How to Treat Craniocerebral Injuries

Abstract

Agents and methods for treating craniocerebral injury (TBI) using annexin A5 or variants thereof.

Description

Detailed Description of the Invention

[0001] This application claims priority to a prior application, filed by the applicant with the United States Patent and Trademark Office on March 4, 2022, bearing patent application serial number 17 / 653,579 and entitled "METHODS OF TREATING A TRAUMATIC BRAIN INJURY," which is incorporated herein by reference in its entirety.

[0002] [Technical field] The present disclosure relates to a method for treating traumatic brain injury (TBI), in particular to a method for treating traumatic brain injury using Annexin A5 or a variant thereof, and the use of Annexin A5 or a variant thereof for treating traumatic brain injury, pharmaceutical compositions and formulations thereof, which belong to the biopharmaceutical field.

[0003] [Background technology] Craniocerebral injury (TBI) is the result caused by the head receiving excessive pressure from blunt impact, shaking or explosion that disrupts brain function. Mild TBI or mTBI subtype represents the portion of TBI that is relatively difficult to diagnose. In this disclosure, mTBI is a subtype of TBI. The terms mild TBI (mTBI) and concussion may be commonly used interchangeably in the field and are associated with post-traumatic stress disorder (PTSD). The severity of head injury may be a temporary change in mental state or consciousness to persistent unconsciousness and amnesia. In cases of severe or multiple concussions, personality changes may have devastating consequences.

[0004] Craniocerebral injuries affect 1.5 to 2 million Americans each year (Center for Disease Control and Prevention, National Center for Injury Prevention and Control, 2003, Vol. 2003). It is estimated that craniocerebral injuries result in 50,000 deaths and 100,000 hospitalizations each year in the United States. Over 80,000 people are disabled each year, of which approximately 17,000 require specialized life support (Kraus (1997) Head Injury, ed. Cooper (Williams & Wilkins Co., Baltimore) pp 1-19; Selecki et al. (1982) Australian & New Zealand Journal of Surgery 52(l):93-102). In addition to the initial damage caused by sudden brain trauma, excessive biomechanical forces can cause a series of secondary adverse events that significantly increase lesion size, morbidity, and mortality over the days to months following the initial injury (MchΛosh et al. (1996) Lab Invest, 74(2):315-42; Stambrook et al. (1990) Can J Surg 33(2):115-8). Despite these serious problems, no effective pharmacological treatment method for human craniocerebral injury has been established. Thus, new methods and therapeutic agents for treating craniocerebral injury are still urgently needed.

[0005] Annexin A5 is a part of the phospholipid-binding protein family, has a molecular weight of about 35 kDa, and can bind to cationic phospholipids (e.g., phosphatidylserine) on the plasma membrane in a calcium-dependent manner. The membrane protein A5 was first found in human placenta in 1970, and its gene is located on chromosome 4q26-q28, has 13 exons and 12 introns, and does not usually enter brain tissue due to its relatively large molecular weight.

[0006] Summary of the Invention To remedy the above-mentioned technical problems, the present disclosure provides a method for treating craniocerebral injury (TBI) in a patient in need thereof, the method comprising: (a) identifying a patient suffering from or suspected of suffering from a craniocerebral injury; (b) administering to the patient a therapeutically effective amount of annexin A5 or a variant thereof.

[0007] The present disclosure provides a method of treating craniocerebral injury (TBI) in a patient in need thereof, the method comprising: (a) identifying a patient suffering from or suspected of suffering from a craniocerebral injury; (b) administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of annexin A5 or a variant thereof.

[0008] In some embodiments, the amino acid sequence contained in annexin A5 or a variant thereof has at least 75% (e.g., 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO:1.

[0009] In some embodiments, the craniocerebral injury is a mild, moderate or severe craniocerebral injury.

[0010] In some embodiments, the craniocerebral injury is due to head impact, penetrating head injury, non-penetrating head injury, a fall, skull fracture, sudden acceleration or deceleration, concussion or contusion.

[0011] In some embodiments, the patient is a mammal, such as a human.

[0012] In some embodiments, annexin A5, its variants or the pharmaceutical composition described above reduces diffuse axonal injury, behavioral impairment, brain tissue damage, brain atrophy, neuronal cell death or apoptosis, microglial activation or neuronal tissue loss in patients suffering from craniocerebral injury.

[0013] In some embodiments, annexin A5, a variant thereof or said pharmaceutical composition increases cerebral blood flow or cerebral glucose uptake in a patient suffering from craniocerebral injury.

[0014] In some embodiments, annexin A5, its variants or the pharmaceutical composition described above alleviates one or more symptoms associated with craniocerebral injury in a patient. In some embodiments, one or more symptoms associated with craniocerebral injury include impaired consciousness level, cognitive impairment, impaired cognitive processing speed, impaired language, impaired motor activity, impaired memory, impaired motor skills, impaired sensory skills, cerebral ischemia, edema, intracranial pressure, hearing loss, tinnitus, headache, epileptic seizures, dizziness, nausea, vomiting, blurred vision, decreased sense of smell or taste, decreased strength or decreased coordination.

[0015] In some embodiments, the first agent, such as annexin A5 or a variant thereof, or the pharmaceutical composition is administered within a time period of up to 4 weeks from the time of the craniocerebral injury. In some embodiments, the first agent is administered within about 0 hours, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after the occurrence of the craniocerebral injury.

[0016] In some embodiments, the therapeutically effective amount of Annexin A5 or a variant thereof is between about 0.01 mg / kg and about 10 mg / kg. In some embodiments, the therapeutically effective amount of Annexin A5 or a variant thereof is between about 0.01 mg / kg and about 0.5 mg / kg. In some embodiments, the therapeutically effective amount of Annexin A5 or a variant thereof is about 0.02 mg / kg, 0.06 mg / kg, or 0.2 mg / kg.

[0017] In some embodiments, the methods of the present disclosure further comprise administering to the patient a second drug or treatment.

[0018] In some embodiments, the second drug or therapy is a cardioprotective drug or therapy, including one or a combination of beta-receptor blockers, diuretics, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers, lipid-lowering therapies, statins, nitrates, antiplatelets, anticlotting agents, anticoagulation agents, and therapeutic applications of the drugs.

[0019] In some embodiments, the step of determining that the patient is suffering from a craniocerebral injury comprises performing a computed tomography (CT) scan or a magnetic resonance imaging (MRI) on the patient.

[0020] In some embodiments, the patient is identified as suffering from craniocerebral injury based on a modified Neurological Severity Score (mNSS), a rotarod test, a Glasgow Coma Scale score, or a Glasgow Outcome Scale score. In some embodiments, the mNSS or Glasgow Coma Scale score is between 3 and 8, between 9 and 12, or between 13 and 15.

