New composition and use

Abstract

The present disclosure relates to compositions comprising monodisperse assemblies of allen oxide synthase (AOS), and in particular to specific compositions and assemblies comprising an AOS enzyme and a polysorbate surfactant. Such compositions are particularly useful for improving biological conditions of oxidative stress.

Description

Technology Field Explanation regarding the sequence list

[0001] The sequence list related to this application is provided in text format instead of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence list is "91902-364496 Sequence Listing_ST26". Field of invention

[0002] The present invention relates to a novel allen oxide composition having enhanced stability, antioxidant activity, and / or biological activity. Furthermore, the present invention relates to a composition and assembly comprising an allen oxide enzyme and a nonionic surfactant that provide enhanced activity and properties compared to existing formulations. The present invention also relates to a method of using said composition and assembly to control excessive oxidative stress of a target. Background Technology background

[0003] The CYP74A enzyme family includes allene oxide synthases (AOS) and consists of fewer than 15 molecules derived from various plants, such as peas, corn, barley, tomatoes, flaxseed, and the biological model plant Arabidopsis. All of these perform AOS functions, and their molecular weight in monomeric form is approximately 55 kDa. These enzymes are unique members of the cytochrome P450 family in that they maintain activity without oxygen or NADPH reductase, possess self-renewal capabilities, and exhibit very rapid reaction rates. AOS enzymes convert fatty acid hydroperoxides, generated by the oxidation of polyunsaturated fatty acids, into unstable epoxides, which are further degraded to produce ketols. Lipid peroxides (LPOs) can be generated by oxidative stimulation or the action of lipoxygenases, and this is the pathway utilized for the production of LPO substrates used in the analysis of AOS enzyme function.

[0004] P450 enzymes, including the AOS family, are generally membrane-bound. For this reason, detergents (e.g., Triton-X 100, Emulphogene) are frequently used during the purification and extraction processes from natural products or molecularly cloned sources to improve enzyme stability in solution. Recently, it has become increasingly clear that without detergents, enzymes may precipitate in solution and lose activity. For example, Hughes et al. (2006) FEBS Letters 580:4188-4194 investigated the effect of detergents on the enzymatic kinetics of allen oxide synthase (CYP74A1) in Arabidopsis thaliana and found that under detergent-free conditions, the enzyme exhibited low activity (k) toward 13-S-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid. cat / K m 0.12 x 10⁷ M -1 s -1 Although ) was shown ,Adding emulsifier micelles significantly increases activity (k cat / K m 5.9 x 107 M -1 s -1 It showed that ) that, that is, the detergent When present, the activity of the enzyme toward the preferred substrate increased nearly 50-fold.

[0005] In fact, an increasing number of research results regarding the effects of detergents on the structure and function of enzymes are being reported in the literature, leading to the conclusion that detergents are essential for enzyme activity. In other words, the addition of detergents significantly increases enzyme activity, particularly improving substrate specificity and reaction rate. The problem to be solved

[0006] Parthenium argentatum Parthenium argentatum AOS enzymes, such as those derived from [source], are well known for their utility as cardioprotective therapies (see, e.g., U.S. Patent 7,157,082 and WO 2015 / 183106; details regarding this background knowledge are incorporated herein by reference). The immediate clinical and / or commercial utility of these and other AOS enzymes lies in the fact that AOS enzymes have been demonstrated in biological systems to act as antioxidant molecules, removing peroxide products and preventing chain oxidation reactions, making them commercially valuable in various applications. However, the resulting enzyme formulations often appear as heterogeneous populations with inconsistent size distribution and stability. Furthermore, certain detergents required for formulation can have adverse effects on biological systems, including sperm and embryo preservation in assisted reproductive technologies, which is an initial product goal for the immediate / early clinical application of existing and next-generation AOS enzymes.

[0007] The demand for AOS enzyme preparations that possess significant enzyme activity and stability while overcoming the limitations of existing methods remains unmet. means of solving the problem Summary of the Invention

[0008] One embodiment described herein is a protein assembly comprising allene oxide synthase, wherein the hydrodynamic diameter of the assembly is about 5 nm to 30 nm. In one aspect, the allene oxide synthase Parthenium argentatum It originates from. In another aspect, the allen oxide synthase comprises an amino acid sequence having at least 80% sequence identity with 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 in this specification is a composition comprising the protein assembly described in this specification. Another aspect described in this specification is a pharmaceutical composition comprising one or more of the protein assemblies described in this specification 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 about 5 nm to 30 nm. Another aspect described herein is a pharmaceutical composition in which one or more polysorbate surfactants are present at a concentration above or above a critical micelle concentration.

[0012] Another aspect described herein is a method for treating ischemic injury in a subject requiring such injury, said method comprising administering to the subject any one of the protein assembly or pharmaceutical composition described herein. In one aspect, said ischemic injury is ischemia-reperfusion injury. In another aspect, said 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, stroke, ischemic stroke, hepatic ischemia, limb ischemia, pulmonary ischemia, renal ischemia, ocular ischemia, and retinal ischemia; and / or the subject is undergoing or has undergone a procedure selected from the group comprising angioplasty, thrombectomy, and coronary artery bypass surgery. In another aspect, this is a method for preserving sperm, wherein the method comprises contacting sperm with any one of the protein assemblies described herein.

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

[0014] Another aspect described herein is a method for improving sperm viability, the method comprising bringing sperm into contact with any one of the protein assemblies described herein.

[0015] Another aspect described herein is a method for improving the viability of an implantable embryo, said method comprising contacting (i) an oocyte or (ii) a fertilized embryo with any one of the protein assemblies or compositions described herein.

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

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

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

[0019] Although various embodiments of the various aspects of the invention discussed above are specified in the detailed description below, the invention is not limited thereto.

[0020] Other aspects of the present invention may become clear through the following description, which is provided as an exemplary description with reference to the accompanying drawings.

[0021] The present invention may also be construed to comprehensively include parts, elements, and features mentioned or indicated individually or collectively in this application specification, and any combination of two or more of such parts, elements, or features; and where a specific component having an equivalent known in the art related to the present invention is mentioned in this specification, such known equivalent is deemed to be included in this specification as if it were individually specified. Brief explanation of the drawing

[0022] Figure 1 illustrates the allen oxide synthase enzyme sequences defined as 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 and SEQ ID NO: 9.

[0023] Figure 2 shows the average enzyme activity of PaAOS cultured with various surfactants at (A) below the corresponding critical micelle concentration and (B) above the corresponding critical micelle concentration (CMC).

[0024] Figure 3 illustrates images of PaAOS cultured with various surfactants observed by negative staining transmission electron microscopy. (A) No surfactant added. (B) 0.000001% Triton X-100 (Tx100) (0.03% of CMC) added. (C) 0.00002% Tween 20 (Tw20) (0.03% of CMC) added. (D) 0.02% Tx100 (approx. 100% of CMC) added. (E) 0.004% Tw20 (60% of CMC) added.

[0025] Figure 4 shows 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 the standard deviation (n=3).

[0026] Fig. 5: (A) shows the native gel electrophoresis results of PaAOS enzymes prepared using Triton X-100, polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), and molecular weight standards (bovine serum albumin (BSA), β-amylase, thyroglobulin), and (B) shows the native gel electrophoresis results of PaAOS enzymes prepared using Triton X-100, polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80), and molecular weight standards (bovine serum albumin (BSA), β-amylase, thyroglobulin).

[0027] Figure 6 shows the effects of allen oxide synthase enzymes formulated with (A) Triton X-100, (B) polysorbate 20 (Tween 20), and (C) polysorbate 80 (Tween 80) on HEK 293 cells. It can be seen that cytotoxicity is significantly reduced in the case of the AOS-polysorbate assembly.

[0028] Figure 7 shows the hydrodynamic diameters of protein samples (A) BSA, (B) thyroglobulin, and (C) PaAOS assembly when the described surfactant is significantly depleted.

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

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

[0031] Figure 10 shows the hydrodynamic diameters of (A) polysorbate 80 (Tween 80) and (B and C) AOS-Tween 80 enzyme-surfactant assemblies.

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

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

[0034] Figure 13 shows the average hydrodynamic diameter of the smallest particle distribution in solution of AOS assemblies formulated with Triton X-100, polysorbate 20 (Tween 20), or polysorbate 80 (Tween 80) above the critical micelle concentration or when the surfactant is substantially depleted, on a volume basis. Error bars represent the standard deviation (n=3).

