Novel compositions and uses

Abstract

The present disclosure is concerned with compositions comprising monodisperse assemblies of allene oxide synthase (AOS), and in particular compositions and assembles comprising AOS enzymes and polysorbate surfactants. Such compositions have improved enzymatic activity that are particularly useful for remediating biological conditions of oxidative stress.

Description

NOVEL COMPOSITIONS AND USESSTATEMENT REGARDING SEQUENCE LISTING

[0001] The Sequence Listing associated with this application is provided in text format in lieu of paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is “91902- 364496 Sequence Listing_ST26.”FIELD OF THE INVENTION

[0002] The present disclosure describes novel allene oxide synthase compositions with improved stability, antioxidant, and / or biological activity. Also described herein are compositions and assemblies comprising allene oxide synthase enzymes and non-ionic surfactants that provide improved activity and properties compared to known formulations. The disclosure further describes methods of use of said compositions and assemblies for controlling undue oxidative stress in a subject.BACKGROUND

[0003] The CYP74A family of enzymes includes allene oxide synthases (AOS) and comprises fewer than 15 molecules from a variety of plant sources including guayule, corn, barley, tomato, flaxseed and Arabidopsis (a model plant in biology), all performing AOS function and having molecular weights in the monomeric form of ~55kDa. The enzymes are atypical members of the Cytochrome P450 family in that they self-regenerate, requiring neither oxygen nor a NADPH reductase for activity and have extraordinary reaction rates. AOS enzymes transform fatty acid hydroperoxides, formed by oxidation of polyunsaturated fatty acids into unstable epoxides which then further degrade into ketols. The lipid peroxides (LPO) can be formed by oxidative challenge or the action of lipoxygenase, a pathway used in the generation of LPO substrate for use in the AOS enzyme function assay.

[0004] P450 enzymes including the AOS family are generally membrane bound. For this reason, detergents including (e.g. Triton-X 100, Emulphogene) are often used in purification / extraction methodologies, whether from natural product or molecularcloned sources to enhance the stability of enzyme in solution. It has become increasingly apparent to those in the art that the absence of detergent can generally be expected to result in precipitation of enzyme from solution and loss of activity. For example, Hughes et al. (2006) FEBS Letters 580:4188-4194, investigated detergent effects on the enzyme kinetics of allene oxide synthase from Arabidopsis thaliana (CYP74A1) and showed that detergent-free enzyme had poor activity against 13 — S — hydroperoxyoctadeca-9Z,11 E,15Z — trienoic acid (kcat / Km0.12 x 107 M'1s'1) this activity was significantly enhanced by the addition of emulphogene micelles (kcat / Km5.9 x 107 M'1s'1). In other words, in the presence of detergent, there was nearly a 50-fold increase in enzyme activity for its preferred substrate.

[0005] Indeed, detergent effects on structure and function of enzymes has been increasingly reported in the literature, and the conclusions reached is that detergent is required for enzyme activity. That is, addition of detergent markedly increases enzyme activity, particularly with respect to substrate specificity and turnover rate.

[0006] AOS enzymes, for example AOS enzymes derived from Parthenium argentatum have known utility as cardioprotective therapies (See, e.g. US 7,157,082, and WO 2015 / 183106, which are herein incorporated by reference with regard to such background teachings). An immediate clinical and / or commercial utility for these and other AOS enzymes is in biological systems where it has been shown that AOS enzymes act as antioxidant molecules to scavenge peroxidation products and prevent chain oxidation reactions, making them commercially valuable for several applications. The resultant enzyme formulations, however, are often produced as heterogeneous populations with inconsistent size distribution and stability.Furthermore, certain detergents required for formulation can have a negative effect in biological systems including, for example, semen and embryo preservation in assisted reproductive technologies, which are initial product targets for immediate / initial clinical application of existing and next generation AOS enzymes.

[0007] There remains an unmet need for AOS enzyme formulations that possess significant enzymatic activity and stability, whilst overcoming the limitations of known methods.SUMMARY OF THE INVENTION

[0008] One embodiment described herein is a protein assembly comprising an allene oxide synthase enzyme wherein the assembly has a hydrodynamic diameter of between about 5 nm to 30 nm. In one aspect the allene oxide synthase enzyme is derived from Parthenium argentatum. In another aspect, the allene oxide synthase enzyme comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from one or more 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.

[0009] In another aspect, the protein assembly further comprises a polysorbate surfactant, wherein the polysorbate surfactant comprises one or more of polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0010] Another aspect described herein is a composition comprising the protein assembly described herein.Another aspect described herein is a pharmaceutical composition comprising one or more of the protein assemblies described herein and a pharmaceutically acceptable carrier.

[0011] In one aspect of the pharmaceutical composition, a portion of the composition comprises a protein assembly having a hydrodynamic diameter of between about 5 nm to 30 nm. Another aspect described herein, is a pharmaceutical composition wherein the one or more polysorbate surfactants comprises a concentration at or above the critical micelle concentration.

[0012] Another aspect described herein is a method for treating ischemic injury in a subject in need thereof, the method comprising administering to the subject any of the protein assemblies or the pharmaceutical compositions described herein. In one aspect, the ischemic injury is ischemia-reperfusion injury. In another aspect, the ischemic injury or ischemia-reperfusion injury is associated with one or more conditions selected from the group comprising coronary artery occlusion, myocardial infarction, angina pectoris, thrombolysis, myocardial ischemia, mesenteric ischemia, cerebrovascular stroke, ischemic stroke, hepatic ischemia, limb ischemia, pulmonary ischemia, kidney ischemia, ocular ischemia and retinal ischemia; and / or the subject is undergoing or has undergone a procedure selected from the group consisting of angioplasty, thrombectomy, and coronary artery bypass surgery.In another aspect, is a method of preserving sperm, wherein the method comprises contacting the sperm with any of the protein assemblies described herein.

[0013] Another aspect described herein is a method for preserving of semen, wherein the method comprises contacting the semen with any of the protein assemblies described herein.

[0014] In another aspect described herein a method for improving sperm viability, wherein the method comprises contacting the sperm with any of the protein assemblies described herein.

[0015] In another aspect described herein a method for improving the percentage of viable implantable embryos wherein the method comprises contacting either: (i) an oocyte; or (ii) a fertilised embryo; and any of the protein assemblies described herein or the any of the compositions described herein.

[0016] Another embodiment described herein is a composition suitable for treating ischemic injury comprising: a. one or more allene oxidase enzymes; b. one or more polysorbate surfactants; c. one or more buffers; and d. one or more or more excipients; wherein the protein assembly has a hydrodynamic diameter of between about 5 nm to 30 nm.

[0017] In another aspect, the one or more polysorbate surfactants comprise polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0018] In another aspect, the one or more polysorbate surfactants comprises a concentration at or above the critical micelle concentration.

[0019] Various embodiments of the different aspects of the invention as discussed above are also set out below in the detailed description of the invention, but the invention is not limited thereto.

[0020] Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

[0021] This disclosure may also be said to broadly consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : describes the allene oxide synthase enzyme sequences defined by SED 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 and SEQ ID NO: 9.

[0023] Figure 2: describes the average enzymatic activity of PaAOS incubated with various surfactants (A) below and (B) above the respective critical micelle concentration.

[0024] Figure 3: describes images of PaAOS when incubated with various surfactants by negative-stain transmission electron microscopy. (A) shows no surfactant addition. (B) shows 0.000001% Triton X-100 (Tx100) (0.03% of CMC). (C) shows 0.00002% Tween 20 (Tw20) (0.03% of CMC). (D) shows 0.02% Tx100 (-100% of CMC). (E) shows 0.004% Tw20 (60% of CMC).

[0025] Figure 4: describes the average enzymatic activity of PaAOS formulated with Triton X-100, polysorbate 20 (Tween 20), and polysorbate 80 (Tween 80), at surfactant concentrations above the critical micelle concentration. Error bars represent standard deviation (n=3).

[0026] Figure 5: (A) describes the native gel electrophoresis of PaAOS enzymes formulated with Triton X-100, polysorbate 20 (Tween 20), and polysorbate 80 (Tween 80), and molecular weight standards (Bovine Serum Albumin (BSA), 0- Amylase, and Thryoglobulin). (B) describes the native gel electrophoresis of PaAOS enzymes formulated with Triton X-100, polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80), and molecular weight standards (Bovine Serum Albumin, 0-Amylase, and Thryoglobulin).

