GB2436255A

GB2436255A - Organ preconditioning, arrest, protection, preservation and recovery (2)

Info

Publication number: GB2436255A
Application number: GB0711805A
Authority: GB
Inventors: Geoffrey Phillip Dobson
Original assignee: Global Cardiac Solutions Pty Ltd
Current assignee: Global Cardiac Solutions Pty Ltd
Priority date: 2002-12-23
Filing date: 2003-12-22
Publication date: 2007-09-19
Anticipated expiration: 2023-12-22
Also published as: GB2436255B; GB0711805D0

Abstract

A composition comprising (i) a local anaesthetic, (ii) an adenosine receptor agonist and (iii) an anti-adrenergic has been found to be particularly useful for the arrest, protection or preservation of a cell, tissue or organ following ischaemia. The preferred active ingredients are (i) lignocaine, (ii) adenosine and (iii) esmolol respectively.

Description

ORGAN PRECONDITIONING, ARREST, PROTECTION, PRESERVATION AND RECOVERY

(2)

Field of the Invention

The present invention relates to a composition for arresting, protecting or preserving a cell, tissue or organ following ischaemia.

Background of the Invention

Globally there are over I million elective open-heart surgery operations performed each year. One to three percent of these patients will die in the operating room, 10% of patients will leave with left ventricular dysfunction and 24% of high risk patients will die within 3 years. Moreover, in patients with elevated blood levels of creatine kinase (CK-MB) immediately following surgery, there is a significantly higher risk of early (first year) and late (3 to 5 years) mortality. Perioperative and post-operative mortality and morbidity are related to iatrogenic ischemia-reperfusion injury during cardiac surgery, and to inadequate myocardial protection.

In 2000, approximately 1.2 million open-heart surgeries were performed worldwide. About 64% of these were coronary artery bypass graft procedures, 24% were heart valve replacement or repair procedures, and about 12% were related to the repair of congenital heart defects1. About 1.2% were neonatal. The majority of open heart surgery operations (over 80%) require cardiopulmonary bypass and elective heart arrest using either a blood or crystalloid cardioplegia solution. During these procedures the heart may be arrested for 3hrs, and a maximum of 4hrs. About 10% of patients undergoing open-heart surgery will have post-operative left-ventricular dysfunction, and up to 30% will have atrial fibrillation following surgery2. 3- 5% of patients die in the operating room and 24% of high risk patients die within 3 years following surgery3. The amount of damage to the heart caused by 3-4hrs is such that the heart is increasingly less likely to recover function, and more likely than not recover after 4hrs arrest.

Currently the majority of cardioplegia solutions used contain high potassium (in excess of 15-20mM).4These include the widely used St Thomas No. 2 Hospital Solution which generally contains 110 mM NaCI, 16 mM KCI, 16 mM MgCl2, 1.2 mM CaC!2 and 10 mM NaHCO3 and has a pH of about 7.8. High potassium solutions usually lead to membrane depolarisation from about -80 to -50mV7.

page 1 Notwithstanding hyperkalemic solutions providing acceptable clinical outcomes, recent evidence suggests that progressive potassium induced depolarisation leads to ionic and metabolic imbalances that may be linked to myocardial stunning, ventricular arrhythmias, ischaemic injury, endothelial cell swelling, microvascular damage, cell death and loss of pump function during the reperfusion period. Infant hearts are even more prone to damage with prolonged cardioplegic arrest from high potassium than adult hearts710.

The major ion imbalances postulated are linked to an increased sodium influx which in turn activates the Na/Ca2 exchangers leading to a rise in intracellular Ca211. Compensatory activation of Na and Ca2 ion pumps then occur, which activate anaerobic metabolism to replenish ATP with a concomitant increase in tissue lactate and fall in tissue pH. Potentially damaging free radical generation and oxidative stress have also been implicated in high potassium arrest and partially reversed by the administration of antioxidants. In some cases, high potassium induced ischaemia has been reported to have damaged smooth muscle and endothelial function which can compromise coronary artery flow8.

In an attempt to minimise ischaemic injury during cardioplegic arrest, an increasing number of experimental studies have employed potassium channel openers instead of high potassium 12 Cardioprotection using nicorandil, aprikalim or pinacidil is believed to be linked to the opening of the potassium channel which leads to a hyperpolarised state, a shortening of the action potential and decreasing Ca2 influx into the cell One shortfall however is that the heart takes the same time or longer to recover with no improvement in function than with high potassium cardioplegic solutions. Another limitation is that pinacidil requires a carrier due to its low solubility in aqueous solutions. The carrier routinely used is dimethyl sulphoxide (DMSO) which is controversial when used in animal or human therapy.

Most investigators, including those who advocate using potassium channel openers, believe that as soon as blood flow is halted and the arrest solution administered, ischaemia occurs and progressively increases with time. To reduce the likelihood of damage, the applicant sought a cardioplegic solution that would place the heart in a reversible hypometabolic state analogous to the tissues of a hibernating turtle, a hummingbird in torpor or an aestivating desert frog. When these animals drop their metabolic rate (some by over 90%), their tissues do not become page 2 progressively ischaemic but remain in a down-regulated steady state where supply and demand are matched. An ideal cardioplegic solution should produce a readily reversible, rapid electrochemical arrest with minimal tissue ischaemia. Ideally, the heart should accumulate low tissue lactate, utilise little glycogen, show minimal changes in high-energy phosphates, cytosolic redox (NAD/NADH) and the bioenergetic phosphorylation (ATP/ADP Pi) ratio and free energy of ATP. There should be little or no change in cytosolic pH or free magnesium, minimal water shifts between the intracellular and extracellular phases, and no major ultrastructural damage to organelles such as the mitochondria. The ideal cardioplegic solution should produce 100% functional recovery with no atrial fibrillation, ventricular arrhythmias, cytosolic calcium overload, or other pump abnormalities. There is no cardioplegic solution currently available which fulfils all these requirements.

lschaemic heart disease is the single leading cause of death in the US and industrialised nations1. Each year, about 1.1 million US people suffer a heart attack, and industry estimates there are over 2.7 million cases globally per annum. About 42% of heart attacks (i.e. 460,000 patients in the USA) are fatal, and half of these occur within the first hour of experiencing symptoms and before the patient reaches the hospital. lschaemia (literally "to hold back blood") is usually defined as an imbalance between blood supply and demand to an organ or tissue and results in deficient oxygen, fuel or nutrient supply to cells. The most common cause of ischaemia is a narrowing of the artery or, in the extreme case, from a blood clot blocking the artery. In 90% of cases a blood clot is usually formed from rupture of an atherosclerotic plaque.

The response of a cell to ischaemia depends upon the time and extent of the deprivation of blood supply. A large percentage of deaths from cardiac ischaemia are due to ventricular fibrillation (VF) associated with profound metabolic, ionic and functional disturbances. Within seconds to minutes of coronary artery occlusion there is a shift from aerobic to anaerobic metabolism, a decrease in high-energy phosphates (phosphocreatine, ATP), glycogen loss, lactate accumulation, tissue acidosis, a rise in intracellular Na and Ca2 and extracellular K as well as changes to the transmembrane potential and ventricular dysfunction. Restoration of coronary flow within 15 mm can lead to full recovery of the heart 13,14 However, it can also stun the myocardium leading to potentially fatal arrhythmias'5. If ischaemia persists page 3 beyond 15 mm, the deprived area of the heart will undergo a progressive loss of ATP, increased Na and Ca2 entry, severe membrane injury, mitochondrial dysfunction, and the closing of gap junctions between cells thereby electrically isolating the damaged cells and eventually, cell death will occur16.

While early reperfusion, or restoration of the blood flow, remains the most effective means of salvaging the myocardium from acute ischaemia, the sudden influx of oxygen paradoxically may lead to further necrosis, ventricular arrhythmias and death619. The extent of "reperfusion injury" has been linked to a cascade of inflammatory reactions including the generation of cytokines, leukocytes, reactive oxygen species and free radicals20.

Reperfusion of ischaemic myocardium is necessary to salvage tissue from eventual death 22.28 However, reperfusion after even brief periods of ischaemia is associated with pathologic changes that represent either an acceleration of processes initiated during ischaemia per Se, or new pathophysiological changes that were initiated after reperfusion. The degree and extent of reperfusion injury can be influenced by inflammatory responses in the myocardium. lschaemia-reperfusion prompts a release of oxygen free radicals, cytokines and other pro-inflammatory mediators that activate both the neutrophils and the coronary vascular endothelium.

