Tick ​​salivary gland polypeptides for the prevention and treatment of encephalitis

Abstract

The present invention provides pharmaceutical compositions for preventing and / or treating encephalitis and related diseases or conditions. [Solution] A pharmaceutical composition comprising proteins and polypeptides from the salivary glands of ticks is provided for use in preventing and / or treating encephalitis and encephalitis-related diseases or conditions. The composition is further used to prevent, reduce, decrease or inhibit neuronal cell death.

Description

[Technical Field]

[0001] This invention relates to proteins and polypeptides containing tick (Ixodes ricinus) salivary gland polypeptides, as well as their use for the prevention and / or treatment of encephalitis. [Background technology]

[0002] Neuroinflammation is defined as an inflammatory response in the brain (i.e., encephalitis) or within the spinal cord. This inflammation is mediated by the production of cytokines, chemokines, reactive oxygen species, and secondary messengers. These mediators are produced by commensal glial cells (microglia and astrocytes), endothelial cells, and peripherally derived immune cells.

[0003] Neuroinflammation or encephalitis can occur after brain injury, for example, in hemorrhagic stroke. Hemorrhagic stroke is caused by bleeding into the brain due to the rupture of blood vessels. Hemorrhagic stroke can be further subdivided into intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). ICH is bleeding into the brain parenchyma, while SAH is bleeding into the subarachnoid space. Although they constitute a minority of stroke cases (10-15%), patients with ICH and SAH often suffer serious disabilities, and these disabilities contribute to a burden of morbidity and mortality comparable to ischemic stroke. The clinical outcome of this disease is poor, with only 20% of patients achieving functional independence 6 months after ICH. The most common cause of ICH is chronic hypertension, but cerebral amyloid angiopathy, anticoagulant therapy, and underlying vascular abnormalities account for a significant proportion of cases.

[0004] ICH not only causes primary brain injury due to the direct mechanical effects of the hemorrhage, but also secondary brain injury (SBI), which is a major cause of poor outcomes after ICH. SBI involves disruption of the blood-brain barrier (BBB) ​​and leads to perihematoma edema (PHE) and brain parenchymal cell death. SBI is particularly associated with neuroinflammation.

[0005] Neuroinflammation associated with ICH involves early activation of commensal microglia, release of pro-inflammatory mediators, and infiltration of systemic inflammatory cells.

[0006] The first innate immune cells activated after ICH and other acute brain injury are microglia (i.e., resident macrophages of the central nervous system). They continuously scan the extracellular brain environment and can be activated within minutes of tissue injury. The number of activated microglia / macrophages peaks at 72 hours and returns to normal levels 3-4 weeks after ICH. Monocyte-derived macrophages infiltrate the perivascular and hematoma areas after microglia are activated. In response to various stimuli, microglia and brain macrophages produce pro-inflammatory cytokines, chemokines, and reactive oxygen species, including TNF-α and IL-1β. In addition to neurotoxic cytokines, chemokines such as CXCL2 produced by microglia have chemotactic activity against neutrophils and thus exacerbate the inflammatory response. Thus, microglia also promote early neuroinflammation by recruiting and activating blood-derived leukocytes, which may exacerbate ICH-induced nerve injury.

[0007] Neutrophils are the fastest leukocyte subtype to infiltrate the hematoma site within 4-5 hours in animal models, peaking at 3 days. In humans, neutrophils infiltrate the hematoma and surrounding brain tissue within the first few days (within 72 hours) after ICH. Neutrophils and neutrophil extracellular traps (NETs), i.e., extracellular reticular structures consisting of nuclear DNA / histones and granule contents released by neutrophils, can damage the brain by, for example, producing reactive oxygen species, releasing pro-inflammatory proteases, and exacerbating neuronal cell death. NETs have also been reported to promote neuroinflammation and nerve damage after SAH, but pharmacological inhibition of NETs reduced inflammatory damage in mice.

[0008] Myeloperoxidase (MPO) is an important inflammatory factor in the myeloid system. MPO is involved in neutrophils and Ly-6C 高Myeloperoxidase is highly expressed in other myeloid cells such as monocytes, macrophages, and microglia. Myeloperoxidase and its active products are involved in the development and progression of intracerebral hemorrhage, including damage to the blood-brain barrier and the brain. Myeloperoxidase can be used as a specific inflammatory marker in the assessment of the appearance and development of vascular disease, and experimental data have shown that inhibition or deficiency of myeloperoxidase has a positive effect on prognosis.

[0009] Over the past few decades, there has been only slight improvement in the case mortality rate for ICH, and most survivors continue to live with severe physical disabilities. Treatment options for ICH are very limited, and there is still no definitive treatment other than supportive care. Therefore, the medical need for drugs that can prevent encephalitis or provide safe and rapid treatment remains unmet.

[0010] The inventors previously demonstrated the role of tick salivary gland polypeptides on neutrophils as part of thrombosis treatment (International Publication No. 2019 / 149906). Here, the inventors surprisingly demonstrated that this polypeptide has anti-inflammatory effects on the brain in a cerebral hemorrhage model without the presence of thrombosis. Advantageously, these anti-inflammatory effects in the brain do not result in increased bleeding volume or edema volume. Therefore, this polypeptide enables the prevention or early treatment of encephalitis without increasing the risk of exacerbating bleeding, which is useful in improving patient clinical outcomes. [Overview of the project]

[0011] Accordingly, the present invention relates to a protein or polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 for use in preventing and / or treating encephalitis in subjects requiring it.

[0012] In some embodiments, the protein or polypeptide comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] In some embodiments, the encephalitis is acquired or spontaneous. In some embodiments, the encephalitis is provoked. In some embodiments, the encephalitis occurs or is more likely to occur after a cerebral hemorrhage. In some embodiments, the encephalitis is post-cerebral hemorrhage.

[0014] In some embodiments, encephalitis is induced by acquired brain injury. In some embodiments, encephalitis is induced by traumatic brain injury. In some embodiments, encephalitis is induced by non-traumatic brain injury.

[0015] In some embodiments, encephalitis is triggered by cerebral hemorrhage, ischemic stroke, seizure, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, or hydrocephalus.

[0016] In some embodiments, the encephalitis is acquired encephalitis. In some embodiments, the acquired encephalitis is induced by traumatic brain injury or infection. In some embodiments, the acquired encephalitis is induced by traumatic brain injury.

[0017] In some embodiments, the encephalitis is spontaneous encephalitis. In some embodiments, spontaneous encephalitis is induced by non-traumatic brain injury or autoimmune disease.

[0018] In some embodiments, the protein or polypeptide is administered to adults at a dose of approximately 200 mg to approximately 20,000 mg per day.

[0019] In some embodiments, the protein or polypeptide is administered within approximately 120 hours, preferably within 96 hours, and more preferably within 72 hours, following the onset of cerebral hemorrhage and / or brain injury. In some embodiments, the protein or polypeptide is administered within 48 hours, 24 hours, 12 hours, 10 hours, 8 hours, or 6 hours, following the onset of cerebral hemorrhage and / or brain injury. In some embodiments, the protein or polypeptide is administered within approximately 6 to 8 hours, following the onset of cerebral hemorrhage and / or brain injury.

[0020] The present invention also relates to a protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 for use in preventing and / or treating encephalitis in subjects requiring it, and which is administered within about 120 hours, preferably within 96 hours, more preferably within 72 hours, after the onset of cerebral hemorrhagic inflammation. In some embodiments, the protein or polypeptide is administered within 48 hours, 24 hours, 12 hours, 10 hours, 8 hours, or 6 hours, after the onset of encephalitis. In some embodiments, the protein or polypeptide is administered within about 6 to about 8 hours, after the onset of encephalitis.

[0021] In some embodiments, the protein or polypeptide is administered continuously, preferably by perfusion. In some embodiments, the protein or polypeptide is administered to the subject immediately after exposure to the risk of developing encephalitis. In some embodiments, the protein or polypeptide is administered continuously over a period of about 48 to 72 hours.

[0022] The present invention also relates to a pharmaceutical composition comprising a protein or polypeptide described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises another therapeutic agent.

[0023] The present invention further relates to a kit comprising a protein or polypeptide or pharmaceutical composition as described herein.

[0024] Another object of the present invention is to administer to a subject in need thereof a protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a therapeutically effective amount, or a pharmaceutical composition comprising said protein or polypeptide, for preventing and / or treating encephalitis in the subject. In some embodiments, encephalitis occurs or is likely to occur after cerebral hemorrhage. In some embodiments, the therapeutically effective amount is a dose of about 200 mg to about 20,000 mg per day for an adult. In some embodiments, the protein or polypeptide is administered to the subject within about 120 hours, preferably within 96 hours, more preferably within 72 hours after the onset of encephalitis and / or after cerebral hemorrhage. In some embodiments, the protein or polypeptide is administered to the subject within about 24 hours, preferably within 15 hours, more preferably within 12 hours after the onset of encephalitis and / or after cerebral hemorrhage.

[0025] Another object of the present invention is to prevent, reduce, decrease and / or inhibit neuronal cell death in a subject in need thereof, comprising administering to the subject a protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a therapeutically effective amount, or a pharmaceutical composition comprising said protein or polypeptide. In some embodiments, neuronal cell death occurs after cerebral hemorrhage. In some embodiments, the protein or polypeptide is administered to the subject within 120 hours, preferably within 72 hours, more preferably within 24 hours, even more preferably within 12 hours after the onset of neuronal cell death and / or cerebral hemorrhage.

[0026] Definitions In the present invention, the following terms have the following meanings.

[0027] "About" preceding a value means ± 10% of said value.

[0028] "Identity" refers to a measure of the identity of a nucleotide or amino acid sequence. Generally, these sequences are aligned to obtain the greatest possible match. "Identity" itself has a recognized meaning in the art and can be calculated using publicly available techniques. While many methods exist for measuring identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to those skilled in the art. Methods for determining identity and similarity have been coded in computer programs. Preferred computer programming methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package.

[0029] As an example, a nucleic acid having a nucleotide sequence that is at least 95% "identical" to a reference nucleotide sequence is intended to be identical to the reference nucleotide sequence, except that the nucleic acid sequence may contain an average of up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleic acid having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or at any location between those terminal positions, and may be scattered individually among the nucleotides in the reference sequence or as one or more consecutive groups within the reference sequence.