[0021] In some embodiments, a patient is identified as suffering from craniocerebral injury if the mNSS or Glasgow Coma Scale score is lower than a reference level, in some embodiments, the reference level is obtained from a reference subject who has not suffered a head injury.

[0022] In some embodiments, the step of identifying a patient suffering from a craniocerebral injury comprises: determining a level of a biomarker in a sample from the patient; comparing the level of the biomarker to a reference level of the biomarker; If the level of the determined biomarker is increased compared to a reference level of the biomarker, the patient is identified as suffering from a craniocerebral injury.

[0023] In some embodiments, the biomarker may be IL-1β, TNF-α, IFN-γ, IL-6, IL-10, IL-8, HMGB1, TGFβ, UCH-L1, NSE, GFAP, S100B, NF proteins (L and H), MBP, Tau, or phosphorylated Tau.

[0024] In some embodiments, the sample is obtained after the patient's head has experienced closed or open head trauma due to physical vibration, blunt impact from an external mechanical or other force, one or more falls, an explosion, blast, solid explosion, gas explosion, or other type of blunt trauma.

[0025] In some embodiments, the sample is selected from a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample.

[0026] In another aspect, the present disclosure further provides a method of reducing the level of a biomarker in a patient with craniocerebral injury, the method comprising: (a) identifying a patient suffering from a craniocerebral injury; (b) administering to the patient a therapeutically effective amount of annexin A5 or a variant thereof, or a pharmaceutical composition comprising a therapeutically effective amount of annexin A5 or a variant thereof; Among these, the biomarkers include cytokines selected from IL-1β, TNF-α and IFN-γ, IL-6, IL-10, IL-8, HMGB1, TGFβ, UCH-L1, NSE, GFAP, S100B, NF proteins (L and H), MBP, Tau and phosphorylated Tau.

[0027] In some embodiments, the therapeutically effective amount of Annexin A5 or a variant thereof is an amount of Annexin A5 or a variant thereof that increases or decreases the level of a biomarker by at least 5%, 10%, 20%, 30%, 40% or 50%.

[0028] The disclosure further provides the use of Annexin A5 or a variant thereof in the manufacture of a medicament.

[0029] The present disclosure further provides a pharmaceutical composition comprising annexin A5 or a variant thereof, preferably a therapeutically effective amount of annexin A5 or a variant thereof.

[0030] In some embodiments, the medicament is for the following uses: Treatment and / or prevention of craniocerebral injuries, for example, where the craniocerebral injuries are mild, moderate or severe craniocerebral injuries; reducing diffuse axonal injury, behavioral impairment, brain tissue damage, brain atrophy, neuronal cell death or apoptosis, microglial activation, or neuronal tissue loss in patients suffering from craniocerebral injury; Increasing cerebral blood flow or cerebral glucose uptake in patients suffering from craniocerebral injury, Relief of one or more symptoms associated with craniocerebral injury in a patient, for example, where the one or more symptoms associated with the craniocerebral injury include impaired level of consciousness, cognitive impairment, impaired cognitive processing speed, impaired language, impaired motor activity, impaired memory, impaired motor skills, impaired sensory skills, cerebral ischemia, edema, intracranial pressure, hearing loss, tinnitus, headache, seizures, dizziness, nausea, vomiting, blurred vision, decreased sense of smell or taste, decreased strength, or decreased coordination; A decrease in said biomarkers in patients suffering from craniocerebral injury, for example, where the biomarkers include cytokines selected from IL-1β, TNF-α and IFN-γ, IL-6, IL-10, IL-8, HMGB1, TGFβ, UCH-L1, NSE, GFAP, S100B, NF proteins (L and H), MBP, Tau and phosphorylated Tau; For example, the drug is in an amount that increases or decreases the level of the biomarker by at least 5%, 10%, 20%, 30%, 40%, or 50%.

[0031] For example, annexin A5 or a variant thereof comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1, or alternatively comprises the amino acid sequence of SEQ ID NO:1.

[0032] In some embodiments, the craniocerebral injury is mild, moderate or severe craniocerebral injury, for example, the craniocerebral injury is due to head impact, penetrating head injury, non-penetrating head injury, fall, skull fracture, sudden acceleration or deceleration, concussion or contusion, for example, the patient is a human, and preferably the drug is in a unit dosage form, for example, the amount of annexin A5 or a variant thereof in the drug is between about 0.01 mg / kg and about 10 mg / kg of patient body weight, for example, between about 0.01 mg / kg and about 0.5 mg / kg of patient body weight, for example, about 0.02 mg / kg, 0.06 mg / kg, or 0.2 mg / kg of patient body weight, for example, the drug is in a unit dosage form, and the amount of annexin A5 or a variant thereof in the unit dosage form is 0.025 mg to 250 mg, for example, 0.1 to 50 mg, 0.1 to 100 mg, 0.1 to 200 mg, mg or 0.1 to 250 mg, alternatively 1 to 50 mg, 1 to 100 mg, 1 to 200 mg or 1 to 250 mg.

[0033] In some embodiments, the drug is administered within a time period of up to 4 weeks from the time of the craniocerebral injury, preferably within about 0 hours, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after the occurrence of the craniocerebral injury. More preferably, the first agent drug is administered to the patient within about 12 hours after the craniocerebral injury, for example, the drug further comprises a cardioprotective drug, for example, the cardioprotective drug is selected from one or a combination of beta-receptor blockers, diuretics, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers, lipid-lowering therapy, statins, nitrates, antiplatelets, anticoagulants, and anticoagulants.

[0034] The above contents are not intended to limit each aspect of the present disclosure. Other aspects of the present disclosure are also described in other parts of the present specification, for example, in the following description for carrying out the invention. The entire present application is intended as a unified and related disclosure. It should be understood that any combination of all features described in this specification should be considered as a technical solution described in the present disclosure, even if they are not simultaneously present in the same sentence, paragraph or chapter of the present application. Other features and advantages of the present disclosure will be described in more detail through the following detailed description. However, these detailed descriptions and specific examples are merely illustrative of specific embodiments of the present disclosure, and it should be understood that various changes and modifications made by those skilled in the art within the spirit and scope of the present disclosure based on these detailed descriptions and specific examples should be included in the scope of the claims of the present application.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A and Figure 1B show the dose-dependent effects of Annexin A5 treatment on neurological outcomes after TBI. Figure 1A shows the results of mNNS, and Figure 1B shows the results of rotarod test (###P<0.001, compared with sham group; *P<0.05, compared with model group; **P<0.01, compared with model group; ***P<0.001, compared with model group; n=10). Among them, mNNS score represents mNNS score, Fall latency represents fall latency, Sham represents sham group, and Model represents model group.

