Compositions and methods for treating central nervous system degeneration

Abstract

The present invention relates to methods of treating chronic degenerative conditions of the central nervous system related to oxidative stress, comprising administering a composition comprising allene oxide synthase (AOS), or a functional equivalent variant thereof.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING CENTRAL NERVOUS SYSTEM DEGENERATION

[0002] Field of Invention

[0003] The present invention relates to methods of treating chronic degenerative conditions of the central nervous system related to oxidative stress. More particularly, the invention relates to methods and uses of allene oxide synthase (AOS) or functionally equivalent variants or derivatives thereof in the treatment of such conditions.

[0004] Background

[0005] Oxidative stress is an excess accumulation of free radicals such as nitrogen oxide (NO-) and reactive oxygen species (ROS) such as superoxide (O?'), hydroxide radical (OH-), and hydroperoxide (HOO-) that arise from an imbalance between the local or systemic production of ROS and a biological systems ability to readily detoxify the reactive intermediates and / or repair the resulting damage. Accumulation of free radicals occurs during this mismatch between the generation and elimination of free radicals causing toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Oxidative stress in the central nervous system can lead to oxidative cell injury, for example damage to neuronal and vascular systems. Cell injury can lead to inflammation and apoptosis.

[0006] Many chronic degenerative conditions of the central nervous system are associated with oxidative stress. Examples of such conditions include dementia, aging, HIV-associated dementia, Alzheimer’s disease, Parkinson’s disease, Motor neurone disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), and Huntington’s disease.

[0007] There are limited treatment options for central nervous system degenerative conditions related to oxidative stress, and fewer that offer a significant benefit to patients. Antioxidant therapy and free radical scavengers are the mainstay approach to counteracting the detrimental effects of oxidative stress. These approaches are dramatically limited by their reaction stoichiometry where one antioxidant or free radical scavenger quenches one free radical species, providing limited attenuation of oxidative cell damage during the propagating free radical cascade. Edaravone is one of the few examples of stoichiometric free radical scavengers to receive clinical approval for use in stroke and amyotrophic lateral sclerosis. However, a minor clinical benefit has been demonstrated in less than 5% of the ALS population. There remains a need for improved approaches to treating neurodegenerative diseases related to oxidative stress.

[0008] AOS 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.

[0009] It is an object of the invention to provide methods of treating chronic degenerative conditions of the central nervous system related to oxidative stress. It is an alternative objective of the invention to provide medicaments for the treatment of such conditions.

[0010] Alternatively, it is an object of the invention to at least provide a useful choice to the public.

[0011] Summary of the Invention

[0012] In one example there is provided a method of treating chronic degenerative conditions of the central nervous system related to oxidative stress in a subject, comprising administering to said subject allene oxide synthase, or a functionally equivalent variant thereof.

[0013] In one aspect the composition comprises a therapeutically effective amount of allene oxide synthase selected from the group 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, or a functionally equivalent variant thereof.

[0014] In one aspect the conditions of the central nervous system related to oxidative stress are neuronal-degenerative conditions.

[0015] In one aspect the conditions of the central nervous system related to oxidative stress are vascular-degenerative conditions.

[0016] In one aspect the conditions of the central nervous system related to oxidative stress are glial cell degenerative conditions. In one aspect the chronic degenerative conditions of the central nervous system are selected from any one or more of the following: dementia, aging, HIV-associated dementia, post-stroke cognitive decline, stroke-induced dementia, postoperative cognitive dysfunction or surgically- induced cognitive impairment. Alzheimer’s disease, Parkinson’s disease, Motor neurone disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), Huntington’s disease. More preferably Alzheimer’s disease, dementia, stroke-indued cognitive impairment, or postoperative cognitive impairment.

[0017] In one aspect allene oxide synthase is administered prophylactically.

[0018] In one aspect allene oxide synthase is administered orally, rectally or by injection, such as cutaneous, subcutaneous, intravenous injection, intrathecal injection, or by direct local injection to the CNS.

[0019] In one aspect the injection is formulated for controlled release.

[0020] In one aspect allene oxide synthase is administered intranasally.

[0021] In another example there is provided there is provided the use of allene oxide synthase, or a functionally equivalent variant thereof, in the manufacture of a medicament for the treatment of chronic degenerative conditions of the central nervous system related to oxidative stress.

[0022] In one aspect the allene oxide synthase is selected from the group 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, or a functionally equivalent variant thereof.

[0023] In one aspect the conditions of the central nervous system related to oxidative stress are neuronal-degenerative conditions.

[0024] In one aspect the conditions of the central nervous system related to oxidative stress are vascular-degenerative conditions.

