Trauma therapy   

FIELD OF THE INVENTION

This invention relates to a method of reducing injury to cells, tissues or organs of a body following trauma, including injury to cells, tissues or organs resulting from shock, stroke, heart conditions or other injuries that may occur as a consequence of trauma.

BACKGROUND OF THE INVENTION

In the western world, many deaths occur suddenly and unexpectedly, particularly as a consequence of trauma. Medically, “trauma” refers to a serious or critical bodily injury, wound, or shock which in some cases may require resuscitation therapy. Trauma is often associated with trauma medicine practiced in hospital (such as in hospital emergency rooms), in emergency transport environments (such as in ambulances), or at out-of-hospital environments where a trauma has occurred, such as domestic or industrial accidents, transport accidents, the battlefield, and terrorist attacks.

Trauma is a leading cause of death among children and all individuals to age 34 years and a major cause of death in the older population resulting in loss of productive life-years with substantial societal costs. This includes deaths resulting from burns, heart attacks, strokes and other cardiovascular events. Deaths can also result from shock or other complications that may occur as a consequence of trauma.

Less than 3% of these unconscious trauma patients will advance to acceptable outcomes. Many survivors require institutional care after 3 months and a significant proportion remain permanently disabled. About 20% of soldiers injured in the battlefield will die, and 90% of deaths occur before reaching a hospital because of shock during emergency transport. More recent statistics suggest that 50% of deaths in potentially treatable combat injuries are due to acute blood loss, making it the leading cause of death on the battlefield.

Shock is a circulatory dysfunction causing decreased tissue oxygenation and accumulation of oxygen debt, which can ultimately lead to multi-organ system failure if left untreated. The most common form of shock in both paediatric and adult trauma patients is hemorrhagic or hypovolemic shock (not enough blood volume). Cardiogenic shock (not enough output of blood by the heart, see below) is also a common form of shock. Shock as a result of blood loss is a frequent complication of trauma. About half of trauma deaths occur during the first hour after injury from a profound compromise in cardiopulmonary and cerebral function. The signs and symptoms of shock include low blood pressure (hypotension), overbreathing (hyperventilation), a weak rapid pulse, cold clammy greyish-bluish (cyanotic) skin, decreased urine flow (oliguria), and mental changes (a sense of great anxiety and foreboding, confusion and, sometimes, combativeness). When blood is lost, the greatest immediate need is replacing the lost volume with blood or blood volume expanders. Provided blood volume is maintained by volume expanders, a trauma patient can safely tolerate very low blood haemoglobin levels, less than one third of a healthy person.

During trauma, the electrical properties of vital organs and tissues cannot be maintained. Falls in resting cell voltage occur during trauma and can lead to the triggering of highly injurious arrhythmias in the heart and activation of systemic inflammatory, coagulative and free radical generating processes that can lead to multiple organ failure and death. During severe haemorrhage, patients become unconscious when the mean arterial perfusion pressure decreases to about 40 mm Hg and the pulse is no longer palpable in the large arteries. When breathing stops and pulsations are no longer palpable, cardiac arrest is assumed. The mortality rate for trauma patients who become pulseless from massive blood loss and undergo emergency department thoracotomy is around 97%.

One form of shock is called “cardiogenic shock”. This may be caused by the failure of the heart to pump effectively due to, for example, damage to the heart muscle (as may result from a large myocardial infarction (heart-attack), disorders of the heart muscle (including rupture), disturbances to the electrical excitation-relaxation (or conduction) system and tamponade. Cardiogenic shock may also be caused by arrhythmias (eg ventricular tachycardia and ventricular fibrillation), cardiomyopathy, cardiac valve problems, ventricular outflow obstruction and the like. Cardiogenic shock is a medical emergency requiring immediate treatment to save the patient\'s life.

One cause of cardiogenic shock is a so-called “heart-attack”. This term is used to refer to a number of different conditions which lead to heart ischaemia, which leads to the death of heart muscle (typically caused by blockage of a coronary artery). The muscle death causes chest pain and electrical instability of the heart muscle tissue. This electrical instability may manifest as “ventricular tachycardia” and “ventricular fibrillation”. Ventricular tachycardia is a tachydysrhythmia originating from a ventricular ectopic focus and characterized by a rate typically greater than 120 beats per minute and must be treated quickly to avoid morbidity or mortality as it may deteriorate rapidly into ventricular fibrillation. Ventricular fibrillation is a condition in which there is chaotic electrical disturbances of the ventricles, such that they no longer beat regularly, nor pump blood effectively, but simply quiver. During ventricular fibrillation the heart muscle is affected by a poor supply of oxygen or by specific heart disorders and the ventricles contract independently of the atria, and some areas of the ventricles contract while others are relaxing, in a disorganized manner. Ventricular fibrillation leads to widespread ischaemia. Unless treated immediately, ventricular fibrillation causes death and is responsible for 75% to 85% of sudden deaths in persons with heart problems. In the USA alone there are nearly 450,000 sudden deaths per year, and in the united kingdom around 70,000-90,000 sudden deaths per year. Ventricular tachycardia and ventricular fibrillation are therefore medical emergencies because if they persist more than a few seconds, the blood circulation will cease, there will be no pulse, no blood pressure and no respiration and death will occur. Typically, medications and procedures at this time are directed towards stabilising the rhythm of the heart and, in the case of the unconscious subject with no measurable pulse, resuscitating the subject by restarting the heart, opening the airways and restoring spontaneous breathing. Amiodarone can be used to treat life-threatening heart arrhythmias, however, the drug can have serious side effects including causing cardiac rhythm irregularities and cardiac arrest itself. Other side effects of amiodarone include lung infiltration, neuropathy, tremors, thyroid disorders, nausea, low blood pressure and liver damage. Effective medications for stabilizing the heart or restarting the heart and restoring the spontaneous circulation in these emergency situations are therefore very limited or non-existent. Noradrenalin or adrenalin (with or without vasopressin) can be used in conjunction with cardiopulmonary resuscitation, however, epinephrine can exacerbate heart contractions and promote heart dysfunction by increasing myocardial oxygen consumption during ventricular fibrillation, as well as eliciting microvascular disorders. If the treatments are successful in stabilising the heart after ventricular tachycardia or ventricular fibrillation, a number of medications are then administered such as oxygen (if available to help breathing), beta-blockers (to help relax the heart), vasodilators (to help deliver more blood to the heart), blood agents (anti-coagulants, anti-platelet agents, thrombolytics and the like) and pain relievers. Apart from a few drugs to treat the heart as well as other tissues and organs, the medications are not directed to treating the cardiac tissue specifically. There is no effective pharmaceutical treatment for the failing heart muscle itself, nor for common ventricular fibrillation. If treated, this is usually treated by electrical shock (cardioversion).

