Treatment of central nervous system injury

Abstract

This invention relates to compositions and methods for improving recovery after traumatic or ischemic central nervous system injury, including the administration of compositions comprising cyclopropenzyme (AOS) or its functionally equivalent variants or derivatives.

Description

Technical Field

[0001] This invention relates to compositions and methods for improving recovery following traumatic or ischemic central nervous system injury (e.g., stroke, traumatic brain injury, spinal cord injury, transient ischemic attack, or retinal vein occlusion). More particularly, this invention relates to compositions and methods for improving recovery following traumatic or ischemic central nervous system injury, using cyclopropenase or a functional equivalent thereof, wherein the cyclopropenase is administered to the patient in need at least approximately 3 hours after the traumatic or ischemic central nervous system injury. Background Technology

[0002] Damage to the brain or spinal cord (known as the central nervous system, CNS) can result from events that reduce blood flow (e.g., stroke, trauma, or neurodegenerative conditions). These types of damage lead to loss of behavioral function with limited recovery.

[0003] Stroke (which is either ischemic or hemorrhagic in nature) is the leading cause of adult disability worldwide in developed countries. There are no therapies specifically designed to promote stroke recovery. Currently, the only stroke treatment is the administration of tissue plasminogen activator (tPA). tPA is a "clot-rupture" drug that does not target stroke recovery but rather the blood vessel blocked in the stroke. tPA must be administered within 4.5 hours of the stroke, as later administration may cause cerebral hemorrhage. There are currently no approved therapies that can be administered later than 4.5 hours after the stroke. Many other neuroprotective therapies under development have similar requirements and need to be administered within a few hours or less of the ischemic or hemorrhagic event. There are currently no approved stroke therapies that target the tissue repair process rather than the blocked blood vessel, or that are effective when administered after a longer period following the injury.

[0004] During a stroke, loss of neurological function typically occurs in two ways. First, acute injury causes complete damage at the center of the injury, resulting in impairment of neural circuits controlling bodily functions such as movement, sensation, language, or memory. Second, the injury causes partial damage to neural circuits adjacent to the site of injury (called the periinfarct cortex), and renders these circuits inoperable. To date, most treatments for CNS injuries and stroke target the first mechanism of damage: preventing the initial injury or cell death (a method known as neuroprotection), rather than attempting to stabilize partially damaged circuits in the brain to promote function. Secondary injury is an indirect consequence of primary injury and involves metabolic and biochemical cascades, including ischemic cascades, which lead to excitotoxicity, acidosis, free radical generation, and disruption of the blood-brain barrier. It arises from a traumatic process that occurs hours to days after the initial injury and is a major contributor to brain damage and death following stroke and traumatic brain injury.

[0005] These mechanisms of secondary injury also support several detrimental effects of other brain injuries, such as concussion, traumatic brain injury, transient ischemic attack, spinal cord injury, retinal vein occlusion, and subarachnoid hemorrhage.

[0006] For example, concussion or mild traumatic brain injury is a transient and clinically detectable alteration of brain function induced by mechanical injury. Evidence of brain abnormalities can be observed several months after the initial trauma following a mild concussion event (when symptoms have largely subsided), thought to be due to cytotoxic edema and reactive glial proliferation; changes in the shape of glial cells in response to damage to the central nervous system.

[0007] Cyclopropene synthase is an enzyme found in a variety of plant and non-mammalian species. It is a member of the cytochrome P450 enzyme superfamily and is involved in the synthesis of certain lipids, fatty acids and biochemical mediators, and catalyzes the production of epoxides (cyclopropenes) from fatty acid (or lipid) hydroperoxides.

[0008] Currently, there are no approved treatments to promote recovery after CNS injury. The primary rehabilitation treatment after CNS injury is physical rehabilitation, including physical therapy as well as occupational and speech therapy. These therapies are expensive, labor-intensive, time-consuming, and have a low success rate. Furthermore, they are not equally available to all patients. Therefore, there is a clear need for effective treatments for such central nervous system injuries that can be administered to patients in need beyond the current 3-hour post-injury period.

[0009] Invention Objective

[0010] One object of the present invention is to provide a method for treating central nervous system injury or chronic deterioration or degeneration following acute or traumatic central nervous system damage in patients. Another object of the present invention is to provide a medicament for treating such conditions.

[0011] Alternatively, one objective of the present invention is to provide at least a useful option for the public. Invention Overview

[0013] The high unmet need for agents that protect the CNS from neurodegeneration or damage caused by trauma and conditions of localized or global cerebral hypoperfusion (e.g., stroke, hemodynamic shock with cardiac arrest, heart failure, or during surgery), or that promote restorative neurological processes following such events, has prompted inventors to search for new methods. The present invention is provided to address at least some of the challenges present with current methods.

[0014] Treatment

[0015] In one instance, a method for improving recovery following central nervous system injury in a subject is provided, comprising administering to the subject a composition comprising cyclopropenase or a functionally equivalent variant or derivative thereof.

[0016] In one aspect, the composition comprises a therapeutically effective amount of cyclopropenase selected from the group consisting 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, or a functionally equivalent derivative or variant thereof.

[0017] In one aspect, the composition comprises a therapeutically effective amount derived from *Gynostemma pentaphyllum* (Gynostemma pentaphyllum). Parthenium argentatum Cyclopropene synthase.

[0018] In one respect, the cyclopropenzyme derived from *Gynostemma pentaphyllum* is SEQ ID NO: 1 or a functionally equivalent derivative or variant thereof.

[0019] In another instance, a method for improving recovery following central nervous system injury in a subject is provided, comprising administering to the subject a composition comprising cyclopropenase or a functionally equivalent variant or derivative thereof, wherein the composition is administered at least approximately 3 hours after the injury.

[0020] 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 after injury.

[0021] In one aspect, the composition is administered to the subject approximately 3 hours after central nervous system injury.

[0022] In another aspect, the composition is administered to the subject at least 3 to about 12 hours after the central nervous system injury.

[0023] In another aspect, the composition is administered to a subject at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days, at least eleven days, at least twelve days, at least thirteen days, or at least fourteen days after central nervous system injury.

[0024] In one aspect, the composition is administered to the subject at least 3 days after the central nervous system injury.

[0025] In another aspect, the composition is administered to the subject at least 3 to about 7 days after the central nervous system injury.

[0026] In another instance, a composition comprising a therapeutically effective amount of cyclopropenase or a functionally equivalent variant or derivative thereof is formulated for administration to the subject orally, intravenously, subcutaneously, intraperitoneally, sublingually, intranasally, inhaled, locally, intrathecally, intraocularly, intramuscularly, intracranially, or systemically.

[0027] In one aspect, a composition comprising a therapeutically effective amount of cyclopropenase or a functionally equivalent variant or derivative thereof is formulated as an aqueous solution for intranasal administration to a subject.

[0028] In another aspect, a composition comprising a therapeutically effective amount of cyclopropenzyme or a functionally equivalent variant or derivative thereof is formulated into an aqueous solution for intranasal administration to a subject, the aqueous solution further comprising a nonionic surfactant.

[0029] In another aspect, the nonionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-X100, or any other pharmaceutically acceptable surfactant.

[0030] In another aspect, the composition comprises a therapeutically effective amount of cyclopropene synthase selected from the group consisting 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, SEQ ID NO: 9, or a functionally equivalent variant or derivative thereof, formulated as an aqueous solution for intranasal administration to a subject, said aqueous solution further comprising a nonionic surfactant selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or Triton-X100.

[0031] In another instance, a composition comprising a therapeutically effective amount of cyclopropenase or a functionally equivalent variant or derivative thereof is formulated for topical administration to the subject.

[0032] In one aspect, the composition comprises a reservoir delivery system containing a therapeutically effective amount of cyclopropenzyme or a functionally equivalent variant or derivative thereof for local administration via intracranial, intrathecal, or intravitreal injection.

[0033] In another aspect, the composition comprises a reservoir delivery system containing a therapeutically effective amount of cyclopropenzyme or a functionally equivalent variant or derivative thereof and a nonionic surfactant for local application via intracranial, intrathecal or intravitreal injection.

[0034] In another aspect, the composition comprises a reservoir delivery system containing a therapeutically effective amount of cyclopropenase or a functionally equivalent variant or derivative thereof and a nonionic surfactant for local administration via intracranial, intrathecal, or intravitreal injection, wherein the reservoir delivery system provides sustained release of the cyclopropenase.

[0035] In one aspect, the reservoir delivery system includes a delivery system selected from nanoparticle formulations, hydrogel formulations, and implantable mechanical delivery systems.

[0036] In another aspect, the reservoir delivery system comprises a hydrogel containing the cyclopropenzyme and additionally a nonionic surfactant.

[0037] In one aspect, the hydrogel comprises a biopolymer.

[0038] In another aspect, the hydrogel comprises one or more materials selected from the group consisting of: alginic acid or a pharmaceutically acceptable salt thereof, hyaluronic acid, gelatin, thiol-modified hyaluronic acid, heparin, thiol-modified heparin, thiol-modified chondroitin sulfate, thiol-modified gelatin, sodium hyaluronate, and acryloyl hyaluronic acid.

[0039] In one instance, the central nervous system injury is selected from the group consisting of: ischemic stroke, hemorrhagic 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.

[0040] Examples of pharmaceutical uses

[0041] Based on one example, the use of cyclopropenase in the preparation of medicaments for treating central nervous system injuries is provided.

[0042] In one aspect, cyclopropenzyme is selected from the group consisting 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, or their functionally equivalent derivatives or variants.

[0043] In one respect, the drug is administered at least approximately 3 hours after the central nervous system injury.

