WO2018100403A1

WO2018100403A1 - Combination of parstatin 1-26 and exenatide

Info

Publication number: WO2018100403A1
Application number: PCT/GR2017/000068
Authority: WO
Inventors: Nikolaos TSOPANOGLOU; Michael Maragoudakis
Original assignee: University Of Patras
Priority date: 2016-12-02
Filing date: 2017-12-04
Publication date: 2018-06-07
Also published as: GR20160100618A

Abstract

The present invention provides a combination comprising (i) parstatin peptide 1 - 26, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof. Said combination is suitable for cardioprotection and for treating and/or preventing ischemia and/or reperfusion injury. Further aspects of the invention relate to pharmaceutical products and pharmaceutical compositions comprising said combinations according to the invention, and methods of treatment using the same.

Description

COMBINATION OF PARSTATIN 1-26 AND EXENATIDE

FIELD OF INVENTION

The present invention provides a combination comprising (i) parstatin peptides, particularly a mammalian parstatin peptide including amino acids 1-26 of full length mammalian parstatin, preferably a human parstatin or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof. Said combinations are suitable for cardioprotection and for treating and/or preventing ischemia and/or reperfusion injury.

Further aspects of the invention relate to pharmaceutical products and pharmaceutical compositions comprising said combinations according to the invention, and methods of treatment using the same.

BACKGROUND Acute myocardial infarction (AMI) is a major cause of mortality and morbidity worldwide. Each year, an estimated 785,000 persons (STEMI 500.000) will have a new AMI in the United States alone and approximately every minute an American will succumb to one (Roger et a/., Circulation, 2012, 125:e2-e220). Every sixth man and every seventh woman in Europe will die from myocardial infarction (ESC Guidelines for the management of acute Ml in patients presenting with ST-elevation, Eur. Heart J., 2012, 33:2569-2619).

Currently, timely myocardial reperfusion using either thrombolytic therapy or primary percutaneous coronary intervention (PPCI) is the choice therapy for acute ST-segment elevation myocardial infarction (STEMI) patients (Worner er a/., Rev Exp Cardiol., 2013, 66: 5-1 1). These interventions limit myocardial infarct (Ml), preserve left-ventricular systolic function and reduce the onset of heart failure. However, mortality remains substantial in these patients with in-hospital mortality ranging between 6 and 14% (Mandelzweig et al., Eur. Heart J., 2006, 27: 2285- 2293). Furthermore, morbidity remains substantial - Ml is the leading cause of chronic heart failure - with about 5 to 6% of patients having a subsequent cardiovascular event by 30 days and re-hospitalisation at 1 year about 2.7%. (Lagerqvist er a/., N. Engl. J. Med., 2014, 371 : 11 1 1-20).

Paradoxically, although myocardial reperfusion is essential for myocardial salvage, providing oxygen and nutrients to the ischemic area comes at a price, as it can in itself induce myocardial injury and cardiomyocyte death - a phenomenon termed "myocardial reperfusion injury", the irreversible consequences of which include microvascular obstruction and myocardial infarction (Yellow and Hausenloy, N. Engl. J. Med., 2007, 357: 1 121-1 135). It is estimated that ischemia-reperfusion injury is responsible for approximately 50% of the final infarct area (Yetgin et a/., Neth. Heart J., 2010; 28, 389-392). Previous attempts to translate cardioprotective therapies (i.e. antioxidants, calcium-channel blockers and anti-inflammatory agents) for reducing reperfusion injury into the clinic have been unsuccessful. (Frohlich et al., Eur. Heart J., 2013, 34: 1714-1724). There is currently no approved effective therapy for preventing myocardial reperfusion injury in reperfused-STEMI patients, making it an important residual target for cardioprotection.

Pioneering work in the 1990s first implicated the mitochondrial permeability transition pore (MPTP) as a critical mediator of lethal myocardial reperfusion injury. The opening of the MPTP (a non-selective channel of the inner mitochondrial membrane) in the first few minutes of reperfusion leads to mitochondrial Ca²⁺ overload, oxidative stress, restoration of a physiological pH, and ATP depletion (Heusch et al, Basic Res Cardiol, 2010, 105: 151-154). These events induce cardiomyocytes death by uncoupling oxidative phosphorylation.

Alterations in membrane proteins by free radicals are among the important factors in the evolution of myocardial reperfusion damage. Large quantities of reactive oxygen species (ROS) lead to overwhelming of the cellular endogenous antioxidant defences. This causes, among other effects, the peroxidation of lipid membranes and loss of membrane integrity which results in necrosis and cell death (Zweier and Talukder, Cardiovasc Res, 2006, 70: 181-190). Re- introduction of abundant oxygen at the onset of reperfusion evokes a burst of additional toxic oxygen derivatives, including superoxide anion, hydroxyl radical and peroxy nitrite, within the first few minutes of reflow. Moreover, oxidative stress also reduces the bioavailability of nitric oxide (vasodilator compound) at reperfusion, and the administration of NO donors is cardioprotective in animal models.

There is presently a need for an effective treatment of reperfusion injury, particularly myocardial reperfusion injury.

STATEMENT OF INVENTION

The present invention provides a new combination which is suitable for prevention or treatment of at least reperfusion injury. The combination and other aspects of the invention provide a treatment which is more efficacious and provides superior clinical outcomes compared to therapies which employ a single active pharmaceutical agent.

A first aspect relates to a combination comprising (i) parstatin peptide including amino acids 1-26 of full length mammalian parstatin, preferably a human parstatin or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof.

A second aspect relates to a pharmaceutical composition comprising (i) parstatin peptide 1-26, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, diluent or excipient. A third aspect relates to a pharmaceutical product comprising (i) parstatin peptide 1-26, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof.

A fourth aspect relates to a combination according to the first aspect or a pharmaceutical composition according to the second aspect for use in the treatment and/or prevention of ischemia and/or reperfusion injury. A fifth aspect relates to a pharmaceutical product according to the third aspect for use in the treatment and/or prevention of ischemia and/or reperfusion injury, wherein (i) and (ii) are for administration simultaneously, sequentially or separately. A sixth aspect relates to a method of treating and/or preventing ischemia and/or reperfusion injury, said method comprising simultaneously, sequentially or separately administering to a subject (i) parstatin peptide 1-26, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof.

A seventh aspect relates to use of (i) parstatin peptide 1-26, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prevention of ischemia and/or reperfusion injury.

An eight aspect relates to the use of a combination comprising (i) parstatin peptide 1-26, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for treating and/or preventing ischemia and/or reperfusion injury in an ex vivo heart organ prior to or during transplantation. DETAILED DESCRIPTION

The preferred embodiments set out below are applicable to any of the above- mentioned aspects of the invention as appropriate.

Parstatin Peptide

As used herein, the term "parstatin peptide" refers to 41-mer peptide of the following sequence: H-Met-Gly-Pro-Arg-Arg-Leu-Leu-Leu-Val-Ala-Ala-Cys-Phe-Ser-Leu-Cys-Gly- Pro-Leu-Leu-Ser-Ala-Arg-Thr-Arg-Ala-Arg-Arg-Pro-Glu-Ser-Lys-Ala-Thr- Asn-Ala-Thr-Leu-Asp-Pro-Arg-NH₂

Parstatin peptide is approximately 4.5 kDa in size and corresponds to cleaved peptide of human PARI (Genbank Accession Number AF019616). Such peptides are naturally generated by cleavage of the N-terminal domain of the protease activated receptor-1 (PARI ). Cleavage and release of the N-terminal domain results in the generation of a new N-terminus on the receptor, activating the receptor. Parstatin peptide has been demonstrated to be effective in the prevention and treatment of myocardial ischemia/reperfusion injury (Strande et al., 2009, Cardiovasc Res, 83: 325-334; Tsopanoglou et al, 2012, Patent No: US8,227,412). Parstatin peptide is demonstrated to work cross species with mouse parstatin having an effect on human cells and tissues, and both mouse and human parstatin having an effect on rat cells and tissue. In addition, the cardioprotective effect was associated with coronary artery vasodilation in the isolated heart, and this vasodilatory effect was confirmed in isolated rat coronary arterioles.

