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US20160166598A1 - A method for treating infection, sepsis and injury - Google Patents

A method for treating infection, sepsis and injury Download PDF

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Publication number
US20160166598A1
US20160166598A1 US14/905,725 US201414905725A US2016166598A1 US 20160166598 A1 US20160166598 A1 US 20160166598A1 US 201414905725 A US201414905725 A US 201414905725A US 2016166598 A1 US2016166598 A1 US 2016166598A1
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composition
sepsis
adenosine
blood
infection
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Geoffrey Phillip Dobson
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Hibernation Therapeutics KF LLC
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Hibernation Therapeutics KF LLC
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Assigned to HIBERNATION THERAPEUTICS A KF LLC reassignment HIBERNATION THERAPEUTICS A KF LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HTS THERAPEUTICS PTY LTD
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Definitions

  • the invention relates to treating infections in a subject, including sepsis and sepsis-related vulnerabilities.
  • the invention also relates to a method for treating infection in a subject by protecting cells, tissues or organs from injury that may result from infection, including sepsis and sepsis-related vulnerabilities.
  • the invention also relates to treating the cardiovascular, renal, respiratory, microvascular and endothelial systems, inflammation, coagulopathy and pain in a subject in need of treatment following infection or injury.
  • the present application claims priority from Australian Provisonal Patent Application Nos. 2013902656, 2013902657, 2013902658, 2013902659 and 2013903644, the entire disclosures of which are incorporated into the present specification by this cross-reference.
  • Drugs can be divided into three categories according to their mechanism of action: i) agents that block bacterial products and inflammatory mediators, ii) modulators of immune function, and iii) immunostimulation (reduce immunosuppression). Drug development could also have an impact on many pathologies involving low levels of inflammatory markets and immune imbalances.
  • the present invention is directed toward overcoming or at least alleviating one or more of the difficulties of the prior art.
  • the invention relates to methods of treating infection in a subject, in particular sepsis or sepsis related vulnerabilities.
  • the invention also relates to compositions which may be used in these methods and pharmaceutical compositions suitable for such treatments.
  • the present invention is directed to a method for treating infection comprising the administration (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic to a subject in need thereof.
  • the method also includes administration of an elevated source of magnesium ions.
  • the method also includes administration of an anti-inflammatory agent and/or metabolic fuel to a subject in need thereof.
  • the present invention is directed to a method for treating sepsis comprising the administration of (i) compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic to a subject in need thereof.
  • the method also includes the administration of an anti-inflammatory agent and/or metabolic fuel to a subject in need thereof.
  • the present invention is directed to a method for treating injury comprising the administration of (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic to a subject in need thereof.
  • the method also includes the administration of an anti-inflammatory agent and/or metabolic fuel to a subject in need thereof.
  • the present invention is directed to a method of reducing infection in a subject by protecting cells, tissues or organs from injury that may result from infection, including sepsis and sepsis-related vulnerabilities comprising administration of compounds (i) and (ii) defined above.
  • the present invention is also directed to use of (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic in the manufacture of a medicament for treating at least one of infection, sepsis and injury in a subject.
  • the present invention is also directed to use of (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic for treating at least one of infection, sepsis and injury in a subject.
  • the present invention is also directed to (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic for use in treating at least one of infection, sepsis and injury in a subject.
  • the infections may result from injury to the subject, including traumatic injury.
  • the present invention is directed to a composition which may be used in treating at least one of infection, sepsis and injury comprising (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic.
  • the composition includes or is administered together with an anti-inflammatory agent and/or metabolic fuel to a subject in need thereof.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • composition is a pharmaceutical composition.
  • the composition may be in the form of a kit in which components (i) and (ii) are held separately.
  • the kit may be adapted to ensure simultaneous, sequential or separate administration of components (i) and (ii) when used in the methods defined in this specification.
  • the anti-inflammatory agent is BOH.
  • the metabolic fuel is citrate.
  • the antiarrhythmic agent is lidocaine.
  • the potassium channel opener, potassium channel agonist and/or adenosine receptor agonist is adenosine.
  • the present invention is directed to a method of inducing a hypotensive state in a subject susceptible to or suffering from at least one of infection, sepsis and injury comprising the administration of a composition including (i) a potassium channel opener or agonist and/or adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic and an elevated source of magnesium ions to the subject.
  • a composition including (i) a potassium channel opener or agonist and/or adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic and an elevated source of magnesium ions to the subject.
  • the composition may also include or be administered with an anti-inflammatory agent and/or metabolic fuel.
  • the present invention is directed to a method of inducing a low pain or analgesic state in a subject susceptible to or suffering from at least one of infection, sepsis and injury comprising the administration of a composition including (i) a potassium channel opener or agonist and/or adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic and an elevated source of magnesium ions to the subject.
  • a composition including (i) a potassium channel opener or agonist and/or adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic and an elevated source of magnesium ions to the subject.
  • the composition may also include or be administered with an anti-inflammatory agent and/or metabolic fuel.
  • the methods for inducing or assisting to induce controlled hypotensive anaesthesia or a low pain or analgesic state are useful for emergency transport, surgery or clinical interventions to reduce blood loss, inflammation and coagulopathy and further injury.
  • This invention is directed to methods for treating infection, sepsis and/or injury.
  • the invention is directed to a method for treating infection comprising the administration of (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic to a subject in need thereof.
  • the composition includes an elevated source of magnesium ions.
  • the method also includes the administration of an anti-inflammatory agent and/or metabolic fuel to a subject in need thereof.
  • the inventor has found that the administration of (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic (preferably including an elevated source of magnesium ions) together with an anti-inflammatory agent and/or metabolic fuel to a subject in need thereof reduces infection in the subject by protecting against injury to cells, tissues and/or organs of the body of the subject.
  • a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist preferably including an elevated source of magnesium ions
  • compositions in this specification may have been infected or suffering from an infection or at risk of developing an infection and the injury to the cells, tissues and/or organs is reduced or minimised by the administration of the composition according to the invention.
  • the infection may be acquired by the subject in hospital or present to the hospital with the infection for treatment.
  • the components of the composition may be administered simultaneously, sequentially or separately depending on the intended use.
  • this composition will be referred to in this specification as the “composition” or “composition useful in methods according to the invention” or “compositions for use” or other similar terms, although there are a number of combinations of components embodying the invention which are compositions useful in the invention.
  • the invention is directed to a method for treating sepsis comprising the administration of (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic to a subject in need thereof.
  • the method also includes administration of an elevated source of magnesium ions.
  • the method also includes administration of an anti-inflammatory agent and/or metabolic fuel to a subject in need thereof.
  • the subject in need thereof may suffering from sepsis or at risk of developing sepsis and the injury to the cells, tissues and/or organs is reduced or minimised by the administration of the composition according to the invention.
  • Severe sepsis is associated with profound cardiovascular dysfunction characterized by hypotension, decreased systemic resistance, altered vascular reactivity to contractile agents and decreased myocardial contractility.
  • Systemic infection depresses heart function and the severity of this myocardial depression correlates with a poor prognosis.
  • Echocardiographic studies suggest that 40% to 50% of patients with prolonged septic shock develop myocardial depression, as defined by a reduced ejection fraction.
  • a circulating myocardial depressant factor in septic shock has long been proposed, and potential candidates for a myocardial depressant factor include circulating factors such as cytokines (TNF-alpha, IL-1beta), lysozyme c, prostanoids, endothelin-1 have direct inhibitory actions on myocyte contractility.
  • cytokines TNF-alpha, IL-1beta
  • lysozyme c lysozyme c
  • prostanoids endothelin-1
  • endothelin-1 have direct inhibitory actions on myocyte contractility.
  • Nitric oxide (NO) has a complex role in sepsis-induced cardiac dysfunction.
  • the concept of septic cardiomyopathy has evolved, which implies alterations in the myocardial phenotype.
  • Sepsis is a severe complication of critically ill patients that is characterized by the systemic inflammatory response syndrome (SIRS) and the early release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF-alpha), IL-1 and IL-6, and together these cytokines contribute to the development of multiple organ dysfunction/failure syndrome (MODS).
  • SIRS systemic inflammatory response syndrome
  • TNF-alpha tumor necrosis factor
  • IL-1 and IL-6 tumor necrosis factor
  • MODS multiple organ dysfunction/failure syndrome
  • systemic inflammatory response mediates its deleterious effects by inducing tissue hypoxia, and cellular injury, either through tissue necrosis or through the induction of programmed cell death or apoptosis.
  • endotoxin or cytokines such as TNF- ⁇ or interleukin-1 (IL-1)
  • IL-1 interleukin-1
  • DIC disseminated intravascular coagulation
  • Septic patients with severe DIC have microvascular fibrin deposition, which can lead to MODS and death.
  • severe bleeding might be the leading symptom (hypocoagulopathy), or even coexisting bleeding and thrombosis.
  • the deranged coagulation, particularly DIC is an important and independent predictor of mortality in patients with severe sepsis.
  • thrombin can elicit many inflammatory responses in microvascular endothelium, loss of control of microvascular thrombin generation due to impaired protein C pathway function probably contributes to microvascular dysfunction in sepsis.
  • activated protein C has been reported to inhibit TNF-alpha elaboration from monocytes and to block leukocyte adhesion to selectins. Thuis the protein C anticoagulant pathway serves as a major system for controlling thrombosis, limiting inflammatory responses, and potentially decreasing endothelial cell apoptosis in response to inflammatory cytokines and ischemia.
  • TNF alpha The primary role of TNF alpha is in the regulation of immune cells.
  • TNF alpha is a cytokine involved in local and systemic inflammation, and along with other cytokines stimulates the acute phase reaction to stress and infection. TNF-alpha also induces activation of coagulation in different pathological states including sepsis.
  • Activated protein C inhibits TNF-alpha production.
  • Activated protein C (and antithrombin) may inhibit the endothelial perturbation induced by cytokines.
  • Antithrombin regulates TNF-alpha induced tissue factor expression on endothelial cells by an unknown mechanism.
  • Activated protein C and antithrombin, and their pathways of regulation may be useful targets for treating coagulation abnormalities associated with sepsis or other inflammation diseases. These sites and pathways inhibit not only coagulation but also involved with the downregulation of anticoagulant activities of endothelial cells.
  • Pro-inflammatory cytokines include tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1 ⁇ , IL-6), Therapies that improve inflammation and coagulation balance with cardiovascular support may reduce “sepsis-associated encephalopathy” and improve outcome and survival.
  • TNF tumor necrosis factor
  • IL-1 ⁇ Interleukin-1 ⁇
  • IL-6 Interleukin-6
  • HAI Healthcare-associated infections
  • CDC Centers for Disease Control and Prevention
  • Infectious complications are frequently reported in critically ill patients (e.g. type 2 diabetes mellitus, chronic obstructive pulmonary disease, chronic heart failure and chronic renal disease). Infections are common after traumatic brain injury, heart attack, cardiac arrest, hemorrhagic shock, non-hemorrhagic shock, surgery and radiation therapy for cancers. Pneumonia occurs in 5-22% and is the most common cause of death in stroke patients. Patients who receive hemodialysis are at a significant risk of developing infections, a leading cause of hospitalization and death in this patient.
  • Adhesions develop as the body attempts to repair itself from infection, surgery, injury (trauma) and radiation. Up to 93 percent of people who have abdominal surgery go on to develop adhesions.
  • Infections after trauma are associated with subsequent cardiac injury and disease including myocardial ischemia and infarction.
  • Infection from bacterial contamination is very common after penetrating abdominal injury when the gut has been perforated and where post-injury antimicrobials are the standard of care.
  • the most common source of infection in community acquired intra-abdominal infections in the Appendix, followed by the colon, the stomach. Dechiscence complicates 5-10% of intra-abdominal bowel anastomoses and is associated with high rates of mortality.
  • the risk for surgical site infection following colon surgery can be as high as 20%.
  • the intestinal tract is engaged in a relationship with a dense and complex microbial ecosystem, the microbiota.
  • Non-penetrating injuries such as from high-energy transfer blunt abdominal impacts can also trigger intra-abdominal infection. These may arise car accidents, sports injuries, projectile impacts, internal hemorrhage, a blast or secondary complications following surgery.
  • the infection can be triggered from local or widespread ischemia to the abdominal organs and particularly to the gut wall, and delays in diagnosis can be fatal.
  • ischemic damage bacterial translocation into the peritoneum can occur resulting in widespread infection and sepsis.
  • a common denominator or mediator network that is a trigger for the sepsis-like symptoms seen in those patients without the diagnosis of sepsis compared to those patients with sepsis.
  • the nature of the common denominator or mediator network is not known, however, treating the cardiovascular and organ systems along with correcting the inflammatory and coagulation systems may benefit both.
  • the present invention therefore bridges this gap and would be beneficial to both patients with sepsis, and those patients without the diagnosis of sepsis but have sepsis-like symptoms.
  • ECMO venoarterial extracorporeal membrane oxygenation
  • Reducing infection, inflammation and coagulation outside the brain may improve postoperative cognitive decline.
  • Postoperative delirium are a major cause of morbidity associated with surgery.
  • POCD occurs in 7-26% of patients undergoing surgery.
  • compositions, methods of treatment and uses involving a composition
  • a composition comprising (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic, together with additional components where applicable.
  • the composition also includes an elevated source of magnesium ions.
  • Acute brain injury results in decreased heart beat oscillations and baroreflex sensitivity indicative of uncoupling of the autonomic and cardiovascular systems.
  • the invention may improve neuroautonomic regulation of heart rate and blood pressure oscillations by reducing dangerous oscillations in the body's normal biorhythms such as in heart rate and blood pressure which implies improved brain function.
  • Improving HR variability and reducing infection, inflammation and coagulation outside the brain may improve brain function including postoperative cognitive decline.
  • Postoperative delirium POCD
  • POCD Postoperative delirium
  • Efferent nerve connections from the vagal nerve to the spleen can be modulated to block experimental septic shock and autoimmune immune models of rheumatoid arthritis.
  • the present invention may act to bring balance to these intricate interactions between the periphery and brain and restore homeostasis
  • the acute phase response is a complex systemic early-defense of the body system activated by infection, injury, trauma, infection, stress, neoplasia, and inflammation.
  • the term ‘acute phase’ was introduced in the 1930s when the first “acute phase” protein, C-reactive protein was discovered early during pneumococcal infection of monkeys and humans.
  • CRP remains an APP of primary interest in humans, where it is a major marker of infection, autoimmune disease, trauma, surgery, malignancy, and necrosis including myocardial infarction.
  • APR serves as a core of the innate immune response involving physical and molecular barriers and responses that serve to prevent infection, clear potential pathogens, initiate inflammatory processes, and contribute to resolution and the healing process.
  • a prominent feature of this early response is the appearance of pro-inflammatory cytokines, particularly IL-6 and the induction of acute phase proteins in the liver and elsewhere such as C-reactive protein, fibrinogen, ⁇ 2 -macroglobulin and other anti-proteinases, which are involved in the restoration of homeostasis.
  • cytokines and growth factors such as transforming growth factor-beta (TGF-b) and TNF ⁇ , are secreted by polymorphonuclear leukocytes (PMN's or neutrophils).
  • TGF-b transforming growth factor-beta
  • PMN's or neutrophils polymorphonuclear leukocytes
  • Systemic inflammation results in a systemic acute phase response.
  • Acute phase proteins are blood proteins primarily synthesized by hepatocytes as part of the acute phase response.
  • APR Acute phase proteins have been well recognized for their application to human diagnostic medicine and have been described to have value in the diagnosis and prognosis of cardiovascular disease, autoimmunity, organ transplant, and cancer treatment.
  • Sepsis is a severe systemic inflammatory response syndrome (SIRS) arising from an infection in response to a pathogen such as bacteria, virus or fungi. It can arise from a traumatic or non-traumatic injury. One in three people die of severe sepsis. Bacterial infections are the most common cause of sepsis. The source of the infection can be any of a number of places throughout the body. Common sites and types of infection that can lead to sepsis include:
  • Septic Shock is an overwhelming whole body response to an infection that leads to life-threatening low blood pressure (shock).
  • Septic shock is a continuum of the clinical manifestations from SIRS to sepsis to severe sepsis to septic shock, which can quickly progress into Multiple Organ Dysfunction Syndrome (MODS).
  • MODS Multiple Organ Dysfunction Syndrome
  • Severe sepsis is characterized by at least one organ dysfunction or hypoperfusion and septic shock describes severe sepsis associated with hypotension that is resistant to adequate fluid resuscitation.
  • Septic shock is a medical emergency and is associated with a high mortality of over 50%.
  • MODS Multiple Organ Dysfunction Syndrome
  • MOF multiple organ failure
  • MSOF multisystem organ failure
  • Battlefield Trauma is defined as combat, wounding, witnessing the wounding or death of fellow soldiers, enemies and civilians and mutilation from small arms aerial bombardment, burns and blasts.
  • military trauma injuries tend to be different from civilian trauma injuries.
  • Infection is also higher from wound contamination caused by particulates (soil, clothing, environmental debris) being forced into injuries from the trauma. Trauma can also arise from the experience of war such as the post-traumatic stress syndrome.
  • Trauma traumatic event include cell, tissue, organ or whole body damage that can occur from a traumatic event.
  • Trauma may appear as the primary injury from the initial traumatic event, and secondary injury which is a time-dependent process progressing from the primary event and may include, but not limited to, injuries from infection, ischemic injury, reperfusion injury with an inflammatory, coagulation and central nervous system regulatory dysfunction.
  • primary injuries (wounds and burns) for war are distinct from peacetime traumatic injuries because these higher velocity projectiles and/or blast devices cause a more severe injury and accompanying wounds are frequently contaminated by clothing, soil, and environmental debris.
  • the secondary injuries share many similarities to the civilian setting with the exception of long evacuation times where complications can arise.
  • Injuries can also occur from a primary non-traumatic (not from a physical or mechanical force) and includes damage resulting from infection, poisoning, tumor, or degenerative disease. Lack of oxygen, glucose, or blood can be considered non-traumatic arising from these causes. Infections can cause encephalitis (brain swelling), meningitis (meningeal swelling), or cell toxicity, as can tumors or poisons. These infections can occur through stroke, heart attack, near-drowning, strangulation or a diabetic coma, poisoning or other chemical causes such as alcohol abuse or drug overdose, infections or tumors and degenerative conditions such as Alzheimer's disease and Parkinson's disease.
  • Traumatic Brain injury is defined as damage to the brain resulting from an external physical or mechanical force, such as that caused by rapid acceleration or deceleration, blast waves, crush, an impact or penetration by a projectile. It can lead to temporary or permanent impairment of cognitive, physical and psychosocial function. In a traumatic injury, damage to nerve tissue is usually focused in one or more areas of the brain at first, although tearing can result in diffuse injury.
  • Non-traumatic Brain Injury is any injury to the brain that does not result from any cause that does not injure the brain using physical or mechanical force, but rather occurs via infection, poisoning, tumor, or degenerative disease.
  • Non-traumatic injury, damage is usually spread throughout the brain and exceptions include tumors and an infection that may remain localised or spreads evenly from one starting point.
  • Hypertonic saline is defined as a saline concentration greater than normal isotonic saline which is 0.9% NaCl (0.154 M). Hypertonic saline has been shown to reduce this bacterial translocation after shock.
  • Mesenteric Ischaemia is a condition characterized by high mortality and occurs when the blood flow to the small intestine is slowed or stopped. Due to the diminished blood flow, the cells in your gut fed by the mesenteric artery are starved for oxygen, and can become damaged and lead to Ileus, adhesions, infection and severe sepsis.
  • Mesenteric venous thrombosis can occur when a blood clot develops in the vein that carries blood away from the intestines and may result from acute or chronic inflammation of the pancreas (pancreatitis), abdominal infection, bowel diseases, such as ulcerative colitis, Crohn's disease or diverticulitis, hypercoagulation disorders, injury (traumatic or non-traumatic) to the abdomen.
  • Ischaemia is defined as reduced blood flow to the entire intestine and can occur in clinical scenarios such as organ transplantation, trauma and cardio-pulmonary bypass, as well as in neonatal necrotizing enterocolitis or persistent ductus arteriosus. Ischemia can lead to inflammation, infection, multiple organ dysfunction and death.
  • Inflammatory bowel disease is an inflammatory disease of the bowel and the cause(s) are not fully known. Genetic, environmental, microbial, and immunologic factors are involved, but the precise mechanisms are obscure. Examples include ulcerative colitis and Crohn's disease. Immunosuppressive therapy can have potentially life-threatening consequences such as infections and reactivations of latent infections like tuberculosis or cytomegalovirus. Treatment often emphasizes a program rather than a drug therapy.