[0044] In one aspect, the drug is administered at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after the central nervous system injury.

[0045] In another respect, the drug is administered at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days, at least eleven days, at least twelve days, at least thirteen days, or at least fourteen days after the central nervous system injury.

[0046] In another respect, the drug should be administered at least 3 days after the central nervous system injury.

[0047] In another respect, the drug is administered at least approximately 3 to approximately 7 days after the central nervous system injury.

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

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

[0050] In another aspect, the drug also contains a nonionic surfactant.

[0051] In one aspect, the nonionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-X100, or any other pharmaceutically acceptable surfactant.

[0052] In another aspect, the medicament comprises a therapeutically effective amount of cyclopropenase selected from the group consisting 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, SEQ ID NO: 9, or a functionally equivalent variant or derivative thereof, formulated as an aqueous solution for intranasal administration, said aqueous solution further comprising a nonionic surfactant selected from the group consisting of: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or Triton-X100.

[0053] Examples of secondary medical uses

[0054] As an example, cyclopropenzyme is provided for use in the treatment of central nervous system injuries.

[0055] In one aspect, cyclopropenzyme is selected from the group consisting 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, or their functionally equivalent derivatives or variants.

[0056] In one respect, cyclopropenzyme should be administered at least approximately 3 hours after the central nervous system injury.

[0057] In one respect, cyclopropenzyme was administered at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after the central nervous system injury.

[0058] In another respect, cyclopropenzyme should be administered at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or at least fourteen days after the central nervous system injury.

[0059] In another aspect, cyclopropenzyme should be administered at least approximately 3 days after the central nervous system injury.

[0060] On the other hand, cyclopropenzyme should be administered at least 3 to 7 days after the central nervous system injury.

[0061] In another aspect, cyclopropenzyme is formulated for oral, subcutaneous, intraperitoneal, intraocular, intrathecal, intramuscular, intravenous, intranasal, or topical application.

[0062] In another aspect, cyclopropenzyme was formulated into an aqueous solution for intranasal administration.

[0063] In another aspect, cyclopropene synthase also contains nonionic surfactants.

[0064] In one aspect, the nonionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-X100, or any other pharmaceutically acceptable surfactant.

[0065] In another aspect, the cyclopropene synthase is selected from the group consisting 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, SEQ ID NO: 9 or their functionally equivalent variants or derivatives, formulated as an aqueous solution for intranasal administration, and further comprising a nonionic surfactant selected from the group consisting of: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or Triton-X100. Attached Figure Description

[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 The diagram illustrates SEQ ID No. 1-9.

[0068] Figure 2 The diagram illustrates the repair mechanism of AOS associated with the formation of lipid peroxides / hydroperoxides during oxidative stress.

[0069] Figure 3 The diagram illustrates the effect of AOS on rescuing neuronal cell death after exposure to hydrogen peroxide.

[0070] Figure 4 The diagram illustrates the effect of AOS on microglial reactivity (measured by IBA1 staining) when administered intranasally as a single dose 3 hours after infection.

[0071] Figure 5 The diagram illustrates the effect of AOS on astrocyte responsiveness (measured by GFAP staining) when administered intranasally as a single dose 3 hours after the stroke. (A) Astrocyte responsiveness immediately adjacent to the stroke. (B) Astrocyte responsiveness at a distance of 600–800 μm from the stroke.

[0072] Figure 6 The diagram illustrates the effect of AOS on pericyte number, showing an increase in the stroke core. (A) Pericyte number in the stroke core. (B) Pericyte number adjacent to the stroke. (C) Pericyte number at a distance of 800 μm from the stroke.

[0073] Figure 7 The diagram illustrates the impact of AOS on motor function, showing an improvement in forelimb asymmetry during the cylinder task.

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

[0075] Figure 9 The diagram illustrates the effect of AOS on astrocyte responsiveness (measured by GFAP staining) when administered via direct local injection 5 days post-infarction. (A) Astrocyte responsiveness immediately adjacent to the stroke. (B) Astrocyte responsiveness at a distance of 600–800 μm from the stroke.

[0076] Figure 10The diagram illustrates the impact of AOS on post-stroke cognitive deficits, demonstrating improvements in spatial memory deficits through an object location recognition task (OLRT). (A) OLRT performance 1 week after infarction; and (B) OLRT performance 4 weeks after infarction.

[0077] definition

[0078] General definition

[0079] Unless otherwise expressly defined, all technical and scientific terms used herein should be regarded as having the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains (e.g., in immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0080] Unless otherwise stated, the recombinant protein and immunological techniques used in this invention are standard procedures well known to those skilled in the art. Such techniques are described and explained in literature from sources such as: J. Perbal, 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); TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); DM Glover and BD Hames (ed.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and FMAusubel et al. (ed.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (ed.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); and JE Coligan et al. (Editor's Note) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0081] In this text, the articles “a” and “an” are used to refer to one or more species (i.e., at least one species) of the grammatical object of the article. As an example, “element” means one or more elements.

[0082] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for either or both of these meanings.

[0083] Throughout this specification, unless otherwise specified or required by context, references to a single step, a composition of substances, a group of steps, or a group of compositions of substances shall be construed as covering one or more (i.e., one or more) of those steps, compositions of substances, groups of steps, or groups of compositions of substances.

[0084] It is intended that references to the ranges of numbers disclosed herein (e.g., 1 to 10) also include references to all relevant numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10), as well as any ranges of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus precisely disclose all subranges of all ranges precisely disclosed herein. These are merely examples of specific intent, and all possible combinations of numerical values ​​between the enumerated minimum and maximum values ​​are considered to be precisely stated in this application in a similar manner.

[0085] Throughout this specification, the word “comprising” or variations thereof, such as “including” or “containing”, shall be understood to imply inclusion of the stated elements, integers or steps, or groups of elements, integers or steps, but not to exclude any other elements, integers or steps, or groups of elements, integers or steps.

[0086] Those skilled in the art will recognize that the invention described herein is susceptible to variations and modifications beyond those specifically described. It should be understood that the invention includes all such variations and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or specified in this specification, as well as any and all combinations or any two or more of said steps or features.

[0087] This invention is not limited to the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this invention, as described herein.

[0088] Any instance described herein should be considered as applicable to any other instance, unless otherwise specified.

[0089] Selected definition

[0090] The term “approximately” is used herein to refer to conditions that vary by as much as 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to explicitly stated conditions (e.g., amount, concentration, time, etc.).

[0091] As used in this article, the term "AOS" refers to cyclopropenzyme used in the context of AOS enzyme activity.

[0092] As used in this article, the term "PaAOS" refers to cyclopropenzyme derived from *Gynostemma pentaphyllum*.

[0093] As used herein, the term "functionally equivalent variant or derivative" means a fragment of a full-length polypeptide, peptide, or protein that retains the activity of the polypeptide, peptide, or protein. As used herein, the term "biologically active fragment" includes deletion mutants and small (poly)peptides (e.g., having 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 consecutive amino acids) that contain the activity of the parent peptide or polypeptide. This type of peptide can be obtained by applying standard recombinant nucleic acid techniques, for example, as described in the following literature: Sambrook et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring HarbourPress, 1989), particularly Sections 16 and 17; Ausubel et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY (John Wiley & Sons, Inc. 1994–1998), particularly Chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995–1997), particularly Chapters 1, 5, and 6. Alternatively, this type of peptide can be synthesized using conventional liquid-phase or solid-phase synthetic techniques. For example, reference can be made to solution synthesis or solid-phase synthesis as described in, for example, the following literature: Atherton and Sheppard, SOLIDPHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989), particularly Chapter 9; or Roberge et al. (1995 Science 269: 202). Alternatively, the peptides of the present invention can be produced by digesting them with proteolytic enzymes such as endoLys-C, endoArg-C, endoGlu-C, and Staphylococcus V8-protease. The digested fragments can be purified by, for example, high-performance liquid chromatography (HPLC).

[0094] As used in this article, the terms “brain injury,” “brain damage,” “brain impairment,” “neuronal injury,” and “neuronal damage” refer to damage to neuronal tissue that impairs the cell viability and / or cell function of the neuronal tissue.

[0095] The term "reservoir delivery system" refers to a system, device, or formulation that can be placed inside the body and provides continuous delivery of a target active agent (e.g., cyclopropenzyme, or its functional derivatives or variants).

[0096] As used herein, the term "derivative" means a polypeptide derived from a base sequence through modification, such as by conjugation or compounding with other chemical moieties or by post-translational modification techniques, as will be understood in the art. The term "derivative" also includes, within its scope, alterations to the parental sequence, including additions or deletions, that provide a functionally equivalent molecule.

[0097] The term "device" refers to a substance or particle that is injected into the brain and slowly releases the desired molecule (cyclopropenzyme).

[0098] The terms “drug,” “active agent,” and “pharmacologically active agent” are used interchangeably herein to refer to any chemical compound, complex, or composition suitable for administration to a subject and having a beneficial biological effect (preferably a therapeutic effect) in the treatment of a disease or abnormal physiological condition. The terms also cover pharmaceutically acceptable and pharmacologically active derivatives of those active agents.