[0035] Figure 14 illustrates the effect of the PaAOS enzyme formulated with polysorbate 80 (Tween 80) on nerve cells damaged by hydrogen peroxide. Specific details for implementing the invention Detailed description

[0036] The present invention relates to a novel allen oxide synthase composition having enhanced stability, antioxidant activity, and / or biological activity. Furthermore, the present invention relates to a composition and assembly comprising an allen oxide synthase enzyme and a nonionic surfactant, which provide enhanced activity and properties compared to conventional formulation methods. The present invention also relates to a method of using said composition and assembly to control excessive oxidative stress of a target.

[0037] Unless otherwise explicitly defined, all technical and scientific terms used herein are deemed to have the same meaning as generally understood by those skilled in the art to which the invention pertains (e.g., immunology, immunohistochemistry, protein chemistry, and biochemistry). In the event of a conflict, the contents of this document, including definitions, shall prevail. Although methods and materials are described below, methods and materials similar or equivalent to those described herein may be used in the practice or testing of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and embodiments disclosed herein are illustrative and should not be construed as restrictive.

[0038] Unless otherwise specified, the recombinant proteins and immunological techniques used herein are standard procedures well known to those skilled in the art. These techniques are described in 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), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al It is described in literature such as ., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0039] In this specification, the articles "a" and "an" are used to indicate that the object of the article is one or more (i.e., at least one). For example, "an element" means one element or more than one element.

[0040] The term "and / or," e.g., "X and / or Y," means "X" and "Y" or "X or Y" and should be regarded as providing explicit support for either one of these meanings.

[0041] Throughout this specification, unless otherwise explicitly stated or otherwise required by the context, references to a single step, material composition, group of steps, or group of material compositions shall be interpreted as including both one and a plurality (i.e., one or more) of such steps, material compositions, group of steps, or group of material compositions.

[0042] References to the range of numbers disclosed in this specification (e.g., from 1 to 10) are intended to include all related numbers within said range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and the range of rational numbers within said range (e.g., from 2 to 8, from 1.5 to 5.5, and from 3.1 to 4.7), and thus all sub-ranges of all ranges explicitly disclosed in this specification are explicitly disclosed. This is merely an example of what is specifically intended, and all possible numerical combinations between the lowest and highest values ​​listed should be deemed to be explicitly described in this application in a similar manner.

[0043] Throughout the specification, the word “comprise” or variations such as “comprises” or “comprises” means to include the specified element, integer, or step, or group of element, integer, or step, and does not mean to exclude other element, integer, or step, or group of element, integer, or step.

[0044] Those skilled in the art will understand that various variations and modifications are possible for the embodiments described herein in addition to those specifically described. It should be understood that this specification includes all such variations and modifications. Furthermore, this specification includes all steps, features, configurations, and assemblies mentioned or indicated individually or collectively in this specification, and all combinations or combinations of two or more of such steps or features.

[0045] The scope of the present invention is not limited by the specific examples described herein, and such examples are provided for illustrative purposes only. As described herein, functionally equivalent products, compositions, and methods are also clearly included within the scope of the present invention.

[0046] Unless otherwise explicitly stated, it is considered to apply to all other examples. Selected definition

[0047] In this specification, the term “about” is used to refer to conditions (e.g., amount, concentration, time, etc.) that vary by up to 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to specific conditions.

[0048] The term "AOS" as used in this specification refers to the allen oxide synthase enzyme used in the context of AOS enzyme activity.

[0049] The term "PaAOS" is Parthenium argentatum It refers to allen oxide synthase derived from.

[0050] As used herein, the term "assembly" refers to a self-assembled structure formed from a collection of molecular aggregates. For example, it refers to protein(s) and surfactant(s) coming together to form a single independent structure.

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

[0052] The term "critical micelle concentration" is used to denote the concentration of a surfactant at which micelles are formed. In other words, micelles are formed when the concentration of the surfactant exceeds this value, and this concentration is called the critical micelle concentration (Translator's note).

[0053] As used herein, terms such as "culture," "culture method," etc., refer to a series of procedures for culturing a population of cells (or a single cell) in vitro under conditions proven to support the growth or maintenance of the cells. Various formats, media, temperature ranges, gas concentrations, etc., that must be defined in a culture system exist in the art. These parameters vary depending on the selected format and the specific requirements of the individual implementing the method disclosed herein. However, determining culture parameters is generally a routine task.

[0054] As used herein, the term “derivative” means a polypeptide derived from a base sequence by modification, for example, by conjugation with other chemical functional groups or formation of complexes, or by post-translational modification techniques as understood in the art. The term “derivative” also includes within its scope modifications, including additions, substitutions, or deletions applied to the parent sequence to produce functionally equivalent molecules.

[0055] In this specification, the terms "detergent" and "surfactant" may be used interchangeably, including their plural forms, and are defined according to generally accepted definitions.

[0056] In this specification, the terms “detergent-depleted” and “detergent-free” may be used interchangeably to refer to enzyme preparations comprising compositions that (e.g.) substantially contain no detergent or contain only a residual amount of detergent. In some examples of enzyme compositions described in this specification, the terms “detergent-depleted” and “detergent-free” mean compositions containing (e.g.) less than 0.025%, 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% of detergent.

[0057] The term “effective dose” as used in relation to the specific uses of the peptides and enzymes described herein means administering to a patient, either as a single dose or as part of a series of doses, an amount of a composition effective for stimulation, prevention, or treatment, as confirmed through clinical trials and evaluations, patient observations, etc. “Effective dose” may also mean a dose that causes a detectable change in biological or chemical activity. Such detectable change may be detected and / or quantified by a person skilled in the art familiar with the relevant mechanisms or processes. Additionally, “effective dose” may mean an amount that maintains a desirable physiological state, that is, an amount that reduces, prevents, or promotes improvement of a severe deterioration of the condition of interest. “Effective dose” may also mean a “therapeutically effective amount.” The effective dose depends on the health and physical condition of the subject, the taxonomic group of the subject, the formulation of the composition, the assessment of the medical situation, and other relevant factors. The amount is expected to be within a relatively broad range that can be confirmed through routine testing.

[0058] As used herein, the terms “fragment” or “functional derivative” refer to a partial sequence of a polypeptide that can be detected using a binder in relation to the polypeptide. These terms may refer to polypeptides, aggregates of polypeptides such as dimers or polymers, fusion polypeptides, polypeptide fragments, polypeptide variants, or derivatives thereof.

[0059] As used herein, the term "hydrodynamic diameter" refers to the diameter of a protein assembly or particle in solution, including all solvation spheres. As used herein, hydrodynamic diameter refers to the diameter of a perfect solid sphere exhibiting the same hydrodynamic friction as the corresponding molecule.

[0060] The term “isolated” applied to polypeptide sequences disclosed herein means a sequence isolated from a natural cellular environment or other naturally occurring biological environment. The isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques. The polypeptide sequence may be prepared through at least one purification step.

[0061] The term "micelle" is used to refer to aggregates or supramolecular assemblies of surfactants (or other similar amphiphilic molecules) dispersed in a liquid, which form a colloidal suspension.

[0062] The terms “patient,” “subject,” “host,” or “individual” used interchangeably in this specification refer to any subject requiring diagnosis, prevention, or treatment, in particular vertebrate subjects, more specifically mammalian subjects, most particularly humans. Suitable vertebrates within the scope of the invention include, but are not limited to, humans, all members of the phylum Chordata including primates, rodents (e.g., mice, rats, guinea pigs), lagomorpha (e.g., rabbits, hares), artiomorpha (e.g., cattle), lamina (e.g., sheep), goata (e.g., goats), swina (e.g., pigs), equida (e.g., horses), canidae (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, canaries, parakeets, etc.), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards, etc.), and fish. The animals in question may require diagnosis, prevention, or treatment, but the aforementioned terms do not imply that symptoms will appear.

[0063] Throughout this specification, the terms “peptide,” “polypeptide,” or “protein” may be used interchangeably and encompass chains of amino acids of any length, including full-length sequences in which amino acid residues are linked by covalent peptide bonds. Polypeptides useful in the invention may be purified natural products or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to polypeptides, aggregates of polypeptides such as dimers or other polymers, fusion polypeptides, polypeptide fragments, polypeptide variants, or derivatives thereof. The polypeptide of the present invention may have a chain length 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. References to other polypeptides of the present invention or other polypeptides described herein should be understood similarly.