  • Ischaemia colitis is caused by inflammation and injury to the large intestine as a result from insufficient blood supply. Some patients with severe gut ischaemia from low blood flow may develop sepsis and become critically ill.
  • hypotensive anaesthesia is the controlled regulation of mean arterial pressures (MAP) that reduces blood loss during surgery or clinical interventions. Studies have shown that if MAP is reduced to 50 mmHg during surgery or interventions the blood loss can reduce by over 50%, which may reduce the need for fluid or blood products. The reduced blood loss also limits dilution and consumption of coagulation factors and subsequent postoperative rebound hypercoagulability. If MAP is maintained at 60 mmHg rather than 50 mmHg, blood loss is about 40% greater. Hypotensive anaesthesia can be induced using either general or regional anaesthesia and enhanced using vasodilators to improve cardiac output.
  • MAP mean arterial pressures
  • Marine Stingers There is an enormous diversity and complexity of venoms and poisons in marine animals. Fatalities have occurred from envenoming by sea snakes, venomous fish (stonefish), cone shells or snails, blue-ringed octopus and jellyfish. There are numerous venomous jellyfish around the pacific rim and Australia. Chironex fleckeri, the box jellyfish, is the most lethal causing rapid cardiorespiratory depression. Carukia barnesi, another small carybdeid leads to the so-called ‘Irukandji’ syndrome which includes delayed pain from severe pain, muscle cramping, vomiting, anxiety, restlessness, sweating and prostration, severe hypertension and acute cardiac failure.
  • the syndromic illness resulting from a characteristic relatively minor sting, develops after about 30 minutes.
  • the mechanisms of actions of their toxins appear to include modulation of neuronal sodium channels leading to massive release of endogenous catecholamines (C. barnesi, A. mordens and M. maxima) and possibly stress-induced cardiomyopathy.
  • endogenous catecholamines C. barnesi, A. mordens and M. maxima
  • stress-induced cardiomyopathy In human cases of severe envenomation, systemic hypertension and myocardial dysfunction are associated with membrane leakage of troponin indicating heart cell death.
  • Clinical management includes parenteral analgesia, antihypertensive therapy, oxygen and mechanical ventilation. The present invention may alleviate some of these symptoms.
  • Tissue 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 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 (ie, myocytes), nerve cells, brain cells or kidney cells.
  • 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
  • Organ 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.
  • 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.
  • the subject 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.
  • a livestock animal eg, sheep, cow or horse
  • laboratory test animal eg, mouse, rabbit or guinea pig
  • a companion animal eg, dog or cat
  • the subject is human.
  • Body The body is the body of a subject as defined above.
  • Treating includes inhibiting, relieving or ameliorating, reducing or preventing.
  • Comprises 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.
  • composition The term “pharmaceutical composition” as used in this specification also includes “veterinary composition”.
  • derivatives refer to variations in the structure of the compounds.
  • the derivatives are preferably “pharmaceutically acceptable derivative” which includes any pharmaceutically acceptable salt, hydrate, ester, ether, amide, active metabolite, analogue, residue or any other compound which is not biologically or otherwise undesirable and induces the desired pharmacological and/or physiological effect.
  • Salts of the compounds are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the specification, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic, toluenesulphonic, benzenes
  • the methods and compositions according to the invention further include magnesium ions, preferably elevated magnesium ions i.e. over normal plasma concentrations.
  • the magnesium is divalent and present at a concentration of 2000 mM or less, 0.5 mM to 800 mM, 10 mM to 600 mM, 15 mM to 500 mM, 20 mM to 400 mM, 20 mM or 400 mM, more preferably 20 mM.
  • Magnesium sulphate and magnesium chloride are suitable sources in particular magnesium sulphate.
  • the inventor has also found that the inclusion of the magnesium ions with (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent may also reduce injury.
  • the effect of the particular amounts of magnesium ions is to control the amount of ions within the intracellular environment. Magnesium ions tend to be increased or otherwise restored to the levels typically found in a viable, functioning cell.
  • composition useful in the methods according to the invention may further include a source of magnesium in an amount for increasing the amount of magnesium in a cell in body tissue.
  • the composition useful in the methods according to the invention includes (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; (ii) an antiarrhythmic agent or a local anaesthetic; and an elevated source of magnesium ions.
  • potassium is present in the composition it will typically be present in an amount at physiological level to ensure that the blood concentration of the subject is less than 10 mM or 3 to 6 mM. This means that when the composition is administered, the cell membrane remains in a more physiological polarised state thereby minimising potential damage to the cell, tissue or organ. High concentrations or concentrations above physiological levels of potassium would result in a hyperkalemic composition. At these concentrations the heart would be arrested alone from the depolarisation of the cell membrane.
  • One advantage of using physiological concentrations of potassium is that it renders the present composition less injurious to the subject, in particular paediatric subjects such as neonates/infants.
  • High potassium has been linked to an accumulation of calcium which may be associated with irregular heart beats during recovery, heart damage and cell swelling. Neonates/infants are even more susceptible than adults to high potassium damage during cardiac arrest. After surgery a neonate/infant's heart may not return to normal for many days, sometimes requiring intensive therapy or life support.
  • component (i) of the composition may be an adenosine receptor agonist. While this obviously includes adenosine itself or derivatives thereof such as CCPA and the like described below, 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 AcadesineTM or AICA-riboside (5-amino-4-imidazole carboxamide ribonucleoside).
  • 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
  • a compound that stimulates endogenous adenosine production such as purine nucleoside analogue AcadesineTM or AICA-riboside (5-amino-4-imidazole carboxamide ribonucleoside).
  • AcadesineTM is desirably administered to produce a plasma concentration of around 50 ⁇ M but may range from 1 ⁇ M to 1 mM or more preferably from 20 to 200 ⁇ M.
  • AcadesineTM 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.
  • Suitable adenosine receptor agonists may be selected from: N 6 -cydopentyladenosine (CPA), N-ethylcarboxamido adenosine (NECA), 2-[p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamido adenosine (CGS-21680), 2-chloroadenosine, N 6 -[2-(3,5-demethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine, 2-chloro-N 6 -cyclopentyladenosine (CCPA), N-(4-am inobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofuranosyl]-adenine (AB-MECA), ([IS-[1a,2b,3b,4a(S*)]]]-4-[7-[[2 ⁇
  • A1 receptor agonists such as N43-(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), or partial agonists such as CVT-2759 and allosteric enhancers such as PD81723.
  • Other agonists 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-naphthoylthiophenes.
  • the A1 adenosine receptor agonist is CCPA.
  • the concentration of adenosine receptor agonist in the composition may be 0.0000001 to 100 mM preferably 0.001 mM to 50 mM and most preferably 0.1 mM to 25 mM. In one embodiment, the concentration of the adenosine receptor agonist in the composition is about 19 mM.
  • the contact concentration of adenosine receptor agonist may be the same or less than the composition concentration set out above.
  • composition is diluted with a pharmaceutically acceptable carrier, including but not limited to blood, saline or a physiological ionic solution, the dosage of the composition may be adapted to achieve the most preferred contact concentrations.
  • a pharmaceutically acceptable carrier including but not limited to blood, saline or a physiological ionic solution
  • component (i) of the composition may be 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.
  • potassium channels are targets for the actions of transmitters, hormones, or drugs that modulate cellular function.
  • the potassium channel openers include the potassium channel agonists which also stimulate the activity of the potassium channel with the same result.
  • 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 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).
  • BK-activators also called BK-openers or BK(Ca)-type potassium channel openers or large-conductance calcium-activated potassium channel
  • Diazoxide and nicorandil are particular examples of potassium channel openers or agonists.
  • 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.
  • concentration of the diazoxide is between about 1 to 200 uM. Typically this is as an effective amount of diazoxide. More preferably, the contact concentration of diazoxide is about 10 uM
  • 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 may further attenuate ischemia/reperfusion injury.
  • 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.
  • 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.
  • Adenosine as well as functioning as an adenosine receptor agonist is also 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 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), analgesic, metabolic agent and possible nitric oxide donor. More recently, adenosine is known to inhibit several steps which can lead to slowing 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 or dormancy. An adenosine analogue, 2-chloro-adenosine, may be used.
  • the potassium channel opener, potassium channel or agonist and/or adenosine receptor agonist has a blood half-life of less than one minute, preferably less than 20 seconds.
  • the concentration of potassium channel opener in the composition may be 0.0000001 to 100 mM, preferably 0.001 mM to 50 mM and most preferably 0.1 mM to 25 mM. In one embodiment, the concentration of the potassium channel opener in the composition is about 19 mM.
  • the contact concentration of potassium channel opener may be the same or less than the composition concentration set out above.
  • composition is diluted with a pharmaceutically acceptable carrier, including but not limited to blood, saline or a physiological ionic solution, the dosage of the composition may be adapted to achieve the most preferred contact concentrations.
  • a pharmaceutically acceptable carrier including but not limited to blood, saline or a physiological ionic solution
  • composition useful in methods according to the invention also includes an antiarrhythmic agent.
  • Antiarrhythmic agents are a group of pharmaceuticals that are used to suppress fast rhythms of the heart (cardiac arrhythmias). The following table indicates the classification of these agents.
  • Repolarisation CLASS Channel effects Time Drug Examples
  • IA Sodium block Prolongs Quinidine, disopyramide, Procaine
  • IB Sodium block Shortens Lidocaine, phenytoin, mexiletine, Tocainide
  • IC Sodium block Unchanged Flecainide Propafenone, moricizine II Phase IV Unchanged Beta-blockers including (depolarising sotalol current); Calcium channel III Repolarising Markedly Amiodarone, Sotalol, Potassium prolongs bretylium Currents
  • IVA AV nodal calcium Unchanged Verapamil, diltiazem block
  • IVB Potassium channel Unchanged Adenosine, ATP openers
  • the antiarrhythmic agent may induce local anaesthesia (or otherwise be a local anaesthetic), for example, mexiletine, diphenylhydantoin, prilocaine, procaine, mepivocaine, quinidine, disopyramide and Class 1B antiarrhythmic agents.
  • local anaesthesia or otherwise be a local anaesthetic
  • mexiletine for example, mexiletine, diphenylhydantoin, prilocaine, procaine, mepivocaine, quinidine, disopyramide and Class 1B antiarrhythmic agents.
  • the antiarrhythmic agent is a class I or class III agent.
  • Amiodarone is a preferred Class III antiarrhythmic agent. More preferably, the antiarrhythmic agent blocks sodium channels. More preferably, the antiarrhythmic agent is a class IB antiarrhythmic agent.
  • Class 1B antiarrhythmic agents include lidocaine or derivatives thereof, for example, QX-314 is a quaternary lidocaine derivative (i.e., permanently charged) and has been shown to have longer-lasting local anesthetic effects than lidocaine-HCl alone.
  • the class 1B antiarrhythmic agent is lidocaine.
  • the terms “lidocaine” and “lidocaine” are used interchangeably.
  • Lidocaine is also known to be 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 lidocaine normally has little effect on atrial tissue, and therefore is ineffective in treating atrial fibrillation, atrial flutter, and supraventricular tachycardias.
  • Lidocaine is also a free radical scavenger, an antiarrhythmic and has anti-inflammatory and anti-hypercoagulable properties.
  • 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.
  • sodium channel blockers include compounds that act to substantially block sodium channels or at least downregulate sodium channels.
  • 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, 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).
  • the composition according to the invention comprises (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic.
  • the antiarrhythmic agent is a local anaesthetic such as lidocaine.
  • the composition also includes a source of elevated magnesium ions.
  • the concentration of antiarrhythmic agent or local anaesthetic in the composition may be 0.0000001 to 100 mM, preferably 0.001 mM to 50 mM and most preferably 0.1 mM to 40 mM.
  • the contact concentration of antiarrhythmic agent or local anaesthetic may be the same or less than the composition concentrations set out above
  • composition is diluted with a pharmaceutically acceptable carrier, including but not limited to blood, saline or a physiological ionic solution, the dosage of the composition may be adapted to achieve the most preferred contact concentrations.
  • a pharmaceutically acceptable carrier including but not limited to blood, saline or a physiological ionic solution
  • the composition according to the invention further includes an anti-inflammatory agent.
  • Anti-inflammatory agents such as beta-hydroxybutyrate (BOH), niacin and GPR109A can act on the GPR109A receptor (also referred to as hydroxyl-carboxylic acid receptor 2 or HCA-2). This receptor is found on immune cells (monocytes, macrophages), adipocytes hepatocytes, the vascular endothelium, and neurones.
  • the composition according to the invention includes (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) an anti-inflammatory agent.
  • the composition includes an elevated source of magnesium ions.
  • the anti-inflammatory agent activates a HCA-2 receptor such as beta-hydroxybutyrate (BOH).
  • BOH beta-hydroxybutyrate
  • Valproic acid is also a suitable anti-inflammatory agent.
  • Valproic acid (VPA) is a histone deacetylase inhibitor that may decrease cellular metabolic needs following traumatic injury.
  • Valproic acid (VPA) has proven to be beneficial after traumatic injury and has been shown to improve survival in lethal models of hemorrhagic shock.
  • VPA also is known to have cytoprotective effects from an increase acetylation of nuclear histones, promoting transcriptional activation of deregulated genes, which may confer multi-organ protection. It may also have beneficial effects in preventing or reducing the cellular and metabolic sequelae of ischemia-reperfusion injury and reduce injury to the endothelium through the TGF- ⁇ and VEGF functional pathways.
  • S1P Sphingosine-1-phosphate
  • further anti-inflammatory therapies have included the administration of aspirin, normal heparin, low-molecular-weight heparin (LMWH), non-steroidal anti-inflammatory agents, anti-platelet drugs and glycoprotein (GP) IIb/IIIa receptor inhibitors, statins, angiotensin converting enzyme (ACE) inhibitor, angiotensin blockers and antagonists of substance P.
  • aspirin normal heparin
  • LMWH low-molecular-weight heparin
  • GP glycoprotein IIb/IIIa receptor inhibitors
  • statins angiotensin converting enzyme (ACE) inhibitor
  • angiotensin blockers antagonists of substance P.
  • protease inhibitors examples include 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 such as aspirin, a glycoprotein (GP) IIb/IIIa receptor inhibitor is abciximab, a statin is pravastatin, an angiotensin converting enzyme (ACE) inhibitor is captopril and an angiotensin blocker is valsartin.
  • LMWH low-molecular-weight heparin
  • non-steroidal anti-inflammatory agent are indomethacin
  • ibuprofen rofecoxib
  • compositions useful in the methods according to the invention to deliver improved management of inflammation and clotting in order to reduce injury to cells, tissues or organs.
  • composition according to the invention may be administered together with any one or more of these agents.
  • protease inhibitors attenuate the systemic inflammatory response in patients undergoing cardiac surgery with cardiopulmonary bypass, 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 are also reduce blood loss and need for blood transfusions in surgical operations such as coronary bypass.
  • the concentration of anti-inflammatory agent in the composition maybe 0.0000001 to 300 mM, preferably 0.001 mM to 50 mM and most preferably 0.1 mM to 10 mM.
  • the contact concentration of anti-inflammatory agent may be the same or less than the composition concentration set out above.
  • composition is diluted with a pharmaceutically acceptable carrier, including but not limited to blood, saline or a physiological ionic solution, the dosage of the composition may be adapted to achieve the most preferred contact concentrations.
  • a pharmaceutically acceptable carrier including but not limited to blood, saline or a physiological ionic solution
  • the composition according to the invention further includes a metabolic fuel.
  • a metabolic fuel In sepsis, inhibition of pyruvate dehydrogenase limits pyruvate conversion to acetyl-coenzyme A, the main substrate that fuels the Krebs cycle to replenish ATP in the cell's powerhouse, the mitochondria.
  • a large part of Acetyl CoA comes from glucose metabolism (glycolysis) however Acetyl CoA can alternatively come from other pathways such as ketone metabolism, which forms acetyl CoA primes the cycle by forming citrate.
  • Alternative energy sources that can bypass glucose as a fuel may also be useful and these include ketones (acetone or acetoacetate) or carboxylic acids (d-beta-hydroxybutryate).
  • Natural hibernating animals produce ketones (and carboxylic acids) during hibernation to replenish the energy currency of the cell (adenosine-5′-triphosphate, ATP) and humans do the same during starvation.
  • Citrate administration may also bypass glucose requirement during insulin resistance and improve outcome.
  • Ketones and citrate have the advantage of not needing insulin to enter the cell and generate ATP in the mitochondria, and thus may replenish the Krebs cycle if acetyl CoA is limiting or when Krebs cycle intermediates are limiting as a result of sepsis.
  • Citrate can also act by lowering the cellular burden of non-esterified fatty acids that have been implicated in mitochondrial dysfunction during sepsis.
  • the metabolic fuel is a citrate.
  • a citrate include citrate and derivatives thereof such as citric acid, salts of citrate, esters of citrate, polyatomic anions of citrate or other ionic or drug complexes of citrate.
  • citrate in its various forms is not included in the composition it can be administered separately in a blood, blood:crystalloid ratio or crystalloid solution and mixed to the preferred level in the composition prior to administration to the body, organ, tissue or cell.
  • the form of citrate includes citrate phosphate dextrose (CPD) solution, magnesium citrate, sodium citrate, potassium citrate or sildenafil citrate, more preferably CPD.
  • CPD citrate phosphate dextrose
  • the composition according to the invention includes (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) a metabolic fuel.
  • the composition also includes a source of elevated magnesium ions.
  • the composition according to the invention may include (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; (ii) an antiarrhythmic agent or a local anaesthetic; (iii) a metabolic fuel; and (iv) an anti-inflammatory agent.
  • the composition also includes a source of elevated magnesium ions.
  • composition according to the invention may include adenosine, lidocaine, in addition to either or both of citrate and BOH.
  • the concentration of a citrate in the composition may be 0.0000001 to 100 mM, preferably 0.001 mM to 50 mM and most preferably 0.1 mM to 10 mM. In one embodiment, the concentration of citrate in the composition is about 2.1 mM.
  • the contact concentration of a citrate may be the same or less than the composition concentration set out above.
  • anti-adrenergics such as beta-blockers, for example, esmolol, atenolol, metoprolol and propranolol could be used in combination with the potassium channel opener, potassium channel agonist and/or adenosine receptor agonist to reduce calcium entry into the cell.
  • the beta-blocker is esmolol.
  • alpha(1)-adrenoceptor-antagonists such as prazosin, could be used instead in combination with the potassium channel opener, potassium channel agonist and/or adenosine receptor agonist to reduce calcium entry into the cell and therefore calcium loading.
  • the antiadrenergic is a beta-blocker.
  • the beta-blocker is esmolol.
  • Na + /Ca 2+ exchange inhibitors may include benzamyl, KB-R7943 (2-4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate) or SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline).
  • Some embodiments of the invention utilise calcium channel blockers which are direct calcium antagonists, the principal action of which is to reduce calcium entry into the cell.
  • Such calcium channel blockers may be selected from three different classes: 1,4-dihydropyridines (eg. nitrendipine), phenylalkylamines (eg. verapamil), and the benzothiazepines (e.g. diltiazem, nifedipine). 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 (see discussion above).
  • Benzothiazepines eg Diltiazem
  • Dihydropyridines eg nifedipine, Nicardipine, nimodipine and many others
  • Phenylalkylamines eg Verapamil
  • Diarylaminopropylamine ethers eg Bepridil
  • Benzimidazole-substituted tetralines eg Mibefradil.
  • L-type calcium channels L-type calcium channels
  • slow channels L-type calcium channels
  • Different classes of L-type calcium channel blockers bind to different sites on the alphal-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.
  • Bepridil is a drug with Na+ and K+ channel blocking activities in addition to L-type calcium channel blocking activities.
  • Mibefradil is a drug with Na+ and K+ channel blocking activities in addition to L-type calcium channel blocking activities.
  • Mibefradil is a drug with Na+ and K+ channel blocking activities in addition to L-type calcium channel blocking activities.
  • Mibefradil is a drug with Na+ and K+ channel blocking activities in addition to L-type calcium channel blocking activities.
  • Mibefradil is a drug with Na+ and K+ channel blocking activities in addition to L-type calcium channel blocking activities.
  • Mibefradil
  • 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.
  • Verapamil is also contraindicated in combination with beta-blockers due to the possibility of AV block or severe depression of ventricular function.
  • mibefradil has negative chronotropic and dromotropic effects.
  • Calcium channel blockers 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 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.
  • 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, HVVTX-I 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
  • indirect ATP-sensitive potassium channel openers eg adenosine, opioids
  • 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, nimodipine, nisoldipine, lercanidipine, telodipine, angizem, altiazem, bepridil, amlodipine, felodipine, isradipine and cavero and other racemic variations.
