Salivary gland polypeptides of dermacentor silvarum for preventing and treating brain inflammation

Abstract

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

Description

TECHNICAL FIELD

[0001] The present invention relates to proteins and polypeptides comprising Ixodes ricinus salivary gland polypeptides, and their use for the prevention and / or treatment of brain inflammation. BACKGROUND

[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 immune cells of peripheral origin.

[0003] Neuroinflammation or brain inflammation can occur after brain injury, such as hemorrhagic stroke. Hemorrhagic stroke is due to the rupture of a blood vessel leading to blood entering the brain. Hemorrhagic stroke can be further subdivided into intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). ICH is an internal hemorrhage of the brain parenchyma, while SAH is a subarachnoid hemorrhage. Although a minority of stroke cases (10-15%), ICH and SAH patients often suffer severe impairment, and the burden of morbidity and mortality caused by these conditions is comparable to ischemic stroke. The clinical outcome of this condition is poor: only 20% of patients achieve functional independence 6 months after ICH. The most common cause of ICH is chronic hypertension, while cerebral amyloid angiopathy, anticoagulant medications and underlying vascular abnormalities account for a significant proportion of cases.

[0004] ICH not only causes primary brain injury through the direct mechanical effects of the hemorrhage, but also causes secondary brain injury (SBI), which is the main cause of poor outcome after ICH. SBI includes the disruption of the blood brain barrier (BBB), which leads to perihematoma edema (PHE) and brain parenchymal cell death. SBI involves, among others, neuroinflammation.

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

[0006] The first innate immune cells to be activated after ICH and other acute brain injuries are microglia, the resident macrophages of the central nervous system. They continuously scan the extracellular brain environment and can be activated within minutes after tissue injury. The number of activated microglia / macrophages peaks at 72 hours and returns to normal levels 3 to 4 weeks after ICH. Upon activation, microglia are infiltrated by monocyte-derived macrophages into the perihematoma and hematoma regions. Under a variety of stimuli, microglia and brain macrophages produce proinflammatory cytokines including TNF-a and IL-1b, chemokines, and reactive oxygen species. In addition to neurotoxic cytokines, chemokines produced by microglia, such as CXCL2, are chemotactic for neutrophils and thus exacerbate the inflammatory response. Thus, microglia also enhance early neuroinflammation by recruiting and activating blood-derived leukocytes, which can exacerbate ICH-induced neuronal damage.

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

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

[0009] In the past few decades, there has been limited improvement in the mortality rate of ICH, and most survivors are left with severe disability. There are very limited treatment options for ICH, and there is currently no clear treatment method other than supportive care. Therefore, there is an unmet medical need for agents that can prevent or safely and quickly treat brain inflammation.

[0010] The inventors have previously demonstrated the effect of Ixodes ricinus salivary gland polypeptide on neutrophils as part of a thrombus treatment (WO2019 / 149906). Here, the inventors surprisingly demonstrate that this polypeptide has an anti-inflammatory effect on the brain in a model of intracerebral hemorrhage and does not cause any thrombus. Advantageously, these anti-inflammatory effects in the brain are not accompanied by an increase in the amount of hemorrhage or in the volume of edema. Thus, this polypeptide can prevent or treat early brain inflammation without increasing the risk of hemorrhage aggravation, which helps to improve the clinical outcome of the patient. SUMMARY

[0011] Thus, the present application 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 the prevention and / or treatment of brain inflammation in a subject in need thereof.

[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 brain inflammation or spontaneous brain inflammation. In some embodiments, the brain inflammation is provoked brain inflammation. In some embodiments, the brain inflammation occurs or is susceptible to occur after a cerebral hemorrhage. In some embodiments, the brain inflammation is accompanied by a cerebral hemorrhage.

[0014] In some embodiments, the brain inflammation is provoked by an acquired brain injury. In some embodiments, the brain inflammation is provoked by a traumatic brain injury. In some embodiments, the brain inflammation is provoked by a non-traumatic brain injury.

[0015] In some embodiments, the brain inflammation is provoked by a cerebral hemorrhage, an ischemic stroke, a seizure, an infection, a brain tumor, a hypoxic brain injury, an autoimmune disease, a toxicity or hydrocephalus.

[0016] In some embodiments, the brain inflammation is acquired brain inflammation. In some embodiments, the acquired brain inflammation is provoked by a traumatic brain injury or an infection. In some embodiments, the acquired brain inflammation is provoked by a traumatic brain injury.

[0017] In some embodiments, the brain inflammation is spontaneous brain inflammation. In some embodiments, the spontaneous brain inflammation is provoked by a non-traumatic brain injury or an autoimmune disease.

[0018] In some embodiments, the protein or polypeptide is administered at a dose of about 200 mg to about 20000 mg per adult per day.

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

[0020] The present application also 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 the prevention and / or treatment of brain inflammation in a subject in need thereof, 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 inflammation. In some embodiments, the protein or polypeptide is administered within 48 hours, within 24 hours, within 12 hours, within 10 hours, within 8 hours, or within 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 polypeptide is administered continuously, preferably by perfusion. In some embodiments, the protein or polypeptide is administered to the subject promptly after exposure to a risk of developing brain inflammation. In some embodiments, the protein or polypeptide is administered continuously for about 48 hours to about 72 hours.

[0022] The present application also relates to a pharmaceutical composition 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 application also relates to a kit comprising a protein or polypeptide or a pharmaceutical composition as described herein.

[0024] Another object of the application is a method for preventing and / or treating brain inflammation in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a 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, or a pharmaceutical composition comprising said protein or polypeptide. In some embodiments, the brain inflammation occurs or is susceptible to occur after a cerebral hemorrhage. In some embodiments, the therapeutically effective amount is a dose of about 200 mg to about 20000 mg per adult human per day. In some embodiments, the protein or polypeptide is administered to said subject within about 120 hours, preferably 96 hours, more preferably 72 hours, after the onset of brain inflammation and / or after a cerebral hemorrhage. In some embodiments, the protein or polypeptide is administered to said subject within about 24 hours, preferably 15 hours, more preferably 12 hours, after the onset of brain inflammation and / or after a cerebral hemorrhage.

[0025] Another object of the application is a method for preventing, reducing, decreasing and / or inhibiting neuronal death in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a 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, or a pharmaceutical composition comprising said protein or polypeptide. In some embodiments, the neuronal death occurs after a cerebral hemorrhage. In some embodiments, the protein or polypeptide is administered to said subject within 120 hours, preferably 72 hours, more preferably 24 hours, even more preferably 12 hours, after the onset of neuronal death and / or cerebral hemorrhage.

[0026] Definitions

[0027] In the present application, the following terms have the following meanings.

[0028] "About" preceding a value indicates plus or minus 10% of the stated value.

[0029] "Identity" refers to a measure of the identity of nucleotide sequences or amino acid sequences. Sequences are aligned to obtain the highest order match. "Identity" has a technically accepted meaning and can be calculated using published techniques. While there are a variety of methods for measuring identity between two nucleic acid sequences or two polypeptide sequences, the term "identity" is well known to those of skill in the art. Methods for determining identity and similarity are codified in computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package.