[0030] "Pharmacologically acceptable excipients" refer to excipients that do not cause adverse reactions, allergic reactions, or other adverse reactions when administered to animals, preferably humans. This includes all solvents, dispersions, coatings, antimicrobial agents, antifungal agents, isotonic agents, and absorption retarders. Pharmaceutically acceptable carriers or excipients refer to all kinds of non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or formulation aids. For administration to humans, formulations must meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA or EMA's Biologics Standards Bureau.

[0031] A polypeptide is any peptide or protein that contains two or more amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., peptide equivalents. Polypeptides generally refer to both short chains called peptides, oligopeptides, or oligomers, and longer chains generally called proteins. Polypeptides may contain amino acids other than the 20 genetically encoded amino acids.

[0032] "Protein" refers to a sequence of more than 100 amino acids and / or a polymer. The proteins of this invention are not limited to products of a specific length. The terms "polypeptide" or "protein" do not refer to, i.e., exclude, post-expression modifications of proteins, such as glycosylation, acetylation, and phosphorylation, as well as other modifications known in the art (both naturally occurring and non-naturally occurring). Such modifications are well described in basic texts and more detailed research papers and a vast amount of research literature. Modifications may occur at any site on the polypeptide or protein, including the peptide backbone, amino acid side chains, and amino or carboxyl terminus. Naturally, the same type of modification may be present in several sites on a given polypeptide or protein to the same or different degrees. Also, a given polypeptide or protein may contain many types of modifications. Polypeptides or proteins may be branched as a result of ubiquitination, or they may be cyclic with or without branching. Cyclic, branched, and branched-cyclic polypeptides or proteins may result from post-translational natural processes or be prepared by synthetic methods. Modifications include amino acid transfer RNA-mediated addition of amino acids to proteins, such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfated, and arginylation, as well as ubiquitination. The protein may be the whole protein or a partial sequence of the protein.

[0033] "Subject" refers to a mammal, preferably a human. In one embodiment, the subject is male. In another embodiment, the subject is female. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, who is awaiting or currently receiving medical care, has been, is currently, or will be the subject of medical treatment in the past or in the future, or is being monitored for the onset of a disease or condition, preferably encephalitis or a disease associated with or induced by encephalitis. In one embodiment, the subject is an adult (e.g., a subject 18 years of age or older). In another embodiment, the subject is a child (e.g., a subject under 18 years of age).

[0034] "Therapeutic effective dose" means a level or amount of a drug intended to (1) delay or prevent a disease or condition, preferably encephalitis or a disease associated with or induced by encephalitis, (2) slow or halt the progression, exacerbation or worsening of one or more symptoms of a disease or condition, preferably encephalitis or a disease associated with or induced by encephalitis, (3) bring about remission of symptoms of encephalitis or a disease or condition, (4) reduce the severity or incidence of a disease or condition associated with or induced by encephalitis, or (5) prevent a disease or condition associated with or encephalitis, without causing significant negative or adverse side effects to the target. In one embodiment, the therapeutic effective dose may be administered before the onset of encephalitis or a disease associated with or induced by encephalitis for prophylactic purposes.

[0035] The terms “to treat,” “to cure,” or “to alleviate” refer to both therapeutic and preventive measures, the purpose of which is to prevent or slow (mitigate) encephalitis or encephalitis-related or induced diseases. Those requiring treatment include individuals already suffering from encephalitis or encephalitis-related or induced diseases, as well as those susceptible to encephalitis or encephalitis-related or induced diseases, or those for whom prevention of encephalitis or encephalitis-related or induced diseases is necessary. A “treatment” for encephalitis or encephalitis-related or induced diseases is considered successful if, after administration of a therapeutic dose of the protein or polypeptide according to the method of the present invention, the patient exhibits one or more observable and / or measurable reductions or absences of the number of pathogenic cells, a reduction in the percentage of total pathogenic cells, and / or some degree of relief of one or more symptoms associated with encephalitis or encephalitis-related or induced diseases, a reduction in morbidity and mortality, and an improvement in quality of life. The above parameters for evaluating the success and improvement of treatment in encephalitis or diseases associated with or induced by encephalitis are readily measurable by standard procedures familiar to physicians. [Modes for carrying out the invention]

[0036] The present invention relates to a protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 for use in preventing and / or treating encephalitis in subjects requiring it. In a specific embodiment, the protein or polypeptide of the present invention is for use in preventing encephalitis in subjects requiring it.

[0037] In some embodiments, the protein or polypeptide for use according to the present invention comprises or consists of a tick salivary gland polypeptide called Ir-CPI or a fragment or variant thereof.

[0038] In some embodiments, the protein or polypeptide for use according to the present invention has at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0039] In some embodiments, the protein or polypeptide for use according to the present invention has at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protein or polypeptide for use according to the present invention has at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0040] As used herein, “at least 75% sequence identity” means at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity.

[0041] In some embodiments, the protein or polypeptide for use according to the present invention comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0042] In some embodiments, the protein or polypeptide for use according to the present invention comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protein or polypeptide for use according to the present invention comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0043] In some embodiments, the protein or polypeptide for use according to the present invention has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0044] In some embodiments, the protein or polypeptide for use according to the present invention has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protein or polypeptide for use according to the present invention has the amino acid sequence of SEQ ID NO: 2.

[0045] In one embodiment, fragments of proteins or polypeptides for use in accordance with the present invention are also included in the present invention. As used herein, the term “fragment” means a polypeptide having an amino acid sequence that is identical in part to, but not all, of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Fragments may be “independent” or they may be included, most preferably as a single continuous region within a larger polypeptide in which they form part or region. Typical examples of polypeptide fragments of the present invention include, for example, fragments with amino acid numbers from about 1 to 20, 21 to 40, 41 to 60, 61 to 80, 81 to 100 and 101 (the position numbers correspond to the amino acid numbers of SEQ ID NO: 1 or SEQ ID NO: 2) to the end of the polypeptide. In this context, “about” includes the particularly listed ranges which are just a few amino acids greater or less than, i.e., 5, 4, 3, 2, or 1 at one or both ends.

[0046] Preferred fragments include, but are not limited to, cleaved polypeptides having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, except for deletions of a series of consecutive residues including an amino terminus or a series of consecutive residues including a carboxyl terminus and / or a transmembrane region, or deletions of two series of consecutive residues, one of which includes an amino terminus and the other including a carboxyl terminus. Fragments characterized by structural or functional attributes such as fragments containing α-helices and α-helix-forming regions, β-sheets and β-sheet-forming regions, turns and turn-forming regions, coils and coil-forming regions, hydrophilic regions, hydrophobic regions, α-amphiphilic regions, β-amphiphilic regions, flexible regions, surface-forming regions, substrate-binding regions, and high antigen index regions are also preferred. Other preferred fragments are biologically active fragments. Biologically active fragments mediate the activity of the protein or polypeptide and include those with similar activity, enhanced activity, or reduced undesirable activity.

[0047] In one embodiment, all of these protein or polypeptide fragments retain some of the biological activity of the protein or polypeptide of the present invention.

[0048] In one embodiment, variants of proteins or polypeptides for use according to the present invention are also included in the present invention. Preferred variants are those that differ from the protein or polypeptide of the present invention by conservative amino acid substitution, i.e., residues are substituted with other residues having similar properties. Typical such substitutions are between Ala, Val, Leu and Ile, between Ser and Thr, between acidic residues Asp and Glu, between Asn and Gln, and between basic residues Lys and Arg or aromatic residues Phe and Tyr. Variants in which several amino acids, i.e., 5 to 10, 1 to 5, or 1 to 2, are substituted, deleted, or added in any combination are particularly preferred.

[0049] In one embodiment, a protein or polypeptide for use according to the present invention has an amino sequence that includes a mutation in which asparagine is replaced with glutamine at the position corresponding to position 54 in the amino acid sequence of SEQ ID NO: 1 in order to prevent N-glycosylation. In one embodiment, a protein or polypeptide of the present invention has an amino sequence that includes or consists of the sequence of SEQ ID NO: 2.

[0050] In one embodiment, proteins or polypeptides for use according to the present invention can be prepared by any preferred method. Examples of peptides prepared by preferred methods include, but are not limited to, isolated naturally occurring polypeptides, recombinant polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

[0051] In some embodiments, the protein or polypeptide for use according to the present invention is a fusion protein comprising (i) a polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) at least one different protein or polypeptide.

[0052] In one embodiment, a protein or polypeptide for use according to the present invention is conjugated to at least one other protein or polypeptide in such a manner that it produces a single protein that retains the biological activity of the protein or polypeptide for use according to the present invention.

[0053] In one embodiment, the polypeptides or proteins of the fusion protein may be condensed in any order. In one embodiment, the polypeptides or proteins are arranged in a single continuous polypeptide chain.

[0054] In one embodiment, the fusion protein of the present invention further comprises at least one peptide linker. In one embodiment, the polypeptides or proteins of the fusion protein of the present invention are linked to each other via one or more peptide linkers.

[0055] In one embodiment, the peptide linker provides greater physical separation between the two parts, thus maximizing the availability of the protein or polypeptide for use according to the present invention. The peptide linker may consist of amino acids to be flexible or more rigid.

[0056] In one embodiment, the peptide linker contains 5 to 50 amino acids, 10 to 50 amino acids, 15 to 50 amino acids, or 20 to 50 amino acids. In another embodiment, the peptide linker contains 6, 7, 8, 9, or 10 to 20 amino acids. In yet another embodiment, the peptide linker contains 2 to 10 amino acids, 5 to 20 amino acids, 10 to 30 amino acids, or 15 to 40 amino acids.

[0057] The peptide linker may be a linker that can be cleaved in vivo or a linker that cannot be cleaved in vivo.

[0058] The fusion protein of the present invention may be obtained, for example, by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, for example, one or more nucleic acids encoding the fusion protein (fragment) described herein are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures.

[0059] Fusion proteins prepared as described herein may be purified by techniques known in the art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a particular protein will be partially determined by factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the fusion protein binds can be used. The purity of the fusion protein can be determined by any of the various well-known analytical methods, including gel electrophoresis and high-pressure liquid chromatography.

[0060] The present invention also relates to the fusion proteins described herein for use in preventing and / or treating encephalitis.

