US20160158280A1 - A method for organ arrest, protection and preservation and reducing tissue injury - Google Patents

A method for organ arrest, protection and preservation and reducing tissue injury Download PDF

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US20160158280A1
US20160158280A1 US14/905,722 US201414905722A US2016158280A1 US 20160158280 A1 US20160158280 A1 US 20160158280A1 US 201414905722 A US201414905722 A US 201414905722A US 2016158280 A1 US2016158280 A1 US 2016158280A1
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citrate
composition
blood
adenosine
surgery
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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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    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
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Definitions

  • the invention relates to a method of inducing organ arrest, maintaining arrest and reanimating in a subject that has suffered a life threatening disease or injury.
  • the invention also relates to a method of inducing cardioplegic arrest, maintenance of arrest and reanimation after arrest in a subject that requires elective normal, high-risk or emergency surgery.
  • the invention also relates to a method of inducing cardioplegic arrest, maintenance of arrest and reanimation in a subject that requires elective or emergency organ transplantation.
  • the invention also relates to a method and composition for preparing a donor subject who has been pronounced clinically dead to protect and harvest the body's organs, tissues and cells for transplantation.
  • the invention also relates to a method for storing organs, tissues and cells that have been harvested from a donor in preparation for a recipient.
  • the invention also relates to a method for rewarming and reanimating the organs after storage prior to implantation into the recipient.
  • the invention also relates to a method for implanting the organ, tissue or cell into the recipient.
  • the invention also relates to a method for reducing the harmful effects of anaesthesia, surgery, clinical interventions and cardiopulmonary bypass on injuring the body's tissues, organs and cells before, during and after the operation during recovery.
  • the invention also includes a method for reducing the harmful effects of surgery or clinical intervention on injuring the organs in the whole body, including the brain.
  • the method and composition of the invention provides a new frontline defense system to protect against injury and therefore improve post-surgery and transplant outcomes.
  • the present application claims priority from Australian Provisional Patent Application Nos. 2013902656, 2013902657, 2013902658, 2013902659 and 2013903644, the entire disclosures of which are incorporated into the present specification by this cross-reference.
  • Coronary artery disease is responsible for approximately one-third of the world's population deaths over 35 years of age and many require the need for cardiac surgery.
  • In-hospital/30-day mortality rates are around 1% for CABG, 5-6% for valve, and 7% for combined CABG and valve surgery.
  • Adult females have up to 1.6 times higher in-hospital mortality rates and higher morbidity than their male counterparts.
  • the present invention provides a method of inducing organ arrest, maintaining arrest and reanimating in a subject that has suffered a life threatening disease or 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; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) a citrate to the subject.
  • the method includes the administration of an elevated source of magnesium ions.
  • the citrate may be a form of citrate 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.
  • CPD citrate phosphate dextrose
  • the administration is in a vial or injectable.
  • the method may further include the administration insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent.
  • the general anaesthetic is propofol.
  • the mitochondrial pore inhibitor is cyclosporine A.
  • the myosin ATPase inhibitor is 2,3-Butanedione Monoxime (BDM).
  • the an anti-inflammatory agent is beta-hydroxy-butryate.
  • the antiarrhythmic agent or local anaesthetic is lidocaine.
  • the potassium channel opener, potassium channel agonist and adenosine receptor agonist is adenosine.
  • the present invention provides a composition which may be used for inducing organ arrest, maintaining arrest and reanimating in a subject that has suffered a life threatening disease or injury comprising (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 citrate.
  • the composition includes an elevated source of magnesium ions.
  • the citrate may be a form of citrate 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.
  • CPD citrate phosphate dextrose
  • the composition is administered in a vial or injectable
  • composition may further contain or be administered with insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent.
  • the general anaesthetic is propofol
  • the mitochondrial pore inhibitor is cyclosporine A
  • the myosin ATPase inhibitor is 2,3-Butanedione Monoxime (BDM)
  • the an anti-inflammatory agent is beta-hydroxy-butryate
  • the antiarrhythmic agent is lidocaine.
  • the potassium channel opener, potassium channel agonist and adenosine receptor agonist is adenosine.
  • the present invention also provides a method for preparing, harvesting, storing organs, tissues and cells, comprising exposing the organ, tissues and cells to (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) citrate.
  • the method includes the administration of an elevated source of magnesium ions.
  • the citrate may be a form of citrate 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.
  • CPD citrate phosphate dextrose
  • the administration is in a vial or injectable
  • the method may further include the administration of insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or anti-inflammatory agent.
  • the general anaesthetic is propofol.
  • the mitochondrial pore inhibitor is cyclosporine A.
  • the myosin ATPase inhibitor is 2,3-Butanedione Monoxime (BDM).
  • the an anti-inflammatory agent is beta-hydroxy-butryate.
  • the antiarrhythmic agent or local anaesthetic is lidocaine.
  • the potassium channel opener, potassium channel agonist and adenosine receptor agonist is adenosine.
  • the present invention also provides a composition which may be used for preparing, harvesting, storing organs, tissues and cells comprising exposing the organ to (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 citrate.
  • the composition includes an elevated source of magnesium ions.
  • the citrate may be a form of citrate 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.
  • CPD citrate phosphate dextrose
  • the composition is administered in a vial or injectable
  • composition may further contain or be administered with insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent.
  • the general anaesthetic is propofol.
  • the mitochondrial pore inhibitor is cyclosporine A.
  • the myosin ATPase inhibitor is 2,3-Butanedione Monoxime (BDM).
  • the an anti-inflammatory agent is beta-hydroxy-butryate.
  • the antiarrhythmic agent or local anaesthetic is lidocaine.
  • the potassium channel opener, potassium channel agonist and adenosine receptor agonist is adenosine.
  • the present invention is directed to a method for reducing the harmful effects of at least one of anaesthesia, surgery, clinical intervention and cardiopulmonary bypass on injuring the tissues, organs and cells of a subject 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; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) a citrate to the subject before, during and/or after the anaesthesia, surgery, clinical intervention and cardiopulmonary bypass.
  • the method includes the administration of an elevated source of magnesium ions.
  • the citrate may be a form of citrate 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.
  • CPD citrate phosphate dextrose
  • compositions 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 “composition for use” or other similar terms, although there are a number of compounds embodying the invention which are compositions useful in the invention.
  • the method administration is in a vial or injectable.
  • the method may further include the administration of insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent.
  • the general anaesthetic is propofol.
  • the mitochondrial pore inhibitor is cyclosporine A.
  • the myosin ATPase inhibitor is 2,3-Butanedione Monoxime (BDM).
  • the an anti-inflammatory agent is beta-hydroxy-butryate.
  • the antiarrhythmic agent or local anaesthetic is lidocaine.
  • the potassium channel opener, potassium channel agonist and adenosine receptor agonist is adenosine.
  • the present invention is directed to a composition which may be used for reducing the harmful effects of at least one of anaesthesia, surgery, clinical intervention and cardiopulmonary bypass on injuring the tissues, organs and cells of a subject before, during and and/or after the anaesthesia, surgery, clinical intervention and cardiopulmonary bypass comprising (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 local anaesthetic; and (iii) a citrate.
  • the composition includes an elevated source of magnesium ions.
  • the citrate may be a form of citrate 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.
  • CPD citrate phosphate dextrose
  • the composition is administered in a vial or injectable
  • composition may further contain or be administered with insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent.
  • the general anaesthetic is propofol.
  • the mitochondrial pore inhibitor is cyclosporine A.
  • the myosin ATPase inhibitor is 2,3-Butanedione Monoxime (BDM).
  • the an anti-inflammatory agent is beta-hydroxy-butryate.
  • the antiarrhythmic agent or local anaesthetic is lidocaine.
  • the potassium channel opener, potassium channel agonist and adenosine receptor agonist is adenosine.
  • the present invention is directed to a method of reducing the harmful effects of surgery or clinical intervention on injuring the organs and brain in the whole body 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; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) a citrate to the subject.
  • the method includes the administration of an elevated source of magnesium ions.
  • the citrate may be a form of citrate 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.
  • CPD citrate phosphate dextrose
  • the administration is in a vial or injectable.
  • the method may further include the administration of insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent.
  • the general anaesthetic is propofol.
  • the mitochondrial pore inhibitor is cyclosporine A.
  • the myosin ATPase inhibitor is 2,3-Butanedione Monoxime (BDM).
  • the an anti-inflammatory agent is beta-hydroxy-butryate.
  • the antiarrhythmic agent or local anaesthetic is lidocaine.
  • the potassium channel opener, potassium channel agonist and adenosine receptor agonist is adenosine.
  • the present invention is directed to a composition which may be used for reducing the harmful effects of surgery or clinical intervention on injuring the organs and brain in the whole body of a subject comprising (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 citrate.
  • the composition includes an elevated source of magnesium ions.
  • the citrate may be a form of citrate 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.
  • CPD citrate phosphate dextrose
  • the composition is administered in a vial or injectable.
  • composition may further contain or be administered with insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent.
  • the general anaesthetic is propofol.
  • the mitochondrial pore inhibitor is cyclosporine A.
  • the myosin ATPase inhibitor is 2,3-Butanedione Monoxime (BDM).
  • the anti-inflammatory agent is beta-hydroxy-butryate.
  • the antiarrhythmic agent or local anaesthetic is lidocaine.
  • the potassium channel opener, potassium channel agonist and adenosine receptor agonist is adenosine.