[0030] By way of illustration, a nucleic acid has a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence means that the nucleotide sequence of such nucleic acid is identical to the reference sequence, except that the nucleic acid sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleic acid having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or a number of nucleotides equal to up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. Such mutations of the reference sequence can occur at positions in the 5' or 3' end of the reference nucleotide sequence or anywhere in between these end positions, or interspersed among the various nucleotides in the reference sequence or in one or more contiguous groups in the reference sequence.

[0031] "Pharmaceutically acceptable excipient" means an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably, a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. A pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient is a non-toxic, solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Formulations for administration to humans should comply with the official standards of the FDA or EMA for sterility, pyrogenicity, general safety and purity of biological products.

[0032] "Polypeptide" means any peptide or protein comprising two or more amino acids connected to each other by a peptide bond or a modified peptide bond, i.e., a peptide isostere. "Polypeptide" refers to both short chains commonly called peptides, oligopeptides or oligomers as well as longer chains commonly called proteins. A polypeptide can contain amino acids other than the 20 gene-encoded amino acids.

[0033] "Protein" refers to a sequence of more than 100 amino acids and / or a multimeric entity. The proteins of the present application are not limited by the particular length of the product. The terms "polypeptide" or "protein" do not refer to, but also do not exclude, post-expression modifications of the protein, e.g., glycosylation, acetylation, phosphorylation, etc., and other natural and non-natural modifications known in the art. These modifications are well described in standard treatises and numerous research articles. Modifications can occur anywhere in a polypeptide or protein, including the peptide backbone, amino acid side chains, and the amino-terminal or carboxyl-terminal ends. It is understood that modifications of the same type can be present in the same or different amounts at various positions in a given polypeptide or protein. Also, a given polypeptide or protein can contain multiple types of modifications. Polypeptides or proteins can be branched due to ubiquitination, they can be cyclic, branched, or unbranched. Cyclic, branched, and branched cyclic polypeptides or proteins can be generated by post-translational natural processes or by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavin, covalent attachment of a hem moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, formation of GPI anchor proteins, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, translocation of an amino acid's amino group into the protein (e.g., arginylation), and ubiquitination. The protein can be an intact protein or a subsequence thereof.

[0034] "Subject" refers to a mammal, preferably a human. In one embodiment, the subject is a man. In another embodiment, the subject is a woman. In one embodiment, the subject can be a "patient", i.e., a warm-blooded animal, more preferably a human, who is awaiting receipt or is receiving medical care, or who has been / is / will be the subject of a medical procedure, or who is being monitored for the development of a disease or condition, preferably a brain inflammation or a disease associated with or triggered by brain inflammation. In one embodiment, the subject is an adult (e.g., a subject 18 years of age or older). In another embodiment, the subject is a child (e.g., a subject 18 years of age or younger).

[0035] A "therapeutically effective amount" means a level or amount of an agent that does not cause significant negative or adverse side effects to the target, which is intended to: (1) delay or prevent a disease or condition, preferably brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation; (2) slow down or stop the progression, aggravation or worsening of one or more symptoms of a disease or condition, preferably brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation; (3) ameliorate a symptom of a disease or condition of brain inflammation or a disease associated with brain inflammation; (4) reduce the severity or incidence of a disease or condition associated with brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation; or (5) prevent a disease or condition associated with brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation. In one embodiment, a therapeutically effective amount is administered prior to the onset of brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation to act prophylactically.

[0036] The terms "treatment" or "treatment" or "alleviation" refer to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation. Those in need of treatment include those who have brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation, as well as those prone to have brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation, or those in whom brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation is to be prevented. A subject or mammal is successfully "treated" for brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation if, after receiving a therapeutic amount of a protein or polypeptide according to the methods of the present application, the patient shows an observable and / or measurable reduction in or absence of one or more of the following: a decrease in the number of pathogenic cells; a decrease in the percentage of the total number of pathogenic cells; and / or an alleviation of one or more symptoms associated with brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation to some extent; a reduction in morbidity and mortality, and an improvement in quality of life issues. The above parameters for assessing successful treatment and amelioration of brain inflammation or a disease associated with brain inflammation or a disease triggered by brain inflammation are readily measurable by routine procedures familiar to a physician. DETAILED DESCRIPTION

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

[0038] In some embodiments, the protein or polypeptide for use according to the application comprises or consists of a Ixodes ricinus salivary gland polypeptide, or a fragment thereof, or a variant thereof, and is therefore referred to as Ir-CPI.

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

[0040] In some embodiments, the protein or polypeptide for use according to the application has at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0041] As used herein, "at least 75% sequence identity" means at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identical.

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

[0043] In some embodiments, the protein or polypeptide for use according to the application comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

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

[0045] In some embodiments, the protein or polypeptide for use according to the application has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0046] In one embodiment, fragments of the proteins or polypeptides according to the use of the present application are also included in the present application. As used herein, the term "fragment" refers to a polypeptide having an amino acid sequence that is identical to a portion but not the whole of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Fragments can be "free-standing" or included in larger polypeptides, which they form part or region of, most preferably a single contiguous region. Representative examples of fragments of the polypeptides of the present application include, for example, fragments from about amino acid position 1 to 20, 21 to 40, 41 to 60, 61 to 80, 81 to 100, and 101 to the end of the polypeptide, the position numbers corresponding to the amino acid position numbers of SEQ ID NO: 1 or SEQ ID NO: 2. In this context, "about" includes the specifically recited range plus or minus a few, 5, 4, 3, 2, or 1 amino acid or minus a few, 5, 4, 3, 2, or 1 amino acid of 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 the deletion of a contiguous series of residues including the amino terminus or a contiguous series of residues including the carboxyl terminus and / or a transmembrane region or the deletion of two contiguous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural attributes or functional attributes, such as fragments comprising alpha-helices and alpha-helix-forming regions, beta- sheets and beta-sheet-forming regions, turns and turn-forming regions, coils and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate-binding regions, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are those fragments that mediate the activity of the Ir-CPI polypeptide, including fragments having similar activity or having increased activity or having decreased undesirable activity.

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

[0049] In one embodiment, variants of the protein or polypeptide for use according to the application are also included in the application. Preferred variants are those which differ from the protein or polypeptide of the application by conservative amino acid substitutions, i.e. those in which a residue is replaced with another residue having similar features. Typical such substitutions are between Ala, Val, Leu and He; between Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gin; and between the basic residues Lys and Arg; or between the aromatic residues Phe and Tyr. Particularly preferred are variants in which a few, 5 to 10, 1 to 5, or 1 to 2 amino acids are substituted, deleted or added in any combination.

[0050] In one embodiment, the protein or polypeptide for use according to the application has an amino acid sequence comprising a mutation that results in the substitution of glutamine for asparagine at a position corresponding to position 54 in the amino acid sequence of SEQ ID NO: 1 to prevent N-glycosylation. In one embodiment, the protein or polypeptide of the application has an amino sequence comprising or consisting of the sequence of SEQ ID NO: 2.

[0051] In one embodiment, the protein or polypeptide for use according to the application can be prepared in any suitable manner. Examples of peptides prepared according to suitable manners include, but are not limited to, isolated naturally occurring polypeptides, recombinantly prepared polypeptides, synthetically prepared 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 for use according to the application is a fusion protein with an amino acid comprising: (i) a polypeptide having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) at least one different protein or polypeptide.

