Treatment of central nervous system injury

Abstract

The present invention relates to compositions and methods for improving recovery after a traumatic or ischemic central nervous system insult, comprising administering a composition comprising allene oxide synthase (AOS), or a functional equivalent variant or derivative thereof.

Description

[0001] TREATMENT OF CENTRAL NERVOUS SYSTEM INJURY

[0002] TECHNICAL FIELD

[0003] The present invention relates to compositions and methods for improving recovery after a traumatic or ischemic central nervous system insult (e.g. a stroke, traumatic brain injury, spinal cord injury, transient ischemic attack or retinal vein occlusion). More particularly, the invention relates to compositions and methods using allene oxide synthase or functional equivalents thereof for improving recovery after a traumatic or ischemic central nervous system insult, wherein the allene oxide synthase is administered, or is to be administered to a patient in need thereof at least about 3-hours after a traumatic or ischemic central nervous system insult.

[0004] BACKGROUND OF THE INVENTION

[0005] Injuries to the brain or spinal cord (termed Central Nervous System, CNS) can result from episodes of reduced blood flow, such as a stroke, trauma, or neurodegenerative disorder. These types of injuries produce a loss of behavioural function with limited recovery.

[0006] Stroke, which is either ischemic or haemorrhagic in nature, is the leading worldwide cause of adult disability in developed countries. There is no therapy that targets the promotion of stroke recovery. At present, the only stroke therapy is the administration of tissue plasminogen activator (tPA). TPA is a "clot busting" drug that does not target stroke recovery, but targets the blood vessels that are obstructed in stroke. TPA must be given within 4.5 hours after stroke because if given later it may cause bleeding into the brain. There is currently no approved therapy that can be given at a time point later than 4.5 hours post stroke. Many other neuroprotective therapies in development have similar requirements, and need to be administered within a few hours or less of the ischemic or haemorrhagic event. There is currently no approved stroke therapy that targets the processes of tissue repair rather than the obstructed vessels, or that are effective when administered after a longer period of time post injury.

[0007] During a stroke the loss of neurological function generally occurs in two ways. First, the acute injury causes complete damage at the centre of insult, resulting in damage to neural circuits that control bodily function, such as movement, sensation, language or memory. Second, the injury causes partial damage to neural circuits that are adjacent to the injury site (termed peri-infarct cortex), and disables the function of these circuits. Most therapies to- date in CNS injury and stroke have been directed towards the first mechanism of damage: that is to prevent the initial injury or cell death (an approach termed neuroprotection), as opposed to attempting to stabilise the partially damaged circuits in the brain to promote function. Secondary injury is an indirect result of the primary injury and involves metabolic and biochemical cascades, including the ischemic cascade leading to excitotoxicity, acidosis, free radical generation, and breakdown of the blood brain barrier. It arises from processes initiated by the trauma that occur within the hours to days following the initial insult and is a major contributor to brain damage and death that occur after stroke and traumatic brain injury.

[0008] These mechanisms of secondary injury also underpin several deleterious effects of other brain insults, such as concussion, traumatic brain injury, transient-ischemic attack, spinal cord injury, retinal vein occlusion, and subarachnoid haemorrhage.

[0009] For example, concussion, or mild traumatic brain injury, is a transient and clinically detectable alternation in brain function imparted by a mechanical insult. After a mild concussive event, evidence of brain abnormalities can be observed months after the initial trauma when symptoms have mostly dissipated which are thought to arise due to cytotoxic oedema and reactive gliosis; a change in glial cells shape in response to damage to the central nervous system.

[0010] Allene oxide synthase is an enzyme that is found in multiple plant and non-mammal species. It is a member of the superfamily of cytochrome P450 enzymes and is involved in the synthesis of certain lipids, fatty acids, and biochemical mediators, and catalyses the production of an epoxide (an allene oxide) from a fatty acid (or lipid) hydroperoxide.

[0011] There are no currently approved treatments that promote recovery after CNS injury. The primary rehabilitative treatment after CNS injury is physical rehabilitation, including physical therapy, and occupational and speech therapy. These therapies are is expensive, labour-intensive, time-consuming and have low levels of success. Additionally they are not available equally to all patients. Thus, there is a clear need for central nervous system injury treatments that are effective and can be administered to a patient in need outside of the current 3-hour period of time post-injury.

[0012] OBJECT OF THE INVENTION

[0013] It is an object of the present invention to provide methods of treating central nervous system injury, or chronic deterioration or degeneration following an acute or traumatic central nervous system insult in a patient. It is an alternative objection of the invention to provide medicaments for the treatment of such conditions.

[0014] Alternatively, it is an object of the invention to at least provide the public with a useful choice.

[0015] SUMMARY OF THE INVENTION

[0016] The high unmet need for agents that protect the CNS from neurodegeneration or injury caused by trauma and conditions of local cerebral or global hypoperfusion (e.g. stroke, haemodynamic shock with cardiac arrest, heart failure, or during surgery), or to promote reparative neurological processes after such an event, have prompted the Inventors to search for new approaches. The present invention is provided to address at least some of the challenges present with the current approaches.

[0017] Methods of treatment

[0018] In one example there is provided a method of improving recovery after a central nervous system injury in a subject, comprising administering to said subject a composition comprising allene oxide synthase, or a functionally equivalent variant or derivative thereof.

[0019] In one aspect the composition comprises a therapeutically effective amount of allene oxide synthase, selected from the group comprising SED ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, or SEQ ID: 9, or a functionally equivalent derivative or variant thereof.

[0020] In one aspect the composition contains a therapeutically effective amount of allene oxide synthase derived from Parthenium argentatum.

[0021] In one aspect allene oxide synthase derived from Parthenium argentatum is SEQ ID: 1, or a functionally equivalent derivative or variant thereof.

[0022] In another example there is provided a method of improving recovery after a central nervous system injury in a subject, comprising administering to said subject a composition comprising a allene oxide synthase, or a functionally equivalent variant or derivative thereof, wherein said composition is administered at least about 3 hours post-injury.

[0023] In one aspect the composition is administered to a subject at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours post-injury.

[0024] In one aspect the composition is administered to said subject about 3 hours post central nervous system injury.

[0025] In another aspect the composition is administered to a subject from at least about 3 hours to about 12 hours post central nervous system injury.

[0026] In another aspect the composition is administered to a subject at least one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days or at least 14 days post central nervous system injury.

[0027] In one aspect the composition is administered to a subject from at least 3 days post central nervous system injury.

[0028] In another aspect the composition is administered to a subject from at least about 3 days to about 7 days post central nervous system injury.

[0029] In another example the composition containing a therapeutically effective amount of allene oxide synthase, or a functionally equivalent variant or derivative thereof, is formulated for oral, intravenous, subcutaneous, intraperitoneal, sublingual, intranasal, inhalation, local, intrathecal, intraocular, intramuscular, intracranial, or systemic administration to the subject.

[0030] In one aspect the composition containing a therapeutically effective amount of allene oxide synthase, or a functionally equivalent variant or derivative thereof, is formulated as an aqueous solution for intranasal administration to a subject.

[0031] In another aspect, the composition containing a therapeutically effective amount of allene oxide synthase, or a functionally equivalent variant or derivative thereof, is formulated as an aqueous solution additionally containing a non-ionic surfactant for intranasal administration to a subject. In another aspect the non-ionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-XlOO, or any other pharmaceutically acceptable surfactant.

[0032] In another aspect the composition contains a therapeutically effective amount of allene oxide synthase selected from the group comprising SEQ ID: 1 SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, SEQ ID: 9, or a functionally equivalent variants or derivatives thereof, is formulated as an aqueous solution additionally containing a non-ionic surfactant selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or Triton-XlOO, for intranasal administration to a subject.

[0033] In another example the composition containing a therapeutically effective amount of an allene oxide synthase enzyme, or a functionally equivalent variant or derivative thereof, is formulated for local administration to the subject.

[0034] In one aspect the composition comprises a depot delivery system containing a therapeutically effective amount of allene oxide synthase, or a functionally equivalent variant or derivative thereof, for local administration via intracranial, intrathecal, or intravitreal injection.

[0035] In another aspect the composition comprises a depot delivery system containing a therapeutically effective amount of allene oxide synthase, or a functionally equivalent variant or derivative thereof, and a non-ionic surfactant, for local administration via intracranial, intrathecal, or intravitreal injection.

[0036] In another aspect the composition comprises a depot delivery system containing a therapeutically effective amount of allene oxide synthase, or a functionally equivalent variant or derivative thereof, and a non-ionic surfactant, for local administration via intracranial, intrathecal, or intravitreal injection, wherein the depot delivery system provides sustained release of said allene oxide synthase.

[0037] In one aspect the depot delivery system comprises a delivery system selected from a nanoparticle formulation, a hydrogel formulation, an implantable mechanical delivery system.

[0038] In another aspect the depot delivery system comprises a hydrogel comprising said allene oxide synthase and additionally a non-ionic surfactant.

[0039] In one aspect the hydrogel comprises a biopolymer. In another aspect the hydrogel comprises one or more materials selected from the group consisting of alginic acid, or a pharmaceutically acceptable salt thereof, hyaluronan, gelatin, thiol-modified hyaluronan, heparin, thiol-modified heparin, thiol-modified chondroitin sulfate, thiol-modified gelatin, a hyaluronan sodium salt, and an acrylated hyaluronic acid.