[0025] In one aspect the conditions of the central nervous system related to oxidative stress are glial cell degenerative conditions. In one aspect the chronic degenerative conditions of the central nervous system are selected from any one or more of the following: dementia, aging, HIV-associated dementia, post-stroke cognitive decline, stroke-induced dementia, postoperative cognitive dysfunction or surgically- induced cognitive impairment. Alzheimer’s disease, Parkinson’s disease, Motor neurone disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), stroke (both ischemic and haemorrhagic), and Huntington’s disease. More preferably Alzheimer’s disease, dementia, stroke-indued cognitive impairment, or postoperative cognitive impairment.

[0026] In one aspect the medicament is administered prophylactically.

[0027] In one aspect the medicament is administered orally, rectally or by injection, such as cutaneous, subcutaneous, intravenous injection, intrathecal injection, or by direct local injection to the CNS.

[0028] In one aspect the injection is formulated for controlled slow release.

[0029] In one aspect the medicament is administered intranasally.

[0030] In one aspect the medicament is administered intranasally to the central nervous system.

[0031] In one aspect the medicament is formulated for intranasal use.

[0032] In another example there is provided a pharmaceutical composition comprising allene oxide synthase, or a functionally equivalent variant thereof for treating chronic degenerative conditions of the central nervous system.

[0033] In one aspect the allene oxide synthase is selected from the group 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, or a functionally equivalent variant thereof.

[0034] In one aspect the conditions of the central nervous system related to oxidative stress are neuronal-degenerative conditions.

[0035] In one aspect the conditions of the central nervous system related to oxidative stress are vascular-degenerative conditions. In one aspect the conditions of the central nervous system related to oxidative stress are glial cell degenerative conditions.

[0036] In one aspect the chronic degenerative conditions of the central nervous system are selected from any one or more of the following: dementia, aging, HIV-associated dementia, post-stroke cognitive decline, stroke-induced dementia, postoperative cognitive dysfunction or surgically- induced cognitive impairment. Alzheimer’s disease, Parkinson’s disease, Motor neurone disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), and Huntington’s disease. More preferably Alzheimer’s disease, dementia, stroke-indued cognitive impairment, or postoperative cognitive impairment.

[0037] In one aspect the pharmaceutical composition is to be administered prophylactically.

[0038] In one aspect the pharmaceutical composition is to be administered orally, rectally or by injection, such as cutaneous, subcutaneous, intravenous injection, intrathecal injection, or by direct local injection to the CNS.

[0039] In one aspect the pharmaceutical composition is an injection formulated for controlled slow release.

[0040] In one aspect the pharmaceutical composition is to be administered intranasally.

[0041] In one aspect the pharmaceutical composition is to be administered nasally to the central nervous system.

[0042] In one aspect pharmaceutical composition is formulated for intranasal use.

[0043] In yet another example there is provided allene oxide synthase, or a functionally equivalent variant thereof, for use in treating chronic degenerative conditions of the central nervous system related to oxidative stress.

[0044] In one aspect the AOS has the amino acid sequence selected from the group 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, or a functionally equivalent variant thereof. In one aspect the conditions of the central nervous system related to oxidative stress are neuronal-degenerative conditions.

[0045] In one aspect the conditions of the central nervous system related to oxidative stress are vascular-degenerative conditions.

[0046] In one aspect the conditions of the central nervous system related to oxidative stress are glial cell degenerative conditions.

[0047] In one aspect the chronic degenerative conditions of the central nervous system are selected from any one or more of the following: dementia, aging, HIV-associated dementia, post-stroke cognitive decline, stroke-induced dementia, postoperative cognitive dysfunction or surgically- induced cognitive impairment. Alzheimer’s disease, Parkinson’s disease, Motor neurone disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), and Huntington’s disease. More preferably Alzheimer’s disease, dementia, stroke-indued cognitive impairment, or postoperative cognitive impairment.

[0048] In one aspect the AOS is to be administered prophylactically.

[0049] In one aspect the AOS is to be administered orally, rectally or by injection, such as cutaneous, subcutaneous, intravenous injection, intrathecal injection, or by direct local injection to the CNS.

[0050] In one aspect the injection is formulated for controlled slow release.

[0051] In one aspect the AOS is administered intranasally to the central nervous system.

[0052] In one aspect the AOS is formulated for intranasal use.

[0053] Further aspects of the invention, which should be considered in all its aspects, will become apparent to those skilled in the art upon reading of the following description which provides at least one example of a practical application of the invention.

[0054] Brief Description of the Drawings

[0055] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0056] Figure 1 illustrates the repair mechanism of AOS in relation to lipid peroxide / hydroperoxide formation during oxidative stress.

