of pixels. The quantity of viable myocardium and collagen was calculated as the percentage of red or blue color-stained pixels, respectively, expressed per unit area of the myocardial section. Scar heterogeneity, a well-recognized arrhythmogenic substrate for re-entrant ventricular arrhythmias, was assessed using a previously validated heterogeneity index. Small values in heterogeneity index indicate multiple small clusters of tissue separated by small distances and large values indicating the opposite. A heterogeneity map of local myofiber clustering portrays a visual representation of the intermingling of viable myocytes and collagen, with color blue to red indicating increased clustering, and yellow showing intermediate clustering.

Polarized light microscopy

To assess the contribution of collagen subtypes I and III to the myocardial scar, 10 infarcted hearts were sectioned and stained with 0.1 % picrosirius red and their relative scar regions imaged using polarized light microscopy. This was carried out on an Olympus VS120 Slide Scanner fitted with a polarized light filter. Collagen subtype was assessed visually by color: orange/red representing type I collagen and yellow or green representing type III collagen.

Second Harmonic Generation (SHG) microscopy

SHG microscopy was used to assess collagen fiber length and orientation. Picrosirius red stained tissue sections of myocardial scar region were imaged using a two-photon microscope (Leica TCS SP8 MP) with a laser excitation wavelength of 880nm, and images collected of the emitted wavelengths 440nm +/- 20nm. Blinded analysis was then performed using Image J software by measuring the length of fibers using the line tool and orientation using the angle tool. Fifty fibers from each image and three images (456pm x 456pm field) from each sample were measured. Atomic Force Microscopy (AFM) characterization

To investigate the mechanical properties of vehicle and rhPDGF-AB-treated hearts, AFM measurements of elasticity in the infarct and border regions were performed. The AFM was mounted on an anti-vibrational table (Flerzan) and operated within an acoustic isolation enclosure (TMC, USA). Tissue samples at room temperature were immersed in PBS, placed in an MFP-3D AFM (Asylum Research) and indented using a SiNi cantilever (Budget Sensors, Bulgaria) having a nominal spring constant of 0.06 N/m, which was checked by a thermal calibration. Samples were indented at a velocity of 2 pm/s and the loading force was kept constant at 2 nN. Force- indentation plots made on a 10x10 pm area were fitted to a Flertz cone model to determine the Young’s elastic modulus, E.

Three-dimensional (3D) human cardiac mesenchymal cell microtissues

Fluman cardiac PDGFRa+ cells were used to make 3D cardiac mesenchymal cell microtissues. Fleart-dyno (96-well device used for functional screening of cardiac organoids) culture inserts were fabricated using standard SU-8 photolithography and polydimethylsiloxane (PDMS) molding practices. For each tissue, a 3.2 pL mixture, containing 32,000 cells, 3.3 mg/mL collagen I and 22% (v/v) Matrigel (growth factor- reduced), was prepared on ice and pipetted into the cell-culture inserts. The mixture was then gelled at 37°C for 75 min prior to the addition of medium (150 pL/tissue) with a complete media change 2 days later. rhPDGF-AB (100 ng/mL) or vehicle was added to the wells daily from day 0 to day 3. The Fleart-Dyno design facilitates the self-formation of tissues around in-built PDMS exercise poles (designed to deform ~0.07 pm/pN). The tissues were imaged on days 0, 1 , 2, 3 and 5 using a Nikon Confocal Microscope at 37°C and the tissue width and distance between the poles were measured using Image J. The endogenous tension was calculated by the difference in the distance between the poles relative to day zero in pm multiplied by 14 pN/pm.

Statistics

Continuous data are presented as mean ± SEM. AFM and organoid data are presented as mean and 95% confidence intervals. Normality was assessed using the Shapiro-Wilk test. Statistical comparisons were performed by unpaired Student’s t-test or one-way ANOVA followed by Holm-Sidak post hoc test to adjust for multiple comparisons. Survival analyses were performed using the Kaplan-Meier method and the log-rank test was applied to determine significance between overall survival between the two groups. P-values <0.05 were considered statistically significant. All analyses were performed using SigmaPlot 12.5 software.