[0027] Figure 6: describes the effect on HEK 293 cells of allene oxide synthase enzymes formulated with (A) Triton X-100, (B) polysorbate 20 (Tween 20), and (C) polysorbate 80 (Tween 80). Note significantly reduced cellular toxicity with AOS- polysorbate assemblies.

[0028] Figure 7: describes the hydrodynamic diameter of protein standards (A) BSA and (B) thyroglobulin, and (C) PaAOS assemblies when substantially depleted of surfactant.

[0029] Figure 8: describes the hydrodynamic diameter of (A) Triton X-100 micelles, (B and C) AOS-Triton X-100 enzyme-surfactant assemblies.

[0030] Figure 9: describes the hydrodynamic diameter of (A) polysorbate 20 (Tween 20) micelles, (B and C) AOS-Tween 20 enzyme-surfactant assemblies.

[0031] Figure 10: describes the hydrodynamic diameter of (A) polysorbate 80 (Tween 80), (B and C) AOS-Tween 80 enzyme-surfactant assemblies.

[0032] Figure 11 : describes the average hydrodynamic diameter of (A) protein controls BSA and thyroglobulin, and (B) surfactant controls Triton X-100, polysorbate 20 (Tween 20), and polysorbate 80 (Tween 80) micelles. Error bars represent standard deviation (n=3).

[0033] Figure 12: describes the average hydrodynamic diameter of the most common particle distributions in solution by volume for AOS assemblies formulated with Triton X-100, polysorbate 20 (Tween 20) or polysorbate 80 (Tween 80) above the critical micelle concentration, or substantially depleted of surfactant. Error bars represent standard deviation (n=3).

[0034] Figure 13: describes the average hydrodynamic diameter of the smallest distinct particle distributions in solution by volume for AOS assemblies formulated with Triton X-100, polysorbate 20 (Tween 20) or polysorbate 80 (Tween 80) above the critical micelle concentration, or substantially depleted of surfactant. Error bars represent standard deviation (n=3).

[0035] Figure 14: describes the effect of PaAOS enzymes formulated with polysorbate 80 (Tween 80) on neuronal cells insulted with hydrogen peroxide.DETAILED DESCRIPTION

[0036] The present disclosure describes novel allene oxide synthase compositions with improved stability, antioxidant, and / or biological activity. Also described herein are compositions and assemblies comprising allene oxide synthase enzymes and non-ionic surfactants that provide improved activity and properties compared to known formulation methods. The disclosure further describes methods of use of said compositions and assemblies for controlling undue oxidative stress in a subject.

[0037] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art to which the inventions belong (for example, in immunology, immunohistochemistry, protein chemistry, and biochemistry). In case of conflict, thepresent document, including definitions, will control. Methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0038] Unless otherwise indicated, the recombinant protein and immunological techniques utilized in the present disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0039] The articles "a" and "an" are used herein to refer to one or more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0040] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0041] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0042] It is intended that reference to a range of numbers disclosed herein (for example 1 to 10) also incorporates reference to all related numbers within that range (for example, 1 , 1.1 , 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range ofrational numbers within that range (for example 2 to 8, 1 .5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0043] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0044] Those skilled in the art will appreciate that the embodiments described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and assemblies referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0045] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0046] Any example described herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise.Selected Definitions

[0047] The term “about” is used herein to refer to conditions (e.g., amounts, concentrations, time etc) that vary by as much as 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% to a specified condition.

[0048] The term “AOS” as used herein refers to an allene oxide synthase enzyme used in the context of AOS enzyme activity.

[0049] The term “PaAOS” as used herein refers to allene oxide synthase as derived from Parthenium argentatum.

[0050] The term “assembly” as used herein refers to a self-assembled structure formed from a group of molecular aggregates. For example, a grouping of protein(s) and surfactant(s) that forms a discrete structure.

[0051] The term “biologically active fragment” as used herein means a fragment of a full-length polypeptide, peptide or protein which fragment retains an activity of the polypeptide, peptide or protein. As used herein, the term “biologically active fragment” includes deletion mutants and small polypeptides that retain the functional activity of the full-length polypeptide, peptide or protein.

[0052] The term “critical micelle concentration” is used to refer to the concentration of surfactants above which micelles form.

[0053] As used herein, “culturing”, “culture” and the like refer to the set of procedures used in vitro where a population of cells (or a single cell) is incubated under conditions which have been shown to support the growth or maintenance of the cells in vitro. The art recognises a wide number of formats, media, temperature ranges, gas concentrations etc. which need to be defined in a culture system. The parameters will vary based on the format selected and the specific needs of the individual who practices the methods herein disclosed. However, it is recognised that the determination of culture parameters is routine in nature.

[0054] The term “derivative” as used herein is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to a parent sequence including additions, substitutions or deletions that provide for functionally equivalent molecules.

[0055] The terms “detergent” and “surfactant” may be used interchangeably in this specification, including reference to plural form, are defined by their commonly used definitions.

[0056] The term “detergent-depleted” and “detergent-free” may be used interchangeably in this specification to refer to (e.g.) enzyme preparations including compositions that contain substantially no detergent or only a residual amount of detergent. In some examples according to the enzyme compositions describedherein, the terms “detergent-depleted” and “detergent-free” mean a composition than contains (e.g.) less than 0.025% detergent, preferably less than 0.020 %, preferably less than 0.015 %, preferably less than 0.010 %, preferably less than 0.005% and preferably less than 0.001% detergent.

[0057] The term “effective amount”, in the context of a particular utility of the peptides and enzymes described herein, is meant the administration of that amount of composition to an individual in need thereof, either in a single dose or as part of a series, that is effective for that stimulation, prevention or treatment, as noted through clinical testing and evaluation, patient observation, and / or the like. An "effective amount" may further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and / or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" may designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and / or promotes improvement in the condition of interest. An "effective amount" may further refer to a “therapeutically effective amount”. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0058] As used in this specification, the term “fragment” or “functional derivative” in relation to a polypeptide is a subsequence of a polypeptide that may be detected (e.g.) using a binding agent. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant or derivative thereof.

[0059] The term “hydrodynamic diameter” as used herein refers to a protein assembly or particle diameter in solution, including all solvation spheres. The hydrodynamic diameter as used herein refers to the diameter of a perfect solid sphere that would exhibit the same hydrodynamic friction as the molecule of interest.

[0060] The term “isolated” as applied to the polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular or other naturally-occurring biological environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, andsynthetic techniques. The polypeptide sequences may be prepared by at least one purification step.

[0061] The term “micelle” is used to refer to an aggregate, or supramolecular assembly, of a surfactant (or other such amphipathic molecules) dispersed in a liquid, forming a colloidal suspension.

[0062] The terms “patient”, “subject”, “host” or “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, most particularly a human, for whom a diagnosis, prophylaxis or therapy is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, humans, any member of the subphylum Chordata including primates, rodents (e.g. , mice rats, guinea pigs), lagomorphs (e.g. , rabbits, hares), bovines (e.g. , cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards etc), and fish. The subject may be in need of diagnosis, prophylaxis or treatment, however, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0063] The terms “peptide” and “polypeptide” or “protein” may be used interchangeably throughout this specification, and encompass amino acid chains of any length, including full length sequences in which amino acid residues are linked by covalent peptide bonds. Polypeptides useful in the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof. Polypeptides herein may contain chain lengths of at least 4 amino acids, at least 5 amino acids, or at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or 23 amino acids. Reference to other polypeptides of the invention or other polypeptides described herein should be similarly understood.

[0064] The term “protein assembly” as used herein refers to the non-covalent supramolecular assembly of allene oxide synthase enzyme to one or more non-ionicsurfactants or micelles. This assembly occurs in a similar way to how the hydrophobic face of an enzyme binds to a cell membrane.

[0065] The term “purified” refers in various embodiments, for example, to at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homogeneity of a polypeptide, for example, in a sample.

[0066] Term “variant” as used herein refers to polypeptide sequences different from the specifically identified sequences. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments described herein are, variants of the polypeptides having biological activities including signal peptide activity or antigenic-binding properties that are the same or similar to those of the parent polypeptides. The term “variant” with reference to polypeptides encompasses all forms of polypeptides as defined herein.