[0064] As used herein, the term “protein assembly” refers to a non-covalent supramolecular assembly of an allen oxide synthase enzyme and one or more nonionic surfactants or micelles. This assembly occurs in a manner similar to how the hydrophobic side of the enzyme binds to a cell membrane.

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

[0066] As used herein, the term “variant” refers to a polypeptide sequence that differs from a specifically identified sequence. A variant may be a naturally occurring allelic variant or a variant that does not occur naturally. Variants may originate from the same species or different species and may include homologues, paralogues, and orthologues. In specific embodiments described herein, a variant of a polypeptide includes cases where the biological activity, including signal peptide activity or antigen-binding properties, is identical or similar to that of the parent polypeptide. With respect to polypeptides, the term “variant” encompasses all forms of polypeptides defined herein.

[0067] The variant polypeptide / protein sequence has 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 with respect to the sequence of the present invention.

[0068] Polypeptide variants also include variants that exhibit similarity to one or more of specifically identified sequences, and such similarity is likely to maintain functional equivalence to those sequences. This includes variants that cannot be reasonably expected to have arisen by random chance.

[0069] The identity and similarity of polypeptide sequences can be verified by comparing them with candidate polypeptide sequences using BLASTP in the bl2seq program (BLAST program suite, version 2.2.18 [April 2008]), which is publicly available at NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq should be used, but the filtering of low-complexity regions should be disabled. Other methods for verifying sequence identity are also known in the field.

[0070] The similarity of polypeptide sequences can be checked using the following UNIX command-line parameters: bl2seq -i peptideseq1 -j peptideseq2 -FF -p blastp. The parameter -FF disables filtering for low-complexity regions. The parameter -p selects an algorithm suitable for the two sequence pairs. This program finds regions of similarity between sequences and reports an "E value" for each region. The E value is the expected number of times such a match would appear by chance in a database of random sequences of a fixed reference size. If the E value is much smaller than 1, it is approximately equal to the probability of such a random match occurring. Variant polypeptide sequences generally exhibit an E value 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, or less than 1 x 10⁻¹⁸ or 1 x 10⁻²¹ when compared to one of the specifically identified sequences. Polypeptide sequence identity can also be calculated using a global sequence alignment program over the entire overlap length between the candidate polypeptide sequence and the target polypeptide sequence. As discussed above, 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.) are also global sequence alignment programs suitable for calculating polypeptide sequence identity. While the use of BLASTP is preferred for determining polypeptide variants according to the present invention, other methods are also known in the art.

[0071] As used herein, the term “sequence identity” refers to the degree of sequence identity within a comparison range, either at the base sequence level or the amino acid level. Accordingly, the “sequence identity percentage” is calculated by comparing two optimally aligned sequences within a comparison range to determine the number of positions in which the same nucleic acid base (e.g., A, T, C, G, U) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ileu, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appears in both sequences, dividing this number of matched positions by the total number of positions within the comparison range (i.e., the range size), and multiplying the result by 100. For the purposes of the present invention,

[0072] The term "sequence identity" can be understood to mean the "match rate" calculated by the DNASIS computer program (version 2.5 for Windows, provided by Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using the standard default values ​​used in the reference manual provided with the software.

[0073] The term "sequence similarity" refers to the percentage of amino acids constituting identical or conservative amino acid substitutions, as defined. Similarity is GAP (Deveraux et al. This can be determined using a sequence comparison program such as the one used in Nucleic Acids Research (12: 387-395) in 1984. In this way, sequences that are similar in length or substantially different from the sequence cited here can be compared by inserting a gap in the alignment, and this gap is determined by, for example, the comparison algorithm used in GAP.

[0074] The terms "treatment," "to treat," and "treating" refer to reversing, alleviating, reducing, slowing, or suppressing the progression of a disease or illness or associated symptoms, and a detailed explanation thereof is specified in this document.

[0075] References to the range of numbers disclosed in this specification (e.g., from 1 to 10) should be interpreted to include all related numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and the range of rational numbers within that range (e.g., from 2 to 8, from 1.5 to 5.5, and from 3.1 to 4.7). Accordingly, all sub-ranges of all ranges explicitly disclosed in this specification are explicitly disclosed. This is merely an example of specific intent, and all possible numerical combinations between the lowest and highest values ​​listed should be deemed to be explicitly described in this application in a similar manner.

[0076] The present invention relates to novel compositions of allen oxide synthase having enhanced stability, enzymatic activity, and antioxidant and / or biological activity. Additionally, this specification describes compositions and assemblies comprising an allen oxide synthase enzyme and a nonionic surfactant that provide enhanced enzymatic activity and properties compared to conventional formulation methods. Furthermore, this specification describes pharmaceutical compositions for controlling excessive oxidative stress in a target or cell, and methods of using said compositions and assemblies.

[0077] The present invention also relates to a method of using the composition and assembly to control excessive oxidative stress in a target.

[0078] Allen oxide synthase (AOS) is a cytochrome P450 plant enzyme with antioxidant activity that can be utilized in various fields, including disease treatment and the preservation of biological materials. However, to utilize these proteins for therapeutic or other purposes, the enzyme must not only be isolated but also incorporated into a composition that maintains or enhances biological activity. Since these enzymes are generally membrane-bound, surfactants (most commonly Triton X-100 and Emulphogene (polyoxyethylene tridecyl ether)) are used for purification and extraction in recombinant production processes. While detergents play an essential role in extracting and purifying proteins from their original environment, care must be taken to maintain the structural stability of the enzyme and minimize toxicity. The present invention is based on the discovery that when the allen oxide synthase enzyme is formulated with nonionic surfactants, particularly nonionic polysorbate surfactants, the enzyme activity and stability are surprisingly and significantly improved compared to other existing surfactants.

[0079] Accordingly, in one embodiment described herein, the protein assembly comprises an allen oxide synthase and a nonionic surfactant. The protein assembly used herein is a higher-order non-covalent supramolecular complex of an allen oxide synthase enzyme and a nonionic surfactant and / or surfactant micelle. In one aspect, the allen oxide synthase used may be derived from a plant. In one aspect, for example, the allen oxide synthase is Arabidopsis thaliana ( Arabidopsis thaliana) or Parthenium argentatum Alternatively, it may originate from other plant species that normally express allen oxide synthase. In another aspect, allen oxide synthase Parthenium argentatum It can be derived from. In another aspect, acetaminophen synthase is Helianthus anus (sunflower: Helianthus annus It can be derived from ).

[0080] In one aspect, allen oxide synthase may be wild-type. In another aspect, allen oxide synthase may be modified or functional variants. When used in relation to polypeptides, "functional variant" refers to a polypeptide that differs from the referenced polypeptide (e.g., differing by at least one conservative amino acid substitution) but retains the major function of the referenced polypeptide. For example, a functional variant of a polypeptide that acts as a transmembrane domain is one that acts as a transmembrane domain as part of that polypeptide. When used in relation to nucleic acids, the term "functional variant" refers to a nucleic acid that differs from the referenced nucleic acid but encodes a polypeptide that has the same major function as the polypeptide encoded by the referenced nucleic acid.

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

[0082] Examples of surfactants disclosed herein include nonionic, anionic, or cationic surfactants. Exemplary surfactants further comprise polyethylene sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide and hydrophobic bases produced by the condensation reaction of propylene oxide and propylene glycol. Nonionic surfactants disclosed herein include, but are not limited to, polysorbate surfactants selected from polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, and polyoxyethylene (20) sorbitan monooleate. In one aspect, the nonionic surfactant comprises one or more of Triton X-100, polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0083] As used herein, the hydrodynamic diameter refers to the diameter of a perfect solid sphere, including all solvation spheres, that exhibits the same hydrodynamic friction as the molecule of interest. Although it is believed that enzyme activity increases with smaller protein assemblies, no specific theory is required. Accordingly, the hydrodynamic diameter of the protein assemblies described herein is 1 nm to about 50 nm. In another aspect, the hydrodynamic diameter of the protein assemblies described herein is 2 nm to about 40 nm. In yet another aspect, the hydrodynamic diameter of the protein assemblies described herein is 3 nm to about 30 nm.

[0084] As used herein, the term “composition” is intended to encompass not only products containing specific amounts of the specific molecules described herein, but also all products directly or indirectly produced by combining specific components in specific amounts. Accordingly, another aspect of the present disclosure is a composition comprising the protein assembly described herein.