  • the present invention also provides use of (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 citrate in the manufacture of a medicament for inducing organ arrest, maintaining arrest and reanimating in a subject that has suffered a life threatening disease or injury; preparing, harvesting, storing organs, tissues and cells; reducing the harmful effects of at least one of anaesthesia, surgery, clinical intervention and cardiopulmonary bypass on injuring the tissues, organs and cells of a subject; or reducing the harmful effects of surgery or clinical intervention on injuring the organs and brain in the whole body of a subject.
  • the present invention also provides use of (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 citrate for inducing organ arrest, maintaining arrest and reanimating in a subject that has suffered a life threatening disease or injury; preparing, harvesting, storing organs, tissues and cells; reducing the harmful effects of at least one of anaesthesia, surgery, clinical intervention and cardiopulmonary bypass on injuring the body's tissues, organs and cells or reducing the harmful effects of surgery or clinical intervention on injuring the organs and brain in the whole body of a subject.
  • the present invention also provides (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 citrate for use in inducing organ arrest, maintaining arrest and reanimating in a subject that has suffered a life threatening disease or injury; preparing, harvesting, storing organs, tissues and cells; reducing the harmful effects of at least one of anaesthesia, surgery, clinical intervention and cardiopulmonary bypass on injuring the body's tissues, organs and cells or reducing the harmful effects of surgery or clinical intervention on injuring the organs and brain in the whole body of a subject.
  • compositions described above further comprise a pharmaceutically acceptable carrier.
  • compositions described above are pharmaceutical compositions such as cardioplegic or cardiopreservation compositions.
  • the composition may be in a form of a kit in which each of components (i) to (iii) are held separately.
  • the kit may be adapted to ensure simultaneous, sequential or separate administration of components (i) to (iii) when used in the methods described above.
  • compositions include the presence of avoiding active oxygenation in some compositions or administered with some compositions to avoid injury to the organ, tissue, cell outside the body (ex vivo) as will be described in the examples.
  • the present invention may also benefit from including or administering a non-protein oxygen and oxygen carriers that include nanoparticles, polymers and/or polymerosomes carriers to protect the organs, tissues and cells in the whole body (in vivo) as well as outside the body (ex vivo).
  • a non-protein oxygen and oxygen carriers that include nanoparticles, polymers and/or polymerosomes carriers to protect the organs, tissues and cells in the whole body (in vivo) as well as outside the body (ex vivo).
  • the invention relates to a method of inducing organ arrest, maintaining arrest and reanimating in a subject that has suffered a life threatening disease or 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; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) a citrate to the subject.
  • the method includes the administration of an elevated source of magnesium ions.
  • the method may further include the administration of insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent to improve function.
  • the invention also relates to compositions for use in these methods and pharmaceutical preparations suitable for such treatments.
  • the present invention also provides a method for preparing, harvesting, storing organs, tissues and cells comprising exposing the organs, tissues and cells to (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 citrate.
  • the method includes the administration of an elevated source of magnesium ions.
  • the method may further include the administration of insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent to improve function.
  • the invention also relates to compositions for use in these methods and pharmaceutical preparation
  • the present invention is directed to a method for reducing the harmful effects of at least one of anaesthesia, surgery, clinical intervention and cardiopulmonary bypass on injuring the tissues, organs and cells of a subject comprising the administration of (i) a compound selected from a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) a citrate before, during and/or after the at least one of anaesthesia, surgery, clinical intervention and cardiopulmonary bypass.
  • the method includes the administration of an elevated source of magnesium ions.
  • the citrate may be a form of citrate.
  • the method may further include the administration of insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and/or an anti-inflammatory agent to improve function.
  • the invention also relates to compositions for use in these methods and pharmaceutical preparations suitable for such treatments.
  • compositions can be administered directly into the subject or isolated organ, tissue or cell using injection, pump device and/or cardiopulmonary bypass machine.
  • the components of the composition may not be administered in the same syringe, vial or bag but can be mixed immediately prior to administration.
  • compositions can be administered into the subject or isolated organ, tissue or cell after being mixed in desired proportions with blood or crystalloid compositions using injection, pump device and/or cardiopulmonary bypass machine.
  • compositions can be administered directly into the subject or isolated organ, tissue or cell using static-storage, bolus, continuous or intermittent infusions via a pump device or machine.
  • compositions can be administered into the subject or isolated organ, tissue or cell after being mixed in desired proportions with blood or crystalloid compositions using static-storage, bolus, continuous or intermittent infusions via a pump device or machine
  • compositions can be administered directly into the subject or isolated organ, tissue or cell as an oxygenated, partially oxygenated or non-oxygenated solution.
  • compositions can be administered into the subject or isolated organ, tissue or cell after being mixed in desired proportions with blood or crystalloid compositions as an oxygenated, partially oxygenated or non-oxygenated solution.
  • compositions can be administered directly into the subject or isolated organ, tissue or cell as microspheres containing different concentrations of gases.
  • compositions can be administered into the subject or isolated organ, tissue or cell after being mixed in desired proportions with blood or crystalloid compositions as microspheres containing different concentrations of gases.
  • compositions can be administered directly into the subject or isolated organ, tissue or cell as a crystalloid, whole blood or whole blood:crystalloid dilutions (e.g 4 parts blood one part crystalloid).
  • compositions can be administered into the subject or isolated organ, tissue or cell after being mixed in desired proportions as a crystalloid, whole blood or whole blood:crystalloid dilutions (e.g 4 parts blood one part crystalloid).
  • compositions can be administered into the subject or isolated organ, tissue or cell loaded into solid lipid nanospheres for improved delivery and to facilitating crossing the blood brain barrier.
  • compositions can be administered into the subject or isolated organ, tissue or cell at a temperature ranging from 0° C. to 37° C.
  • compositions can be administered into the subject or isolated organ, tissue or cell as a bolus followed by constant infusion or a drip.
  • compositions can be administered into the subject via an intravenous or intraosseous drip.
  • the method can be administered into the subject for low or high-risk elective surgery.
  • the method can be administered into the subject for emergency, life-threatening surgery.
  • the method can be administered into the subject who is an adult.
  • the method can be administered into the subject who is a pediatric patient.
  • the method can be administered into the subject who is a neonatal patient.
  • the method can be administered into the subject that in utero whereby both the unborn and the mother are protected.
  • the method can be administered into the subject who has been pronounced clinically brain dead 5 to 15 min prior to organ procurement.
  • the subject may be an adult, youth, child or baby.
  • the method can be administered into the subject who has been pronounced clinically brain dead for organ harvest.
  • the subject may be an adult, youth, child or baby.
  • the method can be administered into the subject prior to On-Pump or Off-pump cardiac surgery to protect the whole body and organs of the body.
  • the subject may be an adult, youth, child or baby
  • the method can be administered into the subject prior to any surgery to protect the whole body and organs of the body.
  • the subject may be an adult, youth, child or baby.
  • the method can be administered into the subject prior to any surgery to protect the brain.
  • the subject may be an adult, youth, child or baby.
  • the treatment can be used over a wide temperature range with or without cardiopulmonary bypass or other extracorporeal life support device.
  • Hypothermia is believed to be protective for the body, particularly the brain, and is used commonly in major surgery (Fukudome and Alam, 2009; Nolan et al., 2012).
  • Another aspect of the invention includes reducing cell injury from improved metabolism in the heart and other organs of the body including the brain and thereby secondary complications that can lead to poor outcomes and death after cardiac surgery (transplant surgery).
  • Another aspect of the invention is to reduce inflammation (systemic and local) that can influence outcome after cardiac surgery (transplant surgery).
  • Another aspect of the invention is to reduce coagulation disorders (systemic and local) that can influence outcome after cardiac surgery (transplant surgery).
  • Another aspect of the invention is directed to reducing the occurrence of post-surgery infection, preventing an immunosuppressive state, reducing inflammation, correcting coagulation disorders and preventing or decreasing postoperative cognitive decline associated with brain injury after surgery.
  • the composition may act to bring balance to these intricate interactions between the periphery and brain and restore homeostasis.
  • Another aspect of the invention is for reducing blood loss during and after cardiac surgery (transplant surgery).
  • Another aspect of the invention is to reduce infection from ischaemia of the bowel from translocation of enteric bacteria to cause infection after cardiac surgery (transplant surgery).
  • Another aspect of the invention is reducing brain damage during and after cardiac surgery (transplant surgery).
  • Another aspect of the invention is reducing pain after cardiac surgery (transplant surgery).
  • Another aspect of the invention is for reducing renal dysfunction during and after cardiac surgery (transplant surgery)
  • a proposed mechanism of the invention includes a whole body improvement of circulation, improved local and CNS control of blood pressure, improved inflammatory and coagulation states and improved tissue oxygenation with multi-organ protection including the brain. Since the medulla in the brainstem is responsible for breathing, heart rate, blood pressure, arrhythmias and the sleep-wake cycle, part of the mechanism may reside in the composition's action in this region of the brain. The specific area may be the nucleus tractus solitaris (NTS), which is the first nucleus in the medulla that receives and integrates sensory information from cardiovascular and pulmonary signals in the body.
  • NTS nucleus tractus solitaris
  • the NTS receives afferent projections from the arterial baroreceptors, carotid chemoreceptors, volume receptors and cardiopulmonary receptors for processing and makes autonomic adjustments along with higher orders of the brain to maintain arterial blood pressure within a narrow range of variation.
  • This central autonomic network consists of three hierarchically ordered circuits or loops: 1) the short-term brainstem-spinal loops, 2) the limbic brain-hypothalamic-brainstem-spinal cord loops mediating anticipatory and stress responses, and 3) the intermediate length hypothalamic-brainstem-spinal cord loops mediating longer-term autonomic reflexes (e.g. involved in temperature regulation).
  • the paraventricular nucleus is one of the most important hypothalamic nucleus of the central autonomic network.