The inflammatory process can lead to endothelial dysfunction, microvascular collapse and blood flow defects, myocardial infarction and apoptosis22.

Pharmacologic anti-inflammatory therapies targeting specific steps have been shown to decrease infarct size and myocardial injury. Adenosine and nitric oxide are two compounds which have been observed to have beneficial effects against such neutrophil-mediated inflammation.

In 1990, Homeister and colleagues aimed to limit reperfusion injury by administering an intravenous bolus of lidocaine (2 mg/kg) in open-chest dogs 1 mm before a 90 mm occlusion of the left circumflex coronary artery and again I mm before reperfusion 29 At reperfusion, adenosine was infused (150 pg/kg/mI/mm) through an intracoronary catheter and continued for 1-hour reperfusion. It was concluded that the sequential treatment of lidocaine and adenosine reduced infarct size 29 In 1996, Vander-Heide and Reimer 30 failed to reproduce these findings mn the same model and concluded that intravenous adenosine therapy (150 pg/kg/mI/mm) page 4 during reperfusion with or without lidocaine pretreatment did not limit infarct size after mm regional ischaemia. In an attempt to clarify the issue, Garratt 31 and Mahaffey, 32 administered lidocaine and adenosine sequentially and separately in humans during balloon angioplasty and thrombolytic therapy respectively, but the results were again conflicting. Garratt and colleagues 31 proposed a potential benefit in 35 patients whereas Mahaffey and colleagues, in the larger AMISTAD trials involving 236 acute myocardial infarction patients, concluded that the presence of lidocaine made no difference to the outcome of adenosine-treated patients in reducing infarct size. Indeed, the clinical outcomes of the adenosine-treated group in the AMISTAD trials tended to be slightly worse than in the placebo group 32 The applicant previously found that the heart can be better protected by using a potassium channel opener and/or an adenosine receptor agonist (preferably adenosine) and a local anaesthetic (preferably lidocaine or lignocaine) to arrest and then preserve the heart under physiological concentrations of potassium. Thus reducing the risk of potassium induced injury to the tissue which prior art high potassium arrest solutions may induce (see WO 00/56145) (the entire disclosure of which is incorporated herein by reference). In this reference these components are administered in a single preparation or simultaneously.

This cardioplegia solution containing the combination of the potassium channel opener and local anaesthetic was shown by the applicant to generally improve functional recovery from arrest of the organ over existing solutions.

This solution provides improved functional recovery of the arrested heart.

However, functional recovery is still decreased with increasing arrest time.

Accordingly, there is a need for a method which further improves functional recovery of an arrested tissue, and/or reduces damage to an arrested tissue, and more particularly after increasing arrest time of the tissue. In particular, there is a need for improved protection of the tissue from damage during arrest.

Also, as stated above, this solution results in the arrest of the heart under physiological potassium concentrations. The arrested heart is then reperfused (i.e., blood flow restored) to recover function. However, there are also risks in further damaging the heart at reperfusion. Accordingly, there is also a need for a method of recovering a tissue from arrest, with improved functional recovery during reperfusion.

page 5 The heart possesses an extraordinary ability to remember' short episodes of sublethal ischaemia-reperfusion (angina) which protects the myocardium and microvascular from a subsequent lethal period of ischaemia (infarction) 41,42 The phenomenon, known as "ischaemic preconditioning" or "preconditioning", is the most powerful means of delaying cell death known. It was first described in 1986 by Murry, Jennings and Reimer who reported an infarct size reduction from 29% to 7% in anaesthetised open-chested dogs after three brief episodes of brief ischaemia followed by 40 mm coronary artery occlusion 41 Since that time, the phenomenon has been described in tissues and organs of most animal models studied including human Two different time frames for preconditioning have been identified; an early "classical" window that lasts I to 3 hrs after the stimulus, and a later "delayed" window which develops over many hours and can last up to 12 to 72 hours 18,36.43,46 The heart can also be protected by preconditioning other organs such as kidney or intestine. This phenomenon is termed "remote preconditioning" . However, most clinicians are reluctant to precondition a patient's diseased heart by temporarily tying off the vessel in the clinical setting. Therefore, there is also a need to develop a pharmacological mimetic or composition for precoriditioning tissue, to protect the tissue from a subsequent period of ischemia without the need to physically tie-off vessels.

Summary of the Invention

This invention is directed towards overcoming, or at least alleviating, one or more of the difficulties or deficiencies associated with the prior art.

In one embodiment, the invention provides a composition for reducing electrical disturbance of a cell's resting membrane potential, the composition comprising an effective amount of a local anaesthetic, an adenosine receptor agonist and an anti-adrenergic.

In another embodiment, the invention provides a composition for reducing damage to an cell, tissue or organ following ischaemia, the composition comprising an effective amount of a local anaesthetic, an adenosine receptor agonist and an anti-adrenergic.

page 6 In another embodiment, the invention provides a composition for preconditioning a cell or tissue during ischaemia or reperfusion, the composition comprising an effective amount of a local anaesthetic, an adenosine receptor agonist and an anti-adrenergic.

In another embodiment, the invention provides a composition for reducing damage to cells, organs and tissues before, during and following a surgical or clinical intervention the composition comprising an effective amount of a local anaesthetic, an adenosine receptor agonist and an anti-adrenergic.

In another embodiment, the invention provides a composition for reducing either or both inflammation and clotting in a tissue or organ the composition comprising an effective amount of a local anaesthetic, an adenosine receptor agonist and an anti-adrenergic.

The compositions of the invention are applicable to any cell, tissue or organ.

Examples include where the cell is a myocyte, endothelial cell, smooth-muscle cell, neutrophil, platelet and other inflammatory cells, or the tissue is heart tissue or vasculature, or the organ is a heart.

In some embodiments, the composition used in these methods further comprises one or more of an antioxidant, ionic magnesium, an impermeant and a metabolic substrate. The composition may also oxygenated. The composition may also be formulated into a medicament by combining with a blood-based or crystalloid (non-cell, non-protein) carrier. In such a medicament, it is desirable in some applications to that the concentrations of one or more of sodium, calcium and chloride are lower than physiological concentrations. Also, it is desirable to use the medicaments at different temperatures, namely: profound hypothermia (0 to 4 degrees Celsius), moderate hypothermia (5 to 20 degrees Celsius), mild hypothermia (20 to 32 degrees Celsius) or normothermia (32 to 38 degrees Celsius).

The components of the medicament or composition may be combined before administration or when the components are administered substantially simultaneously or co-administered.

Detailed Description

The applicant has surprisingly found that the simultaneous delivery of a solution a local anaesthetic together with component(s) as detailed below prior to, page 7 during or following ischaemia markedly reduces cell damage resulting from ischaemia. In particular, continuous administration of a solution (which may be carried in physiological saline or compatible fluid (eg, patient's own blood)) of the components results in significantly less damage to a cell, organ or tissue, such as a heart, than delivery of the components of the composition independently (eg, one component (adenosine) parenterally and the other (lignocaine) in intermittent bolus doses).

The simultaneous delivery of the two components briefly prior to ischaemia, throughout ischaemia and reperfusion shows surprising increased efficacy. In the invention, there is therefore provided a composition for reducing myocardial tissue damage during a heart attack or cardioplegia by delivering the composition to the tissue. The composition also protects myocardial tissue from reperfusion injury, including inflammatory and blood coagulation effects often experienced during reperfusion following an ischaemic event.

The composition also provides a method for reducing infarction size and/or reducing inflammation and blood coagulation responses in myocardial tissue during ischaemia and/or reperfusion.

The composition also provides a method for reducing electrical disturbances in the heart such as atrial or ventricular arrhythmias (including lethal ventricular tachycardia's and ventricular fibrillation) during ischaemia and/or reperfusion.

The composition of the present invention protects the organ after arrest of the organ, with good to excellent recoveries of function after reperfusion.

The invention also provides that use of the composition can extend to many therapeutic applications, including without limitation, cardiovascular diagnosis (including coronary angiography, myocardial scintigraphy, non-invasive diagnosis of dual AV nodal conduction), use in treatment of heart attack, resuscitation therapy, short-term and longterm storage of organs tissues or cells (including graft vessels), use before, prior to, during or following open-heart surgery, angioplasty and other therapeutic interventions.