[0085] The therapeutically effective dose of a protein assembly refers to an amount that produces an effect or influence on the specific condition being treated. The protein assemblies described herein may therefore be administered orally, parenterally, topically, nasally, or via similar routes with a pharmaceutically acceptable carrier using any valid conventional dosage unit form, including immediate-release and sustained-release formulations. In some aspects, the compound is administered intravenously or orally.

[0086] A pharmaceutically acceptable carrier refers to a carrier that is relatively non-toxic and harmless to the patient at the concentration required for the effective action of the active ingredient; therefore, it means that side effects caused by the carrier do not interfere with the beneficial effects of the active ingredient. The amount of a therapeutically effective compound refers to the amount that produces a result or influences the condition being treated.

[0087] In another aspect, the pharmaceutical composition may include one or more buffers. Representative buffers include BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid) buffered saline, bisine (2-(bis(2-hydroxyethyl)amino)acetic acid), carbonate-bicarbonate, CHES (N-cyclohexyl-2-aminoethanesulfonic acid), diethanolamine, EBBS (Earl's equilibrium salt solution), glycine-sodium hydroxide buffer, HEPES ((4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)), HBSS (Hank's equilibrium salt solution), HEPPSO (4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) hydrate), HHBS (Hank's buffer containing HEPES), imidazole-HCl, maleic acid, and 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), trisine (N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), TRIS (tris(hydroxymethyl)aminomethane) and / or trisma (2-amino-2-(hydroxymethyl)-1,3-propanediol), but are not limited thereto. Any suitable buffer may be used, as disclosed in the art.

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

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

[0090] Dispersible powders and granules are suitable for preparing aqueous suspensions. They provide an active ingredient mixed with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Examples of such suspending agents may include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum; As a dispersant or wetting agent, natural phospholipids, e.g., lecithin, or a condensation product of an alkylene oxide and a fatty acid, e.g., polyoxyethylene stearate, or a condensation product of an ethylene oxide and a long-chain aliphatic alcohol, e.g., heptadecaethyleneoxycetanol, or a condensation product of a partial ester derived from an ethylene oxide, a fatty acid, and hexitol, e.g., polyoxyethylene sorbitol monooleate, or a condensation product of a partial ester derived from an ethylene oxide, a fatty acid, and hexitol anhydride, e.g., polyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives, e.g., ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweeteners, e.g., sucrose or saccharin.

[0091] The composition disclosed herein may also be administered parenterally, i.e., as an injectable formulation, subcutaneously, intravenously, intramuscularly, intrathecally, intravitreally, or intraperitoneally in a physiologically acceptable diluent, wherein the pharmaceutical carrier may be a sterile liquid or liquid mixture such as water, physiological saline, aqueous glucose solution and related sugar solution; an alcohol such as ethanol, isopropanol, or hexadecyl alcohol; a glycol such as propylene glycol or polyethylene glycol; a glycerol ketal such as 2,2-dimethyl-1,1-dioxolane-4-methanol; an ether such as poly(ethylene glycol) 400; an oil; a fatty acid; a fatty acid ester or glyceride; or an acetylated fatty acid glyceride; Pharmaceutically acceptable surfactants such as soap or detergent, suspending agents such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or emulsifiers and other pharmaceutical aids may or may not be added.

[0092] A pharmaceutical composition containing an active ingredient may be prepared in a form suitable for oral administration, e.g., tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self-emulsifiers as described in U.S. Patent No. 6,451,339, hard or soft capsules, or syrups or elixirs. A composition for oral administration may be prepared according to methods for preparing pharmaceutical compositions disclosed in the art. Such compositions may contain one or more additives selected from sweeteners, flavorings, colorings, and preservatives to provide a pharmaceutically excellent and palatable formulation. Tablets contain the active ingredient mixed with other non-toxic, pharmaceutically acceptable excipients suitable for tablet production. Such excipients include, for example, inert diluents such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate, or sodium phosphate; Granulators and disintegrants, such as corn starch or alginate; binders, such as PVP, cellulose, PEG, starch, gelatin, or acacia; and lubricants, such as magnesium stearate, stearic acid, or talc. The tablets may be uncoated or coated with an enteric coating or other known techniques to delay disintegration and absorption in the gastrointestinal tract to provide sustained action for a longer period. For example, time-delaying agents such as glyceryl monostearate or glyceryl distearate may be used. These may also be coated with techniques described in U.S. Patents No. 4,256,108; No. 4,166,452; and No. 4,265,874 to form osmotic therapeutic tablets for controlled release.

[0093] For oral administration, the composition may be formulated into solid or liquid formulations, such as, for example, capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, and may be prepared according to methods known in the art for the manufacture of pharmaceutical compositions. The solid unit dosage form may be a general hard or soft gelatin capsule and may contain, for example, a surfactant, a lubricant, and an inert filler such as lactose, sucrose, calcium phosphate, or corn starch.

[0094] Oral formulations may be provided in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin, or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil. Additionally, an emulsion may be prepared using an ingredient that does not mix with water, such as oil, and stabilized with a surfactant such as mono-diglycerides, PEG esters, etc.

[0095] In another aspect, the composition of the present invention may be prepared into a tablet by using a combination of a conventional tablet base, such as lactose, sucrose, or corn starch, a binder, such as acacia, corn starch, or gelatin; a disintegrant, such as potato starch, alginic acid, corn starch, or guar gum, which aids in the breakdown and dissolution of the tablet after administration; a lubricant, such as talc, stearic acid, magnesium, calcium, or zinc stearate, which improves the fluidity of the tablet granules and prevents the tablet material from adhering to the surface of the tablet die and punch; a dye; a coloring agent; and a flavoring agent to improve the aesthetics of the tablet and increase patient acceptance. Excipients suitable for use in oral liquid formulations include water and alcohols, such as ethanol, benzyl alcohol, or polyethylene alcohol, and may be used with or without the addition of pharmaceutically acceptable surfactants, suspending agents, or emulsifiers. Various other materials may be used as coatings or to modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both.

[0096] The pharmaceutical composition disclosed herein may also exist in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, such as liquid paraffin, or a mixture of vegetable oils. Suitable emulsifiers include (1) natural gums, such as acacia gum and tragacanth gum; (2) natural phospholipids, such as soybeans and lecithin; (3) esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and (4) condensation products of said partial esters and ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweeteners and flavoring agents.

[0097] An oily suspension can be prepared by suspending the active ingredient in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or in a mineral oil, such as liquid paraffin. The oily suspension may contain a thickener, such as beeswax, hard paraffin, or cetyl alcohol. Additionally, it may contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweeteners, such as sucrose or saccharin.

[0098] Oral formulations with good palatability can be produced by adding sweeteners and flavoring agents as presented above. Such compositions can be preserved by adding antioxidants such as ascorbic acid.

[0099] Syrups and elixirs can be prepared using sweeteners, such as glycerol, propylene glycol, sorbitol, or sucrose. These preparations may also contain mucosal protective agents, preservatives, flavoring agents, and coloring agents. Oral solutions can be prepared with, for example, cyclodextrin, PEG, and surfactants.

[0100] The compositions disclosed herein may generally contain an active ingredient in solution in an amount of about 0.0001% to about 50% by weight. It may also be advantageous to use preservatives and buffers together. Any suitable preservative may be used with the compositions described herein, including preservatives known in the art, buffers, salts, and antimicrobial agents including but not limited to parabens, as well as other preservatives commonly used in pharmaceutical compositions.

[0101] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension. This suspension may be formulated according to known techniques using the suitable dispersants, wetting agents, and suspending agents mentioned above. The sterile injectable may also be a sterile injectable solution or suspension dissolved in a non-toxic and injectable diluent or solvent, for example, a 1,3-butanediol solution. Available vehicles and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixatives are customarily used as solvents or suspension media. For this purpose, any non-toxic fixative, including synthetic mono- or diglycerides, may be used. Furthermore, fatty acids such as oleic acid are also used in the preparation of the injectable.

[0102] Another formulation used in the method of the present invention utilizes a transdermal delivery device (“patch”). Such transdermal patches may be used to continuously or intermittently inject the composition of the present invention in controlled amounts. The manufacture and use of transdermal patches for drug delivery are well known in the art (e.g., U.S. Patent No. 5,023,252 incorporated herein by reference). Such patches may be manufactured for continuous, pulsed, or on-demand delivery of drugs. For topical use, creams, ointments, jellies, solutions, or suspensions containing the molecules of the present invention are used. Topical application as used herein also includes the use of mouthwashes and gargles.