  • the PVN comprises approximately 21,500 neurones is the “autonomic master controller” and a critical regulator of numerous endocrine and autonomic functions. Regulation of body temperature is also under hypothalamic control of brainstem and spinal autonomic nuclei related to longer-term autonomic reflexes. Activation of sympathetic nervous system is involved in the increase of heat generation and decrease of heat loss: control of thermoregulation muscle tone, shivering, skin blood flow and sweating may be affected.
  • the parvocellular neurons of the PVN are known to be involved in the control of central autonomic outflow. Cholinergic activation of PVN decreases body temperature and cholinergic activation of SON increases body temperature.
  • another possible mechanism of the present invention may be improved heart rate variability, which implicates CNS protection and improved balance of electrical homeostasis. Improvement of heart rate variability during resuscitation from shock also supports the concept of improved CNS function. However, local control of the heart function and blood pressure cannot be ruled out. Acute brain injury results in decreased heart beat oscillations and baroreflex sensitivity indicative of uncoupling of the autonomic and cardiovascular systems. Brain vagal and sympathetic cardiac influences operate on the heart rate in different frequency bands. While vagal regulation has a relatively high cut-off frequency, modulating heart rate both at low and high frequencies, up to 1.0 Hz, sympathetic cardiac control operates only ⁇ 0.15 Hz.
  • heart rate variability The clinical relevance of the information on autonomic cardiac control provided by heart rate variability parameters is supported by the evidence that reduced heart rate variability and baroreflex control of heart rate is associated with increased mortality after myocardial infarction as well as in heart failure patients, and with increased risk of sudden arrhythmic death.
  • the invention may act to bring balance to these intricate interactions between the periphery and brain and restore homeostasis.
  • NO nitric oxide
  • NTS nucleus tractus solitarius
  • compositions, methods of treatment, and methods of manufacturing a medicament for treatment involving a composition which is described as containing (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 citrate.
  • the composition includes an elevated source of magnesium ions.
  • the composition may also include or be administered with at least one of insulin, a general anaesthetic, a mitochondrial pore inhibitor, a myosin ATPase inhibitor and an anti-inflammatory agent to improve function.
  • the invention also relates to compositions for use in these methods and pharmaceutical preparations suitable for such treatments.
  • Arrest is defined as the reversible cessation of activity of the body, organ, tissue or cell. Maintenance of arrest refers to maintaining this cessation of activity. Reanimation refers to restoring its activity after the body, organ, tissue or cell has been arrested.
  • Cardioplegic solution is defined as a solution that temporarily and reversibly stops or arrests the heart from beating usually in diastole.
  • Preservation solution is defined as a solution that temporarily preserves or protects the integrity of the organ, tissue and cell and may include the whole body.
  • preservation solutions have been developed to counteract the detrimental effects of the process of donor organ, tissue cell harvest, storage, cooling, rewarming, reperfusion and implantation into the recipient, and none provide optimal protection.
  • preservation solutions have been designed to specifically target the biochemical and structural changes that occur during this complex process from harvest to reanimation, yet vary considerably in the exact nature and concentrations of their buffers, constituents and/or actives.
  • Cardioplegic versus Preservation Solution These terms are not always easy to distinguish.
  • a cardioplegic solution can be used as a preservation solution and a preservation solution can be used as a cardioplegia.
  • the main reason for this flexibility is because their compositions share high potassium as the arresting agent for the heart and other organs.
  • Custodiol was designed and patented as a preservation solution but it is also used in cardiac surgery as a cardioplegia.
  • Celsior was designed and patented as a preservation solution but it can also be used as a cardioplegia.
  • cardioplegia and preservation have been separated by time with cardioplegia as a method for arrest and protection for shorter times to undertake an operation (1 to 3 hours), and preservation as a method for longer periods (1 to 20 hours) as part of the process between an organ donor and the recipient.
  • a cardioplegia can be used in an operation for up to 11 hours for complex adult or pediatric surgeries, and the current safe time for heart preservation solutions is 4 to 5 hours. Therefore the distinctions between cardioplegia and preservation solutions are often blurred and not always obvious in either composition or use.
  • Crystalloid A substance that in solution can pass through a semipermeable membrane and be crystallized, as distinguished from a colloid.
  • Crystalloid Solutions can be composed of ions, drugs and substrates and other compounds that are beneficial when administered to a tissue, organ or cell in the body (in vivo) (for example, surgery, treatment, clinical intervention or resuscitation) or can be administered outside the body (ex vivo) such as for isolated organs, tissue and cells arrest, protection and preservation.
  • a crystalloid solutions are Krebs Henseleit buffers or modified saline solutions such as Ringers solution, Ringer's Lactate solution, Ringers Acetate solution, Tyrode's salt solution, Hank's buffered salt solution, TRIS-buffered saline or THAM-buffered saline solutions.
  • Crystalloid Cardioplegia is a crystalloid solution for the purpose of arresting, maintaining arrest and reanimating the heart or other organs, tissues or cells.
  • crystalloid cardioplegia examples include the Buckberg Solution, St Thomas Solution, Breshneider's solution and Celsior solution. These crystalloid solutions can be added to blood in different ratios as a cardioplegia such as 8 parts of blood and 1 part of crystalloid or 4 parts of blood and 1 part of crystalloid.
  • the crystalloid composition can also be concentrated in a syringe and added in small amounts for whole blood cardioplegic arrest, maintenance of arrest and reanimation.
  • reversible cell injury can lead to heart dysfunction usually from arrhythmias and/or stunning.
  • Stunning is normally characterised as loss of left pump function during restoration of blood flow following periods of ischaemia. If severe, it can lead to the death of the heart, usually from arrhythmias, even though the heart cells themselves are not initially dead.
  • Irreversible injury by definition arises from actual cell death which may be fatal depending upon the extent of the injury. The amount of cell death can be measured as infarct size. During recovery from cardioplegic arrest, if the conditions are adequate, the heart can be restored substantially to normal function of the tissue by reperfusion, with minimal infarct size.
  • One aspect of the present invention is to reduce injury during and following cardiac surgery (including transplant surgery).
  • Infection Injury and stress associated with cardiac surgery can lead to infections.
  • One aspect of the present invention is to reduce infection during and following cardiac surgery (including transplant surgery).
  • Coronary artery bypass Graft Surgery is a type of surgery that uses conduits or vessels to bypass blockages in the coronary arteries and improves blood flow to the heart as a result of coronary artery disease.
  • the operation can be done with the assistance of cardiopulmonary bypass (on-pump) or without (off-pump). Off-pump is also refereed to as beating heart surgery.
  • cardiopulmonary bypass on-pump
  • the heart is stopped with cardioplegic solution, and is also used to repair or replace heart valves, congenital corrective surgery, aortic reconstructions and many other surgeries.
  • Inter-uterine surgery is surgery performed on the pregnant mother in special cases of life-threatening corrective surgery.
  • On-Pump and Off-Pump Cardiac Surgery has the presence of cardiopulmonary bypass (CPB) assisting the body with oxygenation and support whereas off-pump does not have CPB and the surgery is performed on the beating or moving heart. Both surgeries are pro-inflammatory, alter coagulation and injure many of the organs of the body: Systemic inflammation is a common outcome of cardiac surgery. The inflammatory reaction involving activation of platelets, neutrophils, monocytes, and macrophages with increased blood concentrations of cytokines and leukotrienes. In addition, On-pump and Off-pump surgery causes serious blood clotting abnormalities and coagulopathy: Cardiac surgery promotes bleeding from activation of fibrinolytic pathways.
  • CPB cardiopulmonary bypass
  • Hyperfibrinolysis is a major problem with cardiac surgery and causes blood loss and may require blood products or transfusions.
  • One aspect of the present invention is to arrest the heart and reduce inflammation and correct coagulopathy, and reduce injury during and following surgery with improve post-operative outcomes such as fewer days in hospital and less co-morbidities.
  • High Risk From a practical point of view ‘high-risk’ can probably be defined in two different ways: the first is relevant to an individual and suggests that the risk to a subject that is higher than for a population; the second compares the risk of the procedure in question with the risk of surgical procedures as a whole.
  • a patient or group of patients are at high risk if they carry a number of risk factors after an assessment for a particular operation such as cardiac bypass, valve or corrective surgery that would increase their risk of morbidity and mortality. These patients or group of patients may benefit from high-dependency unit or intensive care unit (ICU) care perioperatively.
  • ICU intensive care unit
  • High-risk patients may have one or more risk factors and include congenital factors, age, redo patients, hypertension, diabetes, overweight, cirrhosis, renal dysfunction, blood disorders, heart failure, and many others. High-risk would include a combination of valve dysfunction, infection and atherosclerosis.
  • a patient requiring aortic reconstruction is high risk. Women are higher risk for complications (mortality and morbidity) than men undergoing all forms of cardiac surgery. Ethnicity is another risk factor. Decreased or depressed heart rate variability is another risk factor and in some cases and independent predictor of death.
  • Diabetes 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.
  • diabetic patients undergoing valve operations have a 5-fold risk of being affected by major lung complications.
  • An emergency patient is one who has not elected or planned to have surgery. Whereas the outcomes from planned elective surgery are well documented, there are less published data describing the results of urgent and emergency surgery where it is generally believed that morbidity and mortality rates are higher. In addition if the emergency patient was also a diabetic or has other risk factors mentioned above this may significantly increase the risk factors of death and post-operative complications or morbidity.
  • Myocardial injury Damage to the myocardium or the heart muscle.
  • One aspect of the present invention is to reduce heart injury and secondary complications following cardiac surgery (including transplant surgery).
  • Brain injury Injury to the brain and includes temporary dementia through to a devastating stroke.
