Haemaphysalis pectinata salivary gland polypeptides for prevention and treatment of brain inflammation

Abstract

The present invention relates to proteins and polypeptides from the salivary glands of sclerophysalis pectinata for use in the prevention and / or treatment of brain inflammation and diseases or conditions associated with brain inflammation, and methods for the prevention and / or treatment of brain inflammation and diseases or conditions associated with brain inflammation. The polypeptides are also useful for preventing, reducing, reducing and / or inhibiting neuronal death.

Description

Technical Field

[0001] This invention relates to proteins and polypeptides containing polypeptides from the salivary glands of the castor tick (Ixodes ricinus), and their use in the prevention and / or treatment of brain inflammation. Background Technology

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

[0003] Neuroinflammation or inflammation of the brain can occur after brain injury, such as in a hemorrhagic stroke. A hemorrhagic stroke is caused by intracerebral hemorrhage due to the rupture of a blood vessel. Hemorrhagic stroke can be further subdivided into intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). ICH is intraparenchymal hemorrhage, while SAH is subarachnoid hemorrhage. Although they account for a minority of stroke cases (10% to 15%), patients with ICH and SAH are often severely impaired, and the morbidity and mortality burden of these conditions is comparable to that of ischemic stroke. The clinical outcomes of this condition are poor: only 20% of patients achieve functional independence within 6 months after ICH. The most common cause of ICH is chronic hypertension, while cerebral amyloid angiopathy, anticoagulants, and underlying vascular abnormalities account for a large proportion of cases.

[0004] ICH not only causes primary brain injury through the direct mechanical effects of hemorrhage, but also secondary brain injury (SBI), a major cause of adverse outcomes after ICH. SBI includes disruption of the blood-brain barrier (BBB), leading to perihematoma edema (PHE) and death of brain parenchymal cells. Among other things, SBI involves neuroinflammatory factors.

[0005] ICH-related neuroinflammation involves early activation of resident microglia, release of pro-inflammatory mediators, and infiltration of systemic inflammatory cells.

[0006] The first innate immune cells activated after ICH and other acute brain 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 of tissue injury. The number of activated microglia / macrophages peaks at 72 hours and returns to normal levels 3 to 4 weeks after ICH. After microglia are activated, monocyte-derived macrophages infiltrate the peri-hematoma and the hematoma area. Under various stimuli, microglia and brain macrophages produce pro-inflammatory cytokines, including TNF-α and IL-1β, chemokines, and reactive oxygen species. In addition to neurotoxic cytokines, chemokines produced by microglia, such as CXCL2, have chemotactic activity towards neutrophils and thus exacerbate the inflammatory response. Therefore, microglia also enhance early neuroinflammation by recruiting and activating blood-derived leukocytes, which may exacerbate neuronal damage induced by ICH.

[0007] Neutrophils are the earliest leukocyte subtype to infiltrate hematoma sites, infiltrating within 4 to 5 hours in animal models and peaking at 3 days. In humans, neutrophils infiltrate the hematoma and surrounding brain tissue in the first few days after ICH (≤72 hours). Neutrophils and neutrophil extracellular traps (NETs) – extracellular network structures composed of nuclear DNA / histones and granular contents released by neutrophils – can damage the brain by, for example, producing reactive oxygen species, releasing pro-inflammatory proteases, and exacerbating neuronal death. In mice, NETs have been reported to promote neuroinflammation and neuronal damage following SAH, while pharmacological inhibition of NETs has been shown to reduce inflammatory damage.

[0008] Myeloperoxidase (MPO) is an important inflammatory cytokine in the bone marrow system. MPO is highly expressed in neutrophils and other myeloid cells, such as Ly-6C-high monocytes, macrophages, and microglia. Myeloperoxidase and its active products are involved in the occurrence and development of cerebral hemorrhage, including its damage to the blood-brain barrier and the brain. As a specific inflammatory marker, myeloperoxidase can be used to assess the occurrence and development of vascular diseases, and experimental data indicate that inhibition or deficiency of myeloperoxidase has a positive prognostic effect.

[0009] Over the past few decades, improvements in ICH mortality rates have been limited, with most survivors suffering severe disabilities. Treatment options for ICH are extremely limited; currently, there are no definitive cures beyond supportive care. Therefore, there is an unmet medical need for agents that can prevent or safely and rapidly treat brain inflammation.

[0010] The inventors have previously demonstrated the effect of castor beetle salivary gland polypeptides on neutrophils as part of thrombosis treatment (WO2019 / 149906). Here, the inventors unexpectedly demonstrate that this polypeptide has an anti-inflammatory effect on the brain in a model of cerebral hemorrhage without causing any thrombosis. Advantageously, these anti-inflammatory effects in the brain are not accompanied by an increase in hemorrhage volume or edema volume. Therefore, this polypeptide can prevent or treat brain inflammation at an early stage without increasing the risk of hemorrhage exacerbation, which helps improve patient clinical outcomes. Summary of the Invention

[0011] Therefore, the present invention relates to a protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1, for use in preventing and / or treating brain inflammation in subjects in need.

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

[0013] In some embodiments, the brain inflammation is acquired or spontaneous. In some embodiments, the brain inflammation is induced. In some embodiments, the brain inflammation occurs or is likely to occur after a brain hemorrhage. In some embodiments, the brain inflammation is accompanied by a brain hemorrhage.

[0014] In some implementations, the brain inflammation is caused by acquired brain injury. In some implementations, the brain inflammation is caused by traumatic brain injury. In some implementations, the brain inflammation is caused by non-traumatic brain injury.

[0015] In some implementations, brain inflammation is caused by cerebral hemorrhage, ischemic stroke, seizure, infection, brain tumor, hypoxic brain injury / hypoxic brain injury, autoimmune disease, toxicity, or hydrocephalus.

[0016] In some implementations, the brain inflammation is acquired brain inflammation. In some implementations, the acquired brain inflammation is caused by traumatic brain injury or infection. In some implementations, the acquired brain inflammation is caused by traumatic brain injury.

[0017] In some implementations, the brain inflammation is spontaneous. In other implementations, spontaneous brain inflammation is caused by non-traumatic brain injury or autoimmune disease.

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

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

[0020] The present invention also relates to the use of a protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1 for the prevention and / or treatment of brain inflammation in subjects of need, wherein 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 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 brain inflammation. In some embodiments, the protein or polypeptide is administered within about 6 hours to about 8 hours after the onset of brain inflammation.

[0021] In some embodiments, the protein or peptide is administered continuously, preferably via perfusion. In some embodiments, the protein or peptide is administered rapidly to the subject after exposure to the risk of brain inflammation. In some embodiments, the protein or peptide is administered continuously for about 48 hours to about 72 hours.

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

[0023] The present invention also relates to kits comprising protein or peptide or pharmaceutical compositions as described herein.

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

[0025] Another object of the present invention is a method for preventing, reducing, decreasing, and / or inhibiting neuronal death in a subject in need, comprising administering to the subject a therapeutically effective amount of a protein or polypeptide or a pharmaceutical composition comprising said protein or polypeptide, said protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, neuronal 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, and even more preferably within 12 hours after the onset of neuronal death and / or cerebral hemorrhage.

[0026] definition

[0027] In this invention, the following terms have the following meanings.

[0028] The word "approximately" before the value indicates a range of plus or minus 10%.

[0029] "Identity" refers to a measure of the similarity between nucleotide or amino acid sequences. Sequence alignment is performed to obtain the highest possible sequence match. "Identity" itself has a technically accepted meaning and can be calculated using publicly available techniques. While various methods exist for measuring the identity between two nucleic acid sequences or two polypeptide sequences, 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 package.

[0030] For illustration, a nucleic acid having a nucleotide sequence that is at least 95% "identical" to a reference nucleotide sequence means that the nucleotide sequence of the nucleic acid is identical to the reference sequence, except that the nucleic acid sequence may include up to five point mutations per 100 nucleotides relative to the reference nucleotide sequence. In other words, to obtain a nucleic acid with a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such mutations in the reference sequence may occur at the 5' or 3' end of the reference nucleotide sequence or at any position between these end positions, or may be scattered among the individual nucleotides in the reference sequence or in one or more adjacent groups in the reference sequence.

[0031] "Pharmaceutically acceptable excipients" refer to excipients that, when administered to animals, preferably humans, will not produce adverse reactions, allergic reactions, or other adverse effects. These include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. Formulations administered to humans should meet the FDA or EMA official standards for sterility, pyrogenicity, general safety, and purity.

[0032] A polypeptide is any peptide or protein containing two or more amino acids linked together by peptide bonds or modified peptide bonds, forming a peptide isostere. The term "polypeptide" can refer to both short chains, commonly called peptides, oligopeptides, or oligomers, and long chains, commonly called proteins. Polypeptides may contain amino acids other than the 20 genetically encoded amino acids.

[0033] "Protein" refers to a sequence and / or multimeric entity of more than 100 amino acids. The proteins of this invention are not limited to a specific length of product. The terms "peptide" or "protein" do not refer to, but do not exclude, post-expression modifications of proteins, such as glycosylation, acetylation, phosphorylation, etc., as well as other naturally occurring and non-natural modifications known in the art. These modifications are well described in the basic context and in more detailed monographs and a large body of research literature. Modifications can occur at any position in a peptide or protein, including the peptide backbone, amino acid side chains, and amino or carboxyl terms. It should be understood that the same type of modification can be present at multiple sites in a given peptide or protein to the same or different degrees. Furthermore, a given peptide or protein can contain a variety of types of modifications. Peptides or proteins may be branched due to ubiquitination; they may be cyclic, branched, or unbranched. Cyclic, branched, and branched cyclic peptides or proteins can be generated by natural post-translational processes or synthesized. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent linkage of flavins, covalent linkage of hem moiety, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cysteine ​​formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI-anchored protein formation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, isopreneation, racemization, selenoylation, sulfidation, transfer RNA-mediated amino acid addition to proteins (e.g., arginylation), and ubiquitination. Proteins can be complete proteins or their subsequences.