[0067] Variant polypeptide / protein sequences may exhibit at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence of the present invention.

[0068] Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences, including those which could not reasonably be expected to have occurred by random chance.

[0069] Polypeptide sequence identity and similarity can be determined by comparing the polypeptide to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.18 [April 2008]]) in bl2seq, which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off. Other methods of determining sequence identity are known in the art.

[0070] The similarity of polypeptide sequences may be examined using the following UNIX command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp. The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an “E value” which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. Variant polypeptide sequences commonly exhibit an E value of less than 1 x 10-5, less than 1 x 10-6, less than 1 x 10-9, less than 1 x 10-12, less than 1 x 10-15, less than 1 x 10-18 or less than 1 x 10-21 when compared with any one of the specifically identified sequences. Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polypeptide sequences using global sequence alignment programs. EMBOSS-needle (available at http: / www.ebi. ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. Use of BLASTP is preferred for use in the determination of polypeptide variants according to the present invention, but other methods are known in the art.

[0071] The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by- amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, lieu, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention.

[0072] The term “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for Windows;available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0073] The term sequence “similarity” refers to the percentage number of amino acids that are identical or constitute conservative amino acid substitutions as defined. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984 Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0074] The term "treatment", "treat", and "treating" refer to reversing, alleviating, mitigating, or slowing the progression of, or inhibiting the progress of, a disorder or disease or symptoms associated with such disorder or disease, and as described in more detail herein.

[0075] It is intended that reference to a range of numbers disclosed herein (for example 1 to 10) also incorporates reference to all related numbers within that range (for example, 1 , 1.1 , 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1 .5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0076] The present disclosure relates to novel compositions of allene oxide synthase enzymes with improved stability, enzymatic activity, antioxidant and / or biological activity. Also described herein are compositions and assemblies comprising allene oxide synthase enzymes and non-ionic surfactants that provide improved enzymatic activity and properties over other known formulation methods. Further described herein are pharmaceutical compositions, methods of use of said compositions and assemblies for controlling undue oxidative stress in a subject or cell.

[0077] The disclosure further relates to methods of use of said compositions and assemblies for controlling undue oxidative stress in a subject.

[0078] Allene oxide synthase (AOS) are cytochrome P450 plant enzymes with antioxidant activity that may be used for numerous applications, including treatmentof some diseases and the preservation of biological materials. However, in order to exploit these proteins for their therapeutic use or other purposes, the enzymes must not only be isolated, but included in compositions that maintain or even enhance their biological activity. Because these enzymes are generally membrane bound, the use of detergents (most commonly Triton X-100 and Emulphogene (polyoxyethylene tridecyl ether)) is employed for their purification and extraction during recombinant production processes. Detergents play an essential role in the extraction and purification of protein from their native environment, but require care to maintain the integrity of the enzyme and minimize toxicity. The present disclosure is predicated on the discovery that formulating allene oxide synthase enzymes with non-ionic surfactants, particularly non-ionic polysorbate surfactants, surprisingly produces substantial improvements in enzymatic activity and stability over other known surfactants.

[0079] Thus, in one embodiment described herein a protein assembly comprising an allene oxide synthase and a non-ionic surfactant. As used herein, a protein assembly is a higher order non-covalent supramolecular complex of the allene oxide synthase enzymes and the non-ionic surfactant and / or surfactant micelles. In one aspect, the allene oxide synthase used may be derived from plants. In one aspect, the allene oxide synthase may be derived from, for example, Arabidopsis thaliana or Parthenium argentatum, or any other plant species that would normally express allene oxide synthase. In another aspect, the allene oxide synthase may be derived from Parthenium argentatum. In another aspect, the allene oxide synthase may be derived from Helianthus annus.

[0080] In one aspect, the allene oxide synthase may be wild-type. In other aspect, the allene oxide synthase may be modified or a functional variant. “Functional variant” when used with reference to a polypeptide refers to a polypeptide that differs from the referenced polypeptide (for example, differing by at least one conservative amino acid substitution) but possesses the primary function of the referenced polypeptide. For example, a functional variant of a polypeptide that serves as a transmembrane domain is a fragment of that polypeptide that also serves as a transmembrane domain. When used with reference to a nucleic acid, the phrase “functional variant” refers to a nucleic acid that differs from the referenced nucleic acid but encodes a polypeptide having the same primary function as the polypeptide encoded by the referenced nucleic acid.

[0081] The allene oxide synthase enzyme may thus be encoded by the amino acid sequences described in Figure 1 . In one another aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 1 , or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1). In one another aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 2, or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2). In another aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 3, or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3). In one aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 4). In yet another aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 5, or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 5). In yet another aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 6, or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 6). In yet another aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 7, or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 7). In yet another aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 8). In yet another aspect, the allene oxide synthase enzyme is encoded by the amino acid sequence of SEQ ID NO: 9, or a functional variant thereof (e.g., a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 9).

[0082] Illustrative surfactants include non-ionic, anionic or cationic surfactants. Exemplary surfactants further include the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The non-ionic surfactants of the present disclosure, include, but are not limited to polysorbate surfactants selected from polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, polyoxyethylene (20) sorbitane monolaurate, polyoxyethylene (20) sorbitane monopalmitate, polyoxyethylene (20) sorbitane monostearate, and polyoxyethylene (20) sorbitane monooleate. In one aspect, the non-ionic surfactant comprises one or more of Triton X-100, polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0083] The hydrodynamic diameter as used herein refers to the diameter of a perfect solid sphere that would exhibit the same hydrodynamic friction as the molecule of interest, including all solvation spheres. Without being bound by any theory, it is believed that smaller protein assembly size correlates to higher enzymatic activity. Thus, in one aspect, the protein assembly described herein has a hydrodynamic diameter of between 1 nm to about 50 nm. In another aspect, the protein assembly described herein has a hydrodynamic diameter of between 2 nm to about 40 nm. In another aspect, the protein assembly described herein has a hydrodynamic diameter of between 3 nm to about 30 nm. In another aspect, the protein assembly described herein has a hydrodynamic diameter of between 3 nm to about 30 nm.

[0084] The term “composition” as used herein is intended to encompass a product comprising the specific molecules described herein, in specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Thus, another aspect of the present disclosure is a composition comprising the protein assembly described herein.

[0085] A therapeutically effective amount of a protein assembly is that amount which produces a result or exerts an influence on the particular condition being treated. The protein assembly described herein may thus be administered with a pharmaceutically-acceptable carrier using any effective conventional dosage unit forms, including, for example, immediate and timed release preparations, orally, parenterally, topically, nasally or the like. In some aspects, the compound is administered intravenously or orally.

[0086] A pharmaceutically acceptable carrier is any carrier which is relatively nontoxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. A therapeutically effective amount of a compound is that amount which produces a result or exerts an influence on the particular condition being treated.

[0087] In another aspect, the pharmaceutical composition may comprise one or more buffers. Exemplary buffers include, but are not limited to, BES (N,N-bis[2- hydroxyethyl]-2-aminoethanesulfonic acid) Buffered Saline, Bicine (2-(Bis(2- hydroxyethyl)amino)acetic acid), Carbonate-Bicarbonate, CHES (N-Cyclohexyl-2- aminoethanesulfonic acid), Diethanolamine, EBBS (Earle’s Balanced Salt Solution), Glycine-Sodium Hydroxide Buffer, HEPES ((4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid), HBSS (Hank’s Balanced Salt Solution), HEPPSO (4- (2-Hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) Hydrate hydrate), HHBS (Hank’s Buffer with HEPES), Imidazole-HCI, Maleic Acid, MES (2-(N- morpholino) ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PBS (Phosphate Buffered Saline), Sodium Borate Buffer, TAE Buffer (Tris Base, Acetic Acid, EDTA), TAE, TBS (Tris Buffered Saline), TE Buffer (Tris EDTA), Tricine (N-(2-Hydroxy-1 ,1-bis(hydroxymethyl)ethyl)glycine), TRIS (tris(hydroxymethyl)aminomethane) and / or Trizma (2-Amino-2-(hydroxymethyl)-1 ,3- propanediol). Buffers are known in the art and any suitable buffer may be used.