  • 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 IIIA and IVA from the funnel web spider Agelelnopsis aperta which selectively blocks R- and P/Q-type calcium channels respectively.
  • GVIA from the snail conus geographus
  • Agelelnopsis aperta which selectively blocks R- and P/Q-type calcium channels respectively.
  • mixed voltage-gated calcium and sodium channel blockers such as NS-7 to reduce calcium and sodium entry and thereby assist cardioprotection.
  • the calcium channel blocker is nifedipine.
  • the methods and composition according to the invention further include an opioid.
  • an opioid particularly D-Pen[2,5]enkephalin (DPDPE), may also result in significantly less damage to the cell, tissue or organ.
  • DPDPE D-Pen[2,5]enkephalin
  • composition according to the invention further includes an opioid.
  • Opioids also known or referred to as opioid agonists, are a group of drugs that inhibit opium (Gropion, 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.
  • opioid peptides 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.
  • opioid agonists include TAN-67, BW373U86, SNC80 ([(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide), (+)BW373U86, DADLE, ARD-353 [4-((2R5S)-4-(R)-4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2,5-dimethylpiperazin-1-ylmethyl)benzoic acid], a nonpeptide delta receptor agonist, DPI-221 [4-((alpha-S) ⁇ alpha-((2S,5R)-2,5-dimethyl-4-(3-fluorobenzyl)-1-piperazinyl)benzyl)-N,N-diethylbenzamide],
  • 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).
  • 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.
  • the heart is capable of synthesising or producing the three types of opioid peptides, namely, enkephalin, endorphin and dynorphin.
  • opioid peptides namely, enkephalin, endorphin and dynorphin.
  • delta and kappa opioid receptors have been identified on ventricular myocytes.
  • 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.
  • 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.
  • Opioids and opioid agonists may be peptidic or non-peptidic.
  • the opioid is selected from enkephalins, endorphins and dynorphins.
  • the opioid is an enkephalin which targets delta, kappa and/or mu receptors. More preferably the opioid is selected from delta-1-opioid receptor agonists and delta-2-opioid receptor agonists.
  • D-Pen [2,5]enkephaiin is a particularly preferred Delta-1-Opioid receptor agonist.
  • the opioid is administered at 0.001 to 10 mg/kg body weight, preferably 0.01 to 5 mg/kg, or more preferably 0.1 to 1.0 mg/kg.
  • compositions according to the invention may further include the use of 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.
  • 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.
  • 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.
  • 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.
  • Substance P an important pro-inflammatory neuropeptide is known to lead to cell oedema and therefore antagonists of substance P may reduce cell swelling.
  • 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.
  • 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 R2R-trans)-4-[3,5-bis(trifluoromethyObenzoyl]-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.
  • glutamate-receptor antagonists AP5/CNQX
  • reactive oxygen species scavengers ascorbate, Trolox(R), dimethylthiourea, tempol(R)
  • 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.
  • 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.
  • 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.
  • energy substrates are sources of reducing equivalents for energy transformations and the production of ATP in a cell, tissue or organ of the body
  • a direct supply of the energy reducing equivalents could be used as substrates for energy production.
  • 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.
  • Beta-hydroxy butyrate is a preferred energy substrate.
  • H 2 S hydrogen sulphide
  • H2S donors eg NaHS
  • the presence of hydrogen sulphide (H 2 S) 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.
  • 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.
  • ATP energy reducing equivalents from energy substrates to energy (ATP) generation and oxygen consumption.
  • hydrogen sulfide may reduce the energy demand of the whole body, organ, tissue or cell which may result in arrest, protection and preservation.
  • very low levels of sulfide down-regulate mitochondria, reduce O 2 consumption and actually increase “Respiratory Control” whereby mitochondria consume less O 2 without collapsing the electrochemical gradient across the inner mitochondrial membrane.
  • hydrogen sulphide (H 2 S) or H 2 S donors may be energy substrates themselves in addition to improving the metabolism of other energy substrates.
  • the invention provides a composition as described above further including hydrogen sulphide or a hydrogen sulfide donor.
  • the compound for minimizing or reducing the uptake of water by the cells in the tissue is PEG.
  • PEG reduces water shifts as an impermeant but also may preserve cells from immune recognition and activation.
  • Impermeant agents such as PEG, sodium gluconate, sucrose, lactobionate and raffinose, trehalose, 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.
  • 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 in the composition. 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 200 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 to 140 mM.
  • the contact concentration of the compound for minimizing or reducing the uptake of water by the cells in the tissue is the same or less than the composition concentration set out above.
  • composition is diluted with a pharmaceutically acceptable carrier, including but not limited to blood, saline or a physiological ionic solution, the dosage of the composition may be adapted to achieve the most preferred contact concentrations.
  • a pharmaceutically acceptable carrier including but not limited to blood, saline or a physiological ionic solution
  • the composition useful in the methods according to the invention may include more than one compound for minimizing or reducing the uptake of water by the cells in the tissue.
  • a combination of impermeants raffinose, sucrose and pentastarch
  • a combination of colloids, and fuel substrates may be included in the composition.
  • the methods and compositions according to the invention may further include a surfactant that has rheologic, anti-thrombotic, anti-inflammatory and cytoprotective properties.
  • surfactants are HCO-60, sodium dodecyl sulfate (SDS), Tween 80, PEG 400, 0.1 to 1% Pluronic 68, F127 and poloxamer 188 (P188).
  • P188 is a surface acting agent with cytoprotective effects of cells, tissues and organs and has been shown to be protective against trauma, electric shock, ischemia, radiation, osmotic stress, heart attack, stroke, burns and haemorrhagic shock.
  • Poloxamer 188 was also associated with potentially beneficial changes in membrane protein expression, reduced capillary leakage, and less hemodilution in pediatric cardiac surgery.
  • Other surfactant-protecting agents such as prostacyclin analog iloprost are also protective and has shown to improve preservation of surfactant function in transplanted lungs.
  • the non-ionic surfactant for minimizing or reducing cell damage for the present invention is P188.
  • the methods and compositions according to the invention may further include a reversible myofilament inhibitor such as 2,3-butanedione monoxime (BDM) to arrest, protect and preserve organ function.
  • BDM 2,3-butanedione monoxime
  • Myosin-actin interactions are present in nearly every cell for transport, trafficking, contraction, cytoskeleton viability.
  • BDM has been shown to improve preservation in skeletal muscle, kidney and renal tubules, lung, and heart.
  • the myosin inhibitor BDM is the choice for reducing cellular demand and minimizing cell damage during injury or ischemia-reperfusion injury.
  • 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 (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic assists in reducing injury and damage.
  • composition useful in the methods 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.
  • 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 and HOE 694.
  • B11 B-513 and T-162559 are other inhibitors of the isoform 1 of the Na+/H+ exchanger.
  • 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.
  • the concentration of the sodium hydrogen exchange inhibitor in the composition is between about 1.0 nM to 1.0 mM. More preferably, the concentration of the sodium hydrogen exchange inhibitor in the composition is about 20 ⁇ M.
  • the contact concentration of the sodium hydrogen exchange inhibitors is the same as or less than the composition set out above.
  • composition is diluted with a pharmaceutically acceptable carrier, including but not limited to blood, saline or a physiological ionic solution, the dosage of the composition may be adapted to achieve the most preferred contact concentrations.
  • a pharmaceutically acceptable carrier including but not limited to blood, saline or a physiological ionic solution
  • composition useful in the methods according to the invention may also 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 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 NAD(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
  • 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.
  • DMPO 5′-5-dimethyl-1-pyrrolione-N-oxide
  • POBN 4-pyridyl-1-oxide-N-t-butylnitrone
  • 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.
  • PBN nitrone radical scavenger alpha-phenyl-tert-N-butyl nitrone
  • MPG N-2-mercaptopropionyl glycine
  • NDGA lipooxygenase inhibitor nordihydroguaretic acid
  • Alpha Lipoic Acid Chondroitin Sulfate
  • L-Cysteine oxypurinol and Zinc.
  • the antioxidant is allopurinol (1H-Pyrazolo[3,4-a]pyrimidine-4-01).
  • 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.
  • the methods and compositions according to the invention include a cellular transport enzyme inhibitor, such as a nucleoside transport inhibitor, for example, dipyridamole, to prevent metabolism or breakdown of components in the composition such as adenosine.
  • a cellular transport enzyme inhibitor such as a nucleoside transport inhibitor, for example, dipyridamole
  • the half life of adenosine in the blood is about 10 seconds so the presence of a medicament to substantially prevent its breakdown will maximise the effect of the composition of the present invention.
  • Dipyridamole is advantageously included in the composition a concentration from about 0.01 ⁇ M to about 10 mM, preferably 0.05 to 100 ⁇ M Dipyridamole and has major advantages with respect to cardioprotection. Dipyridamole may supplement the actions of adenosine by inhibiting adenosine transport and breakdown leading to increased protection of cells, tissues and organs of the body during times of stress. Dipyridamole may also be administered separately for example by 400 mg daily tablets to produce a plasma level of about 0.4 ⁇ g/ml, or 0.8 ⁇ M concentration.
  • compositions may be suitable for administration to the tissue in liquid form, for example, solutions, syrups or suspensions, or alternatively they may be administered as a dry product for constitution with water or other suitable vehicle before use. Alternatively, the composition 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.
  • the invention comprises a composition in tablet form, including nutraceutical or supplement applications and in another form, the invention comprises an aerosol which could be administered via oral, skin or nasal routes.
  • composition useful in the methods 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.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monoole
  • Aqueous suspensions may also contain one or more preservatives, for example benzoates, such as ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example benzoates, such as ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol,
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents.
  • sweetening agents for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents.
  • compositions may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection.
  • 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.
  • composition may also be in the form of a veterinary composition, which may be prepared, for example, by methods that are conventional in the art.
  • veterinary compositions include those adapted for:
  • oral administration external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
  • drenches e.g. aqueous or non-aqueous solutions or suspensions
  • tablets or boluses e.g. aqueous or non-aqueous solutions or suspensions
  • pastes for application to the tongue for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
  • parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced in the udder via the teat;
  • topical applications e.g. as a cream, ointment or spray applied to the skin;
  • intravaginally e.g. as a pessary, cream or foam.
  • each component of the composition 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.
  • Each carrier must be pharmaceutically acceptable such that they are compatible with the components of the composition and not harmful to the subject.
  • the pharmaceutical composition is prepared with liquid carriers such as an ionic solution, for example NaCl or a buffer.
  • 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 or no potassium.
  • the composition has a total potassium concentration of up to about 10 mM, more preferably about 2 to about 8 mM, most preferably about 4 to about 6 mM.
  • the composition according to the invention is hypertonic such as hypertonic saline (NaCl at 3-30%).
  • the composition has a saline concentration greater than normal isontic saline which is 0.9% NaCl (0.154M).
  • magnesium may be used for cell, tissue or organ contact concentrations if desired without substantially affecting the activity of the composition.
  • body fluids e.g. blood or body cavity
  • magnesium concentration in the composition may be as high as 2M (2000 mM) prior to administration into the body.
  • typical buffers or carriers (as discussed above) 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.
  • the invention may also include 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.
  • the calcium present is at a concentration of between 0.1 mM to 0.8 mM, more preferably about 0.3 mM.
  • 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.
  • the pharmaceutically acceptable carrier is a bodily fluid such as blood or plasma. In another embodiment, the pharmaceutically acceptable carrier is crystalloid or blood substitute.
  • compositions according to the invention comprise (i) a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; and (ii) an antiarrhythmic agent or a local anaesthetic and one or more of:
  • a source of magnesium in an amount for increasing the amount of magnesium in a cell in body tissue
  • a pharmaceutically acceptable carrier such as an ionic solution, such as NaCl or a buffer.
  • this composition has two, three or four of the above components.
  • Preferred additional components include one or more of an anti-inflammatory agent, a metabolic fuel such as a citrate, a source of magnesium and a pharmaceutically acceptable carrier such as a buffer.
  • this composition may include more than one of the same component, for example two different potassium channel openers may be present in the composition. It is also contemplated that one component may have more than one function. For example, some calcium antagonists share effects with potassium channel openers.
  • composition useful in the methods according to the invention further including an effective amount of elevated magnesium.
  • the composition useful in the methods according to the invention includes adenosine and lidocaine.
  • this composition further includes a source of elevated magnesium.
  • This composition may optionally include a metabolic fuel, such as a citrate for example CPD.
  • the composition according to the invention includes adenosine and lidocaine.
  • this composition further includes a source of elevated magnesium.
  • This composition may optionally include an anti-inflammatory agent, such as beta-hydroxybutyrate.
  • compositions according to the invention are a combination of adenosine and lidocaine.
  • the composition also includes a source of elevated magnesium.
  • the composition may also include an anti-inflammatory agent, such as beta-hydroxybutyrate, and/or a metabolic fuel, such as a citrate for example CPD.
  • the composition contains 0.1 to 40 mM of adenosine, 0.1 to 80 mM of lidocaine or a salt thereof such as a HCl salt, 0.1 to 2000 mM of a source of magnesium such as a MgSO 4 , 0.1 to 20 mM of a source of citrate such as CPD and 0.9 to 3% of an ionic solution such as NaCl or a buffer.
  • the composition may be administered intravenously or be administered both intravenously and intraperitoneally or directly accessing a major artery such as the femoral artery or aorta in patients who have no pulse from massive exsanguination, or in the carotid artery or another artery during aortic dissection to protect the brain from hypoxia or ischemia.
  • the composition may be administered intravenously and intraperitoneally simultaneously, the perineum acting as, in effect, a reservoir of composition for the bloodstream as well as acting on organs in the vicinity with which it comes into contact.
  • Another rapid route of administration is intraosseously (into the bone). This is particularly suitable for a trauma victim, such as one suffering shock.
  • the composition contains two or more components, these may be administered separately but simultaneously. Substantially simultaneous delivery of the component to the target site is desirable. This may be achieved by pre-mixing the components for administration as one composition, but that is not essential.
  • the invention is directed towards the simultaneous increase in local concentration (for example an organ such as the heart) of the components of the composition.
  • local concentration for example an organ such as the heart
  • the invention may be practised by administering the composition using a perfusion pump, often associated with a procedure known as “miniplegia” or “microplegia”, in which minimal amount of components are titrated by means of a finely adjustable pump directly via a catheter.
  • miniplegia or “microplegia”
  • a protocol utilises miniplegia as described above, where micro amounts are titrated directly to the heart, using the patient's own oxygenated blood.
  • the reference to a “setting” is a measure on the pump, such as a syringe pump, of the amount of substance being delivered directly to the organ, such as a heart.
  • composition may be administered by aerosol.
  • 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 (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.
  • open heart surgery on-pump and off-pump
  • angioplasty balloon and with stents or other vessel devices
  • clot-busters to protect and preserve the cells from injury.
  • the tissue may be contacted by delivering the composition intravenously to the tissue.
  • the composition may be used for blood cardioplegia.
  • the composition may be administered directly as a bolus by a puncture (eg, by syringe) directly to the tissue or organ, particularly useful when blood flow to a tissue or organ is limiting.
  • the composition for arresting, protecting and preserving a tissue may also be administered as an aerosol, powder, solution or paste via oral, skin or nasal routes.
  • composition may be administered directly to the tissue, organ or cell or to exposed parts of the internal body to reduce injury.
  • the composition may be administered by intermittent administration.
  • a suitable administration schedule is a 2 minute induction dose every 20 minutes throughout the arrest period. The actual time periods can be adjusted based on observations by one skilled in the art administering the composition, and the animal/human model selected.
  • the invention also provides a method for intermittently administering a composition for reducing injury to the cell, tissue or organ.
  • 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 composition (or solution) for transporting donor tissues from a donor to recipient.
  • the composition according to the invention is administered in two steps (referred to as “one-two step iv infusion”).
  • the first administration is by bolus followed by drip infusion.
  • the composition is administered in one shot as a bolus or in two steps as a bolus followed by infusion.
  • the dose and time intervals for each delivery protocol may be designed accordingly.
  • the components of the composition according to the invention may be combined prior to administration or administered substantially simultaneously or co-administered.
  • composition may be administered by intravenous, intra-cardiac, intraperitoneal, spinal or cervical epidural.
  • composition useful in the methods according to the invention may be administered with or contain blood or blood products or artificial blood or oxygen binding molecules or solutions to improve the body's oxygen transport ability and survival by helping to reduce hypoxic and ischemic damage from blood loss.
  • the oxygen-containing molecules, compounds or solutions may be selected from natural or artificial products.
  • an artificial blood-based product is perfluorocarbon-based or other haemoglobin-based substitute.
  • Some of the components may be added to mimic human blood's oxygen transport ability such HemopureTM, GelenpolTM, OxygentTM, and PolyHemeTM. Hemopore is based on a chemically stabilized bovine hemoglobin.
  • Gelenpol is a polymerized hemoglobin which comprises synthetic water-soluble polymers and modified heme proteins.
  • Oxygent is a perflubron emulsion for use as an intravenous oxygen carrier to temporarily substitute for red blood cells during surgery.
  • Polyheme is a human hemoglobin-based solution for the treatment of life-threatening blood loss.
  • oxygenation of the body from a variety of ways including but not limited to oxygen gas mixture, blood, blood products or artificial blood or oxygen binding solutions maintains mitochondrial oxidation and this helps preserve the myocyte and endothelium of the organ. Without being bound by any particular mode or theory, the inventor has found that gentle bubbling with 95% 0 2 /5% CO 2 helps maintains mitochondrial oxidation which helps preserve the myocyte and coronary vasculature.
  • composition useful in the methods according to the invention is aerated with a source of oxygen before and/or during administration.
  • the source of oxygen may be an oxygen gas mixture where oxygen is the predominant component.
  • the method according to the invention includes:
  • tissue, cell or organ in contact with the combined composition under conditions sufficient to reduce injury.
  • This method may include the further step of postconditioning the cell, tissue or organ.
  • the oxygen source is an oxygen gas mixture.
  • oxygen is the predominant component.
  • the oxygen may be mixed with, for example CO 2 . More preferably, the oxygen gas mixture is 95% 0 2 and 5% CO 2 .
  • composition useful in the methods of the invention is highly beneficial at about 10° C. but can also be used to prevent injury over a wider temperature range up to about 37° C. Accordingly, the composition may be administered to the cell, tissues or organs 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 20° C. to about 32° C. “Normothermia” is used to describe a tissue at a temperature from about 32° C. to about 38° C., though the normal body temperature is around 37 to 38° C.
  • compositions would also find use as a topical spray or soaked in a gauze soaked and applied to an organ, tissue or cell of the body and has application for surgery and clinical interventions.
  • This application may include a topical aerosol for spraying on surgical incisions or wounds, and around the area of these wounds.
  • the composition could be used for applying to a median sternotomy (sternal incision) in cardiac surgery, and applied during and after the operation to reduce or prevent adhesions from occurring between the underside of sternum area to the underlying heart and other tissues after the operation.
  • sternal incision median sternotomy
  • the composition could be applied to the internal organs during and prior to closing the incision to reduce or prevent adhesions from occurring in the abdominal cavity after surgery.
  • the composition could also be used for incisions made for artery or venous catheterizations. For example, during a cut down and cannulation of the femoral artery or vein the area could be sprayed or soaked and the surgical well with the composition to prevent adhesions from occurring after the incision is closed.
  • Another application would be for harvesting veins or arteries to be used for cardiac surgery as conduits to replace the blocked arteries on the heart in a coronary artery bypass operation.
  • the saphenous vein is exposed from a long incision in the leg and harvested for cardiac surgery, and the area could be sprayed or topically applied on a gauze.
  • the composition would also have an application for less invasive endoscopic harvesting of blood vessels. Topical applications of the composition would also find applications on areas of the heart itself particularly where potential cell fibrosis or injury may occur locally around the region of the heart responsible for arrhythmias or other heart dysfunctions. The whole heart could also be sprayed topically to protect it from any adhesions or dysfunction.
  • the amount of active ingredients present in the composition will depend on the nature of the subject (whole body, isolated organ circuit in the body or isolated cell, organ or tissue ex vivo) and the proposed method of treatment or use.
  • the amount should be effective for the end use, for example, one or more of the components should be present in an amount sufficient to treat infection, sepsis or injury.
  • IV intravenous IV intravenous
  • IA intra-arterial 10 intra-osseous
  • IC intracardiac
  • a Adenosine Adenosine
  • L lidocaine-HCl M
  • Arrest flow 4-7 Cardiac perfusion: 1 to 500 ml/min (0.01to 10 ml/min/kg human) ml/kg/min (A; 1.4 mg/kg; L: 2.9 mg/kg; M: 0.06 g/kg) Non-arrest 1 ml/kg/min of the above BOLUS-INFUSION/DRIP PREVENTATIVE METHOD FOR . . . Bolus As Above Infusion or Drip Surgery, 0.01 to 0.5 to 0.1 to 0.01 to 5 2 g/5 L 1.5 g/5 L 0.9% 0.9% or 3% Pain 20 100 100 mg/kg/min.