[0040] In one example, the central nervous system injury is selected from the group comprising: ischemic stroke, haemorrhagic stroke, white matter stroke, subcortical stroke, retinal vein occlusion, traumatic brain injury, concussion injury or mild-to-moderate traumatic brain injury, and spinal cord injury.

[0041] Swiss-type examples

[0042] According to one example there is provided the use of allene oxide synthase in the manufacture of a medicament for the treatment of central nervous system injury.

[0043] In one aspect allene oxide synthase is selected from the group comprising SED ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, or SEQ ID: 9, or a functionally equivalent derivative or variant thereof.

[0044] In one aspect the medicament is to be administered at least about 3 hours after central nervous system injury.

[0045] In one aspect the medicament is to be administered at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours after central nervous system injury.

[0046] In another aspect the medicament is to be administered at least one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days or at least 14 days after central nervous system injury.

[0047] In another aspect the medicament is to be administered at least 3 days post central nervous system injury. In another aspect the medicament is to be administered from at least about 3 days to about 7 days post central nervous system injury.

[0048] In another aspect the medicament is formulated for oral, subcutaneous, intraperitoneal, intraocular, intrathecal, intramuscular, intravenous, intranasal, intracranial, or local administration.

[0049] In one aspect the medicament is formulated as an aqueous solution for intranasal administration.

[0050] In another aspect the medicament additionally comprises a non-ionic surfactant.

[0051] In one aspect, the non-ionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-XlOO, or any other pharmaceutically acceptable surfactant.

[0052] In another aspect, the medicament comprises a therapeutically effective amount of an allene oxide synthase selected from the group comprising SEQ ID: 1 SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, SEQ ID: 9, or a functionally equivalent variant or derivative thereof, is formulated as an aqueous solution for intranasal administration additionally comprising a non-ionic surfactant selected from the group: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or Triton-XlOO.

[0053] Second medical use examples

[0054] According to one example there is provided allene oxide synthase for use in the treatment of central nervous system injury.

[0055] In one aspect allene oxide synthase is selected from the group comprising SED ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, or SEQ ID: 9, or a functionally equivalent derivative or variant thereof.

[0056] In one aspect allene oxide synthase is to be administered at least about 3 hours after central nervous system injury.

[0057] In one aspect allene oxide synthase is to be administered at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours after central nervous system injury.

[0058] In another aspect allene oxide synthase is to be administered at least one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days or at least 14 days after central nervous system injury.

[0059] In another aspect allene oxide synthase is to be administered at least about 3 days post central nervous system injury.

[0060] In another aspect allene oxide synthase is to be administered from at least about 3 days to about 7 days post central nervous system injury.

[0061] In another aspect allene oxide synthase is formulated for oral, subcutaneous, intraperitoneal, intraocular, intrathecal, intramuscular, intravenous, intranasal, or local administration.

[0062] In another aspect allene oxide synthase is formulated as an aqueous solution for intranasal administration.

[0063] In another aspect allene oxide synthase additionally comprises a non-ionic surfactant.

[0064] In one aspect, the non-ionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-XlOO, or any other pharmaceutically acceptable surfactant.

[0065] In another aspect, the allene oxide synthase is selected from the group comprising SEQ ID: 1 SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, SEQ ID: 9, or a functionally equivalent variant or derivative thereof, is formulated as an aqueous solution for intranasal administration, and additionally comprises a non-ionic surfactant selected from the group: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or Triton-XlOO. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0067] Figure 1 illustrates SEQ ID No. 1 - 9.

[0068] Figure 2: illustrates the repair mechanisms of AOS in relation to lipid peroxide / hydroperoxide formation during oxidative stress.

[0069] Figure 3: illustrates the impact of AOS on rescuing neuronal cell death after insult with hydrogen peroxide.

[0070] Figure 4: illustrates the impact of AOS on microglial reactivity (measured by IBA1 staining) when administered intranasally after 3-hours post-insult as a single dose.

[0071] Figure 5: illustrates the impact of AOS on astrocyte reactivity (measured by GFAP staining) when administered intranasally after 3-hours post-insult as a single dose. (A) Astrocyte reactivity right next to the stroke. (B) Astrocyte reactivity 600-800um away from the stroke

[0072] Figure 6: illustrates the impact of AOS on pericyte numbers demonstrating an increase in the stroke core. (A) Pericyte numbers in the stroke core. (B) Pericyte numbers next to the stroke. (C) Pericyte numbers 800um away from the stroke

[0073] Figure 7: illustrates the impact of AOS on motor function, demonstrating an improvement in forelimb asymmetry on the cylinder task.

[0074] Figure 8: illustrates the impact of AOS on motor function, demonstrating an improvement in forelimb asymmetry on the grid walking task.

[0075] Figure 9: illustrates the impact of AOS on astrocyte reactivity (measured by GFAP staining) when administered via direct local injection 5-days post-infarct (A) Astrocyte reactivity right next to the stroke. (B) Astrocyte reactivity 600- 800um away from the stroke Figure 10: illustrates the impact of AOS on cognitive impairment post-stroke, demonstrating an improvement in spatial memory impairment by object location recognition task (OLRT). (A) OLRT performance 1 week post-infarct and (B) OLRT performance 4 weeks post-infarct.

[0076] DEFINITIONS

[0077] General Definitions

[0078] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art to which the inventions belong (for example, in immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0079] Unless otherwise indicated, the recombinant protein and immunological techniques utilized in the present invention are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0080] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0081] The term "and / or", e.g., "X and / or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0082] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0083] It is intended that reference to a range of numbers disclosed herein (for example 1 to 10) also incorporates reference to all related numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0084] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0085] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0086] The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0087] Any example described herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise.

[0088] Selected Definitions

[0089] The term "about" is used herein to refer to conditions (e.g., amounts, concentrations, time etc) that vary by as much as 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% to a specified condition.

[0090] The term "AOS" as used herein refers to an allene oxide synthase enzyme used in the context of AOS enzyme activity.

[0091] The term "PaAOS" as used herein refers to allene oxide synthase enzyme as derived from Parthenium argentatum.

[0092] The term "functionally equivalent variant or derivative" as used herein means a fragment of a full-length polypeptide, peptide or protein which fragment retains an activity of the polypeptide, peptide or protein. As used herein, the term "biologically active fragment" includes deletion mutants and small poly / peptides, for example of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, contiguous amino acids, which comprise an activity of the parent peptide polypeptide. Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques as, for example, described in Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al. CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. Alternatively, peptides of this type may be synthesised using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989), see particularly Chapter 9, or by Roberge et al. (1995 Science 269: 202). Alternatively, peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques

[0093] The terms "brain injury", "brain insult", "brain damage", "neuronal injury" and "neuronal damage" as used herein refers to injury to neuronal tissues that compromises the cell viability and / or cellular function of the neuronal tissue.

[0094] The term "depot delivery system" refers to a system, device, or formulation that can be placed within the body and that provides sustained delivery of the active agent(s) of interest, e.g. an allene oxide synthase enzyme, or a functional derivative or variant thereof.

[0095] The term "derivative" as used herein is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to a parent sequence including additions, or deletions that provide for functionally equivalent molecules.

[0096] The term "device" means a substance or particle that is injected into the brain and slowly releases the intended molecule, allene oxide synthase.

[0097] The terms "drug", "active agent", and "pharmacologically active agent" are used interchangeably herein to refer to any chemical compound, complex, or composition that is suitable for administration to a subject, and that has a beneficial biological effect, preferably a therapeutic effect in the treatment of a disease or abnormal physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents.

[0098] The term "dosage form" denotes any form of a pharmaceutical composition that contains an amount of active agent sufficient to achieve a therapeutic effect with a single administration, or with multiple administrations. When the formulation is a solution or a suspension, the dosage form is usually one such solution or suspension. The frequency of administration that will provide the most effective results in an efficient manner without overdosing will vary with: (1) the characteristics of the particular drug, including both its pharmacological characteristics and its physical characteristics, such as its solubility and stability;

[0099] The terms "effective amount", "therapeutically effective amount", and "efficacious amount" in the context of a particular utility of the peptides and enzymes described herein, is meant the administration of that amount of composition to an individual in need thereof, either in a single dose or as part of a series, that is effective for that stimulation, prevention or treatment. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0100] As used in this specification, the term "fragment" or "functional derivative" in relation to a polypeptide is a subsequence of a polypeptide that may be detected (e.g.) using a binding agent. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant or derivative thereof.

[0101] The term "hydrogel" refers to a typically crosslinked network of polymer chains (e.g. hyaluronans) that are typically hydrophilic. Hydrogels absorb water and swell. Illustrative examples include, but are not limited to, the hyaluronic acid-based hydrogel, known as HYSTEM® which are crosslinked networks of hyaluronic acid and other thiol-modified macromolecules.