[0088] The pharmaceutical composition may be further characterized by various biophysical characteristics, including pH. In one aspect described herein, the pharmaceutical composition may have a pH of between about 0 to about 14. In another aspect the pharmaceutical composition may have a pH of between about 2 to about 13. In another aspect, the pharmaceutical composition may have a pH of between about 3 to about 11 . In another aspect, the pharmaceutical composition may have a pH of between about 4 to about 10. In another aspect, the pharmaceutical composition may have a pH of between about 5 to about 9. In another aspect, the pharmaceutical composition may have a pH of between about 6 to about 10. In another aspect, the pharmaceutical composition may have a pH of between about 6.1 to about 9.9. In yet another aspect, the pharmaceutical composition may have a pH of between about 6.2 to about 9.8. In another aspect, the pharmaceutical composition may have a pH of between about 6.3 to about 9.7.In another aspect, the pharmaceutical composition may have a pH of between about 6.3 to about 9.6. In another aspect, the pharmaceutical composition may have a pH of between about 6.4 to about 9.5. In another aspect, the pharmaceutical composition may have a pH of between about 6.5 to about 9.5.

[0089] In another aspect, the pharmaceutical composition comprises a salt concentration of between about 1 mM to about 250 mM. In another aspect described herein the pharmaceutical composition comprises a salt concentration of between about 10mM to about 200 mM. In another aspect, the pharmaceutical composition comprises a salt concentration of between about 20mM to about 150 mM. In another aspect the pharmaceutical composition comprises a salt concentration of between about 20mM to about 150 mM. In another aspect the pharmaceutical composition comprises a pl of between about 30mM to about 100 mM.

[0090] Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Such suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n propyl, phydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0091] The compositions of the instant disclosure may also be administered parenterally, that is, subcutaneously, intravenously, intramuscularly, intrathecally, intravitreally, or interperitoneally, as injectable dosages of the compound in a physiologically acceptable diluent with a pharmaceutical carrier which may be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose andrelated sugar solutions; an alcohol such as ethanol, isopropanol, or hexadecyl alcohol; glycols such as propylene glycol or polyethylene glycol; glycerol ketals such as 2,2-dimethyl-1 ,1-dioxolane-4-methanol, ethers such as poly(ethyleneglycol) 400; an oil; a fatty acid; a fatty acid ester or glyceride; or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methycelulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent and other pharmaceutical adjuvants

[0092] The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self-emulsifications as described in U.S. Pat. No. 6,451,339, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. Such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with other non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example PVP, cellulose, PEG, starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated enterically or otherwise by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.

[0093] For oral administration, the compositions may be formulated into solid or liquid preparations such as, for example, capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, and may be prepared according tomethods known to the art for the manufacture of pharmaceutical compositions. The solid unit dosage forms may be a capsule which may be of the ordinary hard- or soft- shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.

[0094] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Additionally, emulsions can be prepared with a non-water miscible ingredient such as oils and stabilized with surfactants such as monodiglycerides, PEG esters and the like.

[0095] In another aspect, the compositions of this disclosure may be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin; disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum; lubricants intended to improve the flow of tablet granulation and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acid, or magnesium, calcium or zinc stearate; dyes; coloring agents; and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance tablets, pills or capsules may be coated with shellac, sugar or both.

[0096] The pharmaceutical compositions of this disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as liquid paraffin or a mixture of vegetable oils. Suitable emulsifying agents may be (1) naturally occurring gums such as gum acacia and gum tragacanth, (2) naturally occurring phosphatides such as soybean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and (4) condensation products of said partial esters with ethylene oxide, for example,polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

[0097] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. The suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p- hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.

[0098] Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0099] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. Oral solutions can be prepared in combination with, for example, cyclodextrin, PEG and surfactants.

[0100] The compositions of this disclosure may typically contain from about 0.0001% to about 50% by weight of the active ingredient in solution. Preservatives and buffers may also be used advantageously. Preservatives are known in the art and any suitable preservative may be used with the compositions described herein, including buffers, salts and antimicrobial agents, including but not limited to parabens and other preservatives generally used with pharmaceutical compositions.

[0101] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3 butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, axed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0102] Another formulation employed in the methods of the present disclosure employs transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the composition of the present disclosure in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art (See, e.g., U.S. Patent No. 5,023,252, incorporated herein by reference). Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. For topical use, creams, ointments, jellies, solutions or suspensions containing the molecules of the present disclosure are employed. As used herein, topical application is also meant to include the use of mouth washes and gargles.

[0103] Methods of delivering the molecules of this disclosure include any number of modes of administering the molecules or pharmaceutical compositions of molecules to the lungs via the mouth or rectum. Modes of administration may include delivery of liquid or powder formulations of pharmaceutical compositions for nasal administration via either passive of active delivery mechanisms. Liquid formulations may be delivered through a variety of mechanisms including vaporization through nasal inhalation, hand actuated nasal devices and mechanical spray pumps. Formulations for such delivery mechanisms may be in the form of propellant containing aerosols or propellant-free inhalable solutions. Mechanical spray pumps may be hand actuated, gas driven or electrical, as in the case of electrically powered nebulizers and atomizers. In one aspect described herein, a propellant-free inhalable solution is administered by nebulizer or direct nasal inhalation.

[0104] Powder formulations may be delivered through mechanical power sprayers, nasal inhalers and nebulizers / atomizers. Prior to delivery, powder formulations may be solubilized in suitable solvents including water and saline solutions. In one aspect described herein the compound may be solubilized in a saline solution. In another aspect described herein a therapeutically effective amount of the compound delivered to the lungs.

[0105] The compositions of the disclosure may also contain other conventional pharmaceutically acceptable compounding ingredients, generally referred to as carriers or diluents, as necessary or desired. Any of the compositions of this disclosure may be preserved by the addition of an antioxidant such as ascorbic acid or by other suitable preservatives. Conventional procedures for preparing such compositions in appropriate dosage forms may be utilized.

[0106] Commonly used pharmaceutical ingredients which may be used as appropriate to formulate the composition for its intended route of administration include: acidifying agents, for example, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid; and alkalinizing agents such as, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, or trolamine.

[0107] Other pharmaceutical ingredients include, for example, but are not limited to, adsorbents (e.g., powdered cellulose and activated charcoal); aerosol propellants (e.g., carbon dioxide, CCI2F2, F2CIC-CCIF2 and CCIF3); air displacement agents (e.g., nitrogen and argon); antifungal preservatives (e.g., benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate); antimicrobial preservatives (e.g., benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal); antioxidants (e.g., ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorus acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite); binding materials (e.g., block polymers, natural and synthetic rubber, polyacrylates, polyurethanes, silicones and styrene-butadiene copolymers); buffering agents (e.g., potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate anhydrous and sodium citrate dihydrate); carrying agents (e.g., acacia syrup, aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, orange syrup, syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection and bacteriostatic water for injection); chelating agents (e.g., edetate disodium and edetic acid); colorants (e.g., FD&C Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red No. 8, caramel and ferric oxide red); clarifying agents (e.g., bentonite); emulsifying agents (includes but are not limited to, acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyethylene 50 stearate); encapsulating agents (e.g., gelatin and cellulose acetate phthalate); flavorants (e.g., anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin); humectants (e.g., glycerin, propylene glycol and sorbitol); levigating agents (e.g., mineral oil and glycerin); oils (e.g., arachis oil, mineral oil, olive oil, peanut oil, sesame oil and vegetable oil);ointment bases (e.g., lanolin, hydrophilic ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white ointment, yellow ointment, and rose water ointment); penetration enhancers (transdermal delivery) (e.g., monohydroxy or polyhydroxy alcohols, saturated or unsaturated fatty alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalin, terpenes, amides, ethers, ketones and ureas); plasticizers (e.g., diethyl phthalate and glycerin); solvents (e.g., alcohol, corn oil, cottonseed oil, glycerin, isopropyl alcohol, mineral oil, oleic acid, peanut oil, purified water, water for injection, sterile water for injection and sterile water for irrigation); stiffening agents (e.g., cetyl alcohol, cetyl esters wax, microcrystalline wax, paraffin, stearyl alcohol, white wax and yellow wax); suppository bases (e.g., cocoa butter and polyethylene glycols (mixtures)); surfactants (e.g., benzalkonium chloride, nonoxynol 10, oxtoxynol 9, polysorbate 80, sodium lauryl sulfate and sorbitan monopalmitate); suspending agents (e.g., agar, bentonite, carbomers, carboxymethylcellulose sodium, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, kaolin, methylcellulose, tragacanth and veegum); sweetening e.g., aspartame, dextrose, glycerin, mannitol, propylene glycol, saccharin sodium, sorbitol and sucrose); tablet anti-adherents (e.g., magnesium stearate and talc); tablet binders (e.g., acacia, alginic acid, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, povidone and pregelatinized starch); tablet and capsule diluents (e.g., dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powdered cellulose, precipitated calcium carbonate, sodium carbonate, sodium phosphate, sorbitol and starch); tablet coating agents (e.g., liquid glucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, cellulose acetate phthalate and shellac); tablet direct compression excipients (e.g., dibasic calcium phosphate); tablet disintegrants (e.g., alginic acid, carboxymethylcellulose calcium, microcrystalline cellulose, polacrillin potassium, sodium alginate, sodium starch glycollate and starch); tablet glidants (e.g., colloidal silica, corn starch and talc); tablet lubricants (e.g., calcium stearate, magnesium stearate, mineral oil, stearic acid and zinc stearate); tablet / capsule opaquants (e.g., titanium dioxide); tablet polishing agents (e.g., carnuba wax and white wax); thickening agents (e.g., beeswax, cetyl alcohol and paraffin); tonicity agents (e.g., dextrose and sodium chloride); viscosity increasing agents (e.g., alginic acid,bentonite, carbomers, carboxymethylcellulose sodium, methylcellulose, povidone, sodium alginate and tragacanth); and wetting agents (e.g., heptadecaethylene oxycetanol, lecithins, polyethylene sorbitol monooleate, polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate).