  • 3% NaCl may be used if brain injury suspected Infusion- 1 to 40 1 to 1 to 50 0.01 to 10 ml/kg/hr with Drip preferred 80 preferred 5 mg/kg/min A: 12 mg/kg; preferred L: 24 mg/kg; M: 12 mg/kg or more hypotension A: 18 mg/kg; L: 36 mg/kg; M: 20 mg/kg 1) Specialized surgery (e.g. shoulder, hip, knee or circulatory arrest. Placement of P: 0.1 to 0.2 mg heart valves via transluminal catheter technique without thoracotomy or P/kg/min (may extracorporal circulation. 2) whole body protection (reduce injury infection, not require P for inflammation, coagulopathy as above) 3) to reduce blood loss during Damage some Control Surgery indications)
  • the concentrations of each component in the composition may be diluted by body fluids or other fluids that may be administered together with the composition.
  • the composition will be administered such that the concentration of each component in the composition contacts the tissue about 100-fold less.
  • containers such as vials that house the composition may be diluted 1 to 100 parts of blood, plasma, crystalloid or blood substitute for administration.
  • FIG. 3 shows a graph comparing TNF-Alpha versus ALM infusion dose.
  • the X-axis refers to the dose of adenosine (A) in the ALM dose with the following combinations being tested: 1) Control animal TNF-alpha with LPS alone infusion; 2) 5 ⁇ g A/10 ⁇ g Lidocaine/5.6 ⁇ g MgSO 4 /kg/min; 3)10 ⁇ g A/20 ⁇ g Lidocaine/5.6 ⁇ g MgSO 4 /kg/min; 4) 300 ⁇ g A/600 ⁇ g Lidocaine/336 ⁇ g MgSO 4 /kg/min. (see example 2)
  • FIG. 6 shows graphs measuring the effect of Adenosine (A), lidocaine (L) and adenosine and lidocaine (AL) on % relaxation (Y axis) of isolated guinea-pig mesenteric artery when intact (square) or denuded (endothelium removed) (diamond)
  • MAP mean arterial pressure on rats following shock and drug induced MAP collapse and spontaneous return (see example 6b)
  • FIG. 9 shows a Graph showing MAP resuscitation following single 3% NaCl ALM single bolus (Group 1)
  • FIG. 10 shows graphs showing bolus alone compared to one-two-step (bolus—infusion) for MAP and heart rate (Group 2)
  • FIG. 12 shows a graph showing the effect of addition of valproic acid
  • FIG. 14 shows ECG traces (A, C and D) and a blood pressure trace (B) showing the effect of ALM with a general anaesthetic from a normal state to whole body arrest.
  • FIG. 15 shows ECG traces (E, F and H) and a blood pressure trace (G) showing the effect of ALM with a general anaesthetic from a normal state to whole body arrest.
  • FIG. 16 shows ECG traces (I and J) and blood pressure traces (K and L) showing the effect of ALM with a general anaesthetic from a normal state to whole body arrest.
  • FIG. 17 shows ECG traces (M and O) and blood pressure traces (N and P) showing the effect of ALM with a general anaesthetic from a normal state to whole body arrest.
  • FIG. 18 shows ECG trace (Q) showing the effect of ALM with a general anaesthetic from a normal state to whole body arrest.
  • FIG. 19 shows ECG traces A and B demonstrating the effect of hemodynamic stabilization with adenosine agonist plus lidocaine and magnesium after extreme blood loss.
  • FIG. 20 shows graphs showing the effect of adenosine and lidocaine solution with different forms of citrate (citrate phosphate dextrose CPD and sodium citrate) and elevated magnesium.
  • FIG. 21 shows graphs showing the effect of adenosine and lidocaine solution with different forms of citrate (citrate phosphate dextrose CPD and sodium citrate) and elevated magnesium.
  • FIG. 22 shows graphs showing the effect of 8 hours of cold (4° C.) continuous perfusion of adenosine and lidocaine solution with and without gentle bubbling (95% O 2 /5% CO 2 ) on functional recovery in the isolated working rat heart
  • FIG. 23 shows graphs showing the effect of adding insulin and melatonin with high and low MgSO 4 to bubbled adenosine and lidocaine solution during 8 hours of constant perfusion at 4° C. in the isolated working rat heart.
  • FIG. 24 shows graphs A and B showing the effect of adenosine and lidocaine solution with sildenafil citrate over 2 hours warm arrest (29° C.) given every 20 minutes (2 min infusion) and 60 min reperfusion.
  • FIG. 25 shows graphs C and D showing the effect of adenosine and lidocaine solution with a sildenafil citrate over 2 hours warm arrest (29° C.) given every 20 minutes (2 min infusion) and 60 min reperfusion.
  • FIG. 26 shows ECG and blood pressure traces before and after inducing hypotensive anesthesia using ALM-CPD (A and B before, C and D after)
  • FIG. 27 shows ECG and blood pressure traces before and after inducing whole body arrest using ALM-CPD (E and F before, G and H after).
  • FIG. 28 shows ECG and blood pressure traces before and after inducing whole body arrest using ALM-CPD (I and J before, K and L after).
  • FIG. 29 shows graphs of the results of the experiments described in Example 46.
  • FIG. 30 shows graphs of the results of the experiments described in Example 46.
  • FIG. 31 shows graphs of the results of the experiments described in Example 46.
  • FIG. 32 shows graphs of the results of the experiments described in Example 46.
  • FIG. 33 shows graphs of the results of the experiments described in Example 46.
  • FIG. 34 shows graphs of the results of the experiments described in Example 46.
  • FIG. 35 shows a schematic diagram of the experimental protocol for Example 47.
  • FIG. 36 shows graphs showing the effect of treatment with adenosine, lidocaine, and Mg2+ (ALM)/adenosine and lidocaine (AL) on mean arterial pressure (MAP) (A) and heart rate (HR) (B).
  • ALM adenosine and lidocaine
  • FIG. 37 shows graphs showing cardiac index (A), stroke volume (B), ejection time (C), and oxygen consumption (Vo 2 ) (D) during both hypotensive resuscitation and after infusion blood.
  • FIG. 38 shows graphs showing cardiac function data during the experiment.
  • Left ventricular (LV) end-systolic pressure (A) and LV end-diastolic pressure (B) measured throughout the course of the experiment.
  • C The maximum positive development of ventricular pressure over time (dP/dtmax) as a marker of cardiac systolic function.
  • D The maximum negative development of ventricular pressure over time (dP/dtmin) as a marker of cardiac diastolic function.
  • FIG. 39 shows graphs showing the renal variables urine output, plasma creatinine, urine protein to creatinine, and urine n-acetyl-8-d-glucosaminide (NAG) to creatinine ratio throughout the course of the experiment.
  • A Urine output measured after 90 min of hemorrhagic shock and then every hour during the remainder of the experiment.
  • B Plasma creatinine as a marker of global kidney function.
  • C Urine protein to urinary creatinine ratio as a marker of glomerular injury.
  • D Urine NAG to urinary creatinine ratio as a marker of proximal tubular injury. Data presented as median (95% Cl).
  • FIG. 40 shows a schematic representation of the in vivo rat protocol of severe polymicrobial sepsis.
  • FIG. 42 shows graphs showing the effect of 0.9% NaCl ALM on the MAP (A) and without the effect of shams (B); SAP (C) and without the effect of shams (D) during 5 hours of CLP in a rat model of polymicrobial sepsis.
  • FIG. 43 shows graphs showing the effect of 0.9% NaCl ALM treatment on HR (A) and without the effect of shams (B). Rectal temperature (C) and without the effect of shams (D) during 5 hours of CLP in a rat model of polymicrobial sepsis.
  • PT aPTT
  • C representative photographs of gross pathophysiologic examinations of the cecum in the shams, saline controls, and ALM-treated rats after 5 hours.
  • the cecal ligation and puncture model is considered the gold standard for sepsis research.
  • toll receptor agonists such as lipopolysaccharide (LPS) toxin model which is only detectible in only a minority of patients with sepsis
  • LPS lipopolysaccharide
  • the cecal ligation model mimics the human disease of ruptured appendicitis or perforated diverticulitis.
  • the cecal model also reproduces the dynamic changes in the cardiovascular system seen in humans with sepsis. In addition, the model recreates the progressive release of pro-inflammatory mediators.
  • the gastrointestinal tract often can be damaged directly from penetrating or blunt trauma, but also from ischemic injury from any kind of major surgery, cardiac arrest, burns, haemorrhage and shock.
  • Ischemic injury poses a significant risk of infection and sepsis because the gut wall becomes leaky and bacteria translocates into the peritoneal cavity resulting in a medical emergency.
  • Reducing the impact of infection from GI injury would also reduce adhesions as infection is one cause of adhesions as the body attempts to repair itself.
  • Adhesions may appear as thin sheets of tissue similar to plastic wrap, or as thick fibrous bands. Up to 93 percent of people who have abdominal surgery go on to develop adhesions.
  • mice Male Sprague Dawley rats (300-450 g) were fed ad libitum with free access to water and housed in a 12-hr light-dark cycle. Animals were not heparinized and anesthetized with an intraperitoneal injection of 100 mg/kg sodium thiopentone (Thiobarb). Anesthetized animals were positioned in the supine position on a custom designed cradle. A tracheotomy was performed and animals were artificially ventilated (95-100 strokes min ⁇ 1 ) on humidified room air using a Harvard Small Animal Ventilator (Harvard Apparatus, Mass., USA). A rectal probe was inserted 5.0 cm and the temperature ranged between 37 and 34° C.
  • the caecum was isolated through midline laparotomy and ligated below ileocaecal valve. It was punctured with 18G needle four times through-and-through (8 holes). The abdominal cavity was surgically closed in 2 layers. Rats were randomly assigned into either control or groups for Example 1 (bolus only) and Example 2 (bolus plus drip infusion).
  • FIG. 1 show that ALM IV bolus ONLY strategy stabilized the cardiovascular system for about 1 hour and preserved body temperature at around 34 C for 3 hours.
  • One-Bolus ALM failed to Sustain Stabilization over 5 hours of polymicrobial infection (sepsis).
  • ALM bolus stabilized the cardiovascular system for about 60 min then failed to protect against collapse and SEPTIC SHOCK over 5 hours of polymicrobial infection.
  • Control animals receive intravenous 0.3 ml bolus 0.9% NaCl and drip infusion (0.4 ml/hr) 0.9% NaCl.
  • Treatment animals received 0.3 ml bolus 0.9% NaCl with 1 mM Adenosine (0.24 mg/kg), 3 mM Lidocaine (0.73 mg/kg, and 2.5 mM MgSO 4 (0.27 mg/kg), and a different composition for drip infusion (0.4 ml/hr) comprising 12 mg/kg/hr Adenosine, 34 mg/kg/hr Lidocaine, and 13.44 mg/kg/hr MgSO 4 in 0.9% NaCl
  • the control and treatment was withdrawn after 4 hr and animals monitored for further 60 min.
  • FIG. 2 (A-E) show that ALM IV bolus infusion one-two treatment strategy stabilizes the cardiovascular system and preserves body temperature regulation during 5 hours of polymicrobial infection (sepsis).
  • TNF alpha The primary role of TNF alpha is in the regulation of immune cells.
  • TNF alpha is a cytokine involved in systemic inflammation, and along with other cytokines stimulates the acute phase reaction to stress and infection. TNF-alpha also induces activation of coagulation in different pathological states including sepsis.
  • Activated protein C inhibits TNF-alpha production.
  • Activated protein C (and antithrombin) may inhibit the endothelial perturbation induced by cytokines.
  • Antithrombin regulates TNF-alpha induced tissue factor expression on endothelial cells by an unknown mechanism.
  • Activated protein C and antithrombin, and their pathways of regulation may be useful targets for treating coagulation abnormalities associated with sepsis or other inflammation diseases. These sites and pathways inhibit not only coagulation but also involved with the downregulation of anticoagulant activities of endothelial cells.
  • a dose response of ALM infusion on inflammation was studied in the swine model of lipopolysaccharide (LPS, an obligatory component of Gram-negative bacterial cell walls) endotoxemia at 90 min infusion (Infusion of LPS for 5 hours 1 ⁇ g/kg/min) into 40 kg female pigs. Pigs were fasted overnight, but allowed free access to water. Anesthesia was induced with midazolam (20 mg) and s-ketamin (250 mg) and maintained with a continuous infusion of fentanyl (60 ⁇ g/kg/h) and midazolam (6 mg/kg/h).
  • LPS lipopolysaccharide
  • the animals were intubated and volume-controlled ventilated (S/5 Avance, Datex Ohmeda, Wis., USA) with a positive end-expiratory pressure of 5 cm H 2 O, Fi02 of 0.35, and a tidal volume of 10 ml/kg. Ventilation rate was adjusted to maintain PaCO 2 between 41-45 mmHg. The body temperature was maintained around 38° C. during the entire study. All animals received normal saline (NS) at a maintenance rate of 10 ml/kg/h during surgery and the baseline period and was increased to 15 ml/kg/h during LPS infusion.
  • NS normal saline
  • the results are shown in FIG. 3 .
  • the Y-axis is TnF-alpha in plasma produced at 90 min in response to the LPS infusion and the X-axis refers to the dose of adenosine (A) in the different ALM doses with the following combinations being tested:
  • the stock composition for infusion (in mM) was 0.075 mM Adenosine, 0.148 mM lidocaine and 0.187 mM MgSO 4 3) 10 ⁇ g A/20 ⁇ g Lidocaine/5.6 ⁇ g MgSO 4 /kg/min over a 4 hour period or 0.6 mg Adenosine per kg/hour, 1.2 mg/kg/hour lidocaine and 0.34 mg MgSO 4 /kg/hr.
  • the stock composition for infusion (in mM) was 0.15 mM Adenosine, 0.296 mM lidocaine and 0.187 mM MgSO 4 4) 300 ⁇ g A/600 ⁇ g Lidocaine/336 ⁇ g MgSO 4 /kg/min over a 4 hour period or 18 mg Adenosine per kg/hour, 36 mg/kg/hour lidocaine and 20 mg MgSO 4 /kg/hr.
  • the stock composition for infusion (in mM) was 4.5 mM Adenosine, 8.88 mM lidocaine and 11 mM MgSO 4 .
  • TNF alpha is a cytokine involved in systemic inflammation, and along with other cytokines stimulates the acute phase reaction to stress and infection. TNF-alpha also induces activation of coagulation in different pathological states including sepsis.
  • the present invention by inhibiting TnF alpha may reduce inflammation and reduce the impact inflammation has on coagulation during infection, sepsis and septic shock. Since adhesions can be caused by infection, the present invention also may reduce the incidence of adhesions.
  • the present invention since inflammation is part of any injury process (traumatic or non-traumatic) particularly as a result of traumatic brain injury, the present invention also may reduce the secondary complications of brain injury. Since inflammation is a result of disease (heart attack, stroke, cardiac arrest, auto-immune diseases, hemorrhagic shock), the present invention also may reduce the complications of disease due to local or systemic inflammation. There is a major unmet need to reduce the impact of infection in health and disease, and to modulate the immune function of the host to reduce the impact of infection or prevent it from progressing into septic shock.
  • Sepsis is a very common complication of almost any infectious disease. There are >1.5 million people develop severe sepsis and septic shock annually in the United States and another 1.5 million people in Europe. Sepsis often develops in the field of co-morbidities like type 2 diabetes mellitus, chronic obstructive pulmonary disease, chronic heart failure and chronic renal disease, trauma, burns and surgery. Despite improvement in medical care, severe sepsis and septic shock remain an unmet medical need. There is a need for new drugs that modulate the immune function of the host to reduce the impact of infection or prevent it from progressing into septic shock.
  • Drugs can be divided into three categories according to their mechanism of action: i) agents that block bacterial products and inflammatory mediators, ii) modulators of immune function, and iii) immunostimulation (reduce immunosuppression). Drug development could also have an impact on many pathologies involving low levels of inflammatory markets and immune imbalances. For example, recent studies suggest that acute and chronic cardiovascular disease is associated with a chronic low-grade inflammation that promotes adverse ventricular remodeling and correlates with disease progression. Several inflammatory mediators, including TNF- ⁇ , IL-1 ⁇ , and IL-6, are involved in cardiac injury subsequent to myocardial ischemia and reperfusion, sepsis, viral myocarditis, and transplant rejection.
  • Severe sepsis defined as sepsis associated with acute organ failure, is a serious disease with a mortality rate of 30-50%. Sepsis always leads to deranged coagulation, ranging from mild alterations up to severe disseminated intravascular coagulation (DIC) (hypercoagulopathy). Septic patients with severe DIC have microvascular fibrin deposition, which often leads to multiple organ failure and death. Alternatively, in sepsis severe bleeding might be the leading symptom (hypocoagulopathy), or even coexisting bleeding and thrombosis. There are no approved drugs for sepsis and currently constitutes a major unmet medical need requiring breakthrough technologies. The deranged coagulation, particularly DIC, is an important and independent predictor of mortality in patients with severe sepsis. The rat model used as an example below is a gold standard to mimic the pathophysiology of severe sepsis in humans.
  • mice Male Sprague Dawley rats (300-450 g) were fed ad libitum with free access to water and housed in a 12-hr light-dark cycle. Animals were not heparinized and anesthetized with an intraperitoneal injection of 100 mg/kg sodium thiopentone (Thiobarb). Anesthetized animals were positioned in the supine position on a custom designed cradle. A tracheotomy was performed and animals were artificially ventilated (95-100 strokes min-1) on humidified room air using a Harvard Small Animal Ventilator (Harvard Apparatus, Mass., USA). A rectal probe was inserted 5.0 cm and the temperature ranged between 37 and 34° C.
  • the caecum was isolated through midline laparotomy and ligated below ileocaecal valve. It was punctured with 18G needle four times through-and-through (8 holes). The abdominal cavity was surgically closed in 2 layers. Rats were randomly assigned into either control or groups for ALM Bolus and Infusion.
  • Control animals receive intravenous 0.3 ml bolus 0.9% NaCl and drip infusion (0.4 ml/hr) 0.9% NaCl.
  • Treatment animals received 0.3 ml bolus 0.9% NaCl with 1 mM Adenosine (0.24 mg/kg), 3 mM Lidocaine-HCl (0.73 mg/kg, and 2.5 mM MgSO 4 (0.27 mg/kg, and a different composition for drip infusion (0.4 ml/hr) comprising 12 mg/kg/hr Adenosine, 34 mg/kg/hr Lidocaine, and 13.44 mg/kg/hr MgSO 4 in 0.9% NaCl
  • control and treatment was withdrawn after 4 hr and animals monitored for further 60 min.
  • composition according to the invention to relax the mesenteric artery and potentially increase blood flow to the gastrointestinal tract.
  • Second order mesenteric artery branches were isolated and mounted in a pressure myograph (see FIG. 4 ) under constant pressure of 60 mmHg and perfusion (luminal flow) of 100 uL/min with Krebs-Henseleit buffer (37° C.). Artery diameter was continuously measured using videomicroscopy (see FIG. 4 ). For the relaxation/vasodilation experiments arteries were equilibrated and then constricted with 10 ⁇ 8 M arginine vasopressin (AVP).
  • AVP arginine vasopressin
  • Adenosine, lidocaine or adenosine-lidocaine together were administered 2) luminally and 2) abluminally and concentration curves were obtained.
  • Stock solutions of adenosine and lidocaine alone or adenosine-lidocaine combined were made in deionized water to 20 mM.
  • a range of volumes were pipetted to provide contact concentrations with the vessel lumen or outer wall that ranged from 0.001 to 1 mM.
  • arteries were dilated using calcium-free solution to obtain 100% relaxation.
  • a number of arteries were denuded by introducing 5 ml air into the lumen with flow rate 1000 ⁇ l/min. The air outflow was then clamped until the intraluminal pressure reached 70 mmHg, flow rate was reduced to 2 ⁇ l/min and the vessel remained pressurized for 10 minutes
  • FIG. 5A shows that adenosine increased relaxation of the isolated intact mesenteric artery in a dose dependent manner, and that at 10 ⁇ M and 100 ⁇ M the effect of adenosine added to the bathing solution surrounding the vessel (abluminal administration) produced significantly more relaxation than if the solution was perfused through the lumen (inside the vessel).
  • FIG. 5B Shows that lidocaine failed to produce relaxation in the isolated intact mesenteric artery and there was no significant difference if the lidocaine was in the lumen or on the outside bathing solution.
