Mite salivary gland polypeptide for preventing and treating encephalitis

Abstract

The present invention relates to proteins and polypeptides from the salivary glands of ticks for their use in preventing and / or treating encephalitis and diseases or conditions associated with encephalitis, and to methods for preventing and / or treating encephalitis and diseases or conditions associated with encephalitis. The polypeptide is further used to prevent, reduce, decrease or inhibit neuronal cell death.

Description

Technical Field

[0001] The present invention relates to proteins and polypeptides containing Ixodes ricinus salivary gland polypeptides and their use for preventing and / or treating encephalitis.

Background Art

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

[0003] Neuroinflammation or encephalitis may occur after brain injury, for example in the case of hemorrhagic stroke. Hemorrhagic stroke is caused by bleeding into the brain due to 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, and SAH is bleeding into the subarachnoid space. Although constituting a minority (10 - 15%) of stroke cases, ICH and SAH patients often suffer from severe disabilities, and these disabilities contribute to a burden of morbidity and mortality comparable to that of ischemic stroke. The clinical outcome of this disease is poor, and only 20% of patients achieve functional independence six 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 bleeding, but also causes secondary brain injury (SBI), which is the main cause of poor outcome after ICH. SBI involves disruption of the blood - brain barrier (BBB), leading to peritumoral edema (PHE) and brain parenchymal cell death. SBI is particularly associated with neuroinflammation.

[0005] Neuroinflammation associated with ICH is accompanied by early activation of resident microglia, release of pro-inflammatory mediators, and infiltration of systemic inflammatory cells.

[0006] The innate immune cells that are first activated after ICH and other acute brain injuries are microglia (i.e., resident macrophages of the central nervous system). They continuously scan the extracellular brain environment and can be activated within minutes after tissue injury. The number of activated microglia / macrophages reaches a peak at 72 hours and returns to normal levels 3 - 4 weeks after ICH. Monocyte-derived macrophages infiltrate the perivascular and hematoma regions after microglia are activated. When stimulated by 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 for neutrophils and thus exacerbate the inflammatory response. Thus, microglia promote early neuroinflammation by recruiting and activating blood-derived leukocytes, which may exacerbate ICH-induced nerve injury.

[0007] Neutrophils are the earliest leukocyte subtype to infiltrate the hematoma site within 4 - 5 hours in animal models and reach a peak 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 network structures composed of nuclear DNA / histones and granule contents released by neutrophils, can damage the brain, for example, by producing reactive oxygen species, releasing pro-inflammatory proteases, and exacerbating neuronal death. NETs have also been reported to promote, for example, neuroinflammation and nerve injury after SAH, but pharmacological inhibition of NETs reduced inflammatory injury in mice.

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

[0009] The improvement in case fatality rate due to ICH in the past few decades has been slight, and most survivors continue to have severe physical disabilities. The treatment options for ICH are very limited, and there is still no definitive treatment other than supportive therapy. Therefore, the medical need for drugs that enable the prevention or safe and rapid treatment of encephalitis is not being met.

[0010] The present inventors have previously demonstrated the role of madani salivary gland polypeptide against neutrophils as part of thrombus treatment (International Publication No. WO 2019 / 149906). Here, the present inventors have surprisingly demonstrated that this polypeptide has an anti-inflammatory effect on the brain without any thrombus in an intracerebral hemorrhage model. Advantageously, these anti-inflammatory effects in the brain are not accompanied by an increase in the amount of bleeding or edema volume. Therefore, this polypeptide enables the prevention or early treatment of encephalitis without increasing the risk of exacerbating bleeding, which is useful for improving the clinical outcome of patients. SUMMARY OF THE INVENTION

[0011] Accordingly, 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 a subject in need thereof.

[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 encephalitis or spontaneous encephalitis. In some embodiments, the encephalitis is induced encephalitis. In some embodiments, the encephalitis occurs or is likely to occur after cerebral hemorrhage. In some embodiments, the encephalitis is post-cerebral hemorrhage.

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

[0015] In some embodiments, the encephalitis is induced 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, the spontaneous encephalitis is induced by non-traumatic brain injury or autoimmune disease.

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

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

[0020] 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 a subject in need thereof, which is administered within about 120 hours, preferably within 96 hours, more preferably within 72 hours after the onset of cerebral hemorrhage 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 hours to about 8 hours after the onset of encephalitis.

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

[0022] The present invention also relates to a pharmaceutical composition comprising the 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 the protein or polypeptide or pharmaceutical composition 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 nucleotide sequence or amino acid sequence identity. Generally, these sequences are aligned to obtain the maximum match. "Identity" itself has the meaning recognized in the art and can be calculated using published techniques. There are many methods for measuring identity between two polynucleotide or polypeptide sequences, and the term "identity" is well known to those skilled in the art. Methods for determining identity and similarity are encoded in computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package.