[0108] Depending on the individual medicaments utilized in a combination therapy for simultaneous administration, they may be formulated in combination (where a stable formulation may be prepared and where desired dosage regimes are compatible) or the medicaments may be formulated separately (for concomitant or separate administration through the same or alternative routes). Allene oxide synthase has been associated with neuroprotective benefit, especially in the context of myocardial infarction, stroke or ischemic injury. Ischemic injuries as contemplated herein include any brain injury or any brain injury resulting from blood clots. Without being bound by any theory, it is believed that a timely administration of allene oxidase synthase blocks the oxidative stress that occurs after any type of ischemic incident, reducing the inflammatory response and cell death. (Mathai, 2012). Ischemia injury may be caused by traumatic event (such as apoplexy or heart attack), and it causes a reduction in blood supply to the tissues.

[0109] Thus, active oxygen species including hydrogen peroxide (H2O2) and lipid peroxides play a major role in ischemia-reperfusion injury. For example, global ischemia in the heart can cause contractile failure in minutes and after 30 minutes of ischemia, there is often a >250% increase in myocardial H2O2Content (Slezak, J., et al., Am. J. Pathol., 1995, 147(3), 772-81). Furthermore, depressed contractile function during the early phase of reperfusion can be accompanies by an approximately 600% increase in myocardial H2O2. The burst of oxygen free radicals during reperfusion of ischemic hearts, is observed generally during several ischemic conditions including for example, but not limited to, cerebrovascular stroke. Allene oxide synthase is known to have a clear benefit to ameliorating ischemia-reperfusion injury during myocardial infarction (Patent No. US7157082; herein incorporated by reference with respect to such background teaching) and cerebrovascular hypoxiaischemia (for example during cerebrovascular stroke, ischemic stroke, traumatic brain injury etc) (Mathai, S., et al., CNS Neurosci. Ther. 2012, 18(11), 887-94; herein incorporated by reference with respect to such background teaching), when administered within a 30-45-minute window of opportunity prior to the onset of reperfusion.

[0110] Thus in another embodiment described herein is a method for treating ischemic injury in a subject in need thereof, the method comprising administering to the subject, any of the compositions described herein. In one aspect, the ischemic injury is ischemia-reperfusion injury. In another aspect, the ischemic injury or ischemia-reperfusion injury is associated with one or more conditions selected from the group comprising coronary artery occlusion, myocardial infarction, angina pectoris, thrombolysis, myocardial ischemia, mesenteric ischemia, cerebrovascular stroke, ischemic stroke, hepatic ischemia, limb ischemia, pulmonary ischemia, kidney ischemia, ocular ischemia and retinal ischemia; and / or the subject is undergoing or has undergone a procedure selected from the group consisting of angioplasty, thrombectomy, and coronary artery bypass surgery. In another aspect, the condition is selected from one or more of coronary artery occlusion, myocardial infarction, angina pectoris, or myocardial ischemia, cerebrovascular stroke, haemorrhagic stroke, and / or ischemic stroke, mesenteric ischemia, hepatic ischemia, limb ischemia, kidney ischemia, ocular ischemia or retinal ischemia.

[0111] Another embodiment described herein is a method for reducing oxidative stress in the brain of a patient, comprising administering to the patient a pharmaceutical composition as described herein.

[0112] The therapeutically effective dosage of the molecules of this disclosure may readily be determined for treatment of each desired indication. The amount of the active ingredient (e.g., allene oxide synthase) to be administered in the treatment of one of these conditions may vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.

[0113] The total amount of the active ingredient to be administered may generally range from about 0.0001 mg / kg to about 10 mg / kg, and preferably from about 0.001 mg / kg to about 10 mg / kg body weight per day. A unit dosage may contain from about 0.05 mg to about 500 mg of active ingredient, and may be administered one or more times per day. The daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous, and parenteral injections, and use of infusion techniques may be from about 0.0001 mg / kg to about 10 mg / kg. The daily rectal dosage regimen may be from 0.0001 mg / kg to 10 mg / kg of total body weight. The transdermal concentration may be that required to maintain a daily dose of from0.0001 mg / kg to 10 mg / kg. The daily inhaled concentration may be that required to maintain a daily dose of from 0.0001 mg / kg to 10 mg / kg.

[0114] The specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age of the patient, the diet of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present disclosure may be ascertained by those skilled in the art using conventional treatment tests.

[0115] An alternative use of the protein assembly and compositions described herein involves the stability of seminal fluid and embryos for use in any reproductive context. Because of the sensitive nature of this biological fluid and cells, freezing and storage conditions may impact the viability of both sperm cells and embryos (including fertilized embryos). Thus, without being bound by any theory, it is believed that sperm cells and / or embryos may be stabilized or enhanced using the protein assembly and compositions described herein. Thus, another embodiment described herein is a method of preserving sperm comprising contacting the sperm with a protein assembly or any of the compositions described herein.

[0116] Another embodiment is a method of extending the life of semen comprising contacting the semen with the protein assembly or compositions described herein. Another embodiment is a method of improving the viability of sperm comprising contacting the sperm with the protein assembly or compositions described herein. In another embodiment is method for improving the percentage or number of implantable embryos, or quality of implantable embryos, or the success rate for implanted embryos, for use in an assisted reproduction technique, wherein the method comprises contacting either: an oocyte; or a fertilised embryo with a protein assembly or the compositions described herein.Non-Limiting Exemplary Embodiments

[0117] 1 . A method for treating ischemic injury in a subject in need thereof, the method comprising administering to the subject any protein assembly of the disclosure or the pharmaceutical composition of the disclosure.

[0118] 2. The method of embodiment 1, wherein the ischemic injury is ischemiareperfusion injury.

[0119] 3. The method of embodiments 1 or 2, wherein the ischemic injury or ischemia-reperfusion injury is associated with one or more conditions selected from the group comprising coronary artery occlusion, myocardial infarction, angina pectoris, thrombolysis, myocardial ischemia, mesenteric ischemia, cerebrovascular stroke, ischemic stroke, hepatic ischemia, limb ischemia, pulmonary ischemia, kidney ischemia, ocular ischemia and retinal ischemia; and / or the subject is undergoing or has undergone a procedure selected from the group consisting of angioplasty, thrombectomy, and coronary artery bypass surgery.

[0120] 4. A method of preserving sperm, wherein the method comprises contacting the sperm with a protein assembly of the disclosure.

[0121] 5. A method for preserving of semen, wherein the method comprises contacting the semen with a protein assembly of the disclosure.

[0122] 6. A method for improving sperm viability, wherein the method comprises contacting the sperm with a protein assembly of the disclosure.