[0103] Methods for delivering molecules disclosed herein include various ways of administering molecules or medicinal compositions of molecules into the lungs via the mouth or rectum. Methods of administration may include delivering medicinal compositions in liquid or powder form for nasal administration via manual or active delivery mechanisms. Liquid formulations may be delivered via various mechanisms including vaporization via nasal inhalation, manual nasal devices, and mechanical nebulizer pumps. The formulations used in these delivery mechanisms may be in the form of aerosols containing a propellant or inhalation solutions without a propellant. Mechanical nebulizer pumps may be manual, gas-driven, or electric, as are electric nebulizers and atomizers. In one aspect described herein, inhalation solutions without a propellant are administered via a nebulizer or direct nasal inhalation.

[0104] The powder formulation may be administered via mechanical nebulizers, nasal inhalers, and nebulizers / nebulizers. Prior to administration, the powder formulation may be dissolved in a suitable solvent, including water and physiological saline solutions. In one aspect described herein, the compound may be dissolved in a physiological saline solution. In another aspect described herein, a therapeutically effective amount of the compound may be delivered to the lungs.

[0105] The compositions disclosed herein may include other conventional pharmaceutically acceptable formulation components, generally referred to as carriers or diluents, where necessary or preferable. The compositions disclosed herein may be preserved by adding antioxidants, such as ascorbic acid, or other suitable preservatives. Conventional methods for preparing such compositions into suitable formulations may be used.

[0106] Commonly used pharmaceutical ingredients that can be appropriately used to formulate a composition for an intended route of administration include, but are not limited to, acidifying agents such as acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid; and alkalizing agents such as ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, or trolamine.

[0107] Other pharmaceutical ingredients include, for example, adsorbents, such as powdered cellulose and activated carbon; aerosol propellants (e.g., carbon dioxide, CCl2F2, F2ClC-CClF2 and CClF3); air displacing agents (e.g., nitrogen and argon); antifungal preservatives (e.g., benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate); and antimicrobial preservatives (e.g., benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercury nitrate and thimerosal); Antioxidants (e.g., ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphate, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite); binders (e.g., block polymers, natural and synthetic rubber, polyacrylates, polyurethanes, silicones, and styrene-butadiene copolymers); buffers (e.g., potassium metaphosphate, potassium monohydrogen phosphate, sodium acetate, anhydrous sodium citrate, and sodium citrate dihydrate); carriers (e.g., acacia syrup, flavor syrup, flavor elixir, cherry syrup, cocoa syrup, orange syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection, and bacteriostatic water for injection); Chelating agents (e.g., disodium edetate and edetic acid); coloring agents (e.g., FD&C Red 3, FD&C Red 20, FD&C Yellow 6, FD&C Blue 2, D&C Green 5, D&C Orange 5, D&C Red 8, caramel and iron oxide red); refining agents (e.g., bentonite); emulsifiers (including, but not limited to, acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyethylene 50 stearate, etc.); encapsulating agents (e.g., gelatin and cellulose acetate phthalate); flavoring agents (e.g., anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin); Humectants (e.g., glycerin, propylene glycol, and sorbitol);Inhibitors (e.g., mineral oil and glycerin); oils (e.g., peanut oil, mineral oil, olive oil, peanut oil, sesame oil, and vegetable oil); ointment bases (e.g., lanolin, hydrophilic ointment, polyethylene glycol ointment, petroleum jelly, hydrophilic petroleum jelly, white ointment, yellow ointment, and rose water ointment); penetration promoters (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, cephalins, 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 perfusion); curing agents (e.g., cetyl alcohol, cetyl ester wax, microcrystalline wax, paraffin, stearyl alcohol, white wax, and yellow wax); suppository bases (e.g., cocoa butter and polyethylene glycol (mixture)); surfactants (e.g., benzalkonium chloride, nonoxynol 10, oxtoxynol 9, polysorbate 80, sodium lauryl sulfate, and sorbitan monopalmitate); Suspensions (e.g., agar, bentonite, carbomer, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, kaolin, methylcellulose, tragacanth, and beeh gum); sweeteners (e.g., aspartame, dextrose, glycerin, mannitol, propylene glycol, sodium saccharin, sorbitol, and sucrose); anti-sticking agents for tablets (e.g., magnesium stearate and talc); binders for tablets (e.g., acacia, alginate, sodium carboxymethylcellulose, compressed sugars, ethylcellulose, gelatin, liquid glucose, methylcellulose, povidone, and pre-gelatinized starch); Tablet and capsule diluents (e.g., dicalcium 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, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetate phthalate, and shellac); tablet direct compression excipients (e.g., dical calcium phosphate); tablet disintegrants (e.g., alginic acid, calcium carboxymethylcellulose, microcrystalline cellulose, potassium polyacrylinate, sodium alginate, sodium starch glycolate, and starch); tablet lubricants (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 opacifiers (e.g., titanium dioxide); tablet abrasives (e.g., carnauba wax and white wax); thickeners (e.g., beeswax, cetyl alcohol, and paraffin); Osmotic pressure regulators (e.g., dextrose and sodium chloride); viscosity enhancers (e.g., alginate, bentonite, carbomer, sodium carboxymethylcellulose, methylcellulose, povidone, sodium alginate and tragacanth); and wetting agents (e.g., heptadecaethylene, oxycetanol, lecithin, polyethylene sorbitol monooleate, polyoxyethylene sorbitol monooleate and polyoxyethylene stearate) may be, but are not limited thereto.;

[0108] Depending on the individual drugs used for concomitant administration in combination therapy, they may be formulated as a combination agent (where a stable formulation can be manufactured and the desired administration regimen is compatible), or they may be formulated separately for concomitant or separate administration via the same or alternative routes. Allen oxide synthase is associated with neuroprotective effects, particularly in relation to myocardial infarction, stroke, or ischemic injury. The ischemic injury considered here includes all types of brain injury or brain injury caused by thrombosis. It is believed, but not bound by any specific theory, that timely administration of allen oxasidase synthase reduces inflammatory responses and apoptosis by blocking oxidative stress occurring after all types of ischemic events (Mathai, 2012). Ischemic injury can occur due to traumatic events (e.g., stroke or myocardial infarction) and results in reduced blood supply to tissues.

[0109] Therefore, reactive oxygen species, including hydrogen peroxide (H2O2) and lipid peroxides, play a significant role in ischemia-reperfusion injury. For example, generalized cardiac ischemia can lead to systolic dysfunction within minutes, and it is common for myocardial H2O2 content to increase by more than 250% after 30 minutes of ischemia (Slezak, J., et al., Am. J. Pathol., 1995, 147(3), 772-81). Furthermore, reduced systolic function during the early stages of reperfusion can be accompanied by an increase of approximately 600% in myocardial H2O2. A rapid increase in oxygen free radicals during reperfusion of an ischemic heart is commonly observed in various ischemic conditions, including stroke. When allan oxide synthase is administered within a window of opportunity of 30–45 minutes prior to the onset of reperfusion, it has been shown to improve ischemia-reperfusion injury in myocardial infarction (U.S. Patent No. US7157082; incorporated herein by reference in relation to this background technology) and cerebrovascular hypoxia-ischemia (e.g., stroke, ischemic stroke, traumatic brain injury, etc.) (Mathai, S., et al. , CNS Neurosci. Ther. 2012, 18(11), 887-94; regarding this background technology, it is known that there are clear advantages to (incorporated herein by reference).

[0110] Accordingly, other embodiments described herein include a method for treating ischemic injury in a subject requiring such injury, comprising administering any one of the compositions described herein to the subject. 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, stroke, ischemic stroke, hepatic ischemia, limb ischemia, pulmonary ischemia, renal ischemia, ocular ischemia, and retinal ischemia; and / or is associated if the subject is undergoing or has undergone a procedure selected from the group comprising 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, stroke, hemorrhagic stroke and / or ischemic stroke, mesenteric ischemia, hepatic ischemia, limb ischemia, renal ischemia, ocular ischemia or retinal ischemia.

[0111] Another embodiment described in this specification is a method for reducing oxidative stress in a patient's brain, comprising administering a pharmaceutical composition as described in this specification to a patient.

[0112] The dosage of the molecules disclosed herein that exhibits therapeutic effects can be easily determined to suit the treatment of each desired indication. The amount of active ingredient (e.g., allen oxide synthase) administered to treat one of these conditions may vary significantly depending on several factors, such as the specific compound and dosage unit used, the method of administration, the duration of treatment, the age and gender of the patient being treated, and the characteristics and severity of the condition being treated.