[0029] By way of illustration, for example, a nucleic acid having a nucleotide sequence with at least 95% "identity" to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the nucleic acid is identical to the reference sequence, except that the nucleic acid sequence may contain an average of at most 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 a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the total number of 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 anywhere between those terminal positions, and may be scattered individually among the nucleotides in the reference sequence or as one or more contiguous groups within the reference sequence.

[0030] "Pharmaceutically acceptable excipient" refers to an excipient that does not cause side effects, allergic reactions or other harmful reactions when administered to animals, preferably humans. It includes all kinds of solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents and absorption delaying agents, etc. Pharmaceutically acceptable carriers or excipients refer to any kind of non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation adjuvants. Preparations for human administration must meet the sterility, pyrogenicity, general safety and purity standards required by the FDA or the Biologicals Standards Directorate of the EMA.

[0031] "Polypeptide" refers to any peptide or protein containing two or more amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., peptide equivalents. "Polypeptide" generally refers to both short chains commonly called peptides, oligopeptides or oligomers and longer chains commonly called proteins. Polypeptides may contain amino acids other than the 20 genetically encoded amino acids.

[0032] "Protein" refers to an amino acid sequence of more than 100 amino acids and / or a multimer. The proteins of the present 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 as well as an enormous research literature. Modifications may occur anywhere in the polypeptide or protein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Of course, the same type of modification may be present at several sites in a given polypeptide or protein to the same or different extents. Also, a given polypeptide or protein may contain many types of modifications. A polypeptide or protein may be branched as a result of ubiquitination and may be cyclic, with or without branches. Cyclic, branched-chain, and branched-chain cyclic polypeptides or proteins may result from post-translational natural processes or may be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, amino acid amino transfer RNA-mediated addition to proteins such as arginylation, and ubiquitination. A protein may be the whole protein or a partial sequence thereof.

[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 is a "patient" who is waiting for or currently receiving medical care, has been or is currently a subject of a medical treatment, or will be a subject in the future, or is being monitored for the occurrence of a disease or condition, preferably encephalitis or a disease associated with or induced by encephalitis, i.e., a warm-blooded animal, more preferably a human. 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 pediatric (e.g., a subject under 18 years of age).

[0034] "Therapeutically effective amount" means the level or amount of an agent that, without causing significant negative or harmful side effects to the subject, (1) delays or prevents a disease or condition, preferably encephalitis or a disease associated with or induced by encephalitis, (2) decelerates or halts 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) brings about remission of the symptoms of encephalitis or a disease or condition associated with encephalitis, (4) reduces the severity or incidence of a disease or condition associated with encephalitis or a disease associated with or induced by encephalitis, or (5) prevents a disease or condition associated with encephalitis or a disease associated with or induced by encephalitis. In one embodiment, the therapeutically effective amount may be administered prior to the onset of encephalitis or a disease associated with or induced by encephalitis for prophylactic treatment.

[0035] The terms "treating" or "treatment" or "alleviating" refer to both therapeutic and prophylactic measures, the purpose of which is to prevent or slow down (mitigate) encephalitis or a disease associated with or induced by encephalitis. Those in need of treatment include those already suffering from encephalitis or a disease associated with or induced by encephalitis, as well as those susceptible to encephalitis or a disease associated with or induced by encephalitis, or those in whom encephalitis or a disease associated with or induced by encephalitis should be prevented. A subject or mammal is considered to have been "treated" successfully for encephalitis or a disease associated with or induced by encephalitis when, after administration of a therapeutically effective amount of the protein or polypeptide according to the method of the present invention, the patient shows one or more observable and / or measurable decreases or absences, such as a decrease in the number of pathogenic cells, a decrease in the proportion of total pathogenic cells, and / or a degree of alleviation of one or more symptoms associated with encephalitis or a disease associated with or induced by encephalitis, a decrease in morbidity and mortality, and an improvement in quality of life issues. The above parameters for evaluating the success and improvement of treatment in encephalitis or a disease associated with or induced by encephalitis are readily measurable by conventional procedures with which physicians are familiar.

DETAILED DESCRIPTION OF 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 a subject in need thereof. In a specific embodiment, the protein or polypeptide of the present invention is for use in preventing encephalitis in a subject in need thereof.

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

[0038] In some embodiments, the protein or polypeptide for use in accordance with 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 in accordance with 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 in accordance with 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 being at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identical.

[0041] In some embodiments, the protein or polypeptide for use in accordance with 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 in accordance with 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 in accordance with 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 in accordance with 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 in accordance with the present invention has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protein or polypeptide for use in accordance with the present invention has the amino acid sequence of SEQ ID NO: 2.

[0045] In one embodiment, fragments of the proteins or polypeptides for use according to 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 not all but part of the same as the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Fragments may be "independent" or may be included within a larger polypeptide of which they form part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the present invention include, for example, fragments from amino acid numbers approximately 1-20, 21-40, 41-60, 61-80, 81-100 and 101 (said position numbers corresponding to the amino acid numbers of SEQ ID NO: 1 or SEQ ID NO: 2) to the end of the polypeptide. "About" in this context includes ranges specifically recited that are larger or smaller by several, i.e., 5, 4, 3, 2 or 1 amino acids at one or both ends.