Results

Administration of PDGF-AB improves survival and cardiac function

As shown in Figure 1 , the administration of PDGF-AB recombinant protein over 5 days has a highly significant positive impact on preserving the survival of pigs subjected to a model of ischaemia reperfusion (l/R) injury (myocardial infarction) tracked over 28 days. Compared to placebo, rhPDGF-AB significantly improved 1 -month survival by 40% (p=0.041 ).

This finding extends to the effect of PDGF-AB on cardiac function as measured by MRI (Figure 2) whereby PDGF-AB improves left ventricular ejection fraction by 1 1.5% (Figure 3 a-b), stroke volume (Figure 4 a-b), and left ventricular end systolic volume (Figure 5 and Figure 6b). Figure 5 demonstrates the effect of PDGF-AB on indexed left ventricular end-diastolic volume (iLVEDV) and indexed left ventricular end- systolic volume (iLVESV) over time. There is a remarkable 1 1.5% increase in LVEF and reduced iLVESV by -0.3ml/kg at 1 month in the PDGF group compared to placebo.

Figure 7 represents an electrophysiology study that induces arrhythmia in paced hearts and is a gold standard measurement of risk of sudden cardiac death. As shown in Figure 7, of those individuals treated with PDGF-AB, only one out of 5 tested presented a positive score reflective of increased risk of sudden death, compared to 3 in the control group. The control group also presented earlier in response to drive train stimuli or extra stimuli. This data indicates that individuals treated with PDGF-AB are

associated with less risk of sudden cardiac death. rhPDGF-AB improves myocardial contractility and energetics with minimal effect on ventricular compliance

Despite cMR being the gold standard for cardiac functional assessment, the derived measures remain load-dependent. Therefore, the inventors performed pressure-volume (PV) loop studies at the day 28 post-MI endpoint to determine intrinsic (load independent) cardiac contractility (Figure 8). Steady-state index of systolic function, the maximal rate of pressure change during systole (dP/dtmax), increased with rhPDGF-AB treatment at day 28 [rhPDGF-AB: 1352 ± 47 mmFIg/s vs vehicle: 1 107 ± 83.3 mmFIg/s; p=0.015] (Figure 8A), indicating improved cardiac contractility.

Contractility assessed during inferior vena cava (IVC) occlusion showed greater contractile function in the rhPDGF-AB group at the day 28 endpoint, including: a steeper slope of the end-systolic pressure-volume relationship (ESPVR) [rhPDGF-AB: 2.48 ± 0.2 mL/mmFIg vs vehicle: 1.64 ± 0.2 mL/mmFIg; p=0.03] (Figures 8B and 8C), and significantly higher preload recruitable stroke work (PRSW) slope [rhPDGF-AB: 72 ± 5 mmFIg vs vehicle: 46 ± 4.9 mmFIg; p=0.004] (Figure 8D), confirming improved

myocardial load-independent contractility with rhPDGF-AB treatment after Ml.

When ascertaining the effects on relaxation kinetics, it was found that the maximal rate of pressure decay (dP/dtmin) was significantly increased in the rhPDGF- AB-treated animals [rhPDGF-AB: -1787 ± 106 mmFIg/s vs vehicle: -1328 ± 96 mmFIg/s; p=0.01 1 ], Figure 8E. Flowever, linear EDPVR slopes [rhPDGF-AB: 0.39 ± 0.06 mL/mmFIg vs vehicle: 0.39 ± 0.04 mL/mmFIg; p=0.93] and tau (time constant of isovolumic LV pressure fall) were comparable between the groups [rhPDGF-AB: 41 ±

1.9 msec vs vehicle: 51 ± 6.5 msec; p=0.08] (Figures 8F and 8G). Together, these data suggest that the rhPDGF-AB group had improved relaxation without altered LV distensibility at the day 28 endpoint. rhPDGF-AB promotes vasculogenesis and arteriolar collateralization post-MI