Damage may also be caused to a heart upon reperfusion. One example of reperfusion damage is when a heart becomes “stunned”. In this condition, the bloodflow has been restored but the heart is functioning abnormally and may result in a further heart-attack (such as ventricular fibrillation) if not treated. Cardiac reanimation inevitably involves reperfusion of the heart with the consequent dangers associated with reperfusion injury, particularly to heart muscle. Where the muscle cells die, this is regarded as an infarction. If blood flow is restored to the cells within a short period of about 15 to 20 minutes the cells may respond to the reperfusion and survive (thus not forming an infarction) but may be “stunned” in the sense that they do not operate normally nor perform their usual function during reperfusion.

In patients who survive resuscitation where the initial event may be less traumatic, they remain at a significant risk from systemic and local inflammatory and immune activation followed by multiple organ dysfunction and failure. Multiple organ failure is believed to be the result of an excessive self-destructive systemic inflammation and immunologic functions, in which hypoxemia, tissue hypoxia/nonviable tissue, micro-organisms/toxins and antigen/antibody complexes may be involved. In particular, the activation of a number of humoral (e.g. complement, coagulation) and cellular systems (endothelium activation, neutrophils, platelets, macrophages) are believed to be involved. Neutrophils play a key role in injury to the lung, heart, kidney, liver, and gastrointestinal tract, often seen after major trauma. As a consequence there is synthesis, expression and release of numerous mediators (toxic oxygen species, proteolytic enzymes, adherence molecules, cytokines), which may produce a generalized inflammation and tissue damage in the body.

The critical core body temperature also can aggravate many of these post-traumatic secondary complications. Below 34° C. mortality increases significantly. Despite this, a number of investigators have suggested a beneficial effect of deliberate hypothermia because this may prolong the “golden hour” of trauma victims by preventing hypoxic organ dysfunction and initiation of the inflammatory response. Organ failure is also the leading cause of death in the postoperative phase after major surgery. An excessive inflammatory response followed by a dramatic depression of cell-mediated immunity after major surgery appears to be responsible for the increased susceptibility to subsequent sepsis.

Resuscitation therapy is generally regarded as any procedure which improves the management of sudden states of life-threatening illnesses or traumatic injuries, such as those from cardiac arrest, respiratory failure, hemorrhagic blood loss, neurological injury, and traumatic injuries to the soft tissues and body skeleton. Generally, resuscitation therapy deals with treating whole body oxygen deprivation. As such, current resuscitation strategies aim to optimize tissue supply and demand ratio and avoid complications of overaggressive volume replacement, which exacerbate haemorrhage, pulmonary oedema, and intracranial hypertension following brain injury.

Resuscitation therapy is very different from treating a localized “big heart attack” or a localized “big stroke”. It involves a complex interplay between multiple organ-tissue responses via poorly understood actions, which separates this science from treatments to preserve particular organs or tissues. Resuscitation is known to involve a complex biological system, with many interactions. These cannot be predicted from study of individual components. Injured organs have secondary effects on other organs, which affects the whole body and can lead to debilitating injuries and death.

Current therapies involve fluid or volume replacement that can either be crystalloid or colloidal. Crystalloids are commonly used for resuscitation therapy because they appear to be safe and help with the negative side effects of coagulation. Crystalloids have been shown to increase coagulation, an effect which seems to be independent of the type of crystalloid used. A crystalloid-induced hypercoagulable state appears to be due to an imbalance between naturally occurring anticoagulants and activated procoagulants. Crystalloids used for volume replacement can be three main types: 1) hypotonic (eg. dextrose in water), 2) isotonic (normal saline or Ringers solution with lactate or acetate) or 3) hypertonic (eg 7.5% saline). Since crystalloids are freely permeable to the vascular membrane, only about 25% remain in the blood compartment and the remainder in the body\'s interstitial and/or intercellular compartment leading to tissue oedema. Crystalloid resuscitation is therefore less likely to achieve adequate restoration of microcirculatory blood flow compared to a colloidal-based volume replacement strategy.

Colloid replacement therapies employ colloids, such as dextran-70, dextran-40, hydroxyethyl starch, pentastarch, lactobionate, sucrose, mannitol and a modified fluid gelatine as artificial colloids, for this purpose. There is much controversy as to the most appropriate solution for volume replacement.