[0061] In one embodiment, the nucleic acid encoding the fusion protein of the present invention may be expressed as a single nucleic acid encoding the entire fusion protein or as multiple (e.g., two or more) nucleic acids that are co-expressed. The polypeptides encoded by the co-expressed nucleic acids may be associated, for example, via disulfide bonds or other means for forming a functional fusion protein.

[0062] In some embodiments, the protein or polypeptide for use according to the present invention prevents and / or treats encephalitis. In some embodiments, the protein or polypeptide for use according to the present invention prevents encephalitis. In some embodiments, the protein or polypeptide for use according to the present invention treats encephalitis.

[0063] As used herein, the term “encephalitis,” used interchangeably with the term “neuroinflammation,” refers to the development of inflammation in the central nervous system, which may include, in particular, the activation of glial cells and the secretion of pro-inflammatory cytokines. The neuroinflammatory process affects neurons, microglia, and macroglia.

[0064] In some embodiments, encephalitis involves or consists of the release of pro-inflammatory mediators in the brain. Non-limiting examples of pro-inflammatory cytokines include interleukins (IL) such as IL-1, IL-2, IL-12, IL-17, and IL-18, as well as IFN-γ and TNF-α.

[0065] In some embodiments, encephalitis includes or consists of early activation of commensal microglia. As used herein, “microglia” refers to macrophages residing in the central nervous system.

[0066] In some embodiments, encephalitis involves or consists of the formation of reactive oxygen species (ROS). Non-limiting examples of ROS include superoxide, hydroxyl radicals, singlet oxygen, nitrous oxide, and peroxides (e.g., hydrogen peroxide). In some embodiments, the formation of ROS induces oxidative stress.

[0067] In some embodiments, encephalitis includes or consists of systemic inflammatory cell infiltration in the brain. In some embodiments, systemic inflammatory cells are selected from the group including or consisting of neutrophils, eosinophils, lymphocytes, plasma cells, and histiocytes. In some embodiments, the systemic inflammatory cells are neutrophils.

[0068] In some embodiments, encephalitis is associated with or induces excitotoxicity. As used herein, the term “excitotoxicity” refers in particular to a state of abnormal stimulation of neurotransmitter receptors in neurons that leads to changes in calcium and energy homeostasis as well as apoptosis.

[0069] In some embodiments, encephalitis is associated with or induces neuronal cell death.

[0070] In one embodiment, encephalitis is localized to a specific area of ​​the brain. Typically, encephalitis is localized to the site of brain injury, such as a cerebral hemorrhage, and / or surrounding it.

[0071] In another embodiment, the encephalitis is not localized to a specific area of ​​the brain. In a particular embodiment, the encephalitis affects the entire brain.

[0072] In some embodiments, encephalitis is not associated with the presence or formation of a thrombus. In some embodiments, encephalitis is not induced by the presence or formation of a thrombus. In some embodiments, encephalitis does not result in the presence or formation of a thrombus. In some embodiments, the proteins or polypeptides described herein are not used to prevent and / or treat thrombosis-related or thrombotic diseases.

[0073] In some embodiments, the encephalitis is induced. In some embodiments, the induced encephalitis is caused by brain injury. In some embodiments, the induced encephalitis is caused by traumatic brain injury. In some embodiments, the encephalitis is induced by cerebral hemorrhage.

[0074] In some embodiments, encephalitis is caused by brain injury. In some embodiments, the brain injury is traumatic brain injury. In some other embodiments, the brain injury is non-traumatic brain injury.

[0075] In some embodiments, encephalitis is caused by acquired brain injury. In some embodiments, the acquired brain injury is traumatic brain injury. In some other embodiments, the acquired brain injury is non-traumatic brain injury.

[0076] In some embodiments, traumatic brain injury includes or is selected from the group consisting of hemorrhage, contusion, concussion, and penetrating brain injury. In some embodiments, traumatic brain injury is hemorrhage.

[0077] In some embodiments, non-traumatic brain injury is selected from the group including or comprising cerebral hemorrhage, ischemic stroke, seizure, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is selected from the group including or comprising cerebral hemorrhage, seizure, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is selected from the group including or comprising cerebral hemorrhage, seizure, infection, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is selected from the group including or comprising cerebral hemorrhage, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is selected from the group including or comprising cerebral hemorrhage, infection, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is cerebral hemorrhage. In some embodiments, non-traumatic brain injury is an autoimmune disease.

[0078] In some embodiments, the infection is selected from the group including or consisting of viral infections, bacterial infections, fungal infections, parasitic infections, and combinations thereof. In some embodiments, the infection is induced by sepsis and / or zepsis. In some embodiments, the infection is a viral infection. In some embodiments, the infection is a bacterial infection. In some embodiments, the infection is a fungal infection. In some embodiments, the infection is a parasitic infection.

[0079] In some embodiments, brain injury is selected from the group including or comprising cerebral hemorrhage, contusion, concussion, penetrating brain injury, ischemic stroke, seizure, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, brain injury is selected from the group including or comprising cerebral hemorrhage, contusion, concussion, penetrating brain injury, seizure, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, brain injury is selected from the group including or comprising cerebral hemorrhage, contusion, concussion, penetrating brain injury, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, brain injury is selected from the group including or comprising cerebral hemorrhage, contusion, concussion, penetrating brain injury, seizure, infection, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus.

[0080] In some embodiments, encephalitis occurs or is likely to occur after traumatic brain injury. In some embodiments, encephalitis occurs or is likely to occur after cerebral hemorrhage. In some embodiments, the protein or polypeptide according to the present invention is intended for use to prevent and / or treat encephalitis after cerebral hemorrhage in subjects requiring it.

[0081] In specific embodiments, the protein or polypeptide of the present invention is intended for use in preventing encephalitis that is likely to occur after brain injury in subjects requiring it. In specific embodiments, the protein or polypeptide of the present invention is intended for use in preventing encephalitis that is likely to occur after cerebral hemorrhage in subjects requiring it.

[0082] In some embodiments, the encephalitis is either acquired encephalitis or spontaneous encephalitis.

[0083] In some embodiments, the encephalitis is acquired encephalitis.

[0084] As used herein, the term “acquired encephalitis” refers to encephalitis described herein in which the cause of the inflammation is external to the organism, such as a mechanical impact to the head, an object penetrating the skull, or an infection.

[0085] In some embodiments, acquired encephalitis is induced by traumatic brain injury or infection.

[0086] In some embodiments, acquired encephalitis is induced by traumatic brain injury.

[0087] In some embodiments, traumatic brain injury is selected from the group including or comprising hemorrhage, contusion, concussion, penetrating brain injury, and anoxic brain injury.

[0088] In some embodiments, traumatic brain injury is a hemorrhage selected from the group including intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, and epidural hemorrhage. In some embodiments, the primary brain injury is intracerebral hemorrhage. In some embodiments, the primary brain injury is subarachnoid hemorrhage. In some embodiments, the primary brain injury is subdural hemorrhage. In some embodiments, the primary brain injury is epidural hemorrhage.

[0089] In some embodiments, traumatic brain injury is a contusion. In some embodiments, traumatic brain injury is a concussion. In some embodiments, traumatic brain injury is a penetrating brain injury. In some embodiments, traumatic brain injury is anoxic brain injury.

[0090] In some embodiments, acquired encephalitis is induced by an infection. In some embodiments, acquired encephalitis is induced by an infection selected from the group including or consisting of viral infections, bacterial infections, fungal infections, parasitic infections, and combinations thereof.

[0091] In some embodiments, acquired encephalitis is induced by sepsis and / or zepsis.

[0092] In some embodiments, acquired encephalitis is induced by a viral infection. Non-limiting examples of viral infections that can lead to encephalitis include infections caused by herpes simplex virus, Epstein-Barr virus, cytomegalovirus, varicella virus, poliovirus, influenza virus, SARS-CoV-2, West Nile virus, enterovirus, chikungunya virus, and dengue virus.

[0093] In some embodiments, acquired encephalitis is induced by bacterial infections. Non-limiting examples of bacterial infections that can lead to encephalitis include infections by bacteria of the genera Treponema, Mycoplasma, Neisseria, Streptococcus, and Listeria.

[0094] In some embodiments, acquired encephalitis is induced by fungal infections. Non-limiting examples of fungal infections that can lead to encephalitis include infections by fungi of the genera Candida, Cladosporium, Cryptococcus, Histoplasma, and Aspergillus.

[0095] In some embodiments, acquired encephalitis is induced by parasitic infections. Non-limiting examples of parasitic infections that can enable encephalitis include infections by parasites of the genera Plasmodium and Toxoplasma.

[0096] In some embodiments, the encephalitis is spontaneous encephalitis.

[0097] As used herein, the term “spontaneous encephalitis” refers to encephalitis described herein that is caused by an endogenous biological condition, such as a spontaneous disease or condition.

[0098] In some embodiments, spontaneous encephalitis is induced by non-traumatic brain injury or autoimmune disease.

[0099] In some embodiments, spontaneous encephalitis is induced by non-traumatic brain injury. In certain embodiments, non-traumatic brain injury is selected from the group including or comprising spontaneous hemorrhage, brain cancer, and stroke.

[0100] In some embodiments, non-traumatic brain injury is non-traumatic idiopathic hemorrhage. In certain embodiments, non-traumatic idiopathic hemorrhage is non-traumatic spontaneous intracerebral hemorrhage or spontaneous subarachnoid hemorrhage. In certain embodiments, non-traumatic idiopathic hemorrhage is non-traumatic spontaneous intracerebral hemorrhage. In certain embodiments, non-traumatic idiopathic hemorrhage is spontaneous subarachnoid hemorrhage.

[0101] In some embodiments, non-traumatic brain injury is brain cancer, such as glioblastoma, oligodendroglioma, and meningioma.

[0102] In some embodiments, non-traumatic brain injury is a stroke. In some embodiments, non-traumatic brain injury is an ischemic stroke.

[0103] In some embodiments, spontaneous encephalitis is induced by an autoimmune disease. In some embodiments, the autoimmune disease is autoimmune encephalitis. In some embodiments, nontraumatic brain injury is induced by an autoimmune disease. In some embodiments, the autoimmune disease is autoimmune encephalitis.

[0104] Encephalitis may be associated with or triggered by other conditions or symptoms, such as neuronal cell death, excitotoxicity, microglial activation, oxidative stress, and infiltration of the brain by systemic inflammatory cells such as neutrophils.