  • One aspect of the present invention is to reduce brain injury including stroke and secondary damage following cardiac surgery (including transplant surgery).
  • Renal Dysfunction Depending upon the type of cardiac surgery, 4 to 40% of patients will have some form of renal dysfunction.
  • One aspect of the present invention is to reduce renal injury and dysfunction following cardiac surgery (including transplant surgery).
  • Shock is defined as a severe hypotensive state when the arterial blood pressure is too low to maintain an adequate supply of blood and oxygen to the body's cells, organs and tissues. Shock is the result of “circulatory collapse” which can be caused from many internal and external sources during or following cardiac or transplant surgery. Shock can be caused by severe allergic reaction or injury (traumatic or non-traumatic) such as brain injury and bleeding.
  • One aspect of the present invention is to reduce the incidence of shock from reducing blood loss during and following cardiac surgery (including transplant surgery).
  • Cardiogenic Shock is a state of end-organ hypoperfusion including brain damage due to cardiac failure. It occurs in 5% to 8% of patients hospitalized with ST-elevation myocardial infarction. Mortality can range from 10% to 80% depending on demographic, clinical, and hemodynamic factors. These factors include age, clinical signs of peripheral hypoperfusion and anoxic brain damage.
  • Haemorrhagic shock can occur after cardiac surgery from blood loss and lead to shock. Brain injury can occur from injury and trauma is often complicated by hemorrhagic shock (HS) and visa versa. Valproic acid (VPA), a histone deacetylase inhibitor, can improve survival after hemorrhagic shock (HS), protect neurons from hypoxia-induced apoptosis, and attenuate the inflammatory response (Jin et al., 2012).
  • One aspect of the present invention is to reduce the incidence of haemorrhagic shock during and following cardiac surgery (including transplant surgery).
  • Sepsis and septic shock The occurrence of sepsis after cardiac surgery is a rare event; however, its occurrence has 80% mortality.
  • One aspect of the present invention is to reduce the infection following cardiac surgery (including transplant surgery).
  • Obstructive Shock is due to obstruction of blood flow outside of the heart. Pulmonary embolism and cardiac tamponade are examples of obstructive shock. Similar to cardiogenic shock.
  • Vasogenic Shock Shock resulting from peripheral vascular dilation produced by factors such as toxins that directly affect the blood pressure to fall; and include anaphylactic shock (allergic reaction) and septic shock (bacterial, viral or fungal).
  • Neurogenic shock is a hypotension that is attributed to the disruption of the autonomic pathways within the spinal cord. Hypotension can lead to brain injury or result from brain, spinal cord or cervical injury.
  • Burn Shock is defined as tissue damage caused by a variety of agents, such as heat, chemicals, electricity, sunlight, or nuclear radiation.
  • the injury a 3-dimensional mass of damaged tissue and can produce massive inflammatory response and coagulopathy and can lead to shock and organ failure including brain damage.
  • Diabetic Shock is a state of hypoglycemia progression and can lead to fainting and eventually to a coma and deaths.
  • Gastrointestinal (GI) complications reported after cardiac surgery occurs in fewer than 5% of cases. However, they are serious events that substantially increase morbidity and may be associated with mortality as high as 67%. Gastrointestinal hemorrhage occurs in 25% of cases, and hyperbilirubinemia in 65% of cases. Mesenteric ischaemia occurs in 14% of cases. Because the hepato-splanchnic circulation consumes 30% of the cardiac output and contains 25% of the total circulating blood volume, improving blood supply to the gut could reduce ischemia, and damage to all GI organs including liver, gut and pancreas.
  • MPs are membrane-derived nano-fragments (0.05 to 1 ⁇ m) that that are shed from virtually all cells in response to stress and include platelets, monocytes, endothelial cells, red blood cells, and granulocytes. Three decades ago they were called “platelet dust”. MPs exist at low concentrations under normal conditions and increased under stress through a membrane reorganization and blebbing processes following cell activation or apoptosis. In a stressed state MPs constitute a storage pool of bioactive effectors that are able to act as intercellular messengers. Microparticles are important as vectors for homeostatic communication between cells (transfer of receptors, organelles and deliver mRNA between cells).
  • microparticles are procoagulant and proinflammatory and can result in organ, tissue and cell injury including linked to atrial fibrillation.
  • MPs can be beneficial in early sepsis then induce deleterious changes and contribute to multi-organ failure. They also are known to be vasodilatory by relaxing vascular smooth muscle.
  • a sustained generation MPs in the plasma or CSF during stress could contribute to a poor clinical outcome during and following surgery. Inhibition of “detrimental” MPs may help to modulate coagulation, inflammation, endothelial function, and permeability in organs, tissues and cells.
  • Mitochondrial permeability transition pore opening plays a critical role in mediating the mitochondrial response to stress and injury and initiation of apoptosis.
  • the relevance of the permeability transition relates to the ‘life or death’ decision of the cell.
  • Two possible protectors of the mitochondrial pore opening and collapse of the mitochondrial membrane potential is cyclosporine A (CsA) and melatonin ((N-acetyl-5-methoxytryptamine)) and erythropoietin has also been shown to keep this pore closed.
  • mitochondrial pore protectors include N-methyl-4-isoleucine cyclosporine (NIM811), 2-aminoethoxydiphenyl borate (2-APB) and alisporivir and sildenafil-citrate.
  • NIM811 N-methyl-4-isoleucine cyclosporine
  • 2-APB 2-aminoethoxydiphenyl borate
  • alisporivir and sildenafil-citrate alisporivir and sildenafil-citrate.
  • P i can either be an important mPTP sensitizer, probably acting by decreasing matrix-free Mg 2+ and/or by the formation of polyphosphates or play a role in desensitizing the mPTP to Ca 2+ (in the presence of CsA, or under circumstances of CyP-D ablation).
  • New approaches aimed at downstream targets such as GSK-3 or the mPTP to bypass potentially defective upstream signalling components might be protective.
  • Ischaemia is defined as mismatch between supply and demand normally as a result of reduced blood flow to the body, organ, tissue or cell or an increased demand from the causing the mismatch of flow to demand. Ischaemia is a flow limitation.
  • Reperfusion is defined as the process of re-establishing blood flow to a whole body, organ, tissue or cell that has had reduced blood flow relative to its demand for a period of time.
  • the reduced blood flow relative to demand is called ischemia (as above).
  • IR injury is a significant contributor to mortality and morbidity in cardiac surgery patients, with females being more susceptible than males. Consequences of IR injury are exacerbated by physiological responses to cardiopulmonary bypass and separately by surgical trauma caused by release of pro-inflammatory mediators. IR injury is also found in off-pump cardiac surgery, percutaneous coronary interventions and in any general surgery. It may be more accurate to redefine reperfusion injury as “post-surgery” injury prevalent in adult and pediatric cardiac surgery. Injury is classified as reversible or irreversible. Reversible injury includes arrhythmias, edema, vascular dysfunction, and contractile stunning expressed as low output syndrome requiring inotropic or mechanical support to maintain acceptable haemodynamics.
  • Irreversible cell death occurs in areas of IP injury Irreversible reperfusion injury includes necrosis and apoptosis. Necrosis involves disruption or disintegration of the cell membrane, and the release of cell contents and large proteins that are used as biomarkers indicative of morphological injury, e.g. creatine kinase (CK, CK-MB) or cardiac troponins (T or I subunits). Peri-operative cell death is substantiated by the release of these biomarkers into the plasma predominantly postoperatively has been reported during cardiac surgery requiring cardiopulmonary bypass and electrochemical arrest.
  • CK creatine kinase
  • T or I subunits cardiac troponins
  • Apoptosis is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Studies of apoptosis have implied that a common metabolic pathway leading to apoptosis can be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation, and infection.
  • 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 cardiopulmonary bypass, as well as in neonatal necrotizing enterocolitis or persistent ductus arteriosus. Ischaemia can lead to inflammation, infection, multiple organ dysfunction and death.
  • Heart Rate Variability is a phenomenon of generation of consecutive heart rate impulses in the different succession via the SA node and believed to have central nervous system control. Altered cardiac autonomic control may play a role in the morbidity and mortality suffered by adults, pediatrics or neonates who undergo surgery for heart disease. Decreased HR variability (loss of control) occurs following surgery and can be due to surgical injury, inflammation and coagulation and infection. Heart rate variability is powerful independent predictor of a poor outcome.
  • One aspect of the present invention is to improve heart rate variability (increased control) and improved neuroautonomic regulation of heart rate and blood pressure oscillations during and following surgery and reduced post-operative complications and length of stay in hospital. Another aspect of the invention is to lessen the decreased heart rate variability after receiving a donor heart, and improve autonomic “reinnervation”.
  • Hypoxia is an oxygen limitation that may be caused by ischaemia or it could be caused by low environmental oxygen such as at high altitude. Blood flow could be maximum at high altitude yet hypoxic damage is caused from an oxygen limitation not a flow limitation.
  • Stunning is the partial loss of organ or tissue function following reperfusion of an organ after ischaemia.
  • the difference between stunning and infarction is that stunning has no apparent cell death just temporary loss of function.
  • Heart stunning leads to low ventricular outputs and may need pharmacological or ventricular assist devices until normal function is restored.
  • Haemodialysis can also induce myocardial stunning in patients with chronic kidney disease.
  • Cold static storage is defined as the storage of an organ, tissue or cell in a cold non-perfused or static environment comprising an ionic composition to reduce injury from cold, hypoxia, ischaemia and reperfusion.
  • Static cold storage is the most prevalent method for organ preservation. The goal is to store the organ, tissue or cell.