Currently there is no optimal fluid composition or fluid resuscitation regimen to treat severe hemorrhagic shock in soldiers on the battlefield or civilians at a natural disaster site or injured from a terrorist attack. Indeed, the majority of approved resuscitation fluids have no intrinsic tissue protection and can trigger life-threatening inflammatory and hypercoagulable imbalances that negatively impact on the resuscitative outcome. A further challenge in designing new drug products and resuscitation therapies, in particular for the military, is hampered by logistical considerations imposed by the combat conditions themselves such as weight and practicability to transport, ease of deployment, administration in low-light environments and stability of drugs in the field, notwithstanding ensuring their safety and clinical effectiveness to increase the survival times of wounded soldiers after prolonged evacuation.

In warfare, bullets and penetrating fragments from exploding munitions frequently cause life-threatening hemorrhage. Acute hemorrhage is the leading cause of mortality in battlefield injuries and responsible for 50% of deaths in potentially treatable combat casualties. One major unmet medical need on the battlefield is how to prevent cardiac destabilization and arrest during severe hemorrhage before control of bleeding is possible. Stabilizing heart and circulatory deficiencies before shock is of paramount importance. Successful treatment of cardiac arrest requires an electrically stable and mechanically viable heart to be re-established. Currently there is no clinically effective method of stabilizing and protecting the heart from fibrillating and arresting before hemorrhagic shock. Indeed, many pharmacological interventions employed to convert the heart to sinus rhythm may unfortunately inflict additional injury and compromise cardiac resuscitability

In those severe traumatic hemorrhagic cases where the heart does not destabilize and arrest, the loss of blood volume, blood pressure and organ perfusion can lead to severe organ ischemia and eventually multiple organ dysfunction and failure (MOF) and death. MOF is the leading cause of mortality secondary to shock (hemorrhage/trauma) and resuscitation, and involves the lungs, kidneys, intestinal tract, pancreas, liver, brain and heart. Importantly, MOF is not an end-point per se but a process involving an overwhelming self-destructive, local and systemic, inflammatory responses and immunologic functions. Despite decades of research, resuscitation fluids restore tissue perfusion, however they have no specific anti-inflammatory, immunosuppression or pro-survival properties. Importantly, the activation of shock-induced inflammatory response occurs during the shock itself, during early crystalloid or colloid-based resuscitation therapy, and during final resuscitation efforts with blood replacement.

It is not known whether protection from injury from trauma could be elicited by a form of artificial hibernation. Natural hibernators possess the ability to lower their metabolic energy demand for days to months. Hibernation, like sleep, is a form of dormancy and helps to keep the animal\'s metabolic supply and demand ratio in balance. Remarkably, no damage occurs during these prolonged “ischemic” states, nor does the cardiac rhythm deteriorate into ventricular fibrillation. However, there is no known method of stimulating a similar response in humans, particularly trauma patients, despite the potential for substantial saving of life or minimising injury.

WO00/56145, WO04/056180 and WO04/056181 describe compositions useful to limit damage to a cell, tissue or organ by administering them in a clinical setting prior to a medical procedure. These compositions are also usually administered following diagnosis of the patient and directly to the cell, tissue or organ. However, much damage or injury to cells, tissues or organs may arise before the patient gets to the hospital and/or at hospital, for example, before substantive medical attention is available or a condition can be diagnosed.

SUMMARY

The present invention is directed toward overcoming or at least alleviating one or more of the difficulties and deficiencies of the prior art.

In one aspect the invention is directed to a method of reducing injury to cells, tissues or organs of a body following trauma by administering a composition to the body following trauma, including: (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; and (ii) a local anaesthetic.

According to this aspect, a further composition comprising components (i) and (ii) may be administered to the body following administration of the composition.

Either composition may include Magnesium cations (divalent) and/or may be hypertonic.

In another aspect the invention is directed to a composition for reducing injury to cells, tissues or organs of a body following trauma including: (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; and (ii) a local anaesthetic. In one embodiment of this aspect, the composition may include divalent magnesium cations and/or may be hypertonic.

DETAILED DESCRIPTION

The invention is directed to improved resuscitation therapies for trauma victims in hospital, emergency transport and out-of-hospital environments. In particular, the invention has application to minimise life-threatening complications of persons suffering injury to cells, tissues or organs resulting from burns, shock, stroke, heart attack or other physical events, including complications from surgical or clinical interventions, as a consequence of trauma. Injured soldiers on the battlefield or civilians at a natural disaster site or injured from a terrorist attack are situations where such treatment may be useful.

The invention applies to protecting, preserving or stabilising key organs such as the heart and brain, other neuronal tissues and cells, renal tissue, lung tissue, muscle tissue, liver and other tissues of the body.

In one form, the invention provides a method of reducing injury to the cells, tissues or organs of a body following trauma by administering a composition to the body following trauma including: (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; and (ii) a local anaesthetic.

In another form of the invention, the invention is directed towards treating tachycardia and/or fibrillation. In one form, the invention treats heart arrhythmias of atrial or ventricular origin, especially ventricular fibrillation. The treatment of tachycardia and/or fibrillation, including ventricular fibrillation and arrhythmias, comprises administering a composition including: (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; and (ii) a local anaesthetic, in amounts effective to arrest a heart. In one embodiment, the amount administered is effective to arrest the heart only momentarily. This is often sufficient to facilitate the heart cardioconverting back to normal rhythm. In an alternate embodiment, the amount administered is effective to substantially down-regulate the beating of the heart for a period of a few beats, before allowing the heart to regain its usual rhythm. The invention also extends to a method for treating tachycardia and/or fibrillation accordingly. Preferably, the composition is administered as a bolus. The administration of the composition is believed to quell the tachycardia and/or fibrillation allowing the heart to cardiovert to a normal and desirable sinus rhythm.