[0099] The term “dosage form” means any form of pharmaceutical composition containing an amount of active agent sufficient to achieve a therapeutic effect with a single or multiple administration. When the formulation is a solution or suspension, the dosage form is typically such a solution or suspension. The frequency of administration that will provide the most effective results without overdose will vary depending on factors such as: (1) the characteristics of the particular drug, including both its pharmacological and physical characteristics, such as its solubility and stability;

[0100] In the context of the specific functions of the peptides and enzymes described herein, the terms "effective amount," "therapeutic effective amount," and "effective dose" refer to the amount of the composition administered, either as a single dose or as part of a series, to an individual in need, the amount of which is effective for the stimulation, prevention, or treatment. The effective amount will vary depending on the health and physical condition of the individual being treated, the individual's taxonomy, the formulation of the composition, medical assessment, and other relevant factors. It is expected that the amount will fall within a relatively wide range that can be determined through routine testing.

[0101] As used herein, the terms “fragment” or “functional derivative” related to peptides are subsequences of peptides that can be detected, for example, using a binding reagent. These terms may refer to peptides, aggregates of peptides such as dimers or multimers, fusion peptides, peptide fragments, peptide variants, or derivatives thereof.

[0102] The term "hydrogel" refers to a typically cross-linked network of polymer chains (e.g., hyaluronic acid) that is typically hydrophilic. Hydrogels absorb water and swell. Illustrative examples include, but are not limited to, hyaluronic acid-based hydrogels called HYSTEM®, which are cross-linked networks of hyaluronic acid and other thiol-modified macromolecules.

[0103] The terms "improved recovery" or "enhanced recovery" refer to the recovery of motor function or improvement in cognitive performance in a subject following central nervous system injury. In some embodiments, improvement in motor recovery or motor function recovery includes improvements in motor coordination, and / or balance, and / or gait, and / or speech. In some embodiments, improvement in cognitive performance includes enhanced and / or improved performance on tasks involving 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 some embodiments, improvement is an improvement in the degree of restored function and / or performance, and / or an improvement in the rate of recovery of function and / or performance.

[0104] The terms “individual,” “subject,” “patient,” and “host” are used interchangeably herein and refer to any mammalian subject for whom a diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a human.

[0105] The term "isolated" as used with respect to the polypeptide sequences disclosed herein refers to a sequence removed from its native cell or other naturally occurring biological environment. Isolated molecules can be obtained by any method or combination of methods, including biochemical, recombinant, and synthetic techniques. The polypeptide sequences can be prepared by at least one purification step.

[0106] The terms “intranasal administration” and “intranasal delivery” refer to the delivery of an effective amount of a target reagent (e.g., cyclopropenzyme, or its functionally equivalent derivatives or variants) to the central nervous system by administering the enzyme to a subject through the nasal cavity.

[0107] The terms “patient,” “subject,” “host,” “individual,” and / or “mammal” are used interchangeably herein to refer to any subject for whom diagnosis, prevention, or treatment is desired, particularly a vertebrate subject, and more particularly a mammalian subject, most particularly a human. Suitable vertebrates falling within the scope of this invention include, but are not limited to: humans, any member of the subphylum Chordata, including primates, rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), sheep (e.g., sheep), goats (e.g., goats), swine (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (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 require diagnosis, prevention, or treatment; however, it will be understood that the foregoing terms do not imply the presence of symptoms. Therefore, both the methods of preparation and the human and veterinary applications of the formulations described herein are taken into consideration.

[0108] The term "pharmaceutically acceptable carrier" means a carrier that is generally safe, non-toxic, and not biologically or otherwise undesirable for use in the preparation of pharmaceutical compositions, and includes carriers acceptable for both veterinary and human pharmaceutical purposes. As used in this specification and claims, a pharmaceutically acceptable carrier includes one or more such carriers.

[0109] The term "pharmaceutical composition" is intended to encompass compositions suitable for administration to subjects (e.g., mammals, especially humans). A "pharmaceutical composition" may be sterile and substantially free of contaminants capable of evoking an undesirable response in the subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions may be designed for administration to subjects or patients in need via a number of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intranasal, inhaled, intradermal, intratracheal, intrathecal, subcutaneous, intravenous, etc.

[0110] The term "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts formed with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or formed with an organic acid, 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, etc. Acids, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheponic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucoconic acid, etc.; or (2) salts formed when the acidic protons present in the parent compound are replaced by metal ions such as alkali metal ions, alkaline earth metal ions or aluminum ions; or coordinated with organic bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc.

[0111] The terms “peptide” and “polypeptide” or “protein” are used interchangeably throughout this specification and cover amino acid chains of any length, including full-length sequences, wherein amino acid residues are linked by covalent peptide bonds. Polypeptides useful in this invention can be purified natural products or can be generated, partially or wholly, using recombinant or synthetic techniques. The term can refer to a polypeptide, an aggregate of polypeptides such as dimers or other polymers, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or a derivative thereof. Polypeptides as used herein can have a chain length of at least 4, at least 5, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all 23 amino acids of the full-length cyclic propionyl synthase described herein. Other polypeptides that refer to this invention or are described herein should be understood similarly.

[0112] The term "purified" as used herein does not require absolute purity. In various embodiments, "purified" means, for example, at least about 80%, 85%, 90%, 95%, 98%, or 99% homogeneity of the peptide in a sample. The terminology should be understood similarly in relation to other molecules and constructs described herein.

[0113] The term “continuous delivery” refers to the delivery of an effective amount of the target reagent (e.g., cyclopropenzyme, or its functionally equivalent derivatives or variants) to a desired site (e.g., infarct cavity) for at least one day, preferably at least three days, at least four days, at least five days, at least six days, more preferably at least one week, at least two weeks, at least five weeks, at least six weeks, at least two months, at least three months, or at least four months.

[0114] As used herein, the term "variant" refers to a polypeptide sequence that differs from a specifically identified sequence, wherein one to six or more amino acid residues are deleted, substituted, or added. Substitutions, additions, or deletions of one, two, three, four, five, or six amino acids are considered. Variants can be naturally occurring allelic variants or non-natural variants. Variants can originate from the same species or from other species and can encompass homologs, paralogs, and orthologs. In some embodiments, variants of the polypeptide useful in this invention have the same or similar biological activities as the parent polypeptide, including signal peptide activity or antigen-binding properties. The term "variant" with respect to polypeptides encompasses all forms of polypeptides as defined herein.

[0115] The variant polypeptide sequences exhibit at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 71%, at least approximately 72%, at least approximately 73%, at least approximately 74%, at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity with the sequences of the present invention. For the polypeptides, identity is found within a comparison window of at least 5 to 7 amino acid positions.

[0116] Peptide variants also include those that exhibit similarity to one or more of the specifically identified sequences, which may maintain the functional equivalence of those sequences, including those that cannot be reasonably expected to occur by random chance.

[0117] Peptide sequence identity and similarity can be determined as follows. The target peptide sequence is compared with candidate peptide sequences using BLASTP (from the BLAST suite, 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 used, except that filtering for low-complexity regions should be turned off.

[0118] Peptide sequence similarity can be checked using the following UNIX command-line arguments: `bl2seq –ipeptideseq1 –j peptideseq2 -FF –p blastp`. The `-FF` parameter disables filtering for low-complexity regions. The `-p` parameter selects an appropriate algorithm for the sequence pairs. This program finds regions of similarity between sequences and reports an "E-value" for each such region, which is the expected number of times one could accidentally see such a match in a database containing random sequences of a fixed reference size. For small E-values ​​(much less than one), this is roughly the probability of such a random match. Variant peptide sequences often exhibit a similarity of less than 1 × 10⁻⁶ when compared to any of the specifically identified sequences. -5 Less than 1×10 -6 Less than 1×10 -9 Less than 1×10 -12 Less than 1×10 -15 Less than 1×10 -18 or less than 1×10 -21 The E-value. Peptide sequence identity can also be calculated using a global sequence alignment program over the entire length of the overlap between the candidate and target peptide sequences. 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) (discussed above) are also suitable global sequence alignment programs for calculating peptide sequence identity. The use of BLASTP is preferred for determining the peptide variants according to the invention.

[0119] As used herein, the term "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-by-amino acid basis within a comparison window. Therefore, the "sequence identity percentage" is calculated as follows: two optimally aligned sequences are compared within a comparison window; the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, U) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ileu, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is determined to produce the number of matching positions; the number of matching positions is divided by the total number of positions in the comparison window (i.e., the window size); and the result is multiplied by 100 to produce the sequence identity percentage. For the purposes of this invention,

[0120] The term “sequence identity” can be understood as referring to the “percentage of matches” calculated by the DNASIS computer program (version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using the standard default values ​​used in the reference manual accompanying the software.

[0121] The term "sequence similarity" refers to the percentage of identical or conserved amino acid substitutions. Similarity can be determined using sequence comparison procedures such as GAP (Deveraux et al., 1984 Nucleic Acids Research 12: 387-395). In this way, sequences of similar or substantially different lengths from those cited herein can be compared by inserting gaps in the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0122] The term "treatment" is used herein to generally refer to achieving a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic in terms of partially or completely stabilizing or curing a disease and / or adverse effects attributable to said disease. As used herein, "treatment" covers any treatment of diseases in mammals (particularly humans) and includes: (a) preventing the occurrence of said disease or symptoms in subjects who may be susceptible to said disease or symptoms but have not yet been diagnosed with it; or (b) suppressing said disease symptoms, i.e., preventing their development; or (c) alleviating said disease symptoms, i.e., causing the remission of said disease or symptoms. Invention Details

[0124] In general, this invention relates to therapies for treating central nervous system injuries, such as stroke, concussion, retinal vein occlusion, or traumatic brain injury, or sequelae resulting from such brain injury, such as, but not limited to, motor, visual, sensory, or cognitive impairments. The invention also includes pharmaceutical compositions for treating such conditions and methods for preparing suitable pharmaceutical compositions.

[0125] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as such embodiments can certainly be varied. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention is defined only by the appended claims.