[0036] [Figure 2] Annexin A5 administration significantly reduced TUNEL-positive cells in the injured rat brain after TBI (###P<0.001, compared with the sham operation group; *P<0.05, compared with the model group, t-test, n=5). Among them, TUNEL positive cells represents the amount of positive cells, Sham represents the sham operation group, and Model represents the model group.

[0037] [Figure 3] Annexin A5 0.2 mg / kg administration significantly reduced GFAP-positive cells in the injured rat brain after TBI (###P<0.001, compared with the sham operation group; *P<0.05, compared with the model group, t-test, n=5). Mean Fluorescence (arbitrary units) represents the mean fluorescence (arbitrary units), Sham represents the sham operation group, and Model represents the model group.

[0038] [Figure 4] The effect of Annexin A5 treatment on neurofunctional outcomes after TBI. (Top) MNS results, (Bottom) Rotarod test results. (###P<0.001, compared with sham operation group; ***P<0.001, compared with model group; n=5). Among them, mNNS score represents mNNS score, Fall latency represents fall latency, Sham represents sham operation group, Model represents model group, A5 One Dose represents administration of only one dose of A5, and Time represents time.

[0039] [Figure 5] The effect of Annexin A5 treatment on neurofunctional outcomes after TBI. (Top) MNS results, (Bottom) Rotarod test results. (###P<0.001, compared with sham operation group; ***P<0.001, compared with model group; n=5). mNNS score: mNNS score, Fall latency: fall latency, Sham: sham operation group, Model: model group, A5 One Dose: one dose of A5, post-TBI: post-TBI.

[0040] [Figure 6] The levels of IL-1β, TNF-α and IFN-γ in brain tissue are shown (###P<0.001, compared to the sham operation group; **P<0.01, compared to the model group; n=5). In this figure, protein represents protein, Sham represents the sham operation group, and Model represents the model group.

[0041] FIG. 7 is a diagram showing the effect of the Annexin A5 solution provided by an embodiment of the present invention on neurological deficit symptoms.

[0042] FIG. 8 is a diagram showing the effect of the Annexin A5 solution provided by an embodiment of the present invention on the area of ​​cerebral infarction.

[0043] FIG. 9 shows the effect of Annexin A5 provided by an embodiment of the present invention on primary cortical neuron OGD injury.

[0044] FIG. 10 is a diagram showing the protective effect of Annexin A5 provided by an embodiment of the present invention against NMDA-induced excitatory injury of primary rat cortical neurons.

[0045] FIG. 11 is a diagram showing the measurement results of Annexin A5 provided by an embodiment of the present invention on the EB content of brain tissue.

[0046] [Mode for carrying out the invention] The embodiments described below with reference to the drawings are illustrative and are used to interpret the present disclosure, but should not be understood as limiting the present disclosure.

[0047] The inventors have unexpectedly discovered that annexin A5 can alleviate one or more symptoms caused by craniocerebral injury (TBI), and thus annexin A5 and its variants represent an effective method for treating craniocerebral injury in patients.

[0048] <Treatment methods for craniocerebral injuries> In one aspect, the present disclosure provides a method of treating craniocerebral injury in a patient in need thereof, the method comprising: (a) identifying a patient suffering from or suspected of suffering from a craniocerebral injury; (b) administering to the patient a therapeutically effective amount of annexin A5 or a variant thereof, or a pharmaceutical composition comprising a therapeutically effective amount of annexin A5 or a variant thereof.

[0049] In some embodiments, the craniocerebral injury is a mild, moderate or severe craniocerebral injury.

[0050] In some embodiments, the craniocerebral injury may be due to a head impact, a penetrating head injury, a non-penetrating head injury, a fall, a skull fracture, an injury caused by sudden acceleration or deceleration, a concussion or a contusion.

[0051] In some embodiments, the patient is a mammal, such as a human.

[0052] In some embodiments, annexin A5 or a variant thereof reduces diffuse axonal injury, behavioral impairment, brain tissue damage, brain atrophy, neuronal cell death or apoptosis, microglial activation, or neuronal tissue loss in patients suffering from craniocerebral injury, hi some embodiments, annexin A5 or a variant thereof reduces diffuse axonal injury or symptoms associated therewith.

[0053] In some embodiments, annexin A5 or a variant thereof increases cerebral blood flow or cerebral glucose uptake in patients suffering from craniocerebral injury.

[0054] In some embodiments, annexin A5 or a variant thereof reduces one or more symptoms associated with craniocerebral injury in a patient. In some embodiments, one or more symptoms associated with craniocerebral injury include impaired level of consciousness, cognitive impairment, impaired cognitive processing speed, impaired language, impaired motor activity, impaired memory, impaired motor skills, impaired sensory skills, cerebral ischemia, edema, intracranial pressure, hearing loss, tinnitus, headache, epileptic seizures, dizziness, nausea, vomiting, blurred vision, decreased sense of smell or taste, decreased strength, or decreased coordination.

[0055] Annexins belong to the phospholipid-binding protein family, are widely expressed, and have many important functions. Annexin A5 is a calcium-dependent phospholipid-binding protein that binds to negatively charged phospholipid surfaces. The molecular weight of Annexin A5 is 35 kDa (see SEQ ID NO: 1), and it is mainly present in the cell membrane and endoplasmic reticulum.

[0056] Radiolabeled annexin A5 can be distributed in the brain as a developing agent.In addition, annexin A5 has the functions of anti-inflammation, anticoagulation, endothelial injury and improving organ dysfunction.Annexin A5 that enters the brain can be used to treat brain diseases, such as craniocerebral injury.

[0057] Annexin A5 that can be used in the disclosed methods includes, but is not limited to, full-length native human annexin A5 polypeptide or variants / fragments thereof. The A5 polypeptide can be provided by any source or method, such as natural isolate or recombinant or synthetic source, or a suitable combination of the above. The polypeptide sequence of annexin A5 can be based on a complete or partial native amino acid sequence or a variant of these complete or partial native amino acid sequences.

[0058] The terms "polypeptide," "peptide," and "protein" may be used interchangeably herein to refer to amino acid polymers of any chain length. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acid insertions. These terms may further include amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation (e.g., conjugation with a labeling component). As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0059] A peptide or polypeptide "fragment" as used herein refers to a peptide, polypeptide or protein that is less than full length. For example, a peptide or polypeptide fragment may have a length or unit length of at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids. For example, the length of a fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more amino acids. There is no upper limit to the size of a peptide fragment. However, in some embodiments, the length of a peptide fragment may be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less than about 250 amino acids.