In a preferred embodiment, the invention comprises the further step of subsequently administering a second composition which includes (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; and (ii) a local anaesthetic, in amounts below that effective to arrest a heart. The purpose of the second composition is to protect the heart and other tissues such as brain, liver, lung and kidney, or assist in doing so. In particular, this embodiment is directed towards reducing reperfusion injury or stunning. As outlined above, reperfusion injury is a common deleterious occurrence upon successfully converting a tachycardic/fibrillating heart to a normal and desirable sinus rhythm. In a preferred embodiment, the second composition is administered as another non-arresting bolus injection or delivered continuously via an intravenous drip or by another delivery device or route.

Without being bound by any theory or mode of action, the inventor has found that the composition according to the invention can be used to place the body, in effect, toward a state of suspended animation like a natural hibernator or to stabilise the body prior to diagnosis or until suitable medical attention can be provided to the trauma victim. The overall protection provided by therapy according to the invention is thought to involve a multi-tiered system from modulating membrane excitability to a multitude of intracellular signalling pathways, including heat shock and pro-survival kinase pathways. A primary focus is on reducing damage to the brain, heart and lungs, because this has been correlated with improved recovery and clinical outcomes. Nonetheless, broad-acting approaches reducing damage throughout the body in a non-specific way are desirable. Proposed mechanisms of the composition of the invention include (i) reduced ion imbalances, in particular sodium and calcium ion loading in the cells, which may help defend the cell\'s voltage when stressed; (ii) attenuation of local and systemic inflammatory response to injury, which is protective in itself to reduce injury as well as reduce secondary effects such as free radical production; and (iii) protection from entering into a hypercoagulable state, ie an anti-clotting or anti-thrombolytic activity. Moreover, it is believed that, in respect of the heart, the invention simultaneously provides improved atrial and ventricular matching of electrical conduction to metabolic demand, which may involve modulation of gap junction communication, and, in respect of the brain, improved brain function. It is also believed that the composition may reduce the body\'s demand for oxygen to varying degrees and thus reduce damage to the body\'s cells, tissues or organs. In another form, the invention provides a composition for reducing injury to cells, tissues or organs of a body following trauma including (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; and (ii) a local anaesthetic. The composition may further include other components as identified below. In some embodiments, the potassium channel opener or agonist and/or adenosine receptor agonist is replaced by another component such as a calcium channel blocker. The composition preferably contains an effective amount of (i) and (ii) for a single dose to reduce injury.

More surprisingly, it has been observed that administration of a composition with arresting or near-arresting concentrations of components (i) and (ii) to a subject experiencing ventricular fibrillation assists the heart to regain normal sinus rhythm without the requirement for electrical shock treatment.

The invention may also be used to treat or inhibit arrhythmias including ventricular fibrillation during or prior to an angiogram test or an exercise test. Similarly it has application during emergency transport of an injured patient and for on-site emergency treatment (ie, at the site of injury or heart-attack such as an airport, sports stadium, hospital, battlefield or disaster site). It can also be used before, during and/or after coronary interventions such as angioplasty, cardiac catheter procedures, or insertion of a pacemaker or leads or a device, or for surgical procedures including paediatric or adult heart surgery, hip, knee, vascular or brain surgery, aortic dissections, carotid endaterectomy or general surgery.

In the embodiments of the invention described above and below, component (i) of the composition may be an adenosine receptor agonist. While this obviously includes adenosine itself, the “adenosine receptor agonist” may be replaced or supplemented by a compound that has the effect of raising endogenous adenosine levels. This may be particularly desirable where the compound raises endogenous adenosine levels in a local environment within a body. The effect of raising endogenous adenosine may be achieved by a compound that inhibits cellular transport of adenosine and therefore removal from circulation or otherwise slows its metabolism and effectively extends its half-life (for example, dipyridamole) and/or a compound that stimulates endogenous adenosine production such as purine nucleoside analogue Acadesine™ or AICA-riboside (5-amino-4-imidazole carboxamide ribonucleoside). Acadesine™ is also a competitive inhibitor of adenosine deaminase (Ki=362 microMolar in calf intestinal mucosa.) Acadesine™ is desirably administered to produce a plasma concentration of around 50 microM (uM) but may range from 1 microM to 1 mM or more preferably from 20 to 200 uM. Acadesine™ has shown to be safe in humans from doses given orally and/or intravenous administration at 10, 25, 50, and 100 mg/kg body weight doses.

In one form of the invention, the composition, and optionally the second composition, also contains divalent magnesium cations. In one embodiment, the concentration of magnesium is up to about 2.5 mM and in another embodiment magnesium is present in higher concentrations, for example up to about 20 mM. The magnesium is present as a physiologically and pharmaceutically acceptable salt, such as for example magnesium chloride and magnesium sulphate.

In another form the composition according to the invention is hypertonic. Preferably the composition contains 7.5% NaCl. The inventor has found that only a small volume of this hypertonic composition may be administered to the subject in need thereof. This is particularly advantageous where the composition according to the invention has application during emergency or for emergency transport. According to this aspect, only a small amount of the composition according to the invention needs to be available, for example, in a medical kit or ambulance. Thus the composition is easier to store and/or transport. This “low volume” composition has unique features of fluid replacement and specific anti-inflammatory, immunosuppression pro-survival properties. The composition according to this aspect of the invention pharmacologically “buys” time for wounded soldiers on the battlefield or civilians in urban “disaster zones” which allow for safer evacuation, triage, and initiation of supportive therapies. The ability of a solution to change the shape or tone of cells by altering their internal water volume is called tonicity (tono=tension). A Hypertonic solution contains a higher concentration of electrolytes than that found in body cells and, therefore, relatively less water in this compartment than inside the body cells. In such a hypertonic environment, osmotic pressure causes water to flow out of the cell into the hypertonic environment. Thus a hypertonic solution creates a hyperosmotic environment and the higher osmotic pressure in this environment relative to the surrounding cells in tissues causes fluid to flow from the cells towards such a system. If too much water is removed in this way, the cell may have difficulty functioning.