[0034] "Object" refers to a mammal, preferably a human. In one embodiment, the object is a man. In another embodiment, the object is a woman. In one embodiment, the object can be a "patient," i.e., a warm-blooded animal awaiting or receiving medical care, or having been / now / will be a subject of medical procedures, or being monitored for the development of a disease or condition, more preferably a human, said disease or condition preferably being a brain inflammation or a disease related to or caused by brain inflammation. In one embodiment, the object is an adult (e.g., an object over 18 years of age). In another embodiment, the object is a child (e.g., an object under 18 years of age).

[0035] "Therapeutic effective dose" refers to the level or amount of an agent that will not cause significant negative or adverse side effects on the target, and is intended to: (1) delay or prevent a disease or condition, preferably a brain inflammation or a disease associated with or caused by a brain inflammation; (2) slow or stop the progression, aggravation or worsening of one or more symptoms of a disease or condition, preferably a brain inflammation or a disease associated with or caused by a brain inflammation; (3) improve the symptoms of a brain inflammation or a disease or condition associated with a brain inflammation; (4) reduce the severity or incidence of a disease or condition associated with a brain inflammation, or a disease associated with or caused by a brain inflammation; or (5) prevent a disease or condition associated with a brain inflammation, or a disease associated with or caused by a brain inflammation. In one embodiment, the therapeutic effective dose is administered prior to the onset of a brain inflammation, or a disease associated with or caused by a brain inflammation, to serve a preventative purpose.

[0036] The terms "treatment," "management," or "relief" refer to therapeutic treatments and preventative or avoidant measures aimed at preventing or alleviating inflammation of the brain, or a condition associated with or caused by inflammation of the brain. Those requiring treatment include individuals who already have inflammation of the brain, or a condition associated with or caused by inflammation of the brain, as well as individuals who are susceptible to inflammation of the brain, or a condition associated with or caused by inflammation of the brain, or individuals who require prevention of inflammation of the brain, or a condition associated with or caused by inflammation of the brain. If, after receiving a therapeutic amount of protein or peptide according to the method of the invention, a patient exhibits an observable and / or measurable reduction or disappearance of one or more of the following, the subject or mammal is successfully "treated" for brain inflammation, or a disease associated with or caused by brain inflammation, including: a reduction in the number of pathogenic cells; a decrease in the percentage of the total number of pathogenic cells; and / or a degree of relief from one or more symptoms associated with brain inflammation, or a disease associated with or caused by brain inflammation; a reduction in morbidity and mortality; and an improvement in quality of life. The parameters described above for assessing the successful treatment and improvement of brain inflammation, or a disease associated with or caused by brain inflammation, are easily measured using routine procedures familiar to physicians. Detailed Implementation

[0037] This invention relates to the use of a protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1 for the prevention and / or treatment of brain inflammation in subjects in need. In a particular embodiment, the protein or polypeptide of the present invention is used for the prevention of brain inflammation in subjects in need.

[0038] In some embodiments, the protein or polypeptide used according to the invention comprises or is composed of a polypeptide from the salivary glands of the castor beetle or a fragment thereof or a variant thereof, and is therefore referred to as Ir-CPI.

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

[0040] In some embodiments, the protein or polypeptide used according to the present invention has at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1. In some embodiments, the protein or polypeptide used according to the present invention has at least 75% sequence identity with the amino acid sequence SEQ ID NO: 2.

[0041] As used in this article, “at least 75% sequence identity” means at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical.

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

[0043] In some embodiments, the protein or polypeptide used according to the present invention comprises a polypeptide having the amino acid sequence SEQ ID NO: 1. In some embodiments, the protein or polypeptide used according to the present invention comprises a polypeptide having the amino acid sequence SEQ ID NO: 2.

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

[0045] In some embodiments, the protein or polypeptide used according to the present invention has the amino acid sequence SEQ ID NO: 1. In some embodiments, the protein or polypeptide used according to the present invention has the amino acid sequence SEQ ID NO: 2.

[0046] In one embodiment, fragments of proteins or polypeptides used according to the invention are also included in the invention. As used herein, the term "fragment" refers to a polypeptide having an amino acid sequence that is a portion, but not entirely, identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Fragments may be "standalone" or contained within a larger polypeptide, forming portions or regions, most preferably a single, continuous region. Representative examples of polypeptide fragments of the invention include, for example, fragments from about 1 to 20, 21 to 40, 41 to 60, 61 to 80, 81 to 100, and 101 to the ends of the polypeptide, the position numbers corresponding to the amino acid numbers of SEQ ID NO: 1 or SEQ ID NO: 2. In this case, "about" includes a particularly enumerated range of several, 5, 4, 3, 2, or 1 more or fewer amino acids than either or both of the two end values.

[0047] Preferred fragments include, but are not limited to, truncated polypeptides having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, except for cases involving a continuous series of residues containing an amino terminus or a continuous series of residues containing a carboxyl terminus and / or the deletion of a transmembrane region or two consecutive series of residues (one containing an amino terminus and the other containing a carboxyl terminus). Fragments characterized by structural or functional properties are also preferred, such as fragments containing α-helices and α-helical forming regions, β-sheets and β-sheet forming regions, rotations and rotation-forming regions, coils and coil-forming regions, hydrophilic regions, hydrophobic regions, α-amphiphilic regions, β-amphiphilic regions, flexible regions, surface-forming regions, substrate-binding regions, and high antigenic index regions. Other preferred fragments are bioactive fragments. Bioactive fragments are those that mediate the activity of Ir-CPI polypeptides, including fragments with similar activity, enhanced activity, or reduced undesirable activity.

[0048] In one embodiment, all these fragments of proteins or polypeptides retain some of the biological activity of the proteins or polypeptides of the present invention.

[0049] In one embodiment, variants of the protein or polypeptide used according to the invention are also included in the invention. Preferred variants are those that differ from the protein or polypeptide of the invention by conserved amino acid substitutions, i.e., those that are substituted with another residue having similar characteristics. Typical such substitutions are between Ala, Val, Leu, and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; between basic residues Lys and Arg; or between aromatic residues Phe and Tyr. Particularly preferred are variants in which several, five to ten, one to five, or one to two amino acids are substituted, deleted, or added in any combination.

[0050] In one embodiment, the protein or polypeptide of the present invention has a mutated amino acid sequence, said mutation causing glutamine to replace asparagine at position 54 in the amino acid sequence corresponding to SEQ ID NO: 1, to prevent N-glycosylation. In one embodiment, the protein or polypeptide of the present invention has an amino sequence comprising or constituting the sequence of SEQ ID NO: 2.

[0051] In one embodiment, the protein or polypeptide used according to the invention can be prepared in any suitable manner. Examples of peptides prepared in a suitable manner include, but are not limited to, isolated naturally occurring polypeptides, recombinant polypeptides, synthetic polypeptides, or polypeptides prepared by a combination of these methods. Methods for preparing these polypeptides are well known in the art.

[0052] In some embodiments, the protein or polypeptide used according to the invention is a fusion protein comprising: (i) a polypeptide having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) at least one different protein or polypeptide.

[0053] In one embodiment, a protein or polypeptide for the purposes described in the invention is linked to at least one other protein or polypeptide in such a manner to generate a single protein that retains the biological activity of the protein or polypeptide for the purposes described in the invention.

[0054] In one embodiment, the peptides or proteins of the fusion protein can be fused in any order. In another embodiment, the peptides or proteins are situated within a single, continuous polypeptide chain.

[0055] In one embodiment, the fusion protein of the present invention further comprises at least one peptide linker. In one embodiment, the polypeptides or proteins of the fusion protein of the present invention are linked to each other through one or more peptide linkers.

[0056] In one embodiment, the peptide linker provides greater physical separation between the two parts, thereby maximizing the usability of the protein or polypeptide for the purposes described in the invention. The linker peptide may be composed of amino acids, making it flexible or more rigid.

[0057] 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 to 20, 7 to 20, 8 to 20, 9 to 20, or 10 to 20 amino acids. In yet 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.

[0058] Peptide linkers can be either cleavable or non-cleavable in vivo.

[0059] The fusion proteins of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant preparation. For recombinant preparation, one or more nucleic acids encoding, for example, the fusion protein (fragment) described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures.

[0060] The fusion proteins prepared as described herein can be purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion-exchange chromatography (IEC), gel electrophoresis, affinity chromatography, and size exclusion chromatography (MSC). The specific conditions used for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens bound to the fusion protein can be used. The purity of the fusion protein can be determined using any of a variety of well-known analytical methods, including gel electrophoresis and high-performance liquid chromatography (HPLC).

[0061] The present invention also relates to fusion proteins as described above for use in the prevention and / or treatment of brain inflammation.

[0062] In one embodiment, the nucleic acid encoding the fusion protein of the present invention may be expressed as a single nucleic acid encoding the entire fusion protein, or as multiple (e.g., two or more) nucleic acids co-expressed. The polypeptides encoded by the co-expressed nucleic acids may bind together, for example, via disulfide bonds or other means, to form a functional fusion protein.

[0063] In some embodiments, the protein or peptide according to the uses described in the present invention prevents and / or treats brain inflammation. In some embodiments, the protein or peptide according to the uses described in the present invention prevents brain inflammation. In some embodiments, the protein or peptide according to the uses described in the present invention treats brain inflammation.

[0064] As used in this article, the terms "brain inflammation" and "neuritis" are used interchangeably, referring to the onset of inflammation in the central nervous system, which may also involve the activation of glial cells and the secretion of pro-inflammatory cytokines. The process of neuroinflammation affects neurons, microglia, and macroglia.

[0065] In some embodiments, brain inflammation includes or constitutes the release of pro-inflammatory mediators in the brain. Non-limiting examples of pro-inflammatory cytokines include interleukins (ILs) such as IL-1, IL-2, IL-12, IL-17, and IL-18; IFN-γ; and TNF-α.

[0066] In some implementations, brain inflammation includes or constitutes the early activation of resident microglia. As used herein, “microglia” refers to resident macrophages in the central nervous system.

[0067] In some embodiments, brain inflammation includes or constitutes the formation of reactive oxygen species (ROS). Non-limiting examples of ROS include superoxide, hydroxyl radicals, singlet oxygen, nitrous oxide, and peroxides (e.g., hydrogen peroxide). In some embodiments, the formation of ROS triggers oxidative stress.