[0060] As used herein, the term "variant" refers to a first composition (e.g., a first molecule) relative to a second composition (e.g., a second molecule, also referred to as a "parent" molecule). A variant molecule may be derived from, isolated from, based on, or homologous to a parent molecule. The term variant can be used to describe a polynucleotide or a polypeptide.

[0061] When applied to polynucleotides, a variant molecule can have complete nucleotide sequence homology with the original parent molecule, or can have less than 100% nucleotide sequence homology with the parent molecule. For example, a variant of a gene nucleotide sequence can be a second nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in nucleotide sequence compared to the original nucleotide sequence. Polynucleotide variants also include polynucleotides that include the entire parent polynucleotide, and also include other fusion nucleotide sequences. Polynucleotide variants also include polynucleotides that are a portion or subsequence of a parent polynucleotide, for example, the present disclosure also includes unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques) of the polynucleotides disclosed herein.

[0062] When applied to proteins, a variant polypeptide can have complete amino acid sequence homology with the original parent polypeptide, or can have less than 100% amino acid homology with the parent protein. For example, an amino acid sequence variant can be a second amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in amino acid sequence compared to the original amino acid sequence.

[0063] Polypeptide variants include polypeptides that comprise the entire parent polypeptide, and also include other fused amino acid sequences. Polypeptide variants also include polypeptides that are portions or subsequences of the parent polypeptide, for example, unique subsequences (as determined, for example, by standard sequence comparison and alignment techniques) of the polypeptides disclosed herein are also included in the present disclosure.

[0064] A "functional variant" of a protein, as used herein, refers to a variant of the protein that at least partially retains the activity of the protein. Functional variants may include variants (which may be insertion, deletion or substitution mutants), including crystalline polymorphs and the like. Functional variants also include fusion products of such protein with another nucleic acid, protein, polypeptide or peptide with which it is not normally associated. Functional variants may be naturally occurring or artificial.

[0065] The variants of Annexin A5 are (i) variants in which one or more amino acid residues are replaced by a conservative or non-conservative amino acid residue, e.g., variants replaced by a conservative amino acid residue, and such substituted amino acid residues may or may not be encoded by the genetic code, (ii) variants in which one or more modified amino acid residues are present, e.g., variants in which one or more residues are present modified by the attachment of a substituent, (iii) variants in which the polypeptides are mutant splice variants of the polypeptides of the present disclosure, (iv) fragments of the polypeptides, and / or (v) variants in which the polypeptides are fused to other polypeptides, e.g., leader sequences or secretion sequences or sequences for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). These fragments include polypeptides generated by proteolytic cleavage (including multi-site proteolysis) of the original sequence. The variants may be post-translational or chemically modified. It should be understood that obtaining and applying these variants is within the capabilities of the skilled artisan based on the teachings herein.

[0066] The algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0) can be used to determine the percentage homology between two amino acid sequences using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at www.GCG.com) can be used to determine the percentage homology between two amino acid sequences using a Blossum62 matrix or a PAM250 matrix, GAP weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0067] Alternatively, the protein sequences of the disclosure can also be used as a "query sequence" to perform a search against public databases, for example to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed to obtain amino acid sequences homologous to the antibody molecules of the disclosure using the XBLAST program (score=50, wordlength=3). To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see www.ncbi.nlm.nih.gov). For purposes of determining protein sequence homology, the values ​​included are those defined by NCBI as "identities" and do not take into account residues that are not conserved but have similar properties.

[0068] In some embodiments, the detectable tag can be conjugated or linked to the N- and / or C-terminus of annexin A5. The detectable tag and the affinity tag can also be separated by one or more amino acids. In some embodiments, the detectable tag can be conjugated or linked to the variant by a cleavable element. In the context of the present disclosure, the term "cleavable unit" refers to a peptide sequence that is easily cleaved by chemical reagents or enzymatic means (e.g., proteases). Proteases may be sequence specific (e.g., thrombin) or have limited sequence specificity (e.g., trypsin). Cleavable units I and II may also be included in the amino acid sequence of the detection tag or polypeptide, especially when the last amino acid of the detection tag or polypeptide is K or R.

[0069] As used herein, the terms "conjugated" or "linked" refer to the joining of two or more entities to form one entity. Conjugates include peptide-small molecule conjugates and peptide-protein / peptide conjugates.

[0070] The term "fusion polypeptide" or "fusion protein" refers to a protein produced by linking two or more polypeptide sequences together. Fusion polypeptides encompassed by the present disclosure include the translation product of a chimeric gene construct in which a nucleic acid sequence encoding a first polypeptide is linked to a nucleic acid sequence encoding a second polypeptide to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins, which are linked by a peptide bond or several peptides. The fusion protein may further include a peptide linker between the two domains.

[0071] In some embodiments, annexin A5 comprises an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, annexin A5 comprises the amino acid sequence of SEQ ID NO:1. (SEQ ID NO:1) AQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDWGDTSGYYQRMLWLLQANRD PDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD.

[0072] The annexin A5 capable of repairing or reducing damage, such as craniocerebral damage, may be a full-length or partial amino acid sequence of a natural human annexin A5 polypeptide, or a variant of the full-length or partial amino acid sequence of a natural human annexin A5 polypeptide. The production or acquisition of these natural annexins or variants may be based on any source or method, such as direct isolation from naturally occurring substances, direct artificial synthesis, or any combination of these methods.

[0073] For example, the sequence homology of the available annexin A5 compared to the natural full-length human annexin A5 is at least 97% or at least 98%, such as at least 98.5%, at least 98.8% or at least 99%, such as at least 99.3% or at least 99.5% or at least 99.6%. These amino acid sequences that show homology compared to the natural full-length human annexin A5 can show one amino acid difference, two amino acid differences, three amino acid differences, four amino acid differences, or even five amino acid differences, six amino acid differences, seven amino acid differences. These amino acid differences may be conservative amino acid substitutions made with the amino acids on the natural full-length human annexin A5 sequence. A "conservative amino acid substitution" may refer to the replacement of an amino acid with a biologically, chemically or structurally similar residue of another amino acid. Structurally similar refers to the amino acid having a side chain of similar length, such as alanine, glycine or serine, or having a side chain of similar size. Chemically similar refers to amino acids that have the same charge or are either hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine, or methionine are substituted for one another. Alternatively, polar amino acids, such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, etc.