[0053] In one embodiment, the protein or polypeptide for use according to the application is linked to at least one other protein or polypeptide in such a way as to generate a single protein that retains the biological activity of the protein or polypeptide for use according to the application.

[0054] In one embodiment, the polypeptides or proteins of the fusion protein can be fused in any order. In one embodiment, the polypeptides or proteins are placed in a single, contiguous polypeptide chain.

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

[0056] In one embodiment, the peptide linker provides greater physical separation between the two moieties, thereby maximizing the availability of the protein or polypeptide for use according to the application. The linker peptide can be composed of amino acids such that it is 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 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] The peptide linker can be an in vivo cleavable linker or a non-cleavable linker.

[0059] The fusion proteins of the application can be obtained, for example, by solid state peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinantly. For recombinant production, one or more nucleic acids encoding, for example, the fusion proteins (fragments) described above are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. These nucleic acids can be readily isolated and sequenced using conventional procedures.

[0060] The fusion proteins prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those with ordinary skill in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the fusion protein binds can be used. The purity of the fusion protein can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0061] The application also relates to the fusion protein for use in preventing and / or treating inflammation of the brain as described above.

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

[0063] In some embodiments, the protein or polypeptide for use according to the application prevents and / or treats brain inflammation. In some embodiments, the protein or polypeptide for use according to the application prevents brain inflammation. In some embodiments, the protein or polypeptide for use according to the application treats brain inflammation.

[0064] As used herein, the term "brain inflammation" is used interchangeably with "neuroinflammation" and refers to the onset of inflammation of the central nervous system, which can involve, among other things, activation of glial cells and secretion of proinflammatory cytokines. The process of neuroinflammation affects neurons, microglia and macroglia.

[0065] In some embodiments, brain inflammation comprises or consists of release of proinflammatory mediators in the brain. Non-limiting examples of proinflammatory cytokines include interleukins (ILs), such as IL-1, IL-2, IL-12, IL-17 and IL-18; IFN-g; and TNF-a.

[0066] In some embodiments, brain inflammation comprises or consists of early activation of resident microglia. As used herein, "microglia" refers to resident macrophages of the central nervous system.

[0067] In some embodiments, brain inflammation comprises or consists of formation of reactive oxygen species (ROS). Non-limiting examples of ROS include superoxide, hydroxyl radical, singlet oxygen, nitric oxide and peroxides (e.g. hydrogen peroxide). In some embodiments, the formation of ROS initiates oxidative stress.

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

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

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

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

[0072] In another embodiment, the brain inflammation is not localized to a specific region or part of the brain. In certain embodiments, the brain inflammation affects the entire brain.

[0073] In some embodiments, the brain inflammation is not associated with the presence or formation of a thrombus. In some embodiments, the brain inflammation is not triggered by the presence or formation of a thrombus. In some embodiments, the brain inflammation does not result in the presence or formation of a thrombus. In some embodiments, the protein or polypeptide described above is not used to prevent and / or treat a disease associated with thrombus formation or a disease triggered by thrombus formation.

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

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

[0076] In some embodiments, the brain inflammation is due to an 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 embodiments, the traumatic brain injury is selected from the group consisting of a hemorrhage, a contusion, a concussion, and a penetrating brain injury. In some embodiments, the traumatic brain injury is a brain hemorrhage.

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

[0079] In some embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, a parasitic infection, and combinations thereof. In some embodiments, the infection is initiated 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, the brain injury is selected from the group consisting of intracerebral hemorrhage, contusion, concussion, penetrating brain injury, ischemic stroke, seizure, infection, brain tumor, hypoxic brain injury / hypoxia brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, the brain injury is selected from the group consisting of intracerebral hemorrhage, contusion, concussion, penetrating brain injury, seizure, infection, brain tumor, hypoxic brain injury / hypoxia brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, the brain injury is selected from the group consisting of intracerebral hemorrhage, contusion, concussion, penetrating brain injury, infection, brain tumor, hypoxic brain injury / hypoxia brain injury, autoimmune disease, toxicity, and hydrocephalus. In some embodiments, the brain injury is selected from the group consisting of intracerebral hemorrhage, contusion, concussion, penetrating brain injury, seizure, hypoxic brain injury / hypoxia brain injury, autoimmune disease, toxicity, and hydrocephalus.

[0081] In some embodiments, the brain inflammation occurs or is prone to occur after a traumatic brain injury. In some embodiments, the brain inflammation occurs or is prone to occur after an intracerebral hemorrhage. In some embodiments, the protein or polypeptide for use according to the application is used for preventing and / or treating brain inflammation accompanying an intracerebral hemorrhage in a subject in need thereof.

[0082] In particular embodiments, the protein or polypeptide of the application is used for preventing brain inflammation prone to occur after a brain injury in a subject in need thereof. In particular embodiments, the protein or polypeptide of the application is used for preventing brain inflammation prone to occur after an intracerebral hemorrhage in a subject in need thereof.

[0083] In some embodiments, the brain inflammation is an acquired brain inflammation or a spontaneous brain inflammation.

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

[0085] As used herein, the term "acquired brain inflammation" refers to a brain inflammation as described hereinabove, wherein the cause of the inflammation is external to the organism, e.g. a mechanical impact to the head, an object penetrating the skull, or an infection.

[0086] In some embodiments, the acquired brain inflammation is initiated by a traumatic brain injury or an infection.

[0087] In some embodiments, the acquired brain inflammation is initiated by a traumatic brain injury.

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

[0089] In some embodiments, the traumatic brain injury is a hemorrhage selected from the group consisting of an intracerebral hemorrhage, a subarachnoid hemorrhage, a subdural hemorrhage, and an epidural hemorrhage. In some embodiments, the primary brain injury is an intracerebral hemorrhage. In some embodiments, the primary brain injury is a subarachnoid hemorrhage. In some embodiments, the primary brain injury is a subdural hemorrhage. In some embodiments, the primary brain injury is an epidural hemorrhage.

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

[0091] In some embodiments, the acquired brain inflammation is initiated by an infection. In some embodiments, the acquired brain inflammation is initiated by an infection selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, a parasitic infection, and combinations thereof.

[0092] In some embodiments, the acquired brain inflammation is initiated by sepsis and / or septicemia.

[0093] In some embodiments, the acquired brain inflammation is initiated 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 virus, Poliovirus, Influenza virus, SARS-CoV-2, West Nile virus, Enterovirus, Chikungunya virus, and Dengue virus.

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

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

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

[0097] In some embodiments, the brain inflammation is an idiopathic brain inflammation.

[0098] As used herein, the term "idiopathic brain inflammation" refers to brain inflammation as described above, wherein the cause of the inflammation is internal to the organism, e.g., triggered by an idiopathic disease or idiopathic condition.

[0099] In some embodiments, the idiopathic brain inflammation is triggered by a non-traumatic brain injury or an autoimmune disease.

[0100] In some embodiments, the idiopathic brain inflammation is triggered by a non-traumatic brain injury. In certain embodiments, the non-traumatic brain injury is selected from the group consisting of idiopathic hemorrhage, brain cancer, and stroke.