The inventors next sought to determine the effects of rhPDGF-AB on the post-MI vasculature. Myocardial sections (5 pm thick) immunostained with vWF and a-SMA were assessed by investigators blinded to the experimental groups. Although the peri- infarct capillary number (vWF+) was significantly increased in the rhPDGF-AB-treated hearts [rhPDGF-AB: 101 ± 16 /mm2 vs vehicle: 56 ± 9 /mm2; p=0.001 ] (Figure 9A), there was no difference in peri-infarct arteriolar density (a-SMA+) between the groups [rhPDGF-AB: 5 ± 3 /mm2 vs vehicle: 5 ± 2 /mm2; p=0.37) (Figure 9B). Standard 2D microscopy vessel quantification may be confounded by inadvertently missing vessels out of the plane of section. The inventors therefore used 3D light sheet microscopy to better understand rhPDGF-AB effects on arteriolar vasculature. 3D tissue (5-10 mm thick) from the peri-infarct zone of experimental animals was cleared using the CUBIC R1 a protocol (Figure 9C), immunostained with a-SMA and then imaged using light sheet microscopy (Figure 9D). Individual filament tracing using IMARIS software was used to identify the origin, branching and terminal points of arterioles. Quantitative analyses showed that rhPDGF-AB enhanced arteriogenesis [rhPDGF-AB: 1919 ± 399 /field vs vehicle: 500 ± 557 /field; p=0.03] (Figure 9E). It also promoted arteriolar branching [rhPDGF-AB: 220 ± 72 /field vs vehicle: 23 ± 20 /field; p=0.03] (Figure 9F) with reduction in mean arteriolar diameter (15 ± 0.1 pm vs 18 ± 0.8 pm; p=0.02) (Figure 9G), suggesting budding and de novo synthesis of microvessels. rhPDGF-AB promotes collagen fiber alignment and wound healing without reduction in overall scar size

Unexpectedly in this study, treatment with rhPDGF-AB did not reduce scar size as assessed by LGE (Figure 10A-B) despite (more importantly) significantly improving LV function. cMR findings were confirmed histologically by quantifying the collagen content of the infarct, border and remote zones using Gomori trichrome staining (Figure 10C). As such, no histological difference in scar size was observed between the two groups. The organization of collagen fibers in the scar may be an important determinant of the anisotropic mechanical properties of the scar. Flence, to determine collagen fiber organization within the peri-infarct zones of experimental animals, the inventors used second-harmonic generation (SFIG) 2-photon microscopy (a label-free collagen imaging technique), Gomori trichrome staining and polarized light microscopy. The inventors found that rhPDGF-AB treatment resulted in directional organization of collagen fibers (Figure 10D). To quantify this observation, the orientation of single collagen fibers relative to one another from representative SFIG images was measured. In the rhPDGF- AB group, over 80% of the collagen fibers were aligned within 10° of the mean orientation with an entire range of distribution of 35° (Figure 10E), demonstrating uniform alignment and therefore increased scar anisotropy.

There is a known changing combination of collagen subtypes within the healing infarcted heart with increasing collagen III expression in the early reparative phase and more collagen I in the later maturation phase. To assess collagen subtypes, we stained peri-infarct sections with picrosirius red and viewed them with polarized light microscopy. rhPDGF-AB treatment resulted in primarily Type I collagen (orange) by day 28 after Ml, suggesting increased scar maturation compared to vehicle treatment, in which Type III collagen (green) was predominant (Figure 10F). To further explore the effects of rhPDGF-AB treatment on the matrix of treated hearts, the inventors next performed nano-indentation using atomic force microscopy (AFM) on LV samples from the infarct and remote zones. There was no significant difference in the stiffness of rhPDGF-AB treated hearts (Young’s elastic modulus, E) compared to vehicle (infarct zone: E = 9 ± 8.3 kPa vs 7 ± 5.5 kPa; p=0.76; border zone: E = 37 ± 18.7 KPa vs 26 ± 10 kPa; p=0.23) at day 28 (Figure 10G). The border zone had an elastic modulus very similar to that of cardiomyocytes but lower than previously reported values in the scar in smaller animal models of Ml, in which the elastic modulus was similar to native tissue (~E = 18 kPa) distal to the ligation.