[0074] The above-mentioned amino acid sequences showing homology or sequences showing conservative amino acid substitutions may be obtained by artificial design and synthesis, or may exist directly in other species in nature. Research has found that the annexin A5 sequence is conserved and shows high homology in different species. The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence of natural human annexin A5, and differs only in that the N-terminal amino acid of the sequence shown in SEQ ID NO: 1 is alanine (A), while the N-terminal of natural human annexin A5 is acetylated alanine. Therefore, the mutation sites and mutated amino acids present in different species shown in Table 1 below are comparisons made using the sequence of SEQ ID NO: 1 as an example, thereby also reflecting the differences between the sequence of annexin A5 and natural human annexin A5 in different species in nature. Compared with the sequence of SEQ ID NO: 1, the mutated amino acid at the third position in Gorilla gorilla is mutated from valine to isoleucine. It has also been reported, for example in J. Mol. Biol. 223 (3), 683-704 (1992) and J. Mol. Biol. 223 (3), 683-704 (1992), that mutations also exist in native human annexins, i.e., the amino acid at position 76 is mutated from glutamic acid to glutamine or from glutamic acid to glycine, compared to native human annexin A5. Table 1 below lists the mutation sites and the corresponding mutated amino acids present in several species, compared to the sequence SEQ ID NO: 1, and all of these sequences may be obtained from NCBI based on the NCBI Access Number, or directly from the corresponding references.

[0075] [Table 1]

[0076] According to an embodiment of the present disclosure, the annexin A5 therein is natural human annexin A5. According to an embodiment of the present disclosure, the annexin A5 therein or its functional equivalent is recombinant human annexin A5 expressed in a prokaryotic expression system. Therefore, the recombinant human annexin A5 can be obtained quickly and efficiently through the prokaryotic expression system, and is not expensive. In addition, the recombinant human annexin A5 has been proven to have no immunotoxicity and very low immunogenicity. The tolerated dose of A5 protein in rats and cynomolgus monkeys is higher than 4500 μg / kg, and even higher than 9000 μg / kg in rats. In some embodiments of the present disclosure, the amino acid sequence of annexin A5 expressed by a prokaryotic expression system is shown as SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence of natural human annexin A5.

[0077] Annexin A5 has been proven to be highly efficient and rapid to obtain, and very safe, with no risk of drug use even at high doses. It has important value in the treatment of craniocerebral injury. In addition, the sequence of Annexin A5 is conserved and has a high degree of homology in different species, and is found to play a similar or homologous role. These highly homologous or conserved Annexin A5 sequences can be used to treat craniocerebral injury or to manufacture craniocerebral injury drugs. For example, in some embodiments of the present disclosure, compared to natural human Annexin A5, the sequence homology of Annexin A5 is greater than 96%, or compared to the sequence shown in SEQ ID NO:1, the sequence homology is greater than 96.5%, 97%, 97.5%, 98%, 98.3%, 98.5%, 98.8%, 99%, 99.3% or 99.5%, for example. In some embodiments, annexin A5 contains one conservative amino acid substitution, two conservative amino acid substitutions, three conservative amino acid substitutions, four conservative amino acid substitutions, or five conservative amino acid substitutions compared to SEQ ID NO: 1. These highly homologous or conservative amino acid substituted annexin A5 can be synthesized by artificial design or codon optimization, or can be isolated or synthesized from natural annexin A5.

[0078] According to some embodiments of the present disclosure, annexin A5 or its functional equivalent is in an injection form. Annexin A5 can be used once or multiple times. Annexin A5 can be administered by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intrathecal injection, nasal spray or oral spray. Annexin A5 may be administered by short-term rapid administration, including but not limited to rapid intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intrathecal injection, nasal spray, oral spray, etc. Annexin A5 may be administered by continuous slow intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intrathecal injection, etc. Considering that annexin A5 belongs to protein drugs, in order to achieve rapid in vivo treatment and high bioavailability, the injection solution is preferably prepared using lyophilized powder or other specific specification injection drugs.

[0079] According to the embodiments of the present disclosure, annexin A5 or its functional equivalent is administered. In some embodiments, the dose of annexin A5 or its variants is 0.01 mg to 500 mg per day, preferably 2 to 200 mg per day. For example, the dose may be 0.01 mg to 500 mg / day or 0.01 mg to 450 mg / day, such as 0.01 mg to 400 mg / day, 0.01 mg to 350 mg / day, 0.01 mg to 300 mg / day, 0.01 mg to 250 mg / day, 0.01 mg to 200 mg / day, etc. Furthermore, in some patients with mild craniocerebral injury, the dose of A5 protein or its variants may be even lower, such as 0.01 mg to 100 mg / day, or 0.01 mg to 10 mg / day.In one example, when the daily dose of annexin A5 is about 0.05 mg to 500 mg, the content of annexin A5 or a variant thereof in a unit dosage form is 0.025 mg to 250 mg, for example, 0.1 to 50 mg, 0.1 to 100 mg, 0.1 to 200 mg, or 0.1 to 250 mg, or 1 to 50 mg, 1 to 100 mg, 1 to 200 mg, or 1 to 250 mg. mg can be adaptively adjusted, examples of which are: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 1, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 1 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 mg.

[0080] By preparing a suitable annexin A5 injection, the treatment of craniocerebral injury can be realized, and it is safe, non-toxic, and has no side effects. The unit dosage form drug refers to the unit dosage form designed when the drug is manufactured into different dosage forms. For example, if the size of the tablet is designed to be 400 milligrams, 400 milligrams is the unit dosage form drug. For example, the drug is designed as an injection, and each injection is packaged separately in a unit dosage form. Usually, the unit dosage form drug is used as a daily dose or a half-day daily dose. The content of annexin A5 in these unit dosage forms can vary between 0.025 and 250 mg, and can be manufactured into a unit dosage form together with other pharma-ceutically acceptable carriers.

[0081] In some embodiments, the therapeutically effective amount of Annexin A5 is between about 0.01 mg / kg and about 10 mg / kg. In some embodiments, the therapeutically effective amount of Annexin A5 is between about 0.01 mg / kg and about 0.5 mg / kg. In some embodiments, the therapeutically effective amount of Annexin A5 is about 0.02 mg / kg, 0.06 mg / kg, or 0.2 mg / kg.

[0082] In some embodiments, the first agent drug, such as the annexin A5, its variant, or the pharmaceutical composition, is administered within a time period of up to 4 weeks from the time of the craniocerebral injury. In some embodiments, the first agent drug is administered within about 0 hours, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after the occurrence of the craniocerebral injury. In some embodiments, the first agent drug is administered to the patient within about 12 hours after the craniocerebral injury. In some embodiments, the first agent drug is administered to the patient about 12 hours (e.g., 12 hours) after the craniocerebral injury. In some embodiments, the annexin A5 administered to the patient is used as a single dose treatment, a three dose QD (once daily) treatment, a QD treatment (e.g., for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days), or a BID (twice daily) treatment (e.g., for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days).

[0083] In some embodiments, the pharmaceutical composition includes a second drug or therapy, such as an anti-inflammatory agent.