Parstatin Peptide 1-26

As used herein, the term "parstatin peptide 1 -26" refers to 26-mer peptide of the following sequence:

H-Met-Gly-Pro-Arg-Arg-Leu-Leu-Leu-Val-Ala-Ala-Cys-Phe-Ser-Leu-Cys-Gly- Pro-Leu-Leu-Ser-Ala-Arg-Thr-Arg-Ala-NH₂ Parstatin peptide is predicted to be less than 41 residues in length because of an initial hydrophobic domain of approximately 21 to 23 amino acids that may represent a putative signal sequence. Indeed, PARI belongs to the small subgroup of G protein-coupled receptors (5-10%) possessing N-terminal signal peptides. Signal peptides have been shown to facilitate export of many proteins across eukaryotic endoplasmic reticulum and are believed to be cleaved-off after mediating the endoplasmatic reticulum targeting/insertion process. In certain embodiments of the invention, a parstatin peptide can include amino acids 1-26, which is also referred to as the hydrophobic fragment of parstatin.

Following structure-function analysis, it has been discovered that the parstatin sequence Met1-Ala26 represents the functional cardioprotective domain of the molecule and the corresponding parstatin fragment 1-26 is much more effective compared to the full-sized peptide 1-41 (Routhu et al, 2010, J Pharmacol Exp Ther, 332: 898-905; Tsopanoglou et al, 2013, Patent No: US8, 389,476). Again parstatin peptide 1-26 is demonstrated to work cross species with human parstatin peptide 1-26 having an effect on rat tissue. Additional observations demonstrated that parstatin peptide 1-26 treatment either before or after ischemia results in an extremely efficacious protection against ischemia- reperfusion injury that depends on antiapoptotic-, NO-, and MPTP-mediated pathways.

Exenatide

As used herein, the term "exenatide" refers to 39-mer peptide of the following sequence:

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu- Ala-Val-Arg-Leu-Phe-lle-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala- Pro-Pro-Pro-Ser-NH₂

Exenatide (synonym is exendin 4) is originally isolated from the saliva of the Gila monster, Heloderma suspectum, by Eng in 1992. It is an insulin secretagogue with glucoregulatory effects similar to the human peptide glucagon-like peptide- 1 (GLP-1 ).

Exenatide mimics human glucagons-like peptide 1 (GLP-1), a gut incretin hormone that is release in response to nutrient intake (Goke et a/., J. Biol. Chem., 1993, 268: 19650-19655). It exerts insulinotropic and insulinomimetic properties via the GLP-1 receptor. GLP-1 receptor is widely expressed in many organs, including heart and vascular endothelium (Bullock et a/., Endocrinology, 1996, 137: 2968-2978; Nystrom er a/., Am J Physiol Endocrinol Metab, 2004, 287: E1209-E1215). Currently, exenatide is approved as an anti-diabetic drug for the treatment of patients with diabetes mellitus type 2.

GLP-1 is ineffective as a therapeutic agent as it has a very short circulating half- life (less than 2 minutes) due to rapid degradation by dipeptidyl peptidase-4. Exenatide is 50% homologous to GLP-1 , but has a 2.4 hours half-life in humans as the dipeptidyl peprtidase-4 cleavage site is absent.

Exenatide enhances glucose-dependent insulin secretion by the pancreatic beta- cell, suppresses inappropriately elevated glucagon secretion, and slows gastric emptying. Exenatide is extremely potent, having a minimum effective concentration of 50pg/mL (12pM) in humans. Current therapies with exenatide involve twice-daily injections (Byetta^®). Also, a slow-release formulation (Bydureon^®) has been approved for once-weekly injection. As used herein a functional derivative or analogue of exenatide may refer to GLP receptor agonists. Suitable functional derivatives or analogues of exenatide of include lixisenatide, albiglutide, liraglutide, taspoglutide and dulaglutide (LY2189265). Combination

In one aspect, the present invention relates to a combination comprising (i) parstatin peptide, preferably a human parstatin peptide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) at least one of exenatide, lixisenatide, albiglutide, !iraglutide, taspoglutide and dulaglutide (LY2189265), or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention relates to a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) at least one of exenatide, lixisenatide, albiglutide, liraglutide, taspoglutide and dulaglutide (LY2189265), or a pharmaceutically acceptable salt thereof. In one embodiment, the present invention relates to a combination of (i) parstatin peptide, preferably a human parstatin peptide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a pharmaceutically acceptable salt thereof. In one embodiment, the present invention relates to a combination of (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a pharmaceutically acceptable salt thereof. The effect of drug combinations is inherently unpredictable and there is often a propensity for one drug to partially or completely inhibit the effects of the other. The present invention is based on the surprising observation that a combination comprising (i) parstatin peptide including amino acids 1-26 of full length mammalian parstatin peptide, preferably a human parstatin peptide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, when administered simultaneously, separately or sequentially, does not lead to any significant or dramatic adverse interaction between the two agents. The unexpected absence of any such antagonistic interaction is critical for clinical applications of the combination.

In one embodiment, the combinations of the active agents of the present invention (see (i) and (ii) above) produce an enhanced effect as compared to either drug administered alone. Furthermore, in another embodiment, the combinations of the active agents of the present invention (see (i) and (ii) above) produce unexpected synergistic effects, for instance, in the treatment and/or prevention of reperfusion injury, particularly myocardial reperfusion injury.

Combination treatments may be evaluated for synergistic effects by analyzing dose-effect data using the median effect model (Chou, T.C. S Talalay, P. (1984) Adv. Enzyme Regul. 22, 27-55. Quantatative analysis of dose-effect

relationships: the combined effects of multiple drugs or enzyme inhibitors, incorporated herein by reference).

Advantageously, a synergistic combination may allow for lower doses of each component to be present, thereby decreasing the toxicity of therapy, whilst producing and/or maintaining the same therapeutic effect. Thus, in a particularly preferred embodiment, each component is present in a sub-therapeutic amount.

The term "sub-therapeutically effective amount" means an amount that is lower than that typically required to produce a therapeutic effect with respect to treatment with each agent alone.

In one embodiment the present invention relates to a synergistic combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) at least one of exenatide, lixisenatide, albiglutide, liraglutide, taspoglutide and dulaglutide (LY2189265), or a pharmaceutically acceptable salt thereof. In one embodiment the present invention relates to a synergistic combination of (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a pharmaceutically acceptable salt thereof.

In one embodiment, the above described combinations comprise at least one further active pharmaceutical ingredient (API).

In one embodiment, the above described combinations may further comprise at least one API selected from an aldosterone antagonist, a beta blocker and a renin-angiotensin inhibitor.

Renin-angiotensin inhibitors include angiotensin converting enzyme inhibitors, angiotensin ΑΤΊ receptor inhibitors and renin inhibitors.

Examples of aldosterone antagonists include spironolactone, eplerenone, canrenone (canrenoate potassium) prorenone (prorenoate potassium) and mexrenone (mexrenoate potassium). Examples of beta-blockers include propranolol, metoprolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol and timolol.

Examples of angiotensin converting enzyme inhibitors include captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, cilazapril, and fosinopril.

Examples of angiotension ΑΤΊ receptor antagonists include losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan and telmisartan. Examples of renin inhibitors include remikiren and aliskiren,

In one embodiment, the above combinations comprise at least one further API selected from spironolactone, eplerenone, canrenone (canrenoate potassium), prorenone (prorenoate potassium), mexrenone (mexrenoate potassium), propranolol, metoprolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, cilazapril, fosinopril, losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan, telmisartan, remikiren and aliskiren.

In another embodiment, the above combinations comprise at least one further API selected from spironolactone, eplerenone, canrenone (canrenoate potassium), carvedilol, metoprolol, losartan, irbesartan, olmesartan, candesartan, valsartan, fimasartan telmisartan. captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, cilazapril, fosinopril, remikiren and aliskiren.

In another embodiment, the above combinations comprise at least one further API selected from spironolactone, eplerenone, canrenone (canrenoate potassium), carvedilol and metoprolol.