[0046] Preferred fragments include, but are not limited to, deletions of a continuous series of residues including the amino terminus or a continuous series of residues including the carboxyl terminus and / or transmembrane region, or cleavage-type polypeptides having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 except for deletions of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural or functional attributes such as alpha helices and alpha helix-forming regions, beta sheets and beta sheet-forming regions, turns and turn-forming regions, coils and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate-binding regions, and regions with high antigenic indices. Other preferred fragments are biologically active fragments. Biologically active fragments mediate the activity of the protein or polypeptide and include those having the same activity or improved or reduced unwanted 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 the 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 proteins or polypeptides of the present invention by conservative amino acid substitutions, i.e., those in which a residue is replaced with another residue having similar properties. Typical such substitutions are between Ala, Val, Leu and Ile, between Ser and Thr, between the acidic residues Asp and Glu, between Asn and Gln and between the basic residues Lys and Arg or between the aromatic residues Phe and Tyr. Variants in which several, i.e., 5 to 10, 1 to 5 or 1 to 2 amino acids are substituted, deleted or added in any combination are particularly preferred.

[0049] In one embodiment, the protein or polypeptide for use according to the present invention has an amino acid sequence comprising a mutation that substitutes glutamine for asparagine 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, the protein or polypeptide of the present invention has an amino acid sequence comprising or consisting of the sequence of SEQ ID NO: 2.

[0050] In one embodiment, the protein or polypeptide for use according to the present invention can be prepared by any suitable method. Examples of peptides prepared according to suitable methods include, but are not limited to, isolated naturally occurring polypeptides, recombinantly produced 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 bound to at least one other protein or polypeptide in such a way as to produce 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 conjugated in any order. In one embodiment, the polypeptides or proteins are arranged within 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 bound to each other via one or more peptide linkers.

[0055] In one embodiment, the peptide linker provides a greater physical separation between the two moieties and thus maximizes the availability of the protein or polypeptide for use according to the present invention. The peptide linker may consist of amino acids such that it is flexible or more rigid.

[0056] In one embodiment, the peptide linker comprises 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 comprises 6, 7, 8, 9 or 10 to 20 amino acids. In another embodiment, the peptide linker comprises 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 cleavable linker or a non-cleavable linker 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 above in this specification are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily isolated and sequenced using conventional procedures.

[0059] The fusion protein 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 are partially determined by factors such as net charge, hydrophobicity, hydrophilicity, etc., 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 a variety of well-known analytical methods including gel electrophoresis and high pressure liquid chromatography.

[0060] The present invention also relates to the fusion protein described above in this specification 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 a plurality of (e.g., two or more) nucleic acids 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 in accordance with the present invention prevents and / or treats encephalitis. In some embodiments, the protein or polypeptide for use in accordance with the present invention prevents encephalitis. In some embodiments, the protein or polypeptide for use in accordance with the present invention treats encephalitis.

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

[0064] In some embodiments, encephalitis comprises 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, IFN-γ, and TNF-α.

[0065] In some embodiments, encephalitis comprises or consists of early activation of resident microglia. As used herein, "microglia" refers to the resident macrophages of the central nervous system.

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

[0067] In some embodiments, encephalitis comprises or consists of infiltration of systemic inflammatory cells into the brain. In some embodiments, the systemic inflammatory cells are selected from the group consisting of or comprising 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 to a state of abnormal stimulation of neurotransmitter receptors in neurons that results in, among other things, changes in calcium and energy homeostasis and apoptosis.

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

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

[0071] In another embodiment, encephalitis is not localized to a specific region of the brain. In certain embodiments, 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 thrombus-related or thrombus-induced diseases.

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

[0074] In some embodiments, encephalitis is caused by a brain injury. In some embodiments, the brain injury is a traumatic brain injury. In some other embodiments, the brain injury is a 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, the traumatic brain injury is selected from the group consisting of or including hemorrhage, contusion, concussion, and penetrating brain injury. In some embodiments, the traumatic brain injury is hemorrhage.

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

[0078] In some embodiments, the infection is selected from the group consisting of or including viral infection, bacterial infection, fungal infection, parasitic infection, and combinations thereof. In some embodiments, the infection is induced by sepsis and / or septicemia. 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, the brain injury is selected from the group consisting of or including 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, the brain injury is selected from the group consisting of or including cerebral hemorrhage, contusion, concussion, penetrating brain injury, seizure, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, the brain injury is selected from the group consisting of or including cerebral hemorrhage, contusion, concussion, penetrating brain injury, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, the brain injury is selected from the group consisting of or including 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 for use in preventing and / or treating encephalitis after cerebral hemorrhage in a subject in need thereof.

[0081] In specific embodiments, the protein or polypeptide of the present invention is for use in preventing encephalitis that is likely to occur after brain injury in a subject in need thereof. In specific embodiments, the protein or polypeptide of the present invention is for use in preventing encephalitis that is likely to occur after cerebral hemorrhage in a subject in need thereof.