[0126] This invention is based on the surprising discovery that cyclopropene synthase, or its functionally equivalent variants, and compositions comprising cyclopropene synthase, or its functionally equivalent variants, can be used to treat central nervous system injuries, such as 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 found that administering cyclopropene synthase to a subject 3 hours after brain injury can promote repair processes in the brain, such as a reduction in neuroinflammation and microglial proliferation, a reduction in reactive astrocyte proliferation, an increase in pericyte number, and an improvement in motor function. These findings are surprising and contradict prior art, which requires cyclopropene synthase to be administered within 45 minutes of brain injury to provide a therapeutic or neuroprotective effect to the subject. Furthermore, although no reduction in stroke volume (a common measure of protection against acute neuronal damage, or "neuroprotection") was observed, the inventors surprisingly found that cyclopropene synthase could be noninvasively administered to the brain of subjects via an intranasal route outside the known window of opportunity, and induced a significant reduction in neuroinflammation and reactive microglial and astrocyte proliferation, increased vascularity and pericyte density, and improved motor function. These effects support the role of cyclopropene synthase in promoting repair processes in the peri-infarct area. Not wanting to be bound by theory, the inventors considered that when administered outside the 45-minute window required for neuroprotection, cyclopropene synthase could attenuate secondary damage following brain injury.

[0127] When referring to AOS, it should be understood to include AOS isolated from any source, including functionally equivalent peptides and proteins, including AOS obtained, for example, through chemical synthesis and / or gene expression techniques. AOS and its functionally equivalent variants may be collectively referred to as AOS herein. Therefore, unless specifically mentioned, reference to AOS used in this invention should be considered to include reference to its functionally equivalent variants.

[0128] The inventors have discovered that administration of AOS protein or its functionally equivalent variants (hereinafter referred to as "AOS" or "AOS protein") is beneficial for CNS damage recovery. In one aspect, said AOS is derived from *Gynostemma pentaphyllum*. This is by no means intended to limit the scope of the invention, but is merely shown for illustrative purposes. Cyclopropene synthase or its functional equivalents from any source may be applicable to this invention.

[0129] As mentioned above, in some instances, cyclopropene synthase can be derived from the rubber plant *Eriocaulon buergerianum* (GenBank CAA55025.5), also known as *Eriocaulon buergerianum* rubber particle protein (RPP), which is part of the CYP74A enzyme family (cyclopropene synthase; AOS). This enzyme family comprises fewer than 15 molecules from a wide variety of plant sources, including *Eriocaulon buergerianum*, maize, barley, tomato, flaxseed, and *Arabidopsis thaliana* (a model plant in biology), all of which perform AOS functions and have a molecular weight of ~55 kDa in monomeric form. These enzymes are atypical members of the cytochrome P450 family because they are self-regenerating (requiring neither oxygen nor NADPH reductase for activity) and exhibit exceptional reaction rates. AOS converts fatty acid hydroperoxides, formed through the oxidation of polyunsaturated fatty acids, into unstable epoxides, which are then further degraded into acetone alcohols. Lipid peroxides (LPOs) can be formed through oxidative attack or the action of lipoxygenases, the latter being a pathway used in the generation of LPO substrates for use in AOS enzyme function assays.

[0130] The compositions disclosed herein

[0131] The cyclopropenzyme synthase preparations and compositions described herein are particularly useful for the treatment of central nervous system injuries, such as, but not limited to, stroke, ischemic stroke, white matter stroke, hemorrhagic stroke, traumatic brain injury, concussion, spinal cord injury and / or retinal vein occlusion.

[0132] 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, and structural and functional variants thereof, said compositions and / or preparations being suitable for pharmaceutical use. Advantageously, these compositions and / or preparations are effective in treating central nervous system injuries (e.g., stroke) when treatment is initiated at least approximately 3 hours to at least approximately 7 days after said injury. Therefore, these compositions and / or preparations are particularly useful for treating central nervous system injuries and can provide substantial benefits exceeding current standards of care.

[0133] In one example, the present invention provides a composition for use in treating central nervous system injury, comprising, consisting of, or substantially consisting of the following amino acid sequences: amino acid sequences 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, and their structural and / or functional variants.

[0134] The cyclopropenzyme compositions described herein can be formulated with biologically or non-biologically acceptable excipients that enhance enzyme viability and activity for use in, for example, biological and non-biological systems. For example, formulation may be carried out in the presence of specific surfactants or detergents; buffers that limit the pH range; ionic strength (i.e., pI); and optionally, sugars / carbohydrates and antimicrobial agents, etc.

[0135] Therefore, in another further aspect, the present invention provides a composition comprising cyclopropenzyme, wherein the composition is formulated for pharmaceutical use, and wherein optionally:

[0136] (i) The surfactant comprises a nonionic surfactant selected from the group consisting of: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton X-100; and / or

[0137] (ii) The pH of the composition is between about 6.0 and about 9.5, including but not limited to pH values ​​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

[0138] (iii) The composition further comprises a buffer solution, including but not limited to BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid) buffer 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)-1-piperazine ethanesulfonic acid), HBSS (Hank's balanced salt solution). HEPES (Hank's buffer with HEPES), HEPPSO (4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) hydrate), HHBS (Hank's buffer with HEPES), imidazole-HCl, 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-1,1-bis(hydroxymethyl)ethyl)glycine), TRIS (tris(hydroxymethyl)aminomethane) and / or Trizma (2-amino-2-(hydroxymethyl)-1,3-propanediol); and / or

[0139] (iv) The pI 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

[0140] (v) The concentration of the cyclopropene synthase is between approximately 0.1 and 100. The values ​​are between ug / mL, including but not limited to approximately 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, 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, 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, and 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, 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, 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

[0141] (vi) The concentration of the cyclopropene synthase is between approximately 0.1 and 500 mg / mL, including but not limited to approximately 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, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 mg / mL. 3.2, 3.3, 3.4, 3.5, 3.6, 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, 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, 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, 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

[0142] (vii) The composition further comprises carbohydrates, including but not limited to sugars such as trehalose and lactose, in amounts from 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 carbohydrates; and / or

[0143] (viii) The composition further comprises an antimicrobial agent, including but not limited to kanamycin, gentamicin, lincomycin, and tylosin tartrate, in an amount from about 0.1 to 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, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, and 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, 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, 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, 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, 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

[0144] (ix) The composition further comprises a polyol, such as glycerol, in an amount from about 0.1 to 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, 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, 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, 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, 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, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20% v / v polyols.

[0145] In certain instances according to the invention, the compositions described herein are substantially surfactant-depleted or surfactant-free and do not further contain, for example, imidazole or residual lipids.

[0146] In other embodiments of the invention, the pI of the compositions described herein is between about 1 and about 250 mM, and is based on the presence of salts including, but not limited to, sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), lithium chloride (LiCl), magnesium chloride (MgCl2), sodium bromide (NaBr), sodium sulfide (Na2S), beryllium chloride (BeCl2), barium fluoride (BaF2), calcium iodide (CaI2), lithium iodide (LiI), cesium fluoride (CsF), potassium sulfate (K2S), cesium oxide (Cs2O), rubidium sulfate (Rb2S), rubidium fluoride (RbF), sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), sodium sulfate (NaHSO4), sodium carbonate (NaHCO3), sodium bicarbonate (Na2CO3), potassium cyanide (KCN), and sodium acetate (CH3COONa).

[0147] In other embodiments, the cyclopropene synthase composition can be formulated for use as a hydrogel (preferably a biopolymer hydrogel) to contain and provide sustained local release of cyclopropene synthase into the central nervous system (e.g., the brain, stroke infarct cavity, peri-infarct region, spinal cord, or eye), thereby providing sustained release of cyclopropene synthase. In some embodiments, the hydrogel can be made from naturally occurring brain proteins and does not elicit a brain response. In some embodiments, the hydrogel can be a HYSTEM® hydrogel (e.g., HYSTEM®-C hydrogel, HYSTEM®-HP hydrogel, etc.).

[0148] In various embodiments, the cyclopropenase may be slowly released over at least one day, or at least three days, or at least five days, or at least one week, or at least two weeks, or at least three weeks, or at least one month, or at least two months, or at least three months, or at least six months. Typically, the cyclopropenase is released directly into target tissues in the body (e.g., peri-infarct tissue, posterior eye, or spinal cord) following an ischemic event (e.g., stroke, traumatic brain injury, retinal vein occlusion, or spinal cord injury) and produces an improvement in recovery from the ischemic event.

[0149] In some embodiments, the use of hyaluronic acid-based hydrogels is considered and preferred. A class of highly suitable hydrogels has been commercially produced and marketed under the name HYSTEM®. In some embodiments, the hydrogel mimics the natural extracellular matrix (ECM) environment and is designed to reproduce the minimum composition necessary to obtain a functional ECM.

[0150] In various embodiments, the individual components of the hydrogel are in situ crosslinkable and can be inoculated with a therapeutic amount of cyclopropene synthase prior to in vivo injection without harming the cyclopropene synthase or the recipient tissue.

[0151] In some embodiments, the hydrogel comprises hyaluronic acid (or functionalized / derivatively derived hyaluronic acid) and gelatin (or functionalized / derivatively derived gelatin). In various embodiments, the hyaluronic acid and / or the gelatin are each thiol-modified, for example, through the use of carbodiimide-mediated hydrazide chemistry. In some embodiments, the gel-forming material is based on chemically modified hyaluronic acid. In some embodiments, the hyaluronic acid matrix for forming the gel is HYSTEM®, HYSTEM®-HP, or HYSTEM®-C. HYSTEM® hydrogels are formed by crosslinking a mixture of these thiolized macromolecules using polyethylene glycol diacrylate (PEGDA) or other suitable crosslinking agents. The gelation rate and hydrogel stiffness can be controlled by varying the amount of crosslinking agent. An important property of the hydrogels used herein (e.g., HYSTEM® hydrogels) in some embodiments is their high water content, typically greater than about 95%, resulting in high permeability to oxygen, nutrients, and other water-soluble metabolites.