Pharmaceutically Acceptable Salts

The active pharmaceutical agents of the present invention can be present as pharmaceutically acceptable salts.

Pharmaceutically acceptable salts of the agents of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al., J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)- alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Enantiomers/Tautomers

The invention also includes where appropriate all enantiomers and tautomers of the active pharmaceutical agents. The man skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.

Stereo and Geometric Isomers

Some of the active pharmaceutical agents of the invention may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).

The present invention also includes all suitable isotopic variations of the active pharmaceutical agents or pharmaceutically acceptable salts thereof. An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 170, 180, 31 P, 32P, 35S, 18F and 36CI, respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

Solvates

The present invention also includes solvate forms of the active pharmaceutical agents of the present invention. The terms used in the claims encompass these forms.

Polymorphs

The invention furthermore relates to active pharmaceutical agents of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation from the solvents used in the synthetic preparation of such compounds. Pharmaceutical Compositions

In another aspect, the present invention relates to a pharmaceutical composition comprising a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, diluent or excipient.

Even though the compounds of the present invention (including their

pharmaceutically acceptable salts) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or non-human animal usage in human and veterinary medicine

respectively. Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients", 2^nd Edition, (1994), edited by A Wade and PJ Weller.

Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's

Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. Examples of routes of administration include parenteral (e.g., intravenous, intramuscular, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration.

In one embodiment, the pharmaceutical composition is for parenteral administration (e.g., intravenous, intramuscular, intradermal, intraperitoneal or subcutaneous).

In another embodiment, the pharmaceutical composition is for intravenous, intramuscular, or subcutaneous administration.

In another embodiment, the pharmaceutical composition is for intravenous administration.

Solutions or suspension used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl-alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compounds into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Pharmaceutical Products

In another aspect, the present invention relates to a pharmaceutical product comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical product is a kit of parts containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course. In one embodiment, the kit comprises separate containers for each active agent. Said containers may be ampoules, disposable syringes or multiple dose vials.

In another embodiment, the kit comprises a container which comprises a combined preparation of each active agent.

The kit may further comprise instructions for the treatment and/or prevention of reperfusion injury.

Medical Uses

In one aspect, the present invention relates to a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of ischemia and/or reperfusion injury.

In another aspect, the present invention relates to a pharmaceutical composition comprising a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of ischemia and/or reperfusion injury.

In another aspect, the present invention relates to a pharmaceutical product comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of ischemia and/or reperfusion injury, wherein (i) and (ii) are for administration simultaneously, sequentially or separately.

In another aspect, the present invention relates to use of (i) parstatin peptide 1- 26, preferably a human parstatin peptide 1 -26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prevention of ischemia and/or reperfusion injury.

In one embodiment, the present invention relates to a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of reperfusion injury.

In another embodiment, the present invention relates to a pharmaceutical composition comprising a combination comprising (i) an insulin modulator and (ii) an immunosuppressive agent for use in the treatment and/or prevention of reperfusion injury.

In another embodiment, the present invention relates to a pharmaceutical composition comprising a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of reperfusion injury. In another embodiment, the present invention relates to a pharmaceutical product comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of reperfusion injury, wherein (i) and (ii) are for administration simultaneously, sequentially or separately.

In another embodiment, the present invention relates to use of (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prevention of reperfusion injury.

In one embodiment, the present invention relates to a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of ischemia.

In another embodiment, the present invention relates to a pharmaceutical composition comprising a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of ischemia.

In another embodiment, the present invention relates to a pharmaceutical product comprising (i) parstatin peptide 1 -26, preferably a human parstatin peptide 1 -26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of ischemia, wherein (i) and (ii) are for administration simultaneously, sequentially or separately.

In another embodiment, the present invention relates to use of (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prevention of ischemia. As used herein, the term "reperfusion injury" refers to the damage to tissue caused when blood supply returns to the tissue after a period of ischemia. The absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation, mitochondrial dysfunction and oxidative damage through the induction of oxidative stress rather than restoration of normal function. Reperfusion injury can occur after a spontaneously occurring event, e.g., arterial blockage, or a planned event, e.g., any of a number of surgical interventions. Myocardial reperfusion injury can occur, for example, after myocardial infarction or as a result of heart transplantation.

In one embodiment, the ischemia and/or reperfusion injury may be ischemia and/or reperfusion injury of the brain, heart, lung, kidney, or other organ/tissue susceptible to ischemia and/or reperfusion injury.

In one embodiment, the ischemia and/or reperfusion injury is ischemia and/or reperfusion injury of the heart, preferably myocardial ischemia and/or myocardial reperfusion injury.

Parstatin peptide 1-26 or a functional derivative or analogue or a

pharmaceutically acceptable salt thereof, and exenatide or a functional derivative or analogue or a pharmaceutically acceptable salt thereof, may be for

administration simultaneously, sequentially or separately (as part of a dosing regime).

As used herein, "simultaneously" is used to mean that the two agents are administered concurrently. As used herein, "sequentially" is used to mean that the active agents are not administered concurrently, but one after the other. Thus, administration

"sequentially" may permit one agent to be administered within 5 minutes, 10 minutes or a matter of hours after the other provided the circulatory half-life of the first administered agent is such that they are both concurrently present in therapeutically effective amounts. The time delay between administrations of the components will vary depending on the exact nature of the components, the interaction there between, and their respective half-lives. In contrast to "sequentially", "separately" is used herein to mean that the gap between administering one agent and the other is significant i.e. the first administered agent may no longer be present in the bloodstream in a therapeutically effective amount when the second agent is administered. In one embodiment, (i) and (ii) are for simultaneous administration.

Methods of Treatment

In another aspect, the present invention relates to a method of treating and/or preventing ischemia and/or reperfusion injury, said method comprising simultaneously, sequentially or separately administering to a subject in need thereof (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention relates to a method of treating and/or preventing reperfusion injury, said method comprising simultaneously, sequentially or separately administering to a subject in need thereof comprising (i) parstatin peptide 1 -26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof. In another embodiment, the present invention relates to a method of treating and/or preventing ischemia, said method comprising simultaneously, sequentially or separately administering to a subject in need thereof (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof. In one embodiment, the method relates to treating and/or preventing ischemia and/or reperfusion injury of the brain, heart, lung, kidney, or other organ/tissue susceptible to ischemia and/or reperfusion injury.

In one embodiment, the method relates to treating and/or preventing reperfusion injury of the brain, heart, lung, kidney, or other organ/tissue susceptible to reperfusion injury.

In one embodiment, the method relates to treating and/or preventing ischemia of the brain, heart, lung, kidney, or other organ/tissue susceptible to ischemia.

In another embodiment, the method relates to treating and/or preventing ischemia and/or reperfusion injury of the heart, preferably myocardial ischemia and/or myocardial reperfusion injury. In another embodiment, the method relates to treating and/or preventing reperfusion injury of the heart, preferably myocardial reperfusion injury.

In another embodiment, the method relates to treating and/or preventing ischemia of the heart, preferably myocardial ischemia.

In one embodiment, the subject is a mammal, more preferably a human. In one embodiment, the method comprises parenterally (e.g., intravenously, intramuscularly, intradermal^, intraperitoneal^ or subcutaneously) administering (i) and (ii) to the subject. In another embodiment, the method comprises intravenously, intramuscularly, or subcutaneously administering (i) and (ii) to the subject.

In another embodiment, the method comprises intravenously administering (i) and (ii) to the subject.

In one embodiment, the claimed combinations are administered to a donor subject and/or a recipient subject prior to, during and/or after heart transplant. For example, in some embodiments the combination may be administered to a first subject from which the heart organ will be removed for transplantation into a second subject. Additionally or alternatively, in some embodiments, the combination is administered to the extracted heart organ, prior to introduction into the second subject. Additionally or alternatively, in some embodiments, the combination therapy is administered to the second subject before, during and/or after heart transplant.

In one embodiment the subject is at risk of (or susceptible to) vessel occlusion injury or cardiac ischemia-reperfusion injury.

In one embodiment, the (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, are administered simultaneously. In one embodiment, the (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof are administered sequentially or separately.