[0101] In some embodiments, the non-traumatic brain injury is a non-traumatic idiopathic hemorrhage. In certain embodiments, the non-traumatic idiopathic hemorrhage is a non-traumatic intracerebral hemorrhage or an idiopathic subarachnoid hemorrhage. In certain embodiments, the non-traumatic idiopathic hemorrhage is a non-traumatic intracerebral hemorrhage. In certain embodiments, the non-traumatic idiopathic hemorrhage is an idiopathic subarachnoid hemorrhage.

[0102] In some embodiments, the non-traumatic brain injury is a brain cancer, e.g., a glioblastoma, oligodendroglioma, and meningioma.

[0103] In some embodiments, the non-traumatic brain injury is a stroke. In some embodiments, the non-traumatic brain injury is an ischemic stroke.

[0104] In some embodiments, the idiopathic brain inflammation is triggered by an autoimmune disease. In some embodiments, the autoimmune disease is autoimmune encephalitis. In some embodiments, the non-traumatic brain injury is triggered by an autoimmune disease. In some embodiments, 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 infiltration of the brain by systemic inflammatory cells such as neutrophils.

[0106] In some embodiments, the prevention and / or treatment of brain inflammation using a protein or polypeptide according to the application also treats and / or alleviates another disease, condition or symptom. In some embodiments, the disease, condition or symptom is associated with brain inflammation. In some embodiments, the disease, condition or symptom is caused by brain inflammation.

[0107] In some embodiments, the protein or polypeptide also prevents and / or treats a disease, condition or symptom associated with brain inflammation. In some embodiments, the protein or polypeptide also prevents and / or treats a disease, condition or symptom caused by brain inflammation.

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

[0109] In some embodiments, the prevention and / or treatment of brain inflammation using a protein or polypeptide according to the application has a positive effect on neuronal cell death. In some embodiments, the prevention and / or treatment of brain inflammation using a protein or polypeptide according to the application has a positive effect on excitotoxicity.

[0110] In some embodiments, the prevention and / or treatment of brain inflammation using a protein or polypeptide according to the application has a positive effect on microglial activation.

[0111] In some embodiments, the prevention and / or treatment of brain inflammation using a protein or polypeptide according to the application has a positive effect on oxidative stress. In some embodiments, the prevention and / or treatment of brain inflammation using a protein or polypeptide according to the application has a positive effect on the formation of reactive oxygen species.

[0112] In some embodiments, the use of the protein or polypeptide according to the application for the prevention and / or treatment of brain inflammation has a positive effect on the infiltration of systemic inflammatory cells to the brain. In certain embodiments the systemic inflammatory cells are selected from the group consisting of neutrophils, eosinophils, lymphocytes, plasma cells and histiocytes. In certain embodiments the systemic inflammatory cells are neutrophils. In some embodiments, the use of the protein or polypeptide according to the application 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 protein or polypeptide for use according to the application is used for the prevention and / or treatment of long-term effects of brain injury. As used herein, the expression "long-term effects of brain injury" refers to the pathophysiological consequences of brain injury, i.e. long-term effects of injury to at least one cell type in the brain, preferably neurons, typically for a week, a month, a year or more than a year. In some embodiments, the protein or polypeptide for use according to the application is used for the prevention and / or treatment of long-term effects of non-traumatic brain injury. In some embodiments, the protein or polypeptide for use according to the application is used for the prevention and / or treatment of long-term effects of traumatic brain injury. In some embodiments, the protein or polypeptide for use according to the application is used for the prevention and / or treatment of long-term effects of traumatic brain injury associated with hemorrhage. In some embodiments, the protein or polypeptide for use according to the application is used for the prevention and / or treatment of long-term effects of brain injury associated with hemorrhage. In some embodiments, the protein or polypeptide for use according to the application is used for the prevention and / or treatment of long-term effects of cerebral hemorrhage.

[0114] The inventors have demonstrated that the administration of the protein or polypeptide of the application does not increase the volume of hemorrhage or the volume of the lesion (edema). This property is advantageous, while the antithrombotic effect of this protein is also unexpected.

[0115] In certain embodiments, the protein or polypeptide of the application is used for the prevention and / or treatment of brain inflammation without increasing the volume of hemorrhage and / or the volume of the lesion (edema). In certain embodiments, the brain inflammation is due to or triggered by cerebral hemorrhage. In one embodiment, the protein or polypeptide of the application is used for the prevention and / or treatment of brain injury following cerebral hemorrhage. In one embodiment, brain injury occurs following hemorrhagic stroke. In one embodiment, the protein or polypeptide of the application is used for the prevention and / or treatment of brain injury with long-term effects following cerebral hemorrhage.

[0116] The present application also relates to a nucleic acid molecule encoding a protein or polypeptide for use according to the application.

[0117] In some embodiments, the nucleic acid is used to prevent and / or treat inflammation of the brain or a disease associated with inflammation of the brain. In some embodiments, the nucleic acid is used to prevent and / or treat inflammation of the brain.

[0118] In some embodiments, 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 a plasmid, naked DNA, cosmid, fosmid, prokaryotic chromosome (e.g., bacterial artificial chromosome), eukaryotic chromosome (e.g., yeast artificial chromosome or artificial chromosome), or a combination thereof.

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

[0121] In some embodiments, the nucleic acid molecule comprises or consists of natural nucleotides, non-natural nucleotides, or a combination thereof. In some embodiments, the nucleic acid molecule comprises or consists of natural nucleotides. In some embodiments, the natural nucleotides are selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil. In some embodiments, the natural nucleotides are selected from the group consisting of adenine, guanine, cytosine, and thymine. In some embodiments, the natural nucleotides are selected from the group consisting of adenine, guanine, cytosine, and uracil. In some embodiments, the nucleic acid molecule comprises or consists 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 application has a nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 3, or has a nucleotide sequence comprising or consisting of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid encoding a protein or polypeptide for use according to the present application has the amino acid sequence of SEQ ID NO: 3.

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

[0125] Another object of the application is a vector comprising one or more nucleic acids encoding a protein or polypeptide of the application, for use in the prevention and / or treatment of brain inflammation and / or a disease associated with brain inflammation. In a preferred embodiment, the vector of the application is an expression vector.

[0126] The application also relates to a composition comprising a protein or polypeptide as described above, one or more nucleic acids or a vector.

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

[0128] In some embodiments, the pharmaceutical composition comprises a protein or polypeptide for use according to the application 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 use in 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 use in 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 can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, 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 further comprises another therapeutic agent.

[0132] In some embodiments, the other therapeutic agent has a positive effect on a disease, disorder or symptom selected from the group consisting of brain inflammation, neuronal cell death, excitotoxicity, microglial cell activation and oxidative stress. In some embodiments, the other therapeutic agent has a positive effect on brain inflammation.

[0133] In some embodiments, the other therapeutic agent is a drug. In some embodiments, the other therapeutic agent is an anti-inflammatory drug, such as minocycline or a hormone inhibitor.

[0134] In some embodiments, the other therapeutic agent comprises or consists of a cellular therapeutic agent, such as a stem cell therapeutic agent.