[0152] In various embodiments, the hydrogel is initially provided as a three-component system comprising: 1) hyaluronic acid (e.g., thiol-modified hyaluronic acid); 2) gelatin (e.g., thiol-modified gelatin); and 3) a linker (e.g., polyethylene glycol diacrylate linker). In a commercially available system (HYSTEM®-C system), these are provided as three components: GLYCOSIL® (thiol-modified hyaluronic acid), GELIN®-S (thiol-modified gelatin), and EXTRALINK® (polyethylene glycol diacrylate). Each component is provided individually in vials as pre-measured, sterile, lyophilized solids that, when dissolved in a physiological buffer (i.e., physiological saline, lactated Ringer's solution, etc.) and mixed together, form a clear, transparent viscoelastic hydrogel at room temperature within approximately 20 minutes. The flexibility (stiffness) of the hydrogel... The pH is 70 ± 20 Pa, similar to that of fat and nerve tissue. The lyophilized hydrogel component does not contain additional salts, resulting in an isotonic gel after dissolution in a physiological buffer solution at a pH of 70 ± 20 Pa. 7.4. In some embodiments, the typical solids percentage of a suitable hydrogel (e.g., HYSTEM®-C hydrogel) is less than 2.0% (w / v), and the EXTRALINK® crosslinker concentration utilizes less than 30% of the available thiol groups on the other components, such that any unreacted acrylate groups of the crosslinker are negligible. Typically, one ml of HYSTEM®-C hydrogel prepared in a physiological buffer contains 4 mg of GLYCOSIL® and 4 mg of GELIN® crosslinked with 4 mg of EXTRALINK® at the ionic strength and pH of the preparation buffer. In some embodiments, for in vivo application, typical hydrogel volumes may vary between 0.1 or 0.5 or 1.0 ml / implantation site and up to 5.0 ml, 4.0 ml or 3.0 ml or 2.0 ml / implantation site, wherein in cases with multiple implantation sites / tissues or organs, the total volume of the hydrogel is up to 20 ml, 15 ml or 10 ml.

[0153] Methods for preparing thiol-modified macromolecules are known in the art. In one method, dithiodipropionylhydrazide (DTP) is coupled to the carboxyl functional group of a macromolecule by reacting it with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), followed by reduction and cleavage of the DTP disulfide bond with dithiothreitol (DTT) to generate a thiol-modified macromolecular DTPH derivative. The thiol-modified product is purified by thorough tangential flow filtration (TFF) using a polyethersulfone membrane with a 10 kDa rearrangement limit. Because the reagents and reaction byproducts are small molecules, this subsequent step yields thiol-modified macromolecules of very high purity. For GLYCOSIL® (the hyaluronic acid component of HYSTEM®-C), prior to thiol modification, the polymer's molecular weight is normalized by controlled alkaline hydrolysis, and the available carbonyl functional groups are increased by carboxylation of the 6′ hydroxyl group of the polymer's glucosamine moiety in a strong base with chloroacetic acid. The gelatin is supplied with thiol-modified gelatin without any pretreatment, and the product is referred to as GELIN®-S.

[0154] In other embodiments, the selected hydrogel can be replaced with another pharmaceutically acceptable hydrogel known in the art.

[0155] polypeptide

[0156] The compositions of the present invention may comprise polypeptides comprising, consisting of, or substantially consisting of the following amino acid sequences: amino acid sequences 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.

[0157] As used in the statements above and in similar statements elsewhere in this specification, the term "comprising" (etc.) means that the polypeptide comprises 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, SEQ ID NO: 8, or SEQ ID NO: 9, and may also include any one or more other elements. Therefore, 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 element is optional and may or may not be present. Other elements may include, for example, additional amino acid residues at any end of the amino acid sequence, and / or other molecules.

[0158] As used in the foregoing statements and similar statements elsewhere in this specification, the term "consistently of..." (etc.) means that the polypeptide comprises 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, SEQ ID NO: 8, or SEQ ID NO: 9, and may also include one or more other elements, provided that those elements do not interfere with or contribute to the activity or function of the peptide. Therefore, 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 other elements are optional and may be present or absent depending on whether they affect the activity or function of the peptide. For example, in the case where the composition “consistently consists of an amino acid sequence corresponding to the sequence SEQ ID NO: 1”, the amino acid sequence may contain additional amino acid residues (e.g., up to 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., protective moieties, such as N-terminal blocking residues (e.g., pyroglutamic acid)), provided that those additional residues or molecules do not substantially modulate the enzymatic properties of the amino acid sequence.

[0159] As used in the statements above and in similar statements elsewhere in this specification, the term "composed of..." (etc.) means that the peptide includes, but is not 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. Therefore, the phrase "composed 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 no other elements (e.g., amino acid residues at any end of said amino acid sequence, or other molecules) may be present.

[0160] As used in the statement "corresponding to the amino acid sequence of SEQ ID NO: 1" and similar statements in this specification, the term "corresponding to" (etc.) means an amino acid sequence that exhibits substantial similarity and / or identity to the sequence defined by SEQ ID NO: 1 and has the desired enzymatic activity. Typically, the peptide exhibits 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.

[0161] In some instances, the amino acid sequence may differ from the sequence SEQ ID NO: 1 by at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 amino acid substitutions, additions, and / or deletions.

[0162] The substituted amino acids can include both conservative and non-conservative substitutions.

[0163] Alternatively or additionally, the substituted or added amino acid may be any non-naturally occurring amino acid or its derivative. Non-naturally occurring amino acids include chemical analogs of the corresponding naturally occurring amino acids. Examples of non-naturally occurring amino acids and their derivatives include, but are not limited to, 4-aminobutyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, tert-butylglycine, leucine, valine, phenylglycine, ornithine, sarcosine, 2-thienylalanine, and / or the D-isomers of amino acids.

[0164] In some instances, a polypeptide comprising, or substantially comprising, 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 is a homolog or isoform of, or a derivative thereof, 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, or exhibits a similarity to, 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. The sequence defined in NO:8 represents at least approximately 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% similarity and / or identity to homologs or isoforms. A “homologous” is a molecule that originates from different species and is related through evolutionary inheritance from a common ancestral DNA sequence. The term “homologous” can be applied to relationships between genes separated by speciation events or by genetic replication events. An “isoform” is a peptide that has the same function as another peptide but is encoded by different polynucleotides and may have minor differences in its sequence.

[0165] A polypeptide comprising, consisting of, or substantially consisting of, 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, or SEQ ID NO: 8 may be a biologically active fragment comprising 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 used herein, "fragment" means a molecule comprising at least about 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, or about 480 consecutive amino acids. The bioactive fragment may have activity associated with the full-length amino acid sequence and / or may have altered activity. "Altered activity" includes enhanced activity or loss of detrimental activity. As described herein, the bioactive fragment may have the ability to convert free or esterified fatty acid peroxides or hydroperoxides into their corresponding epoxides.

[0166] Methods well-known in the art can be used to determine whether an amino acid sequence is, as defined herein, a peptide comprising, or substantially comprising 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, or SEQ ID NO: 8. Prediction methods include algorithms available through lookup references, including the SYFPEITHI and BIMAS algorithms described herein, and those described in US 2010-0168398 (which discusses “statistical” and “structure-related” methods that can be used to predict whether a particular peptide will bind to a class I MHC molecule). “Statistical” methods are typically based on affinity data obtained experimentally, while “structure-related” methods are typically based on available 3D structural information of the MHC molecule. See also Madden, DR et al., 1992 Cell 70: 1035-1048; FaIk, K. et al., 1991 Nature 351: 290-296; Matsumura, M. et al., 1992 Science 257: 927-934; Saper, MA et al., 1991 J. MoI Biol. 219: 277-312; and Latron, F. et al., 1992 Science 257: 964-967. In addition to predictive methods, peptides can be synthesized and their activity tested, 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 those described herein (see, for example, the Examples section).

[0167] The polypeptide comprising, or substantially comprising, 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 or SEQ ID NO: 8 may comprise peptides that have been suitably modified (e.g., by lipid modification) to modify their physicochemical properties.

[0168] The polypeptide comprising, consisting of, or substantially consisting of 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, or SEQ ID NO: 8 can be prepared in recombinant form using standard experimental protocols described, for example, in the following literature: Sambrook et al., MOLECULAR CLONING. A LABORATORY MANUAL (ColdSpring Harbour Press, 1989), particularly sections 16 and 17; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994–1998), particularly chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1994–1998), particularly chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1994–1998). Inc. (1995-1997), especially Chapters 1, 5 and 6.

[0169] Alternatively, the polypeptide comprising, consisting of, or substantially consisting of 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, or SEQ ID NO: 8 may be synthesized, for example, by solution synthesis or solid-phase synthesis as described by Atherton and Sheppard in SOLIDPHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989) or by Roberge et al. (1995 Science 269:202). Synthesis can be performed using, for example, tert-butoxycarbonyl (t-Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) chemistry (see Coligan et al., ibid., Chapter 9.1; Stewart and Young, 1984, SOLID PHASE PEPTIDE SYNTHESIS, 2nd ed., Pierce Chemical Co., Rockford, 111, 1994; and Atherton and Shephard, ibid.).