[0102] The term "improving recovery" or "improved recovery" means an improvement in motor functional recovery or cognitive performance of a subject after central nervous system injury. In certain embodiments, an improvement in motor recovery or motor functional recovery comprises an improvement in motor coordination, and / or balance, and / or gait, and / or speech. In certain embodiments, an improvement in cognitive performance comprises an increase and / or improvement in performance on tasks of mental abilities including, but not limited to, learning, and / or thinking, and / or memory, and / or problem solving, and / or logical reasoning, and / or decision making, and / or attention. In certain embodiments, an improvement is an improvement in the degree of recovered function and / or performance, and / or the rate of recovery of function and / or performance.

[0103] The terms "individual", "subject", "patient", and "host" are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subjects are humans.

[0104] The term "isolated" as applied to the polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular or other naturally-occurring biological environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques. The polypeptide sequences may be prepared by at least one purification step.

[0105] The terms "intranasal administration" and "intranasal delivery" refers to the delivery to the central nervous system an effective amount of an agent of interest, e.g. an allene oxide synthase enzyme, or a functionally equivalent derivative or variant thereof, via administration of said enzyme to a subject through the nasal cavity.

[0106] The terms "patient", "subject", "host", "individual" and / or "mammal" are used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, most particularly a human, for whom a diagnosis, prophylaxis or therapy is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, humans, any member of the subphylum Chordata including primates, rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovine (e.g., cattle), ovine (e.g., sheep), caprine (e.g., goats), porcine (e.g., pigs), equine (e.g., horses), canine (e.g., dogs), feline (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards etc), and fish. The subject may be in need of diagnosis, prophylaxis or treatment; however, it will be understood that the aforementioned terms do not imply that symptoms are present. Accordingly, both human and veterinary applications of the methods and uses of the formulations described herein are contemplated.

[0107] The term "pharmaceutically acceptable carrier" means a carrier that is used in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes carriers that are acceptable for veterinary uses as well as human pharmaceutical use. A pharmaceutically acceptable carrier as used in the specification and claims includes both one and more than one such carrier.

[0108] The term "pharmaceutical composition" is meant to encompass a composition suitable for administering to a subject, such as a mammal, and especially a human. A "pharmaceutical composition" may be sterile and substantially free of contaminants that are capable of eliciting an undesirable response within the subject (e.g. the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intranasal, inhalable, intradermal, intracheal, intrathecal, subcutaneous, intravenous, and the like.

[0109] A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[0110] The terms "peptide" and "polypeptide" or "protein" may be used interchangeably throughout this specification, and encompass amino acid chains of any length, including full length sequences in which amino acid residues are linked by covalent peptide bonds. Polypeptides useful in the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof. Polypeptides herein may have chain lengths of at least 4 amino acids, at least 5 amino acids, or at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or all 23 amino acids of the full-length allene oxide synthase described herein. Reference to other polypeptides of the invention or other polypeptides described herein should be similarly understood.

[0111] The term "purified" as used herein does not require absolute purity. Purified refers in various embodiments, for example, to at least about 80%, 85%, 90%, 95%, 98%, or 99% homogeneity of a polypeptide, for example, in a sample. The term should be similarly understood in relation to other molecules and constructs described herein.

[0112] The term "sustained delivery" refers to the delivery to the desired site (e.g. the infarct cavity) an effective amount of an agent of interest (e.g. an allene oxide synthase enzyme, or a functionally equivalent derivative or variant thereof), for at least one day, preferably for at least 3 days, at least 4 days, at least 5 days, at least 6 days, more preferably for at least 1 week, at least 2 weeks, at least 5 weeks, at least 6 weeks, at least 2 months, at least 3 months, or at least 4 months.

[0113] Term "variant" as used herein refers to polypeptide sequences different from the specifically identified sequences, wherein 1 to 6 or more or amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of one, two, three, four, five or six amino acids are contemplated. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides useful in the invention have biological activities including signal peptide activity or antigenic-binding properties that are the same or similar to those of the parent polypeptides. The term "variant" with reference to polypeptides encompasses all forms of polypeptides as defined herein.

[0114] Variant polypeptide sequences exhibit at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence of the present invention. With regard to polypeptides, identity is found over a comparison window of at least 5 to 7 amino acid positions.

[0115] Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences, including those which could not reasonably be expected to have occurred by random chance.

[0116] Polypeptide sequence identity and similarity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.18 [April 2008]]) in bl2seq, which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

[0117] The similarity of polypeptide sequences may be examined using the following UNIX command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp. The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. Variant polypeptide sequences commonly exhibit an E value of less than 1 x 10-5, less than 1 x 10-6, less than 1 x 10-9, less than 1 x 10-12, less than 1 x 10-15, less than 1 x 10-18 or less than 1 x 10-21 when compared with any one of the specifically identified sequences. Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polypeptide sequences using global sequence alignment programs. EMBOSS-needle (available at http: / www. ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. Use of BLASTP is preferred for use in the determination of polypeptide variants according to the present invention.

[0118] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, lieu, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention,

[0119] The term "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0120] The term "sequence similarity" refers to the percentage number of amino acids that are identical or constitute conservative amino acid substitutions as defined. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984 Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0121] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and / or may be therapeutic in terms of a partial or complete stabilisation or cure for a disease, and / or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; or (b) inhibiting the disease symptom, i.e. arresting its development; or (c) relieving the disease symptom, i.e. causing regression of the disease or symptom. DETAILED DESCRIPTION

[0122] In general terms, the invention relates to therapies for the treatment of central nervous injury, for example stroke, concussion, retinal vein occlusion, or traumatic brain injury, or the sequelae arising from brain injuries thereof, for example but not limited to motor, visual, sensory, or cognitive impairment. The invention also includes pharmaceutical compositions to treat such conditions and methods for preparing suitable pharmaceutical compositions.

[0123] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, or course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0124] The present invention is predicated on the surprising discovery that allene oxide synthase, or functionally equivalent variants thereof, and compositions comprising allene oxide synthase, or functionally equivalent variants thereof, can be used to treat a central nervous system injury, for example a stroke, traumatic brain injury, concussion, or spinal cord injury, when administered to a subject at least 3-hours after said injury. More specifically, the inventors have discovered that allene oxide synthase can be administered to a subject 3-hours post-brain insult to promote reparative processes in the brain, such as a reduction in neuroinflammation and microgliosis, a reduction in reactive astrogliosis, an increase in pericyte numbers and an improvement in motor function. These discoveries are surprising and contradict the known state-of-the-art that allene oxide synthase must be administered within 45 minutes of brain insult to provide a subject with a therapeutic or neuroprotective effect. Moreover, while no decrease in stroke volume was observed (a common measure for protection of acute neuronal damage, or "neuroprotection"), the inventors have surprisingly discovered that allene oxide synthase can be administered to the brain of a subject non-invasively via the intranasal route, outside the known window of opportunity, and induce significant reductions in neuroinflammation and reactive microgliosis and astrogliosis, increase vascular and pericyte density, and improve motor function. These effects support a role for allene oxide synthase in promoting reparative processes within the peri-infarct zone. Without wishing to be bound by theory, the inventors contemplate that allene oxide synthase may attenuate the secondary damage following brain injury when administered outside of the 45-minute window required for neuroprotection. When referring to AOS it should be taken to include AOS isolated from any source including functionally equivalent peptides and proteins including AOS obtained, for example, by chemical synthesis and / or gene expression techniques. AOS and its functionally equivalent variant(s) may be referred to herein collectively as AOS. Accordingly, where not specifically mentioned, references to AOS of use in the invention herein should be taken to include reference to functionally equivalent variants thereof.

[0125] The inventors have found that administration of AOS protein or a functionally equivalent variant thereof (referred to hereinafter as "AOS" or "AOS protein") is beneficial for CNS injury recovery. In one aspect the AOS is sourced from Parthenium argentatum. This is no way intended to limit the scope of the invention, but is shown for demonstrative purposes only. Allene oxide synthase or functional equivalents thereof from any source may be suitable for the invention.

[0126] As mentioned above, in some examples allene oxide synthase may be sourced from the guayule rubber plant (GenBank CAA55025.5), also known as the guayule rubber particle protein (RPP), is part of the CYP74A family of enzymes (allene oxide synthases; AOS). This enzyme family comprises fewer than 15 molecules from a variety of plant sources including guayule, corn, barley, tomato, flaxseed and Arabidopsis (a model plant in biology), all performing AOS function and having molecular weights in the monomeric form of ~55kDa. The enzymes are atypical members of the Cytochrome P450 family in that they selfregenerate, requiring neither oxygen nor a NADPH reductase for activity and have extraordinary reaction rates. AOS transforms fatty acid hydroperoxides, formed by oxidation of polyunsaturated fatty acids into unstable epoxides which then further degrade into ketols. The lipid peroxides (LPO) can be formed by oxidative challenge or the action of lipoxygenase, a pathway used in the generation of LPO substrate for use in the AOS enzyme function assay.

[0127] Compositions of the Disclosure

[0128] The allene oxide synthase enzyme preparations and compositions described herein are particularly useful for the treatment of a central nervous system injury, for example, but not limited to: stroke, ischemic stroke, white matter stroke, haemorrhagic stroke, traumatic brain injury, concussion, spinal cord injury, and / or retinal venous occlusion.