[0135] The present application also relates to a protein or polypeptide, one or more nucleic acids, a vector, a composition or a pharmaceutical composition as described above for use in the manufacture of a medicament for preventing and / or treating brain inflammation.

[0136] Another object of the present application is a medicament comprising a protein or polypeptide, one or more nucleic acids, a vector, a composition or a pharmaceutical composition as described above for preventing and / or treating brain inflammation.

[0137] In one embodiment, the pharmaceutical composition or medicament of the present application comprises a therapeutically effective amount of a protein or polypeptide, one or more nucleic acids or a vector as described above.

[0138] It will be appreciated that the amount of the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application to be used depend on the judgment of the attendant physician, within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the brain inflammation; the activity of the specific agent employed; the specific composition employed, the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific agent employed; the duration of the treatment; drugs used in combination or coincidental with the specific agent employed; factors known to man in the medical arts, and the like. For example, it is well within the skill of the art to start doses of the agent(s) employed at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. A daily dosage of the product can range from about 10 mg to 10,000 mg per adult human per day, preferably from about 100 mg to 5000 mg per adult human per day, more preferably from about 200 mg to about 2000 mg per adult human per day. Preferably, the compositions contain 10 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg and 2000 mg of the active ingredient for a patient of average weight.

[0139] In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the application is administered at a dose of about 2.5 mg / kg / day to about 250 mg / kg / day, preferably at a dose of about 2.5 mg / kg / day to about 100 mg / kg / day, more preferably at a dose of about 2.5 mg / kg / day to about 50 mg / kg / day, more preferably at a dose of about 2.5 mg / kg / day to about 25 mg / kg / day. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the application is administered at a dose of about 5 mg / kg / day to about 50 mg / kg / day. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the application is administered at a dose of about 10 mg / kg / day to about 50 mg / kg / day. In some embodiments, the protein or polypeptide, composition or pharmaceutical composition according to the application 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 application 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 application 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 application is administered at a dose of about 2000 mg per adult per day.

[0141] The inventors have shown the possibility and safety of administering the protein of the application rapidly after the appearance of brain inflammation, brain injury or cerebral hemorrhage. It is particularly beneficial for patients who are at risk of developing a thrombotic event with brain inflammation, brain injury or cerebral hemorrhage, such as bedridden patients. Indeed, within the first 72 hours after the onset of hemorrhage, anticoagulants such as enoxaparin cannot be administered as they increase the risk of hemorrhage. The inventors have demonstrated that, unlike enoxaparin, the risk of hemorrhage does not increase with the protein of the application within 3 days after the onset of hemorrhage (see Example 1). Thus, the protein of the application can be used to treat brain inflammation and / or brain injury within a critical time window to avoid irreversible effects. To obtain the best effect, it is necessary to provide a therapeutic solution for brain inflammation before the inflammatory response begins after the onset of hemorrhage. Current solutions cannot be administered quickly as they increase the risk of hemorrhage. In contrast, the protein or polypeptide of the application does not have this drawback and can thus be administered rapidly 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, occurs within this 72-hour window. The sooner the size of the edema is reduced, the better the outcome for the patient.

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

[0144] Accordingly, in some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered as soon as possible after the onset of the brain haemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered to the subject soon after the brain haemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered within 120 hours, preferably within 96 hours, more preferably within 72 hours, of the onset of the brain haemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered within 48 hours, preferably within 24 hours, more preferably within 12 hours, of the onset of the brain haemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application 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, of the onset of the brain haemorrhage. In other words, in one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered within about 0 hours to about 120 hours, preferably within 96 hours, more preferably within 72 hours, more preferably within 48 hours, more preferably within 24 hours, more preferably within 12 hours, more preferably within 10 hours, more preferably within 9 hours, more preferably within 8 hours, more preferably within 7 hours, more preferably within 6 hours, of the onset of the brain haemorrhage.

[0145] It will be appreciated that the earlier the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered, the better the clinical outcome.

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

[0147] Accordingly, in some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered within about 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 24 hours or 48 hours to about 120 hours, 96 hours or 72 hours of the onset of the brain haemorrhage.

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

[0149] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered within about 24 hours to about 120 hours, preferably about 24 hours to about 96 hours, more preferably about 24 hours to about 72 hours after onset of brain hemorrhage. In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered within about 48 hours to about 120 hours, preferably about 48 hours to about 96 hours, more preferably about 48 hours to about 72 hours after onset of brain hemorrhage.

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

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

[0152] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or medicament according to the application is administered from about 0 hours to about 12 hours, 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, even more preferably from about 0 hours to about 6 hours after the onset of inflammation in the brain and / or after brain injury.

[0153] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or medicament according to the application is administered from about 0 hours to about 12 hours, 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, even more preferably from about 0 hours to about 6 hours after the onset of a cerebral hemorrhage.

[0154] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or medicament according to the application is administered up to 120 hours, preferably up to 96 hours, more preferably up to 72 hours after the onset of inflammation in the brain and / or after brain injury. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or medicament according to the application is administered up to 48 hours, preferably up to 24 hours, more preferably up to 12 hours, even more preferably up to 8 hours, even more preferably up to 7 hours, even more preferably up to 6 hours or less than 6 hours after the onset of inflammation in the brain and / or after brain injury.

[0155] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered up to 120 hours, preferably up to 96 hours, more preferably up to 72 hours after the onset of the cerebral hemorrhage. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered up to 48 hours, preferably up to 12 hours, more preferably up to 8 hours, even more preferably up to 7 hours, even more preferably up to 6 hours or less than 6 hours after the onset of the cerebral hemorrhage.

[0156] For administration to a subject, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application will be formulated for administration to the subject. The protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application can be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term administration as used herein includes injection or infusion techniques subcutaneously, intravenously, intramuscularly, intraocularly, intraarticularly, intrasynovially, intrasternally, intrathecally, intrahepatically, intralesionally and intracranially.

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

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

[0159] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application comprises one or more than one pharmaceutically acceptable vehicle suitable for the formulation for topical administration.

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

[0161] Examples of forms suitable for injection include, but are not limited to, solutions, for example sterile aqueous solutions, dispersions, emulsions, suspensions, solid forms suitable for the preparation of solutions or suspensions upon addition of a liquid before use, for example powders, liposomal forms, etc.

[0162] The sterile injectable form of the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or medicament according to the present application can be either aqueous or oleaginous suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a diluent or dispersing agent, such as carboxymethyl cellulose or similar agents, suitable for use in manufacturing pharmaceutically acceptable dosage forms, including emulsions and suspensions, other surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers, which are commonly used in manufacturing pharmaceutically acceptable solid, liquid, or other dosage forms.

[0163] In one embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or medicament is administered to a subject in need thereof at least once a day. For example, the composition, pharmaceutical composition, or medicament of the present application can be administered once a day, twice a day, or three times a day. In a preferred embodiment, the composition, pharmaceutical composition, or medicament of the present application is administered to a subject in need thereof once a day.

[0164] In another embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition, or medicament is administered to a subject in need thereof at least once a week. For example, the composition, pharmaceutical composition, or medicament of the present application can 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, vector, composition, pharmaceutical composition, or medicament of the present application is administered to a subject in need thereof once a month, twice a month, once every two months, once every two or three months, twice a year, or once a year.