[0084] The anti-inflammatory agents include aceclofenac, acemetacin, acetylsalicylic acid, 5-aminoacetylsalicylic acid, aldehyde polyfenac, amfenac, bendazac, benzoprofen, bermoprofen, bromfenac, 5-bromoacetic acid salicylic acid, butylchloride, butibufen, caffeic acid, carprofen, cromoglycate, ocmetacin, clindanac, clopirac, diclofenac sodium, diflunisal, 3,4-dihydroxybenzoic acid, enfenac acid, etodolac, felbinac, fenbufen, fenclofenac, fendosal, fenoprofen, fentizole, flufenac, flufenamic acid, flunixin, flunoxaprofen, flurbiprofen, 1-hydroxybenzoic acid, benzophenone ... The antineoplastic agent may be selected from the group consisting of naphthoic acid, ibufenac, ibuprofen, indomethacin, indoprofen, isofezolac, isoxepac, ketoprofen, ketorolac, loxoprofen, meclofenamic acid, mefenamic acid, mesalazine, 3,4-methylenedioxycinnamic acid, metiazinic acid, mofezolac, montelukast, mycophenolic acid, naproxen, niflumic acid, olsalazine, oxaproline, oxaprozin, pyrazolic acid, pirprofen, pranoprofen, pritinibic acid, sulindac, suprofen, scoxibacon, tiaprofenic acid, tinozic acid, tolfenamic acid, tolmetin, tropacin, coxibcin, xymoprofen, zaltoprofen, and zomepirac.

[0085] In some embodiments, the methods of the present disclosure further include administering to the patient a second drug or therapy, hi some embodiments, the second drug or therapy is administered before, after, or simultaneously with administration of Annexin A5.

[0086] In some embodiments, the second drug or therapy is a cardioprotective drug or therapy, including one or a combination of the following: beta-receptor blockers, diuretics, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers, lipid-lowering therapy, statins, nitrates, antiplatelets, anticlotting agents, anticoagulation agents, or therapeutic applications of the above drugs.

[0087] In some embodiments, the step of determining that the patient is suffering from a craniocerebral injury comprises performing a computed tomography (CT) scan or a magnetic resonance imaging (MRI) on the patient.

[0088] In some embodiments, the patient is identified as suffering from craniocerebral injury based on a modified Neurological Severity Score (mNSS), a rotarod test, a Glasgow Coma Scale score, or a Glasgow Outcome Scale score. In some embodiments, the mNSS or Glasgow Coma Scale score is between 3 and 8, between 9 and 12, or between 13 and 15.

[0089] In some embodiments, the step of identifying a patient suffering from craniocerebral injury includes determining a level of a biomarker in a sample from the patient, comparing the level of the biomarker to a reference level of the biomarker, and identifying the patient as suffering from craniocerebral injury if the determined level of the biomarker is increased compared to the reference level of the biomarker.

[0090] In some embodiments, the biomarker may be IL-1β, TNF-α, IFN-γ, IL-6, IL-10, IL-8, HMGB1, TGFβ, UCH-L1, NSE, GFAP, S100B, NF proteins (L and H), MBP, Tau or phosphorylated Tau.

[0091] In some embodiments, annexin A5, its variants or the pharmaceutical compositions described above (e.g., a combination of annexin A5 and / or its variants with a second drug or therapy) increases or decreases the level of a biomarker by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250% or 300%.

[0092] In some embodiments, samples are obtained after the patient's head has been subjected to physical vibration, blunt impact from an external mechanical force or other force (resulting in closed or open head trauma), one or more falls, an explosion or blast, or other type of blunt trauma.

[0093] In some embodiments, the sample is selected from a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample.

[0094] In another aspect, the disclosure further provides a method of reducing the level of a biomarker in a patient suffering from craniocerebral injury, the method comprising: (a) identifying a patient suffering from craniocerebral injury; and (b) administering to the patient a therapeutically effective amount of Annexin A5 or a variant thereof, or a pharmaceutical composition comprising a therapeutically effective amount of Annexin A5 or a variant thereof, wherein the biomarker comprises a cytokine selected from IL-1β, TNF-α and IFN-γ, IL-6, IL-10, IL-8, HMGB1, TGFβ, UCH-L1, NSE, GFAP, S100B, NF proteins (L and H), MBP, Tau, and phosphorylated Tau.

[0095] In some embodiments, the pharmaceutical composition includes a second drug or therapeutic agent, such as an anti-inflammatory agent.

[0096] In some embodiments, the methods of the present disclosure further include administering to the patient a second drug or therapy, hi some embodiments, the second drug or therapy is administered before, after, or simultaneously with administration of Annexin A5.

[0097] In some embodiments, annexin A5, its variants or the pharmaceutical compositions described above (e.g., a combination of annexin A5 and / or its variants with a second drug or therapeutic agent) increases or decreases the level of a biomarker by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250% or 300%.

[0098] In some embodiments, a therapeutically effective amount of annexin A5 or a variant thereof is an amount of annexin A5 or a variant thereof that increases or decreases the level of a biomarker by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, or 300%.

[0099] <Composition> In treating craniocerebral injury, a pharmaceutical composition comprising annexin A5 and its variants can be provided. The pharmaceutical composition used according to this method can be prepared in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, annexin A5 can be prepared for administration, for example, by injection, inhalation or insufflation (through the mouth or nose), or by oral, buccal, parenteral or rectal administration. In one embodiment, the agent is administered locally, for example, at the site where target cells are present, for example, by using a patch.

[0100] Pharmaceutical compositions can be prepared for various dosages, including systemic or local administration. Such techniques and formulations can generally be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is selected, including intramuscular, intravenous, intraperitoneal or subcutaneous injection. For injection, the agent can be prepared in a liquid solution, preferably in a physiologically compatible buffer, such as Hank's solution or Ringer's solution. Additionally, these agents can be prepared in solid form and redissolved or suspended immediately prior to use. Pharmaceutical compositions of the present disclosure further include lyophilized forms.

[0101] For oral administration, the pharmaceutical composition can take the form of, for example, a tablet, a smear or a capsule. The tablet, smear or capsule is prepared by conventional means using pharma- ceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium dodecyl sulfate). The tablet can be coated by methods known in the art. The liquid preparation for oral administration can take the form of, for example, a solution, syrup or suspension, or can be presented as a dry product with water or other suitable vehicle immediately before use. Such liquid preparations can be prepared by conventional methods using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or gum arabic), non-aqueous carriers (e.g., cationic oils, oily esters, ethanol or fractionated vegetable oils), and preservatives (e.g., methyl or propyl paraben or sorbic acid). Preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably prepared to control the release of the active compound.