[0129] The present invention is based at least in part on the discovery of enzyme compositions and / or preparations comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 8, or SEQ ID NO 9 as well as structural and functional variants thereof, which compositions and / or preparations are suitable for pharmaceutical use. Advantageously, these compositions and / or preparations are effective in the treatment of a central nervous system injury, such as a stroke, when treatment is initiated between at least about 3 hours and at least about 7 days after said injury. Accordingly, these compositions and / or preparations are particularly useful for the treatment of central nervous system injuries and may provide a substantial benefit over the current standard of care.

[0130] In one example, the present invention provides compositions that comprise, consist or consist essentially of an amino acid sequence corresponding to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 as well as structural and / or functional variants thereof, for use in the treatment of a central nervous system injury.

[0131] The allene oxide synthase compositions described herein may be formulated together with biologically or non-biologically acceptable excipients that enhance enzyme viability and activity for use in (e.g.) biological as well as non-biological systems. For example, formulation in the presence of: a particular surfactant or detergent; buffer limiting pH range; ionic strength (i.e. pl); and which optionally include (e.g.) sugars / carbohyd rates and antimicrobial agents etc.

[0132] Accordingly, in yet a further aspect, the present invention provides a composition comprising an allene oxide synthase enzyme, wherein the composition is formulated for pharmaceutical use, and wherein optionally:

[0133] (i) the surfactant comprises a non-ionic surfactant, selected from the group comprising: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton X-100; and / or

[0134] (ii) the pH of the composition is between about 6.0 and about 9.5, including but not limited to a pH of 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4 and 9.5; and / or

[0135] (iii) the composition further comprises a buffer including, but not limited to, BES

[0136] (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid) Buffered Saline, Bicine (2-(Bis(2-hydroxyethyl)amino)acetic acid), Carbonate-Bicarbonate, CHES (N- Cyclohexyl-2-aminoethanesulfonic acid), Diethanolamine, EBBS (Earle's Balanced Salt Solution), Glycine-Sodium Hydroxide Buffer, HEPES ((4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid), HBSS (Hank's Balanced Salt Solution), HEPPSO (4-(2-Hydroxyethyl)piperazine-l-(2- hydroxypropanesulfonic acid) Hydrate hydrate), HHBS (Hank's Buffer with HEPES), Imidazole-HCI, Maleic Acid, MES (2-(N-morpholino) ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PBS (Phosphate Buffered Saline), Sodium Borate Buffer, TAE Buffer (Tris Base, Acetic Acid, EDTA), TAE, TBS (Tris Buffered Saline), TE Buffer (Tris EDTA), Tricine (N-(2-Hydroxy-l,l- bis(hydroxymethyl)ethyl)glycine), TRIS (tris(hydroxymethyl)aminomethane) and / or Trizma (2-Amino-2-(hydroxymethyl)-l,3-propanediol); and / or

[0137] (iv) the pl of the composition is between about 1 and about 250 mM and includes, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 and 250 mM; and / or

[0138] (v) the concentration of the allene oxide synthase is between about 0.1 and 100 ug / mL including, but not limited to about, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,

[0139] 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,

[0140] 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,

[0141] 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,

[0142] 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,

[0143] 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 ug / mL; and / or

[0144] (vi) the concentration of the allene oxide synthase is between about 0.1 and 500 mg / mL including, but not limited to about, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,

[0145] 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,

[0146] 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,

[0147] 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,

[0148] 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,

[0149] 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, and 500 mg / mL; and / or

[0150] (vii) the composition further comprises a carbohydrate including, but not limited to, saccharides such as trehalose and lactose in an amount of about 1 mM to about 200 mM, including but not limited to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 and 200 mM carbohydrate; and / or

[0151] (viii) the composition further comprises an antimicrobial agent including, but not limited to, kanamycin, gentamicin, lincomycin, tylosin tartrate, in an amount of between about 0.1 and 100 ug / mL including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,

[0152] 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,

[0153] 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,

[0154] 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,

[0155] 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,

[0156] 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 ug / mL; and / or

[0157] (ix) the composition further comprises a polyol such as glycerol in an amount of between about 0.1 and about 20% v / v including but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,

[0158] 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,

[0159] 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,

[0160] 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,

[0161] 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,

[0162] 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20% v / v polyol.

[0163] In certain examples according to the present invention, the compositions described herein are substantially surfactant-depleted or surfactant-free, and do not further contain (e.g.) imidazole or residual lipids.

[0164] In other examples according to the present invention, the pl of the compositions described herein is between about 1 and about 250 mM, and is based on the presence of a salt including, but not limited to, sodium chloride (NaCI), calcium chloride (CaCIz), potassium chloride (KCI), lithium chloride (LiCI), magnesium chloride (MgClz), sodium bromide (NaBr), sodium sulphide (NazS), beryllium chloride (BeCh), barium fluoride (BaFz), calcium iodide (Cah), lithium iodide (lil), caesium fluoride (CsF), potassium sulphate (K2S), caesium oxide (CS2O), rubidium sulphate (Rb2S), rubidium fluoride (RbF), monosodium phosphate (NaP PC ), disodium phosphate (Na2HPO4), sodium sulphate (NaHSC ), sodium carbonate (NaHCCh), sodium bicarbonate (Na2COs), potassium cyanide (KCN), and sodium acetate (O- COONa).

[0165] In other embodiments, the allene oxide synthase compositions may be formulated for use as a hydrogel, preferably a biopolymer hydrogel to contain and provide sustained release of allene oxide synthase locally into the central nervous system, for example, the brain, stroke infarct cavity, peri-infarct zone, spinal cord, or eye, that afford sustained release of allene oxide synthase. In certain embodiments the hydrogel can be made of naturally occurring brain proteins and does not provoke a brain reaction. In some embodiments, the hydrogel can be a HYSTEM® hydrogel (e.g. a HYSTEM®-C hydrogel, a HYSTEM®-HP hydrogel, and the like).

[0166] In various embodiments the allene oxide synthase may be released slowly over at least one day, or over at least 3 days, or over at least 5 days, or over at least one week, or over at least two weeks, or over at least 3 weeks, or over at least one month, or over at least two months, or over at least three months, or over at least six months. Typically allene oxide synthase is released directly into the target tissue in the body (e.g. the peri-infarct tissue, the back of the eye, or the spinal cord) after the ischemic event (e.g. stroke, traumatic brain injury, retinal venous occlusion, or spinal cord injury) and produce an improvement in recovery from the ischemic event.

[0167] In certain embodiments the use of hyaluronan based hydrogels is contemplated and preferred. A family of well-suited hydrogels are produced commercially and marketed under the name HYSTEM®. In certain embodiments the hydrogels mimic the natural extracellular matrix environment (ECM) and are designed to recapitulate the minimal composition necessary to obtain a functional ECM.

[0168] In various embodiments the individual components of the hydrogel are cross-linkable in situ and can be seeded with a therapeutic amount of allene oxide synthase prior to injection in vivo, without compromising the allene oxide synthase or the recipient tissue.

[0169] In certain embodiments the hydrogel comprises a hyaluronan (or a functionalised / derivatised hyaluronan) and a gelatin (or a functionalised / derivatised gelatin). In various embodiments the hyaluronan and / or the gelatin are each thiol-modified, e.g. by using carbodiimide mediated hydrazide chemistry. In some embodiments, the gel-forming material is based on chemically-modified hyaluronic acid. In some embodiments, the gelforming hyaluronic acid matrix is HYSTEM®, HYSTEM®-HP, or HYSTEM®-C. The HYSTEM® hydrogels are formed by crosslinking mixtures of these thiolated macromolecules using polyethylene glycol diacrylate (PEGDA) or other suitable cross-linkers. The rate of gelation and hydrogel stiffness can be controlled by varying the amount of cross-linker. In certain embodiments an important attribute of the hydrogels used herein (e.g., HYSTEM® hydrogels) is their large water content, typically greater than about 95% resulting in high permeability for oxygen, nutrients, and other water-soluble metabolites.

[0170] In various embodiments the hydrogels are initially provided as a three component system comprising 1) a hyaluronan (e.g., a thiol-modified hyaluronan), 2) a gelatin (e.g., a thiol-modified gelatin; and 3) a linker (e.g., a polyethyleneglycoldiacrylate linker). In one commercially available system, the HYSTEM®-C system these are provided as the three components: GLYCOSIL® (thiol-modified hyaluronan), GELIN®-S (thiol-modified gelatin) and EXTRALINK® (polyethyleneglycol diacrylate). The individual components are supplied in vials as pre-measured, sterile, lyophilized solids that, when dissolved in physiologic buffer (i.e. normal saline, Lactated Ringers, etc.) and mixed together, form a clear, transparent viscoelastic hydrogel in approximately 20 minutes at room temperature. The compliance (stiffness) of the hydrogel is ~70+20 Pa which is similar to adipose and neural tissue. The lyophilized hydrogel components contain no extra salts so that upon dissolution in physiologic buffer the resultant gel is isotonic with a pH ~7.4. In certain embodiments the typical percent solids of a suitable hydrogel (e.g., HYSTEM®-C hydrogel is less than 2.0% (w / v) and the EXTRALINK® cross-linker concentration utilizes less than 30% of the available thiol groups on the other components so that any unreacted acrylate groups of the cross-linker are negligible. Typically, one ml of HYSTEM®-C hydrogel formulated in physiologic buffer contains 4 mg of GLYCOSIL® and 4 mg of GELIN® cross-linked with 4 mg of EXTRALINK® at the ionic strength and pH of the formulation buffer. In certain embodiments for in vivo applications in, typical hydrogel volumes may range between 0.1, or 0.5, or 1.0 ml / implant site up to 5.0 ml, 4.0 ml, or 3.0 ml, or 2.0 ml per implant site with a total volume of up to 20 mis, 15 mis, or 10 mis of hydrogel in situations with multiple implant sites per tissue or organ.