  • Continuous perfusion is defined as the uninterrupted flow of either cardioplegia or preservation to arrest, protect and preserve an organ, tissue or cell as opposed to static storage where the organ, tissue or cell is bathed in a solution that is not flowing.
  • Constant infusion requires special equipment for pulsatile perfusion of the organ, controlling flow, temperature and/or oxygenation and can be a simple perfusion box or sophisticated machine apparatus.
  • the advantage of pulsatile perfusion is that it allows for dynamic monitoring of perfusate flow and calculation of vascular resistance, information which, when suboptimal, sometimes leads to donor organ discard. Usually oxygenated solutions are recommended.
  • Hypothermic machine perfusion is a specially designed apparatus that perfused the organ, tissue or cell at cold temperatures.
  • the clinical evidence for the superiority of machine preservation over static cold storage remains uncertain.
  • Normothermic or tepid machine perfusion is a specially designed apparatus that perfused the organ, tissue or cell at warmer temperatures.
  • the clinical evidence for the superiority of warm machine preservation over static cold storage remains uncertain.
  • DCD Donation after circulatory death
  • DCD describes the retrieval of organs for the purposes of transplantation that follows death confirmed using circulatory criteria. The persisting shortfall in the availability of organs for transplantation has prompted many countries to re-introduce DCD schemes not only for kidney retrieval but increasingly for other organs with a lower tolerance for warm ischaemia such as the liver, pancreas, and lungs and heart. DCD contrasts in many important respects to the current standard model for deceased donation, namely donation after brain death (Manara et al., 2012).
  • DBD Brain Dead Donor
  • DGF Delayed Graft Function
  • PGD Primary Graft Dysfunction
  • SIRS systemic inflammatory response syndrome
  • PGF Primary graft failure
  • GME Gaseous microemboli
  • the Emboli Detection and Classification (EDAC) Quantifier has been able to detect and track microemboli in CPB circuits up to 1,000 microemboli per second at flow rates ranging from 0.2 L/min to 6.0 L/min.
  • the deleterious effects of GME are multiple, including damage to the cerebral vascular endothelium, disruption of the blood-brain barrier, complement activation, leukocyte aggregation, increased platelet adherence, and fibrin deposition in the micro-vasculature.
  • Platlelet debris occurs from interactions with the bypass tubing the plasticizer coating of bypass tubing can also act as a toxin.
  • One aspect of the present invention is to reduce tissue injury caused from emboli (gas and debris) in cardiac surgery and dialysis.
  • Gas embolism occurs when a gas, typically air, enters the vasculature. This can occur during a surgical procedure or as a result of a decompression event and the consequences of gas embolism depend on the size of the gas bubbles and their rate of delivery. Bubbles can cause damage in the microcirculation of any organ, obstruct blood vessels, or even air-lock the heart. Despite greater awareness and improved practice, gas microembolism continues to be a serious risk associated with surgical procedures, particularly those involving cardiopulmonary bypass (CPB), and likely contributes to the incidence of cognitive deficit following such surgeries. In addition, the intravascular formation of gaseous microemboli is known to cause decompression illness.
  • CPB cardiopulmonary bypass
  • Decompression Sickness is caused by bubbles in blood or tissue during or after a reduction in environmental pressure (decompression).
  • the bubbles may disrupt cells and cause a loss of function. They may act as emboli and block circulation, as well as cause mechanical compression and stretching of the blood vessels and nerves.
  • the blood-bubble interface may act as a foreign surface, activating the early phases of blood coagulation and the release of vasoactive substances from the cells lining the blood vessels.
  • Surfactants are agents that reduce water tension and at the air bubble-blood interface (microbubbles). Surface tension can be lowered by surfactants at nanomolar concentrations or less.
  • Surfactants can be water-soluble such as biocompatible Pluronic F-68 or nonionic surfactants, which do not ionize in aqueous solutions because their hydrophilic group is of a non-dissociable. Examples on non-ionic surfactants include ethoxylated aliphatic alcohols, polyoxyethylene surfactants and carboxylic esters. Polysorbate 20, 40, 60 and 80 or Tween 20, 40, 60 and 80 are popular non-ionic surfactants. Sometimes these are called lipid nanoparticles. Pluronic F-68 can also prevent accumulation of fat emboli resulting from prolonged cardiopulmonary bypass.
  • One aspect of the present invention is the addition of the composition in the presence of a surfactant to reduce air-bubble blood tension and improve functional outcomes.
  • Carrier The term “carrier” is used herein to describe the use of a delivery vehicle to incorporate a pharmaceutically active agent for the purposes of drug or bioactive delivery.
  • Drug delivery system to a tissue, organ or cell can be: (i) a chemical delivery systems such as lipid-mediated transport; (ii) biological delivery systems, in which pharmaceuticals are re-engineered to cross the cell membrane of a tissue, organ or cell via specific endogenous transporters localized within the capillary endothelium; (iii) disruption of the membrane, for example by modification of tight junctions, which causes a controlled and transient increase in the permeability of capillaries; (iv) the use of molecular Trojan horses, such as peptidomimetic monoclonal antibodies to transport large molecules (e.g. antibodies, recombinant proteins, nonviral gene medicines or RNA interference drugs) and (v) particulate drug carrier systems.
  • a chemical delivery systems such as lipid-mediated transport
  • biological delivery systems in which pharmaceuticals are re-engineered to cross the cell membrane of a tissue, organ or cell via specific endogenous transporters localized within the capillary endothelium
  • disruption of the membrane for example
  • various drug delivery systems e.g. liposomes, microspheres, nanoparticles, nanogels and bionanocapsules
  • microchips and biodegradable polymers have become important in brain tumour therapy. They can be administered intravenous, intracardiac, intraperitnoeal, intraarterial, intramuscular, intraocular, oral, intranasal drug delivery, convection-enhanced diffusion and intrathecal/intraventricular drug delivery systems in a number of media, gels, solutions, bloods, tablets and delivered via patches.
  • the challenge is to develop drug delivery strategies that will allow the passage of boactives through the skin or cell membrane and into the cell in a safe and effective manner.
  • Nanoparticles for drug or gas delivery Many promising pharmaceutical agents have been developed but very few of them ( ⁇ 5%) can be used to treat the organs, tissues and cells.
  • the central nervous system is particularly difficult to access.
  • Drugs, bioactives, vaccines, DNA, RNA, or gases oxygen, carbon dioxide, hydrogen sulphide, nitrogen and others
  • the surface modifications of colloidal delivery systems e.g., with poly(ethylene glycol) may allow access into tissues like the brain and prolong time in the blood circulation for improved efficacy.
  • Nanoparticles are solid colloidal particles ranging in size from 1 to 1000 nm ( ⁇ 1 microm) and are composed of macromolecular material. They can be polymeric (‘smart’ or water soluble polymers) or lipidic nanoparticles.
  • the lipid nanoparticles include: 1) solid lipid nanoparticles 2) nanostructured lipid carriers (NLCs) and/or lipid drug conjugates.
  • Lipid nanoparticles have been shown to be effective drug carries for different drugs such as insulin, sildenafil citrate, amphotericin B and methotrexate and many others.
  • the most common nanoparticles are solid lipid nanoparticles (SLN) meaning they are in solid state at room or body temperature, and these can be coated at surfaces or fabricated with a targeting moiety (bioactive, drug, ion, metabolite, gases), so as to gain access into a tissue.
  • Nanoparticles of about 200 nm in diameter are able to cross the blood brain barrier (BBB) after intravenous administration and can act as drug, gas or bioactive carriers for central nervous system.
  • BBB blood brain barrier
  • SLN's are lipidic in nature they more readily be taken up than ‘non-smart’ polymeric nanoparticles and “loaded with bioactives or gases” may protect the ex vivo organ, tissue or cell during harvest, storage or reperfusion or protect the in vivo organ, tissue or cell within the whole body before or following trauma, disease, infection or during cardiac surgery.
  • the BBB comprises a barrier of complex tight junctions between the endothelial cells of the brain capillaries and serves as a major obstacle for the entry of hydrophilic drugs and the efflux pumps present on its surface restrain the intracellular accumulation of pharmacological moieties in the brain.
  • Solid lipid nanoparticles are also less toxic than ‘non-smart’ polymeric nanoparticles for tissue drug delivery because of their lipidic nature, they have a higher drug loading capacity, and are better suited for large scale production. Smart polmerics could also be used instead of SLN's.
  • One aspect of the present invention is the loading of the composition in a solid lipid nanoparticle to improve functional outcomes.
  • Another aspect of the present invention is using these drug loaded solid lipid nanoparticles target the brain for diagnostics and to reduce injury, reduce infection, inflammation and coagulopathy.
  • DHCA Deep Hypothermic Circulatory Arrest
  • DHCA is a method that involves stopping the heart and cooling the body of the patient and stopping blood circulation.
  • brain injury occurs after around 4 min of circulatory arrest. Cerebral metabolism decreases by 6-7% for every 1° C. decrease in temperature from 37° C.; therefore, brain cooling results in a reduction in oxygen requirements and protection.
  • Circulatory arrest is typically undertaken at 18-20° C. and a range of safe periods for DHCA have been reported at this temperature.
  • DHCA can necessitate prolonged CPB with the associated problems of coagulopathy and cerebral microembolism (see Therapeutic hypothermia).
  • One aspect of the present invention is the use the method and composition to arrest the heart and protect the organs including the brain during DHCA.
  • Therapeutic Hypothermia is defined as “targeted hypothermia” or the active “controlled” cooling of a cell, organ or whole body to reduce injury.
  • the method has clinical applications for arrest, protection and preservation of the brain and heart during cardiac surgery, and has shown to be useful after cardiac arrest or treating an unconscious or coma patient in the out-of-hospital environment.