In one embodiment, the composition comprises adenosine and lignocainein the weight ratio of about 1:2.

page 8 In this application, without being bound by this mode of action, protection is thought to involve a multi-tiered system from modulating membrane excitability to a multitude of intracellular signalling pathways leading to (i) reduced ion imbalances, in particular sodium and calcium ion loading in the cells, (ii) improved atrial and ventricular matching of electrical conduction to metabolic demand, which may involve modulation of gap junction communication, (iii) vasodilation of coronary arteries and (ii) attenuation of the inflammatory response to injury Infusion of the composition during pretreatment and ischaemia and reperfusion provides continuous protection from ischaemic tissue injury including protection from lethal arrhythmias. The protection from localised injury and inflammation can also be obtained when placing a stent into a vessel such as during angioplasty. The composition is also used within a polymer or special coating for a stent for use in any vessel of the body including coronary arteries, carotid arteries, or leg arteries of the body.

The composition according to the invention includes a calcium antagonist such as an indirect calcium antagonist which includes a potassium channel opener.

Potassium channel openers are agents which act on potassium channels to open them through a gating mechanism. This results in efflux of potassium across the membrane along its electrochemical gradient which is usually from inside to outside of the cell. Thus potassium channels are targets for the actions of transmitters, hormones, or drugs that modulate cellular function, It will be appreciated that the potassium channel openers include the potassium channel agonists which also stimulate the activity of the potassium channel with the same result, It will also be appreciated that there are diverse classes of compounds which open or modulate different potassium channels; for example, some channels are voltage dependent, some rectifier potassium channels are sensitive to ATP depletion, adenosine and opiolds, others are activated by fatty acids, and other channels are modulated by ions such as sodium and calcium (i.e.. channels which respond to changes in cellular sodium and calcium). More recently, two pore potassium channels have been discovered and thought to function as background channels involved in the modulation of the resting membrane potential.

Potassium channel openers may be selected from the group consisting of: nicorandil, diazoxide, minoxidil, pinacidil, aprikalim, cromokulim and derivative U-page 9 89232, P-1075 (a selective plasma membrane KATP channel opener), emakalim, YM-934, (+)-7,8-dihydro-6, 6-dimethyl-7-hydroxy-8-(2-oxo-1 -piperidinyl)-6H-pyrano[2,3-f] benz-2, 1, 3-oxadiazole (NIP-i 21), R03 16930, RWJ29009, SDZPCO400, rimakalim, symakalim, YM099, 2-(7,8-dihydro-6,6-dimethyl-6H-[1,4]oxazino[2,3-f][2,1, 3jbenzoxadiazol-8-yl) pyridine N-oxide, 9-(3-cyanophenyl)- 3,4,6,7,9,1 0-hexahydro-i,8-(2H,5H)-acridinedione (ZM244085), [(9R)-9-(4-fluoro-3- 1 25iodophenyl)-2,3, 5,9-tetrahydro-4H-pyrano[3,4-b]thieno[2,3-e]pyrsdin-8(7H)-one...

1,1-dioxide] ([1 251]A-3 12110), (-)-N-(2-ethoxyphenyl)-N'-( 1,2, 3-trimethylpropyl)-2- nitroethene-1,1 -diamine (Bay X 9228), N-(4-benzoyl phenyl)-3, 3, 3-trifluro-2-hydroxy- 2-methylpropionamine (ZD6169), ZD6169 (KATP opener) and ZD0947 (KATP opener), WAY-133537 and a novel dihydropyridine potassium channel opener, A- 278637. In addition, potassium channel openers can be selected from BK-activators (also called BK-openers or BK(Ca)-type potassium channel openers or large-conductance calcium-activated potassium channel openers) such as benzimidazolone derivatives NSOO4 (5-trifluoromethyl-1 -(5-chloro-2-hydroxyphenyl)- 1, 3-dihydro-2H-benzimidazole-2-one), NS 1619 (1,3-dihydro-1 -[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl) -2H-benzimidazol-2-one), NS 1608 (N-(3- (trifi uoromethyl)phenyl)-N'-(2-hydroxy-5-chlorophenyl)urea), BMS-204352, retigabine (also GABA agonist). There are also intermediate (eg. benzoxazoles, chlorzoxazone and zoxazolamine) and small-conductance calcium-activated potassium channel openers.

As mentioned, potassium channel openers may act as indirect calcium antagonists, i.e. they act to reduce calcium entry into the cell by shortening the cardiac action potential duration through the acceleration of phase 3 repolarisation, and thus shorten the plateau phase. Reduced calcium entry is thought to involve L-type calcium channels, but other calcium channels may also be involved.

Some embodiments of the invention utilise direct calcium antagonists, the principal action of which is to reduce calcium entry into the cell. These are selected from at least five major classes of calcium channel blockers as explained in more detail below. It will be appreciated that these calcium antagonists share some effects with potassium channel openers, particularly ATP-sensitive potassium channel openers, by inhibiting calcium entry into the cell.

page 10 Adenosine is particularly preferred as the potassium channel opener or agonist. Adenosine is capable of opening the potassium channel, hyperpolarising the cell, depressing metabolic function, possibly protecting endothelial cells, enhancing preconditioriing of tissue and protecting from ischaemia or damage. Adenosine's actions are complex as the drug has many broad-spectrum properties. Adenosine has been shown to increase coronary blood flow, hyperpolarise the cell membrane, and protect during ischemia and reperfusion 22.Adenosine also acts as a early' and delayed' preconditioning trigger' or agent to protect the heart against ischaemic injury 36,37* Part of adenosine's cardioprotective properties are believed to be activation of one or more of the adenosine receptor subtypes (Al, A2a, A2b and A3) 38 Much of adenosine's protection has been ascribed to Al and A3 receptor activation and their associated transduction pathways leading to preconditioning, protection and preservation of cell integrity It is also known that adenosine, by activating Al receptors, is involved in slowing the sinoatrial nodal pacemaker rate (negative chronotropy), delaying atrioventricular (A-V) nodal impulse conduction (negative dromotropy), reduces atrial contractility (negative inotropy), and inhibits the effect of catecholamines (anti-adrenergic effect) 40, The Al-stimulated negative chronotropic, dromotropic and inotropic effects of adenosine are linked to the drug's action to reduce the activity of adenyl cyclase, to activate the inward rectifier potassium current (lK..Ado), inhibition of phospholipid turnover, activation of AlP-sensitive K channels, inhibits effect of catecholamines on the L-type Ca2 current and activation of nitric oxide synthase in AV nodal cells. A3 receptors have also shown to have direct cardioprotective effects, and A2 receptors have potent vasodilatory and anti-inflammatory actions in response to injury 22,38 Adenosine is also an indirect calcium antagonist, vasodilator, antiarrhythmic, antiadrenergic, free radical scavenger, arresting agent, anti-inflammatory agent (attenuates neutrophil activation), analgesic, metabolic agent and possible nitric oxide donor.

It will be appreciated that anti-adrenergics such as beta-blockers, for example, esmolol, atenolol, metoprolol and propranolol could be used in combination with the potassium channel opener to reduce calcium entry into the cell. Preferably, the beta-blocker is esmolol. Similarly, alpha (1)-adrenoceptor-antagonists such as prazosin, page 11 could be used in combination with the potassium channel opener to reduce calcium entry into the cell and therefore calcium loading.

According to this aspect of the present invention there is provided a composition comprising an effective amount of a ocal anaesthetic, an adenosine receptor agonist and an anti-adrenergic. Preferably, the antiadrenergic is a beta-blocker. Preferably the beta-blocker is esmolol.

Adenosine is also known to indirectly inhibit the sodium-calcium exchanger which would reduce cell sodium and calcium loading. It will be appreciated that inhibitors of the sodium-calcium exchanger would lead to reduced calcium entry and magnify the effect of adenosine. Na/Ca2 exchange inhibitors may include benzamyl, KB-R7943 (2-[4-(4-Nitrobenzyloxy)phenyljethyl]isothiourea mesylate) or SEAO400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline).

Since one of adenosine's properties is to reduce calcium entry and sodium entry in the heart and coronary vascular cells, it will be further appreciated that a compound leading to reduced calcium and sodium entry (or reduce calcium oscillations in the cell) before, during and/orfollowing treatment could be used in combination with adenosine to reduce calcium entry into the cell. Such compounds may be selected from calcium channel blockers from three different classes: 1,4-dihydropyridines (eg. nitrendipine), phenylalkylamines (eg. verapamil), and the benzothiazepines (e.g. diltiazem, nifedipine).

Calcium channel blockers are also called calcium antagonists or calcium blockers. They are often used clinically to decrease heart rate and contractility and relax blood vessels. They may be used to treat high blood pressure, angina or discomfort caused by ischaemia and some arrhythmias, and they share many effects with beta-blockers (see discussion above).