[0171] Methods of making the thiol-modified macromolecules are known in the art. In one approach, dithiobispropanoic dihydrazide (DTP) is coupled to the carboxyl functional groups of the macromolecule by reaction with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) followed by reductive cleavage of the DTP disulfide bonds with dithiothreitol (DTT) to yield the thiol-modified macromolecule DTPH derivatives. The thiol- modified products are purified by exhaustive tangential flow filtration (TFF) using a polyethersulfone membrane with a 10 kDa lower exclusion limit. Since the reagents and reaction by-products are all small molecules, this latter step produces very high purity thiol- modified macromolecules. For GLYCOSIL®, the hyaluronan component of HYSTEM®-C, prior to thiol modification the molecular weight of the polymer is normalized by controlled basic hydrolysis and the available carbonyl functional groups are increased by carboxymethylation of the 6' hydroxyl of the glucosamine moiety of the polymer with chloroacetic acid in strong base. Gelatin is thiol-modified as supplied without any pre-treatment, and the product is called GELIN®-S.

[0172] In other embodiments, the hydrogel of choice can be substituted with another pharmaceutically acceptable hydrogel known in the art. Polypeptides

[0173] The compositions of the present invention may comprise a polypeptide that comprises, consists or consists essentially of an amino acid sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO:9.

[0174] As used in the above statement and in similar statements elsewhere in this specification, the term "comprises" (and the like) means the polypeptide includes the amino acid sequence corresponding to a sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO:9, and may also include any one or more other elements. Thus, the amino acid sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 is a mandatory element, and any other elements are optional and may or may not be present. Other elements may include, for example, additional amino acid residues at either end of the amino acid sequence, and / or other molecules.

[0175] As used in the above statement and in similar statements elsewhere in this specification, the term "consists essentially of" (and the like) means that the polypeptide includes the amino acid sequence corresponding to a sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO:9, and may also include one or more other elements, provided those elements do not interfere with or contribute to the activity or action of the peptide. Thus, the amino acid sequence corresponding to a sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO:9 is a mandatory element, and other elements are optional and may or may not be present, depending upon whether or not they affect the activity or action of the peptide. For example, where a composition "consists essentially of an amino acid sequence corresponding to the sequence SEQ ID NO: 1", the amino acid sequence may comprise additional amino acid residues (e.g., by as much as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more additional residues) at either end of the amino acid sequence, and / or may be conjugated or otherwise associated with other molecules (e.g., a protecting moiety such as an N-terminal blocking residue (e.g., pyroglutamate)), provided those additional residues or molecules do not substantially modulate the enzymatic properties of the amino acid sequence.

[0176] As used in the above statement and in similar statements elsewhere in this specification, the term "consists of" (and the like) means that the peptide includes, and is limited to, the amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO:9. Thus, the phrase "consists of" indicates that the amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO:9 is a mandatory element, and that no other elements (such as amino acid residues at either end of the amino acid sequence or other molecules) may be present.

[0177] As used in the statement "amino acid sequence corresponding to the sequence SEQ ID NO: 1" and similar statements in this specification, the term "corresponding to" or "corresponds to" (and the like) means an amino acid sequence that displays substantial similarity and / or identity to the sequence defined by SEQ ID NO: 1, and that possesses the desired enzymatic activity. In general, the peptide displays at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % similarity and / or identity to the sequence defined by SEQ ID NO: 1.

[0178] In certain examples, the sequence of the amino acid may differ from the sequence SEQ

[0179] ID NO: 1 by at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15,

[0180] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,

[0181] 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

[0182] 64, 65, 66, 67, 68, 69, 70, 71 or 72 amino acid substitutions, additions, and / or deletions.

[0183] Substituted amino acids may include conservative amino acid substitutions, as well as non-conservative substitutions.

[0184] Alternatively, or in addition, substituted amino acids or added amino acids can be any non-naturally occurring amino acids or derivatives thereof. Non-naturally occurring amino acids include chemical analogues of a corresponding naturally occurring amino acid. Examples of non-naturally occurring amino acids and derivatives include, but are not limited to, 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, nor leucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and / or D-isomers of amino acids.

[0185] In some examples, the polypeptide that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

[0186] 8 is or is a derivative of a homolog or isoform of the sequence defined by SEQ ID NO: 1, SEQ

[0187] ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ

[0188] ID NO: 8, or displays at least about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97,

[0189] 98, or 99 % similarity and / or identity to a homolog or isoform of the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. A "homolog" is a molecule from a different species and which is related by descent from a common ancestral DNA sequence. The term "homolog" may apply to the relationship between genes separated by the event of speciation or to the relationship between genes separated by the event of genetic duplication. An "isoform" is a peptide that has the same function as another peptide but which is encoded by a different polynucleotide and may have small differences in its sequence.

[0190] The polypeptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 may be a biologically active fragment of an amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Reference herein to a "fragment" means a molecule which contains at least about 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475 or about 480 contiguous amino acids. The biologically active fragment may have the activity associated with the full-length amino acid sequence and / or may have an altered activity. An "altered activity" includes an enhanced activity or loss of a detrimental activity. The biologically active fragment may have the ability to convert free or esterified fatty acid peroxides or hydroperoxides into their corresponding epoxides, as described herein.

[0191] Methods well known in the art can be used to determine whether an amino acid sequence is a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 as herein defined. The predictive methods include consulting the available algorithms, including SYFPEITHI and BIMAS algorithms described herein, and those described in US 2010-0168398 which discusses both "statistical" and "structure-related" methods that can be used to predict whether a particular peptide will bind to an MHC class I molecule. The "statistical" methods are typically based on experimentally obtained affinity data, whilst the "structure-related" methods are typically based on available 3D structural information of MHC molecules. See also, Madden, D. R. et al, 1992 Cell 70: 1035-1048; Falk, K. et al, 1991 Nature 351: 290- 296; Matsumura, M. et al, 1992 Science 257: 927-934; Saper, M. A. et al, 1991 J. Mol Biol. 219: 277-312; and Latron, F. et al, 1992 Science 257: 964-967. As well as predictive methods, peptides can be synthesised and tested for activity, including in assays such as those known in the art (see for example Fruci, D. et al. 1993 Human Immunology 38(3): 187- 192) and described herein (see, for example, the Examples section).

[0192] The polypeptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 may comprise a peptide that has been suitably modified, for example, by lipid modification to modify its physico-chemical properties.

[0193] The polypeptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 may be prepared in recombinant form using standard protocols as, for example, described in Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.

[0194] Alternatively, the polypeptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 can be synthesised using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989) or by Roberge et al. (1995 Science 269: 202). Syntheses may employ, for example, either t- butyloxycarbonyl (t-Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) chemistries (see Chapter 9.1 of Coligan et al. supra; Stewart and Young, 1984, SOLID PHASE PEPTIDE SYNTHESIS, 2nded. Pierce Chemical Co., Rockford, 111, 1994; and Atherton and Shephard, supra).

[0195] EXAMPLES

[0196] EXAMPLE 1

[0197] Neuroprotective effects of allene oxide synthase in brain cell culture assays.

[0198] The effect of allene oxide synthase (AOS) on neuronal protection was assessed in a cerebellar microexplant culture system in vitro. Two different neuronal injury paradigms were used - the mitochondrial toxin 3-nitropropionic acid, an irreversible inhibitor of succinate dehydrogenase, and hydrogen peroxide, which induces oxidative stress resulting in neuronal cell death.

[0199] Methods

[0200] Cerebellar Microexplant System

[0201] 1. Extraction of cerebellar tissue

[0202] Postnatal day 4 Wistar rats were used for the study. The rats were sacrificed and placed in ice for 1 minute, decapitated and the cerebellum removed and placed on ice. Cerebellum tissue was placed in 1 mL of 0.65% glucose-supplemented PBS (10 uM 65% stock D(+)- glucose / 1 mL PBS) in a large Petri dish, chopped up into smaller sections and triturated with a 1 mL insulin syringe via a 23 G (0.4 mm) needle, and then replaced back into the glucose solution in the large petri dish. The tissue was sieved (through 125 um pore size gauze) and centrifuged twice (2 min at 6000 x g) and transferred into serum-free BSA-supplemented START V medium (Biochrom, Germany). The second centrifugation step was done with 1 mL of START V medium. The microexplants were reconstituted into 500 uL of START V medium and put on ice.