  • the rate and degree of cooling and targeted body temperature is controversial.
  • Profound hypothermia is associated with dysrhythmias (due to loss of potassium), coagulation disturbances, increased plasma viscosity and erythrocyte rigidity, vasoconstriction and microcirculatory damage, metabolic acidosis, hyperglycaemia, and altered drug distribution and elimination.
  • One aspect of the present invention to employ deep hypothermia as a method for protection and improve functional outcomes from cardiac surgery (and transplant surgery).
  • the Krebs cycle also called the citric acid cycle, is a fundamental metabolic pathway in the mitochondria involving eight enzymes essential for energy production through aerobic metabolism.
  • the intermediates include citrate, aconitate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, oxaloacetate, and back to acetyl CoA before another cycle.
  • This cycle involving the intermediates is also an important source of biosynthetic building blocks used in gluconeogenesis, amino acid biosynthesis, and fatty acid biosynthesis.
  • Alpha-ketoglutarate is an important biological compound and can form from glutamate.
  • Krebs' cycle intermediates provide an entry pathway for other metabolites into the cycle and are involved in a variety of important biological actions.
  • One aspect of the present invention is to include a form of citrate in the composition as citrate is a pivotal entry point to the Krebs' cycle and mitochondrial energy functions.
  • Other Krebs Cycle intermediates such as aconitate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, oxaloacetate (or substrates that form Krebs cycle intermediates such as glutamate to form alpha-keto glutarate or aspartate to form fumarate) could be included in the composition but a form of citrate is preferred because it is pivotal to the operation of the cycle.
  • Citrate-phosphate-dextrose (CPD) solutions are used as anti-coagulant solutions intended for a ex vivo single whole blood collection of 500 mL ⁇ 10% CPD solution.
  • CPD solutions There are two forms of CPD solutions one with twice as much dextrose called CP2D where each 100 mL contains: Citric Acid (Monohydrate), USP 0.327 g Sodium Citrate (Dihydrate), USP 2.630 g, Monobasic Sodium Phosphate (Monohydrate), USP 0.222 g Dextrose (Anhydrous), USP 4.640 grams per 100 ml.
  • citrate-phosphate-dextrose has been used as a source of citrate to reduce plasma levels of ionized calcium with the goal to reduce entry into the cell and reduce intracellular calcium overload and cell injury.
  • citrate-phosphate-dextrose solutions as pharmacologic chelators in cardioplegia are controversial as some investigators report a benefit, others report no benefit while others report deleterious effects.
  • One preferred aspect of the present invention is a composition that includes or is administered with CPD for arrest, protection and preservation during and after cardiac surgery (and transplant surgery).
  • Acid Citrate Dextrose Solution (sometimes called Anticoagulant Citrate Dextrose Solution) is a solution of citric acid, sodium citrate and dextrose in water. Two different CPD solutions are available (Solution A and B) as defined by the US Pharmacopeia.
  • Sildenafil citrate is a phosphodiesterase-5 (PDE5) inhibitor that was originally discovered in the search for a novel treatment for chest pain or angina and then it was found to be useful to relax smooth muscle. Sildenafil citrate or analogues lead to vasodilation and increased inflow of blood into the spongy tissue of the penis, causing an erection. Sildenafil citrate also mimics the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.
  • PDE5 phosphodiesterase-5
  • Sildenafil citrate is also believed to prevent the mitochondrial pore from opening via cGMP-dependent protein kinase 1 (PKG1) reducing Ca 2+ overload.
  • cGMP-dependent protein kinase 1 (PKG1) also reduce intracellular Ca 2+ through inhibition of various sarcolemmal Ca 2+ channels.
  • PDE5 inhibitors are tadalafil and vardenafil.
  • Sildenafil and other PDE5s are also known to counteract insulin resistance (IR) in animals and humans.
  • PDE5 inhibitors may also improve systemic endothelial function and protect the myocardium from ischaemia-reperfusion injury.
  • Insulin Hyperglycemia during cardiac surgery is liked to higher mortality. Since its discovery in the 1920s, insulin has been used as an essential therapeutic agent in diabetes for blood glucose management. Hyperglycemia during cardiac surgery can be prevented with insulin. Insulin is also cardioprotective from inhibition of superoxide and peroxynitrite formation in the ischemic myocardium and is known to increase physiological NO production (via eNOS phosphorylation) and thereby increases coronary perfusion. Insulin has other cardioprotective properties such as facilitating myocardial glucose uptake during cold exposure. Insulin also activates ATP citrate lyase, which is the primary enzyme responsible for the synthesis of acetylCoA in the cell cytosol, which can be used for biosynthesis of fatty acids.
  • One aspect of the present invention may include insulin in the composition to regulate blood glucose of the subject for arrest, protection and preservation during and after cardiac surgery (and transplant surgery) or for its in vivo and ex vivo organ, tissue and cellular protective properties.
  • Melatonin N-acetyl-5-methoxytryptamine
  • NO nitric oxide
  • PKC protein kinase C
  • Melatonin-induced cardioprotection is associated with activation of protein kinase B (PKB), extracellular signal-regulated kinase (ERK1/2) (the Reperfusion Injury Salvage Kinase (RISK) pathway) and signal activator and transducer 3 (STAT-3) (the Survivor Activating Factor Enhancement (SAFE) pathway) during reperfusion and inhibition of the mitochondrial permeability transition pore (MPTP).
  • PPKB protein kinase B
  • ERK1/2 extracellular signal-regulated kinase
  • RISK Reperfusion Injury Salvage Kinase
  • STAT-3 signal activator and transducer 3
  • SAFE Survivor Activating Factor Enhancement
  • BDM 2,3-Butanedione Monoxime
  • BDM is a nucleophilic agent, which reduces energy demand by reversibly uncoupling of contractility via affecting both calcium availability and responsiveness of the myofilaments to Ca 2+ .
  • BDM is the well-characterized, low-affinity, non-competitive inhibitor of skeletal muscle myosin-II and also inhibits nonmuscle myosin.
  • BDM is also a vasodilating agent and reduces reperfusion arrhythmias.
  • BDM regulates the Ca 2+ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca 2+ and tissue-dependent manner.
  • Supplementation with 2,3-butanedione monoxime may have clinical utility in improving myocardial contractile function after hypothermic cardioplegic arrest and preservation.
  • One aspect of the present composition may include or administer BDM to reduce demand of the heart and donor organs for protection against injury.
  • Percutaneous aortic valve replacement or Transcatheter Aortic Valve Implantation is a cardiac procedure for a replacement of the aortic valve which is passed through a hole in the groin by a puncture of the femoral artery and advanced up to the ascending aorta of the patient. It substitutes for a more invasive procedure in which the chest is opened. The survival is equivalent, but the risk of stroke is higher.
  • Therapeutic Hypothermia or “targeted hypothermia” is the active “controlled” cooling of a cell, organ or whole body to reduce injury (Tisherman, 2004). It has clinical applications for arrest, protection and preservation of the brain and heart during cardiac surgery, and has shown to be useful after cardiac arrest or treating an unconscious or coma patient in the out-of-hospital environment. The rate and degree of cooling and targeted body temperature is controversial. Deep Hypothermic Circulatory Arrest (DHCA) or hypothermic cardiac standstill is a surgical technique that involves cooling the body of the patient and stopping blood circulation. Mild hypothermia is a core body temperature of 33 to 36° C., moderate is 28 to 32° C., severe is 25 to 28 and deep hypothermia is 20 to 25° C. or below. Extreme therapeutic hypothermia would be below 10° C.
  • 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.
  • 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
  • hyperglycemia and hypoglycemia need to be avoided to prevent aggravation of underlying brain damage.
  • Both hyper- and hypoglycemia have been associated with poor outcome in traumatic brain injury (TBI).
  • Stress insulin resistance high blood glucose is a marker for mortality in traumatic brain injury.
  • the present composition may include or be administered with insulin to regulate glucose levels and reduce tissue and whole body injury.
  • Alternative energy intermediates or substrates that can bypass glucose as a fuel include ketones (acetone or acetoacetate) or carboxylic acids (D-beta-hydroxybutryate).
  • D-beta-hydroxybutryate was reported to suppress lactic acidemia and hyperglycemia via alleviation of glycolysis during hemorrhagic shock in rats.
  • D-beta-hydroxybutryate is converted to acetyl-CoA through pathways separate than glycolysis before entering the Krebs Cycle and preferential utilization of D-beta-hydroxybutryate rather than glucose as an energy substrate might reduce the deleterious accumulation of rising glucose or maintain a normoglycemic state.
  • Ketones have been successfully applied to both rapidly developing pathologies (seizures, glutamate excitotoxicity, hypoxia/ischaemia) and neurodegenerative conditions (Parkinson's disease, Alzheimer's disease) and more recently TBI.
  • the brain's ability to increase its reliance on ketone bodies appears to be a form of cerebral metabolic adaptation. Cerebral shifting to ketone metabolism requires (1) increasing the availability of ketones, (2) increasing cerebral uptake of ketones, and (3) potentially increasing the activity of the necessary enzymes for ketone metabolism.
  • Acetyl CoA the main substrate that fuels the Krebs cycle to replenish ATP in the cell's powerhouse, the mitochondria.
  • 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 as a Krebs cycle intermediate. Citrate administration may also bypass glucose requirement during insulin resistance and improve outcome. Sildenafil citrate has also been shown to counteract insulin resistance (IR) in animals and humans.