Five major classes of calcium channel blockers are known with diverse chemical structures: 1. Benzothiazepines: eg Diltiazem, 2. Dihydropyridines: eg nifedipine, Nicardipine, nimodipine and many others, 3. Phenylalkylamines: eg Verapamil, 4. Diarylaminopropylamine ethers: eg Bepridil, 5. Benzimidazole-substituted tetralines: eg Mibefradil.

The traditional calcium channel blockers bind to L-type calcium channels ("slow channels") which are abundant in cardiac and smooth muscle which helps page 12 explain why these drugs have selective effects on the cardiovascular system.

Different classes of L-type calcium channel blockers bind to different sites on the aiphal-subunit, the major channel-forming subunit (alpha2, beta, gamma, delta subunits are also present). Different sub-classes of L-type channel are present which may contribute to tissue selectivity. More recently, novel calcium channel blockers with different specificities have also been developed for example, Bepridil, is a drug with Na+ and K+ channel blocking activities in addition to L-type calcium channel blocking activities. Another example is Mibefradil, which has T-type calcium channel blocking activity as well as L-type calcium channel blocking activity.

Three common calcium channel blockers are diltiazem (Cardizem), verapamil (Calan) and Nifedipine (Procardia). Nifedipine and related dihydropyridines do not have significant direct effects on the atrioventricular conduction system or sinoatrial node at normal doses, and therefore do not have direct effects on conduction or automaticity. While other calcium channel blockers do have negative chronotropic/dromotropic effects (pacemaker activity/conduction velocity). For example, Verapamil (and to a lesser extent diltiazem) decreases the rate of recovery of the slow channel in AV conduction system and SA node, and therefore act directly to depress SA node pacemaker activity and slow conduction. These two drugs are frequency-and voltage-dependent, making them more effective in cells that are rapidly depolarizing. Verapamil is also contraindicated in combination with beta-blockers due to the possibility of AV block or severe depression of ventricular function. In addition, mibefradil has negative chronotropic and dromotropic effects.

Calcium channel blockers (especially verapamil) may also be particularly effective in treating unstable angina if underlying mechanism involves vasospasm.

Omega conotoxin MVIIA (SNX-111) is an N type calcium channel blocker and is reported to be 100-1 000 fold more potent than morphine as an analgesic but is not addictive. This conotoxin is being investigated to treat intractible pain. SNX-482 a further toxin from the venom of a carnivorous spider venom, blocks R-type calcium channels. The compound is isolated from the venom of the African tarantula, Hysterocrates gigas, and is the first R-type calcium channel blocker described. The R-type calcium channel is believed to play a role in the body's natural communication network where it contributes to the regulation of brain function. Other caictum channel blockers from animal kingdom include Kurtoxin from South African Scorpion, SNX-page 13 482 from African Tarantula, Taicatoxin from the Australian Taipan snake, Agatoxin from the Funnel Web Spider, Atracotoxin from the Blue Mountains Funnel Web Spider, Conotoxin from the Marine Snail, HWTX-l from the Chinese bird spider, Grammotoxin SIA from the South American Rose Tarantula. This list also includes derivatives of these toxins that have a calcium antagonistic effect.

Direct AlP-sensitive potassium channel openers (eg nicorandil, aprikalem) or indirect ATP-sensitive potassium channel openers (eg adenosine, opioids) are also indirect calcium antagonists and reduce calcium entry into the tissue. One mechanism believed for ATP-sensitive potassium channel openers also acting as calcium antagonists is shortening of the cardiac action potential duration by accelerating phase 3 repolarisation and thus shortening the plateau phase. During the plateau phase the net influx of calcium may be balanced by the efflux of potassium through potassium channels. The enhanced phase 3 repolarisation may inhibit calcium entry into the cell by blocking or inhibiting L-type calcium channels and prevent calcium (and sodium) overload in the tissue cell.

Potential targets for the combinational therapy include cardioplegia, management of ischaemic syndromes without or without clot-busters, cardiac surgery (on and off-pump), arrhythmia management, coronary interventions (balloon and stent), preconditioning an organ, tissue or cell to ischaemic stress, longer-term organ or cell preservation, pen-and post-operative pain management, pen-and post operative anti-inflammatory treatments, pen-and post operative anti-clotting strategies, resuscitation therapies, and other related therapeutic interventions.

Calcium channel blockers can be selected from nifedipine, nicardipine, nimodipine, nisoldipine, lercanidipine, telodipine, angizem, altiazem, bepridil, amlodipine, felodipine, isradipine and cavero and other racemic variations.

In addition, it will be appreciated that calcium entry could be inhibited by other calcium blockers which could be used instead of or in combination with adenosine and include a number of venoms from marine or terrestrial animals such as the omega-conotoxin GVIA (from the snail conus geographus) which selectively blocks the N-type calcium channel or omega-agatoxin lIlA and IVA from the funnel web spider Age!elnopsis aperta which selectively blocks R-and P/Q-type calcium channels respectively. There are also mixed voltage-gated calcium and sodium page 14 channel blockers such as NS-7 to reduce calcium and sodium entry and thereby assist cardioprotection.

The composition according to the invention includes an adenosine receptor agonist, It will be appreciated that the adenosine receptor agonists include compounds which act both directly and indirectly on the receptor resulting in activation of the receptor, or mimic the action of the receptor having the same net effect.

Suitable adenosine receptor agonists can be found in the reviews by Linden 3872 72. . 73 6 and colleagues, Hayes and Belardinelli. They may be selected from: N - cyclopentyladenosine (CPA), N-ethylcarboxamido adenosine (NECA), 2-[p-(2- carboxyethyl) phenethylamino5'Nethylcarboxarnjd0 adenosine (CGS-2 1680), 2-chloroadenosine, 2-chloro-N6-cyclopentyladenosine (CCPA), N-(4-aminobenzyl)-9-[5-(rnethylcarbonyl) beta-D-robofuranosyl]-acienine (AB-MECA), ([IS-[ 1 a,2b, 2-thienyl)-1 carboxamide (AM P579), N6-(R)-phenylisopropyladenosine (R-P LA), aminophenylethyladenosine (APNEA) and_cyclohexyladenosine (CHA) 72* Others include full adenosine Al receptor agonists such as lsJ-[3-(R)-tetrahydrofuranylj-6-aminopurine riboside (CVT-51 0), or partial agonists such as CVT-2759 and aflosteric enhancers such as PD81723 7476* Other agonists include N6-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yI)adenosine (TCPA), a very selective agonist with high affinity for the human adenosine Al receptor and allosteric enhancers of Al adenosine receptor includes the 2-amino-3-naphthoylthiophenes 78* CCPA is a particularly preferred adenosine receptor agonist. CCPA an Al adenosine receptor agonist.

Modulation of agonist responses at the Al adenosine receptor can also be achieved indirectly by an irreversible antagonist, receptor-G protein uncoupling and by the G protein activation state Thus any agonist or antagonist which modulates the G protein activation state may be used to mimic adenosine receptor activation.

There is also some evidence that there is some cross-talk between adenosine receptors. Furthermore, there is data suggesting that there are converging pathways and/or receptor cross-talk between adenosine 1 (and perhaps A3) receptors and page 15 deltal-opioid receptor mediated cardioprotection 80 Thus opiold receptor activation may result in identical protection as Al receptor activation. It would be appreciated that opioids could be used in combination with a potassium channel opener or adenosine receptor agonists.

Oploids, also known or referred to as opioid agonists, are a group of drugs that inhibit opium (Gr opion, poppy juice) or morphine- like properties and are generally used clinically as moderate to strong analgesics, in particular, to manage pain, both pen-and post-operatively. Other pharmacological effects of opioids include drowsiness, respiratory depression, changes in mood and mental clouding without loss of consciousness.

Opioids are also believed to be involved as part of the trigger' in the process of hibernation, a form of dormancy characterised by a fall in normal metabolic rate and normal core body temperature. In this hibernating state, tissues are better preserved against damage that may otherwise be caused by diminished oxygen or metabolic fuel supply, and also protected from ischemia reperfusion injury.

There are three types of opioid peptides: enkephalin, endorphin and dynorphin.

Opioids act as agonists, interacting with stereospecific and saturable binding sites, in the heart, brain and other tissues. Three main opioid receptors have been identified and cloned, namely mu, kappa, and delta receptors. All three receptors have consequently been classed in the G-protein coupled receptors family (which class includes adenosine and bradykinin receptors). Opiold receptors are further subtyped, for example, the delta receptor has two subtypes, delta-i and delta-2.