[0203] 2. Culture of cerebellar cells

[0204] Two hours after poly-L-lysine coating, glass slides were washed with Millipore water and airdried. Each slide was placed into a small petri dish (diameter: 35 mm) and 40 uL of START V / cell suspension added. The tissue was incubated for 2 h at 34 °C (settlement period). START V-medium (1 mL) was then added to the petri dish and cultivated at 34 °C in the presence of 5% CO2 in air at 100% humidity for 48 hours.

[0205] 3. Drug application

[0206] For the studies, some explant cultures were exposed to vehicle (PBS buffer) only and acted as controls. In the first study (Study 1), 10 uL of toxin (3-nitropropionic acid, pH 7.4, 0.5 mM) was applied simultaneously with increasing concentrations of AOS enzyme (in PBS, pH 7.4, 10 ng / mL - 20 ng / mL concentrations). In the second study (Study 2), 10 uL toxin (hydrogen peroxide, 0.1 mM, pH 7.4 in Millipore water) was applied simultaneously with increasing concentrations of AOS enzyme (10 ng / mL - 20 ng / mL in PBS, pH 7.4). Study 3 was conducted to confirm the positive results obtained from Study 2. In all studies, drugs were left in contact with the explants for the duration of the study, which was 24 h.

[0207] 4. Determination of drug effect

[0208] After the explants were exposed to drugs (toxin / AOS) for 24 h, the cells were then rinsed in PBS and then fixed in increasing concentrations of paraformaldehyde (PFA) (500 uL, 0.4% PFA, followed by 1.2%, then 3%, and finally 4% PFA). Each fixation step was performed for 3 min. Finally, the microexplants were rinsed in PBS.

[0209] Neurons in the explants were then evaluated for morphology (presence of neurites) and counted as live cells per microscopic field. Four fields displaying highest cell density were counted per cover slip and the data presented as mean + / - standard error of the mean (SEM); n=4 each. Statistical significance was evaluated using a non-paired student t-test. Results

[0210] 1. Study 1 (3-nitropropionic acid injury paradigm)

[0211] Following exposure of cerebellar microexplants to the mitochondrial toxin 3-nitropropionic acid, treatment with AOS enzyme had no effect on neuronal recovery (data not shown).

[0212] 2. Study 2 and 3 (hydrogen peroxide injury paradigm)

[0213] Exposure of cerebellar microexplant to hydrogen peroxide-induced oxidative stress resulted in almost 100% death of cerebellar neurons. However, following treatment with the AOS enzyme neuronal survival was significantly (P < 0.001) increased across all drug concentrations tested (Figure 3). When studies 2 and 3 were pooled, AOS produced a mean neuronal recovery from hydrogen peroxide injury of 23%.

[0214] Discussion

[0215] The study herein demonstrates that AOS can rescue neurons from hydrogen peroxide injury within postnatal cerebellar granule cells organised as cerebellar microexplants to a significant degree.

[0216] These results therefore support the utility of AOS for the ameliorating the impact of central nervous system injuries with surprising efficacy.

[0217] Conclusion

[0218] The lipid peroxidation enzyme can rescue neurons from hydrogen peroxide injury within postnatal cerebellar granule cells organised as cerebellar microexplants. These results suggest AOS is an efficacious treatment option for ischemia-reperfusion central nervous system injuries.

[0219] EXAMPLE 2

[0220] EFFECT OF ALLENE OXIDE SYNTHASE NEUROPROTECTION IN AN IN VIVO MODEL OF MOTOR CORTEX PHOTOTHROMBIC STROKE

[0221] In this study, the inventors sought to investigate whether administration of AOS non- invasively through the intranasal route could be used to improve outcomes of stroke in mice through neuroprotective or neuro-regenerative mechanisms when treated at 3-hours postinfarct, a timepoint previously reported to produce no measurable or statistically significant neuroprotective effects. To evaluate efficacy, the inventors used an in vivo model of focal ischemic stroke and analysed infarct volume, behavioural functional recovery, as well as the potential of AOS to augment gliosis, neurogenesis and vasculogenesis post-stroke.

[0222] Methods

[0223] Animals

[0224] All procedures described in this study were approved and performed in accordance with guidelines on the care and use of laboratory animals set out by the University of Otago Animal Research Committee. Young (2-3 month old) male C57BL / 6 mice (Hercus-Taieri Research Unit, Dunedin, New Zealand) weighing approximately 20-30 g were used as previously described (Reference). The animals were acclimatised for at least 7 days prior to the experiments. All animals were randomly assigned to a treatment group five days post-stroke to ensure that all animals in any given cage received a different treatment (stroke + saline vehicle (n=13); stroke + 10 pg AOS (n=13); stroke + 100 pg AOS (n=12); and stroke + AOSPacific 100 pg (n = ll). All assessments were carried out by observers blinded to the treatment group.

[0225] Photothrombosis model of focal ischemia

[0226] Focal stroke was induced in the mice (total n=60?) by photothrombosis. Under isoflurane anaesthesia (2-2.5% in a 70% N2O I 30% O2 mixture), mice were placed in a stereotactic apparatus, given a dose of Temgesic, and the surgical site was shaved and swabbed with hibitane. The skull was then exposed through a midline incision, cleared of connective tissue, and dried before eye ointment (PolyVisc) was applied to the eyes to prevent them from drying out during the surgical procedure. A cold light source (KL1500 LCD, Zeiss, Auckland, New Zealand) attached to a 40x objective, giving a 2 mm diameter illumination, was positioned as close to the skull as possible, 1.5 mm lateral from Bregma. Rose Bengal solution (0.2 mL, 10 g / L in normal saline, intraperitoneally (i.p.), Signa-Aldrich, Auckland, New Zealand) was then administered. After 5 minutes, the brain was illuminated through the intact skull for 15 minutes while keeping body temperature at 36.9 ± 0.4°C by a heating pad (Harvard apparatus, Holliston, MA, USA) throughout the surgical procedure. Sham surgery mice underwent the same procedure except they received saline injection (100 uL, i.p.) instead of Rose Bengal solution.

[0227] Source of AOS AOS was generated recombinantly. AOS was produced to a purity of >95% and stored at pH 8.3. For in vivo experiments, stocks were directly diluted in PBS to the required concentrations.

[0228] AOS Administration

[0229] AOS (10 uq or 100 pg) was dissolved in sterile isotonic saline. AOS was administered at a low (10 pg) and high (100 pg) dose based on previously published studies (Mathai, S. et al., CNS Neurosci Ther, 2012, 18(11), 887-94). Control animals were injected with saline to serve as controls for AOS. AOS or saline were administered intranasally 3-hours post-stroke. The mice were returned to their home cage after a short recovery period and held under normal housing conditions (12 hour light / dark cycle) with ad libitum access to food and water. Pain relief was administered the following day in the form of temgesic.

[0230] Processing of tissue

[0231] At 14 days post-stroke, animals were deeply anesthetised with pentobarbital and transcardially perfused with 4% paraformaldehyde. Brains were then extracted then cut on a sliding freezing stage microtome in six coronal parallel sets in sections of 30 pm thickness and kept in cryoprotectant at -20 °C).

[0232] Infarct size

[0233] Infarct volume was determined by histological assessment using cresyl violet staining according to a previously published protocol (reference). Infarct volume was quantified using ImageJ (National Institutes of Health, USA) by an observer blinded to the treatment groups, and is based on obtaining measurements from every sixth section through the entire infarct (area in mm2). Infarct volume was quantified as follows: infarct volume mm3= square root of area mm2x section thickness x section interval.

[0234] A total of 11 mice had no stroke visible and were subsequently excluded on the basis that the stroke induction was incomplete (saline vehicle (n=5); 10 pg AOS (n=2); 100 pg (n=2) and AOS2 100 pg (n=2). Therefore, the final number of mice for infarct and behavioural analysis were: saline vehicle (n=8); 10 pg AOS (n=ll); 100 pg AOS (n=10); and AOS(2) 100 pg (n=9). Behavioural assessments

[0235] Animals were tested once on both the grid-walking and cylinder task 7 days prior to surgery to establish baseline performance levels. Recovery of forelimb motor function and forelimb symmetry was determined by both the grid-walking and cylinder tasks, respectively, 7 days and 14 days post-stroke at approximately the same time each day and at the end of their dark cycle. Behaviours were scored by observers who were blind to the treatment group of the animals in the study as previously described (Parker, K., et al., Scientific Reports, 7, 241, 2017).

[0236] Immunofluorescent labelling of GFAP and IBA1

[0237] Immunofluorescent labelling of GFAP and IBA1 was performed XX days post-stroke. Brain sections, every sixth section through the stroke with a thickness of 30 pm, were rinsed in Tris buffered saline (TBS) and transferred into 1% sodium tetraborate in TBS for 20 minutes at room temperature. The sections were blocked for 60 minutes in TBS containing 5% goat and donkey serum with 0.3% Triton X-100 and incubated in TBS with 2% goat and donkey serum and 0.3% Triton X-100 containing primary polyclonal antibodies (add GFAP and IBA1 antibody information) for 24-48 hours at 4 °C. The rinsed sections were incubated for 2 hours at room temperature in the dark and TBS with 2% normal serum and 0.3% Triton X-100 containing appropriate fluorescent secondary antibodies (add secondary antibody information) and nuclear stained Hoechst (1: 1000, Sigma-Alrich) in TBS for 5 minutes at room temperature. Sections were photomicrographed using an inverted montaging microscope (model: Eclipse Ti2, Nikon, Japan) with a lOx objective lens and exported as nd2 files, and 3 sections from each animal were included in the analysis. Changes in GFAP and iBAl staining were investigated at day XX post-stroke in two peri-infarct regions of interest (ROIs, area = 200 pm x 800 pm) 0-200 pm and 600-800 pm from the stroke boarder. Using FUJI Image J software (National Institutes of Health, USA), the integrated density value (IDV) was measured in both ROIs for GFAP and IBAl.