The invention described in this specification largely relates to methods of treatment, and methods of manufacturing a medicament for treatment involving a composition which is described as containing these components (i) and (ii). For convenience, this composition will be referred to in this specification as the “composition of the invention”, although there are a number of combinations of components embodying the invention which are compositions according to the invention. Moreover, as explained particularly in WO00/56145, the components (i) and (ii) may be present in a concentration which arrests, or does not arrest a heart. These two compositions are used in different ways in the invention described in the specification, and are referred to respectively as an “arresting” concentration of the composition and a “non-arresting” concentration of the composition. In one form, the arresting composition contains adenosine and lignocaine, each at greater than 0.1 mM (and preferably below 20 mM). The arresting composition may in some circumstances be referred to as a “cardioplegia solution”. In one form of the non-arresting composition, adenosine and lignocaine are both below 0.1 mM and preferably 50 nM to 95 uM, or more preferably from 1 uM to 90 uM.

In a further form, the invention provides use of (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; and (ii) a local anaesthetic, for the preparation of a medicament for reducing injury to cells, tissues or organs of a body following trauma. The use preferably includes administering the medicament in one or more of the ways set out elsewhere in this specification.

In another form, the invention provides a method of, in effect, placing the body in or toward a hibernating-like state of suspended animation following trauma. This is achieved by administering a composition as described above.

The term “trauma” is used herein in its broadest sense and refers to a serious or critical injury, wound or shock to the body. Trauma may be caused by unexpected physical damage (or injury) to the body as a result of, for example, transport or industrial accidents, birth, surgery, heart attack, stroke, burns, complications due to surgery or other medical interventions etc. Trauma may result from injury to a body, both in a hospital or out of hospital. Trauma is often associated with trauma medicine practiced in hospital (such as in hospital emergency rooms), during emergency transport or at out-of-hospital environments where a trauma has occurred, such as domestic or industrial accidents, transport accidents, the battlefield, and terrorist attacks. In many cases, trauma therapy may also include resuscitation therapy.

The term “tissue” 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 circulatory organs such as the heart, blood vessels and vasculature, 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 ie, myocytes, nerve cells, brain cells or kidney cells. The tissues may come from human or animal donors. The donor organs may also be suitable for xenotransplantation.

The term “organ” is used herein in its broadest sense and refers to any part of the body exercising a specific function including tissues and cells or parts thereof, for example, endothelium, epithelium, blood brain barrier, cell lines or organelle preparations. Other examples include circulatory organs such as the blood vessels, 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 also be understood that the term “comprises” (or its grammatical variants) as used in this specification is equivalent to the term “includes” and should not be taken as excluding the presence of other elements or features.

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 opioids, others are activated by fatty acids, and other channels are modulated by ions such as sodium and calcium (ie. 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-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-121), RO316930, RWJ29009, SDZPCO400, rimakalim, symakalim, YM099, 2-(7,8-dihydro-6,6-dimethyl-6H-[1,4]oxazino[2,3-f][2,1,3]benzoxadiazol-8-yl) pyridine N-oxide, 9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione (ZM244085), [(9R)-9-(4-fluoro-3-125iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno[2,3-e]pyridin-8(7H)-one-1,1-dioxide] ([125I]A-312110), (−)-N-(2-ethoxyphenyl)-N′-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diamine (Bay X 9228), N-(4-benzoyl phenyl)-3,3,3-trifluoro-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 NS004 (5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one), NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one), NS1608 (N-(3-(trifluoromethyl)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. Other compounds that are believed to open KATP channels include Levosimendan and hydrogen sulphide gas (H2S) or the H2S donors (eg sodium hydrosulphide, NaHS).

In addition, potassium channel openers may act as indirect calcium antagonists, ie 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.

Adenosine (6-amino-9-β-D-ribofuranosyl-9H-purine) is particularly preferred as the potassium channel opener. Adenosine is capable of opening the potassium channel, hyperpolarising the cell, depressing metabolic function, possibly protecting endothelial cells, enhancing preconditioning of tissue and protecting from ischaemia or damage. Adenosine is also an indirect calcium antagonist, vasodilator, antiarrhythmic, antiadrenergic, free radical scavenger, arresting agent, anti-inflammatory agent (attenuates neutrophil activation), metabolic agent and possible nitric oxide donor. More recently, adenosine is known to inhibit several steps which can lead to slowing of the blood clotting process. In addition, elevated levels of adenosine in the brain has been shown to cause sleep and may be involved in different forms of dormancy. An adenosine analogue, 2-chloro-adenosine, may be used.

Suitable adenosine receptor agonists may be selected from: N6-cyclopentyladenosine (CPA), N-ethylcarboxamido adenosine (NECA), 2-[p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamido adenosine (CGS-21680), 2-chloroadenosine, N6-[2-(3,5-demethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine, 2-chloro-N-6-cyclopentyladenosine (CCPA), N-(4-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofuranosyl]-adenine (AB-MECA), ([IS-[1a,2b,3b,4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methyl-propyl]amino]-3H-imidazole[4,5-b]pyridyl-3-yl]cyclopentane carboxamide (AMP579), N6—(R)-phenylisopropyladenosine (R-PLA), aminophenylethyladenosine (APNEA) and cyclohexyladenosine (CHA). CCPA is a particularly preferred. Others include full adenosine A1 receptor agonists such as N-[3-(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), or partial agonists such as CVT-2759 and allosteric enhancers such as PD81723. Other agonists may include N6-cyclopentyl-2-(3 phenylaminocarbonyltriazene-1-yl) adenosine (TCPA), a very selective agonist with high affinity for the human adenosine A1 receptor and allosteric enhancers of A1 adenosine receptor includes the 2-amino-3-napthoylthiophenes.