[0170] Example

[0171] Example 1

[0172] Neuroprotective effects of cyclopropenase in brain cell culture assays

[0173] The neuroprotective effects of cyclopropenase synthase (AOS) were evaluated in vitro in a cerebellar microexplant culture system. Two different neuronal injury modalities were used: the mitochondrial toxin 3-nitropropionic acid (an irreversible inhibitor of succinate dehydrogenase); and hydrogen peroxide, which induces oxidative stress leading to neuronal cell death.

[0174] method

[0175] Cerebellar Microexplant System

[0176] 1. Extraction of cerebellar tissue

[0177] Wistar rats, 4 days old, were used for the study. Rats were euthanized and placed on ice for 1 minute, decapitated, and the cerebellum was removed and placed on ice. The cerebellar tissue was placed in 1 mL of PBS supplemented with 0.65% glucose (10 μM 65% stock D(+)-glucose / 1 mL PBS) in a large culture dish, cut into small sections, and homogenized using a 1 mL insulin syringe via a 23 G (0.4 mm) needle, then returned to the glucose solution in the large culture dish. The tissue was sieved (through gauze with 125 μm pores) and centrifuged twice (6000 x g, 2 min), and transferred to serum-free BSA-supplemented START V medium (Biochrom, Germany). The second centrifugation step was performed with 1 mL of START V medium. The microexplants were reconstituted into 500 μL of START V medium and placed on ice.

[0178] 2. Culture of cerebellar cells

[0179] Two hours after poly-L-lysine coating, the slides were washed with Millipore water and air-dried. Each slide was placed in a small culture dish (35 mm in diameter) and 40 μL of START V / cell suspension was added. The tissues were incubated at 34°C for 2 hours (sedimentation period). Then, 1 mL of START V medium was added to the culture dish, and the cells were incubated at 34°C, 5% CO2 in air, and 100% humidity for 48 hours.

[0180] 3. Drug Application

[0181] For the studies, some explant cultures were exposed to loading-only (PBS buffer) and served as controls. In the first study (Study 1), 10 μL of toxin (3-nitropropionic acid, pH 7.4, 0.5 mM) was applied concurrently with a gradually increasing concentration of AOS enzyme (10 ng / mL - 20 ng / mL in PBS, pH 7.4). In the second study (Study 2), 10 μL of toxin (hydrogen peroxide, 0.1 mM, pH 7.4 in Millipore water) was applied concurrently with a gradually increasing concentration 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 which the drug was exposed to the explants for the duration of the study (24 hours) in all studies.

[0182] 4. Measurement of drug effects

[0183] After explants were exposed to the drug (toxin / AOS) for 24 hours, cells were washed in PBS and then fixed in progressively increasing concentrations of paraformaldehyde (PFA) (500 μL, 0.4% PFA, followed by 1.2%, then 3%, and finally 4% PFA). Each fixation step was performed for 3 minutes. Finally, the microexplants were washed in PBS.

[0184] Next, neurons in the explants were evaluated for morphology (presence of neurites) and counted per live cell / microscopic field of view. For each coverslip, four fields of view showing the highest cell density were counted, and the data were presented as mean + / - standard error of the mean (SEM); n=4 for each. Unpaired Student's t-test was used to assess statistical significance.

[0185] result

[0186] 1. Study 1 (3-Nitropropionic acid damage mode)

[0187] After exposing cerebellar microexplants to the mitochondrial toxin 3-nitropropionic acid, treatment with AOS enzyme had no effect on neuronal recovery (data not shown).

[0188] 2. Study 2 and 3 (hydrogen peroxide damage modes)

[0189] Exposure of cerebellar microexplants to hydrogen peroxide-induced oxidative stress resulted in near 100% neuronal death. However, treatment with AOS enzymes significantly increased neuronal survival at all tested drug concentrations (P < 0.001). Figure 3When studies 2 and 3 were combined, AOS produced an average neuronal recovery rate of 23% from hydrogen peroxide damage.

[0190] discuss

[0191] The study in this paper demonstrates that AOS can significantly rescue neurons from hydrogen peroxide damage in postnatal cerebellar granule cells with cerebellar microexplants.

[0192] Therefore, these results support the use of AOS to improve the effects of central nervous system injury, demonstrating surprising efficacy.

[0193] in conclusion

[0194] Lipid peroxidase can rescue neurons from hydrogen peroxide damage in postnatal cerebellar granule cells within tissues of cerebellar microexplants. These results suggest that AOS is an effective therapeutic option for ischemia-reperfusion central nervous system injury.

[0195] Example 2

[0196] Neuroprotective effects of cyclopropene synthase in an in vivo model of motor cortical photochemical thrombotic stroke

[0197] In this study, the inventors sought to investigate whether non-invasive intranasal administration of AOS, when administered 3 hours post-infarction (a time point previously reported to produce no measurable or statistically significant neuroprotective effect), could improve stroke outcomes in mice through neuroprotective or neuroregenerative mechanisms. To evaluate efficacy, the inventors used an in vivo model of focal ischemic stroke and analyzed infarct volume, functional recovery, and the potential of AOS to increase glial proliferation, neurogenesis, and angiogenesis post-stroke.

[0198] method

[0199] animal

[0200] All procedures described in this study were approved and performed in accordance with the guidelines for the care and use of laboratory animals developed by the University of Otago Animal Research Committee. Young (2-3 months old) male C57BL / 6 mice (Hercus-Taieri Research Unit, Dunedin, New Zealand) weighing approximately 20-30 g were used as previously described (reference). Animals were acclimatized for at least 7 days prior to the experiment. Five days after stroke, all animals were randomly assigned to treatment groups to ensure that all animals in any given cage received different treatments (stroke + saline-loaded feed (n=13); stroke + 10 μg AOS (n=13); stroke + 100 μg AOS (n=12); and stroke + AOSPacific 100 μg (n=11). All assessments were performed by observers unaware of the treatment groups.

[0201] Photochemical thrombosis model of focal ischemia

[0202] Focal stroke was induced in mice (total n=60) by photochemical thrombosis. Mice were placed in a stereotactic apparatus under isoflurane anesthesia (2–2.5% in a 70% N₂O / 30% O₂ mixture), given a dose of Temgesic, and the surgical site was shaved and wiped with chlorhexidine. The skull was then exposed through a midline incision, connective tissue was removed and dried, and eye ointment (PolyVisc) was applied to the eyes to prevent them from drying out during the procedure. A cold light source (KL1500 LCD, Zeiss, Auckland, New Zealand) attached to a 40x objective (providing 2 mm diameter illumination) was positioned as close to the skull as possible, 1.5 mm lateral to the anterior fontanelle. Rose red solution (0.2 mL, 10 g / L in physiological saline, intraperitoneally (ip), Signa-Aldrich, Auckland, New Zealand) was then administered. Five minutes later, the brain was illuminated through the entire skull for 15 minutes, while the body temperature was maintained at 36.9 ± 0.4 °C using a heating pad (Harvard apparatus, Holliston, MA, USA) throughout the procedure. Sham-operated mice underwent the same procedure, except they received saline injections (100 uL, ip) instead of a rose red solution.

[0203] The origin of AOS

[0204] Recombinant AOS was generated. AOS was produced to a purity >95% and stored at pH 8.3. For in vivo experiments, the stock solution was diluted directly in PBS to the required concentration.

[0205] AOS application

[0206] AOS (10 μg or 100 μg) was dissolved in sterile isotonic saline. Based on previously published research (Mathai, S. et al., CNS Neurosci Ther, 2012, 18(11), 887-94), AOS was administered at low (10 μg) and high (100 μg) doses. Control animals were injected with saline to serve as controls for AOS. AOS or saline was administered intranasally 3 hours after stroke. After a brief recovery period, mice were returned to their original cages and kept under normal living conditions (12-hour light / dark cycle) with free access to food and water. Pain relief was given in the form of temgesic the following day.

[0207] Organizational processing

[0208] Fourteen days after the stroke, the animals were deeply anesthetized with pentobarbital and perfused with 4% oligooxymethylene via the cardiac route. The brain was then extracted and sectioned into 30 μm thick sections in six coronal parallel groups on a sliding cryostat and held in cryoprotectant at -20°C.

[0209] Infarction size

[0210] Following a previously published experimental protocol (reference), infarct volume was determined using histological assessment with cresol violet staining. Infarct volume was quantified by an observer unaware of the treatment group using ImageJ (National Institutes of Health, USA), and based on measurements obtained from every sixth slice across the entire infarct (in mm). 2 The infarct volume (area) is quantified as follows: infarct volume (mm²) 3 = Area in mm 2 Square root of the slice thickness × inter-slice spacing Separation .

[0211] Eleven mice were found to have no visible stroke and were subsequently excluded based on incomplete stroke induction (saline-loaded (n=5); 10 μg AOS (n=2); 100 μg (n=2); and AOS2 100 μg (n=2)). Therefore, the final number of mice used for infarction and behavioral analysis was: saline-loaded (n=8); 10 μg AOS (n=11); 100 μg AOS (n=10); and AOS2 100 μg (n=9).

[0212] Behavioral assessment

[0213] Seven days prior to surgery, animals were tested once on both grid walking and cylinder tasks to establish baseline performance levels. Recovery of forelimb motor function and forelimb symmetry was determined by both grid walking and cylinder tasks, 7 and 14 days post-stroke, at approximately the same time each day, and at the end of their dark cycles. Behavior was rated by observers unaware of the animals' treatment group in this study, as previously described (Parker, K. et al., Scientific Reports, 7, 241, 2017).