In one embodiment, the (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, are each administered in a therapeutically effective amount with respect to the individual components.

As used herein, the term "therapeutically effective amount" refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, ischemia and/or reperfusion injury or one or more symptoms associated with ischemia and/or reperfusion injury.

In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body, weight and tolerance to drugs. It will also depend on the degree severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic agents.

In one embodiment, the (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, are each administered in a sub- therapeutically effective amount with respect to the individual components. In one embodiment, the (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, are administered prior to reperfusion the subject.

In one embodiment, the (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, are administered during reperfusion of the subject.

In one embodiment, the (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, are administered after reperfusion of the subject.

In one embodiment, the (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, are administered prior to, during and after reperfusion of the subject. In some embodiments of the method, the subject is administered (i) continuously before, during, and after reperfusion of the subject and is administered (ii) as a bolus dose prior to reperfusion.

In some embodiments of the method, the subject is administered (ii) continuously before, during, and after reperfusion of the subject and is administered (i) as a bolus dose prior to reperfusion.

In some embodiments of the method, the subject is administered (i) and (ii) continuously before, during, and after reperfusion of the subject. In some embodiments of the method, the subject is administered (i) and (ii) as a bolus dose prior to reperfusion.

In some embodiments of the method, the subject is administered (i) and (ii) as a bolus dose during reperfusion.

In some embodiments of the method, the subject is administered (i) and (ii) as a bolus dose after reperfusion. As used herein "reperfusion" is the restoration of blood flow to any organ or tissue in which the flow of blood is decreased or blocked. For example, blood flow can be restored to any organ or tissue affected by ischemia or hypoxia. The restoration of blood flow (reperfusion) can occur by any method known to those in the art. For instance, reperfusion of ischemic cardiac tissues may arise from revascularization.

In one embodiment, reperfusion is achieved via a revascularization procedure. In one embodiment, the revascularization procedure is selected from the group consisting of: percutaneous coronary intervention; balloon angioplasty; insertion of a bypass graft; insertion of a stent; directional coronary atheroctomy; treatment with a one or more thrombolytic agent(s); and removal of an occlusion.

In one embodiment, the one or more thrombolytic agents are selected from the group consisting of: tissue plasminogen activator; urokinase; prourokinase;

streptokinase; acylated form of plasminogen; acylated form of plasmin; and acylated streptokinase-plasminogen complex.

Dosage

A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Non-therapeutic Use

In another aspect, the present invention relates to use of a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1 -26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for treating and/or preventing ischemia and/or reperfusion injury in a ex vivo heart organ prior to or during transplantation.

In one embodiment, the present invention relates to use of a combination comprising (i) parstatin peptide 1 -26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for treating and/or preventing reperfusion injury in a ex vivo heart organ prior to or during transplantation.

In one embodiment, the present invention relates to use of a combination comprising (i) parstatin peptide 1-26, preferably a human parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof for treating and/or preventing ischemia in a ex vivo heart organ prior to or during transplantation.

An ex vivo (removed from the body) heart can be susceptible to reperfusion injury due to lack of blood flow. Therefore, the combination of the present invention can be used to prevent reperfusion injury in the removed heart. In some embodiments, the removed heart is placed in a standard buffered solution, such as those commonly used in the art, containing the combination of the invention. For example, a removed heart can be placed in a cardioplegic solution containing exenatide and cyclosporine. The concentration of exenatide and cyclosporine useful in the standard buffered solution can be easily determined by those skilled in the art. Such concentrations may be, for example, between about 0.1 nM to about 10 μΜ, preferably about 1 nM to about 10 μΜ.

EXAMPLES

The present invention is further illustrated by the following example, which should not be construed as limiting in any way.

Example 1. Establishment of a rabbit Model of Acute Myocardial Infarction Injury - a ischemia and reperfusion injury model

Experimental studies suggest that myocardial injury can be reproduced in animal models as a result of ischemia-reperfusion (l/R) insult, as can occur clinically following acute myocardial infarction (AMI) and/or percutaneous coronary intervention/angioplasty (PCI). This study was designed to develop an ischemia/reperfusion animal model in a setting that mimics the clinical scenario of an acute l/R insult.

New Zealand White rabbits were used in this study. The rabbits were males, >8 weeks in age and with a weight between 2.7 and 4.0 Kg. Approval from the Ethical Committee of University of Patras and the veterinary authorities of Western Greece prefecture was obtained before the study was started. Environmental controls in the animal rooms were set to maintain temperature of 22° to 28°C and relative humidity between 30% and 70%. Room temperature and humidity were recorded hourly and monitored daily. There were approximately 10-15 air exchanges per hour in the animal rooms. Photoperiod was 12-hours light and 12-hours dark. Routine daily observations were performed. Certified standard rabbit diet was provided, approximately 180 grams per day from arrival to the facility.

Normal saline (0.9% NaCI) was used as a control. Control articles were given intravenously, under general anaesthesia, in order to mimic the expected route of administration in the clinical setting of acute myocardial infarction and primary percutaneous coronary intervention. Intravenous bolus was administered via a peripheral vein (rabbit auricular vein). The study followed a predetermined sham controlled design. In brief, 18 healthy, acclimatized, male rabbits were randomly assigned to one of two study arms. Arm A (n=4, SHAM) includes sham-operated time-controls treated with normal saline (NS; IV); Arm B (n=1 , CONTROL/PLACEBO) includes l/R animals treated with normal saline (NS; IV).

In l/R group (arm B), rabbits were subjected to 40 min regional ischemia of the heart (coronary occlusion), followed by 120 min (2 h) reperfusion. In all cases, treatments (intravenous bolus administration of NS) were performed 30 min after the onset of ischemia (10 min before the initiation of reperfusion). In all cases, cardiovascular function was monitored both prior to and during ischemia, as well as for up to 120 min (2 h) post-reperfusion. The experiments were terminated 2 h post-reperfusion (end of study); irreversible myocardial injury (infarct size by histomorphometery) at this time-point was evaluated, and was the primary-end- point of the study. The study design is summarized in Table 1.

TABLE 1

Anaesthesia/Surgical Preparation. General anesthesia was induced intramuscularly (IM) with a ketamine (~35-50 mg/Kg) and xylazine (~5-10 mg/Kg) mixture. In order to preserve autonomic function and to maintain anesthesia throughout the experimental procedure, animals received additional anesthesia as required (~10-15 mg ketamine/30 min or ~15-20 mg/Kg sodium pentobarbital). A venous catheter was placed in a peripheral vein (e.g., ear auricular vein) for the administration of additional anesthetics and normal saline/test articles. The effect of anesthesia is assessed by (i) the total abolishment of animal's corneal reflex, (ii) the total abolishment of animal's respiratory centre and the total mechanical ventilation without any resistance to airflow caused by animal's spontaneous breathing, and (iii) the stability of the hemodynamic parameters. A cuffed tracheal , tube was placed via a tracheotomy (ventral midline incision) and used to mechanically ventilate the lungs with a 95% 0₂/5% C0₂ mixture via a volume- cycled animal ventilator (~40 breaths/min with a tidal volume of ~12.5 ml/Kg) in order to sustain PaC0₂ values broadly within the physiological range. Throughout the experimental procedure, electrocardiogram, arterial pressure, cardiac pulses, and capnogram were continuously monitored. A temperature probe was inserted into the rectum and body temperature was maintained using a heating pad. Finally, an arterial catheter was placed in a peripheral artery (e.g., ear) for blood sampling. Subsequently, the animals were placed in left-lateral recumbence and the chest was surgically opened with a left thoracotomy. The chest was opened through the left fourth intercostals space. The beating heart was exposed and the pericardium was incised. Under the atrial appendage, the first large anterolateral branch of the circumflex artery and if necessary, depending on each animal's coronary anatomy, the circumflex artery itself, was circled with a 3-0 silk suture. Coronary artery occlusion in this region normally results in ischemia of a large territory of the anterolateral and apical ventricular wall. The ends of the suture were threaded through a small piece of polyethylene tubing, forming a snare. Ischemia was induced by pulling the thread through the tubing, which was firmly positioned against the coronary arterial wall with the aid of a small clamp. Ischemia resulted in ST elevation on the electrocardiogram and a change in the color (i.e., cyanotic) of the myocardium. After 30 min of ischemia, the animals administered with IV bolus of normal saline; ischemia was continued for additional 10 min (i.e., 40 min total ischemia time) after the treatment. At the end of ischemic period, the coronary snares were released and the previous ischemic myocardium was reperfused for up to 2 hours. It should be noted that in sham-operated animals the vessel snares were manipulated at the time of ischemia-reperfusion onset, but were not either tightened or loosened.