[0105] In some embodiments, the prevention and / or treatment of encephalitis using the protein or polypeptide according to the present invention further treats and / or alleviates another disease, condition, or symptom. In some embodiments, the disease, condition, or symptom is related to encephalitis. In some embodiments, the disease, condition, or symptom is induced by encephalitis.

[0106] In some embodiments, the protein or polypeptide further prevents and / or treats diseases, conditions, or symptoms associated with encephalitis. In some embodiments, the protein or polypeptide further prevents and / or treats diseases, conditions, or symptoms induced by encephalitis.

[0107] In some embodiments, preventing and / or treating encephalitis using the proteins or polypeptides according to the present invention provides a positive effect against another disease, condition, or symptom selected from the group including neuronal cell death, excitotoxicity, microglial activation, oxidative stress, and systemic inflammatory cell infiltration of the brain. In some embodiments, the disease, condition, or symptom is selected from the group including neuronal cell death, excitotoxicity, microglial activation, and oxidative stress. As used herein, the expression “positive effect” is intended to mean that the proteins or polypeptides according to the present invention alleviate, reduce or suppress the symptoms, or partially or completely treat the components of the disease or condition.

[0108] In some embodiments, the use of the protein or polypeptide according to the present invention provides a positive effect against neuronal cell death by preventing and / or treating encephalitis. In some embodiments, the use of the protein or polypeptide according to the present invention provides a positive effect against excitotoxicity by preventing and / or treating encephalitis.

[0109] In some embodiments, the protein or polypeptide according to the present invention provides a positive effect on microglial activation by preventing and / or treating encephalitis.

[0110] In some embodiments, the proteins or polypeptides according to the present invention provide a positive effect against oxidative stress by preventing and / or treating encephalitis. In some embodiments, the proteins or polypeptides according to the present invention inhibit the formation of reactive oxygen species by preventing and / or treating encephalitis.

[0111] In some embodiments, the proteins or polypeptides according to the present invention provide a positive effect against systemic inflammatory cell infiltration of the brain by preventing and / or treating encephalitis. In certain embodiments, the systemic inflammatory cells are selected from the group including or comprising neutrophils, eosinophils, lymphocytes, plasma cells, and histiocytes. In certain embodiments, the systemic inflammatory cells are neutrophils. In some embodiments, the proteins or polypeptides according to the present invention provide a positive effect against systemic inflammatory cell infiltration of the brain by preventing and / or treating encephalitis by preventing and / or treating neutrophil recruitment to the brain and / or the formation of neutrophil extracellular traps in the brain.

[0112] In some embodiments, the protein or polypeptide for use according to the present invention is for preventing and / or treating the long-term effects of brain injury. As used herein, the expression “long-term effects of brain injury” refers to the pathophysiological consequences of brain injury, i.e., injury that affects at least one cell type in the brain, preferably neurons, over a long period, typically a week, a month, a year or more. In some embodiments, the protein or polypeptide for use according to the present invention is for preventing and / or treating the long-term effects of non-traumatic brain injury. In some embodiments, the protein or polypeptide for use according to the present invention is for preventing and / or treating the long-term effects of traumatic brain injury. In some embodiments, the protein or polypeptide for use according to the present invention is for preventing and / or treating the long-term effects of traumatic brain injury associated with bleeding. In some embodiments, the protein or polypeptide for use according to the present invention is for preventing and / or treating the long-term effects of brain injury associated with bleeding. In some embodiments, the protein or polypeptide for use according to the present invention is for preventing and / or treating the long-term effects of cerebral hemorrhage.

[0113] The inventors have demonstrated that administration of the protein or polypeptide of the present invention does not increase bleeding volume or lesion (edema) volume. This property is surprising but advantageous when one is aware of the antithrombotic effect of this protein.

[0114] In certain embodiments, the protein or polypeptide of the present invention is intended for use in preventing and / or treating encephalitis without increasing the amount of bleeding and / or the volume of lesions (edema). In certain embodiments, the encephalitis is caused by or induced by cerebral hemorrhage. In one embodiment, the protein or polypeptide of the present invention is intended for use in preventing and / or treating brain injury after cerebral hemorrhage. In one embodiment, the brain injury occurs after a hemorrhagic stroke. In one embodiment, the protein or polypeptide of the present invention is intended for use in preventing and / or treating the long-term effects of brain injury after cerebral hemorrhage.

[0115] The present invention further relates to nucleic acid molecules encoding proteins or polypeptides for use in accordance with the present invention.

[0116] In some embodiments, nucleic acids are used to prevent and / or treat encephalitis or encephalitis-related diseases.

[0117] In some embodiments, the nucleic acid molecule is either a DNA molecule or an RNA molecule.

[0118] In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is selected from the group including plasmids, naked DNA, cosmids, fosmids, prokaryotic chromosomes (e.g., bacterial artificial chromosomes), eukaryotic chromosomes (e.g., yeast artificial chromosomes or human artificial chromosomes), or combinations thereof.

[0119] In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is messenger RNA (mRNA).

[0120] In some embodiments, the nucleic acid molecule includes or consists of natural nucleotides, non-natural nucleotides, or combinations thereof. In some embodiments, the nucleic acid molecule includes or consists of natural nucleotides. In some embodiments, the natural nucleotides are selected from the group including or consisting of adenine, guanine, cytosine, thymine, and uracil. In some embodiments, the natural nucleotides are selected from the group including or consisting of adenine, guanine, cytosine, and thymine. In some embodiments, the natural nucleotides are selected from the group including or consisting of adenine, guanine, cytosine, and uracil. In some embodiments, the nucleic acid molecule includes or consists of non-natural nucleotides (e.g., chemically modified ones).

[0121] In some embodiments, nucleic acid molecules are single-stranded, double-stranded, or a combination thereof. In some embodiments, nucleic acid molecules are single-stranded. In some embodiments, nucleic acid molecules are double-stranded.

[0122] In some embodiments, the nucleic acid of the present invention has a nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 3 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3. In one embodiment, a nucleic acid encoding a protein or polypeptide for use according to the present invention has the nucleotide sequence described in SEQ ID NO: 3.

[0123] In one embodiment, the nucleic acid encoding the fusion protein of the present invention may be expressed as a single nucleic acid encoding the entire fusion protein or as multiple (i.e., two or more) co-expressed nucleic acids. The polypeptide encoded by the co-expressed nucleic acids may be associated, for example, via disulfide bonds or other means for forming a functional fusion protein.

[0124] Another object of the present invention is a vector comprising one or more nucleic acids encoding the protein or polypeptide of the present invention, and its use for the prevention and / or treatment of encephalitis and / or encephalitis-related diseases. In preferred embodiments, the vector of the present invention is an expression vector.

[0125] The present invention further relates to compositions comprising proteins or polypeptides, one or more nucleic acids or vectors as described herein.

[0126] The present invention further relates to a pharmaceutical composition comprising a protein or polypeptide for use according to the present invention, a nucleic acid for use according to the present invention, a vector for use according to the present invention, or a composition for use according to the present invention, and at least one pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition according to the present invention is for the prevention and / or treatment of encephalitis in a subject requiring it, as described herein.

[0127] In some embodiments, the pharmaceutical composition comprises a protein or polypeptide for use in accordance with the present invention and at least one pharmaceutically acceptable excipient.

[0128] In some embodiments, the pharmaceutical composition comprises a protein or polypeptide having at least 75% sequence identity with the amino acid sequence of Sequence ID No. 1 for use in preventing and / or treating encephalitis, and at least one pharmaceutically acceptable excipient.

[0129] Pharmaceutically acceptable excipients that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0130] In some embodiments, the pharmaceutical composition further includes a different therapeutic agent.

[0131] In some embodiments, further therapeutic agents have a positive effect on diseases, conditions, or symptoms selected from the group including or comprising encephalitis, neuronal cell death, excitotoxicity, microglial activation, and oxidative stress. In some embodiments, further therapeutic agents have a positive effect on encephalitis.

[0132] In some embodiments, the further therapeutic agent is a drug. In some embodiments, the further therapeutic agent is an anti-inflammatory drug such as minocycline or a statin.

[0133] In some embodiments, further therapeutic agents include or consist of cell therapies such as stem cell therapy.

[0134] The present invention further relates to proteins or polypeptides, one or more nucleic acids, vectors, compositions or pharmaceutical compositions described herein for the manufacture of drugs for preventing and / or treating encephalitis.

[0135] Another object of the present invention is a drug comprising a protein or polypeptide, one or more nucleic acids, vectors, compositions or pharmaceutical compositions described herein for the prevention and / or treatment of encephalitis.

[0136] In one embodiment, the pharmaceutical composition or drug of the present invention comprises a therapeutically effective amount of a protein or polypeptide, one or more nucleic acids or vectors described herein.

[0137] Naturally, the total daily dose of the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is determined by the attending physician within the bounds of appropriate medical judgment. A specific therapeutically effective dose level for any particular patient depends on a variety of factors, including the disease and severity of encephalitis being treated, the activity of the specific drug used, the specific composition used, the patient's age, weight, health status, sex and diet, the timing of administration, route of administration and excretion rate of the specific drug used, the duration of treatment, drugs used in combination with or concurrently with the specific drug used, and similar factors well known in the medical field. For example, it is well within the skill of a person skilled in the art to start administration of a drug at a level lower than the dose required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of this product may vary over a wide range, from about 10 to about 10,000 mg per day for adults, preferably 100 to about 5,000 mg, and more preferably about 200 to about 2,000 mg per day for adults. Preferably, the composition contains 10, 50, 100, 250, 500, 1000, and 2000 mg of the active ingredient for dosage adjustment according to the symptoms of the patient being treated. The drug typically contains about 10 to about 10000 mg of the active ingredient, preferably 5 to about 5000, and more preferably about 10 to about 2000 mg of the active ingredient. An effective amount of the drug is usually provided at a dosage level of 0.01 mg / kg to about 100 mg / kg of body weight per day, preferably about 0.05 mg / kg to 40 mg / kg of body weight per day, more preferably about 0.1 mg / kg to 20 mg / kg of body weight per day, and more preferably about 0.2 mg / kg to 1 mg / kg of body weight per day.