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

[0167] In another embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered to a subject in need thereof promptly after exposure to a risk of developing 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 another embodiment, the term "after" means one week or more than one week after exposure.

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

[0169] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered continuously 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 about 12 hours to about 96 hours, preferably about 18 hours to about 84 hours, more preferably about 12 hours to about 72 hours, by perfusion. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered continuously for about 24 hours to about 96 hours, preferably about 48 hours to about 96 hours, preferably about 24 hours to about 96 hours, preferably about 48 hours to about 96 hours, by perfusion. In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered continuously for about 24 hours to about 72 hours, preferably about 48 hours to about 72 hours, preferably about 24 hours to about 72 hours, preferably about 48 hours to about 72 hours, by perfusion.

[0170] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered continuously for at least about 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or 96 hours, preferably by perfusion for at least about 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or 96 hours. In a particular embodiment, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered continuously for at least about 48 hours, preferably by perfusion for at least about 48 hours.

[0171] In some embodiments, the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is administered to the subject at a first dose, then continuously administered by perfusion at the same dose. The first dose is referred to herein as the initial dose, within the scope of the application. In some embodiments, the dose of the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application is greater than or equal to the dose of the perfusion, preferably greater than the dose of the perfusion, in the initial dose.

[0172] The present application also relates to a kit comprising a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application for use in the prevention and / or treatment of brain inflammation.

[0173] In one embodiment, the kit of the application further comprises means for administering the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application to a subject in need thereof.

[0174] In one embodiment, the kit of the application further comprises instructions for administering the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application to the subject.

[0175] The present application also relates to 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 having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a nucleic acid encoding said protein or polypeptide, a vector comprising said nucleic acid, a pharmaceutical composition or medicament comprising said protein or polypeptide, nucleic acid or vector.

[0176] In one embodiment, the method comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the application. In one embodiment, the therapeutically effective dose consists of a dose of about 2.5 mg / kg / day to about 250 mg / kg / day. In one embodiment, the therapeutically effective dose consists of a dose of about 2.5 mg / kg / day to about 100 mg / kg / day. In one embodiment, the therapeutically effective dose consists of a dose of about 2.5 mg / kg / day to about 50 mg / kg / day. In one embodiment, the therapeutically effective dose consists of a dose of about 2.5 mg / kg / day to about 25 mg / kg / day. In one embodiment, the therapeutically effective dose consists of a dose of about 5 mg / kg / day to about 50 mg / kg / day.

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

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

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

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

[0181] In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application from about 0 hours to about 120 hours, preferably from about 0 hours to about 96 hours, more preferably from about 0 hours to about 72 hours after onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application from about 1 hour to about 120 hours, preferably from about 1 hour to about 96 hours, more preferably from about 1 hour to about 72 hours after onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application from about 2 hours to about 120 hours, preferably from about 2 hours to about 96 hours, more preferably from about 2 hours to about 72 hours after onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application from about 3 hours to about 120 hours, preferably from about 3 hours to about 96 hours, more preferably from about 3 hours to about 72 hours after onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury.In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury.

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

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

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

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

[0186] In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application 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 onset of inflammation in the brain and / or after brain injury. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application from about 10 hours to about 24 hours, after onset of inflammation in the brain and / or after brain injury.

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

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

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

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

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

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

[0193] In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the application within about 0 hours to about 12 hours after the onset of the cerebral hemorrhage.

[0194] In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application within about 0 hours to about 24 hours, preferably within about 4 hours to about 20 hours, more preferably within about 8 hours to about 16 hours after the onset of the cerebral hemorrhage. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application within about 10 hours to about 14 hours, preferably within about 10 hours to about 13 hours, more preferably within about 11 hours to about 13 hours after the onset of the cerebral hemorrhage. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application within about 11 hours to about 12 hours after the cerebral hemorrhage.

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

[0196] In one embodiment, the method comprises administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application within about 10 hours to about 16 hours, preferably within about 11 hours to about 14 hours, more preferably within about 11 hours to about 13 hours after the onset of the cerebral hemorrhage.

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

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

[0199] In some embodiments, the method is not for the prevention and / or treatment of a thrombus. In some embodiments, the cerebral inflammation is not associated with the presence or formation of a thrombus.

[0200] In some embodiments, the method comprises the following steps:

[0201] administering to the subject an initial dose comprising a therapeutically effective amount of the protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application, and

[0202] - administering to the subject by perfusion a therapeutically effective amount of a protein or polypeptide, a nucleic acid, a vector, a composition, a pharmaceutical composition or a medicament according to the application,

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

[0204] The present application also relates to a method for preventing and / or treating brain inflammation without increasing the volume of bleeding and / or the volume of the lesion (edema). The present application also relates to a method for preventing and / or treating brain damage after a cerebral hemorrhage, preferably after a hemorrhagic stroke.

[0205] The present application also relates to a method for preventing and / or treating a disease, disorder or symptom associated with brain inflammation, comprising administering to the subject a therapeutically effective amount of a protein or polypeptide, a nucleic acid, a vector, a composition, a pharmaceutical composition or a medicament according to the application. In some embodiments, the method is for preventing and / or treating a disease, disorder or symptom triggered by brain inflammation, comprising administering to the subject a therapeutically effective amount of a protein or polypeptide, a nucleic acid, a vector, a composition, a pharmaceutical composition or a medicament according to the application. In some embodiments, the disease, disorder or symptom is selected from the group consisting of neuronal cell death, excitotoxicity, microglial cell activation and oxidative stress.

[0206] In some embodiments, the method of the application is for alleviating the symptoms of a subject suffering from a traumatic brain injury or a non-traumatic brain injury.

[0207] The present application 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, a nucleic acid, a vector, a composition, a pharmaceutical composition or a medicament according to the application.

[0208] Another object of the present application is 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, a nucleic acid, a vector, a composition, a pharmaceutical composition or a medicament according to the application.

[0209] In one embodiment, the subject in need is a subject suffering from or susceptible to brain inflammation. In one embodiment, the method is for preventing, reducing, decreasing and / or inhibiting neuronal death occurring or susceptible to occur in a subject in need thereof following brain inflammation.

[0210] In one embodiment, the subject in need is a subject suffering from or susceptible to cerebral hemorrhage. In one embodiment, the method is for preventing, reducing, decreasing and / or inhibiting neuronal death occurring or susceptible to occur in a subject in need thereof following cerebral hemorrhage.

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

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

[0213] In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the application within about 0 hours to about 12 hours after onset of neuronal death and / or intracerebral haemorrhage.

[0214] In one embodiment, the method comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the application within about 0 hours to about 24 hours, preferably within about 4 hours to about 20 hours, more preferably within about 8 hours to about 16 hours after onset of neuronal death and / or intracerebral haemorrhage. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the application within about 10 hours to about 14 hours, preferably within about 10 hours to about 13 hours, more preferably within about 11 hours to about 13 hours after onset of neuronal death and / or intracerebral haemorrhage. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the application within about 11 hours to about 12 hours after onset of neuronal death and / or intracerebral haemorrhage.