In one aspect, the composition according to the invention includes an A1 adenosine receptor agonist and a local anaesthetic. CCPA is a particularly preferred A1 adenosine receptor agonist.

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.

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, which could also be used to reduce calcium. Beta-blockers (or beta-adrenergic blocking agents) include atenolol (Tenormin™), propranolol hydrochloride (such as Inderal™), esmolol hydrochloride (Brevibloc™), metoprolol succinate (such as Lopressor™ or Toprol XL™), acebutolol hydrochloride (Sectral™), carteolol (such as Cartrol™), penbutolol sulfate (Levatol™) and pindolol (Visken™).

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 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 alpha1-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 100-1000 fold more potent than morphine as an analgesic but is not addictive. This conotoxin is being investigated to treat intractable 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 Calcium channel blockers from animal kingdom include Kurtoxin from South African Scorpion, SNX-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-1 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 ATP-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.

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

In a preferred form, the potassium channel opener or agonist and/or an adenosine receptor agonist has a blood half-life of less than one minute, preferably less than 20 seconds.

In some embodiments, the composition may include additional potassium channel openers or agonists, for example diazoxide or nicorandil.

The inventor has also found that the inclusion of diazoxide with a potassium channel opener or adenosine receptor agonist and a local anaesthetic reduces injury. Thus in another aspect, the composition according to the invention further includes diazoxide.

Diazoxide is a potassium channel opener and in the present invention it is believed to preserve ion and volume regulation, oxidative phosphorylation and mitochondrial membrane integrity (appears concentration dependent). More recently, diazoxide has been shown to provide cardioprotection by reducing mitochondrial oxidant stress at reoxygenation. At present it is not known if the protective effects of potassium channel openers are associated with modulation of reactive oxygen species generation in mitochondria. Preferably the concentration of the diazoxide is between about 1 to 200 uM. Typically this is as an effective amount of diazoxide. More preferably, the concentration of diazoxide is about 10 uM.

The inventor has also found that the inclusion of nicorandil with a potassium channel opener or adenosine receptor agonist and a local anaesthetic reduces injury. Thus in another aspect, the composition according to the invention further includes nicorandil.

Nicorandil is a potassium channel opener and nitric oxide donor which can protect tissues and the microvascular integrity including endothelium from ischemia and reperfusion damage. Thus it can exert benefits through the dual action of opening KATP channels and a nitrate-like effect. Nicorandil can also reduce hypertension by causing blood vessels to dilate which allows the heart to work more easily by reducing both preload and afterload. It is also believed to have anti-inflammatory and anti-proliferative properties which can further attenuates ischemia/reperfusion injury.

The composition according to the invention also includes a compound for inducing local anaesthesia, otherwise known as a local anaesthetic. The local anaesthetic may be selected from mexiletine, diphenylhydantoin, prilocalne, procaine, mepivocaine, quinidine, disopyramide and Class 1B antiarrhythmic agents such as lignocaine or derivatives thereof, for example, QX-314.

Preferably the local anaesthetic is Lignocaine. In this specification, the terms “lidocaine” and “lignocaine” are used interchangeably. Lignocaine is preferred as it is capable of acting as a local anaesthetic probably by blocking sodium fast channels, depressing 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 lignocaine normally has little effect on atrial tissue, and therefore is ineffective in treating atrial fibrillation, atrial flutter, and supraventricular tachycardias. 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 lignocaine would not completely block the voltage-dependent sodium fast channels, but would down-regulate channel activity and reduce sodium entry. As anti-arrhythmic, lignocaine 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.

As lignocaine acts by primarily blocking sodium fast channels, it will be appreciated that other sodium channel blockers may be used instead of or in combination with the local anaesthetic in the method and composition of the present invention. It will also be appreciated that sodium channel blockers include compounds that act to substantially block sodium channels or at least 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, NW-1029 (a benzylamino propanamide derivative), RS100642, riluzole, carbamazepine, flecainide, propafenone, amiodarone, sotalol, bretylium, 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).

In one aspect, the composition according to the invention consists essentially of (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; and (ii) a local anaesthetic.

In another aspect, the composition according to the invention may further include an opioid. The further addition of an opioid may have similar if not improved effect on the reduction of injury.

Opioids, 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 peri- 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). Opioid receptors are further subtyped, for example, the delta receptor has two subtypes, delta-1 and delta-2.

Cardiovascular effects of opioids are directed within the intact body both centrally (ie, at the cardiovascular and respiratory centres of the hypothalamus and brainstem) and peripherally (ie, 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 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 ischemic 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 instead 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 (natural or synthetic) 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.

Accordingly, the opioid may be selected from enkephalins, endorphins and dynorphins. Preferably the opioid is an enkephalin which targets delta, kappa and/or mu receptors. More preferably the opioid is a delta opioid receptor agonist. Even more preferably the opioid is selected from delta-1-opioid receptor agonists and delta-2-opioid receptor agonists. [D-Pen 2, 5]enkephalin (DPDPE), is a particularly preferred delta-1-opioid receptor agonist.

In another aspect the composition of the invention consists essentially of (i) a potassium channel opener or agonist and/or an adenosine receptor agonist; (ii) a local anaesthetic and (iii) a delta-1-opioid. DPDPE is a particularly preferred delta-1-opioid receptor agonist.

The inventor has found that the inclusion of a compound for minimizing or reducing the uptake of water by a cell in a tissue with a potassium channel opener or adenosine receptor agonist and a local anaesthetic assists in reducing injury to a body, such as a composition comprising sucrose, adenosine and lignocaine.