Cardiovascular effects of opioids are directed within the intact body both centrally (i.e., at the cardiovascular and respiratory centres of the hypothalamus and brainstem) and peripherally (i.e., heart myocytes and both direct and indirect effects on the vasculature). For example, opioids have been shown to be involved in vasodilation. Some of the action of opioids on the heart and cardiovascular system may involve direct opioid receptor mediated actions or indirect, dose dependent non-opioid receptor mediated actions, such as ion channel blockade which has been observed with antiarrhythmic actions of opioids, such as arylacetamide drugs. It is also known that the heart is capable of synthesising or producing the three types of page 16 opioid peptides, namely, enkephalin, endorphin and dynorphin. However, only the delta and kappa opioid receptors have been identified on ventricular myocytes.

Without being bound by any mode of action, opioids are considered to provide cardioprotective effects, by limiting ischaemic damage and reducing the incidence of arrhythmias, which are produced to counter-act high levels of damaging agents or compounds naturally released during ischemia. This may be mediated via the activation of ATP sensitive potassium channels in the sarcolemma and in the mitochondrial membrane and involved in the opening potassium channels. Further, it is also believed that the cardioprotective effects of opioids are mediated via the activation of ATP sensitive potassium channels in the sarcolemma and in the mitochondrial membrane. Thus it is believed that the opioid can be used in stead or in combination with the potassium channel opener or adenosine receptor agonist as they are also involved in indirectly opening potassium channels.

It will be appreciated that the opioids include compounds which act both directly and indirectly on opioid receptors. Opioids also include indirect dose dependent, non-opioid receptor mediated actions such as ion channel blockade which have been observed with the antiarrhythmic actions of opioids.

Local anaesthetic agents are drugs which are used to produce reversible loss of sensation in an area of the body. Many local anaesthetic agents consist of an aromatic ring linked by a carbonyl containing moiety through a carbon chain to a substituted amino group. In general there are 2 classes of local anaesthetics defined by their carbonyl-containing linkage group. The ester agents include cocaine, amethocaine, procaine and chloroprocaine, whereas the amides include prilocaine, mepivacaine, bupivacaine, mexiletine and lignocaine. At high concentrations, many drugs that are used for other purposes possess local anaesthetic properties. These include opioid analgesics, Beta- adrenoceptor antagonists, anticonvulsants (lamotrigine and lifarizine) and antihistamines. The local anaesthetic component of the composition according to the present invention may be selected from these classes, or derivatives thereof, or from drugs than may be used for other purposes.

Preferably, the component possesses local anaesthetic properties also.

Preferably the local anaesthetic is Lignocaine. In this specification "lignociane" and lidocaine" are used interchangeably. Lignocaine is preferred as it is capable of actina as a local anaesthetic probably by blocking sodium fast channels, depressing page 17 metabolic function, lowering free cytosolic calcium, protecting against enzyme release from cells, possibly protecting endothelial cells and protecting against myofilament damage. At lower therapeutic concentrations lidocaine normally has little effect on atrial tissue, and therefore is ineffective in treating atrial fibrillation, atrial flutter, and supraventricular tachycardias 65 Lignocaine is also a free radical scavenger, an antiarrhythmic and has anti-inflammatory and anti-hypercoagulable properties. It must also be appreciated that at non-anaesthetic therapeutic concentrations, local anaesthetics like lidocaine may not completely block the voltage-dependent sodium fast channels, but down-regulate channel activity and reduce sodium entry 82.83 As an anti-arrhythmic, lidocaine is believed to target small sodium currents_that normally continue through phase 2 of the action potential and consequently shortens the action potential and the refractory period 65 Lignocaine is a local anaesthetic which is believed to block sodium fast channels and has anti-arrhythmatic properties by reducing the magnitude of inward sodium current 62-65 The accompanying shortening of the action potential is thought to directly reduce calcium entry into the cell via Ca2 selective channels and Na/Ca2 exchange 65 Recent reports also implicate lignocaine with the scavenging of free radicals such as hydroxyl and singlet oxygen in the heart during reperfusion 66 Associated with this scavenging function, lignocaine may also inhibit phospholipase activity and minimise membrane degradation during ischaemia.

Lignocaine can also depress vascular relaxations by a complex mechanism including poly(ADP-ribose) synthetase enzyme activity, but this effect has recently been shown to be pH dependent with little inhibition occurring below pH 7.2. Lignocaine's vasodilatory effects are believed due to calcium entry blockade that do not appear to involve Na channel blockade or opening of K-channels 67 Lignocaine has also been shown to have a myocardial protective effect and in one study was found to be superior to high potassium solutions. However, these experiments show that lignocaine alone at 0.5, 1.0 and 1.5 mM gave highly variable functional recoveries using the isolated working rat heart. Lignocaine has also been shown to reduce infarct size in the brain and protect against reperfusion injury in the heart. More recently lignocaine has been shown to exhibit a number of pharmacological actions not related to the sodium channel block. For example, recent work has shown that page 18 local anaesthetics, including lignocaine, inhibit inflammatory responses 68,69 They also have beneficial effects in a number of pathological processes dependent on an overly active inflammatory response such as adult respiratory distress syndrome and in ischaemia-reperfusion injury. Intravenous lignocaine has also been shown to be effective in prevention of deep vein thrombosis after elective hip surgery 70 Lignocaine therefore appears to be effective in both attenuating inflammatory and hypercoagulable states (post-operative thrombosis) in the clinical setting Unlike adenosine, lignocaine has not been implicated in the preconditioning of a cell, tissue or organ.

As lignocaine acts as a local anaesthetic by primarily blocking sodium fast channels, it will be appreciated that other sodium channel blockers could be used in combination with the local anaesthetic in the composition of the present invention. It will be appreciated that sodium channel blockers include compounds that substantially block sodium channels and also downregulate sodium channels.

Examples of suitable sodium channel blockers include venoms such as tetrodotoxin, and the drugs primaquine, QX, HNS-32 (CAS Registry # 186086-10-2), NS-7, kappa-opioid receptor agonist U50 488, crobenetine, pilsicainide, phenytoin, tocainide, mexiletine, RS1 00642, riluzole, carbamazepine, flecainide, propafenone, amiodarone, sotalol, imipramine and moricizine, or any of derivatives thereof. Other suitable sodium channel blockers include: Vinpocetine (ethyl apovincaminate); and Beta-carboline derivative, nootropic beta-carboline (ambocarb, AMB).

Lidocaine in addition to being a local anaesthetic also has anti-inflammatory properties. Although the beneficial clinical effect of loca' anaesthetics and the regulation of the immune system remain poorly defined, studies have suggested several mechanisms of action including inhibition of the adhesion of granulocytes to the inflammatory sites, reduction of lysosomal activity, decreased production of superoxide and the suppression of metabolic activation and secretion of LTB4 and IL-I from granulocytes. Lidocaine-related local anaesthetics have been shown to inhibit lymphocyte maturation and proliferation, inhibit the migration of macrophages into tissues, inhibit the expression of CD11b/CDI8 by polymorphonuclear cells, inhibit the adhesion of leucocytes to injured venules and inhibit the LPS-stimulated secretion of LTB4 and IL-i from peripheral blood mononuclear cells. Lidocaine's page 19 actions have also been linked to lidocaine-induced reduction in the release of substance P from nerve terminals.

Since polarisation of the membrane potential of tissue cell is one of the key factors involved in superior arrest, protection and preservation, we reasoned that adenosine and lidocaine may act synergistically to further produce enhanced inhibition of inflammation.