[0138] In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered in a dose of about 2.5 mg / kg / day to about 250 mg / kg / day, preferably about 2.5 mg / kg / day to about 100 mg / kg / day, more preferably about 2.5 mg / kg / day to about 50 mg / kg / day, and more preferably about 2.5 mg / kg / day to about 25 mg / kg / day. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered in a dose of about 5 mg / kg / day to about 50 mg / kg / day. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered in a dose of about 10 mg / kg / day to about 50 mg / kg / day. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered in a dose of about 25 mg / kg / day.

[0139] In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered to adults at a dose of about 20 mg to about 200,000 mg per day, preferably about 20 mg to about 20,000 mg per day, and more preferably about 20 mg to about 10,000 mg per day. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered to adults at a dose of about 200 mg to about 200,000 mg per day, preferably about 200 mg to about 20,000 mg per day, and more preferably about 200 mg to about 10,000 mg per day. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered to adults at a dose of about 2,000 mg per day.

[0140] The inventors have demonstrated the possibility and safety of administering the protein of the present invention immediately after the onset of encephalitis, brain injury, or cerebral hemorrhage. This is particularly advantageous for patients with encephalitis, brain injury, or cerebral hemorrhage who are prone to thrombotic events, such as bedridden patients. In fact, anticoagulants (such as enoxaparin) cannot be administered during the first 72 hours after the onset of bleeding because they increase the risk of bleeding. The inventors have demonstrated that, unlike enoxaparin, the protein of the present invention does not increase the risk of bleeding during the first three days after the onset of bleeding (see Example 1). Therefore, it is possible to treat encephalitis and / or brain injury with the protein of the present invention within a critical timeframe to avoid irreversible effects. For best results, a therapeutic solution to encephalitis must be given before the inflammatory response begins after the onset of bleeding. Current solutions cannot be administered immediately due to the increased risk of bleeding. In contrast, the protein or polypeptide of the present invention does not have such drawbacks and can therefore be administered immediately to reduce the inflammatory response before it causes highly harmful and / or permanent effects.

[0141] Furthermore, the peak of neutrophil infiltration, as well as the rapid growth phase of edema (if present), occurs within this 72-hour period. The faster the edema size decreases, the better the patient's outcome.

[0142] Accordingly, in some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered as soon as possible after the onset of encephalitis and / or brain injury. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered to the subject immediately after exposure to the risk of developing encephalitis. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within 120 hours, preferably within 96 hours, more preferably within 72 hours, after the onset of encephalitis and / or brain injury. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within 48 hours, preferably within 24 hours, more preferably within 12 hours, after the onset of encephalitis and / or brain injury. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within the first 10 hours, preferably within the first 9 hours, more preferably within the first 8 hours, even more preferably within the first 7 hours, and even more preferably within the first 6 hours, after the onset of encephalitis and / or brain injury. In other words, in one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered about 0 to about 120 hours after the onset of encephalitis and / or brain injury, preferably within 96 hours, more preferably within 72 hours, more preferably within 48 hours, more preferably within 24 hours, more preferably within 12 hours, more preferably within 10 hours, more preferably within 9 hours, more preferably within 8 hours, more preferably within 7 hours, and more preferably within 6 hours.

[0143] Accordingly, in some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered as soon as possible after the onset of cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered to the subject immediately after cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within 120 hours, preferably within 96 hours, more preferably within 72 hours, after the onset of cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within 48 hours, preferably within 24 hours, more preferably within 12 hours, after the onset of cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within the first 10 hours, preferably within the first 9 hours, more preferably within the first 8 hours, even more preferably within the first 7 hours, and even more preferably within the first 6 hours, after the onset of cerebral hemorrhage. In other words, in one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 0 to 120 hours after the onset of cerebral hemorrhage, preferably within 96 hours, more preferably within 72 hours, more preferably within 48 hours, more preferably within 24 hours, more preferably within 12 hours, more preferably within 10 hours, more preferably within 9 hours, more preferably within 8 hours, more preferably within 7 hours, and more preferably within 6 hours.

[0144] Naturally, the earlier the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or drug according to the present invention is administered, the better the clinical outcome will be.

[0145] Accordingly, in some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, or 48 hours to approximately 120, 96, or 72 hours after the onset of encephalitis and / or brain injury.

[0146] Accordingly, in some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, or 48 hours to approximately 120, 96, or 72 hours after the onset of cerebral hemorrhage.

[0147] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered about 24 hours to about 120 hours after the onset of encephalitis and / or brain injury, preferably about 24 hours to about 96 hours, more preferably about 24 hours to about 72 hours. In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered about 48 hours to about 120 hours after the onset of encephalitis and / or brain injury, preferably about 48 hours to about 96 hours, more preferably about 48 hours to about 72 hours.

[0148] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 24 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 24 to 96 hours, and more preferably within approximately 24 to 72 hours. In another embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 48 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 48 to 96 hours, and more preferably within approximately 48 to 72 hours.

[0149] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered about 0 to about 72 hours after the onset of encephalitis and / or brain injury, preferably about 2 to about 48 hours, preferably about 4 to about 36 hours, more preferably about 6 to about 12 hours. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered about 4 to about 48 hours after the onset of encephalitis and / or brain injury, preferably about 4 to about 36 hours, more preferably about 4 to about 12 hours, and even more preferably about 4 to about 8 hours. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered about 6 to about 48 hours after the onset of encephalitis and / or brain injury, preferably about 6 to about 36 hours, more preferably about 6 to about 12 hours, and even more preferably about 6 to about 8 hours.

[0150] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 0 to approximately 72 hours after the onset of cerebral hemorrhage, preferably within approximately 2 to approximately 48 hours, preferably within approximately 4 to approximately 36 hours, and more preferably within approximately 6 to approximately 12 hours. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 4 to approximately 48 hours after the onset of cerebral hemorrhage, preferably within approximately 4 to approximately 36 hours, more preferably within approximately 4 to approximately 12 hours, and even more preferably within approximately 4 to approximately 8 hours. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 6 to approximately 48 hours after the onset of cerebral hemorrhage, preferably within approximately 6 to approximately 36 hours, more preferably within approximately 6 to approximately 12 hours, and even more preferably within approximately 6 to approximately 8 hours.

[0151] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered about 0 to 12 hours after the onset of encephalitis and / or brain injury, preferably about 0 to 10 hours, more preferably about 0 to 8 hours, even more preferably about 0 to 7 hours, and even more preferably about 0 to 6 hours.

[0152] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within approximately 0 to 12 hours after the onset of cerebral hemorrhage, preferably within approximately 0 to 10 hours, more preferably within approximately 0 to 8 hours, even more preferably within approximately 0 to 7 hours, and even more preferably within approximately 0 to 6 hours.

[0153] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered up to 120 hours, preferably up to 96 hours, more preferably up to 72 hours, after the onset of encephalitis and / or brain injury. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered up to 48 hours, preferably up to 24 hours, more preferably up to 12 hours, even more preferably up to 8 hours, even more preferably up to 7 hours, and even more preferably up to 6 hours, after the onset of encephalitis and / or brain injury.

[0154] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered up to 120 hours, preferably up to 96 hours, and more preferably up to 72 hours, after the onset of cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered up to 48 hours, preferably up to 24 hours, more preferably up to 12 hours, even more preferably up to 8 hours, even more preferably up to 7 hours, and even more preferably up to 6 hours, after the onset of cerebral hemorrhage.

[0155] The proteins or polypeptides, nucleic acids, vectors, compositions, pharmaceutical compositions, or drugs according to the present invention are formulated for administration to a subject. The proteins or polypeptides, nucleic acids, vectors, compositions, pharmaceutical compositions, or drugs according to the present invention may be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, orally, vaginally, or via implanted reservoir. As used herein, the term administration includes subcutaneous, intravenous, intramuscular, intraocular, intra-articular, intra-sacral, intrasternal, subarachnoid, intrahepatic, intrafocal, and intracranial injection or infusion techniques.

[0156] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or drug according to the present invention is in a form suitable for topical administration.

[0157] Examples of forms suitable for topical administration include, but are not limited to, liquids, pastes, or solid compositions, more specifically aqueous solutions, drops, eye drops, ophthalmic solutions, dispersions, sprays, microcapsules, micro or nanoparticles, polymer patches, or controlled-release patches.

[0158] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention comprises one or more pharmaceutically acceptable carriers for a formulation suitable for administration.

[0159] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is in a form suitable for injection, such as intraocular, intramuscular, subcutaneous, intradermal, transdermal or intravenous injection or infusion.

[0160] Examples of forms suitable for injection include, but are not limited to, solutions such as sterile aqueous solutions, dispersions, emulsions, and suspensions, and solid forms suitable for use in preparing solutions or suspensions by adding liquid before use, such as powders and liposomes.

[0161] The sterile injectable formulations of proteins or polypeptides, nucleic acids, vectors, compositions, pharmaceutical compositions, or drugs according to the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. The sterile injectable formulations may also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents. Acceptable media and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solutions. Sterile non-volatile oils are also conventionally used as solvents or suspension media. For this purpose, any sterile non-volatile oil, including synthetic monoglycerides or diglycerides, can be used. Fatty acids such as oleic acid and their glyceride derivatives are useful in the preparation of injectable formulations, as are naturally pharmaceutically acceptable oils such as olive oil or castor oil, particularly their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in formulations of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tween, Span, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms, may also be used for formulation purposes.

[0162] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered to a subject requiring it at least once a day. For example, the composition, pharmaceutical composition or drug of the present invention may be administered once a day, twice a day or three times a day. In a preferred embodiment, the composition, pharmaceutical composition or drug of the present invention is administered to a subject requiring it once a day.

[0163] In another embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered to a subject requiring it at least once a week. For example, the composition, pharmaceutical composition or drug of the present invention may be administered once a week, twice a week, three times a week, four times a week or up to seven times a week.

[0164] In another embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered to a subject in need therein once a month, twice a month, once every two months, once every two or three months, twice a year or once a year.

[0165] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered to a subject who needs it before being exposed to the risk of developing encephalitis. In one embodiment, the term “~ before” means at least one week before exposure. In another embodiment, the term “~ before” means 5 days, 4 days, 3 days, 2 days or 1 day before exposure. In another embodiment, the term “~ before” means 24 hours, 18 hours, 15 hours, 12 hours, 6 hours, 4 hours, 2 hours or 1 hour before exposure. In yet another embodiment, the term “~ before” means less than one hour before exposure or at the moment of exposure, such as 45 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes before exposure.