[0214] Immunofluorescence labeling of GFAP and IBA1

[0215] Immunofluorescence labeling of GFAP and IBA1 was performed XX days post-stroke. Brain sections (30 μm thick, every sixth section through the stroke) were rinsed in Tris-buffered saline (TBS) and transferred to 1% sodium tetraborate in TBS and incubated at room temperature for 20 minutes. Sections were blocked for 60 minutes in TBS containing 5% goat and donkey serum and 0.3% Triton X-100, and incubated at 4°C for 24–48 hours in TBS containing 2% goat and donkey serum and 0.3% Triton X-100 and containing polyclonal primary antibodies (with added GFAP and IBA1 antibody information). The rinsed sections were incubated in the dark at room temperature in TBS containing 2% normal serum and 0.3% Triton X-100 and containing appropriate fluorescent secondary antibodies (with added secondary antibody information) for 2 hours, and stained with Hoechst (1:1000, Sigma-Alrich) in TBS for 5 minutes at room temperature. Photomicrographs of the sections were taken using an inverted montaging microscope (Eclipse Ti2, Nikon, Japan) with a 10x objective and output as nd2 files. Three sections from each animal were included in the analysis. On day XX post-stroke, changes in GFAP and iBA1 staining were investigated in two target peri-infarct areas (ROIs, area = 200 μm × 800 μm), at distances of 0–200 μm and 600–800 μm from the stroke boundary. Integrated density values ​​(IDV) were measured in both ROIs related to GFAP and IBA1 using FUJI ImageJ software (National Institutes of Health, USA).

[0216] Analysis of vascular characteristics

[0217] Vascular analysis was performed to investigate the effects of biopolymer hydrogels and AOS on post-stroke angiogenesis. Sections were photomicrographed using an inverted image stitching microscope (Eclipse Ti2, Nikon, Japan) with a 10X objective and output as nd2 files. Images were then opened using Fiji ImageJ software (National Institute of Health, USA), and two 200 μm × 800 μm rectangular ROIs were selected using the ROI manager analysis tool in Fiji ImageJ: (1) the core region, (2) the peri-infarct region, and (3) the cortical region approximately 600–800 μm from the stroke boundary. Each of these ROIs was saved as a new JPEG image, and opened in Neurolucida® 360 software (version 2020.1.1, MBF Bioscience, USA) to adjust their scaling before tracing all vascular branches. The total number of vessels, the total length of the traced vessels, and the average size of each vessel were then quantified using Neurolucida® Explorer software. Stroke analysis was performed on at least three slices from each animal to obtain the mean difference between treatment groups.

[0218] result

[0219] Histology: AOS had no effect on infarct volume.

[0220] Mice were given stroke and then treated 3 hours post-stroke with an intranasal dose of either AOS (10 μg or 100 μg) or saline-loaded solution. Infarct volume was assessed 14 days post-stroke using cresol violet staining, and the infarct volume in the loaded treatment group increased from 2.704 mm. 3 Up to 4.963 mm 3 Variation. Assessment of infarct volume did not reveal changes in the group treated with AOS (10 μg: n=6, 2.699 ± 0.85 mm). 3 ;100 μg: n=6, 2.676 ± 0.83 mm 3 ; 100 μg (AOS2): n=6, 2.896± 0.39 mm 3 Compared to the group receiving the load (n=6; 2.896 ± 0.39 mm), any one of them is considered equal to the group receiving the load. 3 The reduction in infarct volume.

[0221] Immunohistochemistry: AOS reduces inflammation after stroke

[0222] To investigate whether AOS reduces reactive astrocyte proliferation and reactive microglial proliferation in stroke animals, we assessed IBA1 using immunofluorescence staining 14 days post-stroke. Figure 4 ) and GFAP ( Figure 5 We observed the expression of AOS (a marker of inflammation) after the animals completed their final behavioral task. We found that 10 μg AOS inhibited reactive astrocyte proliferation in two periinfarct regions (0–200 μm from the stroke boundary (Region 1) and 600–800 μm from the stroke boundary (Region 2)). However, 100 μg and 100 μg Pacific AOS doses only significantly inhibited reactive astrocyte proliferation within Region 1.

[0223] Immunohistochemistry: AOS increases pericytes in the stroke core

[0224] To investigate whether AOS increased pericyte density in the stroke core, region 1, and region 2 in stroke-affected animals, we assessed PDGF-β expression using immunofluorescence staining 14 days post-stroke. We found that 10 μg AOS significantly increased pericyte density in the stroke core, but did not significantly increase pericyte density in adjacent strokes or in the peri-infarct region at a distance of 800 μm from the infarct. Figure 6 Higher doses of AOS (100 μg) also appeared to increase pericyte number, however the results were not significant.

[0225] Behavioral assessment: AOS improves motor function

[0226] Many ischemic strokes result in motor deficits. The inventors evaluated the protective effect of AOS against focal photochemical thrombotic stroke towards the motor cortex, with motor function assessed via grid walking (forelimb function) and cylinder (forelimb asymmetry) tasks at 7 and 14 days post-stroke. The grid walking task revealed a significant increase in forelimb motor function in stroke animals receiving AOS compared to those receiving a feed at both 7 and 14 days and at all doses. Figure 7 ).

[0227] The cylinder task showed that at both 7 and 14 days post-stroke, compared to pre-stroke motor function, the feed group exhibited significant forelimb motor deficits on the contralateral side of the stroke hemisphere (time spent on the left forelimb vs. time spent on the right forelimb: 50% pre-stroke, 32% at 7 days post-stroke, and 31% at 14 days post-stroke). Compared to the feed group, treatment with AOS at 10 μg, 100 μg, and 100 μg Pacific AOS resulted in significant reductions in motor deficits at both 7 days post-stroke (time spent on the left forelimb relative to the right forelimb: 40% for 10 μg, 37% for 100 μg, and 41% for 100 μg Pacific) and 14 days post-stroke (time spent on the left forelimb relative to the right forelimb: 39% for 10 μg, 40% for 100 μg, and 38% for 100 μg Pacific). Figure 8 ).

[0228] Discussion and Conclusion

[0229] This study demonstrates that treatment with AOS 3 hours after central nervous system injury (e.g., 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 known limitations in the field: AOS treatment for ischemic brain injury is only effective when administered within a narrow 45-minute window following the ischemic event. Furthermore, this study demonstrates that AOS can be non-invasively delivered to the central nervous system via an intranasal route to produce substantial therapeutic improvements.

[0230] Example 3

[0231] The effect of cyclopropene synthase on neurological recovery in an in vivo model of photochemical thrombotic stroke in the prefrontal cortex

[0232] This invention seeks to further investigate whether the 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 analyzed the potential of AOS to increase post-stroke cognitive deficits.

[0233] method

[0234] Animals: Young (2-3 months old) male C57BL / 6 mice weighing approximately 20-30 g were used as previously described (REF). Animals were acclimatized for at least 7 days prior to the experiment. All animals were randomly assigned to treatment groups to ensure that all animals in any given cage received different treatments (stroke + saline feed; stroke + 10 μg AOS; stroke + 100 μg AOS; and stroke + 100 μg AOS (second batch)). All assessments were performed by observers unaware of the treatment groups.

[0235] Photochemical thrombosis model of focal ischemia: Focal stroke was induced in the prefrontal cortex of mice via photochemical thrombosis. Mice were placed in a stereotactic apparatus under isoflurane anesthesia (2–2.5% in a 70% N₂O / 30% O₂ mixture), given a dose of Temgesic, and the surgical site was shaved and wiped with chlorhexidine. The skull was then exposed through a midline incision, connective tissue was removed, and the area was dried. Eye ointment (PolyVisc) was applied to the eyes to prevent them from drying out during the procedure. A cold light source (KL1500 LCD, Zeiss) attached to a 40x objective lens (providing 2 mm diameter illumination) was positioned as close to the skull as possible, 1.2 mm anterior to the anterior fontanelle. A rose red solution (0.2 mL, in 10 g / L physiological saline, intraperitoneally) was then administered. Five minutes later, the brain was illuminated through the entire skull for 15 minutes, while the body temperature was maintained at 37°C using a heating pad throughout the procedure. Sham-operated mice underwent the same procedure, except that they received an injection of saline (100 μL, intraperitoneally) instead of a rose red solution.

[0236] AOS administration: Under aseptic conditions, AOS (100 μg) was dissolved in sterile isotonic saline and combined with a hyaluronic acid / heparan sulfate proteoglycan biopolymer hydrogel (HyStem-C, Biotime Inc, Alameda, CA, USA). The AOS dissolved in HyStem-C was administered at 100 μg / dose according to the manufacturer's instructions. In short, AOS was added to the HyStem-C / Gelin-S mixture (component 1 of the hydrogel), followed by Extralink (component 2 of the hydrogel) at a 4:1 ratio. The impregnated HyStem-C mixture was injected into the stroke cavity immediately after preparation using a 30-gauge needle attached to a Hamilton syringe at stereotactic coordinates of 1.2 mm AP, 0 mm ML, and 0.75 mm DV. Mice received Temgesic on the day of surgery and the following day during all surgical procedures. ®Buprenorphine hydrochloride was administered as pain relief. HyStem-C without AOS was injected into control animals as a control for AOS. AOS or controls were administered directly and locally to the periinfarct cortex five days after stroke, as previously described by Houlton et al. (Houlton, J. et al., Int. J. Mol. Sci. 2022, 23, 4817). After a brief recovery period, mice were returned to their original cages and maintained under normal living conditions (12-hour light / dark cycle) with free access to food and water. Pain relief was administered in the form of Temgesic the following day.