Hemodynamic variables and rectal temperature were monitored and recorded at 8 predetermined time-points: post-anesthesia (i.e., normal), post-surgical preparation and just before ischemia initiation (i.e., baseline), 30 (vehicle/test articles administration) and 40 min (end of ischemia) of ischemia, as well as at 30, 60, 90, and 120 min post reperfusion. In addition, in order to determine/quantify the degree of irreversible myocardial injury (i.e., infarction) resulting from the l/R insult, cardiac biomarkers as well as infarct area were evaluated. Blood Samples. Venous (<3 mL) whole blood samples were collected for the evaluation of myocardial injury via cardiac biomarker analyses at two data- collection time-points: baseline, and 120 min post-reperfusion. Cardiac troponin-l (cTnl), as the most valuable and reliable clinically used biomarker, was measured. cTnl was determined with a biochemical analyzer (Triage® Cardiac Panel, Alere San Diego, Inc. CA, USA).

Histopathology/Histomorphometery. At the completion of the protocol, irreversible myocardial injury (i.e., infarction) resulting from the l/R insult was evaluated. In brief, after the end of reperfusion, hearts were excised, mounted on an apparatus, and perfused with normal saline for 2 min for blood removal. Then the coronary ligature was retightened at the same site and 5 mL of green fluorescent polymer microsphere solution (8 mg/ml; diameter 3-8 μιτι; Fluoro-Max™, Thermo Scientific, CA, USA) was infused for the separation of the normally perfused area (no ischemic area) and to delineate the myocardial area-at-risk (AAR, ischemic area) during ischemia. Hearts were kept at -20°C for 24 hours and sectioned perpendicular to its long axis (from apex to base) into 3 mm thick slices. Subsequently, the slices were incubated for 20 min in 2% triphenyl-tetrazolium- chrolide (TTC, Sigma, St. Louis, MO, USA) at 37°C, and fixed in a 10% non- buffered formalin solution. Following fixation, the total left ventricular area (LV), the area-at-risk (AAR), and the infarct area (IF) were delineated/measured digitally. With a wavelength of 366 nm UV light, it was separated in each slide the ischemic area (AAR) from infarcted zone (IF) and no ischemic area. All areas were traced onto an acetate sheet. The tracings were subsequently imported into an image analysis program (Image J; National Institutes of Health), and computer-assisted planometry was performed to determine the overall size of the left ventricular (LV), the area-at-risk (AAR) and the infarct (IF). For each slide, the AAR was expressed as a percentage of the LV area (AAR/LV), and the IF was expressed as a percentage of the AAR (IF/AAR). In all cases, quantitative histomorphometery was performed by personnel blinded to the treatment assignment/study-design.

Data analysis/Statistics. Obtained data from the above experiment procedure were tabulated and calculated using Excel work sheets. Statistical analysis was performed with SPSS version 21.0 (SPSS Inc., IL, USA). Values are presented as mean ± standard deviation (SD). Statistical comparisons of numeric variables among the three groups were analyzed using the one-way analysis of variance (ANOVA) model with Bonferroni correction analysis. A calculated P-value of less than 0.05 was considered to be statistically significant.

Animal Observations/Results. The LV size and AAR size were comparable in sham and l/R animals, indicating that the initial left ventricular and ischemic injury did not differ significantly between the groups. Whereas cardiac troponin I (cTnl) release was similar at baseline among groups (<0.05 ng/mL), l/R rabbits exhibited an increase in cTnl blood level at 2 h of reperfusion averaging 137.8±98 ng/mL, when compared with sham animals (9.5 ± 8.9 ng/mL, PO.001 vs. controls). Table 2 presents data showing cardiac troponin I levels, the ratios of area of risk to left ventricular, infarcted area to left ventricular, and infarcted area to area of risk for each of group used in this study.

TABLE 2 Histopathology Results of Study Animals

These results show that in a rabbit model the acute myocardial ischemia and reperfusion was able to significantly produce a myocardial infarct size compared to the sham group. In the sham rabbits in which no ischemia/reperfusion was performed, the size of the myocardial infarct area was reduced by 95% relative to the infarct size noted in control animals. These results indicate that myocardial injury can be reproduced in a rabbit model as a result of ischemia-reperfusion (l/R) insult, as can occur clinically following acute myocardial infarction and/or percutaneous coronary intervention/angioplasty. As such, the rabbit model of acute myocardial infarction injury was demonstrated to be standardized, reliable and reproducible and is useful in methods at preventing and/or treating ischemia- reperfusion injury in mammalian subjects. Example 2. Intravenous Parstatin Peptide 1-26 Dose-Response Treatment after Ischemia in a Rabbit Model of Acute Myocardial Infarction Injury

Experimental studies suggest that treatment with parstatin peptide 1-26 can attenuate/mitigate myocardial injury resulting from an ischemia-reperfusion (l/R) insult as can occur clinically following acute myocardial infarction (AMI) and/or percutaneous coronary intervention/angioplasty (PCI). Parstatin peptide 1-26 (IV bolus), when given prior to the onset of reperfusion, may have myocardial sparing effects. This study was designed to test the cardioprotective effects of parstatin peptide 1-26 in a setting that mimics the clinical scenario of an acute l/R insult. The study was conduct under the general hypothesis that treatment with parstatin peptide 1-26 after the onset of ischemia (but prior to reperfusion) would attenuate irreversible myocardial injury (i.e., infarct size). The effects of several doses of parstatin peptide 1-26 in protecting against an acute myocardial infarction injury in a rabbit model were investigated. By this Example, the myocardial protective effect of parstatin peptide 1-26 was demonstrated in the rabbit model of acute myocardial infarction injury described in the Example 1.

Parstatin peptide 1-26, a 26mer lipophilic peptide, synthesized by Biosynthesis S.A. (Lewisville, Texas, USA) and provided as lyophilized powder. Parstatin peptide 1-26 reconstituted in dimethyl sulfoxide (DMSO) and dosing solutions were prepared in normal saline just before the administration to the rabbits. Normal saline (0.9% NaCI) was used as a control.

The test/control articles were given intravenously, under general anaesthesia, in order to mimic the expected route of administration in the clinical setting of acute myocardial infarction and primary percutaneous coronary intervention. Intravenous bolus was administered via a peripheral vein (rabbit auricular vein).

The study followed a predetermined placebo controlled design. In brief, 64 healthy, acclimatized, male rabbits were randomly assigned to one of seven study arms. Arm A (n=14, CONTROL/PLACEBO) includes l/R animals treated with normal saline (NS; IV); Arm B (n=4, PARSTATIN 1-26, 30pg/Kg) includes l/R animals treated with parstatin peptide 1-26 at dose of 30Mg/Kg; Arm C (n=5, PARSTATIN 1-26, 10pg/Kg) includes l/R animals treated with parstatin peptide 1- 26 at dose of 10pg/Kg; Arm D (n=11 , PARSTATIN 1-26, 3Mg/Kg) includes l/R animals treated with parstatin peptide 1-26 at dose of 3 g/Kg; Arm E (n=8, PARSTATIN 1-26, i Mg/Kg) includes l/R animals treated with parstatin peptide 1- 26 at dose of i Mg/Kg; Arm F (n=14, PARSTATIN 1-26, O.l Mg/Kg) includes l/R animals treated with parstatin peptide 1-26 at dose of 0.1 g/Kg; Arm G (n=8, PARSTATIN 1-26, 0.0l Mg/Kg) includes l/R animals treated with parstatin peptide 1 -26 at dose of 0.01 pg/Kg. .