[0113] The total amount of the active ingredient to be administered is generally in the range of about 0.0001 mg to about 10 mg per kg of body weight, preferably in the range of about 0.001 mg to about 10 mg per kg of body weight per day. A single dose may contain about 0.05 mg to about 500 mg of the active ingredient and may be administered at least once a day. When using injectable administration and drip infusion methods, including intravenous injection, intramuscular injection, subcutaneous injection, and parenteral injection, the daily dose may be in the range of about 0.0001 mg to about 10 mg per kg of body weight. When administered rectally, the daily dose may be in the range of 0.0001 mg to 10 mg per kg of body weight. When absorbed transdermally, the blood concentration may be set to the concentration required to maintain the daily dose in the range of 0.0001 mg to 10 mg per kg of body weight. The daily inhalation concentration may be the concentration required to maintain a daily dose of 0.0001 mg / kg to 10 mg / kg.

[0114] The specific initial and continuous dosages applied to each patient vary depending on the characteristics and severity of the condition as determined by the attending physician, the activity of the specific compound used, the patient's age, diet, time of administration, route of administration, drug elimination rate, combination drug therapy, etc. The preferred treatment method and frequency of administration of the compounds disclosed herein can be determined by a person skilled in the art through conventional treatment trials.

[0115] Another use of the protein assemblies and compositions described herein is to maintain the stability of sperm and embryos in all reproductive situations. Due to the sensitive nature of these bodily fluids and cells, freezing and storage conditions can affect the viability of sperm cells and embryos (including fertilized eggs). Therefore, it is believed that the protein assemblies and compositions described herein can be used to stabilize sperm cells and / or embryos or to enhance their viability, but the invention is not bound by any specific theory. Accordingly, another embodiment described herein is a sperm preservation method comprising bringing sperm into contact with a protein assembly or one of the compositions described herein.

[0116] Another embodiment is a method for extending the lifespan of semen, comprising contacting semen with a protein assembly or composition described herein. Another embodiment is a method for improving the viability of sperm, comprising contacting sperm with a protein assembly or composition described herein. Another embodiment is a method for improving the ratio or number of implantable embryos, the quality of implantable embryos, or the success rate of implanted embryos for use in assisted reproductive technology, comprising contacting an oocyte or a fertilized embryo with a protein assembly or composition described herein. Non-limiting exemplary implementation example

[0117] 1. A method for treating ischemic injury in a subject requiring treatment for ischemic injury, wherein the method comprises administering to the subject a protein assembly disclosed herein or a pharmaceutical composition disclosed herein.

[0118] 2. Method in Embodiment 1, wherein the ischemic injury is ischemia-reperfusion injury.

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

[0120] 4. A method for preserving sperm, wherein the method comprises the step of contacting sperm with a protein assembly disclosed herein.

[0121] 5. A method for preserving semen, wherein the method comprises the step of contacting the semen with the protein assembly disclosed above.

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

[0123] 7. A method for improving the survival rate of implantable embryos, wherein the method comprises: (i) oocyte; or (ii) modified embryo A method comprising contacting with a protein assembly or composition disclosed in this specification.

[0124] 8. As a composition suitable for the treatment of ischemic injury: a. One or more allen oxidase enzymes; b. One or more polysorbate surfactants; c. One or more buffers; and d. One or more excipients; A composition comprising, wherein the hydrodynamic diameter of the protein assembly is about 5 nm to 30 nm.

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

[0126] 10. A composition according to Embodiment 9, wherein one or more polysorbate surfactants are included at a concentration greater than or equal to the critical micelle concentration. Examples Variant allen oxide synthase enzyme

[0127] Variant allen oxide synthase (AOS) was prepared containing non-conserved amino acid substitutions in residues predicted to be important for enzyme structure / function and activity, such as those playing important roles in substrate binding, heme binding, etc. All sequences in Fig. 1 possess multiple non-conserved amino acid substitutions in major residues compared to known AOS enzymes, particularly SEQ ID NO: 2 (Fig. 1). Example 1: Activity of an allene oxide synthase assembly containing a nonionic surfactant

[0128] Parthenium argentatum Allen oxide synthase (PaAOS) activity k obs )silver 13, a natural temperament ( S)- Measurements were performed using hydroperoxylinoleic acid (13(S)-HpODE), and the effect of adding nonionic surfactants on enzyme activity was evaluated. The purpose of this study is to investigate the effects of structurally different nonionic surfactants on enzyme activity, considering the surfactant dependence of the AOS enzyme, and thereby to find the optimal combination of AOS enzyme and surfactant that maximizes enzyme activity and minimizes surfactant toxicity.

[0129] As mentioned above, AOS E. coli It was prepared and purified through recombinant expression (Li et al. , PNAS, 2008, 105(37), 13883-13888). The initial panel of nonionic and zwitterionic surfactants showed generality in protein and enzyme purification (Triton X-100 (Tx-100) and n-dodecyl-β-D-maltoside (DDM)), or Oryza sativa ( Oryza sativa Effect on use or activity in the extraction / solubilization of ) derived AOS enzymes (Tween 20 (Tw 20), also known as polysorbate 20; Szymczyk et al., 2018), PaAOS (3-[(3-colamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); Rodi et al., 2014), and Arabidopsis thaliana It was selected based on (emulpogen (Emul) or polyoxyethylene (10) tridecyl ether; used to first demonstrate the detergent dependence of AOS enzyme activity in Hughes et al., 2006). The characteristics of the initial surfactant panel are shown in Table 1. Table 1 method

[0130] The effect on PaAOS enzymatic reaction kinetics and concentration dependence were measured through activity assays using the natural substrate 12(S)-HpODE. Enzyme stock cultured with detergent was used in the study. Specific activity above and below the critical micelle concentration (CMC) of the corresponding surfactant was measured to determine whether the surfactant dependence of AOS requires micelle formation for activity. The PaAOS substrate 13(S)-HpODE was obtained in situ from linoleic acid (LA) and lipoxagenase (LOX:L 7395, Signa-Aldrich) in situ ) LA was prepared. After diluting LA in an equal volume of ethanol, the ethanol solution was diluted 500-fold in a 10 mM sodium tetraborate (pH 9.0) solution and incubated for 1 hour. The obtained solution was further diluted 15-fold to make a 0.05 mg / mL LOX (2.5 mM KPO4, pH 7.4) solution, and 10 The reaction was carried out for 10 minutes. Afterwards, the solution was centrifuged at 1048 g for 10 minutes using a 30 kDa centrifugation filter device to remove LOX from the 13(S)-HpODE solution.