[0068] In some embodiments, brain inflammation includes or constitutes infiltration of systemic inflammatory cells in the brain. In some embodiments, the systemic inflammatory cells are selected from neutrophils, eosinophils, lymphocytes, plasma cells, and histiocytes. In some embodiments, the systemic inflammatory cells are neutrophils.

[0069] In some implementations, brain inflammation is associated with or triggers excitotoxicity. As used herein, the term "excitotoxicity" refers to a state of abnormal stimulation of neurotransmitter receptors in neurons, which, among other things, leads to alterations in calcium and energy homeostasis and apoptosis.

[0070] In some implementations, brain inflammation is associated with or triggers neuronal cell death.

[0071] In one implementation, the brain inflammation is located in a specific region or site of the brain. Typically, the brain inflammation is located at and / or around the site of a brain injury, such as a brain hemorrhage.

[0072] In another implementation, the brain inflammation is not located in a specific area or part of the brain. In some implementations, the brain inflammation affects the entire brain.

[0073] In some embodiments, brain inflammation is unrelated to the presence or formation of a thrombus. In some embodiments, brain inflammation is not caused by the presence or formation of a thrombus. In some embodiments, brain inflammation does not lead to the presence or formation of a thrombus. In some embodiments, the proteins or peptides described above are not used for the prevention and / or treatment of diseases related to or caused by thrombus formation.

[0074] In some embodiments, the brain inflammation is induced brain inflammation. In some embodiments, the induced brain inflammation is caused by brain injury. In some embodiments, the induced brain inflammation is caused by traumatic brain injury. In some embodiments, the brain inflammation is caused by cerebral hemorrhage.

[0075] In some implementations, brain inflammation is due to brain injury. In some implementations, the brain injury is traumatic brain injury. In some other implementations, the brain injury is non-traumatic brain injury.

[0076] In some embodiments, the brain inflammation is due to acquired brain injury. In some embodiments, the acquired brain injury is a traumatic brain injury. In some other embodiments, the acquired brain injury is a non-traumatic brain injury.

[0077] In some implementations, traumatic brain injury is selected from hemorrhage, contusion, concussion, and penetrating brain injury. In some implementations, traumatic brain injury is cerebral hemorrhage.

[0078] In some embodiments, non-traumatic brain injury is selected from cerebral hemorrhage, ischemic stroke, seizure, infection, brain tumor, hypoxic / hypoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is selected from cerebral hemorrhage, seizure, infection, brain tumor, hypoxic / hypoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is selected from cerebral hemorrhage, seizure, infection, hypoxic / hypoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is selected from cerebral hemorrhage, infection, brain tumor, hypoxic / hypoxic brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, non-traumatic brain injury is cerebral hemorrhage. In some embodiments, non-traumatic brain injury is an autoimmune disease.

[0079] In some embodiments, the infection is selected from viral infections, bacterial infections, fungal infections, parasitic infections, and combinations thereof. In some embodiments, the infection is caused 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.

[0080] In some embodiments, brain injury is selected from cerebral hemorrhage, contusion, concussion, penetrating brain injury, ischemic stroke, seizure, infection, brain tumor, hypoxic / hypoxic brain injury, autoimmune disease, toxicity, and hydrocephalus.

[0081] In some embodiments, brain inflammation occurs or is prone to occur following traumatic brain injury. In some embodiments, brain inflammation occurs or is prone to occur following cerebral hemorrhage. In some embodiments, the protein or polypeptide used according to the invention is used to prevent and / or treat brain inflammation associated with cerebral hemorrhage in subjects of need.

[0082] In certain embodiments, the proteins or peptides of the present invention are used in subjects who require the prevention of brain inflammation that is predisposed to occur after brain injury. In particular embodiments, the proteins or peptides of the present invention are used in subjects who require the prevention of brain inflammation that is predisposed to occur after cerebral hemorrhage.

[0083] In some implementations, the brain inflammation is either acquired or spontaneous.

[0084] In some implementations, the brain inflammation is acquired brain inflammation.

[0085] As used herein, the term “acquired brain inflammation” refers to brain inflammation as described above, where the cause of the inflammation is external to the organism, such as mechanical impact to the head, object penetrating the skull, or infection.

[0086] In some implementations, acquired brain inflammation is caused by traumatic brain injury or infection.

[0087] In some implementations, acquired brain inflammation is caused by traumatic brain injury.

[0088] In some embodiments, traumatic brain injury is selected from hemorrhage, contusion, concussion, penetrating brain injury, and hypoxic brain injury.

[0089] In some implementations, traumatic brain injury is selected from hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, and epidural hemorrhage. In some implementations, primary brain injury is cerebral hemorrhage. In some implementations, primary brain injury is subarachnoid hemorrhage. In some implementations, primary brain injury is subdural hemorrhage. In some implementations, primary brain injury is epidural hemorrhage.

[0090] In some embodiments, traumatic brain injury is a contusion. In some embodiments, traumatic brain injury is a concussion. In some embodiments, traumatic brain injury is a penetrating brain injury. In some embodiments, traumatic brain injury is a hypoxic-ischemic brain injury.

[0091] In some implementations, acquired brain inflammation is caused by an infection. In some implementations, acquired brain inflammation is caused by an infection selected from viral infections, bacterial infections, fungal infections, parasitic infections, and combinations thereof.

[0092] In some implementations, acquired brain inflammation is caused by sepsis and / or septicemia.

[0093] In some implementations, acquired brain inflammation is caused by a viral infection. Non-limiting examples of viral infections that can cause brain inflammation include infections caused by herpes simplex virus, Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, poliovirus, influenza virus, SARS-CoV-2, West Nile virus, enterovirus, chikungunya virus, and dengue virus.

[0094] In some implementations, acquired brain inflammation is caused by a bacterial infection. Non-limiting examples of bacterial infections that can cause brain inflammation include infections caused by bacteria of the genera *Treponema*, *Mycoplasma*, *Neisseria*, *Streptococcus*, and *Listeria*.

[0095] In some implementations, acquired brain inflammation is caused by a fungal infection. Non-limiting examples of fungal infections that can cause brain inflammation include infections caused by fungi of the genera Candida, Cladosporium, Cryptococcus, Histoplasma, and Aspergillus.

[0096] In some implementations, acquired brain inflammation is caused by parasitic infection. Non-limiting examples of parasitic infections that can cause brain inflammation include infections caused by parasites of the genera Plasmodium and Toxoplasma.

[0097] In some implementations, the brain inflammation is spontaneous brain inflammation.

[0098] As used herein, the term “spontaneous brain inflammation” refers to brain inflammation as described above, where the cause of the inflammation is internal to the organism, for example, caused by an autonomous disease or symptom.

[0099] In some implementations, spontaneous brain inflammation is caused by non-traumatic brain injury or autoimmune disease.

[0100] In some implementations, spontaneous brain inflammation is triggered by non-traumatic brain injury. In some implementations, non-traumatic brain injury is selected from spontaneous hemorrhage, brain cancer, and stroke.

[0101] In some embodiments, non-traumatic brain injury is non-traumatic spontaneous hemorrhage. In some embodiments, non-traumatic spontaneous hemorrhage is non-traumatic spontaneous intracerebral hemorrhage or spontaneous subarachnoid hemorrhage. In some embodiments, non-traumatic spontaneous hemorrhage is non-traumatic spontaneous intracerebral hemorrhage. In some embodiments, non-traumatic spontaneous hemorrhage is spontaneous subarachnoid hemorrhage.

[0102] In some implementations, non-traumatic brain injury is brain cancer, such as glioblastoma, oligodendroglioma, and meningioma.

[0103] In some implementations, non-traumatic brain injury is stroke. In some implementations, non-traumatic brain injury is ischemic stroke.

[0104] In some implementations, spontaneous brain inflammation is caused by an autoimmune disease. In some implementations, the autoimmune disease is autoimmune encephalitis. In some implementations, non-traumatic brain injury is caused by an autoimmune disease. In some implementations, the autoimmune disease is autoimmune encephalitis.

[0105] Brain inflammation can be associated with or caused by other conditions or symptoms, such as neuronal cell death, excitotoxicity, microglial activation, oxidative stress, and systemic inflammatory cell infiltration of the brain, such as neutrophils.

[0106] In some embodiments, using the proteins or peptides according to the invention to prevent and / or treat brain inflammation can also treat and / or alleviate another disease, condition, or symptom. In some embodiments, the disease, condition, or symptom is related to brain inflammation. In some embodiments, the disease, condition, or symptom is caused by brain inflammation.

[0107] In some embodiments, the protein or peptide also prevents and / or treats diseases, conditions, or symptoms associated with brain inflammation. In some embodiments, the protein or peptide also prevents and / or treats diseases, conditions, or symptoms caused by brain inflammation.

[0108] In some embodiments, the use of the protein or peptide according to the invention to prevent and / or treat brain inflammation has a positive effect on another disease, condition, or symptom selected from neuronal cell death, excitotoxicity, microglial activation, oxidative stress, and systemic inflammatory cell infiltration of the brain. In some embodiments, the disease, condition, or symptom is selected from neuronal cell death, excitotoxicity, microglial activation, and oxidative stress. As used herein, the expression "positive effect" means that the protein or peptide according to the invention alleviates, reduces, or inhibits symptoms, or partially or completely treats a part of the disease or condition.

[0109] In some embodiments, the use of the proteins or peptides according to the invention has a positive effect on preventing and / or treating brain inflammation and neuronal cell death. In some embodiments, the use of the proteins or peptides according to the invention has a positive effect on preventing and / or treating brain inflammation and excitotoxicity.

[0110] In some embodiments, the use of proteins or peptides according to the invention to prevent and / or treat brain inflammation has a positive effect on microglial cell activation.

[0111] In some embodiments, the use of proteins or peptides according to the invention to prevent and / or treat brain inflammation has a positive effect on oxidative stress. In some embodiments, the use of proteins or peptides according to the invention to prevent and / or treat brain inflammation has a positive effect on the formation of reactive oxygen species.