In all groups, rabbits were subjected to 40 min regional ischemia of the heart (coronary occlusion), followed by 120 min (2 h) reperfusion. In all cases, treatments (intravenous bolus administration of NS or different doses of parstatin peptides 1-26) were performed 30 min after the onset of ischemia (10 min before the initiation of reperfusion). In all cases, cardiovascular function was monitored both prior to and during ischemia, as well as for up to 120 min (2 h) post- reperfusion. The experiments were terminated 2 h post-reperfusion (end of study); irreversible myocardial injury (infarct size by histomorphometery) at this time-point was evaluated, and was the primary-end-point of the study. The study design is summarized in Table 3.

TABLE 3

Anaesthesia/Surgical Preparation. As described in Example 1. Blood Samples. As described in Example 1. Histopathology/Histomorphometery. As described in Example 1. Data analysis/Statistics. As described in Example 1.

Animal Observations/Results. The LV size and AAR size were comparable in control- and parstatin 1-26-treated animals, indicating that the initial left ventricular and ischemic injury did not differ significantly between the groups. Administration of parstatin peptide 1-26 resulted in dose-response effects in infarct size compared to the control. Whereas cardiac troponin I (cTnl) release was similar at baseline among all groups (<0.05 ng/mL), parstatin 1-26 rabbits at dose of O.l g Kg exhibited an statistically significant decrease in cTnl blood level at 2 h of reperfusion averaging 96.3±110 ng/mL, when compared with control animals (137.8±98 ng/mL, P<0.05 vs. controls). Other doses of parstatin peptide 1-26 did not have significant effect in cTnl levels.

Table 4 presents data showing cardiac troponin I levels, the ratios of area of risk to left ventricular, infarcted area to left ventricular, and infarcted area to area of risk for each of group used in this study.

TABLE 4

_ro_pu

The results show that in a standardized rabbit model of acute myocardial ischemia and reperfusion injury, parstatin peptide 1-26 when administrated in a dose of O.^g/Kg, as an IV bolus at 10 min before the initiation of reperfusion, was able to significantly reduce myocardial infarct size compared to the control group. At this dose, the size of the myocardial infarct area was reduced by 41% relative to the infarct size noted in control animals. These results indicate that parstatin peptide 1-26 treatment prevents the occurrence of symptoms of acute cardiac ischemia-reperfusion injury. As such, parstatin peptide 1-26 is useful in methods at preventing and/or treating ischemia-reperfusion injury in mammalian subjects.

Example 3. Effects of Intravenous Exenatide Treatment after Ischemia in a rabbit Model of Acute Myocardial Infarction Injury

Experimental studies suggest that treatment with exenatide can attenuate/mitigate myocardial injury resulting from an ischemia-reperfusion (l/R) insult as can occur clinically following acute myocardial infarction (AMI) and/or percutaneous coronary intervention/angioplasty (PCI). Exenatide (0.150 pg/Kg IV bolus), when given prior to the onset of reperfusion, may have myocardial sparing effects. This study was designed to test the cardioprotective effects of exenatide in a setting that mimics the clinical scenario of an acute l/R insult. The study was conduct under the general hypothesis that treatment with exenatide after the onset of ischemia (but prior to reperfusion) would attenuate irreversible myocardial injury (i.e., infarct size). The effects of exenatide in protecting against an acute myocardial infarction injury were investigated. The myocardial protective effect of exenatide was demonstrated in the_rabbit model of acute myocardial infarction injury described in the Example 1. Exenatide peptide, the synthetic version of exendin-4, is the active ingredient of Byetta^® [10Mg/injected dose (40μΙ), Eli Lilly Nederland B.V.], which is presently approved for the treatment of Type II diabetes. Exenatide of Byetta^® was used as the test article. Dosing solutions (0.15 g/Kg of exenatide in of normal saline) were prepared just before the administration to the rabbits. Normal saline (0.9% NaCI) was used as a control.

The study followed a predetermined placebo controlled design. In brief, 28 healthy, acclimatized, male rabbits were randomly assigned to one of two study arms. Arm A (n=14 CONTROL/PLACEBO) includes l/R animals treated with normal saline (NS; IV); Arm B (n=14, EXENATIDE, 0.15 g/ g) includes l/R animals treated with exenatide.

Both groups (arm A and B), rabbits were subjected to 40 min regional ischemia of the heart (coronary occlusion), followed by 120 min (2 h) reperfusion. In all cases, treatments (intravenous bolus administration of NS or exenatide) were performed 30 min after the onset of ischemia (10 min before the initiation of reperfusion). In all cases, cardiovascular function was monitored both prior to and during ischemia, as well as for up to 120 min (2 h) post-reperfusion. The experiments were terminated 2 h post-reperfusion (end of study); irreversible myocardial injury (infarct size by histomorphometery) at this time-point was evaluated, and was the primary-end-point of the study. The study design is summarized in Table 5.

TABLE 5

Anaesthesia/Surgical Preparation. As described in Example 1.

Blood Samples. As described in Example 1.

Histopathology/Histomorphometery. As described in Example 1.

Data analysis/Statistics. As described in Example 1.

Animal Observations/Results. The LV size and AAR size were comparable in sham-, control- and exenatide-treated animals, indicating that the initial left ventricular and ischemic injury did not differ significantly between the groups. Administration of exenatide resulted in decreased infarct size compared to the control. Whereas cardiac troponin I (cTnl) release was similar at baseline among groups (<0.05 ng/mL), exenatide rabbits exhibited an statistically significant reduction in cTnl blood level at 2 h of reperfusion averaging 53.4±63.9 ng/mL, when compared with control animals (137.8±98 ng/mL, P<0.01 vs. controls).

Table 6 presents data showing cardiac troponin I levels, the ratios of area of risk to left ventricular, infarcted area to left ventricular, and infarcted area to area of risk for each of group used in this study.

TABLE 6

Histopathology Results of Study Animals

These results show that in a standardized rabbit model of acute myocardial ischemia and reperfusion injury, exenatide when administrated as an IV bolus (0.15 g/Kg) at 10 min before the initiation of reperfusion was able to significantly reduce myocardial infarct size compared to the control group. In the rabbits in which there was a definable response to treatment, the size of the myocardial infarct area was reduced by 43% relative to the infarct size noted in control animals. These results indicate that exenatide treatment prevents the occurrence of symptoms of acute cardiac ischemia-reperfusion injury. As such, exenatide is useful in methods at preventing and/or treating ischemia-reperfusion injury in mammalian subjects.

Example 4. Effects of Intravenous Cyclosporine Treatment after Ischemia in a rabbit Model of Acute Myocardial Infarction Injury

Experimental studies suggest that treatment with cyclosporine-A (CsA) can attenuate/mitigate myocardial injury resulting from an ischemia-reperfusion (l/R) insult as can occur clinically following acute myocardial infarction (AMI) and/or percutaneous coronary intervention/angioplasty (PCI). CsA (2.5 mg/Kg IV bolus), when given prior to the onset of reperfusion, may have myocardial sparing effects. This study was designed to test the cardioprotective effects of CsA in a setting that mimics the clinical scenario of an acute l/R insult. The study was conduct under the general hypothesis that treatment with CsA after the onset of ischemia (but prior to reperfusion) would attenuate irreversible myocardial injury (i.e., infarct size). The effects of CsA in protecting against an acute myocardial infarction injury were investigated. The myocardial protective effect of cyclosporine was demonstrated in the rabbit model of acute myocardial infarction injury described in the Example 1.

CsA is the active ingredient of Sandimmun® (50mg/ml, Novartis Pharma A.G., Switzerland), which is presently approved as immunosuppressant treatment. CsA of Sandimmun® was used as the test article. Dosing solutions (2.5 mg/Kg of CsA in normal saline) were prepared just before the administration to the rabbits. Normal saline (0.9% NaCI) was used as a control.