[0123] 7. A method for improving the percentage of viable implantable embryos wherein the method comprises contacting either:(i) an oocyte; or(ii) a fertilised embryo; with a protein assembly of the disclosure or a compositions of the disclosure.

[0124] 8. A composition suitable for treating ischemic injury comprising: a. one or more allene oxidase enzymes; b. one or more polysorbate surfactants; c. one or more buffers; and d. one or more or more excipients; wherein the protein assembly has a hydrodynamic diameter of between about 5 nm to 30 nm.

[0125] 9. A composition of the disclosure, wherein the one or more polysorbate surfactants comprise polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0126] 10. The composition of embodiment 9. wherein the one or more polysorbate surfactants comprises a concentration at or above the critical micelle concentration.EXAMPLESVARIANT ALLENE OXIDE SYNTHASE ENZYMES

[0127] Variant allene oxide synthase enzymes were made comprising nonconservative amino acid substitutions at residues predicted to be important to enzyme structure / function and activity such as, for example, residues which play an important role in substrate binding, heme-binding etc. The sequences in Figure 1 all possess several non-conservative amino acid substitutions at key residues, relative to known AOS enzymes, primarily SEQ ID NO: 2 (Figure 1).EXAMPLE 1 : ACTIVITY OF ALLENE OXIDE SYNTHASE ASSEMBLIES COMPRISING NON-IONIC SURFACTANTS

[0128] Parthenium argentatum allene oxide synthase (PaAOS) activity (Obs) was measured with the native substrate, 13(S)-hydroperoxylinoleic acid (13(S)-HpODE) to assess the impact of non-ionic surfactants on enzymatic activity following retrospective surfactant addition. The aim of this study was to investigate, given the known detergent dependency of AOS enzymes, how structurally different non-ionic surfactants impact the enzymatic activity, and to consequently optimise the combination of AOS enzyme and surfactant for maximal enzymatic activity and minimal surfactant toxicity.

[0129] AOS was prepared and purified by recombinant expression in E. Coli as previously described (Li et al., PNAS, 2008, 105(37), 13883-13888). An initial panel of non-ionic and zwitterionic surfactants were selected based on commonality for protein and enzyme purification (Triton X-100 (Tx-100) and n-dodecyl-p-D-maltoside (DDM)) or their use in extraction / solubilisation or impact on the activity of AOS enzymes from Oryza sativa (Tween 20 (Tw 20), also known as polysorbate 20; Szymczyk et al., 2018) PaAOS (3-[(3-Cholamidopropyl) dimethylammonio]-1- propanesulfonate (CHAPS); Rodi et al., 2014), and Araabidopsis thaliama (Emulphogene (Emul) or polyoxyethylene (10) tridecyl ether; which was first used to demonstrate a detergent dependency of AOS enzyme activity in Hughes et al., 2006). The properties of the initial surfactant panel are shown in Table 1.Table 1Detergent Polarity CMC mM %w / vTxlOO Non-ionic 0.55 0.032Tw20 Non-ionic 0.06 0.007CHAPS Zwitterionic 4 0.25Emul Non-ionic 0.125 0.003DDM Non-ionic 0.15 0.006Methods

[0130] The effect and concentration-dependency of detergent on PaAOS enzymatic rate was determined using activities assays suing the native substrate, 12(S)- HpODE. Detergent-incubated enzyme stocks were used for the study. The specific activity above and below the critical micelle concentration (CMC) of the corresponding surfactant was measured to determine whether the surfactantdependency of AOS requires micelle formation for activity. The PaAOS substrate 13(S)-HpODE was generated in situ horn linoleic acid (LA) and lipoxagenase (LOX:L7395, Signa-Aldrich). LA was diluted in an equivalent volume of ethanol, followed by a 500-fold dilution of the ethanolic solution into 10 mM sodium tetraborate, pH 9.0, and incubated for 1 hour. The resulting solution was the further diluted 15-fold into 0.05 mg / mL LOX (2.5 mM KPO4, pH 7.4) and left to react for 10 minutes. The solution was subsequently centrifuged at 1048 g for 10 minutes with a 30 kDa centrifugal filter unit to remove LOX from the 13(S)-HpODE solution.

[0131] Specific enzymatic activity of PaAOS was assessed by adding 10 pL of a 0.005 mg / mL enzyme sample to 490 pL of substrate in a quartz cuvette and measuring the decline in UV-Visible absorbance (A 234 nm) over a 30 second period. Samples were measured in triplicate and at ambient temperature. Change in absorbance per second was converted to observed activity (ObS; s-1) to reflect the number of substrate molecules turned over per second to adjust for molar enzyme concertation. The extinction coefficient of 13(S)-HpODE is 24,500. Retrospective addition of surfactant was performed to determine the impact of different surfactantson enzymatic function of PaAOS. Experiments were conducted using surfactant concentrations as outlined in Table 2. Samples were prepared by diluting PaAOS (containing residual Triton X-100 from enzyme purification) to a final concentration of 0.005 mg / mL in Tris buffer (20 mM, pH 8.2) with the specified detergent and concentration. Activity assays were then performed as described above. Negative stain transmission electron microscopy was performed on PaAOS incubated with different retrospective surfactant additions. Samples were adsorbed onto a glow- discharged carbon- and parlodium-coated 400 mesh copper grid and stained with 2% uranyl acetate. Grids were imaged using a Tecnai TF-20 FEG-TEM (Thermo Fisher Scientific), operating at 200 kV. Images were recorded using a Gatan Ultrascan 1000 CCD camera operating under Gatan Digital Micrograph v 19.3 software.Table 2: Surfactant / Detergent concentrations used for experimentation.Results and DiscussionDuring the course of investigation, it was found that in all cases, the enzymatic activity (turnover rate) of PaAOS was substantially lower when the concentration of surfactant was below the critical micelle concentration (CMC), irrespective of the surfactant used. No difference in enzymatic activity was observed between any surfactant when the surfactant concentration was substantially below the CMC (Figure 2 A). Little difference in the enzymatic activity of PaAOS was observed with retrospective addition of non-ionic surfactants Triton X-100, DDS, or Emulphogene (Emul), or with zwitterionic surfactant CHAPS. However, surprisingly, it was observed that the inclusion of polysorbate 20 (Tween 20, Tw20) resulted insubstantial increase in enzymatic activity, suggesting a synergistic enhancement in activity with the inclusion of polysorbate micelles (Figure 2 B). Figure 2 shows a comparison of AOS enzymes with different non-ionic and zwitterionic surfactants. In (A) (below the CMC) they all have equivalent activity and in (B) they have enzymatic activity above the CMC but the enymatic activity of the polysorbate formulation is significantly increased over the other surfactants. Thus, while the skilled artisan would have expected a difference in activity between surfactant concentration above and below the CMC, based on the data for the other non-ionic surfactant examples, the skilled artisan would also anticipate that the enzymatic activity would also be (greater) but equivalent regardless of surfactant choice as long as it is above the CMC. The polysorbate was found to be more enzymatically active than the others surfactants suggesting a synergistic interaction. Thus, Without wishing to be bound by theory, it is believed that this synergistic enhancement in enzyme turnover rate may arise through improved interactions between the AOS enzyme and polysorbate micelle, due to the difference in headgroup substructure and packing parameters of polysorbate micelles compared with the other known surfactants. Transmission electron microscopy of PaAOS bound to Triton X-100 and polysorbate 20 (Tween 20; Tw20) at surfactant concentrations corresponding to either 0.03% or 60% of the critically micelle concentration (CMC) (Figure 3). Figure 3 suggests concentrations greatly below the surfactant CMC, larger protein aggregates of approximately 20-50 nm in diameter are visible. These larger protein aggregates formed with low surfactant concentrations correspond to significantly reduced enzymatic activity relative to activity at concentrations greater than the CMC. In contrast, as the surfactant concentration approaches that of the CMC, the protein aggregates become much smaller and monodisperse, suggesting the presence of more uniform, oligomeric AOS enzymes, which corresponded to greater enzymatic activity. It was noted that generally enzymatic activity tended to plateau between with surfactant concentrations approaching the CMC to those in excess of the CMC, relative to that observed with sub-CMC concentrations of surfactant. Therefore, the results suggest that non-aggregated, monodisperse assemblies of AOS produce the highest enzymatic activity, and that the formulation of AOS with polysorbate surfactants further enhances enzymatic activity over other known surfactants through a synergistic surfactant-protein interaction.