[0112] In some embodiments, the use of the protein or peptide according to the invention for the prevention and / or treatment of brain inflammation has a positive effect on the infiltration of systemic inflammatory cells into the brain. In some embodiments, the systemic inflammatory cells are selected from neutrophils, eosinophils, lymphocytes, plasma cells, and histiocytes. In some embodiments, the systemic inflammatory cells are neutrophils. In some embodiments, the use of the protein or peptide according to the invention for the prevention and / or treatment of brain inflammation inhibits the recruitment of neutrophils to the brain and / or the formation of neutrophil extracellular traps.

[0113] In some embodiments, the proteins or peptides of the uses described in this invention are used to prevent and / or treat the long-term effects of brain injury. As used herein, the term "long-term effects of brain injury" refers to the pathophysiological consequences of brain injury, i.e., damage that has a long-term effect on at least one cell type, preferably neurons, in the brain, typically for one week, one month, one year, or more than one year. In some embodiments, the proteins or peptides of the uses described in this invention are used to prevent and / or treat the long-term effects of non-traumatic brain injury. In some embodiments, the proteins or peptides of the uses described in this invention are used to prevent and / or treat the long-term effects of traumatic brain injury. In some embodiments, the proteins or peptides of the uses described in this invention are used to prevent and / or treat the long-term effects of hemorrhage-related traumatic brain injury. In some embodiments, the proteins or peptides of the uses described in this invention are used to prevent and / or treat the long-term effects of hemorrhage-related brain injury. In some embodiments, the proteins or peptides of the uses described in this invention are used to prevent and / or treat the long-term effects of cerebral hemorrhage.

[0114] The inventors have demonstrated that the application of the proteins or polypeptides of this invention does not increase bleeding or lesion (edema) volume. This property is advantageous, and the antithrombotic effect of this protein is also unexpected.

[0115] In some embodiments, the proteins or peptides of the present invention are used to prevent and / or treat brain inflammation without increasing the amount of bleeding and / or the volume of lesions (edema). In some embodiments, the brain inflammation is due to or caused by brain hemorrhage. In one embodiment, the proteins or peptides of the present invention are used to prevent and / or treat brain injury following brain hemorrhage. In one embodiment, brain injury occurs after a hemorrhagic stroke. In one embodiment, the proteins or peptides of the present invention are used to prevent and / or treat brain injury with long-term effects following brain hemorrhage.

[0116] The present invention also relates to nucleic acid molecules encoding proteins or polypeptides for the purposes described in the present invention.

[0117] In some implementations, nucleic acids are used to prevent and / or treat brain inflammation or diseases related to brain inflammation.

[0118] In some implementations, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0119] In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is selected from plasmids, naked DNA, granules, F granules, prokaryotic chromosomes (e.g., bacterial artificial chromosomes), eukaryotic chromosomes (e.g., yeast artificial chromosomes or artificial chromosomes), or combinations thereof.

[0120] In some implementations, the nucleic acid molecule is an RNA molecule. In other implementations, the nucleic acid molecule is messenger RNA (mRNA).

[0121] In some embodiments, the nucleic acid molecule comprises or is composed of natural nucleotides, non-natural nucleotides, or combinations thereof. In some embodiments, the nucleic acid molecule comprises or is composed of natural nucleotides. In some embodiments, the natural nucleotides are selected from adenine, guanine, cytosine, thymine, and uracil. In some embodiments, the natural nucleotides are selected from adenine, guanine, cytosine, and thymine. In some embodiments, the natural nucleotides are selected from adenine, guanine, cytosine, and uracil. In some embodiments, the nucleic acid molecule comprises or is composed of non-natural nucleotides (e.g., chemically modified).

[0122] In some embodiments, the nucleic acid molecule is a single-stranded nucleic acid molecule, a double-stranded nucleic acid molecule, or a combination thereof. In some embodiments, the nucleic acid molecule is a single-stranded nucleic acid molecule. In some embodiments, the nucleic acid molecule is a double-stranded nucleic acid molecule.

[0123] In some embodiments, the nucleic acid of the present invention has a nucleotide sequence comprising or constituting the sequence of SEQ ID NO: 3, or a nucleotide sequence comprising or constituting a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid encoding a protein or polypeptide for the use described in the present invention has the amino acid sequence of SEQ ID NO: 3.

[0124] In one embodiment, the nucleic acid encoding the fusion protein of the present invention can be expressed as a single nucleic acid encoding the entire fusion protein, or as multiple (i.e., two or more) nucleic acids co-expressed. The polypeptides encoded by the co-expressed nucleic acids can bind together, for example, via disulfide bonds or other means, to form a functional fusion protein.

[0125] Another object of the present invention is a vector comprising one or more nucleic acids encoding the protein or polypeptide of the present invention for the prevention and / or treatment of brain inflammation and / or diseases related to brain inflammation. In a preferred embodiment, the vector of the present invention is an expression vector.

[0126] The present invention also relates to compositions comprising the protein or polypeptide as described above, one or more nucleic acids or carriers.

[0127] The present invention also relates to pharmaceutical compositions comprising a protein or polypeptide for use in the present invention, a nucleic acid for use in the present invention, a carrier for use in the present invention, or a composition for use in the present invention, and at least one pharmaceutically acceptable excipient. As described above, in one embodiment, the pharmaceutical composition according to the present invention is used for the prevention and / or treatment of brain inflammation in subjects of need.

[0128] In some embodiments, the pharmaceutical composition comprises a protein or polypeptide for the purpose described in the invention, and at least one pharmaceutically acceptable excipient.

[0129] In some embodiments, the pharmaceutical composition comprises a protein or polypeptide and at least one pharmaceutically acceptable excipient for the prevention and / or treatment of brain inflammation, said protein or polypeptide comprising a polypeptide having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1. In some embodiments, the pharmaceutical composition comprises a protein or polypeptide and at least one pharmaceutically acceptable excipient for the prevention and / or treatment of brain inflammation, said protein or polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0130] Pharmaceutically acceptable excipients that may 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), mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (such as sodium carboxymethyl cellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0131] In some embodiments, the pharmaceutical composition also includes another therapeutic agent.

[0132] In some embodiments, the other therapeutic agent has a positive effect on diseases, conditions, or symptoms selected from brain inflammation, neuronal cell death, excitotoxicity, microglia activation, and oxidative stress. In some embodiments, the other therapeutic agent has a positive effect on brain inflammation.

[0133] In some implementations, another therapeutic agent is a drug. In some implementations, another therapeutic agent is an anti-inflammatory drug, such as minocycline or an anti-inflammatory hormone.

[0134] In some implementations, another therapeutic agent includes or constitutes a cell therapeutic agent, such as a stem cell therapeutic agent.

[0135] The present invention also relates to proteins or polypeptides, one or more nucleic acids, carriers, compositions or pharmaceutical compositions as described above for the manufacture of medicaments for the prevention and / or treatment of brain inflammation.

[0136] Another object of the present invention is a medicament comprising the protein or polypeptide, one or more nucleic acids, a carrier, a composition or a pharmaceutical composition as described above, for the prevention and / or treatment of brain inflammation.

[0137] 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 carrier as described above.

[0138] It should be understood that the total daily dosage of the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the severity of the disease being treated and the inflammation of the brain; the activity of the specific reagent used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific reagent used; the duration of treatment; drugs used in combination with or by chance with the specific reagent used; and factors well-known in the medical field. For example, a technique well-known in the art is to start the reagent at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range, from about 10 mg to 10,000 mg per adult per day, preferably from about 100 mg to 5,000 mg per adult per day, and more preferably from about 200 mg to about 2,000 mg per adult per day. Preferably, the composition contains 10 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg, and 2000 mg of the active ingredient for dose adjustment according to symptoms in treating patients. The drug typically contains about 10 mg to about 10,000 mg of the active ingredient, preferably 5 mg to about 5,000 mg, more preferably about 10 mg to about 2,000 mg of the active ingredient. The effective amount of the drug is typically supplied daily at a dose level of about 0.01 mg / kg body weight to about 100 mg / kg body weight, preferably about 0.05 mg / kg body weight to 40 mg / kg body weight daily, more preferably about 0.1 mg / kg body weight to 20 mg / kg body weight daily, and even more preferably about 0.2 mg / kg body weight to 1 mg / kg body weight daily.

[0139] In some embodiments, the protein or peptide, composition, or pharmaceutical composition according to the 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, and even more preferably about 2.5 mg / kg / day to about 25 mg / kg / day. In some embodiments, the protein or peptide, composition, or pharmaceutical composition according to the invention is administered at a dose of about 5 mg / kg / day to about 50 mg / kg / day. In some embodiments, the protein or peptide, composition, or pharmaceutical composition according to the invention is administered at a dose of about 10 mg / kg / day to about 50 mg / kg / day. In some embodiments, the protein or peptide, composition, or pharmaceutical composition according to the invention is administered at a dose of about 25 mg / kg / day.

[0140] In some embodiments, the protein or polypeptide, composition, or pharmaceutical composition according to the invention is administered at a dose of about 20 mg to about 200,000 mg per adult per day, preferably about 20 mg to about 20,000 mg per adult per day, more preferably about 20 mg to about 10,000 mg per adult per day. In some embodiments, the protein or polypeptide, composition, or pharmaceutical composition according to the invention is administered at a dose of about 200 mg to about 200,000 mg per adult per day, preferably about 200 mg to about 20,000 mg per adult per day, more preferably about 200 mg to about 10,000 mg per adult per day. In some embodiments, the protein or polypeptide, composition, or pharmaceutical composition according to the invention is administered at a dose of about 2,000 mg per adult per day.

[0141] The inventors have demonstrated the possibility and safety of rapidly administering the proteins of the present invention following the occurrence of brain inflammation, brain injury, or cerebral hemorrhage. This is particularly beneficial for patients with brain inflammation, brain injury, or cerebral hemorrhage who are prone to thrombotic events, such as bedridden patients. In fact, anticoagulants (such as enoxaparin) cannot be administered within the first 72 hours after a hemorrhage, as they increase the risk of bleeding. The inventors have demonstrated that, unlike enoxaparin, the risk of bleeding does not increase with the addition of the protein of the present invention within 3 days after a hemorrhage (see Example 1). Therefore, the proteins of the present invention can be used within a critical time window to treat brain inflammation and / or brain injury to avoid irreversible effects. For optimal results, a treatment solution for brain inflammation must be provided before the onset of the inflammatory response after a hemorrhage. Current solutions cannot be administered quickly because they increase the risk of bleeding. In contrast, the proteins or peptides of the present invention do not have this disadvantage and can therefore be rapidly administered to reduce the inflammatory response before it causes excessive damage and / or permanent effects.