[0131] The specific enzyme activity of PaAOS was evaluated by adding 10 μL of an enzyme sample at a concentration of 0.005 mg / mL to 490 μL of substrate contained in a quartz cuvette and measuring the decrease in UV-visible absorbance (A 234 nm) over 30 seconds. The sample was measured three times at room temperature. The change in absorbance per second is the observed activity ( k obs ; s -1 Convert to ) The number of substrate molecules converted per second was reflected, and the molar concentration of the enzyme was corrected. The quenching coefficient of 13(S)-HpODE is 24,500. Various surfactants Surfactants were retrospectively added to determine the effect on the enzymatic function of PaAOS. Experiments were performed using the surfactant concentrations presented in Table 2. Samples were prepared by diluting PaAOS (containing Triton X-100 remaining from the enzyme purification process) to a final concentration of 0.005 mg / mL in Tris buffer (20 mM, pH 8.2) of the specified detergent and concentration. Activity analysis was performed as described above. Negative staining transmission electron microscopy (TEM) analysis was performed on PaAOS cultured with various surfactants. Samples were adsorbed onto glow discharge-treated carbon and PaAOS onto a rhodium-coated 400-mesh copper grid and stained with 2% uranyl acetate. The grids were imaged using a Tecnai TF-20 FEG-TEM (Thermo Fisher Scientific) operating at 200 kV. Images were recorded using a Gatan instrument: an Ultrascan 1000 CCD camera operating with Gatan Digital Micrograph v 19.3 software. Results and Discussion During the study, it was found that in all cases, the enzymatic activity (turnover rate) of PaAOS was significantly lower when the surfactant concentration was below the critical micelle concentration (CMC). This was independent of the type of surfactant used. When the surfactant concentration was much lower than the CMC, no difference in enzymatic activity was observed among any surfactants (Fig. 2A). There was also almost no difference in the enzymatic activity of PaAOS when nonionic surfactants such as Triton X-100, DDS, or Emulphogene (Emul), or amphoteric surfactants such as CHAPS, were added. However, surprisingly, a significant increase in enzymatic activity was observed when polysorbate 20 (Tween 20, Tw20) was included, suggesting an activity-enhancing effect due to the addition of polysorbate micelles (Fig. 2B). Fig. 2 compares the activity of the AOS enzyme with respect to various nonionic and amphoteric surfactants. (A) At concentrations below CMC, all surfactants exhibit equivalent activity, and (B) they exhibit enzymatic activity above CMC, but the enzymatic activity of polysorbate formulations is significantly increased compared to other surfactants. Therefore, a person skilled in the art would have expected a difference in activity between surfactant concentrations above and below CMC; however, based on data regarding other examples of nonionic surfactants, a person skilled in the art would also have expected that enzymatic activity would be equivalent (though greater) regardless of surfactant selection as long as CMC is exceeded. The fact that polysorbate shows higher enzymatic activity than other surfactants suggests a synergistic effect. Therefore, speculating without being bound by theory, it is thought that this synergistic increase in enzymatic turnover rate occurs because the interaction between the AOS enzyme and polysorbate micelles is improved due to the head group substructure and packing parameters of polysorbate micelles being different from other known surfactants.Transmission electron microscope images of PaAOS bound to Triton X-100 and polysorbate 20 (Tween 20; Tw20) were observed under conditions where the surfactant concentration corresponded to 0.03% or 60% of the critical micelle concentration (CMC) (Fig. 3). As shown in Fig. 3, larger protein aggregates with a diameter of approximately 20–50 nm were observed at surfactant concentrations much lower than the CMC. These large protein aggregates formed at low surfactant concentrations indicate a significant decrease in enzyme activity compared to the activity observed at concentrations higher than the CMC. Conversely, as the surfactant concentration approached the CMC, the protein aggregates became much smaller and more monodisperse, suggesting the presence of more uniform oligomeric forms of AOS enzymes, which is associated with higher enzyme activity. In general, enzyme activity tended to saturate compared to the activity observed at concentrations below the CMC from the range where the surfactant concentration approached the CMC to the range where it exceeded the CMC. Therefore, these results suggest that non-aggregated, monodisperse AOS aggregates exhibit the highest enzymatic activity, and that AOS formulations containing polysorbate surfactants further enhance enzymatic activity compared to other conventional surfactants through a synergistic effect between the surfactant and the protein.

[0132] The combination of polysorbate surfactants and AOS enzymes above critical micelle concentrations resulted in an unexpected enhancement of enzyme activity compared to other known similar nonionic or zwitterionic surfactants. Furthermore, at surfactant concentrations approaching or exceeding the critical micelle concentration, AOS formed more uniform AOS-surfactant assemblies, and these assemblies were generally observed to aggregate more significantly and exhibit higher enzyme activity than their surfactant-depleted counterparts. Taken together, these findings support a novel composition having improved AOS activity compared to other known methods, which is useful for controlling excessive oxidative stress in the target. Example 2: Activity, structure, and toxicity of an allene oxide synthase assembly containing a polysorbate surfactant

[0133] As an unexpected enhancement in enzyme activity was observed when AOS was formulated with polysorbate 20 (Tween 20), the effect of polysorbate surfactants was investigated. After purifying the enzyme using various detergents, the activity of the AOS-surfactant formulations was evaluated. Activity was OD 234 Activity was measured by the decrease in absorbance at the maximum absorbance of linoleic acid hydroperoxide (the natural substrate of AOS), and 1 unit (U) of activity was defined as the amount of enzyme required to decrease the OD by 1 under standard analytical conditions (1 min at 22°C). Tween 20, Tween 40, Tween 60, and Tween 80, which represent the smallest to largest molecular weights in the Tween / polysorbate surfactant family, were used for formulation and activity evaluation. The Tween 20, Tween 40, Tween 60, and Tween 80 formulations exhibited statistically equivalent activity and, surprisingly, showed significantly higher activity than AOS formulated with Triton X-100 (Tables 3A and 3B; Fig. 4).

[0134] Native PAGE was performed to evaluate the differences in the natural forms of the purified AOS proteins. As shown in Figures 5A and 5B, formulation of AOS using Triton or Tween / polysorbate surfactants produces AOS enzymes with different natural structures. It should be noted that both the charge and molecular size of the proteins are factors determining the behavior of the proteins in these gel systems. In general, the use of Tween / polysorbate surfactants surprisingly produces a much denser and more 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 evaluated using the WST-1 cell proliferation assay with HEK 293 cells. Surfactant screening was performed to determine the toxicity profiles of three surfactants (Triton X-100 (Fig. 6A), Tween 80 (Fig. 6B), and Tween 20 (Fig. 6C)) on HEK 293T cells. Plated cells were treated with various detergent concentrations ranging from 0.001% to 0.05%, and cell viability was measured 24 hours after treatment using WST-1 cell proliferation reagent (OD). 450 (Reflects cell number). Triton X-100 OD at concentrations of 0.0075% or higher 450Significant cytotoxicity was exhibited, as evidenced by the decrease in value. HEK 293T cells showed much higher resistance to Tween-based detergents, and toxicity was observed only at Tween 80 at the highest concentration tested (0.05%). No toxicity was observed at Tween 20. Rather, surprisingly, Tween 20 increased the viability of HEK 293T cells at low concentrations (0.001–0.02%). In summary, AOS enzymes formulated and stabilized with polysorbate surfactants surprisingly produced smaller, more aggregated AOS-surfactant assemblies with substantially higher enzymatic activity. Furthermore, polysorbate surfactants exhibit substantially lower toxicity to cells compared to their Triton X-100 counterparts. Taken together, the inventors have prepared a novel AOS enzyme composition that meets desired criteria in terms of activity, stability, polydispersity, and safety for human or veterinary pharmaceutical use. Example 3: Light scattering irradiation of AOS-surfactant assembly

[0135] The inventors conducted a light dynamic scattering investigation to study the hydrodynamic diameter and polydispersity of the AOS-surfactant assembly. The size distribution of the AOS-surfactant assembly was measured by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano. The hydrodynamic diameter (HD) and polydispersity index (PDI) were measured at 25°C. For this purpose, 150 μL of the AOS-surfactant suspension was placed in a 12 mm square disposable polystyrene cuvette with a light path length of 10 mm and added to 2 mL of phosphate buffer solution. Light scattering was measured at a backscattering angle of 173°. Bovine serum albumin (BSA) and thyroglobulin were used as control proteins with known size distributions. A summary of the results is presented in Table 3 and Figures 7–13.

[0136] The study results show that the AOS-surfactant assembly has a significantly smaller hydrodynamic diameter compared to the surfactant-removed complex, which is consistent with the results of previously performed negative staining transmission electron microscopy (TEM) image analysis of AOS-surfactant and surfactant-removed complexes. Along with the phenomenon of increased enzymatic activity in complexes where the surfactant concentration is above the corresponding critical micelle concentration (CMC), these results provide new insights into the AOS-surfactant structure-function relationship and suggest that suspensions with an average hydrodynamic diameter of less than 30 nm are optimal for providing formulations with higher enzymatic activity than suspensions with larger diameters.

[0137] Along with the increase in enzymatic activity of assemblies containing surfactant concentrations exceeding the corresponding critical micelle concentration (CMC), these results provide new insights into the AOS-surfactant structure-function relationship. Thus, smaller assemblies with a diameter of less than approximately 30 nm in at least one dimension surprisingly exhibit higher catalytic activity than larger assemblies. Example 4: Usefulness of an AOS-surfactant assembly for protecting neurons from oxidative neuronal damage and ischemia-reperfusion injury

[0138] The inventors investigated the utility of an AOS-polysorbate assembly for protecting brain tissue from oxidative damage as a model of ischemia-reperfusion injury using a hydrogen peroxide-induced neurological injury paradigm.