[0132] The combination of AOS enzymes with a polysorbate surfactant at or above the critical micelle concentration resulted in an unexpected enhancement in enzymatic activity compared with other known and similar non-ionic or zwitterionic surfactants. Furthermore, it was generally observed that at surfactant concentrations approaching or above the critical micelle concentration, AOS formed more uniform AOS -surfactant assemblies that displayed higher enzymatic activity than their larger aggregated and surfactant-depleted counterparts. Taken together, these findings support a novel composition with improved AOS activity over other known methods with utility in controlling undue oxidative stress in a subject.EXAMPLE 2: ACTIVITY, STRUCTURE, AND TOXICITY OF ALLENE OXIDE SYNTHASE ASSEMBLIES COMPRISING POLYSORBATE SURFACTANTS

[0133] Due to the unexpected enhancement in enzymatic activity observed when AOS was formulated with polysorbate 20 (Tween 20), the impact of polysorbate surfactants was investigated. The activity of AOS-surfactant preparations was assessed following the purification of enzyme using different detergents. Activity was assessed as a drop in optical density at OD234 (the absorbance maxima of AOS native substrate linoleic acid hydroperoxide), with one unit (U) of activity being defined as the amount of enzyme required to cause a drop in OD of 1 under standard assay conditions (1 minute at 22°C). Tween 20, Tween 40, Tween 60 and Tween 80 were chosen for formulation and activity assessment as representative detergents of the tween / polysorbate surfactant family representing those with the smallest and largest molecular weight of the family respectively. It was observed that Tween 20, Tween 40, Tween 60 and Tween 80 formulations produced statistically equivalent activity that was surprisingly and substantially greater than AOS formulated with Triton X-100 (Table 3A and 3B; Figure 4).Table 3A: Enzymatic activity (U / mg) of AOS-surfactant assemblies formulated with Triton X-100 (Tx100), Polysorbate / Tween 20 (Tw20), or Polysorbate / Tween 80 (Tw80). Standard deviation (Std Dev) and Standard Error of the Mean (SEM) represent n=3.Table 4B: Enzymatic activity (U / mg; U / s) of AOS-surfactant assemblies formulated with Triton X-100 (Tx100), Polysorbate / Tween 20 (Tw20), Polysorbate / Tween 40 (Tw40), Polysorbate / Tween 60 (Tw60) or Polysorbate / Tween 80 (Tw80).

[0134] Native PAGE was performed to assess differences in the native form of purified AOS protein. As shown in Figure 5A and 5B, formulation of AOS with Triton or Tween / polysorbate surfactants generate AOS enzyme with different native assembly. It is noteworthy here that both protein charge and molecule size are determinant of how a protein will behave in these gel systems. In general, the use of Tween / polysorbate surfactants surprisingly result in a much more consolidated and uniform / monodisperse form of AOS with a reduced overall (average) molecular weight. The toxicity of AOS-surfactant assemblies formulated with Triton or Tween / polysorbate surfactants was assessed in a WST-1 cell proliferation assay using HEK 293 cells. Detergent screens were performed on HEK 293T to determine the toxicity profile of three detergents: Triton X-100 (Figure 6A), Tween 80 (Figure 6B), and Tween 20 (Figure 6C). Plated cells were treated with a range of detergent concentrations from 0.001% - 0.05% and cell viability was determined at 24 hours after treatment using WST-1 cell proliferation reagent (Od4so reflects cell number). Triton X-100 demonstrated significant cellular toxicity at concentrations of 0.0075% and above, as evidenced by the decreased in OD450 values. HEK 293T cells were much more tolerant of Tween detergents with toxicity only evident with Tween 80 at the highest concentration tested (0.05%). No toxicity was observed with Tween 20. In fact, surprisingly, Tween 20 increased the viability of HEK 293T cells at low concentrations (0.001 - 0.02%). In summary, AOS enzymes formulated and stabilised with polysorbate surfactants surprisingly produced smaller and more consolidated AOS-surfactant assemblies with substantially greater enzymatic activity. Moreover, polysorbate surfactants are substantially less toxic to cells than their Triton X-100 counterpart. Taken together, the Inventors have produced novelcompositions of AOS enzymes that meet the desired criteria in terms of activity, stability, polydispersity, and safety, for human or veterinary pharmaceutical use.Example 3: LIGHT SCATTERING INVESTIGATIONS OF AOS-SURFACTANT ASSEMBLIES

[0135] The Inventors undertook light dynamic scattering investigations to study the hydrodynamic diameter and polydispersity of AOS-surfactant assemblies.The size distribution of the AOS-surfactant assemblies was analysed by dynamic light scattering (DLS) on a Malvern Zetasizer Nano. The hydrodynamic diameter (HD) and polydispersity index (PDI) were measured at 25° C by adding 150 pL of the AOS- surfactant suspension to 2 mL of phosphate buffered saline in a 12 mm square disposable polystyrene cuvette with a path length of 10 mm. Light scattering was measured with a backscattering angle of 173°. Bovine serum albumin (BSA) and thyroglobulin were used as control proteins of known size distribution. A summary of the results is shown in Table 3 and Figures 7 - 13.Table 5: Average hydrodynamic diameter of protein standards (bovine serum albumin and thyroglobulin), surfactants (Triton-X100, Tween 20, and Tween 80), and AOS-surfactant assemblies produced by different expression systems (A, B and C) as measured by dynamic light scattering. Data represents average by particle volume (nm) and standard deviation (nm; n=3).Table 6: Average hydrodynamic diameter of AOS-surfactant assemblies produced by different expression systems (A, B and C) as measured by dynamic light scattering by smallest particle distribution in suspension. Data represents average by particle volume (nm) and standard deviation (nm; n=3).Assembly Hydrodynamic Standard Min Average MaxPreparation Diameter (nm) Deviation (nm) (nm) (nm)A_AOS with Tw80 12.1 6 6.1 12.1 18.1(0.1%) P1A_AOS with Tx100 52.5 15.2 37.3 52.5 67.7(<0.05%) P1B_AOS with Tw20 17.1 16 1.1 17.1 33.1(0.1%) P1B_AOS with Tw20 11 .7 27 9 11 7 14.4C AOS Tw80 12.3 5.4 6.9 12.3 17.7(0.1%) P1C AOS Tw20 14.3 3.4 10.9 14.3 17.7(0.1%) P1A_Detergent 25.7 17 8.7 25.7 42.7Depleted AOS P1

[0136] The results demonstrate that AOS-surfactant assemblies present with a significantly smaller hydrodynamic diameter than their detergent-depleted counterpart, which is in agreement with the previous negative stain TEM imaging of AOS-surfactant and surfactant-depleted assemblies. Taken together with the increased enzymatic activity of assemblies containing surfactant concentrations above the corresponding CMC, these findings provide a novel insight into AOS- surfactant structure function relationship, suggesting that suspensions with an average hydrodynamic diameter of less than 30 nm are optimal for providing enzyme preparations with higher activity than their larger counterparts.

[0137] Taken together with the increased enzymatic activity of assemblies containing surfactant levels above the corresponding CMC, these findings provide novel insights into AOS-surfactant structure-function relationships. Thus, smaller assemblies with at least one dimension with a diameter of less than approximately 30 nm are surprisingly more catalytic than their larger counterparts.Example 4: UTILITY OF AOS-SURFACTANT ASSEMBLIES FOR NEUROPROTECTION AGAINST OXIDATIVE NEURONAL CELL DAMAGE AND ISCHEMIA-REPERFUSION INJURY

[0138] The Inventors studied the utility of AOS-polysorbate assemblies for the protection of brain tissue against oxidative damage as a model of ischemiareperfusion injury using a hydrogen peroxide-induced neuronal injury paradigm.