[0142] Furthermore, the peak of neutrophil infiltration and the rapid growth phase of edema (if present) occur within this 72-hour window. The earlier the size of the edema decreases, the better the patient's outcome.

[0143] Therefore, in some embodiments, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered as soon as possible after the onset of brain inflammation and / or brain injury. In some embodiments, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered rapidly to the subject after exposure to the risk of developing brain inflammation. In some embodiments, the protein or peptide, nucleic acid, carrier, 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 brain inflammation and / or brain injury. In some embodiments, the protein or peptide, nucleic acid, carrier, 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 brain inflammation and / or brain injury. In some embodiments, the protein or peptide, nucleic acid, carrier, 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 brain inflammation and / or brain injury. In other words, in one embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the invention is applied from about 0 hours to about 120 hours after the onset of brain inflammation and / or brain injury, preferably within 96 hours, more preferably within 72 hours, more preferably within 48 hours, more preferably within 24 hours, more preferably within 12 hours, more preferably within 10 hours, more preferably within 9 hours, more preferably within 8 hours, more preferably within 7 hours, and more preferably within 6 hours.

[0144] Therefore, in some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered as soon as possible after the onset of cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is rapidly administered to the subject after cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, carrier, 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 cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, carrier, 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 cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, carrier, 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 cerebral hemorrhage. In other words, in one embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the invention is administered within about 0 hours to about 120 hours after the onset of cerebral hemorrhage, preferably within 96 hours, more preferably within 72 hours, more preferably within 48 hours, more preferably within 24 hours, more preferably within 12 hours, more preferably within 10 hours, more preferably within 9 hours, more preferably within 8 hours, more preferably within 7 hours, and more preferably within 6 hours.

[0145] It should be understood that the earlier the proteins or polypeptides, nucleic acids, carriers, compositions, pharmaceutical compositions or drugs according to the present invention are administered, the better the clinical outcomes will be.

[0146] Therefore, in some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the invention is administered from 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 after the onset of brain inflammation and / or brain injury to about 120 hours, 96 hours or 72 hours.

[0147] Therefore, in some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the invention is administered from 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 after the onset of cerebral hemorrhage to about 120 hours, 96 hours or 72 hours.

[0148] In one embodiment, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered within about 24 hours to about 120 hours after the onset of brain inflammation and / or brain injury, preferably within about 24 hours to about 96 hours, more preferably within about 24 hours to about 72 hours. In another embodiment, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered within about 48 hours to about 120 hours after the onset of brain inflammation and / or brain injury, preferably within about 48 hours to about 96 hours, more preferably within about 48 hours to about 72 hours.

[0149] In one embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered within about 24 hours to about 120 hours after the onset of cerebral hemorrhage, preferably about 24 hours to about 96 hours, more preferably about 24 hours to about 72 hours. In another embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered within about 48 hours to about 120 hours after the onset of cerebral hemorrhage, preferably about 48 hours to about 96 hours, more preferably about 48 hours to about 72 hours.

[0150] In some embodiments, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered from about 0 hours to about 72 hours after the onset of brain inflammation and / or brain injury, preferably from about 2 hours to about 48 hours, more preferably from about 4 hours to about 36 hours, and even more preferably from about 6 hours to about 12 hours. In some embodiments, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered from about 4 hours to about 48 hours after the onset of brain inflammation and / or brain injury, preferably from about 4 hours to about 36 hours, more preferably from about 4 hours to about 12 hours, and even more preferably from about 4 hours to about 8 hours. In some embodiments, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered from about 6 hours to about 48 hours after the onset of brain inflammation and / or brain injury, preferably from about 6 hours to about 36 hours, more preferably from about 6 hours to about 12 hours, and even more preferably from about 6 hours to about 8 hours.

[0151] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered from about 0 hours to about 72 hours after the onset of cerebral hemorrhage, preferably from about 2 hours to about 48 hours, more preferably from about 4 hours to about 36 hours, and even more preferably from about 6 hours to about 12 hours. In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered from about 4 hours to about 48 hours after the onset of cerebral hemorrhage, preferably from about 4 hours to about 36 hours, more preferably from about 4 hours to about 12 hours, and even more preferably from about 4 hours to about 8 hours. In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered from about 6 hours to about 48 hours after the onset of cerebral hemorrhage, preferably from about 6 hours to about 36 hours, more preferably from about 6 hours to about 12 hours, and even more preferably from about 6 hours to about 8 hours.

[0152] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the invention is applied about 0 hours to about 12 hours after the onset of brain inflammation and / or brain injury, 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, and even more preferably about 0 hours to about 6 hours.

[0153] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the invention is administered from about 0 hours to about 12 hours after the onset of cerebral hemorrhage, preferably from about 0 hours to about 10 hours, more preferably from about 0 hours to about 8 hours, even more preferably from about 0 hours to about 7 hours, and even more preferably from about 0 hours to about 6 hours.

[0154] In some embodiments, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered for a maximum of 120 hours, preferably a maximum of 96 hours, and more preferably a maximum of 72 hours after the onset of brain inflammation and / or brain injury. In some embodiments, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered for a maximum of 48 hours, preferably a maximum of 24 hours, more preferably a maximum of 12 hours, even more preferably a maximum of 8 hours, even more preferably a maximum of 7 hours, even more preferably a maximum of 6 hours or less after the onset of brain inflammation and / or brain injury.

[0155] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered at most 120 hours, preferably at most 96 hours, more preferably at most 72 hours after the onset of cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered at most 48 hours, preferably at most 12 hours, more preferably at most 8 hours, even more preferably at most 7 hours, even more preferably at most 6 hours or less after the onset of cerebral hemorrhage.

[0156] For administration to a target, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention will be formulated for administration to the target. The protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention can be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, orally, vaginally, or via 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.

[0157] In one embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is in a form suitable for topical application.

[0158] Suitable forms for topical application include, but are not limited to, liquid compositions, paste compositions or solid compositions, and more specifically, aqueous solutions, drops, eye drops, eye washes, dispersants, sprays, microcapsules, microparticles or nanoparticles, polymer patches or controlled-release patches.

[0159] In one embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention comprises one or more pharmaceutically acceptable carriers suitable for topical application.

[0160] In one embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the invention is in a form suitable for injection, for example, for intraocular, intramuscular, subcutaneous, intradermal, transdermal or intravenous injection or infusion.

[0161] Examples of injectable forms include, but are not limited to, solutions such as sterile aqueous solutions, dispersants, emulsions, suspensions, and solid forms suitable for preparing solutions or suspensions, such as powders, liposomes, etc., which are added before use.

[0162] The aseptic injectable form of the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the present invention can be an aqueous suspension or an oil suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Aseptic injectable formulations can also be aseptic injectable solutions or aseptic injectable suspensions in non-toxic, parenteral-acceptable diluents or solvents. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixed oils are commonly used as solvents or suspension media. For this purpose, any unadded fixed oil containing synthetic monoglycerides or diglycerides can be used. Fatty acids such as oleic acid and its glyceride derivatives can be used as pharmaceutically acceptable natural oils for the preparation of injectables, such as olive oil or castor oil, particularly their polyoxyethylated versions. These oil solutions or oil suspensions may also contain diluents or dispersants of long-chain alcohols, such as carboxymethyl cellulose or similar dispersants, which are commonly used to formulate pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers, are typically used to manufacture pharmaceutically acceptable solids, liquids, or other dosage forms that may also be used in formulations.

[0163] In one embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug is administered to the recipient at least once daily. For example, the compositions, pharmaceutical compositions, or drugs of the present invention may be administered once daily, twice daily, or three times daily. In a preferred embodiment, the compositions, pharmaceutical compositions, or drugs of the present invention are administered to the recipient once daily.

[0164] In another embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug is administered to a subject in need at least once a week. For example, the compositions, pharmaceutical compositions, or drugs 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.

[0165] In another embodiment, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug of the present invention is administered to a subject of need once a month, twice a month, once every two months, once every two or three months, twice a year or once a year.

[0166] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered to a subject in need prior to exposure to a risk of brain inflammation. In one embodiment, the term "prior" means at least one week prior to exposure. In another embodiment, the term "prior" means five days, four days, three days, two days, or one day prior to exposure. In another embodiment, the term "prior" means 24 hours, 18 hours, 15 hours, 12 hours, 6 hours, 4 hours, 2 hours, or 1 hour prior to exposure. In yet another embodiment, the term "prior" means less than one hour prior to exposure, such as 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or at the moment of exposure.

[0167] In another embodiment, the protein or peptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is rapidly administered to a subject of need after exposure to a risk of brain inflammation. In one embodiment, the term "after" means 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 45 minutes after exposure. In another embodiment, the term "after" means 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 15 hours, 18 hours, or 14 hours after exposure. In another embodiment, the term "after" means 1 day, 2 days, 3 days, 4 days, or 5 days after exposure. In yet another embodiment, the term "after" means one week or more after exposure.

[0168] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the invention is administered continuously, typically by perfusion.

[0169] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is continuously administered 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, preferably 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, carrier, composition, pharmaceutical composition, or drug according to the invention is continuously administered for about 24 hours to about 96 hours, preferably about 48 hours to about 96 hours, preferably by perfusion for about 24 hours to about 96 hours, more preferably about 48 hours to about 96 hours. In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is continuously administered for about 24 hours to about 72 hours, preferably about 48 hours to about 72 hours, more preferably by perfusion for about 24 hours to about 72 hours, more preferably about 48 hours to about 72 hours.

[0170] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered continuously for at least about 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours, preferably by perfusion. In certain embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered continuously for at least about 48 hours, preferably by perfusion.

[0171] In some embodiments, the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is administered to the subject at a first dose, followed by continuous administration at the same dose via perfusion. Within the scope of the invention, the first dose is referred to herein as the initial dose. In some embodiments, the dose of the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention is greater than or equal to, preferably greater than, the perfused dose in the initial dose.