[0166] In another embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered to a subject who requires it after exposure to a risk of developing encephalitis. In one embodiment, the term “~after” means 5, 10, 15, 30, or 45 minutes after exposure. In another embodiment, the term “~after” means 1, 2, 4, 6, 12, 15, 18, or 14 hours after exposure. In another embodiment, the term “~after” means 1, 2, 3, 4, or 5 days after exposure. In another embodiment, the term “~after” means one week or more after exposure.

[0167] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered sequentially, typically by perfusion.

[0168] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered continuously, preferably by perfusion, for about 12 to about 96 hours, preferably about 18 to about 84 hours, and more preferably about 12 to about 72 hours. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered continuously, preferably by perfusion, for about 24 to about 96 hours, and more preferably about 48 to about 96 hours. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered continuously, preferably by perfusion, for about 24 to about 72 hours, and more preferably about 48 to about 72 hours.

[0169] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered continuously, preferably by perfusion, for at least about 12, 24, 36, 48, 72, or 96 hours. In certain embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered continuously, preferably by perfusion, for at least 48 hours.

[0170] In some embodiments, an initial dose of the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or drug according to the present invention is administered to a subject and then perfused for continuous administration. Within the scope of the present invention, the initial dose is referred to as the initial dose. In some embodiments, the dose of the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or drug according to the present invention in the initial dose is higher than or equal to the dose administered during perfusion, and preferably higher.

[0171] The present invention also relates to a kit comprising a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention for the prevention and / or treatment of encephalitis.

[0172] In one embodiment, the kit of the present invention further includes means for administering a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention to a subject requiring it.

[0173] In one embodiment, the kit of the present invention further includes instructions for administering a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention to the subject.

[0174] The present invention further relates to a method for preventing and / or treating encephalitis in a subject requiring it, comprising administering to a subject a therapeutically effective amount of a protein or polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a nucleic acid encoding such a protein or polypeptide, a vector containing such a nucleic acid, or a pharmaceutical composition comprising such a protein or polypeptide, nucleic acid or vector or drug according to the present invention.

[0175] In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a target. In one embodiment, the therapeutically effective dose is approximately 2.5 mg / kg / day to approximately 250 mg / kg / day. In one embodiment, the therapeutically effective dose is approximately 2.5 mg / kg / day to approximately 100 mg / kg / day. In one embodiment, the therapeutically effective dose is approximately 2.5 mg / kg / day to approximately 50 mg / kg / day. In one embodiment, the therapeutically effective dose is approximately 2.5 mg / kg / day to approximately 25 mg / kg / day. In one embodiment, the therapeutically effective dose is approximately 5 mg / kg / day to approximately 50 mg / kg / day.

[0176] In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 120 hours, preferably within 96 hours, more preferably within 72 hours, after the onset of encephalitis and / or brain injury. In other words, in one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject from about 0 hours to about 120 hours, preferably within 96 hours, more preferably within 72 hours, after the onset of encephalitis and / or brain injury.

[0177] In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 48 hours, preferably within 24 hours, more preferably within 15 hours, and even more preferably within 12 hours, after the onset of encephalitis and / or brain injury. In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 10 hours, within 8 hours, or within 6 hours, after the onset of encephalitis and / or brain injury. In some embodiments, the protein or polypeptide is administered approximately 6 to 8 hours after the onset of encephalitis and / or brain injury.

[0178] In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 120 hours, preferably within 96 hours, and more preferably within 72 hours, after the onset of cerebral hemorrhage. In other words, in one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to approximately 120 hours, preferably within 96 hours, and more preferably within 72 hours, after the onset of cerebral hemorrhage.

[0179] In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 48 hours, preferably within 24 hours, more preferably within 15 hours, and even more preferably within 12 hours, after the onset of cerebral hemorrhage. In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 10 hours, within 8 hours, or within 6 hours, after the onset of cerebral hemorrhage. In some embodiments, the protein or polypeptide is administered approximately 6 to 8 hours after the onset of cerebral hemorrhage.

[0180] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 0 to 120 hours after the onset of encephalitis and / or brain injury, preferably about 0 to 96 hours, more preferably about 0 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 1 to 120 hours after the onset of encephalitis and / or brain injury, preferably about 1 to 96 hours, more preferably about 1 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 2 to 120 hours after the onset of encephalitis and / or brain injury, preferably about 2 to 96 hours, more preferably about 2 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 3 hours to about 120 hours after the onset of encephalitis and / or brain injury, preferably about 3 hours to about 96 hours, more preferably about 3 hours to about 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 4 hours to about 120 hours after the onset of encephalitis and / or brain injury, preferably about 4 hours to about 96 hours, more preferably about 4 hours to about 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 5 hours to about 120 hours after the onset of encephalitis and / or brain injury, preferably about 5 hours to about 96 hours, more preferably about 5 hours to about 72 hours. In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 6 to 120 hours, preferably about 6 to 96 hours, and more preferably about 6 to 72 hours, after the onset of encephalitis and / or brain injury.In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 7 to 120 hours, preferably about 7 to 96 hours, more preferably about 7 to 72 hours, after the onset of encephalitis and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 8 to 120 hours, preferably about 8 to 96 hours, more preferably about 8 to 72 hours, after the onset of encephalitis and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 9 to 120 hours, preferably about 9 to 96 hours, more preferably about 9 to 72 hours, after the onset of encephalitis and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 10 to 120 hours, preferably about 10 to 96 hours, and more preferably about 10 to 72 hours, after the onset of encephalitis and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 12 to 120 hours, preferably about 12 to 96 hours, and more preferably about 12 to 72 hours, after the onset of encephalitis and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 24 to 120 hours, preferably about 24 to 96 hours, and more preferably about 24 to 72 hours, after the onset of encephalitis and / or brain injury. In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 48 to 120 hours, preferably about 48 to 96 hours, and more preferably about 48 to 72 hours, after the onset of encephalitis and / or brain injury.

[0181] In one embodiment, the method comprises administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 0 to 120 hours, preferably about 0 to 36 hours, more preferably about 0 to 12 hours, after the onset of encephalitis and / or brain injury. In one embodiment, the method comprises administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 0 to 36 hours, preferably about 0 to 24 hours, more preferably about 0 to 12 hours, after the onset of encephalitis and / or brain injury.

[0182] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 0 to 24 hours, preferably about 0 to 20 hours, more preferably about 0 to 16 hours, after the onset of encephalitis and / or brain injury. In another embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 0 to 15 hours, preferably about 0 to 14 hours, more preferably about 0 to 13 hours, after the onset of encephalitis and / or brain injury.

[0183] In certain embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject approximately 0 to 12 hours after the onset of encephalitis and / or brain injury.

[0184] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 0 to 24 hours, preferably about 4 to 20 hours, more preferably about 8 to 16 hours, after the onset of encephalitis and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 10 to 14 hours, preferably about 10 to 13 hours, more preferably about 11 to 13 hours, after the onset of encephalitis and / or brain injury. In a specific embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 11 to 12 hours, after the onset of encephalitis and / or brain injury.

[0185] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 2 to 24 hours, preferably about 4 to 24 hours, and more preferably about 8 to 24 hours, after the onset of encephalitis and / or brain injury. In a specific embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 10 to 24 hours, after the onset of encephalitis and / or brain injury.

[0186] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject about 10 to 16 hours, preferably about 11 to 14 hours, more preferably about 11 to 13 hours, after the onset of encephalitis and / or brain injury.

[0187] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject approximately 12 hours after the onset of encephalitis and / or brain injury.

[0188] In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject up to 24 hours, preferably up to 15 hours, more preferably up to 12 hours, after the onset of encephalitis and / or brain injury.

[0189] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 0 to 96 hours, and more preferably within approximately 0 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 1 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 1 to 96 hours, and more preferably within approximately 1 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 2 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 2 to 96 hours, and more preferably within approximately 2 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 3 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 3 to 96 hours, and more preferably within approximately 3 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 4 hours to approximately 120 hours after the onset of cerebral hemorrhage, preferably within approximately 4 hours to approximately 96 hours, and more preferably within approximately 4 hours to approximately 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 5 hours to approximately 120 hours after the onset of cerebral hemorrhage, preferably within approximately 5 hours to approximately 96 hours, and more preferably within approximately 5 hours to approximately 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 6 hours to approximately 120 hours after the onset of cerebral hemorrhage, preferably within approximately 6 hours to approximately 96 hours, and more preferably within approximately 6 hours to approximately 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 7 hours to approximately 120 hours after the onset of cerebral hemorrhage, preferably within approximately 7 hours to approximately 96 hours, and more preferably within approximately 7 hours to approximately 72 hours.In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 8 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 8 to 96 hours, and more preferably within approximately 8 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 9 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 9 to 96 hours, and more preferably within approximately 9 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 10 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 10 to 96 hours, and more preferably within approximately 10 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 12 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 12 to 96 hours, and more preferably within approximately 12 to 72 hours. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 24 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 24 to 96 hours, and more preferably within approximately 24 to 72 hours. In another embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 48 to 120 hours after the onset of cerebral hemorrhage, preferably within approximately 48 to 96 hours, and more preferably within approximately 48 to 72 hours.

[0190] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 120 hours, preferably within approximately 0 to 36 hours, and more preferably within approximately 0 to 12 hours, after the onset of cerebral hemorrhage. In another embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 36 hours, preferably within approximately 0 to 24 hours, and more preferably within approximately 0 to 12 hours, after the onset of cerebral hemorrhage.

[0191] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 24 hours, preferably within approximately 0 to 20 hours, and more preferably within approximately 0 to 16 hours, after the onset of cerebral hemorrhage. In another embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 15 hours, preferably within approximately 0 to 14 hours, and more preferably within approximately 0 to 13 hours, after the onset of cerebral hemorrhage.

[0192] In certain embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 12 hours after the onset of cerebral hemorrhage.

[0193] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 24 hours, preferably within approximately 4 to 20 hours, and more preferably within approximately 8 to 16 hours, after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 10 to 14 hours, preferably within approximately 10 to 13 hours, and more preferably within approximately 11 to 13 hours, after the onset of cerebral hemorrhage. In a specific embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 11 to 12 hours, after the onset of cerebral hemorrhage.