[0057] Figure 2 illustrates SEQ ID No. 1-9.

[0058] Figure 3 illustrates the neuroprotective effect of AOS on primary rodent cortex cells in an oxidative stress paradigm when assaulted with hydrogen peroxide (H2O2).

[0059] Figure 4 illustrates the impact of AOS on neuroinflammation (IBA1) in a focal photothromobic model of ischemic stroke (motor cortex) when administered intranasally.

[0060] Figure 5 illustrates the impact of AOS on reactive astrogliosis (GFAP) in a focal photothromobic model of ischemic stroke (motor cortex) when administered intranasally.

[0061] Figure 6 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.

[0062] Figure ?: illustrates the impact of AOS on astrocyte reactivity (measured by GFAP staining) in the white matter, infralimbic and peri-infarct zones when administered via direct local injection 5-days post-infarct.

[0063] Figure 8: illustrates the impact of AOS vascularisation and angiogenesis when administered via direct local injection 5-days post-infarct.

[0064] Detailed Description of Preferred Embodiments

[0065] In general terms, the invention relates to therapies for treatment of chronic degenerative conditions of the central nervous system related to oxidative stress. The invention also includes pharmaceutical compositions of use to treat such conditions and methods of treatment. 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.

[0066] 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.

[0067] 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 self-regenerate, requiring neither oxygen nor a NADPH reductase for activity and have extraordinary reaction rates. AOS enzymes transform 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.

[0068] Many chronic degenerative conditions of the central nervous system are associated with oxidative stress. Examples of such conditions include dementia, aging, post-stroke cognitive impairment, post-stroke dementia, post-operative cognitive dysfunction, post-operative dementia, HIV-associated dementia, Alzheimer’s disease, Parkinson’s disease, Motor neurone disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), and Huntington’s disease. Treatment of these conditions by administration of AOS is to be considered an aspect of the current invention. One of the detrimental effects of oxidative stress in the central nervous system is oxidative cell injury, for example neuronal, glial cell and vascular damage. Cell injury due to oxidative stress can lead to apoptosis (programmed cell death) and inflammation.

[0069] One of the mechanisms known to lead to cell damage is the formation of lipid peroxides / hydroperoxides by free radicals such as nitrogen oxide and other reactive oxygen species (ROS) associated with oxidative stress. Lipid peroxides / hydroperoxides can spontaneously generate additional peroxides by chain propagation reactions. The accumulation of these lipid peroxides in the central nervous system can lead to severe oxidative cell injury.

[0070] While not wishing to be bound by theory, it is believed the action of AOS breaks the chain propagation of peroxides and therefore acts as a form of repair mechanism (see Figure 1 ). It is believed AOS converts lipid peroxides / hydroperoxides to lipid epoxides. The lipid epoxides are then thought to undergo decomposition to ketols, therefore breaking the chain reaction.

[0071] If left untreated lipid peroxides / hydroperoxides are thought to trigger apoptosis (programmed cell death) and ferroptosis (cell death triggered by lipid peroxidation). A further aspect of the invention should therefore be considered to be a method of treating chronic degenerative conditions of the central nervous system related to apoptosis or ferroptosis comprising the step of administering AOS protein a functionally equivalent variant thereof to a subject.

[0072] Lipid peroxides / hydroperoxides, apoptosis and oxidative stress have also been associated with inflammation. Accordingly a still further aspect of the invention should be considered to be a method of treating chronic degenerative conditions of the central nervous system related to inflammation comprising the step of administering AOS protein or a functionally equivalent variant thereof to a subject.

[0073] In a preferred embodiment the conditions related to oxidative stress which may be treated with AOS are vascular-degenerative conditions. Oxidative stress is one of the major causes and mechanisms of vascular damage / degeneration. The role of vascular degeneration and dysfunction in brain aging has been suggested by clinical research that demonstrates the reduction of cerebral flow and volume from middle age. Therefore a further preferred embodiment of the invention is the use of AOS to treat / mitigate the effects of aging on the central nervous system. In addition AOS is particularly suited to treatment of chronic degenerative condition because it is an enzyme that functions long after administration.

[0074] Further, virtually all antioxidant enzymes, such as catalase and superoxide dismutase, also generate a secondary pro-oxidant radical species that requires a second enzyme to remove it. AOS is believed to act against lipid hydroperoxides and is able to act alone, without a second enzyme.