[0172] The present invention also relates to kits comprising proteins or polypeptides, nucleic acids, carriers, compositions, pharmaceutical compositions or drugs according to the present invention for the prevention and / or treatment of brain inflammation.

[0173] In one embodiment, the kit of the present invention further comprises tools for applying proteins or peptides, nucleic acids, carriers, compositions, pharmaceutical compositions or drugs to a subject in need according to the present invention.

[0174] In one embodiment, the kit of the present invention further includes instructions for applying the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the present invention to the subject.

[0175] The present invention also relates to a method for preventing and / or treating brain inflammation in a subject in need, comprising administering to the subject a therapeutically effective amount of a protein or polypeptide, a nucleic acid encoding the protein or polypeptide, a carrier comprising the nucleic acid, a pharmaceutical composition or drug comprising the protein or polypeptide, the nucleic acid or the carrier, wherein the protein or polypeptide has at least 75% sequence identity with the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.

[0176] In one embodiment, the method includes administering to a subject a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention. In one embodiment, the therapeutically effective dose is composed of a dose of about 2.5 mg / kg / day to about 250 mg / kg / day. In one embodiment, the therapeutically effective dose is composed of a dose of about 2.5 mg / kg / day to about 100 mg / kg / day. In one embodiment, the therapeutically effective dose is composed of a dose of about 2.5 mg / kg / day to about 50 mg / kg / day. In one embodiment, the therapeutically effective dose is composed of a dose of about 2.5 mg / kg / day to about 25 mg / kg / day. In one embodiment, the therapeutically effective dose is composed of a dose of about 5 mg / kg / day to about 50 mg / kg / day.

[0177] In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within 120 hours, preferably within 96 hours, and more preferably within 72 hours after the onset of brain inflammation and / or brain injury. In other words, in one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject from about 0 hours to about 120 hours, preferably within 96 hours, and more preferably within 72 hours after the onset of brain inflammation and / or brain injury.

[0178] In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within 48 hours, preferably within 24 hours, more preferably within 15 hours, and even more preferably within 12 hours after the onset of brain inflammation and / or brain injury. In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within 10 hours, 8 hours, or 6 hours after the onset of brain inflammation and / or brain injury. In some embodiments, the protein or peptide is administered within approximately 6 to approximately 8 hours after the onset of brain inflammation and / or brain injury.

[0179] In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide, or pharmaceutical composition according to the invention, to the subject within 120 hours, preferably within 96 hours, and more preferably within 72 hours after the onset of cerebral hemorrhage. In other words, in one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide, or pharmaceutical composition according to the invention, to the subject within about 0 hours to about 120 hours, preferably within 96 hours, and more preferably within 72 hours after the onset of cerebral hemorrhage.

[0180] In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within 48 hours, preferably within 24 hours, more preferably within 15 hours, and even more preferably within 12 hours after the onset of cerebral hemorrhage. In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within 10 hours, 8 hours, or 6 hours after the onset of cerebral hemorrhage. In some embodiments, the protein or polypeptide is administered within about 6 hours to about 8 hours after the onset of cerebral hemorrhage.

[0181] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject from about 0 to about 120 hours, preferably from about 0 to about 96 hours, and more preferably from about 0 to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject from about 1 to about 120 hours, preferably from about 1 to about 96 hours, and more preferably from about 1 to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject from about 2 to about 120 hours, preferably from about 2 to about 96 hours, and more preferably from about 2 to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject from about 3 to about 120 hours, preferably from about 3 to about 96 hours, and more preferably from about 3 to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 4 hours to about 120 hours, preferably from about 4 hours to about 96 hours, more preferably from about 4 hours to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 5 hours to about 120 hours, preferably from about 5 hours to about 96 hours, more preferably from about 5 hours to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 6 hours to about 120 hours, preferably from about 6 hours to about 96 hours, more preferably from about 6 hours to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 7 hours to about 120 hours, preferably from about 7 hours to about 96 hours, more preferably from about 7 hours to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 8 hours to about 120 hours, preferably from about 8 hours to about 96 hours, more preferably from about 8 hours to about 72 hours after the onset of brain inflammation and / or brain injury. In another embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 9 hours to about 120 hours, preferably from about 9 hours to about 96 hours, more preferably from about 9 hours to about 72 hours after the onset of brain inflammation and / or brain injury.In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 10 hours to about 120 hours, preferably from about 10 hours to about 96 hours, more preferably from about 10 hours to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 12 hours to about 120 hours, preferably from about 12 hours to about 96 hours, more preferably from about 12 hours to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 24 hours to about 120 hours, preferably from about 24 hours to about 96 hours, more preferably from about 24 hours to about 72 hours after the onset of brain inflammation and / or brain injury. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 48 hours to about 120 hours, preferably from about 48 hours to about 96 hours, more preferably from about 48 hours to about 72 hours after the onset of brain inflammation and / or brain injury.

[0182] In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 0 to 120 hours after the onset of brain inflammation and / or brain injury, preferably from about 0 to 36 hours, more preferably from about 0 to about 12 hours. In another embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 0 to 36 hours after the onset of brain inflammation and / or brain injury, preferably from about 0 to about 24 hours, more preferably from about 0 to about 12 hours.

[0183] In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 0 to about 24 hours, preferably from about 0 to about 20 hours, more preferably from about 0 to about 16 hours after the onset of brain inflammation and / or brain injury. In another embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 0 to about 15 hours, preferably from about 0 to about 14 hours, more preferably from about 0 to about 13 hours after the onset of brain inflammation and / or brain injury.

[0184] In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject from about 0 to about 12 hours after the onset of brain inflammation and / or brain injury.

[0185] In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 0 to about 24 hours, preferably from about 4 to about 20 hours, more preferably from about 8 to about 16 hours after the onset of brain inflammation and / or brain injury. In another embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 10 to about 14 hours, preferably from about 10 to about 13 hours, more preferably from about 11 to about 13 hours after the onset of brain inflammation and / or brain injury. In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to a subject from about 11 to about 12 hours after the onset of brain inflammation and / or brain injury.

[0186] In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject from about 2 hours to about 24 hours, preferably from about 4 hours to about 24 hours, more preferably from about 8 hours to about 24 hours after the onset of brain inflammation and / or brain injury. In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject from about 10 hours to about 24 hours after the onset of brain inflammation and / or brain injury.

[0187] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject about 10 to 16 hours, preferably about 11 to 14 hours, more preferably about 11 to 13 hours after the onset of brain inflammation and / or brain injury.

[0188] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject approximately 12 hours after the onset of brain inflammation and / or brain injury.

[0189] In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject up to 24 hours, preferably up to 15 hours, more preferably up to 12 hours after the onset of brain inflammation and / or brain injury.

[0190] In one embodiment, the method includes administering a therapeutically effective amount of the protein, polypeptide, or pharmaceutical composition according to the invention to the subject within about 0 to about 120 hours, preferably within about 0 to about 96 hours, and more preferably within about 0 to about 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein, polypeptide, or pharmaceutical composition according to the invention to the subject within about 1 to about 120 hours, preferably within about 1 to about 96 hours, and more preferably within about 1 to about 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein, polypeptide, or pharmaceutical composition according to the invention to the subject within about 2 to about 120 hours, preferably within about 2 to about 96 hours, and more preferably within about 2 to about 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein, polypeptide, or pharmaceutical composition according to the invention to the subject within about 3 to about 120 hours, preferably within about 3 to about 96 hours, and more preferably within about 3 to about 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 4 hours to approximately 120 hours, preferably within approximately 4 hours to approximately 96 hours, and more preferably within approximately 4 hours to approximately 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 5 hours to approximately 120 hours, preferably within approximately 5 hours to approximately 96 hours, and more preferably within approximately 5 hours to approximately 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 6 hours to approximately 120 hours, preferably within approximately 6 hours to approximately 96 hours, and more preferably within approximately 6 hours to approximately 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 7 hours to approximately 120 hours, preferably within approximately 7 hours to approximately 96 hours, and more preferably within approximately 7 hours to approximately 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 8 to about 120 hours, preferably within about 8 to about 96 hours, and more preferably within about 8 to about 72 hours after the onset of cerebral hemorrhage. In another embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 9 to about 120 hours, preferably within about 9 to about 96 hours, and more preferably within about 9 to about 72 hours after the onset of cerebral hemorrhage.In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 10 to 120 hours, preferably within approximately 10 to 96 hours, and more preferably within approximately 10 to 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 12 to 120 hours, preferably within approximately 12 to 96 hours, and more preferably within approximately 12 to 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 24 to 120 hours, preferably within approximately 24 to 96 hours, and more preferably within approximately 24 to 72 hours after the onset of cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 48 to 120 hours, preferably within approximately 48 to 96 hours, and more preferably within approximately 48 to 72 hours after the onset of cerebral hemorrhage.

[0191] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 0 to about 120 hours, preferably within about 0 to about 36 hours, and more preferably within about 0 to about 12 hours after the onset of cerebral hemorrhage. In another embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 0 to about 36 hours, preferably within about 0 to about 24 hours, and more preferably within about 0 to about 12 hours after the onset of cerebral hemorrhage.

[0192] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 0 to about 24 hours, preferably within about 0 to about 20 hours, and more preferably within about 0 to about 16 hours after the onset of cerebral hemorrhage. In another embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 0 to about 15 hours, preferably within about 0 to about 14 hours, and more preferably within about 0 to about 13 hours after the onset of cerebral hemorrhage.

[0193] In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 0 to approximately 12 hours after the onset of cerebral hemorrhage.

[0194] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within 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 cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 10 to about 14 hours, preferably within about 10 to about 13 hours, more preferably within about 11 to about 13 hours after the onset of cerebral hemorrhage. In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 11 to about 12 hours after the onset of cerebral hemorrhage.

[0195] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 2 hours to about 24 hours, preferably about 4 hours to about 24 hours, and more preferably about 8 hours to about 24 hours after the onset of cerebral hemorrhage. In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 10 hours to about 24 hours after the onset of cerebral hemorrhage.

[0196] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within 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 cerebral hemorrhage.