  • FIG. 5C shows that adenosine-lidocaine together increased relaxation of the isolated intact mesenteric artery in a dose dependent manner. In contrast to adenosine alone ( FIG. 5A ) the greater relaxation from abluminal administration was not significantly different over the range of AL studied.
  • This example tests the effect of 0.9% NaCl ALM on correcting hypocoagulopathy (or reducing bleeding) and reducing blood clot retraction (strengthening the clot from breaking down) after asphyxial cardiac arrest with “sepsis-like” cardiac syndrome.
  • Post-cardiac arrest recovery is characterized by high levels of circulating cytokines and adhesion molecules, the presence of plasma endotoxin, and dysregulated leukocyte production of cytokines: a profile similar to that seen in severe sepsis. Coagulation abnormalities occur consistently after successful resuscitation, and their severity is associated with mortality.
  • a 0.5 mL bolus ALM contained 1.8 mM Adenosine, 3.7 mM Lidocaine-HCl and 4.0 mM MgSO 4 .
  • In the 0.5 ml there were 0.48 mg adenosine, 1.0 mg lidocaine-HCl and 2.4 mg MgSO 4 . This was also equivalent to a bolus of 1.44 mg/kg adenosine, 3.0 mg/kg lidocaine-HCl and 7.2 mg/kg MgSO 4 .
  • ROTEM Rotational Thromboelastometry
  • ROTEM Tem International, Kunststoff, Germany
  • ROTEM provides a real-time evaluation of the viscoelastic properties of whole blood in health and disease. Parameters include time to initiation of the clot, early clot formation kinetics, clot firmness and prolongation, clot fibrin-platelet interactions and clot lysis.
  • Venous whole blood was obtained at baseline, following cardiac arrest, and at 120 min following ROSC or in those animals that failed to attain ROSC in the first 2 to 5 min of attempts.
  • a volume of 1.8 ml blood was drawn into a 2.0 ml BD vacutainer containing citrate-phosphate-dextrose solution. After warming the blood at 37° C.
  • EXTEM, INTEM and FIBTEM viscoelastic analysis was performed within 30 minute of blood withdrawal.
  • the EXTEM test is extrinsically activated by thromboplastin (tissue factor) whereas INTEM test is activated by the contact phase (as in aPTT).
  • FIBTEM is activated as in EXTEM with the addition of cytochalasin D, which inhibits platelet glycoprotein (GP) IIb/IIIa receptors.
  • the FIBTEM test thus provides information about the effect of fibrin polymerization on clot strength and is independent of platelet involvement. The following parameters were measured in EXTEM, INTEM and FIBTEM.
  • Clotting time or the time from start of measurement until a clot amplitude of 2 mm
  • CFT clot formation time
  • MCF maximum clot firmness
  • the alpha angle (a) was also measured and represents the angle between baseline and a tangent at the maximum clot slope and clot amplitude (amplitude at 5 to 30 min) in mm over a 30 min period.
  • the lysis index (LI, %) was estimated as the ratio of clot firmness (amplitude at 30 or 60 min) divided by MCF times 100.
  • MCE maximum clot elasticity
  • PT Prothrombin Time
  • aPTT Activated Partial Thromboplastin Time
  • the blood remaining from ROTEM analysis was centrifuged at room temperature and the plasma removed, snap frozen in liquid nitrogen, and stored at ⁇ 80° C. until use.
  • PT and aPTT were measured using a coagulometer (Trinity Biotech, Ireland) as described by Letson and colleague. These standard plasma coagulation tests reflect the kinetics of first fibrin formation with no information from platelet contributions.
  • the PT is a measure of the integrity of the extrinsic and final common pathways analogous to EXTEM CT (CFT).
  • the aPTT is a measure of the integrity of the intrinsic and final common pathways analogous to INTEM CT (CFT)
  • Table 2 below provides a summary of the Major Coagulation Changes over 2 hours of sustained return of spontaneous circulation (ROSC) in the rat model of 8 min asphyxial cardiac hypoxia and arrest.
  • ROSC spontaneous circulation
  • ROTEM lysis index decreased during cardiac arrest, implying hyperfibrinolysis.
  • Control ROSC survivors displayed hypocoagulopathy (prolonged EXTEM/INTEM CT, CFT, PT, aPTT), decreased maximal clot firmness (MCF), lowered elasticity and lowered clot amplitudes but no change in lysis index.
  • ALM corrected these coagulation abnormalities at 120 min post-ROSC.
  • Small bolus of 0.9% NaCl ALM improved survival and hemodynamics and corrected prolonged clot times and clot retraction compared to controls.
  • FIG. 7 shows representative ROTEM traces for the different groups asphyxial cardiac hypoxia and arrest (AB), 0.9% NaCl at 120 min (CD), 0.9% NaCl ALM at 120 min (EF), and in four controls that failed to achieve ROSC (GH).
  • ALM administration prevents clot retraction (prevents a decrease in clot amplitude) thus making it a stronger clot to reduce bleeding.
  • ALM's ability to correct clot strength (amplitudes) may be significant because point-of-care low clot strength is an independent predictor of massive transfusion, and coagulation-related mortality within 15 min following the resuscitation of trauma patients.
  • reduced or weak clot strength before hospital admission has been shown to be independently associated with increased 30-day mortality in trauma patients.
  • ALM fully corrected clot strength, maximum clot elasticity (MCE) and MCE platelet (P ⁇ 0.05) (Table 2) compared to saline controls implies that ALM provides more favorable conditions for a stronger, denser fibrin network with higher elastic modulus (Table 1) and possibly higher thrombin concentrations compared with saline control.
  • ALM appears to alleviate the sepsis-like changes in clot abnormalities after asphyxial cardiac hypoxia and arrest.
  • the left femoral vein and artery was cannulated using PE-50 tubing for drug infusions and blood pressure monitoring (UFI 1050 BP coupled to a MacLab) and the right femoral artery was cannulated for bleeding.
  • Lead II electrocardiogram (ECG) leads were implanted subcutaneously on the left and right front legs and grounded to the back leg. The chest was opened and the heart was exposed to observe the effect the treatment in addition to the hemodynamic and ECG measurements. Rats were stabilized for 10 minutes prior to whole body arrest.
  • Estimated blood volume of 650 g rat is ⁇ 39.47 ml. The animal was not bled or in shock.
  • concentrations of the actives in mM are 3.75 mM Adenosine, 7.38 mM lidocaine-HCl, 833 mM MgSO 4 and 3.71 mM propofol.
  • the composition When expressed in mg/kg animal the composition includes 1.5 mg/kg adenosine, 3 mg/kg lidocaine-HCl and 125 mg/kg MgSO 4 and 1 mg/kg propofol.
  • the rat After an intravenous bolus of ALM/propofol the rat underwent circulatory collapse within 10 sec. The blood pressure fell to zero and the heart rate fell to zero. The heart rate returned after ⁇ 4 min. Began chest compressions at 6 min for 2 min only then again at 15 min and pressure increased. Within 10 min the hemodynamics returned to normal. The animal was monitored for 2 hours and hemodynamics were stable and following the experiment an autopsy showed no signs of ischemia to the heart, lungs, kidneys or gastrointestinal tract.
  • ECG acceleratory ‘blips’ see FIGS. 12C and 12D . More regular pattern started at 1 min 40 sec (HR ⁇ 35 bpm). Still coordinated transient pressure increase (trace not shown). During this time period noticed paws twitching and twitching in abdominal region
  • mice Male Sprague Dawley rats (300-400 g) were fed ad libitum with free access to water and housed in a 12-hr light-dark cycle. Animals were anesthetized with an intraperitoneal (IP) injection of 100 mg/kg sodium thiopentone (Thiobarb). After Thiobarb anesthesia, rats were positioned in the supine position on a custom designed cradle. A tracheotomy was performed and the animals artificially ventilated at 90-100 strokes per min on humidified room air using a Harvard Small Animal Ventilator (Harvard Apparatus, Mass., USA) to maintain blood pO 2 , pCO 2 and pH in the normal physiological range.
  • IP intraperitoneal
  • Thiobarb sodium thiopentone
  • Rectal temperature was monitored using a rectal probe inserted 5 cm from the rectal orifice before, during and following shock and resuscitation, and previous experiments show the temperature ranges between 37 to 34° C.
  • the left femoral vein and artery was cannulated using PE-50 tubing for drug infusions and blood pressure monitoring (UFI 1050 BP coupled to a MacLab) and the right femoral artery was cannulated for bleeding.
  • Lead II electrocardiogram (ECG) leads were implanted subcutaneously on the left and right front legs and grounded to the back leg. Rats were stabilized for 10 minutes prior to blood withdrawal.
  • Hemorrhagic shock was induced by withdrawing blood from the femoral artery at an initial rate of ⁇ 1 ml/min then decreasing to ⁇ 0.4 ml/min over 20 min. Initially blood was withdrawn slowly into a 10 ml heparinized syringe (0.2 ml of 1000 U/ml heparin) to reduce MAP to between 35 and 40 mmHg. If MAP increased, more blood was withdrawn to maintain its low value, and the process was continued over a 20 min period. The Thiobarb animal was left in shock for 60 min with frequent checking to ensure the MAP remains between 35 to 40 mmHg. After 60 min shock the animal was injected with an IV 0.5 ml bolus of hypertonic saline with ALM.
  • total volume injected IV was 0.5 ml made up to 7.5% NaCl.
  • concentrations in mM in 0.5 ml bolus were 1.5 mM adenosine, 1.48 mM lidocaine-HCl and 333 mM MgSO 4 , and 1270 mM NaCl.
  • Example 6a A single 0.5 ml bolus resulted in a collapse in blood pressure but not heart rate. Having a heart rate and no pressure development is termed pulseless electrical activity (PEA) or electromechanical dissociation. After 1 min 50 sec, there were electrical amplitude spikes in voltage and these occurred after every 7 seconds, and within 20 seconds the blood pressure rose and after 2 min 30 sec the pressure was surprisingly 1.7 times higher than when the treatment was first administered.
  • PDA pulseless electrical activity
  • electromechanical dissociation electromechanical dissociation
  • Heart rate variability is the physiological phenomenon of variation in the time interval between heartbeats. Heart rate and rhythm are largely under the control of the autonomic nervous system whereby the baroreflex continually adjusts heart rate to blood pressure via changes in vagal (parasympathetic) activity. In this way the arterial baroreflex also affects arrhythmogenesis and whole body hemodynamic stability. Thus sympathetic activation can trigger malignant arrhythmias, whereas vagal activity may exert a protective effect. Baroreflex sensitivity is quantified in ms of RR interval prolongation for each mmHg of arterial pressure increase. In the analysis of HR variability, there is a time domain and a frequency domain of analysis.
  • the time domain indices include SDNN, SADNN, NN50, pNN50, RMSSD, SDSD.
  • the most commonly used are the average heart rate and the standard deviation of the average R-R intervals (SDNN) calculated over a 24-hour period or 5 min R-R period (SADNN).
  • SDNN mostly reflects the very-low-frequency fluctuation in heart rate behavior).
  • NN50 is the number of pairs of successive beat to beat (NN) that differ by more than 50 ms or when expressed as a percentage (pNN50).
  • the RMSSD is the square root of the mean squared differences of successive R-R intervals, and the SDSD is the standard deviation of successive differences of R-R intervals. These time domain measures are recognized to be strongly dependent on the vagal (parasympathetic) modulation with a low value indicating lower vagal tone. In contrast to SDNN, RMSSD is a short-term variation of heart rate and correlates with high frequency domain of heart rate variability reflecting fluctuations in HR associated with breathing.
  • Frequency domain analysis is traditionally understood to indicate the direction and magnitude of sympatho-vagal balance of heart rate variability. It is obtained by dividing the heart rate signal into its low and high frequency bands and analyze the bands in terms of their relative intensities (power).
  • the LF or low frequency band (0.04 to 0.15 Hz) is involved with oscillations related to regulation of blood pressure and vasomotor tone.
  • the HF or high frequency band (0.15 to 0.4 Hz) reflects the effects of respiration on heart rate (i.e. in respiratory frequency range).
  • the LF band reflects primarily sympathetic tone
  • the HF band reflects parasympathetic tone
  • the ratio LF/HF is viewed as an index of sympatho-vagal balance.
  • the LF/HF ratio is much more complex than originally thought and it appears to be restricted to the estimation of parasympathetic influences on heart rate.
  • An increase or decrease in the LF/HF ratio appears to reflect more on the different dominating parasympathetic oscillation inputs that determine blood pressure and vagal tone relative to those inputs involved in regulating fluctuations in HR associated with breathing (respiratory sinus arrhythmia).
  • Sympathetic inputs would undoubtedly contribute to in vivo sympatho-vagal balance, however, it cannot be directly interpreted from the indices that are currently used to examine the time and frequency domains of heart rate variability. Direct analysis of baroreflex sensitivity may be more informative combined with HR variability analysis.
  • mice Male Sprague Dawley rats (300-400 g) were fed ad libitum with free access to water and housed in a 12-hr light-dark cycle. Animals were anesthetized with an intraperitoneal (IP) injection of 100 mg/kg sodium thiopentone (Thiobarb). After Thiobarb anesthesia, rats were positioned in the supine position on a custom designed cradle. A tracheotomy was performed and the animals artificially ventilated at 90-100 strokes per min on humidified room air using a Harvard Small Animal Ventilator (Harvard Apparatus, Mass., USA) to maintain blood pO 2 , PCO 2 and pH in the normal physiological range.
  • IP intraperitoneal
  • Thiobarb sodium thiopentone
  • Rectal temperature was monitored using a rectal probe inserted 5 cm from the rectal orifice before, during and following shock and resuscitation, and previous experiments show the temperature ranges between 37 to 34° C.
  • the left femoral vein and artery was cannulated using PE-50 tubing for drug infusions and blood pressure monitoring (UFI 1050 BP coupled to a MacLab) and the right femoral artery was cannulated for bleeding.
  • Lead II electrocardiogram (ECG) leads were implanted subcutaneously on the left and right front legs and grounded to the back leg. Rats were stabilized for 10 minutes prior to blood withdrawal.
  • Hemorrhagic shock was induced by withdrawing blood from the femoral artery at an initial rate of ⁇ 1 ml/min then decreasing to ⁇ 0.4 ml/min over 20 min (40-50% blood loss). Initially blood was withdrawn slowly into a 10 ml heparinized syringe (0.2 ml of 1000 U/ml heparin) to reduce MAP to between 35 and 40 mmHg. If MAP increased, more blood was withdrawn to maintain its low value, and the process was continued over a 20 min period. The animal was left in shock for 60 min with frequent checking to ensure the MAP remains between 35 to 40 mmHg.
  • the ability of the invention to be employed for hypotensive resuscitation was examined in number of experiments, and it was found that survival for delayed retrieval times could only be achieved by an intravenous bolus followed by an intravenous infusion (one-two treatment strategy). A single intravenous bolus or a bolus followed by a bolus was not sufficient to prevent circulatory collapse and death after haemorrhagic shock.
  • FIG. 9 Group 1: Bolus alone: ALM treatment animal received intravenous 0.3 ml bolus 3.0% NaCl (508 mM, 0.045 g/kg) with 1 mM Adenosine (0.24 mg/kg), 3 mM Lidocaine (0.73 mg/kg), and 2.5 mM MgSO 4 (0.27 mg/kg).
  • FIG. 10 Group 2 Bolus alone vs Bolus and infusion:
  • ALM treatment animal received intravenous 0.3 ml bolus 3.0% 0.4 NaCl with 1 mM Adenosine (0.24 mg/kg), 3 mM Lidocaine (0.73 mg/kg), and 2.5 mM MgSO 4 (0.27 mg/kg) and after 60 min and an infusion of 1 ml/kg/hr 0.9% NaCl+3 mg/kg Adenosine+6 mg/kg Lidocaine+3.36 mg/kg MgSO 4 .
  • composition administered per kg body weight per hour comprised 11.23 mM adenosine, 22 mM lidocaine-HCl and 28 mM MgSO 4 .
  • This example shows that an ALM treatment animal that received an intravenous 1 ml bolus of 7.5% NaCl ALM (1 mM Adenosine, 3 mM Lidocaine HCl; 2.5 mM MgSO 4 ) followed by a second 0.5 ml bolus of 7.5% NaCl ALM (1 mM Adenosine (0.24 mg/kg), 3 mM Lidocaine HCl (0.73 mg/kg); 2.5 mM MgSO 4 (0.27 mg/kg)) at 90 min did not improve survival.
  • the examples provide evidence that a intravenous single bolus of 3% or 7.5% hypertonic saline with ALM treatment or a bolus-bolus administration are not adequate for sustained hypotensive resuscitation following a period of shock induced by bleeding. Survival requires the administration of a bolus followed by an intravenous infusion, which is equivalent to a bolus then a drip.
  • This example is clinically (or venterinarily) relevant because long delays can occur to reach the patient or subject in prehospital or military settings. Long delays can also occur in Rural and Remote Medical hospitals or environments. The results also pertain to the battlefield environment where small expeditionary teams routinely operate in austere and hostile environments and have access to limited medical supplies and where evacuation times may be many hours to days depending upon location.
  • MAP mean arterial pressure
  • RPP peak arterial systolic pressure times heart rate (index of myocardial O2 consumption)
  • SDNN indicates standard deviation of normal to normal R-R intervals, where R is the peak of a QRS complex (heartbeat)
  • NN50 is the number of pairs of successive beat to beat (NN) that differ by more than 50 ms.
  • ALM In the frequency domain, ALM also reduced LF by 54% and HF by 31% relative to 7.5% NaCl controls, again implying a reduced parasympathetic input to heart rate variability at both low and high frequencies.
  • the 33% lower LF/HF ratio in the ALM treated animals than controls would suggest either the drug 1) decreased parasympathetic control of MAP and vagal tone or 2) increased the regulating the effect of respiration on heart rate, or both compared to 7.5% NaCl alone. Since the animals were actively ventilated at ⁇ 90 strokes per min and heart rate was not different between groups, it appears the fall in LF/HF ratio is due to the drugs action to decrease the parasympathetic input on MAP and vagal tone to increase stability in heart rate.
  • Rectal temperature was monitored using a rectal probe inserted 5 cm from the rectal orifice before, during and following shock and resuscitation, and previous experiments show the temperature ranges between 37 to 34° C.
  • the left femoral vein and artery was cannulated using PE-50 tubing for drug infusions and blood pressure monitoring (UFI 1050 BP coupled to a MacLab) and the right femoral artery was cannulated for bleeding.
  • Lead II electrocardiogram (ECG) leads were implanted subcutaneously on the left and right front legs and grounded to the back leg. Rats were stabilized for 10 minutes prior to blood withdrawal.
  • Hemorrhagic shock was induced by withdrawing blood from the femoral artery at an initial rate of ⁇ 1 ml/min then decreasing to ⁇ 0.4 ml/min over 20 min. Initially blood was withdrawn slowly into a 10 ml heparinized syringe (0.2 ml of 1000 U/ml heparin) to reduce MAP to between 35 and 40 mmHg. If MAP increased, more blood was withdrawn to maintain its low value, and the process was continued over a 20 min period. The animal was left in shock for 60 min with frequent checking to ensure the MAP remains between 35 to 40 mmHg.
  • ALM treatment animal received intravenous 0.3 ml bolus 3.0% NaCl with 1 mM Adenosine (0.24 mg/kg), 3 mM Lidocaine (0.73 mg/kg), and 2.5 mM MgSO 4 (0.27 mg/kg) with 50 mM beta-hydroxy butyrate (D-isomer, 4.7 mg/kg).
  • ALM with BHB “kick” started around 15 min and continued through 60 min resuscitation.
  • Beta-hydroxy butyrate was added to the hypotensive resuscitation fluid because it is known to bind to the GPR109A receptor on immune cells (monocytes and macrophages) and the vascular endothelium to have a direct anti-inflammatory effect. This example shows that Beta-hydroxy butyrate did not compromise hemodynamic support of hypotensive resuscitation.
  • VPA valproic acid
  • MgSO 4 (0.27 mg/kg). with administration of valproic acid (VPA) (231 mM in 0.3 ml or 30 mg/kg body weight) raised MAP in the hypotensive range from 40 to 55 mmHg over 60 min after hemorrhagic shock.
  • the example further demonstrates that administering an intravenous infusion of 0.9% NaCl ALM protected the animal from suffering circulatory collapse. This provides evidence that the addition of valproic acid in a bolus followed by an infusion or drip maintained hemodynamics, and that histone deacetylase inhibitors may be useful for protecting the brain and other organs of the body during delayed retrieval from the prehospital or military setting to definitive care.