This indicated that the changes in the scar mainly affected the anisotropy and bulk properties of the scar or, because these measurements were performed on frozen, thawed samples, contributions from the live cells were missing. Given the cardiac functional and collagen distribution results detailed above, we hypothesized that rhPDGF-AB may have been changing the scar environment through mesenchymal stromal cells. To test this, the inventors generated microtissues from human cardiac mesenchymal cells sorted for the PDGFRa+/CD90+/CD31 - fraction (data not shown). rhPDGF-AB treatment increased the rate of tissue condensation and tensile force generation (Figure 10H). Interestingly, these effects occurred mainly at days 0-3, which is consistent with the short duration of rhPDGF-AB treatment (days 0-7) after Ml in pigs having a sustained benefit at day 28. Taken together, these data suggest that rhPDGF- AB promotes post-MI wound healing by accelerating scar maturation and contractile force transmission without increased stiffness of the scar matrix. rhPDGF-AB promotes early survival and reduces arrhythmogenicity

In our study, 9 of 36 (25%) animals died during balloon occlusion or reperfusion (before randomization). After randomization, there were 6 deaths (1 in the rhPDGF-AB group and 5 in the vehicle group) over the course of the study. Post-mortem

examination revealed that a femoral bleed was the cause of death in 2 animals (1 in the rhPDGF-AB group and 1 in the vehicle group). All 4 remaining deaths occurred in the vehicle-treated group, within 1 week of Ml. These deaths were classified as sudden cardiac deaths (SCD) (after exclusion at necropsy of other causes such as HF, a cardiac bleed or myocardial wall rupture), suggesting that rhPDGF-AB improved early survival from arrhythmia. The Kaplan-Meier curve shows a 40% improvement in early survival from presumed arrhythmic deaths in the rhPDGF-AB-treated group (log rank, p=0.04) compared to vehicle (Figure 1 ).

Programmed electrical stimulation (PES) has been shown to strongly correlate with spontaneous arrhythmias. PES was performed in a subgroup of animals at the day 28 endpoint to identify animals at risk of SCD post-MI. PES revealed a significant reduction in inducible ventricular tachycardia (VT) [rhPDGF AB: 1/5 (20%) vs. vehicle: 3/3 (100%); p=0.04] associated with reduced number of extrastimuli required for VT induction (Figure 1 1 A) in the rhPDGF-AB group. This suggests abatement of late arrhythmogenicity following rhPDGF-AB treatment. The high rates of arrhythmogenicity seen with placebo-treated animals are consistent with previous observations in porcine models.

In lieu of these arrhythmic findings, the inventors investigated whether

differences in the anatomic substrate for ventricular arrhythmias was present between the groups. In rhPDGF-AB treated animals, the architectural organisation of myofibers interspersed with collagen were less heterogeneous (lower heterogeneity index) than placebo-treated animals in in both scar core [rhPDGF-AB: 0.49 ± 0.01 /mm2 vs vehicle: 0.56 ± 0.01 /mm2; p=0.048] and scar border regions [rhPDGF-AB: 0.49 ± 0.01 /mm2: vs vehicle: 0.51 ± 0.01 /mm2; p=0.03] (Figure 1 1 B). Conversely, vehicle-treated hearts showed considerable heterogeneity of putative conducting myofibers colocalised to collagen, which provides the substrate for a re-entry circuit and a milieu for ventricular arrhythmias and subsequent SCD (Figure 1 1 C). Together, these results suggest that rhPDGF-AB decreases ventricular arrhythmias and SCD by decreasing the

heterogeneity of post-MI scar composition.

To investigate potential mechanisms by which PDGF-AB could aid force transduction we treated human cardiac mesenchymal cell microtissues with PDGF-AB and demonstrated that proteins (including Tenascin C, ITGB1 and aSMA) known to influence mechanotransduction are up-regulated by PDGF-AB (Figure 12A-B).

The inventors herein demonstrate that intravenous infusion of rhPDGF-AB after reperfusion of the occluded coronary artery that caused myocardial infarction (Ml): 1 ) significantly improves left ventricular ejection fraction (LVEF) with reduced left ventricular (LV) end-systolic volume; 2) increases scar anisotropy (high fiber alignment) without affecting overall scar size or stiffness; 3) promotes angiogenesis and arteriolar collateralization; 4) improves cumulative survival by reducing early SCD; and 5) decreases inducible ventricular arrhythmias by attenuating myocardial scar

heterogeneity.

These results demonstrate that PDGF-AB recombinant protein has a highly significant benefit on key clinical parameters of cardiac function. These findings are significant in so far as being the first demonstration of the therapeutic potential of PGDF-AB for the treatment of conditions associated with ischaemia injury, such as Ml. Significantly, the data demonstrates clinical relevance in a large animal model.

Claims

1. A method of improving cardiac function in an individual having sub optimal

cardiac function comprising administering to the individual a therapeutically effective amount of PDGF-AB or a functional derivative thereof, thereby improving cardiac function in the individual.