  • the rationale for the inclusion of magnesium in cardioplegic solutions is fivefold: (i) for its negative inotropic effect; (ii) to prevent ischaemia-induced magnesium loss; (iii) to influence cellular ionic movements including act as a calcium blocker, (iv) to reduce arrhythmias, and (v) improves blood flow to the heart and vasospasm.
  • Preservation temperature alters the effects of magnesium. Results obtained from animal models suggest that elevated magnesium (16 mM) is beneficial to the hypothermic preservation of hearts with extracellular type solutions, especially when calcium is elevated in the solution formulation.
  • Elevation of extracellular Mg has also been shown to reduce the intracellular sodium ion activity and this decline in [Na]i can be related to the negative inotropic properties of Mg.
  • Mg is also important in control of arterial tone and blood pressure via pressure via regulation of vascular membrane Mg 2+ -Ca 2+ exchange sites.
  • the arterial blood pressure elevation appears to be inversely related to the level of ionized intracellular and plasma Mg 2+ .
  • MgSO4 Infusion of MgSO4 into the brain via the internal carotid artery has been reported to alleviate cerebrovasospasms and produces dose-dependent lowering of systolic and diastolic blood pressure as well as dose-dependent vasodilatation of arterioles (17-30 micron) and venules (18-40 micron) in the cerebral microcirculation.
  • 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, 20 mM to 400 mM, 20 mM or 400 mM.
  • Magnesium sulphate and magnesium chloride are a suitable source, 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; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) a citrate 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.
  • potassium is present in the composition it will typically be present in an amount to ensure that the blood concentration in the subject is at physiological levels such as less than 10 mM or 3 mM 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 to pediatric 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-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofuranosyl]-adenine (AB-MECA), ([IS-[1 a,2b,3b,4a(S*)]]-4-[7-[[2 ⁇
  • A1 receptor agonists such as N-[3-(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), or partial agonists such as CVT-2759 and allosteric enhancers such as PD81723.
  • Other agonists 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 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
  • 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 the group consisting of: nicorandil, diazoxide, minoxidil, pinacidil, aprikalim, cromokulim and derivative U-89232, P-1075 (a selective plasma membrane KATP channel opener), emakalim, YM-934, (+)-7,8-dihydro-6,6-dimethyi-7-hydroxy-8-(2-oxo-1-piperidinyl)-6H-pyrano[2,3-1] benz-2,1,3-oxadiazole (NIP-121), R0316930, RWJ29009, SDZPC0400, rimakalim, symakalim, YM099, 2-(7,8-dihydro-6,6-dimethyl-6H-[1,4]oxazino[2,3-f][2,1,3]benzoxadiazol-8-yl) pyridine N-oxide, 9-(3-cyanophenyl)-3,4,6,
  • 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
  • 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 ischaemia 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 ischaemia/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 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 or agonist in the composition may be from 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 or agonist 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 a Krebs Cycle metabolic intermediate.
  • the citrate include a form of a citrate 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
  • Citrate comes in different forms such as citric acid, salts of citrate (Na + , Mg 2+ , K + salts), esters of citrate (triethyl citrate), polyatomic anions of citrate (ammonium citrate) or other ionic or drug complexes of citrate (e.g. sildenafil citrate).
  • citrate can be used as a component in buffer solutions such as sodium citrate, citric acid with sodium chloride to maintain a neutral 7.0 pH.
  • Other buffers may use a mixture of sodium citrate and citric acid for buffer pH between 3.0 and 6.2.
  • Citrate can be in powder or liquid form in a reaction tube for collecting blood or other fluids for coagulation tests (3.6% citrate), as well as in blood transfusion bags. Citrate is also part of Citrate Phosphate Dextrose (CPD) Solution (see below).
  • CPD Citrate Phosphate Dextrose
  • Citrate is also a mild chelator of divalent ions such as calcium but it is not as strong as EDTA.
  • a number of studies have shown the use of citrate to chelate Ca 2+ in cardioplegia is detrimental for the perfused isolated working rat heart, especially with high Mg 2+ . Notably, these workers were unable to demonstrate a protective effect of citrate's ability to lower Ca 2+ in crystalloid cardioplegia and reported a significant loss of functional recovery.
  • Citrate is also an intermediate in the Krebs cycle. Citrate can also be transported out of the mitochondria and into the cytoplasm, and broken down into acetyl CoA for free fatty acid (FFA) synthesis.
  • FFA free fatty acid
  • citrate allosterically regulates the enzyme acetyl CoA carboxylase, which catalyzes acetyl-CoA into Malonyl CoA, the first commitment step in fatty acid synthesis.
  • High concentrations of cytosolic citrate can inhibit glycolysis (PFK-1).
  • 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 citrate; (iv) a Krebs Cycle metabolic intermediate; and (v) an anti-inflammatory agent.
  • a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist e.g., an antiarrhythmic agent or a local anaesthetic
  • a citrate e.g., a citrate
  • iv a Krebs Cycle metabolic intermediate
  • an anti-inflammatory agent e.g., a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist
  • an antiarrhythmic agent or a local anaesthetic e.g., a citrate, iii
  • 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.
  • 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 lodicaine-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; (ii) an antiarrhythmic agent or local anaesthetic; and (iii) a citrate.
  • a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist e.g., a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist
  • an antiarrhythmic agent or local anaesthetic e.g., a citrate
  • the composition includes an elevated source of magnesium ions.
  • the antiarrhythmic agent is a local anaesthetic such as lidocaine.
  • 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. In one embodiment, the concentration of antiarrythmic agent or local anaesthetic in the composition is about 37 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.
  • Valproic acid is also a suitable anti-inflammatory agent.
  • VPA is a short-chain branched fatty acid with anti-inflammatory neuro-protective and exon-remodelling effects.
  • Valproic acid 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 ischaemia-reperfusion injury and reduce injury to the endothelium through the TGF- ⁇ and VEGF functional pathways.
  • SIP Sphingosine-1-phosphate
  • 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; (iii) a citrate; and (iv) an anti-inflammatory agent.
  • a compound selected from at least one of a potassium channel opener, a potassium channel agonist and an adenosine receptor agonist e.g., an antiarrhythmic agent or a local anaesthetic
  • a citrate e.g., a citrate
  • an anti-inflammatory agent e.g., an anti-inflammatory agent that is administered to the composition.
  • the composition includes an elevated source of magnesium ions.
  • the anti-inflammatory agent is beta-hydroxybutyrate (BOH).
  • 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 may be 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
  • 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 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, HWTX-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 compositions 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 Krebs Cycle metabolic intermediate supply, and also protected from ischaemia 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 for example 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-diethylcarbamoyl phenyl)(3-hydroxyphenyl)methyl)-2,5-dimethylpiperazin-1-ylmethyl)benzoic acid], and 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 ischaemia. 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 are 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 a 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 [(2R-trans)-4-[1-[3,5-bis(trifluoromethyi)benzoyl]-2-(phenylmethyl)-4-piperidinyl]-N-(2,6-dimethylphenyl)-1-acetamide (S)-Hydroxybutanedioate] is another specific, active neurokinin-1 (NK(1)) receptor antagonist with subnanomolar affinity for the human NK(1) receptor (K(i): 0.45 nM) and over 200-fold selectivity toward NK(2) and NK(3) receptors.
  • Antagonists of neurokinin receptors 2 (NK-2) that may also reduce cell swelling include SR48968 and NK-3 include SR142801 and SB-222200.
  • Blockade of mitochondrial permeability transition and reducing the membrane potential of the inner mitochondrial membrane potential using cyclosporine A has also been shown to decrease ischaemia-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 ischaemia, reperfusion and trauma.
  • 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 polyethylene glycol (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 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.
  • the dosage of the composition may be adapted to achieve the most preferred contact concentrations.
  • 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, ischaemia, 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 ischaemia-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; (ii) an antiarrhythmic agent or a local anaesthetic; and (iii) a citrate 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 ischaemia 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 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 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, 0-phenanthroline, dithiocarbamate, selegilize and desferrioxamine (Desferal), an iron chelator, has been used in experimental infarction models, where it exerted some level of antioxidant protection.
  • 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 in 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;
  • 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.
  • 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 2.0 M (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 ischaemia 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.
  • 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; (iii) a citrate 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 for example 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, antioxidant, a source of magnesium and a pharmaceutically acceptable carrier such as a buffer. It is also contemplated that 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.
  • composition useful in the methods according to the invention which includes adenosine, lidocaine, a citrate such as CPD and a pharmaceutically acceptable carrier.
  • This composition may optionally include a source of magnesium.
  • the composition according to the invention includes adenosine, lidocaine, a citrate and a pharmaceutically acceptable carrier.
  • This composition may optionally include an anti-inflammatory agent, such as beta-hydroxybutyrate.
  • compositions according to the invention are a combination of adenosine, lidocaine, a citrate and a pharmaceutically acceptable carrier.
  • the composition may also include an anti-inflammatory agent, such as beta-hydroxybutyrate and/or a source of magnesium.
  • 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 mMof a source of magnesium such as MgSO 4 , 0.1 to 20 mM of a citrate such as CPD and 0.9% to 3% of an ionic solution such as NaCl or a buffer.
  • compositions When the composition is used for organ tissue, cell or whole body, arrest, maintaining arrest or organ harvest arrest higher concentrations of magnesium may be used, such as 300 to 500 mM or 400 mM.
  • concentrations of magnesium When the composition is being used to reanimate a subject, organ tissue, or to prepare, harvest or store organs, tissues or cells not requiring arrest, or to reduce the harmful effects of at least one of anaesthesia, surgery, clinical intervention and cardiopulmonary bypass, lower concentrations of magnesium may be used, such as 30 mM or less than 20 mM.
  • the method of the present invention involves contacting a tissue with the composition for a time and under conditions sufficient for reducing injury to the cell, tissue or organ.