[0194] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 2 to 24 hours, preferably within approximately 4 to 24 hours, and more preferably within approximately 8 to 24 hours, after the onset of cerebral hemorrhage. In a specific embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 10 to 24 hours, after the onset of cerebral hemorrhage.

[0195] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within about 10 to about 16 hours, preferably within about 11 to about 14 hours, and more preferably within about 11 to about 13 hours, after the onset of cerebral hemorrhage.

[0196] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 12 hours after the onset of cerebral hemorrhage.

[0197] In some embodiments, the method comprises administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject preferably 12 hours, preferably up to 15 hours, and more preferably up to 12 hours after the onset of cerebral hemorrhage.

[0198] In some embodiments, this method is not intended to prevent and / or treat thrombosis. In some embodiments, encephalitis is not associated with the presence or formation of thrombosis.

[0199] In some embodiments, this method The process involves administering an initial dose comprising a therapeutically effective amount of the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or drug according to the present invention to the target, The process involves perfusion containing a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention. Includes, Here, the initial dose is either higher than or equal to the perfusion dose.

[0200] The present invention further relates to a method for preventing and / or treating encephalitis without increasing the amount of bleeding and / or the volume of lesions (edema). The present invention further relates to a method for preventing and / or treating brain injury after cerebral hemorrhage, preferably after hemorrhagic stroke.

[0201] The present invention further relates to a method for preventing and / or treating encephalitis-related diseases, conditions, or symptoms, comprising administering a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or drug according to the present invention to a subject. In some embodiments, the method is for preventing and / or treating encephalitis-induced diseases, conditions, or symptoms, comprising administering a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or drug according to the present invention to a subject. In some embodiments, the disease, condition, or symptom includes or is selected from the group consisting of neuronal cell death, excitotoxicity, microglial activation, and oxidative stress.

[0202] In some embodiments, the method of the present invention is used to alleviate symptoms in subjects suffering from traumatic or non-traumatic brain injury.

[0203] The present invention further relates to a method for reducing inflammation in a subject, comprising administering a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention to the subject.

[0204] Another object of the present invention is a method for preventing, reducing, decreasing and / or inhibiting neuronal cell death in a subject requiring it, comprising administering a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention to the subject.

[0205] In one embodiment, the subjects requiring it are those who have or are prone to encephalitis. In one embodiment, the method is for preventing, reducing, decreasing and / or inhibiting neuronal cell death that occurs or is prone to occurring after encephalitis in subjects requiring it.

[0206] In one embodiment, the subjects requiring this method are those who have or are prone to cerebral hemorrhage. In one embodiment, the method is for preventing, reducing, decreasing and / or inhibiting neuronal cell death that occurs or is prone to occurring after cerebral hemorrhage in subjects requiring this method.

[0207] In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 120 hours, preferably within 96 hours, more preferably within 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 48 hours, preferably within 24 hours, more preferably within 15 hours, and even more preferably within 12 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In some embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within 10 hours, within 8 hours, or within 6 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In some embodiments, the protein or polypeptide is administered approximately 6 to 8 hours after the onset of neuronal cell death and / or cerebral hemorrhage.

[0208] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 120 hours, preferably within approximately 0 to 96 hours, and more preferably within approximately 0 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 1 to 120 hours, preferably within approximately 1 to 96 hours, and more preferably within approximately 1 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 2 to 120 hours, preferably within approximately 2 to 96 hours, and more preferably within approximately 2 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 3 to 120 hours, preferably approximately 3 to 96 hours, and more preferably approximately 3 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 4 to 120 hours, preferably approximately 4 to 96 hours, and more preferably approximately 4 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 5 to 120 hours, preferably approximately 5 to 96 hours, and more preferably approximately 5 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 6 to 120 hours, preferably within approximately 6 to 96 hours, and more preferably within approximately 6 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage.In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 7 to 120 hours, preferably approximately 7 to 96 hours, and more preferably approximately 7 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 8 to 120 hours, preferably approximately 8 to 96 hours, and more preferably approximately 8 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 9 to 120 hours, preferably approximately 9 to 96 hours, and more preferably approximately 9 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method comprises administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 10 to 120 hours, preferably within approximately 10 to 96 hours, and more preferably within approximately 10 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In another embodiment, the method comprises administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 12 to 120 hours, preferably within approximately 12 to 96 hours, and more preferably within approximately 12 to 72 hours, after the onset of neuronal cell death and / or cerebral hemorrhage.

[0209] In certain embodiments, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 12 hours after the onset of neuronal cell death and / or cerebral hemorrhage.

[0210] In one embodiment, the method comprises administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 0 to 24 hours, preferably within approximately 4 to 20 hours, more preferably within approximately 8 to 16 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In one embodiment, the method comprises administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 10 to 14 hours, preferably within approximately 10 to 13 hours, more preferably within approximately 11 to 13 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In a specific embodiment, the method comprises administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 11 to 12 hours, after neuronal cell death and / or cerebral hemorrhage.

[0211] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 2 to 24 hours, preferably within approximately 4 to 24 hours, and more preferably within approximately 8 to 24 hours, after the onset of neuronal cell death and / or cerebral hemorrhage. In a specific embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 10 to 24 hours, after the onset of neuronal cell death and / or cerebral hemorrhage.

[0212] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within about 10 to about 16 hours, preferably within about 11 to about 14 hours, and more preferably within about 11 to about 13 hours, after the onset of neuronal cell death and / or cerebral hemorrhage.

[0213] In one embodiment, the method involves administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention to a subject within approximately 15 hours, preferably within 12 hours, of the onset of neuronal cell death and / or cerebral hemorrhage.

[0214] A further object of the present invention is a method for preventing and / or treating a disease associated with, accompanying or resulting from neuronal cell death in a subject requiring such treatment, comprising administering a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention to the subject.

[0215] The present invention further relates to proteins or polypeptides, nucleic acids, vectors, compositions, pharmaceutical compositions or drugs according to the present invention for use in preventing and / or treating diseases associated with, accompanying or resulting from neuronal cell death in subjects requiring such treatment.

[0216] In one embodiment, diseases associated with, accompanying, or causing neuronal cell death include, or are selected from, Alzheimer's disease, Parkinson's disease, Huntington's disease, ischemic stroke, hemorrhagic stroke, prion disease (Creutzfeldt-Jakob disease), amyotrophic lateral sclerosis, motor neuron disease, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, epilepsy, brain tumor, stroke of unknown cause, brainstem stroke, transient ischemic attack, Batten disease, Friedreich's ataxia, Alexander disease, chronic traumatic encephalopathy, cerebellar stroke, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis.

[0217] In one embodiment, diseases associated with, accompanying, or causing neuronal cell death include, or are selected from, ischemic stroke, hemorrhagic stroke, prion disease (Creutzfeldt-Jakob disease), amyotrophic lateral sclerosis, motor neuron disease, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, stroke of unknown cause, brainstem stroke, transient ischemic attack, Batten disease, Friedreich's ataxia, Alexander disease, chronic traumatic encephalopathy, cerebellar stroke, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis.

[0218] In one embodiment, diseases associated with, accompanying, or causing neuronal cell death include, or are selected from, prion diseases (Creutzfeldt-Jakob disease), amyotrophic lateral sclerosis, motor neuron diseases, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, Batten disease, Friedreich's ataxia, Alexander disease, chronic traumatic encephalopathy, cerebellar stroke, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis.

[0219] In one embodiment, diseases associated with, accompanying, or causing neuronal cell death include, or are selected from, prion diseases (Creutzfeldt-Jakob disease), amyotrophic lateral sclerosis, motor neuron diseases, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, Batten disease, Friedreich's ataxia, Alexander disease, chronic traumatic encephalopathy, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis. [Brief explanation of the drawing]

[0220] [Figure 1] Figures 1A–1D are sets of box plots showing lesion volume and hemorrhage measured on days 1 and 3 after induction of intracerebral hemorrhage by injection of collagenase into the right striatum of mice. Mice were treated with either Ir-CPI (injection + perfusion), enoxaparin (enoxaparin injection + NaCl perfusion), or PBS alone (injection + perfusion). Figures 1A and 1B show lesion volume on days 1 and 3, respectively. Figures 1C and 1D show hemorrhage on days 1 and 3, respectively. Boxes show median and quartiles. Whiskers show minimum and maximum values. Individual values ​​are plotted. Thick lines show the mean. Day 1 had N=8 mice per group. Day 3 had n=7 in the PBS group and n=6 in the Ir-CPI and enoxaparin groups. *p<0.05 (Kruskal-Wallis test including Dunn's multiple comparison test). [Figure 2]Figures 2A-2C are a set of box plots showing the quantification of mean labeled cells in the hemorrhagic area (anterior / injection site / posterior location) 3 days after induction of intracerebral hemorrhage. Mice were treated with either Ir-CPI (injection + perfusion), enoxaparin (enoxaparin injection + NaCl perfusion), or PBS alone (injection + perfusion). Figure 2A shows the number of cells positive for MPO immunostaining. Figure 2B shows the number of cells (neutrophils) positive for Ly6G immunostaining. Figure 2C shows the number of cells (neutrophils releasing NETs) positive for Ly6G, H3Cit, and MPO co-immunostaining. The boxes show the median and quartiles. The whiskers show the minimum and maximum values. Individual values ​​are plotted. The thick line shows the mean. N=5 mice per group. *p<0.05 (unpaired t-test). [Figure 3] This box plot shows the quantification of NET-releasing neutrophils (number of cells positive for Ly6G, H3Cit, and MPO co-immunostaining) at the collagenase injection site 3 days after induction of intracerebral hemorrhage. Mice were treated with either Ir-CPI (injection + perfusion), enoxaparin (enoxaparin injection + NaCl perfusion), or PBS alone (injection + perfusion). The boxes show the median and quartiles. The whiskers show the minimum and maximum values. Individual values ​​are plotted. The thick line shows the mean. N=5 mice per group. *p<0.05 (unpaired t-test). [Figure 4] Figures 4A and 4B are a set of box plots showing that Ir-CPI reduces neuronal cell death in the brains of mice with intracranial hemorrhage in the hemorrhagic region (anterior region, injection site, and posterior region mean) (Figure 4A) and the injection site (Figure 4B). The boxes show the median and quartiles. The thick line shows the mean. The whiskers show the minimum and maximum values. Individual data are shown [on day 3, n=7 mice (PBS / PBS), n=6 mice (Ir-CPI / Ir-CPI or enoxaparin / NaCl)]. P-values ​​were calculated using the Kruskal-Wallis test, including Dunn's multiple comparison test. *p<0.05. [Examples]

[0221] The present invention is further illustrated by the following examples. Example 1: Assessment of bleeding risk of Ir-CPI in a mouse model of intracranial hemorrhage. material and method Mouse model of brain hemorrhage Intracerebral hemorrhage (ICH) was induced in adult male Swiss mice by injection of a standardized amount of bacterial collagenase into the right striatum. Collagenase injection disrupts the extracellular matrix in the basement membrane.