2. The method of claim 1 wherein the sub optimal cardiac function is caused by, or associated with a cardiovascular event that causes cardiac tissue ischemia or infarction.

3. The method of claim 2 wherein the cardiovascular event is selected from the

group consisting of: unstable angina, coronary artery spasm, coronary artery embolism, coronary artery dissection, and coronary artery spasm.

4. The method of claim 2 or claim 3 wherein the infarction is acute myocardial

infarction.

5. The method of any one of claims 2 to 4 wherein the PDGF-AB is administered after the cardiovascular event.

6. The method of any one of the preceding claims wherein the PDGF-AB is

administered after cardiac tissue infarction, or administered to infarcted tissue, or administered to fibrotic infarcted tissue.

7. The method of any one of claims 2 to 6 wherein the PDGF-AB is not administered prior to the cardiovascular event.

8. The method of any one of the preceding claims wherein the PDGF-AB is not administered prior to cardiac tissue infarction.

9. The method of any one of the preceding claims wherein PDGF-AB administration prevents a further increase in sub optimisation of cardiac function.

10. The method of any one of the preceding claims wherein PDGF-AB administration restores cardiac function to normal cardiac function with respect to the individual profile.

1 1. The method of any one of the preceding claims wherein the administration of PDGF-AB leads to an improvement in left ventricular ejection fraction, thereby leading to an improvement in cardiac function.

12. The method of any one of the preceding claims wherein the administration of PDGF-AB leads to an improvement in stroke volume, thereby leading to an improvement in cardiac function.

13. The method of any one of the preceding claims wherein the administration of PDGF-AB leads to an improvement in left ventricular end systolic volume, thereby leading to an improvement in cardiac function.

14. The method of any one of the preceding claims wherein the administration of PDGF-AB leads to a reduction in ventricular tachycardia or arrhythmia, thereby leading to an improvement in cardiac function.

15. The method of any one of the preceding claims wherein the administration of PDGF-AB leads to an increase in anisotropy or alignment of collagen fibre in myocardial scar tissue, thereby leading to an improvement in cardiac function.

16. The method of any one of the preceding claims, wherein the improvement in

cardiac function is assessed by electrocardiography (ECG) or MRI.

17. A method for increasing anisotropy or alignment of collagen fibre in myocardial scar tissue of an individual comprising administering a therapeutically effective amount of PDGF-AB or a functional derivative thereof to infarcted myocardial tissue of the individual requiring said treatment, thereby increasing anisotropy or alignment of collagen fibre.

18. A method of remodelling left ventricular cardiac tissue associated with infarction or ischemia in an individual comprising administration of PDGF-AB to an individual having ischemic or infarcted left ventricular cardiac tissue, thereby remodelling the left ventricular cardiac tissue.

19. A method of improving survival in an individual who has suffered a cardiovascular event comprising administering to the individual a therapeutically effective amount of PDGF-AB or a functional derivative thereof, thereby improving survival in the individual.

20. The method according to claim 19, wherein the survival is disease - free survival.

21. The method according to claim 19, wherein the survival is progression - free

survival.

22. The method according to claims 20 or 21 , wherein the improvement in survival is associated with an improvement in cardiac function.

23. The method according to any one of the preceding claims, wherein PDGF-AB is a recombinant protein.

24. The method of any one of the preceding claims wherein the PDGF-AB is administered from less than 1 minute to 120 hours after a cardiovascular event, or 30 minutes to 72 hours after tissue infarction or up to 240 hours.

25. The method of anyone of the preceding claims wherein the PDGF-AB is

administered once.

26. The method of any one of the preceding claims wherein the PDGF-AB is

administered in an amount of 0.2 to 200 mg/day.

27. The method of any one of the preceding claims wherein the PDGF-AB is

administered by intravenous infusion.

28. The method of any one of the preceding claims wherein the PDGF-AB is

administered over a period of 1 second to 120 hours.

29. The method of any one of the preceding claims wherein the PDGF-AB is

administered together with a further compound selected from the group consisting of: an anti-inflammatory, an anti-fibrotic, a stimulator of cardiomyocyte

dedifferentiation, proliferation or migration, or a regulator of extracellular matrix deposition or composition.

30. The method of any one of the preceding claims wherein the individual is a human.