[0075] In some cases AOS it is beneficial to administer AOS prophylactically. For example where there is a family history of a chronic degenerative condition of the central nervous system, it may be beneficial to administer AOS prior to the appearance of noticeable symptoms, as initial vascular damage may not be noticeable without extensive testing. AOS is also useful in delaying degeneration of the central nervous system associated with aging. In this case, it is beneficial to administer AOS prior to the appearance of significant symptoms associated with aging of the central nervous system. Oxidative stress is believed to be an early event of degenerative cascades, which is an on-going process during degeneration of the central nervous system. The ability of AOS to mitigate oxidative stress makes it particularly suited to prophylactic use, early use, or prior to the appearance of significant / noticeable symptoms.

[0076] The preferred compositions and methods of treatment will thus include controlled slow release options. Treatments of such conditions are likely to be relatively long term and generally not a single / immediate treatment. Therefore controlled slow release options allow for reduced numbers of treatments which will be more convenient for the user. This will also most likely improve uptake of the treatment by the user.

[0077] A therapeutically effective amount of AOS is administered in accordance with the invention in form of a composition including AOS and pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabiliser. The pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabiliser should be non-toxic and should not detrimentally interfere with the efficacy of the AOS. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, such as cutaneous, subcutaneous, or intravenous injection. A “therapeutically effective amount”, is to be understood as an amount of an AOS that is sufficient to show retardation of the progress of the chronic condition. In some cases there may be cessation of the progress of the chronic condition. However, in the case of some conditions, for example aging, cessation would not be expected. The actual amount, rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment is within the responsibility of general practitioners and other medical doctors.

[0078] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such as gelatin.

[0079] For intravenous, cutaneous or subcutaneous injection, the AOS will be in the form of a parenterally acceptable solution which has a suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride injection, Ringer’s injection, Lactated Ringer’s injection. Preservatives, stabilisers, buffers antioxidants and / or other additives may be included as required.

[0080] For intranasal administration, the AOS will be in the form of an aqueous, hydroalcoholic, nonaqueous, suspension, or emulsion suitable for intranasal administration to a patient which has a suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride. Preservatives, stabilisers, buffers, antioxidants, and / or other additives such as penetration enhancers (e.g. compounds to improve absorption or penetration, such as chitosan, ) and mucoadhesive agents (e.g. hydroxypropyl carboxymethylcellulose, polyethylene glycol) may be included as required.

[0081] For direct local injection, the AOS will be in the form of an aqueous hydrogel (also called an aquagel) preparation for administration to, for example, the spinal cord, ventricles, or the brain (e.g., the stroke or infarction core), of a patient, which has a suitable pH, viscosity, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable hydrogel preparation using, for example, hyaluronic acid (e.g. HyStem™), alginate, chitosan, or heparan based hydrogels. Preferably, the hydrogel preparation used comprises a thiol-modified hyaluronan, thiol-modified gelatin, hyaluronan, gelatin, heparin, thiol-modified heparin, thiol- modified chondroitin sulfate, a hyaluronan sodium salt, and / or an acrylated hyaluronic acid. Preservatives, stabilisers, buffers, antioxidants, and / or other additives such as penetration enhancers (e.g. compounds to improve absorption or penetration, such as chitosan, ) and mucoadhesive agents (e.g. hydroxypropyl carboxymethylcellulose, polyethylene glycol) may be included as required.

[0082] The compositions, methods of treatment and uses of the invention optionally comprises one or more additional active compounds in addition to AOS protein or a functionally equivalent variant thereof. Preferably, the one or more additional active compounds are therapeutically active compounds, for example in the form of an additional therapeutic compound for co-delivery with the AOS protein or a functionally equivalent variant thereof.

[0083] In one embodiment of the invention uses AOS protein as described 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, or a functionally equivalent variant of this protein.

[0084] The phrase “functionally equivalent variants” as used herein, includes those peptides or proteins having one or more (for example, 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5): deletions, additions and / or substitutions; while substantially retaining the desired function of the protein. In one particular embodiment, the amino acid substitutions are conservative amino acid substitutions.

[0085] The functionally equivalent variants will have anti-oxidant activity. In one particular embodiment they will have the ability to convert lipid peroxides / hydroperoxides to lipid epoxides. In one particular embodiment, they will have the ability to convert lipid peroxides / hydroperoxides to lipid epoxides at the lipid / cel I membrane interface.

[0086] It should be appreciated that a “functionally equivalent variant” may have a level of activity higher or lower than the protein of which it is a variant. In various embodiments of the invention a functionally equivalent variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the level of activity of the protein of which it is a variant. It should be appreciated that the term “recovery”, “functional recovery”, “improved recovery” or “improving recovery” means an improvement in motor function or cognitive performance of a subject or a patient afflicted with a neurodegenerative disease. As an example, an improvement in motor function may comprise an improvement in motor coordination, and / or balance, and / or gait, and / or speech. As another example, an improvement in cognitive performance may comprise an increase and / or improvement in performance 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. It certain examples, an improvement is an improvement in the degree of recovered function and / or performance, and / or the rate of recovery or improvement of function and / or performance.