  • the composition may for example be infused or administered as a bolus intravenous, intracoronary or any other suitable delivery route as pre-treatment for protection during a cardiac intervention such as open heart surgery (on-pump and off-pump), angioplasty (balloon and with stents or other vessel devices) and as with clot-busters (anti-clotting drug or agents).
  • 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 ischaemia.
  • 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.
  • composition according to the invention may be used with crystalloid cardioplegia to minimise injury to a tissue.
  • a composition could be administered to provide localised arrest of the target tissue as well as protection during reperfusion and postconditioning.
  • composition may be delivered according to one of or a combination of the following delivery protocols: intermittent, continuous and one-shot. Accordingly, in another aspect of the invention, the composition may be administered as a single dose of the composition.
  • the administration is in one shot as a bolus or in two steps as a bolus followed by infusion
  • 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 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 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% O 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:
  • nutrient molecules selected from the group consisting of blood, blood products, artificial blood and a source of oxygen;
  • compositions optionally aerating the composition with the oxygen (for example, in the case of isolated organs) or combining the nutrient molecules with the composition, or both; and
  • 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% O 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.
  • the amount of active ingredients 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 arrest, protect or preserve the body, organs, tissues, cells or cell organelles.
  • Heart Circuit 2 to 10 Cardiac perfusion 1 to 500 ml/min (0.01to 10 ml/min/kg human) ml/kg/min
  • Arrest flow 4-7 ml/kg/min (A; 1.4 mg/kg; L: 2.9 mg/kg; M: 0.06 g/kg)
  • P Saline
  • 3% NaCl may be used if brain injury suspected Infusion- 1 to 40 1 to 80 1 to 50 0.01 to 10 ml/kg/hr with Drip preferred preferred preferred 5 mg/kg/min A: 12 mg/kg; 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 P: 0.1 to 0.2 mg of heart valves via transluminal catheter technique without thoracotomy or extracorporal P/kg/min (may circulation. 2) whole body protection (reduce injury infection, inflammation, not require P for coagulopathy as above) 3) to reduce blood loss during Damage Control Surgery some indications) indicates data missing or illegible when filed
  • A Adenosine
  • L lidocaine-HCl
  • M Magnesium Sulphate
  • Insulin IU international units per Liter
  • BHB beta-hydroxy butyrate
  • CyA Cyclosporin A.
  • BDM 2,3-Butanedione Monoxime (BDM); polyethylene glycol (PEG); dextran-40; bovine serum albumin (BSA); Lactobionate; Poloxamer 188 surfactant P188; and Pluronic F-68 surfactant
  • 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. 1 shows graphs showing measurement of (A) Heart Rate; (B) MAP; (C) Systolic Pressure; (D) Diastolic Pressure; (E) Temperature against Time (min) in Rat Polymicrobial Bacterial Infection Model: Single Bolus Intravenous Treatment only for Rat ALM Bolus v's Control.
  • FIG. 2 shows graphs showing measurement of (A) Heart Rate; (B) MAP; (C) Systolic Pressure; (D) Diastolic Pressure; (E) Temperature against Time (min) in Rat Polymicrobial Bacterial Infection Model: One-Two Intravenous Treatment Delivery over 5 hours for Rat ALM Bolus v's Control. (see example 1)
  • 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. 4 shows a flow diagram of videomicroscopy procedure described in Example 4.
  • FIG. 5 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 added in the lumen (luminal—square) or in the bathing solution (abluminal—diamond).
  • 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)
  • FIG. 7 shows 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 return of spontaneous circulation (ROSC) (GH). (See example 5)
  • 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 A: MAP and B: heart rate (Group 2)
  • FIG. 11 shows graphs showing bolus-bolus for MAP (Group 3). (See example 7)
  • FIG. 12 shows a graph showing the effect of addition of valproic acid
  • FIG. 13 shows a graph showing MAP resuscitation following single NaCl ALM bolus in the presence of L-NAME.
  • 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).
  • MAP mean arterial pressure
  • HR heart rate
  • 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- ⁇ -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% CI).
  • FIG. 40 shows a schematic representation of the in vivo rat protocol of severe polymicrobial sepsis.
  • FIG. 41 shows a table showing the effect of 0.9% NaCl ALM on hemodynamics and rectal temperature during 5 hours following CLP in a rat model of severe 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 detectable 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 18 G 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).
  • Control animals receive intravenous 0.3 ml bolus 0.9% NaCl and treatment groups was 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), in 0.9% NaCl.
  • 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, FiO2 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:
  • 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 18 G 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.
  • PT prothrombin times (extrinsic clotting pathway begins with tissue factor and believed to be the initiator of clotting in vivo)
  • aPTT activated partial thromboplastin time in contrast to the PT, measures the activity of the intrinsic and common pathways of coagulation.
  • thromboplastin in this test refers to the formation of a complex formed from various plasma clotting factors which converts prothrombin to thrombin and the subsequent formation of the fibrin clot.
  • 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
  • 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.
  • Two min of chest compressions at 6 min after the bolus injection increased blood pressure to 25 mmHg with extremely low pulse pressure, a state normally characterized as severe life-threatening shock.
  • the heart rate was 115 bpm. This example demonstrates that the treatment can arrest the whole body and may include the brain with unexpected and surprising near-full hemodynamic recovery after 15 min.
  • FIGS. 12A-Q This is also shown in FIGS. 12A-Q .
  • 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.
  • Time Domain The time domain measures of HR variability as calculated by statistical analyses (means and variance) from the lengths of successive R-R intervals in the ECG and considered reliable indices of cardiac parasympathetic activity.
  • the time domain indices include SDNN, SADNN, NN50, pNN50, RMSSD, SDSD.
  • SDNN standard deviation of the average R-R intervals
  • SADNN standard deviation of the average R-R intervals
  • the 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.
  • 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.
  • 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% 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 .
  • Group 3 Bolus-Bolus treatment 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 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.
  • 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.
  • 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.
  • Without being limited to mechanism is appears that 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.
  • 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 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.
  • Treatment is 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), citrate (1 mM), BHB (4 mM).
  • the bolus and infusion will commence 5 min before percutaneous endovascular repair. Infusion rate will begin at 10 ml/min/kg and increased to produce hypotensive anaesthetized state to reduce blood loss.
  • 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.
  • Postpartum hemorrhage is the leading cause of maternal mortality and disability, particularly in under-resourced areas. PPH is defined as bleeding from the genital tract (500 ml or more) after childbirth.
  • the first line therapy for severe PPH includes transfusion of packed cells and fresh-frozen plasma in addition to uterotonic medical management and surgical interventions.
  • Obstetric haemorrhage is associated with hemodynamic instability, inflammatory activation and coagulopathy and these women patients have a higher incidence of infection.
  • Postpartum uterine sepsis is believed to arise from an ascending infection caused by colonizing vaginal flora. The incidence of infection (post-partum endometritis or infection of the decidua) after vaginal delivery is 0.9 and 3.9% and as high as12-51% after Caesarean section.
  • 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.
  • PVL periventricular leukomalacia
  • necrosis more often coagulation
  • 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 lrukandji. 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
  • FIG. 20A-C Effect adenosine and lignocaine solution with two forms of citrate and elevated magnesium on aortic flow, coronary flow and heart rate after 2 hours of warm (tepid) heart arrest in the working rat heart. Function monitored for 60 min reperfusion.
  • 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
  • Adenosine (A9251 >99% purity) and all other chemicals were obtained from Sigma Chemical Company (Castle Hill, NSW).
  • Lidocaine hydrochloride was purchased as a 2% solution (ilium) from the local Pharmaceutical Supplies (Lyppard, Queensland). Hearts were rapidly removed from anaesthetised rats and placed in ice-cold heparinised modified KH buffer.
  • 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 PO2 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)
  • FIG. 22A-D 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
  • 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 buffer was filtered using a one micron (1 ⁇ M) membrane and was not recirculated.
  • the concentration of adenosine in the solution was 0.4 mM.
  • the concentration of lidocaine in the solution was 1 mM.
  • This solution of modified Krebs Henseleit buffer, adenosine and lidocaine is referred to below as the cardioplegia preservation solution.
  • 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 ).
  • FIG. 23A-D The effect of adding melatonin and insulin with low and high MgSO 4 to bubbled adenosine and lidocaine solution during 8 hours of constant perfusion at 4° C. in the isolated working rat heart.
  • 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% O 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 ).
  • the same trends were seen in the functional recovery of coronary flow (CF) ( FIG. 22B ), heart rate (HR) ( FIG. 22C ) and cardiac output (CO) ( FIG. 22D ).
  • 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% O 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.
  • FIG. 24 The effect of adenosine and lidocaine solution with 0.3 mg/L sildenafil citrate over 2 hours warm arrest (29° C.) given every 20 minutes (2 min infusion) and 60 min reperfusion in the working rat heart
  • 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)
  • 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). Results: Data are presented in Table 2.
  • ISVR Indexed systemic vascular resistance Polarized ALM arrest with Depolarizing INSULIN 4:1 Parameter (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 p ⁇ 0.001 red-packed cells Transfusions of Lower Higher Significant p ⁇ 0.001 fresh-frozen 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.
  • Method The method is that described in detail by Cypel and colleagues (Cypel et al., 2011).
  • Ninety patients (10 per group) will be recruited after obtaining the hospital's internal review board protocol approval and patient or family consent for the study.
  • Lungs will be perfused for 4 hours in the ex-vivo lung perfusion (EVLP) Organ Care System (OCS). Lungs will be considered suitable for transplantation if 1) during EVLP the PO2:FiO2 ratio (ie.
  • 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.

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