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

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

[0084] As used herein, the term "acquired encephalitis" refers to the encephalitis described above in this specification, where 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 infectious disease.

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

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

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

[0088] In some embodiments, the traumatic brain injury is a hemorrhage selected from the group consisting of or 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, the traumatic brain injury is a contusion. In some embodiments, the traumatic brain injury is a concussion. In some embodiments, the traumatic brain injury is a penetrating brain injury. In some embodiments, the traumatic brain injury is anoxic brain injury.

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

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

[0092] In some embodiments, encephalitis is induced by viral infections. Non-limiting examples of viral infections that can cause encephalitis include infections 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, encephalitis is induced by bacterial infections. Non-limiting examples of bacterial infections that can cause encephalitis include infections by bacteria of the genera Treponema, Mycoplasma, Neisseria, Streptococcus, and Listeria.

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

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

[0096] In some embodiments, encephalitis is autoimmune encephalitis.

[0097] As used herein, the term "autoimmune encephalitis" refers to encephalitis as described herein that is caused by something endogenous to the organism, for example, resulting from an autoimmune disease or condition.

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

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

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

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

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

[0103] In some embodiments, autoimmune encephalitis is induced by an autoimmune disease. In some embodiments, the autoimmune disease is autoimmune encephalitis. In some embodiments, the non-traumatic brain injury is induced by an autoimmune disease. In some embodiments, the autoimmune disease is autoimmune encephalitis.

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

[0105] In some embodiments, by preventing and / or treating encephalitis using the protein or polypeptide according to the present invention, another disease, condition, or symptom is further treated and / or alleviated. In some embodiments, the disease, condition, or symptom is associated with 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 with the protein or polypeptide according to the present invention has a positive effect on another disease, condition or symptom selected from the group consisting of or including neuronal cell death, excitotoxicity, microglial activation, oxidative stress and infiltration of the brain by systemic inflammatory cells. In some embodiments, the disease, condition or symptom is selected from the group consisting of or including neuronal cell death, excitotoxicity, microglial activation and oxidative stress. As used herein, the expression "positive effect" is intended to mean that the protein or polypeptide according to the present invention reduces, decreases or suppresses the symptoms, or partially or completely treats the components of the disease or condition.

[0108] In some embodiments, preventing and / or treating encephalitis with the protein or polypeptide according to the present invention has a positive effect on neuronal cell death. In some embodiments, preventing and / or treating encephalitis with the protein or polypeptide according to the present invention has a positive effect on excitotoxicity.

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

[0110] In some embodiments, preventing and / or treating encephalitis with the protein or polypeptide according to the present invention has a positive effect on oxidative stress. In some embodiments, preventing and / or treating encephalitis with the protein or polypeptide according to the present invention inhibits the formation of reactive oxygen species.

[0111] In some embodiments, preventing and / or treating encephalitis with the protein or polypeptide according to the present invention has a positive effect on the infiltration of the brain by systemic inflammatory cells. In certain embodiments, the systemic inflammatory cells are selected from the group consisting of or including neutrophils, eosinophils, lymphocytes, plasma cells, and histiocytes. In certain embodiments, the systemic inflammatory cells are neutrophils. In some embodiments, preventing and / or treating encephalitis with the protein or polypeptide according to the present invention inhibits the recruitment of neutrophils 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 of time, typically one week, one month, one 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 hemorrhage. 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 hemorrhage. 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 intracerebral hemorrhage.

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

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

[0115] The present invention further relates to a nucleic acid molecule encoding a protein or polypeptide for use according to the present invention.

[0116] In some embodiments, the nucleic acid is for preventing and / or treating encephalitis or a disease associated with encephalitis. In some embodiments, the nucleic acid is for preventing and / or treating encephalitis.

[0117] In some embodiments, the nucleic acid molecule is 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 consisting of or comprising a plasmid, naked DNA, cosmid, fosmid, prokaryotic chromosome (e.g., bacterial artificial chromosome), eukaryotic chromosome (e.g., yeast artificial chromosome or human artificial chromosome), 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 comprises or consists of natural nucleotides, unnatural nucleotides, or a combination thereof. In some embodiments, the nucleic acid molecule comprises or consists of natural nucleotides. In some embodiments, the natural nucleotides are selected from the group consisting of or comprising adenine, guanine, cytosine, thymine, and uracil. In some embodiments, the natural nucleotides are selected from the group consisting of or comprising adenine, guanine, cytosine, and thymine. In some embodiments, the natural nucleotides are selected from the group consisting of or comprising adenine, guanine, cytosine, and uracil. In some embodiments, the nucleic acid molecule comprises or consists of unnatural nucleotides (e.g., chemically modified ones).