  • VPA also is known to have cytoprotective effects from an increase acetylation of nuclear histones, promoting transcriptional activation of deregulated genes, which may confer multi-organ protection.
  • mice Male Sprague Dawley rats (300-400 g) were fed ad libitum with free access to water and housed in a 12-hr light-dark cycle. Animals were anesthetized with an intraperitoneal (IP) injection of 100 mg/kg sodium thiopentone (Thiobarb). After Thiobarb anesthesia, rats were positioned in the supine position on a custom designed cradle. A tracheotomy was performed and the animals artificially ventilated at 90-100 strokes per min on humidified room air using a Harvard Small Animal Ventilator (Harvard Apparatus, Mass., USA) to maintain blood pO 2 , PCO 2 and pH in the normal physiological range.
  • IP intraperitoneal
  • Thiobarb sodium thiopentone
  • Rectal temperature was monitored using a rectal probe inserted 5 cm from the rectal orifice before, during and following shock and resuscitation, and previous experiments show the temperature ranges between 37 to 34° C.
  • the left femoral vein and artery was cannulated using PE-50 tubing for drug infusions and blood pressure monitoring (UFI 1050 BP coupled to a MacLab) and the right femoral artery was cannulated for bleeding.
  • Lead II electrocardiogram (ECG) leads were implanted subcutaneously on the left and right front legs and grounded to the back leg. Rats were stabilized for 10 minutes prior to blood withdrawal.
  • Hemorrhagic shock was induced by withdrawing blood from the femoral artery at an initial rate of ⁇ 1 ml/min then decreasing to ⁇ 0.4 ml/min over 20 min. Initially blood was withdrawn slowly into a 10 ml heparinized syringe (0.2 ml of 1000 U/ml heparin) to reduce MAP to between 35 and 40 mmHg. If MAP increased, more blood was withdrawn to maintain its low value, and the process was continued over a 20 min period. The animal was left in shock for 60 min with frequent checking to ensure the MAP remains between 35 to 40 mmHg. Anaesthetized, ventilated male Sprague-Dawley Rat 336 g (estimated blood volume 20.93 ml)
  • MAP mean arterial pressure
  • the large pulse pressure (difference between systolic and diastolic arterial pressure) indicates a high heart stroke volume despite the body's circulation being maintained at these low arterial pressures.
  • the addition of the adenosine agonist placed the animal in a deep sleep with protection.
  • the Example suggests lowering the level of [CCPA] for and provide a bolus and further treatment in form of continuous infusion.
  • mice Male Sprague Dawley rats (300-400 g) were fed ad libitum with free access to water and housed in a 12-hr light-dark cycle. Animals were anesthetized with an intraperitoneal (IP) injection of 100 mg/kg sodium thiopentone (Thiobarb). After Thiobarb anesthesia, rats were positioned in the supine position on a custom designed cradle. A tracheotomy was performed and the animals artificially ventilated at 90-100 strokes per min on humidified room air using a Harvard Small Animal Ventilator (Harvard Apparatus, Mass., USA) to maintain blood pO 2 , PCO 2 and pH in the normal physiological range.
  • IP intraperitoneal
  • Thiobarb sodium thiopentone
  • Rectal temperature was monitored using a rectal probe inserted 5 cm from the rectal orifice before, during and following shock and resuscitation, and previous experiments show the temperature ranges between 37 to 34° C.
  • the left femoral vein and artery was cannulated using PE-50 tubing for drug infusions and blood pressure monitoring (UFI 1050 BP coupled to a MacLab) and the right femoral artery was cannulated for bleeding.
  • Lead II electrocardiogram (ECG) leads were implanted subcutaneously on the left and right front legs and grounded to the back leg. Rats were stabilized for 10 minutes prior to blood withdrawal.
  • Hemorrhagic shock was induced by withdrawing blood from the femoral artery at an initial rate of ⁇ 1 ml/min then decreasing to ⁇ 0.4 ml/min over 20 min. Initially blood was withdrawn slowly into a 10 ml heparinized syringe (0.2 ml of 1000 U/ml heparin) to reduce MAP to between 35 and 40 mmHg. If MAP increased, more blood was withdrawn to maintain its low value, and the process was continued over a 20 min period. The animal was left in shock for 60 min with frequent checking to ensure the MAP remains between 35 to 40 mmHg. If MAP deviated from this range either shed blood was re-infused or further blood was withdrawn.
  • L-NAME N ⁇ -nitro-L-arginine methyl ester hydrochloride
  • NO nitric oxide
  • FIG. 13 shows that the addition of 30 mg/kg L-NAME to 7.5% NaCl/ALM totally abolished MAP resuscitation during the hypotensive period.
  • the addition of L-NAME led to ventricular dysrhythmia with each animal experiencing an average of 65.5 ⁇ 1.5 arrhythmic episodes.
  • ALM cannot resuscitate in the presence of the NOS inhibitor L-NAME indicating the involvement of NOS & or NO in some way.
  • ALM operates as a NO-dependent, ‘pharmacological switch’ which releases a natural “handbrake” on the shocked heart to gently raise MAP and improve whole body protection and stabilization, including brain.
  • ALM operates as a NO-dependent, ‘pharmacological switch’ which releases a natural “handbrake” on the shocked heart to gently raise MAP and improve whole body protection and stabilization, including brain.
  • NO through site-specific and differential modulation of neuronal activity affects cardiac function.
  • the nucleus tractus solitari (NTS) receives input from baroreceptors that is processed in this and other regions of the brain and eventually expressed with altered cardiac and whole body functions.
  • ALM may modulate CNS function to improve heart and multi-organ protection from hemodynamic, anti-inflammatory and coagulation correction mechanisms during shock states, and other forms or injury (traumatic and non-traumatic), burns, sepsis, infection and stress and disease states. This may be one of the underlying mechanisms of action of the invention.
  • CPB cardiopulmonary bypass
  • hypothermic circulatory arrest temporary interruption of brain circulation
  • transient cerebral hypoperfusion transient cerebral hypoperfusion
  • manipulations on the frequently atheromatic aorta A combination of antegrade and retrograde cerebral perfusion has also been shown to be useful for brain protection during aortic reconstruction.
  • hypothermic circulatory arrest occurs when the systemic body temperature is around 20° C. for up to 30 min. It is during this time the surgeon performs the aortic repair and the brain must be protected.
  • the brain is normally perfused with cold oxygenated whole blood or blood:fluid dilutions (e.g. 4 parts blood: 1 part fluid) at temperatures 20 to 25° C. and as low as 6 to 15° C.
  • cold oxygenated whole blood or blood:fluid dilutions e.g. 4 parts blood: 1 part fluid
  • the operative mortality for aortic arch replacement ranges from 6% to 23%, the incidence of permanent neurological dysfunction from 2% to 16%, and the incidence of temporary neurological dysfunction from 5.6% to 37.9%.
  • the aim of the study is to test the protective effect of ALM and a general anesthetic on the brain, with and without an inflammatory such as beta-hydroxybutyrate (BHB) and brain fuel citrate.
  • the vehicle can be whole blood, whole blood; crystalloid dilutions or crystalloid alone and isotonic or hypertonic with respect to saline.
  • the hypothesis that will be tested is selective cerebral perfusion with blood containing a bolus of 10 ml ALM Propofol (1 mg adenosine; 2 mg Lidocaine-HCl and 0.3 g MgSO 4 , 1 mg/kg propofol) administered via the innominate and left common carotid arteries (Di Eusanio, M., et al, 2003, J.
  • Cerebral perfusion aims for a flow of 10 ml/kg body wt/min which is normally adjusted to maintain a radial arterial pressure of between 40 to 70 mm Hg.
  • Cerebral monitoring is achieved by means of a right radial arterial pressure line, electroencephalography, regional oxygen saturation in the bilateral frontal lobes with near-infrared spectroscopy, and transcranial Doppler ultrasonographic measurement of the blood velocity of the middle cerebral arteries
  • Brain damage biomarkers such as neurofilament (NF), S100 ⁇ , glial fibrillary acidic protein (GFAP), and ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1) neuron-specific enolase (NSE)).
  • Brain ischemia will be assessed using blood lactate levels and pH.
  • Inflammation will be assessed using select markers (e.g. IL-1, IL-6, IL-12, tumor necrosis factor-alpha), and coagulopathy using coagulometry (aPTT, PT) and visco-elastic ROTEM analysis.
  • aPTT, PT coagulometry
  • Temporary neurological deficit, 30-day mortality and mortality-corrected permanent neurological dysfunction will be assessed. The 30-day mortality will include any death that occurred from the intraoperative period until the 30 th postoperative day.
  • Secondary end points will be perioperative complications and perioperative and postoperative times, intubation times.
  • This example will demonstrate one aspect of the invention, which is to protect the brain using non-arrest levels of the composition in bolus and constant infusion.
  • An arm may be included where the doses are raised to examine another aspect of the invention to arrest the brainstem (and higher centres) during circulatory arrest for aortic reconstructions or large intracranial aneurysm surgeries. This example would also be applicable for pediatric and neonatal circulatory arrest interventions and surgeries.
  • Abdominal aortic rupture is a highly lethal event, claiming about 15,000 lives each year.
  • open surgical repair with thoracotomy has been the mainstay of treatment, yet this surgery is associated with up to 50% perioperative mortality.
  • Minimally invasive endovascular stent grafts has become popular and while still remaining a high-risk procedure with high mortality, it has been used with great success in the elective repair of aortic aneurysms.
  • Hypotensive anaesthesia may also be protective to reduce blood loss, however, the brain must be protected.
  • the aim of the study is to test the protective effect of intravenous infusion of ALM with and without an inflammatory such as beta-hydroxybutyrate (BHB) and brain fuel citrate 5 min before and during minimally invasive endovascular stent grafts in the elective repair of aortic aneurysms.
  • BHB beta-hydroxybutyrate
  • the hypothesis that will be tested is that intravenous bolus and infusion of 3% NaCl ALM with citrate (1 mM) and BHB (4 mM) will result in 1) targeted systemic hypotension to reduce bleeding, and 2) protect the body and organs (e.g.
  • the primary end points will be biomarkers for the clinical diagnosis of brain injury, inflammatory markers, coagulopathy, temporary neurological deficit, 30-day mortality and mortality-corrected permanent neurological dysfunction.
  • the 30-day mortality included any death that occurred from the intraoperative period until the 30 th postoperative day.
  • Secondary end points will be perioperative complications and perioperative and postoperative times, intubation times.
  • the data will demonstrate one aspect of the invention to protect the brain and organs of the body using non-arrest levels of the composition administered as bolus and infusion.
  • the aim of the study is to provide a bolus and infusion of ALM immediately following parturition and haemorrhage.
  • An intravenous ALM bolus (0.3 mg/kg adenosine; 0.6 mg/kg Lidocaine-HCl and 0.03 g/kg MgSO 4 ) followed by intravenous infusion of ALM (Adenosine; 0.2 mg/kg/min. Lidocaine-HCl; 0.4 mg/kg/min and MgSO 4 ; 0.224 g/kg/min) at a flow rate of 10 ml/kg/min would be investigated.
  • ALM therapy will correct coagulopathy, reduce bleeding and improve whole body function following childbirth such as improved hemodynamics, inflammation and reduce the incidence of infection.
  • ALM therapy compared to no treatment will correct coagulopathy and reduce post-partum complications and treatment for hemorrhage.
  • a second study will be performed investigating the ALM therapy administered before parturition for complicated pregnancy/delivery cases to protect both the mother and baby. The data will demonstrate one aspect of the invention to protect the mother and organs of the body using non-arrest levels of the composition administered as bolus and infusion.
  • Operative factors that contribute to brain injury in both pediatric and adult cardiac surgery include poor perfusion, anesthetic-induced brain toxicity, cardiopulmonary bypass-mediated inflammation, ischemia-reperfusion injury, thromboembolic events, and glucose, electrolyte and acid-based disturbances.
  • the early postoperative period is also a highly vulnerable time for injury because of poor perfusion, free radical and oxidant damage, cyanosis, inflammation, coagulopathy, abnormal vascular reactivity, hyperthermia, endocrine abnormalities and poor glycemic control and insulin-resistance including pyruvate dehydrogenase inhibition.
  • Postoperative variables such as cyanosis, low systolic and diastolic blood pressures, low cardiac output, and prolonged periods of poor cerebral O 2 saturation.
  • the aim of the study is twofold: 1) to investigate the effect of intra-arterial ALM bolus and infusion 5 to 15 min and brain protection before beginning and continued throughout the surgical procedure, and 2) a second intravenous bolus and infusion 5 to 15 min and during circulatory arrest throughout the whole body where appropriate.
  • the hypothesis is that the ALM therapy improves 1) brain and 2) whole body function compared to vehicle controls, including cardiac, renal and lung functional improvement. The therapy will reduce inflammation, reduce coagulation disturbances and lead to less whole body ischemia.
  • the surgical method for neonatal aortic arch reconstruction is described by Malhotra and Hanley and references therein (Malhotra and Hanley, 2008).
  • the intravenous whole body bolus-infusion will commence before cardiopulmonary bypass and cooling. Cardiopulmonary bypass will be initiated and once adequate venous drainage confirmed, the patient will be cooled to 22° C. to 24° C. for a minimum.
  • the arch vessels will then be prepared for cerebral perfusion.
  • the innominate artery, the left carotid artery, and the left subclavian artery are each individually clamped with atraumatic neurovascular clips to ensure uniform cooling of the central nervous system.
  • ALM bolus and infusion will commence at least 5 min before the operation at a flow rate of ⁇ 30 ml/kg/min to generate sufficient cerebral pressures for optimal protection.
  • the whole body ALM bolus-intravenous infusion can be lowered and continued for further stabilization in the intensive care unit.
  • intravenous bolus and infusion to whole body
  • intra-arterial bolus and infusion to brain circuit.
  • the whole body infusion may have to be stopped as circulation is stopped and re-started.
  • the doses would include ALM bolus (0.3 mg/kg adenosine; 0.6 mg/kg Lidocaine-HCl and 0.03 g/kg MgSO 4 ) followed by intravenous infusion of ALM (Adenosine; 0.2 mg/kg/min. Lidocaine-HCl; 0.4 mg/kg/min and MgSO 4 ; 0.224 g/kg/min) at 10 ml/min/kg (whole body), and arterial flow to the brain adjusted to meet the flow requirements according to surgeon preference.
  • Brain protection in neonates will include near infrared spectroscopy (NIRS), transcranial Doppler (TCD), electroencephalography (EEG), and serum measurement of S100B protein.
  • NIRS near infrared spectroscopy
  • TCD transcranial Doppler
  • EEG electroencephalography
  • serum measurement of S100B protein Whole body protection will be assessed using routine haemodynamic measurements, cardiac output, ultrasound volume relaxation parameters of left ventricular function, troponins, inflammatory markers and coagulopathy. 30-day mortality and infection rates will be recorded.
  • the data will demonstrate one aspect of the invention to protect the brain, heart, kidney and lungs using non-arrest levels of the composition.
  • Post-operative infections include sepsis, wound infection, mediastinitis, endocarditis, and pneumonia and any of these conditions contributes to prolonged LOS and increased hospital costs.
  • Increased risk factors for major infections were age, reoperation, preoperative length of stay longer than 1 day, preoperative respiratory support or tracheostomy, genetic abnormality, and medium or high complexity score.
  • An intravenous bolus of ALM and infusion/drip will begin prior to placing the patient on CPB the cardiac surgery and continued throughout the surgery.
  • the hypothesis is that the one-two ALM treatment will induce whole body protection from reducing inflammation and coagulopathy and improve cardiac function (lower troponin and lactate) and reduce infection.
  • the bolus and drip will also improve brain and renal function following surgery and reduce hospital length of stay. The results will be compared with historical controls and with vehicle infusion.
  • IL-6 interleukin-6
  • IL-8 tumor necrosis factor alpha
  • PMN-E polymorphonuclear elastase
  • CRP C-reactive protein
  • WBC white blood cell count
  • NC neutrophil count
  • IL6 has recently been associated with acute kidney injury within the first 24 hours after pediatric cardiac surgery.
  • Coagulation status will be assessed using ROTEM. Cardiac troponins will be measured during and following surgery including 12 hours and 24 hours post-operative times. Brain function will be assessed using blood markers and cerebral oximetry and transcranial Doppler ultrasonographic measurement of the blood velocity of the middle cerebral arteries.
  • the data will demonstrate that the intravenous bolus and drip or infusion will confer perioperative protection including improved whole body post-operative cardiac, renal and neural function and blunting of the inflammatory response and restoring coagulation leading to lower intensive care and hospital room stays.
  • ECMO extracorporeal membrane oxygenation
  • the ALM therapy can be continued at a lower dose for whole body stabilization.
  • the therapy will be shown to be a central component in the management neonatal, paediatric and adult patients, and the critically ill suffering a traumatic and non-traumatic injury.
  • Carotid endarterectomy is a procedure used to prevent stroke by correcting blockage in the common carotid artery, which delivers blood to the brain.
  • Endarterectomy is the removal of material from the inside of the vessel causing the blockage.
  • the surgeon opens the artery and removes the blockage.
  • Many surgeons lay a temporary bypass or shunt to ensure blood supply to the brain during the procedure.
  • the procedure may be performed under general or local anaesthetic.
  • the shunts may take 2.5 minutes and ischemic cerebral signals (flat wave) in electroencephalographic can occur soon after insertion of the shunt.
  • the mean shunting time can be around 1 hour for the operation to take place. Damage the brain and other organs can occur during the procedure.
  • New ischemic lesions on diffusion-weighted magnetic resonance imaging are detected in 7.5% of patients after carotid endarterectomy. Twenty patients will be recruited after obtaining the hospital's internal review board protocol approval and patient consent for the study. The aim of the present study is to provide an arterial ALM bolus and infusion with and without propofol prior to placing the shunt, and continued for 60 min or as long as the operation takes. Diffusion-weighted magnetic resonance imaging will be conducted to examine if there are reduced lesions compared to saline or blood controls. The data will demonstrate one aspect of the invention to protect the brain, heart, kidney and lungs of the body using non-arrest levels of the composition involving a bolus and infusion. This is one aspect of the invention showing the clinical advantage of the bolus and drip (infusion) ALM treatment therapy on brain and whole body protection.
  • One of the further challenges of the arthroscopic procedures is the need for controlled hypotension during anaesthesia to lessen intra-articular haemorrhage and thereby provide adequate visualisation to the surgeon, and reduced local and systemic inflammation coagulopathy for the patient.
  • Bones bleed at normal blood pressure and the shoulder is highly vascularized and this area is difficult if not impossible to use a tourniquet. Achievement of optimal conditions necessitates several interventions and manipulations by the anaesthesiologist and the surgeon, most of which directly or indirectly involve maintaining intra-operative blood pressure (BP) control.
  • BP blood pressure
  • the aim of our study is: 1) to examine the effect of ALM injectable applications or topical sprays at select times within the joint to reduction of local adhesions, reduce local inflammation and reduce local coagulopathy and pain following surgical or arthroscopic repair of the rotator cuff. 2) to examine the effect of intravenous whole body ALM dose and infusion, with and without proprofol, to induce a hypotensive state to reduce bleeding during the surgery, and to protect the whole body from the trauma of surgery with reduced inflammation and coagulation and reduced pain.
  • ALM bolus-infusion therapy will assist in inducing a whole body hypotensive anaesthesia to reduce bleeding, which would also be applicable for other types of interventions and surgery including knee surgery and the intravenous bolus-infusion will protect distal areas once a tourniquet at the knee is applied and released every 30 min.
  • results of the study will demonstrate one aspect of the invention to protect the joint from stiffness and the whole body using non-arrest levels of the composition involving a bolus and infusion, and another aspect of the invention to facilitate hypotensive state for anesthesia with reduced blood loss.
  • Opening of the pericardial cavity during cardio-thoracic surgical operations promotes inflammation, coagulopathy, injury and adhesions.
  • Postsurgical intrapericardial adhesions may complicate the technical aspects of reoperations from injury to the heart and great vessels as well as perioperative bleeding.
  • the rate of inadvertent injury ranged from 7% to 9%.
  • Closing the chest (sternum) also has a risk of infection and adhesions.
  • Sternal wound infections are a life-threatening complication after cardiac surgery associated with high morbidity and mortality. Deep sternal wound infection is also termed mediastinitis after median sternotomy occurs in 1 to 5% of patients and the associated mortality rate in the literature ranges from 10 to 47%.
  • the present invention will show that intravenous ALM bolus and infusion during the operation during or following the surgery will lower infection rate and incidence of adhesions following surgery.
  • the second aim is to show that ALM in a syringe applied topically or by spray or other means of delivery to the area during, prior to closure of the wound, or following closure of the wound will reduce adhesions, promote healing and reduce infection following cardiac surgery.