[0087] Skilled persons will readily be able to assess function and determine the level of activity of a protein or functionally equivalent variants of use in the invention, based on the information contained herein, and using techniques known in the art. However, by way of example, one can determine anti-oxidant activity using the methods described in Pinchuk et al, Chemistry and Physics of Lipids 164 (2001) 42-48 or using a commercially available assay kit available thought Sigma-Aldrich the details of which are set out in Example 1 . .

[0088] As used herein “conservative amino acid substitution(s)” should be taken broadly to mean substitution of amino acids that have similar biochemical properties. Persons skilled in the art will appreciate appropriate conservative amino acid substitutions based on the relative similarity between different amino acids, including the similarity of the amino-acid side chain substituents (for example, their size, charge, hydrophilicity, hydrophobicity and the like). By way of example, a conservative substitution includes substitution of one aliphatic amino acid for another aliphatic amino acid, substitution of an amino acid with a hydroxyl- or sulphur- containing side chain with another amino acid with a hydroxyl- or sulphur-containing side chain, substitution of an aromatic amino acid with another aromatic amino acid, substitution of a basic amino acid with another basic amino acid, or substitution of an acidic amino acid with another acid amino acid. By way of further example, “conservative amino acid substitution(s)” include: substitution of Glycine, Alanine, Valine, Leucine, or Isoleucine, one for another substitution of Serine, Cysteine, Theronine, or Methionine, one for another substitution of Phenylalanine, Tyrosine, or Tryptophan, one or another substitution of Histidine, Lysine, or Arginine, one for another substitution of Aspartic acid, Glutamic acid, Asparagine or Glutamine, one for another Functionally equivalent variants will preferably retain at least 70%, 80%, 90%, 95% or 99% amino acid sequence similarity to an AOS specifically referred to herein (for example AOS on Genbank CAA55025.2). In one embodiment, the functionally equivalent variant has at least 70%, 80% 90%, 95% or 99% sequence identity with an AOS protein specifically referred to herein.

[0089] Functionally equivalent variants may be composed of L-amino acids, D-amino acids or a mixture thereof and may include non-naturally occurring amino acids.

[0090] A protein or peptide of use in the invention may be isolated from natural sources, or derived by chemical synthesis (for example, fmoc solid phase peptide synthesis as described in Fields GB, Lauer-Fields JL, Liu RQ and Barany G (2002) Principles and Practice of Solid-Phase peptide Synthesis; Grant G (2002) Evaluation of the Synthetic Product. Synthetic Peptides, A User’s Guide, Grant GA, Second Edition, 93-219; 220-291 , Oxford University Press, New York) or genetic expression techniques, methods for which are readily known. Standard recombinant DNA and molecular cloning techniques are described for example in: Sambrook, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Silhavy et al., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and, Ausubel et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-lnterscience (1987). The inventor’s also contemplate production of a protein or peptide of use in the invention by an appropriate transgenic animal, microbe, or plant.

[0091] Those of general skill in the art to which the invention relates will readily be able to identify a variety of nucleic acids which encode the proteins and functionally equivalent variants of use in the invention on the basis of the amino acid sequences provided herein, the genetic code and the understood degeneracy therein.

[0092] In one embodiment, the protein or functionally equivalent variant thereof may be connected to one or more additional compounds. For example, they may be connected to one or more additional compounds that aids the function or activity of the protein, protects the protein from degradation, otherwise improves its half-life, aids in isolation and / or purification of the protein during manufacture (for example ubiquitin, a his-tag, or biotin), or assists with cell membrane translocation or cell-specific targeting. The one or more additional compounds may be of any desirable nature and include, for example, peptides, nucleic acids, lipids and carbohydrates.

[0093] The compounds may be connected to the protein, or synthesised as a part of a construct, using any appropriate means which allows the protein to retain at least a level of its desired function. The word “connected” or like terms should be taken broadly to encompass any form of attachment, bonding, fusion or association between the peptide and the at least one compound (for example, but not limited to, covalent bonding, ionic bonding, hydrogen bonding, aromatic stacking interactions, amide bonds, disulfide bonding, chelation) and should not be taken to imply a particular strength of connection. The protein and the at least one compound may be connected in an irreversible or a reversible manner, such that upon administration the protein is released from the compound.