[0238] Analysis of blood vessel characteristics

[0239] Blood vessel analysis was performed to investigate the effect of biopolymer hydrogel and AOS on post-stroke angiogenesis. The sections were photomicrographed using an inverted montaging microscope (model: Eclipse Ti2, Nikon, Japan) with a lOx objective lens and exported as nd2 files. Images were then opened using Fiji ImageJ software (National Institute of Health, USA) and two 200 pm x 800pm rectangle shaped ROIs: (1) core, (2) peri-infarct and (3) a cortical region approximately 600-800pm away from the stroke border were chosen using the ROI manager analysis tool in Fiji ImageJ software. Each of the ROIs were saved as a new jpeg image and images opened in Neurolucida® 360 software (version 2020.1.1, MBF Bioscience, USA) to adjust their scaling before all vascular branches were traced. The total number of vessels, total length of the traced blood vessels and average size of each vessel was then quantified using in Neurolucida® Explorer software. At least three sections from each animal were analysed through the stroke to get an average difference between treatment groups.

[0240] Results

[0241] Histology: AOS has no effect on infarct volume

[0242] Mice were given a stroke then treated with either an intranasal dose of AOS (10 pg or 100 pg) or saline vehicle 3-hours post-stroke. Infarct volume was assessed 14 days post-stroke using cresyl violet staining and ranged from 2.704 mm3TO 4.963 mm3in the vehicle-treated group. Assessment of infarct volume did not reveal a reduction in infarct volume in any of the groups treated with AOS (10 pg: n=6, 2.699 ± 0.85mm3; lOOpg: n = 6, 2.676 ± 0.83 mm3; lOOpg (AOS2): n = 6, 2.896 ± 0.39 mm3) compared to the group that received vehicle (n=6; 2.896 ± 0.39 mm3).

[0243] Immunohistochemistry: AOS reduces inflammation post-stroke

[0244] To investigate whether AOS reduced reactive astrogliosis and reactive microgliosis in the stroke animals, we assessed IBA1 (Figure 4) and GFAP (Figure 5) expression (markers of inflammation) using immunofluorescent labelling 14 days post-stroke, after the animals had completed the final behavioural tasks. We found that lOpg AOS dampened reactive astrogliosis in two peri-infarct zones, 0-200pm (zone 1) and 600-800pm (zone 2) from the stroke border. However, lOOpg and lOOpg Pacific AOS doses only dampened reactive astrogliosis to significant levels within zone 1.

[0245] Immunohistochemistry: AOS increases pericytes in the stroke core

[0246] To investigate whether AOS increased pericytes in the stroke core, zone 1, and zone 2 in the stroke animals, we assessed PDGF-beta expression using immunofluorescent labelling 14 days post-stroke. We found that 10 pg AOS significantly increased pericyte density in the stroke core, but not in the peri-infarct zone adjacent to the stroke, or at 800 |_im from the infarct (Figure 6). The higher doses of AOS (100 pg) appeared to also increase pericyte numbers, however the results were non-significant. Behavioural assessment: AOS improves motor function

[0247] Many ischemic strokes result in motor impairments. The inventors assessed the protective effects of AOS on a focal photothrombic stroke to the motor cortex, with motor function assessed via grid-walking (forelimb function) and cylinder (forelimb asymmetry) tasks at 7 days and 14 days post-stroke. The grid-walking task revealed a significant increase in forelimb motor function in stroke animals receiving AOS compared with stroke animals receiving vehicle at both 7- and 14-day timepoints, and at all doses (Figure 7).

[0248] The cylinder task revealed a significant forelimb motor deficit contralateral to the hemisphere with the stroke in the vehicle group compared to pre-stroke motor function at both 7 days and 14 days post-stroke (time spent on left forelimb vs right: 50% pre-stroke, 32% 7 days post-stroke, 31% 14 days post-stroke). Treatment with AOS at lOpg, lOOpg, and lOOpg Pacific AOS resulted in a significant reduction in motor impairment at both 7 days compared to vehicle group (time spent on left forelimb relative to right: 40% for lOpg, 37% for lOOpg, 41% for lOOpg Pacific) and 14 days post-stroke (time spent on left forelimb relative to right: 39% for lOpg, 40% for lOOpg, 38% for lOOpg Pacific) (Figure 8).

[0249] Discussion and Conclusion

[0250] The study herein demonstrates that treatment with AOS at 3-hours following a central nervous system injury (e.g. a stroke) can lead to significant improvements in motor function and upregulation of brain repair processes following a single intranasal dose. This finding is surprising and contradicts the limitations known in the art: that treatment ischemic brain injury with AOS is only effective when given within a narrow 45-minute window following the ischemic event. Furthermore, the study herein demonstrates that AOS can be delivered non- invasively to the central nervous system through the intranasal route to produce a substantial therapeutic improvement.

[0251] EXAMPLE 3

[0252] EFFECT OF ALLENE OXIDE SYNTHASE ON NEUROLOGICAL RECOVERY IN AN IN VIVO MODEL OF PREFRONTAL CORTEX PHOTOTHROMBIC STROKE

[0253] The Inventions sought to further investigate whether administration of AOS is effective in treating post-stroke degeneration of the central nervous system. To evaluate this, the Inventors used an in vivo model of focal ischemic stroke in the prefrontal cortex as a model of post-stroke dementia, and analysed the potential of AOS to augment post-stroke cognitive impairment.

[0254] Methods

[0255] Animals: young (young (2-3 month old) male C57BL / 6 mice weighing approximately 20-30 g are used as previously described (REF). Animals are acclimatised for at least 7 days prior to experiments. All animals are randomly assigned to a treatment group to ensure that all animals in any given cage received a different treatment (stroke + saline vehicle; stroke + 10 pig AOS; stroke + 100 pig AOS; and stroke + 100 pig AOS (second batch)). All assessments are carried out by observers blinded to the treatment group.

[0256] Photothrombosis model of focal ischemia: Focal stroke is induced to the prefrontal cortex of mice by photothrombosis. Under isoflurane anaesthesia (2-2.5% in a 70% N2O I 30% O2 mixture), mice were placed in a stereotactic apparatus, given a dose of Temgesic, and the surgical site is shaved and swabbed with hibitane. The skull is then exposed through a midline incision, cleared of connective tissue, and dried. Eye ointment (PolyVisc) is applied to the eyes to prevent them from drying out during the surgical procedure. A cold light source (KL1500 LCD, Zeiss) attached to a 40x objective, giving a 2 mm diameter illumination, is positioned as close to the skull as possible, 1.2 mm anterior to Bregma. Rose Bengal solution (0.2 mL, 10 g / L in normal saline, intraperitoneally) was then administered. After 5 minutes, the brain is illuminated through the intact skull for 15 minutes while maintaining body temperature at 37 °C with a heating pad throughout the surgical procedure. Sham surgery mice undergo the same procedure except they receive a saline injection (100 jiL intraperitoneal) instead of Rose Bengal solution.

[0257] AOS Administration: Under sterile conditions, AOS (100 pig) is dissolved in sterile isotonic saline and combined with a hyaluronan / heparan sulfate proteoglycan biopolymer hydrogel (HyStem-C, Biotime Inc, Alameda, CA, USA). AOS is administered at 100 pig / dose dissolved in HyStem-C as per the manufacturer's instructions. Briefly, AOS is added to the HyStem- C / Gelin-S mix (component 1 of hydrogel), followed by the addition of Extralink (component 2 of the hydrogel) in a 4: 1 ratio. The impregnated HyStem-C mix is injected immediately after preparation into the stroke cavity using a 30-guage needle attached to a Hamilton syrine at stereotaxic coordinates 1.2 mm AP, 0 mm ML, and 0.75 mm DV. During all surgical procedures, mice receive Temgesic® (Buprenorphine hydrochloride) as pain relief on the day of surgery as well as the following day. Control animals are injected with HyStem-C without AOS as controls for AOS. AOS or control are administered to directly and locally to the periinfarct cortex at five days post-stroke as previously described by Houlton et al. (Houlton, J., et al., Int. J. Mol. Sci. 2022, 23, 4817). The mice are returned to their home cage after a short recovery period and held under normal housing conditions (12 hour light / dark cycle) with ad libitum access to food and water. Pain relief is administered the following day in the form of temgesic.

[0258] Tissue processing: At 14 days post-stroke, animals are deeply anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde. Brains are then extracted and cut on a sliding freezing stage microtome in six coronal parallel sets in sections of 30 pirn thickness and kept in cryoprotectant at -20 °C.