[0197] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 12 hours after the onset of cerebral hemorrhage.

[0198] In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide, or pharmaceutical composition according to the invention, to the subject after the onset of cerebral hemorrhage, preferably 12 hours later, preferably up to 15 hours later, more preferably up to 12 hours later.

[0199] In some implementations, the method is not used for the prevention and / or treatment of thrombosis. In some implementations, brain inflammation is not related to the presence or formation of thrombosis.

[0200] In some implementations, the method includes the following steps:

[0201] - Administering to the subject an initial dose comprising a therapeutically effective amount of the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention, and

[0202] - Administering a therapeutically effective amount of the protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention to the subject via perfusion.

[0203] The initial dose is greater than or equal to the perfusion dose.

[0204] The present invention also relates to methods for preventing and / or treating brain inflammation without increasing the amount of bleeding and / or the volume of the lesion (edema). The present invention further relates to methods for preventing and / or treating brain injury following cerebral hemorrhage, preferably brain injury following hemorrhagic stroke.

[0205] The present invention also relates to methods for preventing and / or treating diseases, conditions, or symptoms associated with brain inflammation, comprising administering to a subject a therapeutically effective amount of a protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention. In some embodiments, the method is used for preventing and / or treating diseases, conditions, or symptoms caused by brain inflammation, comprising administering to a subject a therapeutically effective amount of a protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition, or drug according to the invention. In some embodiments, the disease, condition, or symptom is selected from neuronal cell death, excitotoxicity, microglia activation, and oxidative stress.

[0206] In some embodiments, the method of the present invention is used to alleviate symptoms in subjects suffering from traumatic or non-traumatic brain injury.

[0207] The present invention also 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, carrier, composition, pharmaceutical composition, or drug according to the invention.

[0208] Another object of the present invention is a method for preventing, reducing, decreasing and / or inhibiting neuronal death in a desired subject, comprising administering to the subject a therapeutically effective amount of a protein or polypeptide, nucleic acid, carrier, composition, pharmaceutical composition or drug according to the present invention.

[0209] In one implementation, the subject in need is a subject who has or is susceptible to brain inflammation. In one implementation, the method is used to prevent, reduce, decrease, and / or inhibit neuronal death that occurs or is susceptible to in the subject in need following brain inflammation.

[0210] In one implementation, the subject in need is a person who has or is susceptible to cerebral hemorrhage. In one implementation, the method is used to prevent, reduce, decrease, and / or inhibit neuronal death that occurs or is susceptible to following cerebral hemorrhage in the subject in need.

[0211] In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within 120 hours, preferably within 96 hours, more preferably within 72 hours after neuronal death and / or cerebral hemorrhage. In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within 48 hours, preferably within 24 hours, more preferably within 15 hours, and even more preferably within 12 hours after neuronal death and / or cerebral hemorrhage. In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within 10 hours, 8 hours, or 6 hours after neuronal death and / or cerebral hemorrhage. In some embodiments, the protein or peptide is administered within about 6 hours to about 8 hours after neuronal death and / or cerebral hemorrhage.

[0212] In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 0 to about 120 hours, preferably within about 0 to about 96 hours, and more preferably within about 0 to about 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 1 to about 120 hours, preferably within about 1 to about 96 hours, and more preferably within about 1 to about 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 2 to about 120 hours, preferably within about 2 to about 96 hours, and more preferably within about 2 to about 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 3 to about 120 hours, preferably within about 3 to about 96 hours, and more preferably within about 3 to about 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within approximately 4 hours to approximately 120 hours, preferably within approximately 4 hours to approximately 96 hours, and more preferably within approximately 4 hours to approximately 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within approximately 5 hours to approximately 120 hours, preferably within approximately 5 hours to approximately 96 hours, and more preferably within approximately 5 hours to approximately 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within approximately 6 hours to approximately 120 hours, preferably within approximately 6 hours to approximately 96 hours, and more preferably within approximately 6 hours to approximately 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within approximately 7 hours to approximately 120 hours, preferably within approximately 7 hours to approximately 96 hours, and more preferably within approximately 7 hours to approximately 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 8 hours to about 120 hours, preferably within about 8 hours to about 96 hours, and more preferably within about 8 hours to about 72 hours after the onset of neuronal death and / or cerebral hemorrhage.In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within approximately 9 to 120 hours, preferably within approximately 9 to 96 hours, and more preferably within approximately 9 to 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within approximately 10 to 120 hours, preferably within approximately 10 to 96 hours, and more preferably within approximately 10 to 72 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within approximately 12 to 120 hours, preferably within approximately 12 to 96 hours, and more preferably within approximately 12 to 72 hours after the onset of neuronal death and / or cerebral hemorrhage.

[0213] In some embodiments, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 0 to approximately 12 hours after neuronal death and / or onset of cerebral hemorrhage.

[0214] In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 0 to about 24 hours, preferably within about 4 to about 20 hours, and more preferably within about 8 to about 16 hours after the onset of neuronal death and / or cerebral hemorrhage. In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 10 to about 14 hours, preferably within about 10 to about 13 hours, and more preferably within about 11 to about 13 hours after the onset of neuronal death and / or cerebral hemorrhage. In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 11 to about 12 hours after the onset of neuronal death and / or cerebral hemorrhage.

[0215] In one embodiment, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 2 hours to about 24 hours, preferably within about 4 hours to about 24 hours, and more preferably within about 8 hours to about 24 hours after neuronal death and / or the onset of cerebral hemorrhage. In some embodiments, the method includes administering a therapeutically effective amount of the protein or peptide or pharmaceutical composition according to the invention to the subject within about 10 hours to about 24 hours after neuronal death and / or the onset of cerebral hemorrhage.

[0216] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within about 10 to about 16 hours, preferably within about 11 to about 14 hours, and more preferably within about 11 to about 13 hours after the onset of neuronal death and / or cerebral hemorrhage.

[0217] In one embodiment, the method includes administering a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the invention to the subject within approximately 15 hours, preferably within 12 hours, following the onset of neuronal death and / or cerebral hemorrhage.

[0218] Another object of the present invention is a method for preventing and / or treating diseases involving neuronal death, diseases related to neuronal death, or diseases caused by neuronal death in subjects in need, comprising administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the present invention.

[0219] The present invention also relates to proteins or polypeptides, nucleic acids, carriers, compositions, pharmaceutical compositions or drugs according to the invention for use in the prevention and / or treatment of diseases involving neuronal death, diseases related to neuronal death or diseases caused by neuronal death.

[0220] In one implementation, the disease involving neuronal death, the disease associated with neuronal death, or the disease caused by neuronal death is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, ischemic stroke, hemorrhagic stroke, prion disease (Kreutzfeldt-Jacob disease), amyotrophic lateral sclerosis, motor neuron disease, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, epilepsy, brain tumor, cryptogenic stroke, brainstem stroke, transient ischemic attack, Batten disease, Friedreich ataxia, Alexander disease, chronic traumatic encephalopathy, minor stroke, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis.

[0221] In one implementation, the disease involving neuronal death, the disease associated with neuronal death, or the disease caused by neuronal death is selected from ischemic stroke, hemorrhagic stroke, prion disease (Kreutzfeldt-Jacob disease), amyotrophic lateral sclerosis, motor neuron disease, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, cryptogenic stroke, brainstem stroke, transient ischemic attack, Batten disease, Friedreich ataxia, Alexander disease, chronic traumatic encephalopathy, minor stroke, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis.

[0222] In one implementation, the disease involving neuronal death, related to neuronal death, or caused by neuronal death is selected from prion disease (Kreutzfeldt-Jacob disease), amyotrophic lateral sclerosis, motor neuron disease, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, Batten disease, Friedreich ataxia, Alexander disease, chronic traumatic encephalopathy, minor stroke, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis.

[0223] In one implementation, the disease involving neuronal death, related to neuronal death, or caused by neuronal death is selected from prion disease (Kreutzfeldt-Jacob disease), amyotrophic lateral sclerosis, motor neuron disease, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, Batten disease, Friedreich ataxia, Alexander disease, chronic traumatic encephalopathy, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis. Attached Figure Description

[0224] Figures 1A to 1D This is a set of box plots showing the lesion volume versus hemorrhage amount measured on days 1 and 3 after induced cerebral hemorrhage by injection of collagenase into the right striatum of mice. Mice were treated with Ir-CPI (injection + perfusion), enoxaparin (injection + perfusion with NaCl), or PBS alone (injection + perfusion). Figure 1A and Figure 1B The lesion volumes on day 1 and day 3 are shown respectively. Figure 1C and Figure 1D Bleeding amounts on days 1 and 3 are shown separately. Boxes show the median and quartiles. Whiskers indicate the minimum and maximum values. Independent values ​​are plotted. Thick lines represent the mean. On day 1, N=8 mice per group. On day 3, n=7 for the PBS group, n=6 for the Ir-CPI group and the enoxaparin group. *p<0.05 (Kruskal-Wallis test and Dunn's multiple comparison test).

[0225] Figures 2A to 2C This is a set of box plots showing the quantification of average labeled cells in the hemorrhage area (anterior / injection site / posterior site) on day 3 after induced cerebral hemorrhage. Mice were treated with Ir-CPI (injection + perfusion), enoxaparin (injection + perfusion with NaCl), or PBS alone (injection + perfusion). Figure 2A The number of MPO-immunostaining cells is shown. Figure 2B The number of Ly6G immunostaining positive cells (neutrophils) is shown. Figure 2CThe number of cells positive for Ly6G, H3Cit, and MPO immunostaining (neutrophils releasing NETs) is shown. Boxes indicate the median and quartiles. Whiskers indicate the minimum and maximum values. Independent values ​​are plotted. Thick lines represent the mean. N=5 mice per group. *p<0.05 (unpaired t-test).

[0226] Figure 3 This is a set of box plots showing the quantitative number of neutrophils releasing NET at the collagenase injection site on day 3 after induced cerebral hemorrhage (number of cells positive for Ly6G, H3Cit, and MPO immunostaining). Mice were treated with Ir-CPI (injection + perfusion), enoxaparin (injection + perfusion with NaCl), or PBS alone (injection + perfusion). Boxes show the median and quartiles. Whiskers show the minimum and maximum values. Independent values ​​are plotted. Thick lines represent the mean. N=5 mice per group. *p<0.05 (unpaired t-test).