[0094] The at least one compound may be connected to the protein at its N-terminus, its C-terminus or at any other location.

[0095] It should be appreciated that while the protein and at least one compound may be connected directly to one another, linker molecules could also be used.

[0096] Example 1 - Neuroprotection of AOS against hydrogen peroxide injury in primary excised rat cortex.

[0097] The effect of allene oxide synthase (AOS) on neuronal protection was assessed in a cerebellar microexplant culture system in vitro. A hydrogen peroxide-induced neuronal injury paradigm was used which induces oxidative stress resulting in neuronal cell death.

[0098] Methods

[0099] Cerebellar Microexplant System

[0100] 1. Extraction of cerebellar tissue

[0101] 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 urn 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.

[0102] 2. Culture of cerebellar cells

[0103] Two hours after poly-L-lysine coating, glass slides were washed with Millipore water and airdried. Each side was placed into a small petri dish (diameter: 35 mm) and 40 uL of STAART V / cell suspension added. The tissue was incubated for 2 hours 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.

[0104] 3. Drug application

[0105] 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 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 2 was conducted to confirm the positive results obtained from Study 1. In each study 10 nM glypromate was included as a positive control. In all studies, drugs were left in contact with the explants for the duration of the study, which was 24 hours.

[0106] 4. Determination of drug effect

[0107] After the explants were exposed to drugs (toxin / AOS) for 24 hours, 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.

[0108] 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.

[0109] Results

[0110] 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). In contrast, treatment with 10 nM glypromate (positive control) resulted in little or no neuroprotection. AOS produced a mean neuronal recovery from hydrogen peroxide injury of 23%. In comparison, glypromate only produced a 4% recovery from injury (Figure 3).

[0111] Discussion and Conclusion

[0112] The study herein demonstrates that AOS enzymes are able to rescue neurons from hydrogen peroxide injury within postnatal cerebellar granule cells organised as cerebellar microexplants to a significant degree. Furthermore, treatment with AOS resulted in an approximately 5.75x increase in neuronal survival compared with treatment with glypromate (positive control) in this oxidative stress-induced neuronal injury paradigm.

[0113] Glypromate, or N-terminal glycine-proline-glutamate (GPE), is a bioactive neurotripeptide that is naturally cleaved from insulin-like growth factor 1 (IGF-1 ) by an acid protease that mimics the action of IGF-1 signalling and confers neuroprotective effects. Glypromate and closely related analogues (for example trofinetide and NNZ-2591 ) have shown broad-spectrum neuroprotective activity and mitigate glutamate excitotoxicity in a number of neurodegenerative and neuroinflammatory disorders and animal models, including for example Alzheimer’s disease (Biomolecules, 2021 , 11 (1 ), 126); Parkinson’s disease; amyotrophic lateral sclerosis (ALS); peripheral neuropathy; ischemic stroke; ischemia-reperfusion injury; traumatic brain injury; postoperative cognitive impairment, Autism spectrum disorder; Fragile X Syndrome; Friedreich’s ataxia; Huntington’s disease; cognitive impairment and epilepsy.

[0114] The superior neuroprotective activity of AOS when compared with glypromate in the H2O2 insult model suggests a neuroprotective role for AOS across a wide range of neurodegenerative and neuroinflammatory conditions where oxidative stress plays a fundamental role.

[0115] Example 2 - Impact of AOS on focal photothromobic stroke in mice when administered intranasally.

[0116] The Inventions sought to investigate whether administration of AOS enzymes non-invasively through the intranasal route is effective in treating degenerative diseases of the central nervous system related to oxidative stress. To evaluate this route of administration, the Inventors used an in vivo model of focal ischemic stroke and analysed the potential of AOS to augment gliosis poststroke.

[0117] Methods

[0118] Animals: young (young (2-3 month old) male C57BL / 6 mice weighing approximately 20-30 g are used as previously described (Clarkson, A. N., et al., Nature, 2010, 468 (7321 ), 305-309; Leurs, U., PNAS, 2021 , 118 (31 ), e2108079118). 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 pg AOS; stroke + 100 pg AOS; and stroke + 100 pg AOS (second batch)). All assessments are carried out by observers blinded to the treatment group.

[0119] Photothrombosis model of focal ischemia: Focal stroke is induced in the 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, 1.5 mm lateral from 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 pL intraperitoneal) instead of Rose Bengal solution.

[0120] AOS Administration: AOS ( 10 or 100 pg) is dissolved in sterile isotonic saline. AOS is administered at low (10 pg) or high (100 pg) dose. Control animals are injected with saline to serve as controls for AOS. AOS or saline are administered intranasally 3-hours after the stroke. 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.