Thus in a further aspect, the composition according to the invention may further include at least one compound for minimizing or reducing the uptake of water by a cell in the cell, tissue or organ.

A compound for minimizing or reducing the uptake of water by a cell in the tissue tends to control water shifts, ie, 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.

Compounds for minimizing or reducing the uptake of water by a cell in a tissue are typically impermeants or receptor antagonists or agonists. 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 [(2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo[2.2.2.)-octan-3-amine (CP-96,345)] and L-733,060 or [(2S,3S)3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine]. R116301 or [(2R-trans)-4-[1-[3,5-bis(trifluoromethyl)benzoyl]-2-(phenylmethyl)-4-piperidinyl]-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, beta-hydroxy butyrate 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. In one embodiment, the at least one compound for minimizing or reducing the uptake of water by the cells in the tissue is an energy substrate. The energy substrate helps with recovering metabolism. The 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. Given that energy substrates are sources of reducing equivalents for energy transformations and the production of ATP in a cell, tissue or organ of the body, it will be appreciated that a direct supply of the energy reducing equivalents could be used as substrates for energy production. For example, a supply of either one or more or different ratios of reduced and oxidized forms of nicotinamide adenine dinucleotide (e.g. NAD or NADP and NADH or NADPH) or flavin adenine dinucleotides (FADH or FAD) could be directly used to supply bond energy for sustaining ATP production in times of stress. Preferably, beta-hydroxy butyrate is added to the composition of the invention for treatment of trauma or reducing injury.

In addition to providing energy substrates to the whole body, organ, tissue or cell, improvements in metabolising these substrates may occur in the presence of hydrogen sulphide (H2S) or H2S donors (eg NaHS). The presence of hydrogen sulphide (H2S) or H2S donors (eg NaHS) may help metabolise these energy substrates by lowering energy demand during arrest, protect and preserve the whole body, organ, tissue or cell during periods of metabolic imbalance such ischemia, reperfusion and trauma. Concentrations of Hydrogen sulfide above 1 microM (10-6 M) concentration can be a metabolic poison that inhibits respiration at Respiratory Complex IV, which is part of the mitochondrial respiratory chain that couples metabolising the high energy reducing equivalents from energy substrates to energy (ATP) generation and oxygen consumption. However, it has been observed at lower concentrations, below 10-6 M (eg 10-10 to 10-9M), hydrogen sulfide may reduce the energy demand of the whole body, organ, tissue or cell which may result in arrest, protection and preservation. In other words, very low levels of sulfide down-regulate mitochondria, reduce O2 consumption and actually increase “Respiratory Control” whereby mitochondria consume less O2 without collapsing the electrochemical gradient across the inner mitochondrial membrane. Thus there are observations that a small amount of sulfide, either directly or indirectly, may close proton leak channels and better couple mitochondrial respiration to ATP production more tightly, and this effect may improve the metabolism of high energy reducing equivalents from energy substrates. There is also the possibility that a sulphur cycle exists between the cell cytosol and mitochondria in mammals, including humans, providing the sulphur concentration is low. The presence of a vestige sulphur cycle would be consistent with current ideas on the evolutionary origin of mitochondria and their appearance in eukaryote cells from a symbiosis between a sulfide-producing host cell and a sulfide-oxidizing bacterial symbiont. Thus, hydrogen sulphide (H2S) or H2S donors (eg NaHS) may be energy substrates themselves in addition to improving the metabolism of other energy substrates. Accordingly, in one form, the invention provides a composition as described above further including hydrogen sulphide or a hydrogen sulfide donor.

In one embodiment, the at least one 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 too large 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.

In another embodiment, the at least one compound for minimizing or reducing the uptake of water by the cells in the tissue is a colloid. Suitable colloids include, but not limited to, Dextran-70, 40, 50 and 60, hydroxyethyl starch and a modified fluid gelatin. A colloid is a composition which has a continuous liquid phase in which a solid is suspended in a liquid. Colloids can be used clinically to help restore balance to water and ionic distribution between the intracellular, extracellular and blood compartments in the body after an severe injury. Colloids can also be used in solutions for organ preservation. Administration of crystalloids can also restore water and ionic balance to the body but generally require greater volumes of administration because they do not have solids suspended in a liquid. Thus volume expanders may be colloid-based or crystalloid-based

Preferably, the concentration of the compound for minimizing or reducing the uptake of water by the cells in the tissue is between about 5 to 500 mM. Typically this is an effective amount for reducing the uptake of water by the cells in the tissue. More preferably, the concentration of the compound for reducing the uptake of water by the cells in the tissue is between about 20 and 100 mM. Even more preferably the concentration of the compound for reducing the uptake of water by the cells in the tissue is about 70 mM.

In a further embodiment, the composition according to the invention may include more than one compound for minimizing or reducing the uptake of water by the cells in the tissue. For example, a combination of impermeants (raffinose, sucrose and pentastarch) may be included in the composition or even a combination of colloids, and fuel substrates may be included in the composition.

The composition according to the invention may be hypo, iso or hyper osmotic.

The inventor has also found that the inclusion of a compound for inhibiting transport of sodium and hydrogen ions across a plasma membrane of a cell in the tissue with a potassium channel opener or adenosine receptor agonist and a local anaesthetic assists in reducing injury.

Thus in another aspect, the composition according to the invention further includes a compound for inhibiting transport of sodium and hydrogen ions across a plasma membrane of a cell in the tissue.

The compound for inhibiting transport of sodium and hydrogen across the membrane of the cell in the tissue is also referred to as a sodium hydrogen exchange inhibitor. The sodium hydrogen exchange inhibitor reduces sodium and calcium entering the cell.