Antioxidants are commonly enzymes or other organic substances that are capable of counteracting the damaging effects of oxidation in the tissue. The antioxidant component of the composition according to the present invention may be selected from one or more of the group consisting of: allopurinol, carnosine, histidine, Coenzyme Q 10, n-acetyl-cysteine, superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GP) modulators and regulators, catalase and the other metalloenzymes, NADPH and AND(P)H oxidase inhibitors, glutathione, U-74006F, vitamin E, Trolox (soluble form of vitamin E), other tocopherols (gamma and alpha, beta, delta), tocotrienols, ascorbic acid, Vitamin C, Beta-Carotene (plant form of vitamin A), selenium, Gamma Linoteic Acid (GLA), alpha-lipoic acid, uric acid (urate), curcumin, bilirubin, proanthocyanidins, epigallocatechin gallate, Lutein, lycopene, bioflavonoids, polyphenols, trolox(R), dimethylthiourea, tempol(R), carotenoids, coenzyme Q, melatonin, flavonoids, polyphenols, aminoindoles probucol and nitecapone, 21-aminosteroids or lazaroids, sulphydryl-containing compounds (thiazolidine, Ebselen, dithiolethiones), and N-acetylcysteine. Other antioxidants include the ACE inhibitors (captopril, enalapril, lisinopril) which are used for the treatment of arterial hypertension and cardiac failure on patients with myocardial infarction. ACE inhibitors exert their beneficial effects on the reoxygenated myocardium by scavenging reactive oxygen species. Other antioxidants that could also be used include beta-mercaptopropionylglycine, 0-phenanthroline, dithiocarbamate, selegilize and desferrioxamine (Desferal), an iron chelator, has been used in experimental infarction models, where it exerted some level of antioxidant protection. Spin trapping agents such as 5'-5-dimethyl-1-pyrrolione-N-oxide (DMPO) and (a-4-pyridyl-1 -oxide)-N-t-butylnitrone (POBN) also act as antioxidants. Other antioxidants include: nitrone radical scavenger alpha-phenyl-tert-N-butyl nitrone (PBN) and derivatives PBN (including disuiphur derivatives); N-2-mercaptopropionyl glycine (MPG) a specific scavenger of the OH page 20 free radical; lipooxygenase inhibitor nordihydroguaretic acid (NDGA); Alpha Lipoic Acid; Chondroitin Sulfate; L-Cysteine; oxypurinol and Zinc.

Preferably, the antioxidant is allopurinol (1 H-Pyrazolo[3,4-a]pyrimidine-4-oI).

Allopurinol is a competitive inhibitor of the reactive oxygen species generating enzyme xanthine oxidase. Allopurinol's antioxidative properties may help preserve myocardial and endothelial functions by reducing oxidative stress, mitochondriat damage, apoptosis and cell death.

In addition, protease inhibitors attenuate the systemic inflammatory response in patients undergoing cardiac surgery with cardiopulmonary bypasss, and other patients where the inflammatory response has been heightened such as AIDS or in the treatment of chronic tendon injuries. Some broad spectrum protease inhibitors such as aprotinin also reduce blood loss and need for blood transfusions in surgical operations such as coronary bypass.

In another embodiment of the present invention there is provided a composition according to the present invention, further including an effective amount of a sodium hydrogen exchange inhibitor. The sodium hydrogen exchange inhibitor reduces sodium and calcium entering the cell.

The sodium hydrogen exchange inhibitor may be selected from one or more of the group consisting of amiloride, cariporide, eniporide, triamterene and EMD 84021, EMD 94309, EMD 96785 and HOE 642 and T-162559 (inhibitors of the isoform I of the Na/H exchanger). Preferably, the sodium hydrogen exchange inhibitor is amiloride. Amiloride inhibits the sodium proton exchanger (Na4/H exchanger, also often abbreviated NHE-1) and reduces calcium entering the cell. During ischaemia excess cell protons (or hydrogen ions) are exchanged for sodium via the Na/H4 exchanger.

In yet another embodiment of the present invention there is provided a composition according to the present invention, further including an effective amount of: a source of magnesium in an amount for increasing the amount of magnesium in a cell in the tissue; and a source of calcium in an amount for increasing the amount of calcium in a cell in the tissue.

page 21 Elevated magnesium and low calcium has been associated with protection during ischaemia and reoxygenation of the organ. The action is believed due to decreased calcium loading.

Preferably the magnesium is present at a concentration of between 0.5mM to 20mM, more preferably about 2.5mM. Preferably the calcium present is at a concentration of between 0.1mM to 2.5mM, more preferably about 0.3mM.

The composition may include an effective amount of elevated magnesium.

The composition according to the invention may also include an impermeant or a compound for minimizing or reducing the uptake of water by a cell in a tissue.

Compounds for minimizing or reducing the uptake of water by a cell in a tissue are typically impermeants or receptor antagonists or agonists.

A compound for minimizing or reducing the uptake of water by a cell in the tissue tends to control water shifts, i.e., the shift of water between the extracellular and intracellular environments. Accordingly, these compounds are involved in the control or regulation of osmosis. One consequence is that a compound for minimizing or reducing the uptake of water by a cell in the tissue reduces cell swelling that is associated with Oedema, such as Oedema that can occur during ischemic injury.

An impermeant according to the present invention may be selected from one or more of the group consisting of: sucrose, pentastarch, hydroxyethyl starch, raffinose, mannitol, gluconate, lactobionate, and colloids. Colloids include albumin, hetastarch, polyethylene glycol (PEG), Dextran 40 and Dextran 60. Other compounds that could be selected for osmotic purposes include those from the major classes of osmolytes found in the animal kingdom including polyhydric alcohols (polyols) and sugars, other amino acids and amino-acid derivatives, and methylated ammonium and sulfonium compounds.

Cell swelling can also result from an inflammatory response which may be important during organ retrieval, preservation and surgical grafting. Substance P, an important pro-inflammatory neuropeptide is known to lead to cell oedema and therefore antagonists of substance P may reduce cell swelling. Indeed antagonists of substance P, (-specific neurokinin-1) receptor (NK-1) have been shown to reduce inflammatory liver damage, i.e., oedema formation, neutrophil infiltration, hepatocyte apoptosis, and necrosis. Two such NK-1 antagonists include CP-96,345 or page 22 [(2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxypheny-methyl) 1 -azabicyclo(2.2. 2.)- octan-3-amine (C P-96,345)J and L-733,060 or [(2S,3S)3-([3,5- bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperjdjne]. RI 16301 or [(2R-trans)-4- [1 -[3, 5-bis(trifluoromethyl)benzoyl]-2-(phenylmethyl)-4-pipericiinyl]...N...(2, 6 dimethylphenyl)-1 -acetamide (S)-Hydroxybutanedioate] is another specific, active neurokinin-1 (NK(1)) receptor antagonist with subnanomolar affinity for the human NK(1) receptor (K(i): 0.45 nM) and over 200-fold selectivity toward NK(2) and NK(3) receptors. Antagonists of neurokinin receptors 2 (NK-2) that may also reduce cell swelling include SR48968 and NK-3 include SR142801 and SB-222200. Blockade of mitochondrial permeability transition and reducing the membrane potential of the inner mitochondrial membrane potential using cyclosporin A has also been shown to decrease ischemia-induced cell swelling in isolated brain slices. In addition glutamate-receptor antagonists (AP5/CNQX) and reactive oxygen species scavengers (ascorbate, Trolox(R), dimethylthiourea, tempol(R)) also showed reduction of cell swelling. Thus, the compound for minimizing or reducing the uptake of water by a cell in a tissue can also be selected from any one of these compounds.

It will also be appreciated that the following energy substrates can also act as impermeants. Suitable energy substrate can be selected from one or more from the group consisting of: glucose and other sugars, pyruvate, lactate, glutamate, glutamine, aspartate, arginine, ectoine, taurine, N-acetyl-beta-lysine, alanine, proline and other amino acids and amino acid derivatives, trehalose, floridoside, glycerol and other polyhydric alcohols (polyols), sorbitol, myo-innositol, pinitol, insulin, alpha-keto glutarate, malate, succinate, triglycerides and derivatives, fatty acids and carnitine and derivatives.

Preferably the compound for minimizing or reducing the uptake of water by the cells in the tissue is sucrose. Sucrose reduces water shifts as an impermeant.

Impermeant agents such as sucrose, lactobionate and raffinose are toolarge to enter the cells and hence remain in the extracellular spaces within the tissue and resulting osmotic forces prevent cell swelling that would otherwise damage the tissue, which would occur particularly during storage of the tissue. -Preferab'y, the concentration of the compound for minimizing or reducing the uptake of water by the cells in the tissue is between about 5 to 500mM. Typically this is an effective amount for reducing the uptake of water by the cells in the tissue.

page 23 More preferably, the concentration of the compound for reducing the uptake of water by the cells in the tissue is between about 20 and 100mM. Even more preferably the concentration of the compound for reducing the uptake of water by the cells in the tissue is about 70mM.

The term utissue) is used herein in its broadest sense and refers to any part of the body exercising a specific function including organs and cells or parts thereof, for example, cell lines or organelle preparations. Other examples include conduit vessels such as arteries or veins or circulatory organs such as the heart, respiratory organs such as the lungs, urinary organs such as the kidneys or bladder, digestive organs such as the stomach, liver, pancreas or spleen, reproductive organs such as the scrotum, testis, ovaries or uterus, neurological organs such as the brain, germ cells such as spermatozoa or ovum and somatic cells such as skin cells, heart cells (i.e., myocytes), nerve cells, brain cells or kidney cells.