[0121] 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 pm thickness and kept in cryoprotectant at -20 °C.

[0122] Infarct size: Infarct volume is determined by histological assessment using cresyl violet staining according to a standard protocols. Infarct volume is quantified using Imaged 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.

[0123] Immunofluorescent labelling of GFAP and IBA1 : Immunofluorescent labelling of GFAP and IBA1 is performed 14 days post-stroke. Brain sections, every sixth section through the stroke with a thickness of 30 pm, 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. Images are taken with an inverted montaging microscope (model: Eclipse Ti2, Nikon, Japan) with a 10x 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 14 days 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 Fiji Imaged software (National Institute of Health, USA), the integrated density value (IDV) is measured in both ROIs for GFAP and IBA1 .

[0124] Results

[0125] Immunohistochemistry: To investigate whether AOS administered intranasally to the central nervous system can reduce reactive astrogliosis and reactive microgliosis in the stroke animals, GFAP and IBA1 expression (markers of neuroinflammation) were assessed using immunofluorescent labelling 14 days after the infarct. It is observed that intranasal administration of AOS significantly reduces microgliosis (IBA1 ) (Figure 4) and reactive astrogliosis (GFAP) (Figure 5) in the stroke core and peri-infarct zone. These findings demonstrate that AOS significantly reduces neuroinflammation. Discussion and Conclusion

[0126] The study herein demonstrates that intranasal administration of AOS enzymes is an effective delivery mechanism to significantly rescue neurons and reduce 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.

[0127] The superior neuroprotective activity of AOS when compared with glypromate in the H2O2 insult model, combined with the reduction in microgliosis, astrogliosis, and effective delivery of AOS enzymes to the CNS demonstrated intranasally 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.

[0128] Example 3 - Impact of AOS on post-stroke cognitive impairment in a focal photothrombic prefrontal cortex model.

[0129] The Inventions sought to further investigate whether administration of AOS enzymes is effective in treating degenerative diseases of the central nervous system related to oxidative stress. To evaluate this, the Inventors used an in vivo model of focal ischemic stroke in the prefrontal cortex as a model of dementia, and analysed the potential of AOS to augment post-stroke cognitive impairment.

[0130] Methods

[0131] Animals: young (2-3 month old) male C57BL / 6 mice weighing approximately 20-30 g are used as previously described (Zhou, L. Y. Y., et al., Behav. Brain Res. 2016 (296), 373-378; Houlton, J., et al., Neurobiol. Learn Mem., 2021 (177), 107355). 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 pg AOS; stroke + 100 pg AOS; and stroke + 100 pg AOS (second batch)). All assessments are carried out by observers blinded to the treatment group. 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% N20130% 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 pL intraperitoneal) instead of Rose Bengal solution.

[0132] AOS Administration: Under sterile conditions, AOS (100 pg) 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 pg / dose dissolved in HyStem-C as per the manufacturers 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 syringe 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 peri-infarct cortex at five days poststroke 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.

[0133] 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 pm thickness and kept in cryoprotectant at -20 °C. Infarct size: Infarct volume is determined by histological assessment using cresyl violet staining according to a standard protocols. Infarct volume is quantified using Imaged 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.

[0134] 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 pm, 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. Images are taken with an inverted montaging microscope (model: Eclipse Ti2, Nikon, Japan) with a 10x 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 pm x 800 pm) 0-200 pm and 600-800 pm from the stroke boarder. Using FUJI Image J software, the integrated density value (IDV) is measured in both ROIs for GFAP and IBA1.

[0135] 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 poststroke) 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 10% ethanol and water between behavioural runs to prevent the presence of confounding odours.

[0136] Vascular analysis: 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 10x objective lens and exported as nd2 files. Images were then opened using Fiji Imaged 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 Imaged 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.

[0137] Results

[0138] Behavioural analysis: The therapeutic potential of AOS is assessed by comparing the performance of sham and stroke animals on OLRT. Consistent with previous reports (XX YY), 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 contract, treatment with AOS significantly prevents cognitive decline and impaired spatial working memory (Figure 6).

[0139] Immunohistochemistry: To confirm an associated reduction in reactive astrogliosis and reactive microgliosis in the stroke animals, GFAP and IBA1 expression (markers of reactive astrogliosis and neuroinflammation) were assessed using immunofluorescent labelling 35 days after the infarct. It is observed that AOS significantly reduces reactive gliosis (Figure 7). These findings demonstrate that AOS significantly reduces neuroinflammation.