The test/control articles were given intravenously, under general anaesthesia, in order to mimic the expected route of administration in the clinical setting of acute myocardial infarction and primary percutaneous coronary intervention. Intravenous bolus was administered via a peripheral vein (rabbit auricular vein). The study followed a predetermined placebo controlled design. In brief, 28 healthy, acclimatized, male rabbits were randomly assigned to one of two study arms. Arm A (n=14, CONTROL/PLACEBO) includes l/R animals treated with normal saline (NS; IV); Arm B (n=14, CYCLOSPORINE, 2.5mg/Kg, IV) includes l/R animals treated with CsA.

In both groups (arm A and B), rabbits were subjected to 40 min regional ischemia of the heart (coronary occlusion), followed by 120 min (2 h) reperfusion. In all cases, treatments (intravenous bolus administration of NS or CsA) were performed 30 min after the onset of ischemia (10 min before the initiation of reperfusion). In all cases, cardiovascular function was monitored both prior to and during ischemia, as well as for up to 120 min (2 h) post-reperfusion. The experiments were terminated 2 h post-reperfusion (end of study); irreversible myocardial injury (infarct size by histomorphometery) at this time-point was evaluated, and was the primary-end-point of the study. The study design is summarized in Table 7.

TABLE 7

Group Study Group (no. of animals) Ischemia Time Reperfusion

Time

Group

Anaesthesia/Surgical Preparation. As described in Example 1. Blood Samples. As described in Example 1.

Histopathology/Histomorphometery. As described in Example 1. Data analysis/Statistics. As described in Example 1. Animal Observations/Results. The LV size and AAR size were comparable in control- and CsA-treated animals, indicating that the initial left ventricular and ischemic injury did not differ significantly between the groups. Administration of CsA resulted in decreased infarct size compared to the control. Whereas cardiac troponin I (cTnl) release was similar at baseline among groups (<0.05 ng/mL), cyclosporin rabbits exhibited an statistically significant reduction in cTn_ff D_{i (}%_{) it}erennl blood level at 2 h of reperfusion averaging 93.3±55.6 ng/mL, when compared _{I f}F_/AARrom with

C_ltonro

control animals (137.8±98 ng/mL, P<0.05 vs. controls).

S_{iifi i}gncancen IF_/AAR versus

Table 8 presents data showing cardiac troponin I levels, the ratios of area of risk C_ltonro to left ventricular, infarcted area to left ventricular, and infarcted area to area of risk for each of group used in this study.

TABLE 8 Histopathology Results of Study Animals

CONTROL 137.8±98 60.13114.7 19.9±11.1 34.3±14.2

n=14

These results show that in a standardized rabbit model of acute myocardial ischemia and reperfusion injury, CsA when administrated as an IV bolus (2.5mg/Kg) at 10 min before the initiation of reperfusion was able to significantly reduce myocardial infarct size compared to the control group. In the rabbits in which there was a definable response to treatment, the size of the myocardial infarct area was reduced by 41% relative to the infarct size noted in control animals. These results indicate that CsA treatment prevents the occurrence of symptoms of acute cardiac ischemia-reperfusion injury. As such, CsA is useful in methods at preventing and treating ischemia-reperfusion injury in mammalian subjects.

Example 5. Effects of Combined Parstatin Peptide 1-26 and Cyclosporine Treatment in a rabbit Model of Acute Myocardial Infarction Injury

This study was designed to test the cardioprotective effects of combined parstatin peptide 1-26 and CsA in a setting that mimics the clinical scenario of an acute l/R insult. The study was conduct under the general hypothesis that combined treatment with parstatin peptide 1-26 and CsA after the onset of ischemia would attenuate irreversible myocardial injury in a more efficacious manner and provides superior clinical outcomes compared to therapies which employ a single active agents. The combined effects of parstatin peptide 1 -26 and cyclosporine in protecting against an acute myocardial infarction injury were investigated. This example demonstrates that the combined parstatin peptide 1-26 and cyclosporine treatment does not provide a more pronounced myocardial protective effect compared to parstatin peptide 1-26 or cyclosporine treatment alone. The myocardial protective effect of parstatin peptide 1-26 and cyclosporine was investigated in the rabbit model of acute myocardial infarction injury described in the Example 1. Parstatin peptide (Biosynthesis S.A., Lewisville, Texas, USA) and cyclosporine A [CsA, Sandimmun® (50mg/ml) Novartis Pharma A.G., Switzerland] are used as the test articles, either in monotherapy or in combined therapy. Dosing solutions of parstatin peptide 1-26 (0.1 g Kg in of normal saline) and/or CsA (2.5 mg/Kg in normal saline) were prepared just before the administration to the rabbits. Normal saline (0.9% NaCI) was used as a control.

The test/control articles were given intravenously, under general anaesthesia, in order to mimic the expected route of administration in the clinical setting of acute myocardial infarction and primary percutaneous coronary intervention. Intravenous boluses were administered via a peripheral vein (rabbit auricular vein).

The study followed a predetermined placebo controlled design. In brief, 53 healthy, acclimatized, male rabbits were randomly assigned to one of four study arms. Arm A (n=14, CONTROL/PLACEBO) includes l/R animals treated with normal saline (NS; IV); Arm B (n=14, PARSTATIN 1-26, 0.1 Mg/Kg) includes l/R animals treated with parstatin peptide 1-26; Arm C (n=14, CYCLOSPORINE, 2.5mg/Kg, IV) includes l/R animals treated with CsA; Arm D (n=11 , PARSTATIN 1-26 + CYCLOSPORINE, IV) includes l/R animals treated with parstatin peptide and CsA.

In all groups (arm A, B, C and D), rabbits were subjected to 40 min regional ischemia of the heart (coronary occlusion), followed by 120 min (2 h) reperfusion. In all cases, treatments (intravenous bolus administration of NS or parstatin peptide 1-26 or CsA or parstatin peptide 1-26 plus CsA) were performed 30 min after the onset of ischemia (10 min before the initiation of reperfusion). In all cases, cardiovascular function was monitored both prior to and during ischemia, as well as for up to 120 min (2 h) post-reperfusion. The experiments were terminated 2 h post-reperfusion (end of study); irreversible myocardial injury (infarct size by histomorphometery) at this time-point was evaluated, and was the primary-end-point of the study. The study design is summarized in Table 9.

TABLE 9

Histopathology/Histomorphometery. As described in Example 1. Data analysis/Statistics. As described in Example 1. Animal Observations/Results. The LV size and AAR size were comparable in all- treated animals, indicating that the initial left ventricular and ischemic injury did not differ significantly between the groups. Although the administration of parstatin peptide 1-26 or CsA resulted in a statistically significant decreased of infarct size compared to the control, the administration of the combination of parstatin peptide 1-26 and CsA does not resulted in a superior decrease of infarct size. On the contrary, the combined treatment of parstatin peptide 1-26 and CsA resulted in a no statistically significant effect in infarct size compared to control (26.05±14.5 versus 34.3±14.2; P>0.05). Similarly, whereas parstatin peptide 1- 26 or CsA as monotherapies reduced cTnl release by 23% and 32%, respectively, the combined therapy of parstatin peptide 1-26 and cyclosporine increased cTnl release by 26% (17.38±14, F>0.05). Table 10 presents data showing cardiac troponin I levels, the ratios of area of risk to left ventricular, infarcted area to left ventricular, and infarcted area to area of risk for each of group used in this study. TABLE 10 Histopathology Results of Study Animals

These results show that in a standardized rabbit model of acute myocardial ischemia and reperfusion injury, the combined therapy of parstatin peptide 1-26 and CsA when administrated as an IV bolus (O.l pg/Kg and 2.5mg/Kg, respectively) at 10 min before the initiation of reperfusion was not able to reduce myocardial infarct size in more pronounced manner compared to parstatin peptide 1-26 or CsA monotherapy groups. On the contrary, the combined therapy was demonstrated to abolish and reverse the cardioprotective effects of parstatin peptide 1-26 or CsA monotherapies. Accordingly, these results demonstrate that in therapy of myocardial reperfusion injury the combined administration of cyclosporine and parstatin peptide 1-26 is not beneficial and us such combined parstatin peptide 1-26 and CsA is not useful in methods at preventing and treating ischemia-reperfusion injury in mammalian subjects. Example 6. Effects of Combined Parstatin Peptide 1-26 and Exenatide Treatment in a rabbit Model of Acute Myocardial Infarction Injury

This study was designed to test the cardioprotective effects of combined parstatin peptide 1-26 and exenatide in a setting that mimics the clinical scenario of an acute l/R insult. The study was conduct under the general hypothesis that combined treatment with parstatin peptide 1-26 and exenatide after the onset of ischemia would attenuate irreversible myocardial injury in a more efficacious manner and provides superior clinical outcomes compared to therapies which employ a single active agents. The combined effects of parstatin peptide 1-26 and exenatide in protecting against an acute myocardial infarction injury were investigated. This example demonstrates that the combined parstatin peptide 1- 26 and exenatide treatment provides a more pronounced myocardial protective effect compared to parstatin peptide 1-26 or exenatide treatment alone. The myocardial protective effect of parstatin peptide 1-26 and exenatide was investigated in the rabbit model of acute myocardial infarction injury described in the Example 1.