[0222] After anesthesia and positioning of the jugular vein catheter, the mouse was positioned in the sphinx position within a stereotactic frame. Under sterile conditions, the skin over the skull was incised to a length of approximately 1 cm. The skull was cleaned and the location of the bregma was identified. The injection coordinates (right striatum: bregma +0.5 mm; medial-lateral -2.5 mm; dorsal-ventral -3 mm) were then calculated from the bregma based on a brain map. A hole was made in the skull, and the tip of a glass needle was inserted into the right striatum using a micromanipulator. A standardized amount (0.04 IU in 0.05 μL of sterile 0.9% NaCl) of bacterial collagenase (collagenase from Clostridium histolyticum, reference no. C2399, Sigma-Aldrich (registered)) was slowly injected. The needle was then slowly withdrawn, and the cranial skin was sutured.

[0223] Therapeutic administration Immediately after stroke induction, a bolus of treatment (PBS, Ir-CPI, or enoxaparin) was administered. Subsequently, continuous perfusion (PBS or Ir-CPI) was performed for 72 hours using an ALZET® osmotic pump while maintaining a constant plasma concentration of the drug. Animals that received a bolus of enoxaparin (Lovenox®; Sanofi Aventis) (20 mg / kg) after ICH induction were subjected to continuous perfusion of physiological saline (0.9% NaCl) for 72 hours. Ir-CPI (batch: DP B3012503) was injected as an IV bolus of 30.7 mg / kg, followed by continuous IV perfusion at 8.3 mg / kg / h for 72 hours. Mice to be injected with the medium were given an IV bolus of PBS, followed by continuous perfusion of PBS for 72 hours.

[0224] Magnetic resonance imaging Magnetic resonance imaging (MRI) was used to evaluate intracerebral hemorrhage and lesion volume 24 hours and 3 days (D3) after stroke onset. The MRI system used was a 7T-Pharmascan MRI system (Bruker BioSpin MRI, Ettlinger, Germany) equipped with volume transmitting and surface receiving coils and operated by Paravision (copyright) V6.

[0225] Images were analyzed using ImageJ v1.52 (NIH, Bethesda, USA). The research assistant responsible for image analysis was not informed of the group assignments. T2 * Brain hemorrhage was measured by analyzing images. For each mouse in each session, the hemorrhagic area (appearing as a low-signal area on the image) was measured on a slice. For each animal, the amount of bleeding was calculated as follows: sum of measured area × slice thickness. The amount of bleeding was in mm. 3 It was represented as follows.

[0226] T2W images were analyzed to determine the lesion volume. For each mouse in each session, the lesion area (appearing as a high-signal area on the image) was measured on a slice. For each animal, the amount of bleeding was estimated as follows: sum of measured area × slice thickness. The amount of bleeding was in mm. 3 It was represented as follows.

[0227] result In contrast to treatment with enoxaparin (low molecular weight heparin, LMWH), ir-CPI treatment did not increase lesion (edema) volume or bleeding in the striatum on days 1 and 3 compared to the control group (PBS) (Figures 1A-1D). Therefore, ir-CPI perfused for 72 hours is safer than enoxaparin.

[0228] Example 2: Anti-inflammatory effect of Ir-CPI in a mouse model of intracerebral hemorrhage material and method immunostaining method Immunostaining was performed on brain slices to evaluate neutrophil infiltration and the expression of their NETs. Brain slices were obtained from the mice previously used to measure the effect of treatment on lesion volume and bleeding volume (see Example 1). MPO is a marker expressed by activated neutrophils, NETs, microglia, and monocytes / macrophages. Ly6G is a specific marker for neutrophils. To evaluate and identify neutrophils that release extracellular traps (NETs), simultaneous staining of Ly6G (anti-mouse Ly-6G antibody, clone 1A8, rat monoclonal IgG2a - Cat No. 60031 STEMCELL (trademark)), H3Cit (anti-histone H3 (citrulline R2+R8+R17) antibody - Cat No. ab5103 ABCAM), MPO (anti-myeloperoxidase antibody - Cat No. AF3667 BIO-TECHNE (registered trademark)), and DNA (DAPI) was performed. The research assistant responsible for the analysis was not informed about the group assignment.

[0229] Results In contrast to the mice administered with PBS, a significant decrease in the number of cells expressing MPO (a marker for activated neutrophils, NETs, microglia, monocytes / macrophages) was observed in the Ir-CPI treated mice in the entire bleeding area (average of the anterior region - injection site - posterior region) (Figure 2A). Furthermore, Ir-CPI significantly reduced neutrophil infiltration (Ly6G + cells) compared to the mice administered with PBS (Figure 2B). Furthermore, when evaluating the entire bleeding area, a tendency for a decrease in the number of neutrophils (Ly6G + H3Cit + MPO + cells) that release NETs was observed in the Ir-CPI and enoxaparin groups (Figure 2C).

[0230] Furthermore, when evaluating only the injection site, neutrophils (Ly6G + H3Cit + MPO +A significant decrease in cells was observed (Figure 3). Enoxaparin did not have a statistically significant effect on these cells.

[0231] Example 3: Effects of Ir-CPI on neuronal cell death material and method Degenerated neurons were evaluated and located by staining brain slices with Fluoro-Jade® C (TR-100-FJ, Tebu-Bio). Brain sections were sectioned into coronal slices (10 μm slices) using a cryostat. Three slices were used per animal for staining, corresponding to the collagenase injection site, 600 μm anterior to the injection site, and 600 μm posterior to the injection site.

[0232] Slides containing frozen tissue sections were first immersed in a basic alcohol solution of 1% sodium hydroxide in 80% ethanol for 5 minutes. They were then rinsed with 70% ethanol for 2 minutes, then with distilled water for 2 minutes, and incubated in a 0.06% potassium permanganate solution for 10 minutes. After rinsing with water for 2 minutes, these slides were transferred to a solution of Fluoro-Jade® C (TR-100-FJ, Tebu-Bio) in the dark for 10 minutes. Appropriate dilution was performed by preparing a 0.01% stock solution containing the dye in distilled water. This working solution was stored at 4-8°C for up to 4 hours.

[0233] To label the nuclear DNA, 4',6-diamidino-2-phenylindole (DAPI; 0.01% staining solution D) was added to Fluoro-Jade® staining solution C. These slides were then rinsed three times with distilled water for 1 minute per change. Excess water was drained onto a paper towel, and the slides were then air-dried in the dark using a blade heater at 50°C for at least 5 minutes. The air-dried slides were then bleached with xylene for at least 1 minute and coated with the non-fluorescent mounting agent Fluoromount-G® (00-4958-02; Invitrogen®).

[0234] Using an epifluorescence microscope (Leica DM6 B), Fluoro-Jade® C was visualized using blue light or a 488 nm laser (excitation peak: 495 nm; emission peak: 521 nm), and the blue nuclear labeling provided by DAPI was visualized using ultraviolet excitation (emission peak: 340 nm; excitation peak: 488 nm). Images were acquired, and neuronal cell death was qualitatively assessed by analyzing four regions per slice using ImageJ software (version 1.52a). These analyses were blinded to group assignment by research assistants.

[0235] result Neurodegeneration in the brains of mice treated with PBS, Ir-CPI, or enoxaparin was evaluated using Fluoro-Jade® C staining.

[0236] In the bleeding area (average of anterior, injection site, and posterior regions), Ir-CPI reduced the average number of degenerated neurons compared to PBS-treated mice (Figure 4A). At the injection site (Figure 4B), Ir-CPI significantly reduced the number of degenerated neurons compared to PBS-treated control mice (p<0.05).

[0237] Conversely, enoxaparin treatment did not have a significant effect on neuronal cell death (p>0.05) (Figures 4A and 4B).

Claims

1. A pharmaceutical composition comprising a protein or polypeptide having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, for use in preventing and / or treating encephalitis that occurs or is likely to occur after cerebral hemorrhage in subjects requiring such use, The aforementioned pharmaceutical composition is administered within 120 hours after the onset of cerebral hemorrhage. Pharmaceutical composition.

2. The pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered within 72 hours after the onset of cerebral hemorrhage.

3. The pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered within 48 hours after the onset of cerebral hemorrhage.

4. The pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered within 24 hours after the onset of cerebral hemorrhage.

5. The pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered within 12 hours after the onset of cerebral hemorrhage.

6. The pharmaceutical composition for use according to claim 1, wherein the protein or polypeptide comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

7. The pharmaceutical composition for use according to claim 1, wherein the protein or polypeptide comprises a polypeptide having the amino acid sequence of SEQ ID NO:

2.

8. The pharmaceutical composition for use according to claim 1, wherein the bleeding is intracerebral hemorrhage.

9. The pharmaceutical composition for use according to claim 1, wherein the protein or polypeptide is administered to an adult in a dose of about 200 mg to about 20,000 mg per day.

10. The pharmaceutical composition for use according to claim 1, wherein the subject is administered an initial dose of the protein or polypeptide, and then perfused with the protein or polypeptide for continuous administration.

11. The pharmaceutical composition for use according to claim 10, wherein the initial dose is higher than the dose in perfusion.

12. The pharmaceutical composition for use according to claim 10, wherein the protein or polypeptide is administered by perfusion over a period of about 12 to about 96 hours, preferably about 24 to about 72 hours.

13. The pharmaceutical composition for use according to claim 1, wherein the protein or polypeptide is administered in a dose of about 2.5 mg / kg / day to about 250 mg / kg / day.

14. The pharmaceutical composition for use according to claim 1, wherein the protein or polypeptide is administered in a dose of about 2.5 mg / kg / day to about 25 mg / kg / day.

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