[0139] Active oxygen species including hydrogen peroxide (H2O2) and lipid peroxides play a major role in ischemia-reperfusion injury. For example, global ischemia in the heart can cause contractile failure in minutes and after 30 minutes of ischemia, there is often a >250% increase in myocardial H2O2 content (Slezak, J., et al., Am. J. Pathol., 1995, 147(3), 772-81). Furthermore, depressed contractile function during the early phase of reperfusion can be accompanies by an approximately 600% increase in myocardial H2O2. The burst of oxygen free radicals during reperfusion of ischemic hearts, is observed generally during several ischemic conditions including for example, but not limited to, cerebrovascular stroke. Allene oxide synthase is known to have a clear benefit to ameliorating ischemia-reperfusion injury during myocardial infarction (Patent No. US7157082; herein incorporated by reference with respect to such background teaching) and cerebrovascular hypoxia-ischemia (for example during cerebrovascular stroke, ischemic stroke, traumatic brain injury etc) (Mathai, S., et al., CNS Neurosci. Ther. 2012, 18(11), 887-94; herein incorporated by reference with respect to such background teaching), when administered within a 30-45-minute window of opportunity prior to the onset of reperfusion.Methods

[0140] Cerebellar Microexplant System1 . Extraction of cerebellar tissue

[0141] Postnatal day 4 Wistar rats were used for the study. The rats were sacrificed and placed in ice for 1 minute, decapitated, and the cerebellum removed and placed on ice. Cerebellum tissue was placed in 1 mL of 0.65% glucose-supplemented PBS (10 uM 65% stock D(+)-glucose / 1 mL PBS) in a large Petri dish, chopped up into smaller sections and triturated with a 1 mL insulin syringe via a 23 G (0.4 mm) needle, and then replaced back into the glucose solution in the large petri dish. The tissue was sieved (through 125 urn pore size gauze) and centrifuged twice (2 min at 6000 x g) and transferred into serum-free BSA-supplemented START V medium (Biochrom, Germany). The second centrifugation step was done with 1 mL of STARTV medium. The microexplants were reconstituted into 500 uL of START V medium and put on ice.2. Culture of cerebellar cells

[0142] Two hours after poly-L-lysine coating, glass slides were washed with Millipore water and air-dried. Each side was placed into a small petri dish (diameter: 35 mm) and 40 uL of STAART V / cell suspension added. The tissue was incubated for 2 hours at 34 °C (settlement period). START V-medium (1 mL) was then added to the petri dish and cultivated at 34 °C in the presence of 5% CO2 in air at 100% humidity for 48 hours.3. Drug application

[0143] For the studies, some explant cultures were exposed to vehicle (PBS buffer) only and acted as controls. In the first study (Study 1), 10 uL toxin (hydrogen peroxide, 0.1 mM, pH 7.4 in Millipore water) was applied simultaneously with increasing concentrations of AOS enzyme (10 ng / mL- 20 ng / mL in PBS, pH 7.4). Study 2 was conducted to confirm the positive results obtained from Study 1 . In all studies, drugs were left in contact with the explants for the duration of the study, which was 24 hours.4. Determination of drug effect

[0144] After the explants were exposed to drugs (toxin / AOS) for 24 hours, the cells were then rinsed in PBS and then fixed in increasing concentrations of paraformaldehyde (PFA) (500 pL, 0.4% PFA, followed by 1.2%, then 3%, and finally 4% PFA). Each fixation step was performed for 3 min. Finally, the microexplants were rinsed in PBS.

[0145] Neurons in the explants were then evaluated for morphology (presence of neurites) and counted as live cells per microscopic field. Four fields displaying highest cell density were counted per cover slip and the data presented as mean + / - standard error of the mean (SEM); n=4 each. Statistical significance was evaluated using a non-paired student t-test.Results

[0146] Exposure of cerebellar microexplant to hydrogen peroxide-induced oxidative stress resulted in almost 100% death of cerebellar neurons. However, following treatment with the AOS enzyme neuronal survival was significantly (P < 0.001) increased across all drug concentrations tested (Figure 14). AOS produced a mean neuronal recovery from hydrogen peroxide injury of 23%.Discussion and Conclusion

[0147] The study herein demonstrates that AOS enzymes can rescue neurons from hydrogen peroxide injury within postnatal cerebellar granule cells organised as cerebellar microexplants to a significant degree.

[0148] These results therefore support the utility of novel AOS-polysorbate assemblies with improved activity for the amelioration of ischemia-reperfusion injury. The inventors contemplate that these novel assemblies will have utility for the treatment of several ischemia disorders, including for example, but not limited to coronary artery occlusion, myocardial infarction, cerebrovascular stroke, ischemic stroke, hepatic ischemia, limb ischemia, pulmonary ischemia, kidney ischemia, mesenteric ischemia, retinal and ocular ischemia, and during a number of procedures including, but not limited to angioplasty, thrombectomy, and coronary artery bypass surgery.Example 5: PROPHETIC EXAMPLE - STABILITY OF SEMINAL FLUID AND EMBRYOS FOR REPRODUCTIVE CONTEXTFurther experiments will be conducted to determine the stability of seminal fluid and embryos in the various formulations as described herein. The seminal fluid and embryos will be tested for their viability and quality after being frozen and thawed in the formulations described herein.***The disclosure has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components or parameters may be varied or modified to acertain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. The invention also includes all of the steps, features, compositions and molecules referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of said steps or features.Titles, headings, or the like are provided to enhance the readers comprehension of this document and should not be read as limiting the scope of the invention.The entire disclosures of all applications, patents, and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents, and publications in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that they constitute valid prior art or form any part of the common general knowledge in any country in the world.

Claims

CLAIMS1 . A protein assembly comprising an allene oxide synthase enzyme wherein the assembly has a hydrodynamic diameter of between about 5 nm to 30 nm.

2. The protein assembly of claim 1 , wherein the allene oxide synthase enzyme is derived from Parthenium argentatum^3. The protein assembly of claims 1 to 2, wherein the allene oxide synthase enzyme comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from one or more 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.

4. The protein assembly of claim 1 to 3, further comprising a polysorbate surfactant, wherein the polysorbate surfactant comprises one or more of polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

5. A composition comprising the protein assembly of claims 1 to 4.

6. A pharmaceutical composition comprising one or more of the protein assembly of claims 1 to 4, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein a portion of the composition comprises a protein assembly having a hydrodynamic diameter of between about 5 nm to 30 nm.

8. The pharmaceutical composition of claims 6 to 7, wherein the one or more polysorbate surfactants comprises a concentration at or above the critical micelle concentration.

9. A protein assembly of claims 1 to 4 or the pharmaceutical composition according to claim 6 for use of in the treatment of ischemic injury in a subject in need thereof.

10. The protein assembly or pharmaceutical composition for use according to claim 9, wherein the ischemic injury is ischemia-reperfusion injury.

11. The protein assembly or pharmaceutical composition for use according to claims 9 to 10, wherein the ischemic injury or ischemia-reperfusion injury is associated with one or more conditions selected from the group comprising coronary artery occlusion, myocardial infarction, angina pectoris, thrombolysis, myocardial ischemia, mesenteric ischemia, cerebrovascular stroke, ischemic stroke, hepatic ischemia, limb ischemia, pulmonary ischemia, kidney ischemia, ocular ischemia and retinal ischemia; and / or the subject is undergoing or has undergone a procedure selected from the group consisting of angioplasty, thrombectomy, and coronary artery bypass surgery.

12. A protein assembly according to any of claims 1 to 4 for use in a method of preserving sperm, wherein the method comprises contacting the sperm with the protein assembly.

13. A protein assembly according to any of claims 1 to 4 for use in a method for preserving of semen, wherein the method comprises contacting the semen with the protein assembly.

14. A protein assembly according to any of claims 1 to 4 for use in a method for improving sperm viability, wherein the method comprises contacting the sperm with the protein assembly.

15. A protein assembly according to claims 1 to 4 or the compositions of claims 5 to 7 for use in a method for improving the percentage of viable implantable embryos wherein the method comprises contacting either:(i) an oocyte; or(ii) a fertilised embryo; with the protein assembly or the compositions.

16. A composition suitable for use in the treatment of ischemic injury comprising: a. one or more allene oxidase enzymes; b. one or more polysorbate surfactants; c. one or more buffers; and d. one or more or more excipients;wherein the protein assembly has a hydrodynamic diameter of between about 5 nm to 30 nm.

17. The composition of claim 16, wherein the one or more polysorbate surfactants comprise polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

18. The composition of claim 17, wherein the one or more polysorbate surfactants comprises a concentration at or above the critical micelle concentration.

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