[0139] Reactive oxygen species, including hydrogen peroxide (H2O2) and lipid peroxides, cause ischemia - It plays a significant role in reperfusion injury. For example, generalized cardiac ischemia can cause systolic dysfunction within minutes, and it is common for myocardial H₂O₂ content to increase by more than 250% after 30 minutes of ischemia (Slezak, J. et al.,Am. J. Pathol., 1995, 147(3), 772-81). Furthermore, reduced systolic function during the early stages of reperfusion can be accompanied by an increase of approximately 600% in myocardial H2O2. A rapid increase in oxygen free radicals during reperfusion of the ischemic heart is commonly observed in various ischemic conditions, including stroke. Allen oxide synthase is known to have a clear benefit in mitigating ischemia-reperfusion injury during myocardial infarction (U.S. Patent No. 7157082; incorporated herein by reference in relation to such background technology) and cerebrovascular hypoxic-ischemia (e.g., stroke, ischemic stroke, traumatic brain injury, etc.) when administered within an opportunity window of 30 to 45 minutes prior to the onset of reperfusion (Mathai, S., et al., CNS Neurosci. Ther. 2012, 18(11), 887-94; incorporated herein by reference in relation to such background technology). method

[0140] Cerebellar microimplant system 1. Extraction of cerebellar tissue

[0141] Four-day-old Wistar rats were used in the study. After sacrificing the rats, they were cooled on ice for 1 minute; the heads were decapitated, and the cerebellum was extracted and placed on ice. Cerebellar tissue was placed in 1 mL of PBS supplemented with 0.65% glucose (10 μM 65% D(+)-glucose stock solution / 1 mL PBS) in a large Petri dish. The tissue was chopped and finely ground using a 1 mL insulin syringe equipped with a 23 G (0.4 mm) needle. The ground tissue was then placed back into the glucose solution in the large Petri dish. The tissue was filtered through 125 μm gauze, centrifuged twice (at 6000 xg for 2 minutes), and transferred to serum-free START V medium supplemented with BSA (Biochrom, Germany). The second centrifugation was performed using 1 mL of START V medium. The microscopic sections were reconstituted in 500 μL of START V medium and stored on ice. 2. Cerebellar cell culture

[0142] Two hours after poly-L-lysine coating, the glass slides were washed with Millipore distilled water and air-dried. Each slide was placed in a small Petri dish (diameter: 35 mm), and 40 μL of STAART V / cell suspension was added. The tissues were stabilized by incubating at 34°C for 2 hours. Then, 1 mL of START V medium was added to the Petri dish, and the samples were incubated for 48 hours under conditions of 34°C, 5% CO2, and 100% humidity. 3. Drug Administration

[0143] In this study, some tissue cultures were exposed only to the solvent (PBS buffer) and used as a control group. In the first study (Study 1), 10 μL of toxin (0.1 mM hydrogen peroxide, pH 7.4, Millipore distilled water) was administered simultaneously while increasing the AOS enzyme concentration (10 ng / mL - 20 ng / mL, PBS, pH 7.4). Study 2 was conducted to confirm the positive results obtained in Study 1. In all studies, the drug was maintained in contact with the tissue cultures for the entire 24-hour study period. 4. Measurement of drug effects

[0144] After exposing tissue sections to a drug (toxin / AOS) for 24 hours, they were rinsed with PBS and fixed with gradually increasing concentrations of paraformaldehyde (PFA) (500 μL, 0.4% PFA, followed by 1.2%, 3%, and finally 4% PFA). Each fixation step was performed for 3 minutes. Finally, the microscopic tissue sections were rinsed with PBS.

[0145] The morphology of neurons within tissue sections (presence of neurites) was evaluated, and the number of viable cells per microscopic field was counted. The four fields with the highest cell density per coverslip were selected for counting, and data were expressed as mean ± standard error of the mean (SEM) (n=4 each). Statistical significance was evaluated using a non-paired t-test. result

[0146] As a result of exposing cerebellar microtissues to hydrogen peroxide-induced oxidative stress, nearly 100% of cerebellar neurons died. However, after treatment with the AOS enzyme, neuronal survival rates increased significantly (P < 0.001) at all drug concentrations (Fig. 14). AOS demonstrated an average neuronal recovery rate of 23% from hydrogen peroxide damage. Discussion and Conclusion

[0147] This study shows that the AOS enzyme can protect neurons from hydrogen peroxide damage to a significant extent in postnatal cerebellar granule cells composed of cerebellar microtissue cultures.

[0148] Therefore, these results support the utility of a new AOS-polysorbate assembly with enhanced activity in alleviating ischemia-reperfusion injury. The inventors anticipate that this new assembly may be useful in the treatment of various ischemic diseases, including coronary artery occlusion, myocardial infarction, stroke, ischemic stroke, hepatic ischemia, limb ischemia, pulmonary ischemia, renal ischemia, mesenteric ischemia, retinal and ocular ischemia, as well as in various procedures including angioplasty, thrombectomy, and coronary artery bypass surgery. Example 5: Predictive Example - Stability of semen and embryo in a reproductive context Additional experiments are performed to confirm the stability of semen and embryos in the various formulations described herein. Semen and embryos will be tested for viability and quality after freezing and thawing in the formulations described herein. *** The present disclosure is described by reference to specific preferred embodiments so that the reader may practice the invention without excessive experimentation. However, those skilled in the art will readily recognize that many of the components or parameters may be changed or modified to some extent or replaced with known equivalents without departing from the scope of the invention. Such modifications and equivalents should be understood as being incorporated herein by reference as individually described. The invention also includes all steps, features, compositions, and molecules mentioned or indicated in the specification, individually or collectively, and any combination of any two or more of said steps or features. Titles, headings, etc. are provided to help readers better understand this specification and should not be interpreted as limiting the scope of the invention. The entire contents of all applications, patents, and published documents cited above and below are incorporated herein by reference. However, no reference to any applications, patents, or published documents mentioned herein shall be construed as an acknowledgment or implied that they constitute valid prior art or are part of common sense in any country in the world.

Claims

Claim 1 A protein assembly comprising allen oxide synthase, wherein the hydrodynamic diameter of the assembly is about 5 nm to 30 nm. Claim 2 In paragraph 1, the allen oxide synthase enzyme is Parthenium argentatum (Parthenium argentatum A protein assembly derived from ). Claim 3 A protein assembly according to claims 1 and 2, wherein the allen oxide synthase enzyme comprises an amino acid sequence having at least 80% sequence identity with 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. Claim 4 A protein assembly according to claims 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. Claim 5 A composition comprising the protein assembly of claims 1 to 4. Claim 6 A pharmaceutical composition comprising one or more of the protein assemblies of claims 1 to 4 and a pharmaceutically acceptable carrier. Claim 7 A pharmaceutical composition according to claim 6, wherein a portion of the composition comprises a protein assembly having a hydrodynamic diameter of about 5 nm to 30 nm. Claim 8 A pharmaceutical composition according to claims 6 and 7, wherein one or more polysorbate surfactants are present at a concentration greater than or equal to the critical micelle concentration. Claim 9 A protein assembly of claims 1 to 4 or a pharmaceutical composition of claim 6, wherein the protein assembly or pharmaceutical composition is to be used to treat a subject requiring treatment for ischemic injury. Claim 10 In paragraph 9, the protein assembly or pharmaceutical composition, wherein the ischemic injury is ischemia-reperfusion injury. Claim 11 In claim 9 or 10, 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, renal ischemia, ocular ischemia, and retinal ischemia; and / or the subject is a protein assembly or pharmaceutical composition that is undergoing or has undergone a procedure selected from the group comprising angioplasty, thrombectomy, and coronary artery bypass surgery. Claim 12 A protein assembly used in a sperm preservation method according to any one of claims 1 to 4, wherein the sperm preservation method comprises contacting sperm with the protein assembly. Claim 13 A protein assembly used in a semen preservation method according to any one of claims 1 to 4, wherein the semen preservation method comprises contacting semen with a protein assembly. Claim 14 A protein assembly used in a method for improving sperm viability in any one of claims 1 to 4, wherein the method for improving sperm viability comprises contacting sperm with the protein assembly. Claim 15 A protein assembly according to claims 1 to 4 or a composition according to claims 5 to 7, used in a method for improving the viability of an implantable embryo, said method for improving the viability of an implantable embryo comprising: (i) an oocyte; or (ii) contacting a fertilized embryo with the protein assembly or composition. Claim 16 A composition suitable for use in treating ischemic injury, comprising: a. one or more allen oxidase enzymes; b. one or more polysorbate surfactants; c. one or more buffers; and d. one or more excipients; wherein the hydrodynamic diameter of the protein assembly is about 5 nm to 30 nm. Claim 17 A composition according to claim 16, wherein one or more polysorbate surfactants comprise polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. Claim 18 A composition according to claim 17, wherein one or more polysorbate surfactants are present at a concentration greater than or equal to the critical micelle concentration.

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