[0102] Pharmaceutical compositions that may be oxidized and lose biological activity, particularly those in liquid or semi-solid form, can be manufactured in a nitrogen gas atmosphere or sealed in oxygen gas-free capsules and / or foil packaging (e.g., Capsugel™).

[0103] For administration by inhalation, the drug can be conveniently delivered in the form of an aerosol spray from a pressurized pack or atomizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., gelatin capsules and cartridges) for use in an inhaler or insufflator can be prepared in the form of a powder mix containing the drug and a suitable powder matrix (e.g., lactose or starch).

[0104] The pharmaceutical compositions can be prepared for parenteral administration by injection, for example, bolus injection or continuous infusion. Injection preparations can be provided in unit dosage form, for example, in ampoules or multi-dose containers, and preservatives are added. These agents can be in the form of suspensions, solutions or emulsions in oily or aqueous carriers, and can contain formulation aids such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form, for mixing with a suitable carrier (for example, sterile pyrogen-free water) before use. These agents can also be prepared into rectal compositions, for example, suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides.

[0105] In addition to the formulations described above, pharmaceutical compositions can also be prepared into long-acting formulations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the agent can be prepared using suitable polymeric or hydrophobic materials (e.g., as an emulsion in acceptable oil) or ion exchange resins, or as a weakly soluble derivative (e.g., as a weakly soluble salt). The controlled release formulation also includes a patch, such as a transdermal patch. The patch can be used with an ultrasound therapy device, which deploys ultrasound waves with a unique waveform combination to introduce drug molecules that are not normally effectively delivered transdermally into the skin.

[0106] In some embodiments, pharmaceutical compositions (including cosmetic formulations) may contain from about 0.00001 to 100% by weight, such as from 0.001 to 10% or from 0.1% to 5% of one or more of the agents described herein.

[0107] The pharmaceutical compositions described herein usually contain topical formulation adjuvants suitable for topical administration, and can also be incorporated into topical formulations containing any such materials known in the art.Topical carriers can be selected to provide the composition or analogues in the desired form, such as ointments, emulsions, creams, microemulsions, gels, oils, solutions, and can be composed of naturally occurring or synthetically derived materials.Preferably, the carrier selected does not adversely affect the active agent or other components of the topical formulation.Examples of suitable topical carriers used herein include water, alcohol and other non-toxic organic solvents, glycerin, mineral oil, silicone, petrolatum, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like.

[0108] In some embodiments, the formulation may be a colorless, odorless ointment, emulsion, cream, microemulsion, and gel. The pharmaceutical composition may be incorporated into an ointment, which is a semi-solid formulation that is usually based on petrolatum or other petroleum derivatives. As will be understood by those skilled in the art, the particular ointment base used provides optimal drug delivery and preferably also provides other desired properties, such as emollients. As with other carriers or carriers, the ointment base should be inert, stable, non-irritating, and non-sensitizing. As interpreted by Remington, ointment bases can be classified into four types: oil-based bases, emulsified bases, emulsion bases, and water-soluble bases. For example, oil-containing ointment bases include vegetable oils, fats obtained from animals, and semi-solid hydrocarbon compounds obtained from petroleum. Emulsified ointment bases, also called absorbent ointment bases, may contain small amounts of water or not, and include, for example, hydroxystearic acid sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsion ointment bases are water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Exemplary water-soluble ointment bases are made with polyethylene glycols (PEGs) of different molecular weights, see Remington's book above for more information.

[0109] In some embodiments, the pharmaceutical composition can be incorporated into an emulsion, which is a formulation that is usually applied to the skin surface without friction and is usually a liquid or semi-liquid formulation in which solid particles containing the active agent are present in a water or alcohol base. The emulsion is usually a solid suspension that can include a liquid oil emulsion of the oil-in-water type. Emulsions are preferred formulations for treating large areas of the body because they allow for easier application of more liquid components. They usually require breaking up the insoluble matter in the washing solution. Emulsions usually contain a suspending agent to create a better dispersion, as well as compounds to position and hold the active agent in contact with the skin, such as methylcellulose, sodium carboxymethylcellulose, and the like. An exemplary emulsion formulation used in conjunction with this method includes propylene glycol mixed with hydrophilic petrolatum, and is available, for example, under the trademark Aquaphor™ from Beiersdorf, Inc. (Norwalk, Conn.).

[0110] In some embodiments, the pharmaceutical composition can be incorporated into a cream, which is usually a viscous liquid or semisolid emulsion, oil-in-water or water-in-oil. The cream base can be water-washed and contains an oil phase, an emulsifier and an aqueous phase. The oil phase usually consists of petrolatum and a fatty alcohol, such as hexadecyl or stearyl alcohol, and the aqueous phase usually (but not necessarily) exceeds the oil phase in volume and usually contains a moisturizer. As described in the Remington book above, the emulsifier in a cream formulation is usually a nonionic, anionic, cationic or amphoteric surfactant.

[0111] In some embodiments, the pharmaceutical composition may be incorporated into a microemulsion, which is typically a thermodynamically stable, transparent dispersion of two isotropic immiscible liquids (e.g., oil and water) stabilized by an interfacial film of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9). For the preparation of a microemulsion, a surfactant (emulsifier), a co-surfactant (co-emulsifier), an oil phase and an aqueous phase are required. Suitable surfactants include any surfactant that can be used to prepare emulsions, for example, emulsifiers commonly used in the preparation of creams. Co-surfactants (or "co-emulsifiers") are typically selected from polyglycerol derivatives, glycerol derivatives and fatty alcohols. Preferred emulsifier / co-emulsifier combinations are usually, but not necessarily, selected from the group consisting of glyceryl monostearate and polyoxyethylene stearate, polyethylene glycol and palm ethylene glycol stearate, caprylic and capric triglycerides, and oleoyl polyethylene glycol glyceride.The aqueous phase not only contains water, but also usually further contains buffer, glucose, propylene glycol, polyethylene glycol, preferably relatively low molecular weight polyethylene glycol (e.g., PEG 300 and PEG 400) and / or glycerin, etc., and the oil phase usually contains, for example, fatty acid esters, modified vegetable oils, silicone oils, mixtures of mono-, di-, and triglycerides, monoesters and diesters of PEG (e.g., oleoyl polyethylene glycol glyceride), etc.