[0215] In one embodiment, the method comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the application within about 2 hours to about 24 hours, preferably within about 4 hours to about 24 hours, more preferably within about 8 hours to about 24 hours after onset of neuronal death and / or intracerebral haemorrhage. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a protein or polypeptide or pharmaceutical composition according to the application within about 10 hours to about 24 hours after onset of neuronal death and / or intracerebral haemorrhage.

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

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

[0218] A further object of the application is a method for preventing and / or treating a disease involving neuronal death, a disease associated with neuronal death or a disease triggered by neuronal death in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the protein or polypeptide or pharmaceutical composition according to the application.

[0219] The application also relates to a protein or polypeptide, nucleic acid, vector, composition, pharmaceutical composition or medicament according to the application for use in the prevention and / or treatment of a disease involving neuronal death, a disease associated with neuronal death or a disease triggered by neuronal death.

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

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

[0222] In one embodiment, the disease involving, associated with or caused by neuronal death is selected from the group consisting of prion disease (Creutzfeldt-Jakob disease), amyotrophic lateral sclerosis, motor neuron disease, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, Batten disease, Friedreich's ataxia, Alexander disease, chronic traumatic encephalopathy, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis.

[0223] In one embodiment, the disease involving, associated with or caused by neuronal death is selected from the group consisting of prion disease (Creutzfeldt-Jakob disease), amyotrophic lateral sclerosis, motor neuron disease, spinal muscular atrophy, spinocerebellar ataxia, traumatic brain injury, multiple sclerosis, frontotemporal dementia, Batten disease, Friedreich's ataxia, Alexander disease, chronic traumatic encephalopathy, acute hepatic encephalopathy, acute carbon monoxide poisoning, encephalitis, meningitis, acute necrotizing encephalopathy, and acute disseminated encephalomyelitis. BRIEF DESCRIPTION OF DRAWINGS

[0224] Figures 1A-1D is a set of box plots showing the lesion volume measured at day 1 and day 3 after intracerebral hemorrhage induced by collagenase injection in the right striatum of mice versus the amount of hemorrhage. Mice received Ir-CPI (injection + perfusion), enoxaparin (injection of enoxaparin + perfusion of NaCl) or PBS alone (injection + perfusion) treatment. Figure 1A and Figure 1B shows the lesion volume at day 1 and day 3, respectively. Figure 1C and Figure 1D shows the amount of hemorrhage at day 1 and day 3, respectively. Box shows median and quartiles. Whiskers show minimum and maximum. Independent values are plotted out. Thick line represents the mean. N = 8 mice per group at day 1. n = 7 for PBS group, n = 6 for Ir-CPI and enoxaparin groups at day 3. * p < 0.05 (Kruskal-Wallis test with Dunn's multiple comparison test).

[0225] Figures 2A-2C is a set of box plots showing the quantification of the average labeled cells in the hemorrhagic area (anterior position / injection site / posterior position) at day 3 after intracerebral hemorrhage induction. Mice received Ir-CPI (injection + perfusion), enoxaparin (injection of enoxaparin + perfusion of NaCl) or PBS alone (injection + perfusion) treatment. Figure 2A shows the number of cells positive for MPO immunostaining. Figure 2B shows the number of cells positive for Ly6G immunostaining (neutrophils). Figure 2CThe number of cells positive for Ly6G, H3Cit and MPO immunocolocalization (NET-releasing neutrophils) is shown. Boxes show median and quartiles. Whiskers show minimum and maximum values. Independent values are plotted. Thick lines represent mean values. N=5 mice per group. *p<0.05 (unpaired T test).

[0226] Figure 3 is a set of boxplots showing quantification of NET-releasing neutrophils at collagenase injection site 3 days after intracerebral hemorrhage (number of cells positive for Ly6G, H3Cit and MPO immunocolocalization). Mice received Ir-CPI (injection + perfusion), enoxaparin (injection enoxaparin + perfusion NaCl) or PBS alone (injection + perfusion) treatment. Boxes show median and quartiles. Whiskers show minimum and maximum values. Independent values are plotted. Thick lines represent mean values. N=5 mice per group. *p<0.05 (unpaired T test).

[0227] Figures 4A-4B is a set of boxplots showing that Ir-CPI reduced neuronal death in the hemorrhagic area (mean of the anterior, injection site and posterior regions) Figure 4A ) of the brain and at the injection site Figure 4B ) of mice with intracerebral hemorrhage. Boxes represent median and quartiles. Thick lines represent mean values. Whiskers represent minimum and maximum values. Independent data are shown [n=7 mice (PBS / PBS), n=6 mice (Ir-CPI / Ir-CPI or enoxaparin / NaCl) at day 3]. p values were calculated using the Kruskal-Wallis test with Dunn’s multiple comparison test. *p<0.05.

[0228] EMBODIMENT

[0229] The present application is further illustrated by the following examples.

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

[0231] MATERIALS AND METHODS

[0232] Mouse model of intracerebral hemorrhage

[0233] Intracerebral hemorrhage (ICH) was induced in adult male Swiss mice by injection of a standard amount of bacterial collagenase into the right striatum. Injection of collagenase leads to disruption of the extracellular matrix of the basal lamina.

[0234] After anesthesia and jugular catheterization, mice were positioned in the sphinx position in a stereotaxic frame. Under sterile conditions, the skin was incised about one centimeter long on the top of the skull. The skull was cleaned to locate the Bregma point. Then the injection coordinates were calculated from the Bregma point according to the brain atlas (right striatum: +0.5 mm from Bregma; mediolateral -2.5 mm; dorsoventral -3 mm). A skull hole was drilled and a glass needle tip was inserted into the right striatum using a micromanipulator. A standard volume (0.04 IU of 0.5 μΐ of sterile 0.9% NaCl solution) of bacterial collagenase (collagenase from Clostridium histolyticum, ref C2399, Sigma-Aldrich) was slowly injected. Then the needle was slowly removed and the skull skin was sutured. ). Then the needle was slowly removed and the skull skin was sutured.

[0235] Administration of treatment

[0236] A bolus (PBS, Ir-CPI or enoxaparin) was administered immediately after stroke was induced. Then, a continuous perfusion (PBS or Ir-CPI) was performed for 72 hours using osmotic pumps, ensuring constant plasma levels of the drug. Animals receiving a bolus of enoxaparin (Clexane® Sanofi Aventis) (20 mg / kg) after stroke was induced received a continuous perfusion of a saline solution (0.9% NaCl) for 72 hours. Clexane® Sanofi Aventis) (20 mg / kg) received a continuous perfusion of a saline solution (0.9% NaCl) for 72 hours. Ir-CPI (lot DP B3012503) was injected intravenously as a bolus at 30.7 mg / kg and then as a continuous intravenous perfusion at 8.3 mg / kg / hour for 72 hours. Vehicle-injected mice received a bolus of PBS and then a continuous perfusion of PBS for 72 hours.

[0237] Magnetic resonance imaging

[0238] The amount of intracerebral hemorrhage and lesion volume was assessed using magnetic resonance imaging (MRI) 24 hours and 3 days (D3) after stroke onset. The MRI device was a 7T-Pharmascan MRI system (Bruker BioSpin MRI GmbH, Ettlinger, Germany) equipped with a volume transmission coil and a surface reception coil, operated by V6.