  • the Box Jellyfish (also known as the sea wasp or sea stinger) is the only known coelenterate that is lethal to humans.
  • the venom has cardiotoxic, neurotoxic and dermatonecrotic components. It is injected by hundreds of thousands of microscopic stings over a wide area of the body and on the trunk. Absorption into the circulation is rapid. Each sting arises from the discharge of a nematocyst.
  • the central rod of the microbasic mastigphore carries the venom, and is like a microscopic spear, which is impaled, on contact, into the victim by a springy protein.
  • Other jellyfish may cause a similar syndrome such as Irukandji. When stung, the pain is absolutely excruciating and can lead to shock and death.
  • Systemic magnesium in slow boluses of 10-20 mMol, may attenuate pain and hypotension.
  • ALM will produce greater pain relief and whole body physiological support by reducing the devastating effect of the catecholamine storm compared with magnesium alone.
  • ALM adenosine, lignocaine with 10-20 mM magnesium sulphate
  • the working rat heart is considered the gold standard model for translation research in cardioplegia and preservation solutions for cardiac surgery or heart storage for transplantation.
  • a new concept of polarized arrest and protection for surgical cardioplegia employing a composition of adenosine and lidocaine in a physiological Krebs-Henseleit ionic solution (Dobson, 2004, 2010).
  • adenosine and lidocaine in a normokalemic solution arrested the heart by ‘clamping’ the myocyte's diastolic membrane potential at around ⁇ 80 mV and was accompanied by a fall in oxygen consumption of over 95% (Dobson, 2004).
  • Rats Male Sprague-Dawley rats (350-450 g) were obtained from James Cook University's breeding colony. Animals were fed ad libitum and housed in a 12-hour light/dark cycle. On the day of experiment, rats were anaesthetised with an intraperitoneal injection of Thiobarb (Thiopentone Sodium; 60 mg/kg body wt) and the hearts were rapidly excised as described in Dobson and Jones (Dobson, 2004). Rats were handled in compliance with James Cook University Guidelines (Ethics approval number A1084), and with the ‘Guide for Care and use of Laboratory Animals’ from the National Institutes of Health (NIH Publication No. 85-23, revised 1985, and PHS Publication 1996).
  • Thiobarb Thiobarb
  • heart preparation, attachment and perfusion are described in by Dobson and Jones (Dobson, 2004) and Rudd and Dobson (Rudd and Dobson, 2009). Briefly, hearts were attached to a Langendorff apparatus and perfused at a pressure head of 90 cm H 2 O (68 mmHg). The pulmonary artery was cannulated for collection of coronary venous effluent and O 2 consumption measurements. For working mode operation, a small incision was made in the left atrial appendage and a cannula inserted and sutured.
  • the heart was then switched from Langendorff to the working mode by switching the supply of perfusate from the aorta to the left atrial cannula at a hydrostatic pressure of 10 cm H 2 O (pre-load) and an afterload of 100 cm H 2 O (76 mmHg).
  • Hearts were stabilized for 15 minutes and pre-arrest data recorded before converting back to Langendorff mode prior to inducing normothermic arrest.
  • Heart rate, aortic pressure, coronary flow and aortic flow were measured prior to and following 6 hour arrest and cold static storage (see FIG. 14 ).
  • Aortic pressure was measured continuously using a pressure transducer (ADI Instruments, Sydney, Australia) coupled to a MacLab 2e (ADI Instruments). Systolic and diastolic pressures and heart rate were calculated from the pressure trace using the MacLab software.
  • compositions are Compositions:
  • Krebs buffer Hearts were perfused in the Langendorff and working modes with a modified Krebs-Henseleit crystalloid buffer containing 10-mmol/L glucose, 117 mmol/L sodium chloride, 5.9-mmol/L potassium chloride, 25-mmol/L sodium hydrogen carbonate, 1.2-mmol/L sodium dihydrogenphosphate, 1.12-mmol/L calcium chloride (1.07-mmol/L free calcium ion), and 0.512-mmol/L magnesium chloride (0.5-mmol/L free magnesium ion), pH 7.4, at 37_C.
  • the perfusion buffer was filtered with a 1-mm membrane and then bubbled vigorously with 95% oxygen and 5% carbon dioxide to achieve a P02 greater than 600 mm Hg.
  • the perfusion buffer was not recirculated.
  • Adenosine lidocaine magnesium (ALM) with 2% CPD (20 ml/L cardioplegia)
  • the heart is arrested for a total time of 2 or 4 hours and arrest is ensured by a flush of cardioplegia every 18 min.
  • the method of intermittent cardioplegic delivery has been previously described by Dobson and Jones (Dobson, 2004).
  • Arrest in the Langendorff mode was induced by a 5-minute infusion of cardioplegic solution (50-100 mL) comprising 200 ⁇ M (0.2 mM or 53.4 mg/L) adenosine plus 500 ⁇ M (0.5 mM or 136 mg/L) lidocaine-HCL.
  • the amount of A and L in mg in 100 ml over a 5 min period would be 5.34 mg adenosine and 13.6 mg Lidocaine-HCl or 1.07 mg adenosine per min and 2.72 mg/min lidocaine-HCl. Since the heart weighs around 1 gm in mg/min/kg this would be equivalent to 13.6 g/min/kg heart adenosine and 2.72 kg/min/kg heart lidocaine-HCl. through the aorta at 37° C. and a constant pressure of 68 mm Hg. After arrest, the aorta was cross-clamped at the completion of infusion with a plastic atraumatic aortic clip.
  • Cardioplegia was replenished every 18 minutes with a 2-min infusion comprising 200 ⁇ M (0.2 mM or 53.4 mg/L) adenosine plus 500 ⁇ M (0.5 mM or 136 mg/L) lidocaine-HCL, after which the crossclamp was reapplied.
  • the heart was switched immediately to the working mode and reperfused with oxygenated, glucose-containing Krebs-Henseleit buffer at 37° C.
  • the heart temperature during intermittent arrest ranged from 35° C. during delivery to about 25° C. before the next delivery (average 28°-30° C.), as directly measured and discussed by Dobson and Jones (Dobson, 2004).
  • Example 20a This example is the same as Example 20a but differs by arresting the heart for 4 hours not 2 hours. After 4 hours arrest ALM (2% CPD)
  • the adenosine and lidocaine solution is also versatile as a preservation solution at both cold static storage (4° C.) and warmer intermittent perfusion (28-30° C.) compared with FDA approved solution Celsior.
  • the inventor published this information in the Journal of Thoracic and Cardiovascular Surgery in 2009 (Rudd and Dobson, 2009). In 2010, the inventor also showed that reperfusing the heart for 5 min with warm, oxygenated polarizing adenosine and lidocaine arrest following 6 hours cold static storage led to significantly higher recoveries in cold adenosine and lidocaine and Celsior hearts and it was proposed that this new reperfusion strategy may find utility during cold-to-warm ‘wash’ transitions and implantation of donor hearts.
  • the inventor further reported that the adenosine and lidocaine cardioplegia could preserve the heart over 8 hours in cold static storage with a 78% return of cardiac output using normokalemic, polarizing adenosine and lidocaine at twice their concentrations (0.4 and 1 mM respectively) in glucose-Krebs-Henseleit solution with melatonin and insulin as ancillary or additional agents.
  • This new adenosine and lidocaine preservation solution with ancillary agents returned 78% of cardiac output (CO) was significantly higher than 55% CO for AL cardioplegia, 25% CO for Celsior and 4% CO for Custodiol (HTK) preservation solutions after 8 hours cold static storage (4° C.).
  • CO cardiac output
  • compositions Gentle Bubbling Adenosine and Lidocaine Solution and 5 Min Rewarm:
  • the 2.5 L glass bottle with the cardioplegia preservation solution was not actively bubbled itself.
  • gentle bubbling was required occurred in the vertical 30 cm long glass oxygenation chamber which delivered the cardioplegia to the isolated heart via the aorta and coronary artery ostia: ie retrograde Langendorff perfusion.
  • the temperature-controlled chamber was filled with cardioplegia preservation solution and single gas tubing with a special stainless steel aerator at the end sitting at the bottom of the chamber prior to being delivered to the heart.
  • Gentle bubbling was defined as a gas flow adjusted to deliver a few bubbles per sec in the chamber with 95% O 2 /5% CO 2 . In those cases were no bubbling was required the tubing was clamped off.
  • the perfusion buffer was filtered using a one micron (1 ⁇ M) membrane and then bubbled vigorously 95% O 2 /5% CO 2 to achieve a pO 2 greater than 600 mmHg. The perfusion buffer was not recirculated.
  • FIG. 22 shows that this was not the case.
  • FIG. 22 shows that gently bubbling of the adenosine and lidocaine (lignocaine) preservation cold cardioplegia over the 8 hour cold perfusion period led to no aortic flow after 15 min reperfusion ( FIG. 22A ).
  • no active bubbling led to nearly 90% return of aortic flow or pump function. This result shows that gentle bubbling severely damages the heart to pump fluid from the left ventricle.
  • gentle bubbling reduces coronary flow to 40% recovery of baseline compared to 90% for no-bubbling. This result indicates that gentle bubbling may damage the coronary vasculature that leads to a reduced recovery of flow from vasoconstriction.
  • gentle bubbling led to a cardiac output (AF+CF) of less than 10% baseline indicating major damage to the heart's ability to function as a pump, whereas no bubbling of the adenosine and lidocaine preservation cardioplegia led to around 90% full recovery after 8 hours of constant perfusion at 4° C. ( FIG. 22C ).
  • compositions are Compositions:
  • Adenosine and lidocaine cardioplegia solution with melatonin and insulin (ALMI):
  • the rewarm solutions were the same solutions as the continuous infusion solutions but hearts were slowly rewarmed for 20 min in Langendorff mode by slowly heating the solutions to 37° C. and vigorously bubbled with 95% 0 2 /5% CO 2 to achieve a pO 2 greater than 600 mmHg and the solutions were not recirculated. This vigorous bubbling is in direct contrast to the gentle bubbling during 8 hours of perfusion (few bubbles per sec).
  • the Custodiol-HTK solution contained 15 mmol/L NaCl, 9 mmol/L, KCl, 4.0 mmol/L MgCl 2 , 0.015 mmol/L CaCl 2 , 1.0 mmol/L alpha-ketoglutarate, 180 mmol/L histidine, 18 mmol/L histidine-HCl, 30 mmol/L mannitol, and 2 mmol/L tryptophan.
  • Example 21(a) Equally surprising as Example 21(a) was the finding that adding melatonin and insulin to constant perfusion adenosine and lidocaine preservation cardioplegia largely abolished the damaging effects of gentle bubbling on aortic flow.
  • FIG. 22A perfusing the heart with a solution of adenosine and lidocaine that had gentle bubbling resulted in zero aortic flow.
  • the addition of melatonin and insulin with gentle bubbling led to 80% return of aortic flow ( FIG. 22A ) compared to 90% with adenosine and lidocaine without bubbling ( FIG.
  • FIG. 22A implying that melatonin and insulin did not fully correct the damage but surprisingly reversed much of it after 8 hours of cold constant infusion and 60 min normothermic reperfusion ( FIG. 22A ).
  • the addition of 16 mM MgSO 4 along to melatonin and insulin did not add further improvement with a 70% return of aortic flow compared to 80% with melatonin and insulin.
  • Krebs Henseleit (KH) buffer alone only returned around 20% of aortic flow and FDA-approved preservation cardioplegia—custodial-HTK could not generate aortic flow ( FIG. 22A ).
  • adenosine and lidocaine preservation cardioplegia alone without gentle bubbling gave the highest return of aortic flow and cardiac output which implies superior left ventricular pump function than any cardioplegia group with different additives.
  • Left ventricular pump function is a key parameter in assessing the success of donor heart storage and the success of cardiac function after heart transplantation or implantation.
  • Hearts were rapidly removed from anaesthetised rats and placed in ice-cold heparinised modified KH buffer. Details of anesthesia, ethics approvals, heart preparation, attachment and perfusion are described in Rudd and Dobson (2009).
  • the perfusion buffer was filtered using a one micron (1 ⁇ M) membrane and then bubbled vigorously with 95% O 2 /5% CO 2 to achieve a pO 2 greater than 600 mmHg. The perfusion buffer was not recirculated.
  • the perfusion buffer was filtered using a one micron (1 ⁇ M) membrane and then bubbled vigorously with 95% O 2 /5% CO 2 to achieve a pO 2 greater than 600 mmHg. The perfusion buffer was not recirculated.
  • the adenosine and lidocaine with low calcium and high magnesium (AL (Low Ca 2+ :High Mg 2+ )) solution contained (0.2 mM) adenosine plus 0.5 mM lidocaine in 10 mmol/L glucose containing Modified Krebs Henseleit (LowCa 2+ :HighMg 2+ ) buffer (pH 7.7 at 37° C.) The solution was filtered using 0.2 ⁇ M filters and maintained at 37° C. The arrest solution was not actively bubbled with 95% 0 2 /5% CO 2 hence the higher pH.
  • the average pO 2 of the AL solution was 140 mmHg and the pCO 2 was 5-10 mmHg.
  • Cyclosporine A improves cardiac output by 1.5 times following 6 hours cold static storage. Cyclosporine A may be a possible additive to the ALM cardioplegia/preservation solution for the arrest, protection and preservation of organs, cells and tissues.
  • Rat Hearts were rapidly removed from anaesthetised rats and placed in ice-cold heparinised modified KH buffer. Details of anesthesia, ethics approvals, heart preparation, attachment and perfusion methods are described in Dobson and Jones (Dobson, 2004).
  • the adenosine and lidocaine solution was made fresh daily and contained 200 ⁇ M (0.2 mM or 53.4 mg/L) adenosine plus 500 ⁇ M (0.5 mM or 136 mg/L) lidocaine-HCL (arrest and 2 min infusion every 20 min is the same as example 20)
  • the concentration of sildenafil citrate 3 mg/L (6.3 micromolar).
  • AL sildenafil produces 85% cardiac output and 100% heart rate after 2 hours warm arrest.
  • Rat Hearts were rapidly removed from anaesthetised rats and placed in ice-cold heparinised modified KH buffer. Details of anesthesia, ethics approvals, heart preparation, attachment and perfusion methods are described in Dobson and Jones (Dobson, 2004).
  • the adenosine and lidocaine solution was made fresh daily and contained 200 ⁇ M (0.2 mM or 53.4 mg/L) adenosine plus 500 ⁇ M (0.5 mM or 136 mg/L) lidocaine-HCL (arrest and 2 min infusion every 20 min is the same as example 20)
  • AL BDM recovers 105% heart rate after 2 hours warm arrest and 51% cardiac output.
  • Rat Hearts were rapidly removed from anaesthetised rats and placed in ice-cold heparinised modified KH buffer. Details of anesthesia, ethics approvals, heart preparation, attachment and perfusion methods are described in Dobson and Jones (Dobson, 2004).
  • the adenosine and lidocaine solution was made fresh daily and contained 200 ⁇ M (0.2 mM or 53.4 mg/L) adenosine plus 500 ⁇ M (0.5 mM or 136 mg/L) lidocaine-HCL (arrest and 2 min infusion every 20 min is the same as example 20)
  • Example 28 Sixty consecutive patients undergoing isolated aortic valve replacement were randomly allocated to adenosine-lidocaine-magnesium with insulin in the concentrations and dosages described in Example 28 (30 patients) or standard 4:1 blood DA (30 patients) according to “Buckberg-protocol”. Coronary sinus blood was sampled for lactate release preoperatively (T0) and after reperfusion (T1). Myocardial specimens from right atrium were analyzed for high-energy phosphate content, energy charge, activation of pro-survival kinases Akt and ERK1/2, and cardiomyocyte apoptosis (TUNEL-assay) at T0 vs T1. Spontaneous recovery of sinus rhythm (SRSR) at aortic declamping was also recorded.
  • SRSR sinus rhythm
  • Polarising arrest with ALM and insulin preserves myocardial high-energy phosphates and energy charge, and activates pro-survival kinases Akt and ERK resulting in attenuated apoptosis.
  • PA is superior to DA at the myocellular level.
  • Diabetes mellitus affects 230 million people worldwide. Diabetes is a well-recognized independent risk factor for mortality and morbidity due to coronary artery disease. When diabetic patients need cardiac surgery, either CABG or valve operations, the presence of diabetes represents an additional risk factor for these major surgical procedures. Diabetic patients undergoing CABP have, on the basis of the relative risk evaluation, a 5-fold risk for renal complications, a 3.5-fold risk for neurological dysfunction, a double risk of being hemotransfused, reoperated or being kept 3 or more days in the ICU in comparison with non-diabetic patients. Moreover, diabetic patients undergoing valve operations have a 5-fold risk of being affected by major lung complications.
  • MAPAS adenosine/lidocaine with insulin
  • Buck-Group 4:1-Buckberg cardioplegia (30 patients; Buck-Group).
  • MAPAS composition was 10.4 mg Adenosine, 43 mg Lidocaine-HCl and 3.5 g MgSO 4 in 40 ml w1 mM Adenosine, 4 mM Lidocaine-HCl and 350 mM MgSO 4 in 40 ml) with insulin.
  • HOT SHOT No K + in ALM(I) vs 9 mM K + in Buckberg (Additive 50 ml/L of blood cardioplegia) Contact concentrations therefore for ALM are 15 ⁇ M A, 60 ⁇ M L and 5.25 mM MgSO 4
  • Troponin-I and lactate were sampled from coronary sinus at reperfusion (T1), and from peripheral blood preoperatively (T0), at 6 (T2), 12 (T3) and 48 (T4) hours.
  • Hemodynamic monitoring derived cardiac index (CI), left ventricular dP/dt, cardiac-cycle efficiency (CCE), indexed systemic vascular resistances (ISVR) and central venous pressure (CVP) preoperatively (T0), at ICU-arrival (T1), after 6 (T2) and 24 (T3) hours.
  • Echocardiographic wall motion score index investigated the systolic function, E-wave (E), A-wave (A), E/A, peak early-diastolic TDI-mitral annular-velocity (Ea), E/Ea the perioperative diastolic function preoperatively (T0) and at 96 hours (T1).
  • ISVR Indexed systemic vascular resistance Polarized ALM Depolarizing arrest with 4:1 Parameter INSULIN (MAPAS) arrest Significant Blood Lactate Lower Higher Yes P ⁇ 0.001 at reperfusion Troponin-1 Lower Higher Yes P ⁇ 0.001 Cardiac index Higher Lower Yes P ⁇ 0.001 Left dp/dT Higher Lower Yes P ⁇ 0.001 Cardiac cycle Improved Yes p ⁇ 0.001 efficiency ISVR Not different Not different Not Significant Central venous Not different Not different Not Significant pressure systolic function Higher Lower Yes p ⁇ 0.001 Hemodynamic Higher Lower Yes p ⁇ 0.001 profile Transfusions of Lower Higher Significant red-packed cells p ⁇ 0.001 Transfusions of Lower Higher Significant fresh-frozen p ⁇ 0.001 plasma, ICU-stay and Lower Higher Higher
  • Modified microplegia ALM with Insulin cardioplegia improved myocardial protection in high-risk diabetic patients referred to CABG surgery for unstable angina.
  • cardiopulmonary bypass for surgical cardiac procedures is characterized by a whole-body inflammatory reaction and coagulation imbalances due to the trauma of surgery, contact of blood through nonendothelialized surfaces which can activate specific (immune) and nonspecific (inflammatory) and coagulative responses Q. These responses are then related with postoperative injury to many body systems, like pulmonary, renal or brain injury, excessive bleeding and postoperative sepsis.
  • Example 27 Repeat the above clinical trial in Example 27 but with a form of citrate present with the ALM with insulin cardioplegia. With groups with ALM insulin with CPD and a separate group with ALMI and sildenafil citrate.
  • ALM cardioplegia with a form of citrate (CPD or sildenafil citrate) will improve cardiac function, reduce inflammation and reduce coagulation disurbances with less brain and renal injury.
  • cardiopulmonary bypass for surgical cardiac procedures is characterized by a whole-body inflammatory reaction and coagulation imbalances due to the trauma of surgery, contact of blood through nonendothelialized surfaces which can activate specific (immune) and nonspecific (inflammatory) and coagulative responses. These responses are then related with postoperative injury to many body systems, like pulmonary, renal or brain injury, excessive bleeding and postoperative sepsis. Microparticles are known to contribute to activation of the complement system in patients undergoing cardiac surgery and may be linked to brain and organ injury.
  • ALM insulin cardioplegia with a form of citrate (CPD or sildenafil citrate) will improve cardiac function and reduce microparticles, reduce inflammation and reduce coagulation disurbances with less brain and renal injury.