[0121] In some embodiments, the nucleic acid molecule is single-stranded, double-stranded, or a combination thereof. In some embodiments, the nucleic acid molecule is single-stranded. In some embodiments, the nucleic acid molecule is 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 to SEQ ID NO: 3. In one embodiment, the nucleic acid encoding a protein or polypeptide for use according to the present invention has the nucleotide sequence set forth 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 a plurality of (i.e., two or more) nucleic acids co-expressed. The polypeptides encoded by the co-expressed nucleic acids may associate, 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 preventing and / or treating encephalitis and / or diseases associated with encephalitis. In a preferred embodiment, the vector of the present invention is an expression vector.

[0125] The present invention further relates to a composition comprising the protein or polypeptide described above in the present specification, one or more nucleic acids or vectors.

[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 preventing and / or treating encephalitis in a subject in need thereof as described above in the present specification.

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

[0128] In some embodiments, the pharmaceutical composition comprises 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, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a protein or polypeptide having the amino acid sequence set forth in SEQ 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, protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, salts or electrolytes such as sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., sodium carboxymethyl cellulose), polyethylene glycol, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, polyethylene glycol, and lanolin.

[0130] In some embodiments, the pharmaceutical composition further comprises another therapeutic agent.

[0131] In some embodiments, the additional therapeutic agent has a positive effect on a disease, condition or symptom selected from or consisting of encephalitis, neuronal cell death, excitotoxicity, microglial activation and oxidative stress. In some embodiments, the additional therapeutic agent has a positive effect on encephalitis.

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

[0133] In some embodiments, the additional therapeutic agent comprises or consists of a cell therapy such as stem cell therapy.

[0134] The present invention further relates to the protein or polypeptide, one or more nucleic acids, vectors, compositions or pharmaceutical compositions described above herein for the manufacture of a medicament for preventing and / or treating encephalitis.

[0135] Another object of the present invention is a medicament comprising a protein or polypeptide, one or more nucleic acids, a vector, a composition or a pharmaceutical composition as described hereinabove for preventing and / or treating encephalitis.

[0136] In one embodiment, the pharmaceutical composition or medicament of the present invention comprises a therapeutically effective amount of a protein or polypeptide, one or more nucleic acids or a vector as described hereinabove.

[0137] Of course, the total daily dosage 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 scope of appropriate medical judgment. The specific therapeutically effective dosage level for any particular patient depends on the disease being treated and the severity of encephalitis, the activity of the specific agent used, the specific composition used, the patient's age, weight, health status, gender and diet, the time of administration, route of administration and excretion rate of the specific agent used, the duration of treatment, the drugs combined with or used simultaneously with the specific agent, and various factors including similar factors well known in the medical field. For example, it is well within the skill of those in the art to start administering the agent at a level lower than the dosage required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of this product may vary widely from about 10 to about 10,000 mg per day for an adult, preferably 100 to about 5,000, more preferably about 200 to about 2,000 mg per day for an adult. Preferably, the composition contains active ingredients of 10, 50, 100, 250, 500, 1000 and 2,000 mg for dosage adjustment according to the symptoms of the patient being treated. The drug typically contains from about 10 to about 10,000 mg of active ingredient, preferably 5 to about 5,000, more preferably about 10 to about 2,000 mg of 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 even 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 at 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, even 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 at 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 at 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 at 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 at a dose of about 20 mg to about 200,000 mg per day for an adult, preferably about 20 mg to about 20,000 mg per day for an adult, more preferably about 20 mg to about 10,000 mg per day for an adult. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered at a dose of about 200 mg to about 200,000 mg per day for an adult, preferably about 200 mg to about 20,000 mg per day for an adult, more preferably about 200 mg to about 10,000 mg per day for an adult. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the present invention is administered at a dose of about 2000 mg per day for an adult.

[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. It is particularly advantageous for patients having encephalitis, brain injury or cerebral hemorrhage, such as bedridden patients, who are prone to thromboembolic events. In fact, within the first 72 hours after the onset of bleeding, anticoagulants (such as enoxaparin) increase the risk of bleeding, and thus they cannot be administered. The inventors have demonstrated that, unlike enoxaparin, the risk of bleeding is not increased by the protein of the present invention within 3 days after the onset of bleeding (see Example 1). Therefore, in order to avoid irreversible effects, it is possible to treat encephalitis and / or brain injury with the protein of the present invention within an important time frame. For the best results, a therapeutic solution for encephalitis must be given before the inflammatory response starts after the onset of bleeding. Current solutions cannot be administered immediately because of the increased risk of bleeding for them. In contrast, the protein or polypeptide of the present invention does not have such drawbacks and thus can be administered immediately to reduce the inflammatory response before causing very harmful and / or permanent effects.

[0141] Furthermore, the peak of neutrophil infiltration and, if present, the rapid growth phase of edema also occur within this 72-hour period. The earlier the size of the edema decreases, the better the patient outcome.

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

[0143] Thus, 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 intracerebral 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 intracerebral 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 intracerebral 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 intracerebral 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, even more preferably within the first 6 hours after the onset of intracerebral 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 about 0 hours to about 120 hours, 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, more preferably within 6 hours after the onset of intracerebral hemorrhage.