[0259] Infarct size: Infarct volume is determined by histological assessment using cresyl violet staining according to a standard protocols. Infarct volume is quantified using ImageJ software by an observer blinded to the treatment groups, and is based on obtaining measurements from every sixth section through the entire infarct (area in mm2). Infarct volume is quantified as follows: infarct volume mm3= square root of area mm2x section thickness x section interval.

[0260] Immunofluorescent labelling of GFAP and IBA1: Immunofluorescent labelling of GFAP and IBA1 is performed 35 days post-stroke. Brain sections, every sixth section through the stroke with a thickness of 30 pirn, are rinsed in Tris buffered saline (TBS) and transferred into 1% sodium tetraborate in TBS for 20 minutes at room temperature. The sections are blocked for 60 minutes in TBS containing 5% goat and donkey serum with 0.3% Triton X-100 and incubated in TBS with 2% goat and donkey serum and 0.3% Triton X-100 containing primary polyclonal antibodies for IBA1 or GFAP for 24-48 hours at 4 °C. The rinsed sections are incubated for 2 hours at room temperature in the dark and TBS with 2% normal serum and 0.3% Triton X-100 containing appropriate fluorescent secondary antibodies and nuclear stained Hoechest (1: 1000, Sigma-Aldrich) in TBS for 5 minutes at room temperature. Sections were photomicrographed using an inverted montaging microscope (model: Eclipse Ti2, Nikon, Japan) with a lOx objective lens and exported as nd2 files, and 3 sections from each animal were included in the analysis. Changes in GFAP and IBA1 staining are investigated at 30 days post-stroke in two peri-infarct regions of interest (ROIs, area = 200 pirn x 800 pirn) 0-200 pirn and 600-800 pirn from the stroke boarder. Using FUJI Image J software, the integrated density value (IDV) is measured in both ROIs for GFAP and IBA1.

[0261] Behavioural analysis: The object location recognition task (OLRT) is widely used to evaluate spatial working memory in rodents and reliably identifies relayed-onset impairments in mice exposed to bilateral prefrontal cortex strokes. OLRT testing is conducted at the same time of day at one- and four- week timepoints to minimise variability. All testing is recorded via overhead cameras before being analysed by a blinded researcher on the software TopScan (CleverSys Inc). On the day prior to OLRT testing, animals are placed into the centre of the OLRT arena (400 x 400 x 200 mm, plexiglass) without any objects and are allowed to roam freely for ten minutes to habituate to the arena and testing room. The following day (Day 8 and Day 29 post-stroke) animals undergo the OLRT to evaluate spatial memory. Mice are initially placed in the centre of the arena that contain two identical objects placed in two neighbouring corners (80 mm from the corner walls) for a period of 10 minutes. Immediately following this pre-test phase, mice are returned to their home cage for one hour and the arenas are cleaned. Mice are then placed back into the arena for a testing period of three- minutes, in which the location of one of the objects is moved into the opposing corner. Object exploration is defined when the mice were pointing towards (within a 20 mm perimeter around the object) and sniffing an object. Periods where the mice are either standing or climbing on the objects were excluded from the final analysis. The duration spent in the novel location was assessed as the ratio between total time interacting with one object relative to the total time interacting with both objects. Consistent with what has previously been demonstrated, ceramic bear salt-shakers and soft drink cans were used as OLRT objects at one- and four- weeks post-stroke recovery, respectively. The arenas and all objects are cleaned with 30% ethanol between behavioural runs to prevent the presence of confounding odours.

[0262] Results

[0263] Immunohistochemistry: To confirm an associated reduction in reactive astrogliosis and reactive microgliosis in the stroke animals, GFAP and IBA1 expression (markers of neuroinflammation) were assessed using immunofluorescent labelling 35 days after the infarct. It is observed that AOS significantly reduces reactive gliosis (Figure 9). These findings demonstrate that AOS significantly reduces neuroinflammation.

[0264] Behavioural analysis: The therapeutic potential of AOS is assessed by comparing the performance of sham and stroke animals on OLRT. Consistent with previous reports, a two- way ANOVA assessing the exploratory preference of vehicle and AOS treated animals at one- week post-stroke fails to identify any treatment, stroke, or interaction effects. At four-weeks post-stroke, however, a significant overall effect of stroke only was observed demonstrating impaired spatial working memory. In contrast, treatment with AOS significantly prevents cognitive impairment at 4 weeks post stroke (Figure 10). There was no significant difference in cognitive ability and memory between AOS treated and non-stroked (control) animals. Discussion and Conclusion

[0265] The study herein demonstrates that treatment with AOS is effective in treating cognitive impairment and neuroinflammation after brain insult. GFAP and IBA1 are well established markers of neuroinflammation and reactive gliosis associated with several neurodegenerative diseases, including for example Alzheimer's disease, dementia, stroke, stroke-induced cognitive decline, postoperative cognitive dysfunction, HIV-associated dementia, Parkinson's disease, Motor neurone diseases, amyotrophic lateral sclerosis (ALS, Lou Gehring's disease), Huntington's disease. Reductions in these neuroinflammatory markers have a known association with disease improvement.

[0266] The strong neuroprotective activity of AOS combined with the reduction in microgliosis, astrogliosis, the effective intranasal delivery of AOS to the CNS, and the substantial improvement in cognitive impairment in the object location recognition task demonstrated herein suggests that AOS may be an effective treatment across a wide range of neurodegenerative and neuroinflammatory conditions where oxidative stress plays a fundamental role, including for example Alzheimer's disease, dementia, stroke, stroke- induced cognitive decline, postoperative cognitive dysfunction, HIV-associated dementia, Parkinson's disease, Motor neurone diseases, amyotrophic lateral sclerosis (ALS, Lou Gehring's disease), Huntington's disease.

Claims

CLAIMS1. A method of improving recovery post central nervous system injury in a subject, comprising administering to said subject a composition comprising allene oxide synthase, or a functionally equivalent variant or derivative thereof.

2. The method of claim 1, wherein allene oxide synthase is selected from the group comprising SED ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, or SEQ ID: 9, or functionally equivalent derivatives or variants thereof.

3. The method of claim 1 or claim 2, wherein the composition is administered at least about 3 hours post central nervous system injury.

4. A method according to anyone of claims 1 to 3, wherein the composition is administered to a subject at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours post-injury.

5. A method according to any one of claims 1 to 4, wherein the composition is administered to a subject at least about one day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days or at least about 14 days post central nervous system injury.

6. A method according to any one of claims 1 to 5, wherein the composition is formulated for oral, intravenous, subcutaneous, intraperitoneal, sublingual, intranasal, inhalation, local, intrathecal, intraocular, intramuscular, intracranial, or systemic administration to the subject.

7. A method according to claim 6, where the composition is formulated as an aqueous solution for intranasal administration to a subject.

8. A method according to any one of claims 1 to 7, wherein the composition additionally comprises a non-ionic surfactant.

9. A method according to claim 8, wherein the non-ionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-XlOO, or any other pharmaceutically acceptable surfactant.

10. A method according to any one of claims 1 to 9, wherein allene oxide synthase is derived from Parthenium argentatum.

11. A method according to claim 10, wherein allene oxide synthase from Parthenium argentatum is SEQ ID: 1.

12. A method according to any one of claims 1 to 11, wherein the central nervous system injury is selected from ischemic stroke, haemorrhagic stroke, white matter stroke, subcortical stroke, retinal vein occlusion, traumatic brain injury, concussion injury or mild-to-moderate traumatic brain injury, and spinal cord injury.

13. The use of allene oxide synthase, or a functionally equivalent variant or derivative thereof, in the manufacture of a medicament for the treatment of central nervous system injury.

14. The use of claim 13, wherein allene oxide synthase is selected from the group comprising SED ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5, SEQ ID: 6, SEQ ID: 7, SEQ ID: 8, or SEQ ID: 9, or functionally equivalent derivatives or variants thereof.

15. The use of claim 13 or 14, wherein the medicament is to be administered at least about 3 hours post central nervous system injury.

16. The use according to any one of claims 13 to 15, wherein the medicament is to be administered at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours post-injury.

17. The use according to any one of claims 13 to 16, wherein the medicament is to be administered at least one day, at least about one day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11days, at least about 12 days, at least about 13 days or at least about 14 days post central nervous system injury.

18. The use according to any one of claims 13 to 17, wherein the medicament is formulated for oral, subcutaneous, intraperitoneal, intraocular, intrathecal, intramuscular, intravenous, intranasal, intracranial, or local administration.

19. The use according to claim 18, wherein the medicament is formulated as an aqueous solution for intranasal administration.

20. The use according to any one of claims 13 to 19, wherein the medicament additionally comprises a non-ionic surfactant.

21. The use according to claim 20, where the non-ionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-XlOO, or any other pharmaceutically acceptable surfactant.

22. The use according to any one of claims 13 to 21, wherein allene oxide synthase is derived from Parthenium argentatum.

23. The use according to claim 22, wherein allene oxide synthase from Parthenium argentatum is SEQ ID: 1.

24. The use according to any one of claims 1 to 11, wherein the central nervous system injury is selected from ischemic stroke, haemorrhagic stroke, white matter stroke, subcortical stroke, retinal vein occlusion, traumatic brain injury, concussion injury or mild-to-moderate traumatic brain injury, and spinal cord injury.

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