It will be understood that the term comprises" or its grammatical variants as used in this specification and claims is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

The composition of the present invention is particularly useful in preconditioning, arresting, protecting and/or preserving the heart during open-heart surgery including heart transplants. Other applications include reducing heart damage before, during or following cardiovascular intervention which may include a heart attack, "beating heart' surgery, angioplasty or angiography. For example, the composition could be administered to subjects who have suffered or are developing a heart attack and used at the time of administration of blood clot-busting drugs such as streptokinase. As the clot is dissolved, the presence of the composition may protect the heart from further injury such as reperfusion injury. The composition may be particularly effective as a cardioprotectant in those portions of the heart that have been starved of normal flow, nutrients and/or oxygen for different periods of time.

For example, the composition may be used to treat heart ischaemia which could be pre-existing or induced by cardiovascular intervention.

In a preferred embodiment the composition according to the present invention is a cardioplegic and/or cardioprotectant composition.

page 24 In a preferred embodiment of the present invention it is preferred to aerate the composition with a source of oxygen before and/or during use. The source of oxygen may be an oxygen gas mixture where oxygen is the predominant component. The oxygen may be mixed with, for example CO2. Preferably, the oxygen gas mixture is 95% 02 and 5% CO2.

It is considered that the oxygenation with the oxygen gas mixture maintains mitochondrial oxidation and this helps preserve the myocyte and endothelium of the tissue.

In another aspect of the present invention there is provided a method for preconditioning, arresting, protecting and/or preserving a tissue including: providing in a suitable container a composition according to the invention and a source of oxygen; aerating the composition with the oxygen; and placing the tissue in contact with the composition under conditions sufficient to precondition arrest, protect and/or preserve thereof.

In another embodiment of the present invention there is provided use of a composition according to the invention for preconditioning, arresting, protecting and/or preserving a tissue, wherein the composition is aerated with the oxygen and contacts the organ.

Preferably the oxygen source is an oxygen gas mixture. Preferably oxygen is the predominant component. The oxygen may be mixed with, for example CO2. More preferably, the oxygen gas mixture is 95% 02 and 5% CO2 Preferably the composition is aerated before andlor during contact with the tissue.

Preferably the composition according to this aspect of the invention is in liquid form. Liquid preparations of the composition may take the form of, for example, solutions, syrups, or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives and energy sources.

page 25 While the present invention is particularly advantageous in preconditioning, arresting, protecting and/or preserving an organ while intact in the body of a subject, for example in the treatment of the heart in circumstances of myocardial infarction or heart attack, it will also be appreciated that the present invention may also be used to arrest, protect and/or preserve isolated organs.

The subject from which the tissue is to be preconditioned, arrested, protected and/or preserved may be a human or an animal such as a livestock animal (eg, sheep, cow or horse), laboratory test animal (eg, mouse, rabbit or guinea pig) or a companion animal (eg, dog or cat), particularly an animal of economic importance.

Use of the composition involves contacting a tissue with the composition, for a time and under conditions sufficient for the tissue to be preconditioned, arrested, protected and/or preserved.

The composition may be infused or administered as a bolus intravenous, intracoronary or any other suitable delivery route as pre-treatment for protection during a cardiac intervention such as open heart surgery (on-pump and off-pump), angioplasty (balloon and with stents or other vessel devices) and as with clot-busters (ant-clotting drug or agents).

The composition can also be infused or administered as a bolus intravenous, intracoronary or any other suitable delivery route for protection during cardiac intervention such as open heart surgery (onpump and off-pump), arigioplasty (balloon and with stents or other vessel devices) and as with clot-busters to protect and preserve the cells from injury.

The composition may also be infused or administered as a bolus intravenous, intracoronary or any other suitable delivery route for protection following a cardiac intervention such as open heart surgery (on-pump and off-pump), angioplasty (balloon and with stents or other vessel devices) and as with clot-busters to protect and preserve the cells from injury.

Accordingly, the tissue may be contacted by delivering the composition according to the invention intravenously to the tissue. This involves using the blood as a vehicle for delivery to the tissue. In particular, the composition according to the invention may be used for blood cardioplegia.

page 26 Alternatively, the composition may be delivered directly to the tissue for affecting the viability of the tissue. In particular, the composition according to the invention may be used for crystalloid cardioplegia.

The composition according to the invention may be delivered according to one of or a combination of the following delivery protocols: intermittent, continuous and bolus. An example of such protocols is illustrated in Figure 1A.

The dose and time inteivals for each delivery protocol may be designed accordingly. For example, a composition according to the invention may be delivered as a bolus to the tissue to initially arrest the tissue. A further composition according to the invention may then be administered continuously to maintain the tissue in an arrested state. Yet a further composition according to the invention may be administered continuously to reperfuse the tissue or recover normal function.

The composition can of course also be used in continuous infusion with both normal and injured tissues or organs, such as heart tissue. Continuous infusion also includes static storage of the tissue, whereby the tissue is stored in a composition according to the invention, for example the tissue may be placed in a suitable container and immersed in a solution according to the invention for transporting donor tissues from a donor to recipient.

The dose and time intervals for each delivery protocol may be designed accordingly. For example, a composition according to the invention may be delivered as a one-shot to the tissue to initially arrest of the tissue. A further composition according to the invention may then be administered continuously to maintain the tissue in an arrested state. Yet a further composition according to the invention may be administered continuously to reperfuse the tissue or recover normal function.

As mentioned previously, the composition according to the invention may be used at a temperature range selected from one of the following: from about 0 C to about 5 C, from about 5 C to about 20 C, from about 20 C to about 32 C and from about 32 C to about 38 C. It is understood that "profound hypothermia" is used to describe a tissue at a temperature from about 0 C to about 5 C. Moderate hypothermia" is used to describe a tissue at a temperature from about 5 C to about 20 C. "Mild hypothermia" is used to describe a tissue at a temperature from about page 27 20 C to about 32 C "Normothermia" is used to describe a tissue at a temperature from about 32 C to about 38 C.

The composition according to the present invention is highly beneficial at about 10 C but can also arrest preserve and protect over a wider temperature range up to about 37 C. In contrast, the majority of present day arrest and preservation solutions operate more effectively at lower temperatures the longer arrest times using St Thomas No. 2 solution may only be achieved when the temperature is lowered, for example, to a maximum of 4 C. Moreover, the composition according to the invention may be used at a temperature range selected from the following: 0 C to 5 C, 5 C to 20 C, 20 C to 32 C and 32 C to 38 C.

While it is possible for each component of the composition to contact the tissue alone, it is preferable that the components of the composition be provided together with one or more pharmaceutically acceptable carriers, diluents, adjuvants and/or excipients. Each carrier, diluent, adjuvant and/or excipient must pharmaceutically acceptable such that they are compatible with the components of the composition and not harmful to the subject. Preferably, the composition is prepared with liquid carriers, diluents, adjuvants and/or excipients.

The composition according to the invention may be suitable for topical administration to the tissue. Such preparation may be prepared by conventional means in the form of a cream, ointment, jelly, solution or suspension.

The composition may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition according to the invention may be formulated with suitable polymeric or hydrophobic materials (eg, as an emulsion in an acceptable oil or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

A preferred pharmaceutically acceptable carrier is a buffer having a pH of about 6 to about 9, preferably about 7, more preferably about 7.4 and/or low concentrations of potassium, for example, up to about 10mM, more preferably about 2 to about 8 mM, most preferably about 4 to about 6mM. Suitable buffers include Krebs-Henseleit which generally contains 10mM glucose, 117 mM NaCl, 5.9 mM KCI, 25 mM NaHCO3, 1.2 mM NaH2PO4, 1.12 mMCaCI2 (free Ca21.07mM) and 0.512 mM page 28 MgCI2 (free Mg20.5mM), St. Thomas No. 2 solution, Tyrodes solution which generally contains 10mM glucose, 126 mM NaCI, 5.4 mM KCI, 1 mM CaCI2, 1 mM MgCI2, 0.33 mM NaH2PO4 and 10 mM HEPES (N-[2-hydroxyethyl]piperazine-N'-[2- ethane suiphonic acidj, Fremes solution, Hartmanns solution which generally contains 129 NaCI, 5 mM KCI, 2 mM CaCI2 and 29 mM lactate and Ringers-Lactate.

Other naturally occurring buffering compounds that exist in muscle that could be also used in a suitable ionic environment are carnosine, histidine, anserine, ophidine and balenene, or their derivatives. Very recent studies have suggested that the process&s of inflammation and thrombosis are linked through common mechanisms.