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

[0146] Thus, in some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered within about 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 24 hours or 48 hours to about 120 hours, 96 hours 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, preferably about 24 hours to about 96 hours, more preferably about 24 hours to about 72 hours after the onset of encephalitis and / or after brain injury. 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, preferably about 48 hours to about 96 hours, more preferably about 48 hours to about 72 hours after the onset of encephalitis and / or after brain injury.

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

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

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

[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 hours to about 12 hours, preferably about 0 hours to about 10 hours, more preferably about 0 hours to about 8 hours, even more preferably about 0 hours to about 7 hours, even more preferably about 0 hours to about 6 hours after the onset of encephalitis and / or after brain injury.

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

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

[0155] For use in administration to a subject, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is formulated for administration to a subject. The protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention may be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, intraorally, vaginally or by an implanted reservoir. As used herein, the term administration includes subcutaneous, intravenous, intramuscular, intraocular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional 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, liquid, paste or solid compositions, more particularly 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, for example for 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, suspensions, and solid forms suitable for use in preparing a solution or suspension by adding a liquid before use, such as powders and liposome forms.

[0161] The protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the present invention may be an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injection preparation may also be a sterile injection solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable media and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. Also, sterile non-volatile oils are conventionally used as solvents or suspending media. For this purpose, any sterile non-volatile oil containing synthetic monoglycerides or diglycerides can be used. Fatty acids such as oleic acid and their glyceride derivatives are useful in the preparation of injection preparations, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersing agents such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation 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 solid, liquid or other dosage forms may also be used for the purposes of the formulation.

[0162] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the present invention is administered to a subject in need thereof at least once a day. For example, the composition, pharmaceutical composition or medicament of the present invention may be administered once, twice or three times a day. In a preferred embodiment, the composition, pharmaceutical composition or medicament of the present invention is administered to a subject in need thereof 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 in need thereof 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 thereof 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 in need thereof before being exposed to the risk of developing encephalitis. In one embodiment, the term "before" means at least one week before the exposure. In another embodiment, the term "before" means 5 days, 4 days, 3 days, 2 days or 1 day before the 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 the exposure. In another embodiment, the term "before" means less than 1 hour before the exposure such as 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes before the exposure or at the moment of the 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 in need thereof after being exposed to the risk of developing encephalitis. In one embodiment, the term "after" means 5 minutes, 10 minutes, 15 minutes, 30 minutes or 45 minutes after the exposure. In another embodiment, the term "after" means 1, 2, 4, 6, 12, 15, 18 or 24 hours after the exposure. In another embodiment, the term "after" means 1, 2, 3, 4 or 5 days after the exposure. In another embodiment, the term "after" means 1 week or more after the exposure.

[0167] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is administered continuously, 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 preferably administered continuously by perfusion for about 12 hours to about 96 hours, preferably about 18 hours to about 84 hours, more preferably about 12 hours 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 preferably administered continuously by perfusion for about 24 hours to about 96 hours, preferably about 48 hours 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 preferably administered continuously by perfusion for about 24 hours to about 72 hours, preferably about 48 hours 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 preferably administered continuously by perfusion for at least about 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or 96 hours. In certain embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention is preferably administered continuously by perfusion for at least 48 hours.

[0170] In some embodiments, a first 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 it is perfused for continuous administration. Within the scope of the present invention, the first 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 at the initial dose is higher than or the same as the dose during perfusion, preferably higher than that.

[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 preventing and / or treating encephalitis.

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

[0173] In one embodiment, the kit of the present invention further comprises 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 in need thereof, comprising administering to the 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 comprising such a nucleic acid, 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 comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the present invention. In one embodiment, the therapeutically effective dose consists of a dose of about 2.5 mg / kg / day to about 250 mg / kg / day. In one embodiment, the therapeutically effective dose consists of a dose of about 2.5 mg / kg / day to about 100 mg / kg / day. In one embodiment, the therapeutically effective dose consists of a dose of about 2.5 mg / kg / day to about 50 mg / kg / day. In one embodiment, the therapeutically effective dose consists of a dose of about 2.5 mg / kg / day to about 25 mg / kg / day. In one embodiment, the therapeutically effective dose consists of a dose of about 5 mg / kg / day to about 50 mg / kg / day.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0194] In one embodiment, the method includes administering to a subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention within about 2 hours to about 24 hours, preferably within about 4 hours to about 24 hours, more preferably within about 8 hours to about 24 hours after the onset of intracerebral hemorrhage. In certain embodiments, the method includes administering to a subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the present invention within about 10 hours to about 24 hours after the onset of intracerebral hemorrhage.

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

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

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

[0198] In some embodiments, the method is not for preventing and / or treating thrombosis. In some embodiments, encephalitis is not associated with the presence or formation of thrombosis.