[0239] ​Images were analyzed using ImageJ v1.52 (NIH, Bethesda, USA). The study assistant in charge of the image analysis was blinded to the allocation of the groups. T2* images were analyzed to measure brain hemorrhage. For each mouse in each stage, its hemorrhagic area - shown as a low signal area on the images - was measured on the slices. For each animal, the hemorrhagic volume was calculated as follows: sum of the measured areas x slice thickness. The hemorrhagic volume was expressed in mm 3 .

[0240] T2W images were analyzed to determine the lesion volume. For each mouse in each stage, its lesion volume - shown as a high signal area on the images - was measured on the slices. For each animal, the hemorrhagic volume was estimated as follows: sum of the measured areas x slice thickness. The hemorrhagic volume was expressed in mm 3 .

[0241] Results

[0242] The group treated with Ir-CPI did not increase the lesion (edema) volume or the hemorrhagic volume of the striatum at day 1 and day 3 compared to the control group (PBS), in contrast to the group treated with enoxaparin (low molecular weight heparin, LMWH) ( Figures 1A-1D ). Thus, in contrast to enoxaparin, the infusion of Ir-CPI for 72 hours was safe.

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

[0244] Materials and methods

[0245] Immunostaining

[0246] Brain slices were immunostained to evaluate neutrophil infiltration and expression of their NETs. Brain slices were obtained from mice previously used to measure the effect of the treatment on the lesion volume and the hemorrhagic volume (see Example 1). MPO is a marker expressed by activated neutrophils, NETs, microglia and monocytes / macrophages. Ly6G is a specific marker of neutrophils. Staining was performed simultaneously with Ly6G (Anti-mouse Ly-6G antibody, clone 1A8, Rat Monoclonal IgG2a - Cat. # 60031 STEMCELL TM ), H3Cit (Anti-Histone H3 (Citrullinated R2+R8+R17) antibody - Cat. # ab5103 ABCAM), MPO (Anti-Mycloperoxidase antibody - Cat. # AF3667 BIO- ) and DNA (DAPI) to evaluate and determine the neutrophils releasing extracellular traps (NETs). The study assistant in charge of the analysis was blinded to the allocation of the groups.

[0247] Results

[0248] In the whole bleeding area (mean of the pre-area - injection site - post-area), a significant decrease of cells expressing MPO, a marker of activated neutrophils, NETs, microglia, monocytes / macrophages, was observed in Ir-CPI treated mice compared to PBS administered mice ( Figure 2A ). In addition, Ir-CPI significantly decreased the infiltration of neutrophils (Ly6G+ cells) compared to PBS administered mice ( Figure 2B ). Nevertheless, when evaluating the whole bleeding area, a trend of decrease in the number of neutrophils releasing NETs (Ly6G+H3Cit+MPO+ cells) was observed in Ir-CPI and enoxaparin groups ( Figure 2C ).

[0249] In addition, when evaluating only the injection site, a significant decrease of neutrophils releasing NETs (Ly6G+H3Cit+MPO+ cells) was observed in Ir-CPI treated mice compared to PBS administered mice ( 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 subjected to C staining (TR-100-FJ, Tebu-Bio) to evaluate and localize degenerating neurons. The brain was cut into coronal sections (10 pm sections) with a cryostat. Three (3) sections per animal were used for staining, 3 sections corresponding to the collagenase injection site, 600 pm before this injection site, 600 pm after this injection site.

[0253] The slides with frozen tissue sections were first immersed in a solution of 80% ethanol containing 1% sodium hydroxide for 5 minutes. They were then rinsed in 70% ethanol for 2 minutes, in distilled water for 2 minutes, then incubated in a solution of 0.06% potassium permanganate for 10 minutes. After rinsing in water for 2 minutes, the sections were transferred to a solution of C (TR-100-FJ, Tebu-Bio) for 10 minutes in the dark. The appropriate dilution was made by preparing a 0.01% dye stock solution in distilled water. The working solution was stored at 4-8°C for a maximum of 4 hours.

[0254] To label the nuclear DNA, 4',6-diamidino-2-phenylindole (DAPI; 0.01% staining solution D) was added to the in solution C. Sections were then rinsed in distilled water 3 times for 1 minute each. Excess water was blotted onto a paper towel and sections were air-dried on a slide warmer at 50°C for at least 5 minutes in the absence of light. The air-dried sections were then bleached with xylene for at least 1 minute and then coated with non-fluorescent mounting medium Fluoromount-G TM (00-4958-02; Invitrogen TM ).

[0255] Using an epifluorescence microscope (Leica DM6 B), sections were observed using blue light or a 488 nm laser (excitation peak: 495 nm; emission peak: 521 nm) for C and the blue nuclear labeling imparted by DAPI was observed by ultraviolet light excitation (emission peak: 340 nm; excitation peak: 488 nm). Images were acquired and 4 regions of each section were analyzed using Image J software (version 1.52a) to qualitatively assess neuronal death. The study assistant responsible for analysis was blinded to the group assignment.

[0256] Results

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

[0258] Ir-CPI reduced the average number of degenerating neurons in the hemorrhagic areas (anterior, injection site, and posterior, averaged) compared to mice treated with PBS ( Figure 4A ). Ir-C significantly reduced the number of degenerating neurons compared to control mice receiving PBS at the injection site ( Figure 4B ).

[0259] In contrast, enoxaparin treatment did not have a significant effect on neuronal death (p>0.05) Figure 4A and Figure 4B ).

Claims

1. Use of the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the preparation of a medicament for the prevention and / or treatment of inflammation of the brain that occurs or is susceptible to following cerebral hemorrhage in subjects of need. The polypeptide is administered within 120 hours after the onset of cerebral hemorrhage.

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

3. The use according to claim 1, wherein the polypeptide is administered within 24 hours after the onset of cerebral hemorrhage.

4. The use according to claim 1, wherein the bleeding is intracerebral hemorrhage.

5. Use of a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the preparation of a medicament for the prevention and / or treatment of brain inflammation in subjects in need, wherein the brain inflammation occurs or is prone to occur after brain injury, and the polypeptide is administered within 120 hours after the onset of brain injury.

6. The use according to claim 5, wherein the polypeptide is administered within 72 hours after the onset of brain injury.

7. The use according to claim 1 or 5, wherein the polypeptide is administered to the subject at an initial dose, and then the polypeptide is continuously administered via perfusion.

8. The use according to claim 7, wherein the initial dose is greater than the infused dose.

9. The use according to claim 1 or 5, wherein the polypeptide is administered by perfusion for 12 to 96 hours.

10. The use according to claim 1 or 5, wherein the polypeptide is administered by perfusion for 24 to 72 hours.

11. The use according to claim 1 or 5, wherein the polypeptide is administered at a dose of 2.5 mg / kg / day to 250 mg / kg / day.

12. The use according to claim 1 or 5, wherein the polypeptide is administered at a dose of 2.5 mg / kg / day to 25 mg / kg / day.

13. The use according to claim 1 or 5, wherein the polypeptide is contained in a pharmaceutical composition, and wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

14. The use according to claim 1 or 5, wherein the polypeptide is contained in the kit.

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