Parstatin peptide (Biosynthesis S.A., Lewisville, Texas, USA) and exenatide [Byetta^® (lOpg/injected dose (40μΙ), Eli Lilly Nederland B.V.] are used as the test articles, either in monotherapy or in combined therapy. Dosing solutions of parstatin peptide 1-26 (O.l g/Kg in of normal saline) and/or exenatide (0.15 g/Kg in normal saline) were prepared just before the administration to the rabbits. Normal saline (0.9% NaCI) was used as a control.

The study followed a predetermined placebo controlled design. In brief, 56 healthy, acclimatized, male rabbits were randomly assigned to one of four study arms. Arm A (n=14, CONTROL/PLACEBO) includes l/R animals treated with normal saline (NS; IV); Arm B (n=14, PARSTATIN 1-26, O.l g/ g IV) includes l/R animals treated with parstatin peptide 1-26; Arm C (n=14, EXENATIDE, 0.15 g/Kg IV) includes l/R animals treated with exenatide; Arm D (n=14, PARSTATIN 1-26+EXENATIDE, IV) includes l/R animals treated with parstatin peptide 1-26 and exenatide.

In all groups (arm A, B, C and D), rabbits were subjected to 40 min regional ischemia of the heart (coronary occlusion), followed by 120 min (2 h) reperfusion. In all cases, treatments (intravenous bolus administration of NS or parstatin peptide 1-26 or exenatide or parstatin peptide 1-26 plus exenatide) were performed 30 min after the onset of ischemia (10 min before the initiation of reperfusion). In all cases, cardiovascular function was monitored both prior to and during ischemia, as well as for up to 120 min (2 h) post-reperfusion. The experiments were terminated 2 h post-reperfusion (end of study); irreversible myocardial injury (infarct size by histomorphometery) at this time-point was evaluated, and was the primary-end-point of the study. The study design is summarized in Table 11.

TABLE 11

Animal Observations/Results. The LV size and AAR size were comparable in all- treated animals, indicating that the initial left ventricular and ischemic injury did not differ significantly between the groups. Although the administration of parstatin peptide 1-26 or exenatide resulted in decreased infarct size compared to the control, the administration of the combination of parstatin peptide 1-26 and exenatide resulted in a superior decrease of infarct size. Similarly, whereas parstatin peptide 1 -26 or exenatide reduced cTnl release by 23% and 61 %, respectively, the combined therapy of parstatin peptide 1-26 and exenatide reduced cTnl release by 74% (35.6±24.2, PO.001).

Table 12 presents data showing cardiac troponin I levels, the ratios of area of risk to left ventricular, infarcted area to left ventricular, and infarcted area to area of risk for each of group used in this study. TABLE 12 Histopathology Results of Study Animals

G_rou_p

^(/Ln^gm

n=14

These results show that in AAR^/LV a standardized rabbit model of acute myocardial ischemia and reperfusion injury, the combined therapy of parstatin peptide 1-26 and exenatide when administrated as an IV bolus (O.l g/Kg and 0.15Mg/Kg, respectively) at 10 min before the initiation of reperfusion was able to reduce myocardial infarct size in more pronounc ^IF/LVed manner compared to parstatin peptide 1-26 or exenatide monotherapy grouSD⁾ⁱp means. Accordingly, these results demonstrate that in therapy of myocardial reperfusion injury the combined administration of parstatin peptide 1-26 and exenatide ^IF/AAR is more beneficial and advantageous and provides superior clinical outcome against therapies either with parstatin peptide 1-26 or exenatide alone. As such, combined parstatin peptide 1-26 and exenatide is exceptionally useful in methods at preventing and

f^f% D^{i () it}een^rn

treating ischemia-reperfusion injury in mammalian subjects. ^IF/AAR ^from

C^lton^ro

Remarks S^{fiii ig}ncancen

Various modifications and variations of the invention will be apparent to those ^IF/AAR ve^rsus

C^lton^ro skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

This invention includes all modifications and equivalents of the subject matter recited in the paragraphs appended hereto as permitted by applicable law.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law). The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

Claims

1. A combination comprising (i) parstatin peptide 1-26, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, wherein parstatin peptide 1-26 functional derivative or analogue is at least 95% identical to amino acids of SEQ ID NO:1 ; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, wherein the exenatide functional derivative or analogue is a GLP-1 receptor agonist .

2. A pharmaceutical composition comprising a combination according to claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

3. A pharmaceutical product comprising (i) parstatin peptide 1-26, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, wherein parstatin peptide 1-26 functional derivative or analogue is at least 95% identical to amino acids of SEQ ID NO:1; and (ii) exenatide, or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof, wherein the exenatide functional derivative or analogue is a GLP-1 receptor agonist.

4. A combination according to claim 1 or a pharmaceutical composition according to claim 2 for use in treatment and/or prevention of ischemia and/or reperfusion injury, wherein the ischemia and/or reperfusion injury is ischemia and/or reperfusion injury of the brain, heart, lung, kidney, preferably myocardial ischemia and/or myocardial reperfusion injury.

5. A combination or a pharmaceutical composition for use according to claims 1 and 2 wherein (i) and (ii) are administered intravenously during, before or after reperfusion..

6. A combination or a pharmaceutical composition for use according to claims 4 and 5 wherein (i) and (ii) are each for administration in a therapeutically effective amount with respect to the individual components or in a subtherapeutic amount with respect to the individual components.

7. A pharmaceutical product according to claim 3 for use in the treatment and/or prevention of ischemia and/or reperfusion injury, wherein (i) and (ii) are for administration simultaneously, sequentially or separately and wherein the ischemia and/or reperfusion injury is ischemia and/or reperfusion injury of the brain, heart, lung, kidney, preferably myocardial ischemia and/or myocardial reperfusion injury

8. A pharmaceutical product for use according to claim 7 wherein (i) and (ii) are for administration intravenously during, before or after reperfusion.

9. A pharmaceutical product for use according to claims 7 and 8 wherein (i) and (ii) are for simultaneous administration or wherein (i) is for administration prior to sequential or separate administration of (ii) or wherein (ii) is for administration prior to sequential or separate administration of (i).

10. A pharmaceutical product for use according to any one of claims 7 to 9 wherein (i) and (ii) are each for administration in a therapeutically effective amount with respect to the individual components or in a subtherapeutic amount with respect to the individual components

11. Use of (i) parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof wherein parstatin peptide 1-26 functional derivative or analogue is at least 95% identical to amino acids of SEQ ID NO:1 ; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof wherein the exenatide functional derivative or analogue is a GLP-1 receptor agonist in the manufacture of a medicament for the treatment and/or prevention of ischemia and/or reperfusion injury.

12. Use of a combination comprising (i) parstatin peptide 1-26 or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof wherein parstatin peptide 1-26 functional derivative or analogue is at least 95% identical to amino acids of SEQ ID NO:1 ; and (ii) exenatide or a functional derivative or analogue, or a pharmaceutically acceptable salt thereof wherein the exenatide , functional derivative or analogue is a GLP-1 receptor agonist for treating and/or preventing ischemia and/or reperfusion injury in a ex-vivo heart organ prior to or during transplantation.