Preferably the compound for inhibiting transport of sodium and hydrogen across the membrane of the cell in the tissue may be selected from one or more of the group consisting of Amiloride, EIPA(5-(N-entyl-N-isopropyl)-amiloride), cariporide (HOE-642), eniporide, Triamterene (2,4,7-triamino-6-phenylteride), EMD 84021, EMD 94309, EMD 96785, EMD 85131, HOE 694. B11 B-513 and T-162559 are other inhibitors of the isoform 1 of the Na+/H+ exchanger.

Preferably, the sodium hydrogen exchange inhibitor is Amiloride (N-amidino-3,5-diamino-6-chloropyrzine-2-carboximide hydrochloride dihydrate). Amiloride inhibits the sodium proton exchanger (Na+/H+ exchanger also often abbreviated NHE-1) and reduces calcium entering the cell. During ischemia excess cell protons (or hydrogen ions) are believed to be exchanged for sodium via the Na+/H+ exchanger.

Preferably, the concentration of the compound for inhibiting transport of sodium and hydrogen across the membrane of the cell in the tissue is between about 1.0 nM to 1.0 mM. More preferably, the concentration of the compound for inhibiting transport of sodium and hydrogen across the membrane of the cell in the tissue is about 20 uM.

The inventor has also found that the inclusion of antioxidant with a potassium channel opener or adenosine receptor agonist and a local anaesthetic. Thus in another aspect, the composition of the present invention may further include an antioxidant.

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 Linoleic 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, O-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 disulphur derivatives); N-2-mercaptopropionyl glycine (MPG) a specific scavenger of the OH free radical; lipooxygenase inhibitor nordihydroguaretic acid (NDGA); Alpha Lipoic Acid; Chondroitin Sulfate; L-Cysteine; oxypurinol and Zinc.

Preferably, the antioxidant is allopurinol (1H-Pyrazolo[3,4-a]pyrimidine-4-ol). 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, mitochondrial damage, apoptosis and cell death. Preferably, the concentration of the antioxidant is between about 1 nM to 100 uM.

The inventor has also found that the inclusion of particular amounts of calcium and magnesium ions with a potassium channel opener or adenosine receptor agonist and a local anaesthetic reduces injury. The effect of the particular amounts of calcium and magnesium ions is to control the amount of ions within the intracellular environment. Calcium ions tend to be depleted, exported or otherwise removed from the intracellular environment and magnesium ions tend to be increased or otherwise restored to the levels typically found in a viable, functioning cell.

Thus in another aspect, the composition according to the invention further includes a source of magnesium in an amount for increasing the amount of magnesium in a cell in body tissue. Preferably the magnesium is present at a concentration of between 0.5 mM to 20 mM, more preferably about 2.5 mM. It will be appreciated that these concentrations refer to the effective concentration of the magnesium in the composition that contacts the tissue, organ or cell.

In addition, typical buffers or carriers (which are discussed in more detail below) in which the composition of the invention is administered typically contain calcium at concentrations of around 1 mM as the total absence of calcium has been found to be detrimental to the cell, tissue or organ. In one form, the invention also includes using carriers with low calcium (such as for example less than 0.5 mM) so as to decrease the amount of calcium within a cell in body tissue, which may otherwise build up during injury/trauma/stunning. As described in the present invention, elevated magnesium and low calcium has been associated with protection during ischemia and reoxygenation of an organ. The action is believed to be due to decreased calcium loading. Preferably the calcium present is at a concentration of between 0.1 mM to 0.8 mM, more preferably about 0.3 mM.

In one embodiment, the composition includes elevated divalent magnesium ions. Magnesium sulphate and magnesium chloride is a suitable source.

In the case of a human subject requiring treatment, the following alternative compositions with corresponding concentrations of Adenosine(Ado), Lignocaine (Lido) and magnesium sulphate are provided, without limitation:

Ado Lido MgSO4 7 H2O I 2.25 mM 1.844 mM 243.4 mM II 3.74 mM 3.688 mM 243.4 mM III 3.74 mM 7.376 mM 243.4 mM IV 5.61 mM 3.688 mM 243.4 mM V 5.61 mM 7.376 mM 243.4 mM VI 22.5 mM 18.44 mM 243.4 mM VII 37.4 mM 36.88 mM 243.4 mM VIII 37.4 mM 73.76 mM 243.4 mM IX 56.1 mM 36.88 mM 243.4 mM X 56.1 mM 73.76 mM 243.4 mM

The concentrations of each respective active ingredient in these compositions refer to the concentrations in the composition before administration. It will be appreciated that the concentrations may be diluted by body fluids or other fluids that may be administered together with the composition. Typically, the composition will be administered such that the concentration of these ingredients at the tissue is about 100-fold less than the concentrations in the table above. For example, containers (such as vials) of such a composition may be diluted 1 to a 100 parts of blood, plasma, crystalloid or blood substitute for administration.

In one embodiment, the composition according to the invention includes Adenosine and Lignocaine. Typically, the concentration of Adenosine and Lidocaine in the composition is between about 1 mM to 100 mM. The final concentration of these components once administered may be between about 0.1 mM to 10 mM.

In another embodiment, the composition includes a cellular transport enzyme inhibitor, such as dipyridamole, to prevent metabolism or breakdown of components in the composition.

In a further aspect, the invention provides a composition including a local anaesthetic and one or more of: potassium channel opener; adenosine agonist; opioid; at least one compound for reducing uptake of water; sodium hydrogen exchange inhibitor; antioxidant; and a source of magnesium in an amount for increasing the amount of magnesium in a cell in body tissue.

Preferably, this composition has two, three or four of the above. Preferred compounds for these components are listed above.

In another embodiment, the invention provides a composition including a potassium channel opener and/or an adenosine agonist and one or more of: local anaesthetic; opioid;

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