Therefore, it is believed that understanding of the processes of inflammation will help with better management of thrombotic disorders including the treatment of acute and chronic ischaemic syndromes. In the clinical and surgical settings, a rapid response and early intervention to an organ or tissue damaged from ischemia, can involve both anti-inflammatory and anti-clotting therapies. In addition to protease inhibitors which have been shown to attenuate the inflammatory response, further anti-inflammatory therapies have included the administration of aspirin, normal hepariri, low-molecular-weight heparin (LMWH), non-steroidal anti-inflammatory agents, anti-platelet drugs and glycoprotein (GP) Ub/Illa receptor inhibitors, statins, angiotensin converting enzyme (ACE) inhibitor and angiotensin blockers. Examples of protease inhibitors are indinavir, nelfinavir, ritonavir, lopinavir, amprenavir or the broad-spectrum protease inhibitor aprotinin, a low-molecular-weight heparin (LMWH) is enoxaparin, non-steroidal anti-inflammatory agent are indomethacin, ibuprofen, rofecoxib, naproxen or fluoxetine, an anti-platelet drug is Ciopidogrel, a glycoprotein (GP) lb/lila receptor inhibitor is abciximab, a statin is pravastatin, an angiotensin converting enzyme (ACE) inhibitor is captopril and an angiotensin blocker is valsartin.

Another example of an anti-platelet drug is Aspirin.

In another preferred embodiment of the present invention there is also provided a reperfusion solution which is administered after arrest particularly long-term arrest, protection and preservation, together with the solution according to the invention.

Preferably, the reperfusion solutions comprises Krebs Henseleit buffer.

Preferably, the reperfusion solution is provided at 37 C.

page 29 The composition according to the invention may also include an energy substrate. The energy substrate helps with recovering metabolism. The energy substrate can be selected from one or more components selected from the group consisting of: glucose and other sugars, pyruvate, lactate, glutamate, glutamine, aspartate, arginine, ectoine, taurine, N-acetyl-beta-lysine, alanine, proline and other amino acids and amino acid derivatives, trehalose, floridoside, glycerol and other polyhydric alcohols (polyols), sorbitol, myo-innosotil, pinitol, insulin, alpha-keto glutarate, malate, succinate, trigylcerides and derivatives, fatty acids and carnitine and derivatives..

Throughout this specification, unless stated otherwise, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge, or any combination thereof, at the priority date, was part of the common general knowledge.

Description of Figures

The invention will now be described with reference to the following example.

This example is not to be construed as limiting in any way. In this description, there are the following figures.

Figure IA. illustrates diagrammatically the experimental design for combinatorial therapy after regional ischemia at varying concentrations.

Figure lB. contains Table 1, being the results of functional parameters of isolated working rat hearts during pre-arrest and reperfusion following 30 minute arrest with a composition according to the invention including Adenosine (200nM), Lidocaine (500uM) and Esmolol (lOOuM) (in 10 mM glucose containing Krebs Henseleit, pH 7.55 delivered intermittently at 37 C).

Figure 2. contains Table 2, being the results of functional parameters of isolated working rat hearts during pre-arrest and reperfusion following 30 minute arrest with a composition according to the invention including Adenosine (200mM), Liguocaine (500uM) and Esmolol (lOuM). (in 10 mM glucose containing Krebs Henseleit, pH 7.60 delivered intermittently at 37 C) Figure 3. contains Table 3, being the results of functional parameters of isolated working rat hearts during pre-arrest and reperfusion following 30 minute arrest with a page 30 composition according to the invention including adenosine (20mM), lidocaine (500uM) and esmolot (lOOuM) (in 10 mM glucose containing Krebs Henseleit, pH 7.51 delivered intermittently at 37 C).

Examples

Example 1: Effect of Adenosine and Lignocaine with Esmolol on functional recovery of the rat heart after arrest This example demonstrates the effect of esmolol, an antiadrenergic, together with Adenosine and Lignocaine on functional recovery after a period of arrest using intermittent perfusion.

Hearts from adult whistler rats (350g) were prepared using the method described below. Intermittent retrograde perfusion was performed under a constant pressure head of 7OmmHg after hearts were switched back from the working mode to the Lagendorif mode. After stabilisation, the hearts were arrested using either: (I) Adenosine (200uM) and Lignocaine (500uM) plus Esmolol (lOOuM); (ii) Adenosine (200uM) and Lignocaine (500uM) PIUS Esmolol (buM); (iii) Adenosine (2OuM) and Lignocaine (500uM) PIUS Esmolot (1 OOuM).

Solutions containing these compounds were provided in Krebs Henseleit (lOnM glucose, pH 7.55 @ 37 C). The aorta was then crossctamped and the heart left to sit arrested for 5 mins, after which the clamp was released and 2 mins of arrest solution delivered from a pressure head of 7OmmHg. The clamp was replaced and this procedure continued for l8mins arrest time then 30mins arrest time. The recovery results are shown in Table 1 (Figure 1B), Table 2 (Figure 2) and Table 3 (Figure 3).

This example demonstrates improved functional recovery of the heart after 30mins arrest, providing superior protection during arrest and recovery of the heart.

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Claims

Claims 1. A composition comprising an effective amount of a local

anaesthetic; an adenosine receptor agonist; and an anti-adrenergic.

2. A composition according to claim 1 wherein the anti-adrenergic is selected from beta-blockers, such as esmolol, atenolol, metoprolol and propranolol and alpha( 1)-adrenoceptor-antagonists such as prazosin.

3. A composition according to claim 1 or 2 wherein the composition further includes a sodium hydrogen exchange inhibitor selected from amiloride, cariporide, eniporide, triamterene and EMD 84021, EMD 94309, EMD 96785, HOE 642 and T-162559.

4. A composition according to any of claims 1-3 wherein the composition further comprises an agent selected from normal or low-molecular-weight heparin (such as enoxaparin), non-steroidal anti-inflammatory agents (such as indomethacin, ibuprofen, rofecoxib, naproxen, celecoxib or fluoxetine), an anti-platelet drug (such as Clopidogrel), platelet glycoprotein (GP) JIb/lila receptor inhibitors (such as abciximab), statins (such as pravastatin), angiotensin converting enzyme (ACE) inhibitors (such as captopril) and angiotensin blockers (such as valsartin).

5. A composition according to any one of the preceding claims wherein the adenosine receptor agonist is selected from N6-cyclopentyladenosine (CPA), N-ethytcarboxamido adenosine (NECA), 2-[p-(2-carboxyethyl)phenethyl-amino-5'-N-ethyicarboxamido adenosine (CGS21680), 2-chioroadenosine, N6-[2-(3,5-demethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine, 2-chloro-N6-cyclopentyladenosine (CC PA), N-(4-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofuranosyl]-adenine (AB-MECA), ([IS-El a, 2b, -methyl-propyl]amino]-3H-imidazole[4, 5-b]pyridyl-3-yl]cyclopentane carboxamide (AM P579), N6-(R)-phenylisopropyladenosine (R-PLA), aminophenylethyladenosine (APNEA) and cyctohexyladenosine (CHA), page 50 adenosine Al receptor agonists (such as N-[3-(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510)), CVT-2759 and allosteric enhancers such as PD8 1723, N6-cyclopentyt-2-(3-phenylaminocarbonyltriaZefle-l -yl)adenosine (TCPA), and allosteric enhancers of Al adenosine receptor, such as 2-amino-3-naphthoylthiophenes.

6. A composition according to claim 5 wherein the adenosine receptor agonist is an Al adenosine receptor agonist, preferably CCPA.

7. A composition according to any one of the preceding claims wherein local anaesthetic is selected from mexiletine, diphenyl hydantoin prilocaine, procaine, mepivacaine, Class lB antiarrhythmic agents, such as lignocaine or derivatives thereof (eg QX-314), and sodium channel blockers such as tetrodotoxin, primaquine, OX, HNS-32 (CAS Registry # 186086-10-2), NS- 7, kappa-opiold receptor agonist U50 488, crobenetine, pilsicainide, phenytoin, tocainide, NW-1029 (a benzylamino propanamide derivative), RS100642, riluzole, carbamazepine, flecainide, propafenone, amiodarone, sotalol, imipramine and moricizine, and any derivatives thereof.

8. A composition according to any one of the preceding claims wherein the components are administered substantially simultaneously or co-administered.

9. A composition according to any one of the preceding claims wherein the composition is aerated with a source of oxygen before use, during use or both.

10. A composition according to claim 10, wherein the source of oxygen is an oxygen gas mixture.

11. A composition according to any one of the preceding claims wherein the composition is used at a temperature range selected from about 32CC to about 38C.

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