[0140] Vascular analysis: To investigate where AOS administration intranasally to the central nervous system can improve vascularisation following focal ischemic stroke, blood vessel analysis was performed to assess post-stroke angiogenesis. It was observed that treatment with AOS significantly alters cortex and peri-infarct vascularisation, leading to a significant improvement in blood vessel numbers, vessel lengths and vessel volumes (Figure 8).

[0141] Discussion and Conclusion

[0142] The study herein demonstrates that treatment with AOS enzymes 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 and glial cell degenerative conditions, 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. The study further demonstrates that treatment with AOS significantly improves vascularisation, a key feature for the treatment of vascular-degenerative conditions.

[0143] The superior neuroprotective activity of AOS when compared with glypromate in the H2O2 insult model, combined with the reduction in microgliosis, astrogliosis, the effective intranasal delivery of AOS enzymes 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. General

[0144] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of “including, but not limited to”.

[0145] The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

[0146] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour in any country in the world.

[0147] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

[0148] Wherein the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

[0149] It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the scope of the invention.

Claims

CLAIMS1. A method of treating chronic degenerative conditions of the central nervous system related to oxidative stress in a subject, comprising administering to said subject allene oxide synthase, or a functionally equivalent variant thereof.

2. The method according to claim 1 wherein allene oxide synthase selected from the group 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, or functionally equivalent variants thereof.

3. The method of claim 1 or claim 2 wherein the conditions of the central nervous system related to oxidative stress are neuronal-degenerative conditions.

4. The method of claim 1 or claim 2 wherein the conditions of the central nervous system related to oxidative stress are vascular-degenerative conditions.

5. The method of claim 1 or claim 2 wherein the conditions of the central nervous system related to oxidative stress are glial cell degenerative conditions.

6. A method according to anyone of claims 1 to 5 wherein the chronic degenerative conditions of the central nervous system are selected from any one or more of the following: dementia, aging, HIV-associated dementia, post-stroke cognitive decline, stroke-induced dementia, postoperative cognitive dysfunction or surgically-induced cognitive impairment. Alzheimer’s disease, Parkinson’s disease, Motor neurone disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), Huntington’s disease, Alzheimer’s disease, dementia, stroke- indued cognitive impairment, or postoperative cognitive impairment.

7. A method according to any one of claims 1 to 6 wherein said allene oxide synthase is administered prophylactically.

8. A method according to any one of claims 1 to 7 wherein allene oxide synthase is administered orally, rectally or by injection.

9. A method according to claim 8 wherein said injection is cutaneous, subcutaneous, intravenous injection, intrathecal injection, or by direct local injection to the CNS.

10. A method according to claim 8 or claim 9 wherein said injection is formulated for controlled release.

11. A method according to any one of claims 1 to 7 wherein allene oxide synthase is administered intranasally.

12. The use of allene oxide synthase, or a functionally equivalent variant thereof, in the manufacture of a medicament for the treatment of chronic degenerative conditions of the central nervous system related to oxidative stress.

13. The use of claim 11 wherein said allene oxide synthase is selected from the group 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, or functionally equivalent variants thereof.

14. The use of claim 12 or claim 13 wherein the conditions of the central nervous system related to oxidative stress are neuronal-degenerative conditions.

15. The use of claim 12 or claim 13 wherein the conditions of the central nervous system related to oxidative stress are vascular-degenerative conditions.

16. The use of claim 12 or claim 13 wherein the conditions of the central nervous system related to oxidative stress are glial cell degenerative conditions.

17. The use of any one of claims claim 12 to 16 wherein the chronic degenerative conditions of the central nervous system are selected from any one or more of the following: dementia, aging, HIV-associated dementia, post-stroke cognitive decline, stroke-induced dementia, postoperative cognitive dysfunction or surgically-induced cognitive impairment. Alzheimer’s disease, Parkinson’s disease, Motor neurone disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease), Huntington’s disease, Alzheimer’s disease, dementia, stroke-indued cognitive impairment, or postoperative cognitive impairment.

18. The use of any one of claims claim 12 to 17 wherein the AOS is administered prophylactically.

19. The use of any one of claims claim 12 to 18 wherein the AOS is administered orally, rectally or by cutaneous, subcutaneous, intravenous injection, intrathecal injection, or by direct local injection to the CNS.

20. The use of claim 19 wherein the injection is formulated for controlled slow release.

21. The use of any one of claims claim 12 to 18 wherein, the AOS is administered intranasally.

22. A pharmaceutical composition comprising allene oxide synthase, or a functionally equivalent variant thereof for treating chronic degenerative conditions of the central nervous system.

24. The composition of claim 22 wherein that AOS comprises the amino acid sequence selected from the group 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, or a functionally equivalent variant thereof.

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