[0199] In some embodiments, the method · comprises administering to a subject a loading dose comprising a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the invention; · and perfusing the subject with a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the invention, wherein the loading dose is 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 the lesion (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 a disease, condition or symptom associated with encephalitis, comprising administering to a subject a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the invention. In some embodiments, the method is for preventing and / or treating a disease, condition or symptom induced by encephalitis, comprising administering to a subject a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the invention. In some embodiments, the disease, condition or symptom is selected from the group consisting of or including neuronal cell death, excitotoxicity, microglial activation and oxidative stress.

[0202] In some embodiments, the methods of the invention are used to alleviate the symptoms of a subject suffering from traumatic or non-traumatic brain injury.

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

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

[0205] In one embodiment, the subject in need thereof is a subject having or prone to having encephalitis. In one embodiment, the method is for preventing, reducing, decreasing and / or inhibiting neuronal cell death that occurs or is likely to occur after encephalitis in a subject in need thereof.

[0206] In one embodiment, the subject in need thereof is a subject having or prone to having cerebral hemorrhage. In one embodiment, the method is for preventing, reducing, decreasing and / or inhibiting neuronal cell death that occurs or is likely to occur after cerebral hemorrhage in a subject in need thereof.

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

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

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

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

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

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

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

[0214] Yet another object of the present invention is a method for preventing and / or treating a disease associated with, attendant upon, or caused by neuronal cell death in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention.

[0215] The present invention further relates to a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or drug according to the present invention for use in preventing and / or treating a disease associated with, attendant upon, or caused by neuronal cell death in a subject in need thereof.

[0216] In one embodiment, the disease associated with, attendant upon, or caused by neuronal cell death is selected from the group consisting of or including 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, the disease associated with, attendant upon, or caused by neuronal cell death is selected from the group consisting of or including 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, the disease associated with, accompanying, or caused by neuronal death is selected from the group consisting of or including prion disease (Creutzfeldt-Jakob disease), amyotrophic lateral sclerosis, motor neuron disease, 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, the disease associated with, accompanying, or caused by neuronal death is selected from the group consisting of or including prion disease (Creutzfeldt-Jakob disease), amyotrophic lateral sclerosis, motor neuron disease, 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 Description of the Drawings

[0220]

Figure 1

Figure 2

Figure 3

Figure 4

Example

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

[0222] After anesthesia and positioning of the jugular vein catheter, the mice were positioned in the sphinx position in a stereotaxic fixation frame. Under aseptic conditions, the skin on top of the skull was incised about 1 centimeter in length. The skull was cleaned and the position of bregma was identified. Next, injection coordinates (right striatum: bregma +0.5 mm; medial-lateral -2.5 mm; dorsal-ventral -3 mm) were calculated based on the brain map from bregma. 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 0.0.5 μL of sterile 0.9% NaCl) of bacterial collagenase (collagenase derived from Clostridium histolyticum, reference number C2399, Sigma-Aldrich (registered)) was slowly injected. Then, the needle was slowly removed and the skull skin was sutured.

[0223] Therapeutic administration Immediately after stroke induction, a treatment (either PBS, Ir-CPI or enoxaparin) bolus was administered. Then, continuous perfusion (PBS or Ir-CPI) was performed for 72 hours using an ALZET (registered trademark) osmotic pump while maintaining a constant plasma concentration of the drug. Animals receiving a bolus of enoxaparin (Lovenox (registered trademark); Sanofi Aventis) (20 mg / kg) after ICH induction received continuous perfusion of physiological saline (0.9% NaCl) for 72 hours. After injecting Ir-CPI (batch: DP B3012503) as an IV bolus of 30.7 mg / kg, IV continuous perfusion was performed at 8.3 mg / kg / h for 72 hours. An IV bolus of PBS was administered to mice injected with the vehicle, and then continuous perfusion of PBS was performed for 72 hours.

[0224] Magnetic resonance imaging Using magnetic resonance imaging (MRI), intracerebral hemorrhage and lesion volume were evaluated 24 hours and 3 days (D3) after the onset of stroke. The MRI apparatus was a 7T-Pharmascan MRI system (Bruker BioSpin MRI, Ettlinger, Germany) equipped with a volume transmitter and a surface receiver coil 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 unaware of the group assignment. T2 * Images were analyzed to measure intracerebral hemorrhage. For each mouse in each session, the hemorrhage area (appearing as a hypointense area on the image) was measured on the slice. For each animal, the hemorrhage volume was calculated as follows: the sum of the measured area × slice thickness. The hemorrhage volume was expressed in mm 3 as shown.

[0226] T2W images were analyzed to determine the lesion volume. For each mouse in each session, the lesion area (appearing as a hyperintense area on the image) was measured on the slice. For each animal, the hemorrhage volume was estimated as follows: the sum of the measured area × slice thickness. The hemorrhage volume was expressed in mm 3 as shown.