[0227] Figures 4A to 4B This demonstrates that Ir-CPI reduced the average value of the hemorrhage area in the brain of mice with cerebral hemorrhage (anterior region, injection site, and posterior region). Figure 4A and injection site () Figure 4B A set of box plots showing neuronal death. Boxes represent the median and quartiles. Thick lines represent the mean. Whisker lines represent the minimum and maximum values. Independent data are shown [day 3, n=7 mice (PBS / PBS), n=6 mice (Ir-CPI / Ir-CPI or enoxaparin / NaCl)]. p-values ​​were calculated using the Kruskal-Wallis test and Dunn's multiple comparison test. *p<0.05.

[0228] Example

[0229] The present invention is further illustrated by the following embodiments.

[0230] Example 1: Risk assessment of Ir-CPI in a mouse model of intracerebral hemorrhage

[0231] Materials and methods

[0232] Mouse model of cerebral hemorrhage

[0233] In adult male Swiss mice, intracranial hemorrhage (ICH) was induced by injecting a standard amount of bacterial collagenase into the right striatum. The injection of collagenase resulted in the destruction of the extracellular matrix of the basal layer.

[0234] After anesthesia and jugular duct localization, the mouse was positioned at the Sphinx location in a stereotactic frame. Under aseptic conditions, a skin incision was made approximately one centimeter long at the top of the skull. The skull was cleaned to locate the anterior fontanelle (Bregma). The injection coordinates were then calculated from the anterior fontanelle based on a brain atlas (right striatum: anterior fontanelle +0.5 mm; medial suture -2.5 mm; skull (dura mater) plane downwards -3 mm). A hole was drilled in the skull, and a glass needle tip was inserted into the right striatum using a micromanipulator. A standard volume (0.04 IU in 0.5 μL of sterile 0.9% NaCl solution) of bacterial collagenase (collagenase from Clostridium histolyticum, reference C2399, Sigma-Aldrich®) was slowly injected. The needle was then slowly removed, and the skull skin was sutured.

[0235] Application of treatment

[0236] Immediately after stroke initiation, administer a bolus injection (PBS, Ir-CPI, or enoxaparin). Then, continuously perfuse (PBS or Ir-CPI) for 72 hours using an ALZET® osmotic pump to ensure constant plasma drug levels. Animals receiving an enoxaparin bolus injection (Lovenox®; Sanofi Aventis) (20 mg / kg) after ICH initiation were continuously perfused with saline solution (0.9% NaCl) for 72 hours. Ir-CPI (batch DP B3012503) was administered via intravenous bolus injection at 30.7 mg / kg, followed by continuous intravenous perfusion at 8.3 mg / kg / hour for 72 hours. Mice injected with the drug carrier received an intravenous bolus injection of PBS, followed by continuous PBS perfusion for 72 hours.

[0237] Magnetic resonance imaging

[0238] Magnetic resonance imaging (MRI) was used to assess the amount of intracranial hemorrhage and lesion volume at 24 hours and 3 days (D3) after stroke. The MRI device was a 7T-Pharmascan MRI system (Bruker BioSpin MRI GmbH, Ettlinger, Germany), equipped with a volume transfer coil and a surface receiving coil, operated by Paravision© V6.

[0239] Images were analyzed using ImageJ v1.52 (NIH, Bethesda, USA). The research assistants responsible for image analysis were unaware of group assignments. T2* images were analyzed to measure cerebral hemorrhage. For each mouse at each stage, the hemorrhage area—shown as a low-signal region on the image—was measured on a slice. For each animal, the hemorrhage volume was calculated as: sum of measured areas × slice thickness. Hemorrhage volume is expressed in mm. 3 express.

[0240] T2W images were analyzed to determine lesion volume. For each mouse at each stage, the lesion volume—shown as a high-signal area on the image—was measured on a slide. For each animal, hemorrhage was estimated as follows: sum of measured areas × slide thickness. Hemorrhage volume is expressed in mm. 3 express.

[0241] result

[0242] Compared to the control group (PBS), the Ir-CPI treatment group did not show an increase in striatal lesion (edema) volume or hemorrhage on days 1 and 3, unlike the group treated with enoxaparin (low molecular weight heparin, LMWH). Figures 1A to 1D Therefore, unlike enoxaparin, Ir-CPI perfusion for 72 hours is safe.

[0243] Example 2: Anti-inflammatory effect of Ir-CPI in a mouse model of cerebral hemorrhage

[0244] Materials and methods

[0245] Immunostaining

[0246] Immunostaining was performed on brain slices to assess neutrophil infiltration and NET expression. Brain slices were obtained from mice previously used to measure the effect of treatment on lesion volume and hemorrhage (see Example 1). MPO is a marker expressed by activated neutrophils, NETs, ​​microglia, and monocytes / macrophages. Ly6G is a neutrophil-specific marker. Ly6G (anti-mouse Ly-6G antibody, clone 1A8, rat monoclonal IgG2a – catalog number #60031 STEMCELL™), H3Cit (anti-histone H3 (citrulline R2+R8+R17) antibody – catalog number #ab5103 ABCAM), and MPO (anti-myeloperoxidase antibody – catalog number #AF3667BIO-TECHNE) were also used. ® The cells were stained with DNA (DAPI) to assess and identify neutrophils releasing extracellular traps (NETs). The research assistants responsible for the analysis were unaware of the group assignments.

[0247] result

[0248] In the entire hemorrhage area (average of the pre-injection site-post-hemorrhagic zone), unlike mice treated with PBS, a significant reduction in MPO-expressing cells was observed in mice treated with Ir-CPI. MPO is a marker of activated neutrophils, NETs, ​​microglia, and monocytes / macrophages. Figure 2A Furthermore, compared with mice administered PBS, Ir-CPI significantly reduced neutrophil infiltration (Ly6G+ cells). Figure 2B Nevertheless, when assessing the entire hemorrhage area, a decreasing trend in the number of neutrophils releasing NET (Ly6G+H3Cit+MPO+ cells) was observed in both the Ir-CPI and enoxaparin groups. Figure 2C ).

[0249] Furthermore, when only the injection site was assessed, a significant reduction in the number of neutrophils releasing NET(Ly6G+H3Cit+MPO+ cells) was observed in mice treated with Ir-CPI compared to mice administered PBS. Figure 3 Enoxaparin had no statistically significant effect on these cells.

[0250] Example 3: Effects of Ir-CPI on neuronal death

[0251] Materials and methods

[0252] Brain sections were stained with Fluoro-Jade® C (TR-100-FJ, Tebu-Bio) to assess and locate degenerated neurons. The brain was cut into coronal sections (10 μm sections) using a cryostat. Three (3) sections were stained for each animal, corresponding to the collagenase injection site, 600 μm anterior to the injection site, and 600 μm posterior to the injection site.

[0253] First, immerse the slides containing frozen tissue sections in an 80% ethanol solution containing 1% sodium hydroxide for 5 minutes. Then rinse with 70% ethanol for 2 minutes, rinse with distilled water for 2 minutes, and incubate in a 0.06% potassium permanganate solution for 10 minutes. After rinsing with water for 2 minutes, transfer the sections to a Fluoro-Jade® C (TR-100-FJ, Tebu-Bio) solution in the dark for 10 minutes. Dilute appropriately by preparing a 0.01% dye stock solution in distilled water. The working solution can be stored at 4°C to 8°C for a maximum of 4 hours.

[0254] To label nuclear DNA, 4',6-diamidinyl-2-phenylindole (DAPI; 0.01% staining solution D) was added to Fluoro-Jade® staining solution C. The sections were then rinsed three times with distilled water for one minute each time. Excess water was drained onto paper towels, and the sections were air-dried in the dark at 50°C on a leaf heater for at least 5 minutes. The air-dried sections were then bleached with xylene for at least one minute and then coated with the non-fluorescent sample mounting agent Fluoromount-G™ (00-4958-02; Invitrogen™).

[0255] Fluoro-Jade® C was observed using epifluorescence microscopy (Leica DM6 B) with blue light or a 488 nm laser (excitation peak: 495 nm; emission peak: 521 nm), and DAPI-conferred blue nuclear labeling was observed with UV excitation (emission peak: 340 nm; excitation peak: 488 nm). Images were acquired and analyzed in four regions of each slice using ImageJ software (version 1.52a) to qualitatively assess neuronal death. The research assistants responsible for the analysis were unaware of the group assignments.

[0256] result

[0257] Fluoro-Jade® C staining was used to assess neuronal degeneration in the brains of mice treated with PBS, Ir-CPI, or enoxaparin.

[0258] In the hemorrhage area (average of anterior, injection site, and posterior), Ir-CPI reduced the average number of degenerated neurons compared to mice treated with PBS. Figure 4A At the injection site ( Figure 4B Compared with control mice receiving PBS, Ir-C significantly reduced the number of degenerated neurons (p < 0.05).

[0259] 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 brain inflammation occurring or susceptible to occurring 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 an 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 brain inflammation is caused by acquired brain damage.

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

5. The protein or polypeptide for use according to any one of claims 1 to 3, wherein the brain inflammation 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 brain inflammation is caused by cerebral hemorrhage, ischemic stroke, epileptic seizure, infection, brain tumor, hypoxic brain injury / 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 adult per day.

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 brain inflammation, wherein the protein or polypeptide is administered within about 120 hours, preferably within 96 hours, and more preferably within 72 hours after the onset of brain inflammation.

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

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

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

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

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 of claim 13, further comprising another therapeutic agent.

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

16. A method for preventing and / or treating brain inflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein or polypeptide or a pharmaceutical composition comprising the protein or polypeptide, wherein the protein or polypeptide comprises a polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

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

18. The method according to claim 16 or 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 brain inflammation and / or brain injury.

19. A method for preventing, reducing, decreasing and / or inhibiting neuronal death in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein or polypeptide or a pharmaceutical composition comprising the protein or polypeptide, wherein the protein or polypeptide comprises a polypeptide having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

20. The method of claim 19, wherein the neuronal death occurs following a brain 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 death and / or cerebral hemorrhage.

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