  • CPD citrate
  • sildenafil citrate a form of citrate
  • Pulmonary preservation for transplantation is associated with inflammation, endothelial cell injury and surfactant dysfunction. Inflammation and the induction of the primary immune response are important in arresting an organ and in lung preservation and can be assessed by measuring tumor necrosis factor alpha (TNF ⁇ ), interleukin-6 (IL-6) and receptor for advanced glycation endproducts (RAGE) in bronchoalveolar lavage fluid.
  • TNF ⁇ tumor necrosis factor alpha
  • IL-6 interleukin-6
  • RAGE receptor for advanced glycation endproducts
  • the study's goal is to assess the effect of ALM cardioplegia/preservation solutions on lung function following 12 and 24 hour cold storage and compare with Celsior and low phosphate dextran solution (e.g. Perfadex, Vitrolife) and Lifor (LifeBlood Medical Inc, NJ).
  • Celsior and low phosphate dextran solution e.g. Perfadex, Vitrolife
  • Lifor LifeBlood Medical Inc, NJ
  • ALM CPD ALM CPD
  • BALF Bronchoalveolar lavage fluid
  • the ALM preservation solutions will lead to no deaths after storage and implantation compared to Celsior or low potassium dextran, and Lifor storage solutions after both 12 and 24 hours.
  • a second finding will be that ALM groups will have significantly less pulmonary vascular resistance index, and less sequestration of neutrophils compared to Celsior or low potassium dextran, and Lifor storage solutions after both 12 and 24 hours. Improvement in surfactant activity will also be evident in the ALM solutions and improved haemodynamics over 5 hours post storage and transplant.
  • ALM cardioplegia preservation with sildenafil citrate or CPD will be superior to standard of care solutions and FDA approved Celsior and Perfadex (or Vitrolife), or Lifor for cold lung storage and implantation.
  • EVLP excreted ex-vivo lung perfusion
  • the aim of this study was to assess the feasibility of transplanting high-risk donor lungs using ALM solutions and comparing with Celsior and low potassium dextran solutions (Perfadex, Vitrolife) or Lifor (LifeBlood Medical) at 29-30° C. for lung preservation.
  • Lungs will be considered suitable for transplantation if 1) during EVLP the P02:Fi02 ratio (ie. the partial pressure of oxygen ex vivo (P02) to the fraction of inspired oxygen (Fi02) of 350 mm Hg or more) and 2) if deterioration from baseline levels of all three physiological measurements (pulmonary vascular resistance, dynamic compliance, and peak inspiratory pressure) was less than 15% while the lungs were ventilated with the use of a tidal volume of 7 ml per kilogram of donor body weight and a rate of 7 breaths per minute during the perfusion period.
  • the primary end point will be graft dysfunction 72 hours after transplantation. Secondary end points will be 30-day mortality, bronchial complications, duration of mechanical ventilation, and length of stay in the intensive care unit and hospital.
  • ALM solution with a form of citrate will have an improved functional after recovery in ex vivo perfused lungs for 4 hours at tepid temperatures from high-risk donors at tepid temperatures compared to Celsior, Perfadex, Vitrolife or Lifor solutions.
  • Oxygen loaded lipid-coated perfluorocarbon microbubbles have been prepared for oxygen delivery; these oxygen-enriched microbubbles have been tested in a rat model of anemia and the results showed that it maintained the rat's survival at very low hematocrit levels.
  • the oxygen release kinetics could be enhanced after nanobubble insonation with ultrasound at 2.5 MHz. It has previously been shown that oxygen-filled nanobubbles were prepared using perfluoropentan as core and dextran sulphate, a polysaccharide polymer, as shell the dextran nanobubbles were able to release oxygen in hypoxic condition.
  • Example 31 The study is the same design as Example 31 differing only in the ALM groups with a form of citrate and oxygen loaded nanoparticle and solutions perfused lungs at normothermic (tepid) temperatures for 4 hours.
  • Oxygen-filled nanobubbles were prepared using perfluoropentan as core and dextran sulphate, a polysaccharide polymer, as shell (Cavalli et al., 2009). Polyvinylpyrrolidone (PVP) was added to the shell as a stabilizing agent. Methods same as Example 31 and 5 ALM groups (50 lungs).
  • ALM with a form of citrate with oxygen-loaded nanoparticles ex vivo perfused lungs for 4 hours from high-risk donors at tepid temperatures have equivalent or improved functional after recovery of lungs compared with ALM solutions without nanoparticles.
  • Transplanted lungs are subjected to injuries including the event causing death of the donor, the inflammatory cascade in brain death, resuscitation of the donor and management in the intensive-care unit and on ventilation.
  • injury related to organ harvest, preservation (storage or perfusion), transport, and implantation injury Once implanted from donor to recipient, ischaemia-reperfusion injury is followed by immunological attack of the foreign organ by the recipient host. For optimum short-term and long-term results, a composition and method is needed to prevent injury at all these stages. Organ preservation thus begins in the donor. Cerebral injury and brain death also is associated with apparent hypercoagulation and poor organ outcome.
  • the aim of this study is to examine the effect of ALM citrate infusions in the validated pig model of intracranial hemorrhage and brain death.
  • Pigs will be divided into 8 groups of 10 pigs per group and the solutions will be infused 5 min before organ harvest after pronounced brain death and the catecholamine storm.
  • the following metrics will include inflammatory markers TnF alpha, IL6, epinephrine, lactate, pH, hemodynamics, cardiac function prior to harvest and coagulopathy. Immediately following harvest; tissues will be prepared for histology and tissue fluorescence studies examining tissue injury.
  • ALM citrate treated body after brain death will lead to less damage to tissues reduce coagulopathy and better prepare the organ, tissue or cell for cold storage, cold perfusion or warm perfusion than Celsior or low Potassium dextran and Lifor solutions prior to implantation into a recipient animal.
  • transient cognitive dysfunction or delirium which can last for up to 5 years, and 2%-13% patients will have a stroke.
  • Four to 40% of patients will have some form of renal dysfunction and perioperative bleeding is a common complication of cardiac surgery with excessive bleeding occuring in 20% of patients, and 5-7% will lose in excess of 2 L within the first 24 h postoperatively. It has been estimated that about 50% of blood loss is due to identifiable surgical bleeding, and the other 50% is due to a complex hypocoagulopathy associated with surgical trauma and cardiopulmonary bypass.
  • Brain injury in the form of temporary or permanent neurological dysfunction also remains a major cause of morbidity and mortality following aortic arch surgery or large intracranial aneurysm surgeries in both adults and pediatric and neonate patients.
  • the aim of the study is to test the protective effect of ALM with sildenafil citrate, ALM citrate beta-hydroxy butyrate and ALM citrate-propofol loaded into nanospheres and without nanospheres on brain function.
  • the vehicle will include whole blood.
  • ALM bolus will be (1 mg adenosine; 2 mg Lidocaine-HCl and 0.3 g MgSO 4 ) and ALM infusion Adenosine; 0.2 mg/kg/min. Lidocaine-HCl; 0.4 mg/kg/min and MgSO 4 ; 0.224 g/kg/min.
  • Sildenafil 1 mg/L, propofol 1 mg/kg; BHB (4 mM blood concentration). 10 ml Bolus administered via the innominate and left common carotid arteries (Di Eusanio et al., 2003) followed by infusion 10 ml/kg/min in whole blood.
  • Surgical Methods and Cerebral Perfusion 72 patients (8 per group) will be recruited after obtaining the hospital's internal review board protocol approval and patient consent for the study.
  • the methods for aortic arch surgery and dissection are described by Kruger et al., (Kruger et al., 2011) and Misfield and others (Misfeld et al., 2012), and references therein.
  • Cerebral perfusion aims for a flow of 10 ml/kg body wt/min which is normally adjusted to maintain a radial arterial pressure of between 40 to 70 mm Hg (Di Eusanio et al., 2003).
  • Cerebral monitoring is achieved by a right radial arterial pressure line, electroencephalography, regional oxygen saturation in the bilateral frontal lobes with near-infrared spectroscopy, and transcranial Doppler ultrasonographic measurement of the blood velocity of the middle cerebral arteries.
  • Primary and Secondary Endpoints will include brain damage biomarkers such as neurofilament (NF), S100 ⁇ , glial fibrillary acidic protein (GFAP), and ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1) neuron-specific enolase (NSE)) (Yokobori et al., 2013).
  • Brain ischemia will be assessed using blood lactate levels and pH.
  • Inflammation will be assessed using select markers (e.g. IL-1, IL-6, IL-12, tumor necrosis factor-alpha), and coagulopathy using coagulometry (aPTT, PT) and visco-elastic ROTEM analysis.
  • aPTT coagulometry
  • the 30-day mortality will include any death that occurred from the intraoperative period until the 30 th postoperative day. Secondary end points will be perioperative complications and perioperative and postoperative times, intubation times.
  • This example will demonstrate one aspect of the invention, which is to protect the brain using non-arrest levels of the composition in bolus and constant infusion with and without nanoparticles.
  • An arm may be included where the doses are raised to examine another aspect of the invention to arrest the brainstem (and higher centres) during circulatory arrest for aortic reconstructions or large intracranial aneurysm surgeries. This example would also be applicable for pediatric and neonatal circulatory arrest interventions and surgeries.
  • kidney preservation is to reduce damage to the kidney from pre-harvest to implantation, and of particular interest is the time for the kidney to provide adequate renal function, reducing the need for dialysis, the primary purpose of the transplant.
  • One key factor is effective graft washout of blood remnants before ischemia cold storage. The presence of blood remnants and cellular debris may contribute to impaired blood flow and injury upon reperfusion.
  • An effective washout of the kidney by the preservation solution prevents cell swelling, formation of interstitial edema, and excessive cellular acidosis, injury and potentially graft failure.
  • Kidneys were harvested from Australian Yorkshire pigs (35-40 Kg) from a local abattoir in Charters Towers. Animals were sacrificed using a captive bolt stunner as per the Humane Slaughter Act and then exsanguinated. Kidneys were removed surgically and placed in a dish for approximately 15 minutes of warm ischaemia for preparation. The renal artery, vein and ureter were identified and clipped to avoid accidental damage, while excess peri-renal connective tissue and the renal capsule were removed. Kidneys were then flushed with 700-800 mls of preservation solution held at a 1 m pressure head.
  • kidneys were weighed and placed in a zip-lock plastic bag containing 200-250 mls of the same preservation solution then stored at 4° C. for 12 hours in an ice-filled polystyrene retrieval box. Kidney weights were recorded 1) prior to, 2) following flushing and again 3) following the 12 hour cold static storage (CSS). For quantitative evaluation of the washout, the remaining red blood cells were counted in specimens of the corticomedullary junction. In a blinded manner, counting of RBCs was performed in ten randomly selected fields of hematoxylin and eosin (H&E)—stained sections
  • Adenosine at 4 mM and Lidocaine at 8 mM with 4% BSA and 0.5% Dextran had significantly lower weight gains than HTK before and after 12 hours storage.
  • Stem cells are pluripotent, self-renewing cells found in all multicellular organisms. In adult mammals, stem cells and progenitor cells act as a repair system for the body, replenishing tissues. The key is that stem cells have the potential to develop into many different kinds of human tissue cells. They remain ‘quiescent’ as undifferentiated cells within tissues or organs as long as tissue homeostasis does not require generation of new cells. Here, they can renew themselves or differentiate into some or all major specialized cell types that make up the tissue or organ. This ‘quiescent’ state, one reversible cell cycle withdrawal, has long been viewed as a dormant state with minimal basal activity.
  • NSC Neural stem cells
  • the first function is to actively maintain the quiescent state, indicating that this is not simply a state of dormancy but in fact under active regulation.
  • the second is to prime the cells for activation, a process that is characterized by the upregulation of multiple cellular processes necessary for cells to enter the cell cycle and begin the process of differentiation.
  • Neural stem cells are not only a valuable tool for the study of neural development and function, but an integral component in the development of transplantation strategies for neural disease. Regardless of the source material, similar techniques are used to maintain NSC in culture and to differentiate NSC toward mature neural lineages.
  • distinct cell membrane voltage controls are found in many precursor cell systems and cancer cells, which are known for their proliferative and differentiation capacities, respectively.
  • NSC neural precursor cells
  • neural precursor cells can be separated into neuronal and glial restricted precursors and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS.
  • Cells will be preserved in different culture solutions with and without ALM sildenafil citrate, ALM citrate phosphate dextrose (CPD), ALM with CPD and cyclosporine A or ALM with erythropoietin, glyceryl trinitrate and zoniporide and quiescent and differentiation will be examined after 12 and 24 hours.
  • Membrane potentials will be performed using the methods described in Sundelacruz et al. (Sundelacruz et al., 2009).
  • mice Male Sprague Dawley rats (300-450 g) were fed ad libitum with free access to water and housed in a 12-hr light-dark cycle. Animals were not heparinized and anesthetized with an intraperitoneal injection of 100 mg/kg sodium thiopentone (Thiobarb). Anesthetized animals were positioned in the supine position on a custom designed cradle. A tracheotomy was performed and animals were artificially ventilated (95-100 strokes min ⁇ 1 ) on humidified room air using a Harvard Small Animal Ventilator (Harvard Apparatus, Mass., USA). Femoral artery and vein cannulations were performed on the left leg for drug pressure monitoring and drug infusions. A lead II ECG was attached via ECG wires. A rectal probe was inserted 5.0 cm and the temperature ranged between 37 and 34° C.
  • a 0.2 ml bolus intravenous injection of a composition comprising 0.2 mg adenosine, 0.4 mg lidocaine-HCl and 200 mg MgSO 4 in 0.9% saline and 0.1% citrate phosphate dextrose (CPD) was administered to a rat. No propofol was in this composition.
  • the concentration of each of the components in the composition was as follows, adenosine 3.75 mM, lidocaine-HCl 7.38 mM, MgSO 4 833 mM, and citrate 3.4 mM.
  • the dosage of each of the components administered to the animal was as follows, adenosine 0.6 mg/kg, lidocaine-HCl 1.2 mg/kg, MgSO 4 600 mg/kg, and citrate 0.6 mg/kg.
  • the baseline heart rate, blood pressure and mean arterial blood pressure (MAP) of the animal was HR 339 bpm, BP 159/113 mmHg, MAP 129 mmHg, Temp 36.7° C. (see FIGS. 26A and B).
  • MAP mean arterial pressure
  • MAP mean arterial pressure
  • a MAP a fall of 48% from baseline
  • a heart rate fell from 339 to 288 beats per min (a 15% fall in heart rate from baseline) (see FIGS. 26C and D).
  • hypotension is often defined as either: mean arterial blood pressure (MAP) decrease of >40% and MAP ⁇ 70 mm Hg. This hypotensive state was maintained for over 10 min.
  • MAP mean arterial blood pressure
  • Example 2 In the same animal as Example 1, after 10 min, a 0.1 ml bolus intravenous injection of the composition comprising 0.1 mg adenosine, 0.2 mg lidocaine-HCl 200 mg MgSO 4 , and propofol in 0.9% saline and 0.1% citrate phosphate dextrose (CPD) was administered.
  • the concentration of each of the components in the composition was as follows, adenosine 3.75 mM, lidocaine-HCl 7.38 mM, MgSO 4 1666 mM, citrate 3.40 mM and propofol 18.5 mM.
  • the dosage of each of the components administered to the animal in this step was as follows, adenosine 0.6 mg/kg, lidocaine-HCl 1.2 mg/kg, MgSO 4 600 mg/kg, citrate 0.3 mg/kg and propofol 1 mg/kg.
  • the baseline heart rate, blood pressure and mean arterial blood pressure (MAP) of the animal was HR 320 bpm, BP 137/95 mmHg, MAP 108 mmHg, Temp 37.0° C. (See FIGS. 27E and F).
  • the blood pressure and heart rate immediately dropped to near zero (not shown) and after 3 min the MAP was 12 and heart rate 191 beats per min (3 min post-bolus: HR 191 bpm, BP 15/11 mmHg, MAP 12 mmHg, Temp 36.6° C., see FIGS. 27G and H)).
  • a composition comprising 1.25 g Adenosine, 2.5 g Lidocaine HCl, 1.25 g MgSO 4 2% CPD in 250 ml of 0.9% NaCl is provided.
  • the concentration of each of the components in the composition was as follows, adenosine 18.71 mM, lidocaine-HCl 36.92 mM, MgSO 4 20 mM, and citrate 2.1 mM.
  • composition may be administered by IV infusion at the following rates:
  • the IV administration could increase to 1 ml/kg/hr or higher, or lower than 0.1 ml/kg/hr.
  • the above mentioned dosages of adenosine used during the infusion are substantially reduced compared to the dosages of adenosine typically used during major surgery, such as when adenosine is used as an analgesic.
  • the above mentioned dosages of magnesium used during the infusion are substantially reduced compared to the dosages of magnesium typically used during major surgery, such as when magnesium is used during cardiac surgery.
  • a composition comprising adenosine, lidocaine, MgSO 4 2% CPD in 250 ml of 0.9% NaCl is provided.
  • concentration of each of the components in the composition may be as follows;
  • Citric Acid (Monohydrate), 0.327 g
  • the composition may be administered by a bolus to the blood to provide a contact concentration at the heart.
  • a bolus of the composition is diluted up to 1 L of blood to provide the following heart contact concentrations:
  • Table 12 below describes the blood flow rates and ALM-CPD solution sequence used in the treatment of both adult and pediatric patients with ALM-CPD solution.
  • Oxygenated whole blood is provided to the patient at a flow rate as indicated in column 2 of the Table.
  • the whole blood is combined with ALM-CPD solution through a Y-adapter just prior to administration.
  • the Polar Shot is supplied to the Y-adapter by either a Quest MPS system or a syringe pump.
  • a warm solution of ALM-CPD solution is administered for 1 minute at different flow rates for adult and pediatric patients as described in the Table. After the warm solution is administered, a cold solution of ALM-CPD solution is administered for 3 minutes.
  • the contact concentrations for induction, maintenance and reanimation between the two methods of delivery are the same or similar.
  • the data in Table 12 may be changed by the skilled person to suit their own preferences. For example, Instead of warm induction some skilled persons may prefer colder induction temperatures and the range could be between 2 and 32° C. Some skilled persons may also prefer warm thoughout induction and maintenance and higher concentrations of polarshot may be required for maintenance and more frequent intermittent infusions (i.e. every 20 min).
  • ALM-CPD solution solution is provided to the patient to maintain arrest (maintenance).
  • the time interval between administering doses of ALM-CPD solution during maintenance and the amount of ALM-CPD solution administered during maintenance is to be determined between the surgeon and perfusionist, although the Table below provides a guide as to the volume per minute recommended during maintenance.
  • Quest MPS is the Quest MPS2 Myocardial Protection System which is a patented device to delivery cardioplegia to the heart
  • Tetralogy of Fallot is a rare, complex heart defect. It occurs in about 5 out of every 10,000 babies and equal incidence in males and females. Tetralogy of Fallot involves four heart defects:1) ventricular septal defect (VSD), 2) pulmonary stenosis 3) Right ventricular hypertrophy, 4) overriding aorta where the aorta is located between the left and right ventricles, directly over the VSD. As a result, oxygen-poor blood from the right ventricle flows directly into the aorta instead of into the pulmonary artery. Tetrology of Fallot leads to death if not surgically repaired as not enough blood is able to reach the lungs and body.
  • Rectal temperature was monitored using a rectal probe inserted 5 cm from the rectal orifice before, during and following shock and resuscitation, and previous experiments show the temperature ranges between 37 to 34° C.
  • the left femoral vein and artery was cannulated using PE-50 tubing for drug infusions and blood pressure monitoring (UFI 1050 BP coupled to a MacLab) and the right femoral artery was cannulated for bleeding.
  • Lead II electrocardiogram (ECG) leads were implanted subcutaneously on the left and right front legs and grounded to the back leg. Rats were stabilized for 10 minutes prior to blood withdrawal.
  • Hemorrhagic shock was induced by withdrawing blood from the femoral artery at an initial rate of ⁇ 1 ml/min then decreasing to ⁇ 0.4 ml/min over 20 min. Initially blood was withdrawn slowly into a 10 ml heparinized syringe (0.2 ml of 1000 U/ml heparin) to reduce MAP to between 35 and 40 mmHg. If MAP increased, more blood was withdrawn to maintain its low value, and the process was continued over a 20 min period.
  • the amounts of ALM in mg/kg rat are 0.24 mg/kg adenosine, 0.73 mg/kg lidocaine-HCl and 0.27 mg/kg MgSO 4 . After administration of 0.3 ml bolus hemodynamics was monitored over a 60 min period.
  • **ALM is adenosine, lidocaine and magnesium are the identical concentrations in bolus administered in controls and present invention

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