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

[0228] Example 2: Anti-inflammatory effect of Ir-CPI in an intracerebral hemorrhage mouse model Materials and methods 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 (TM)), H3Cit (anti-histone H3 (citrulline R2+R8+R17) antibody - Cat No. ab5103 ABCAM), MPO (anti-myeloperoxidase antibody - Cat No. AF3667 BIO-TECHNE (R)) 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 area - injection site - posterior area) (Figure 2A). Furthermore, Ir-CPI significantly reduced neutrophil infiltration (Ly6G + cells) compared to the mice administered with PBS (Figure 2B). Additionally, 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: Effect of Ir-CPI on neuronal death Materials and methods Fluoro-Jade (R) C staining (TR-100-FJ, Tebu-Bio) was performed on brain slices to evaluate and identify degenerated neurons. The brain was sectioned into coronal slices (10-μm slices) using a cryostat. Three slices per animal were used for staining, corresponding to the collagenase injection site, 600 μm anterior and 600 μm posterior to the injection site.

[0232] Slides with frozen tissue sections were first immersed in a basic alcohol solution of 1% sodium hydroxide in 80% ethanol for 5 minutes. Then they were rinsed with 70% ethanol for 2 minutes, with distilled water for 2 minutes, and then 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 (R) 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 nuclear DNA, 4′,6-diamidino-2-phenylindole (DAPI; 0.01% staining solution D) was added to the Fluoro-Jade (R) staining solution C. Then these slides were rinsed with distilled water, changed three times for 1 minute per change. Excess water was drained onto a paper towel, and then these slides were air-dried in the dark by a blade heater at 50 °C for at least 5 minutes. Then the air-dried slides were bleached with xylene for at least 1 minute and then coated with a non-fluorescent mounting agent Fluoromount-G (TM) (00-4958-02; Invitrogen (TM)).

[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 by ultraviolet excitation (emission peak: 340 nm; excitation peak: 488 nm). Images were acquired, and four regions per slice were analyzed using Image J software (version 1.52a) to qualitatively evaluate neuronal death. These analyses were performed blindly with respect to the group assignment by the research assistant.

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

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

[0237] Conversely, enoxaparin treatment had no significant effect on neuronal death (p > 0.05) (Figure 4A and Figure 4B).

Claims

1. 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 that occurs or is likely to occur after cerebral hemorrhage in a subject in need thereof.

2. The protein or polypeptide 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.

3. The protein or polypeptide for use according to any one of claims 1 to 2, wherein the encephalitis is induced by acquired brain injury.

4. The protein or polypeptide for use according to any one of claims 1 to 3, wherein the encephalitis is induced by traumatic brain injury.

5. The protein or polypeptide for use according to any one of claims 1 to 3, wherein the encephalitis is induced by non-traumatic brain injury.

6. The protein or polypeptide for use according to any one of claims 1 to 5, wherein the encephalitis is induced by cerebral hemorrhage, ischemic stroke, seizure, infection, brain tumor, hypoxic / anoxic brain injury, autoimmune disease, toxicity or hydrocephalus.

7. The protein or polypeptide for use according to any one of claims 1 to 6, wherein the protein or polypeptide is administered at a dose of about 200 mg to about 20,000 mg per day for an adult.

8. 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, which is administered within about 120 hours, preferably within 96 hours, more preferably within 72 hours after the onset of encephalitis.

9. The encephalitis occurs or is likely to occur after brain injury, and the protein or polypeptide is administered within about 120 hours, preferably within 96 hours, more preferably within 72 hours after the onset of brain injury. The protein or polypeptide for use according to claim 8.

10. The encephalitis occurs or is likely to occur after cerebral hemorrhage, and the protein or polypeptide is administered within about 120 hours, preferably within 96 hours, more preferably within 72 hours after the onset of cerebral hemorrhage. The protein or polypeptide for use according to claim 8 or 9.

11. The protein or polypeptide is administered within about 15 hours, preferably within 12 hours after the onset of encephalitis, trauma or hemorrhage. The protein or polypeptide for use according to any one of claims 8 to 10.

12. The protein or polypeptide is preferably administered continuously by perfusion. The protein or polypeptide for use according to any one of claims 8 to 11.

13. A pharmaceutical composition comprising a protein or polypeptide for use according to any one of claims 1 to 12 and at least one pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13, further comprising another therapeutic agent.

15. A kit comprising a protein or polypeptide for use according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 or 14.

16. A method for preventing and / or treating encephalitis 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 the protein or polypeptide.

17. The method according to claim 16, wherein the therapeutically effective amount is a dosage of about 200 mg to about 20,000 mg per day for an adult.

18. The method according to claim 16 or claim 17, wherein the protein or polypeptide is administered to the subject within about 120 hours, preferably within 72 hours, more preferably within 24 hours, even more preferably within 12 hours after encephalitis and / or brain injury.

19. A method for preventing, reducing, decreasing and / or inhibiting 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 the protein or polypeptide.

20. The method according to claim 19, wherein the neuronal cell death occurs after intracerebral hemorrhage.

21. The method according to claim 19 or 20, wherein the protein or polypeptide is administered 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 intracerebral hemorrhage.

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