[0237] Tissue preparation: 14 days post-stroke, animals were deeply anesthetized with pentobarbital and perfused with 4% oligooxymethylene via the cardiac route. The brain was then extracted and sectioned into 30 μm thick sections in six coronal parallel groups on a sliding cryostat microtome, and held in cryoprotectant at -20°C.

[0238] Infarct size: Infarct volume was determined histologically using cresol violet staining, according to standard experimental protocol. Infarct volume was quantified by an observer unaware of the treatment group using ImageJ software, based on measurements obtained from every sixth slice across the entire infarct (in mm). 2 The infarct volume (area) is quantified as follows: infarct volume (mm²) 3 = surface area mm 2 Square root × slice thickness × slice interval .

[0239] Immunofluorescence labeling of GFAP and IBA1: Immunofluorescence labeling of GFAP and IBA1 was performed 35 days post-stroke. Brain sections (30 μm thick, every sixth section through the stroke) were rinsed in Tris-buffered saline (TBS) and transferred to 1% sodium tetraborate in TBS and incubated at room temperature for 20 minutes. Sections were blocked for 60 minutes in TBS containing 5% goat and donkey serum and 0.3% Triton X-100, and incubated at 4°C for 24–48 hours in TBS containing 2% goat and donkey serum and 0.3% Triton X-100 and polyclonal primary antibodies against IBA1 or GFAP. Rinsed sections were incubated in the dark at room temperature in TBS containing 2% normal serum and 0.3% Triton X-100 and appropriate fluorescent secondary antibodies for 2 hours, and stained with Hoechest (1:1000, Sigma-Aldrich) in TBS for 5 minutes at room temperature. The sections were photomicrographed using an inverted montaging microscope (Eclipse Ti2, Nikon, Japan) with a 10x objective and output as nd2 files, with three sections from each animal included in the analysis. At 30 days post-stroke, changes in GFAP and IBA1 staining were investigated in two target peri-infarct areas (ROIs, area = 200 μm × 800 μm), at distances of 0–200 μm and 600–800 μm from the stroke boundary. Integrated density values ​​(IDV) were measured in both ROIs related to GFAP and IBA1 using FUJIImage J software.

[0240] Behavioral Analysis: The Object Location Recognition Task (OLRT) is widely used to evaluate spatial working memory in rodents and has reliably identified relay deficits in mice exposed to bilateral prefrontal cortical stroke. OLRT testing was performed at the same time of day at one and four-week time points to minimize variability. All tests were recorded by an uninformed researcher using the software TopScan (CleverSys Inc.) via an overhead camera prior to analysis. The day before the OLRT test, animals were placed in the center of an OLRT arena (400×400×200 mm, plexiglass) without any objects and allowed to roam freely for ten minutes to acclimatize to the arena and testing chamber. On the second day (days 8 and 29 post-stroke), animals underwent OLRT to evaluate spatial memory. Initially, mice were placed in the center of an arena containing two identical objects (placed in two adjacent corners (80 mm from the corner walls)) for a period of 10 minutes. Immediately after this pre-test phase, mice were returned to their original cages one hour later, and the arena was cleaned. The mice were then placed back into the experimental chamber for a three-minute test period during which one of the objects was moved to the opposite corner. Object exploration was defined as the time the mouse pointed to (within a 20 mm perimeter surrounding the object) and sniffed it. The time spent standing or climbing on the object was excluded from the final analysis. The duration spent in the new location was assessed as the ratio of the total time spent interacting with one object to the total time spent interacting with both objects. Consistent with previous findings, ceramic bear salt bottles and soft drink cans were used as OLRT objects during one-week and four-week post-stroke recovery, respectively. The experimental chamber and all objects were cleaned with 30% ethanol between behavioral runs to prevent the presence of confusing odors.

[0241] result

[0242] Immunohistochemistry: To confirm the associated reduction in reactive astrocyte proliferation and reactive microglial proliferation in stroke animals, immunofluorescence staining was used to assess GFAP and IBA1 expression (markers of neuroinflammation) 35 days post-infarction. AOS was observed to significantly reduce reactive glial proliferation (…). Figure 9 These findings demonstrate that AOS significantly reduces neuroinflammation.

[0243] Behavioral analysis: The therapeutic potential of AOS was evaluated by comparing the performance of sham-operated and stroke-treated animals on OLRT. Consistent with previous reports, a two-factor ANOVA assessing the exploratory preferences of animals treated with feed and AOS one week post-stroke failed to identify any treatment, stroke, or interaction effects. However, at four weeks post-stroke, only a significant overall effect of stroke was observed, demonstrating impaired spatial working memory. Conversely, treatment with AOS significantly prevented cognitive deficits at four weeks post-stroke. Figure 10 There were no significant differences in cognitive abilities and memory between animals treated with AOS and non-stroke (control) animals.

[0244] Discussion and Conclusion

[0245] The study in this article demonstrates that treatment with AOS is effective in treating cognitive deficits and neuroinflammation following brain injury. GFAP and IBA1 are well-established biomarkers of neuroinflammation and reactive gliosis associated with several neurodegenerative diseases, including, for example, Alzheimer's disease, dementia, stroke, stroke-induced cognitive decline, postoperative cognitive impairment, HIV-related dementia, Parkinson's disease, motor neuron disease, amyotrophic lateral sclerosis (ALS, Lou Gehring's disease), and Huntington's disease. Reductions in these neuroinflammatory biomarkers have a known association with disease improvement.

[0246] The strong neuroprotective activity of AOS demonstrated in this paper, combined with the reduction in microglial and astrocyte proliferation, efficient intranasal delivery of AOS to the CNS, and substantial improvement in cognitive deficits in object location recognition tasks, suggest that AOS could be an effective treatment for a wide range of neurodegenerative and neuroinflammatory conditions in which oxidative stress plays a fundamental role, including, for example, Alzheimer's disease, dementia, stroke, stroke-induced cognitive decline, postoperative cognitive impairment, HIV-related dementia, Parkinson's disease, motor neuron disease, amyotrophic lateral sclerosis (ALS, Lou Gehring's disease), and Huntington's disease.

Claims

1. A method for improving recovery after central nervous system injury in a subject, comprising administering to the subject a composition comprising cyclopropenase or a functionally equivalent variant or derivative thereof.

2. The method according to claim 1, wherein the cyclopropenzyme is selected from the group consisting 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, or functionally equivalent derivatives or variants thereof.

3. The method according to claim 1 or claim 2, wherein the composition is administered at least approximately 3 hours after central nervous system injury.

4. The method according to any one of claims 1 to 3, wherein the composition is administered to the subject at least approximately 4 hours, at least approximately 5 hours, at least approximately 6 hours, at least approximately 7 hours, at least approximately 8 hours, at least approximately 9 hours, at least approximately 10 hours, at least approximately 11 hours, at least approximately 12 hours, at least approximately 13 hours, at least approximately 14 hours, at least approximately 15 hours, at least approximately 16 hours, at least approximately 17 hours, at least approximately 18 hours, at least approximately 19 hours, at least approximately 20 hours, at least approximately 21 hours, at least approximately 22 hours, at least approximately 23 hours, or at least approximately 24 hours after the injury.

5. The method according to any one of claims 1 to 4, wherein the composition is administered to the subject at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, at least about ten days, at least about eleven days, at least about twelve days, at least about thirteen days, or at least about fourteen days after the central nervous system injury.

6. The method according to any one of claims 1 to 5, wherein the composition is formulated for administration to the subject orally, intravenously, subcutaneously, intraperitoneally, sublingually, intranasally, by inhalation, locally, intrathecally, intraocularly, intramuscularly, intracranially, or systemically.

7. The method of claim 6, wherein the composition is formulated into an aqueous solution for intranasal administration to a subject.

8. The method according to any one of claims 1 to 7, wherein the composition further comprises a nonionic surfactant.

9. The method of claim 8, wherein the nonionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-X100 or any other pharmaceutically acceptable surfactant.

10. The method of any one of claims 1 to 9, wherein the cyclopropene synthase is derived from Parahyalosphenia glabra (P. glabra). Parthenium argentatum ).

11. The method of claim 10, wherein the cyclopropenzyme from *Gynostemma pentaphyllum* is SEQ ID NO:

1.

12. The method according to any one of claims 1 to 11, wherein the central nervous system injury is selected from ischemic stroke, hemorrhagic 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. Use of cyclopropenase or its functionally equivalent variants or derivatives in the preparation of medicaments for the treatment of central nervous system injury.

14. The use according to claim 13, wherein the cyclopropenzyme is selected from the group consisting 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, or functionally equivalent derivatives or variants thereof.

15. The use according to claim 13 or 14, wherein the drug is administered at least approximately 3 hours after the central nervous system injury.

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

17. The use according to any one of claims 13 to 16, wherein the drug is administered at least one day, at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, at least about ten days, at least about eleven days, at least about twelve days, at least about thirteen days, or at least about fourteen days after the 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 topical application.

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

20. The use according to any one of claims 13 to 19, wherein the drug further comprises a nonionic surfactant.

21. The use according to claim 20, wherein the nonionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton-X100 or any other pharmaceutically acceptable surfactant.

22. The use according to any one of claims 13 to 21, wherein the cyclopropenzyme is derived from hygrophila var. gracilis.

23. The use according to claim 22, wherein the cyclopropenzyme from *Gynostemma pentaphyllum* is SEQ ID NO:

1.

24. The use according to any one of claims 1 to 11, wherein the central nervous system injury is selected from ischemic stroke, hemorrhagic 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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