[0112] In some embodiments, the pharmaceutical composition can be incorporated into a gel formulation, which is usually a suspension of small inorganic particles (two-phase system) or a semi-solid system of large organic molecules essentially uniformly distributed throughout the carrier solution (single-phase gel). For example, the active agent, the carrier solution and a suitable gelling agent, such as tragacanth (2-5%), sodium alginate (2-10%), gelatin (2-15%), methylcellulose (3-5%), sodium carboxymethylcellulose (2-5%), carbomer (0.3-5%) or polyvinyl alcohol (10-20%) can be mixed together until a characteristic semi-solid product is produced. Other suitable gelling agents include methylhydroxycellulose, polyoxyethylene-polyoxyethylene, hydroxyethylcellulose and gelatin. Gels usually use a carrier solution that includes water, although alcohols and oils can also be used as the carrier solution.

[0113] Various additives known to those skilled in the art may be included in formulations such as topical formulations. Illustrative examples of additives include, but are not limited to, solubilizers, skin penetration enhancers, opacifiers, preservatives (e.g., antioxidants), gelling agents, buffers, surfactants (especially nonionic and amphoteric surfactants), emulsifiers, emollients, thickeners, stabilizers, moisturizers, colorants, fragrances, and the like. Particularly preferred are solubilizers and / or skin penetration enhancers, as well as emulsifiers, emollients, and preservatives. An optimal topical formulation comprises about 2% to 60% by weight, preferably 2% to 50% by weight, solubilizers and / or skin penetration enhancers, 2% to 50% by weight, preferably 2% to 20% by weight, emulsifiers, 2% to 20% by weight, emollients, and 0.01 to 0.2% by weight, of which the active agent and carrier (e.g., water) make up the remainder of the formulation. Skin penetration enhancers are used to facilitate the passage of therapeutic levels of an active agent through a reasonably sized unbroken area of ​​skin. Suitable enhancers are known in the art and include, for example, lower alkanols such as methanol, ethanol, and 2-propanol, dimethyl sulfoxide (DMSO), decyl methyl sulfoxide (C 10MSO) and alkyl methyl sulfoxides such as tetradecyl methyl sulfoxide, pyrrolidones such as 2-pyrrolidone, N-methyl-2-pyrrolidone and N-(hydroxyethyl)pyrrolidone, urea, N,N-diethyl-m-toluamide, C 2-6 Other solvents such as alkanediols, dimethylformamide (DMF), N,N-dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol, as well as 1-substituted azepan-2-ones, particularly 1-n-dodecylcycloazepan-2-one (Laurocapram, Azone from Whitby Research Incorporated, Richmond, VA). RTM (available under the trademarks of

[0114] Examples of solubilizers include, but are not limited to, hydrophilic ethers such as diglycol monoethyl ether (ethoxydiglycol, available under Transcutol™) and diethylene glycol monoethyl ether oleate (available under Softcutol™), polyethylene castor oil derivatives such as polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, polyethylene glycols, particularly relatively low molecular weight polyethylene glycols such as PEG 300 and PEG 400, and polyethylene glycol derivatives such as PEG-8 caprylate / caprate glyceryl (available under Labrasol™), alkyl methyl sulfoxides such as DMSO, pyrrolidones such as 2-pyrrolidone and N-methyl-2-pyrrolidone, and DMA. Many solubilizers can also be absorption enhancers. A single solubilizer can be incorporated into the formulation, or a mixture of solubilizers can be incorporated therein.

[0115] Suitable emulsifiers and co-emulsifiers include, but are not limited to, those emulsifiers and co-emulsifiers as described with respect to microemulsion formulations. Emollients include propylene glycol, glycerin, isopropyl myristate, propylene glycol-2 (PPG-2) myristyl ether propionate, and the like.

[0116] In some embodiments, the formulation may include active agents such as anti-inflammatory agents, analgesics, antibacterial agents, antifungal agents, antibiotics, vitamins, antioxidants, and sunscreens common in sunscreen cream formulations, including, but not limited to, anthranilates, benzophenones (especially benzophenone-3), camphor derivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoylmethanes (e.g., butyl methoxydibenzoylmethane), para-aminobenzoic acid (PABA) and its derivatives, and salicylates (e.g., octyl salicylate). In some topical formulations, the active agent is present in an amount ranging from about 0.25% to 75% by weight of the formulation, preferably from about 0.25% to 30% by weight of the formulation, more preferably from about 0.5% to 15% by weight of the formulation, and most preferably from about 1.0% to 10% by weight of the formulation. The topical skin treatment composition may be packaged in a suitable container to suit its viscosity and expected use by the consumer. For example, a cleansing solution or cream can be packaged in a bottle or rollerball applicator, or a propellant-driven aerosol device, or a container equipped with a pump suitable for finger operation. If the composition is a cream, it can simply be stored in a non-deformable bottle or squeeze container, such as a tube or a can with a lid. The composition may also be contained in a capsule, such as described in U.S. Pat. No. 5,063,507. Thus, a sealed container containing a cosmetically acceptable composition is also provided.

[0117] In some embodiments, pharmaceutical formulations for oral or parenteral administration are provided, where the formulation may include an active compound containing microemulsion as described above, and may include alternative pharma- ceutically acceptable carriers, carriers, excipients, etc. that are particularly suited for oral or parenteral administration. Alternatively, the active compound containing microemulsion may be administered orally or parenterally, essentially as described above, without modification.

[0118] <Definition> To aid in the understanding of the compositions and methods according to the present disclosure, the present specification provides some clear definitions to facilitate a clear disclosure of each aspect of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0119] As used herein, a "patient" refers to humans and non-human animals. Examples of non-human animals include all vertebrates, such as mammals, such as non-human mammals, non-human primates (especially higher primates), dogs, rodents (e.g., mice or rats), guinea pigs, cats, and rabbits, as well as non-mammals, such as birds, amphibians, reptiles, etc. In one embodiment, the patient is a human. In another embodiment, the patient is an animal suitable as a laboratory animal or disease model.

[0120] As used herein, "treatment" refers to the administration of a compound or agent to a patient suffering from a disease for the purpose of curing, alleviating, ameliorating, delaying the onset of the disease, preventing or ameliorating the disease, symptoms of the disease, disease states secondary to the disease, or susceptibility to the disease.

[0121] "Effective amount" or "therapeutically effective amount" refers to an amount of a compound or drug capable of producing a medically desirable result in the subject being treated. Treatment methods can be performed in vivo or ex vivo, alone or in combination with other drugs or therapies. A therapeutically effective amount can be administered in one or multiple administrations, applications, or doses, and is not limited to a particular formulation or route of administration.

[0122] As used herein, the term "in vitro" refers to events that take place not inside a multicellular organism, but in an artificial environment, such as a test tube or reaction vessel, cell culture, etc.

[0123] As used herein, the term "in vivo" refers to events that take place within a multicellular organism, such as a non-human animal.

[0124] The term "disease" as used herein is generally synonymous and may be used interchangeably with the terms "disorder" and "condition" (e.g., in a medical context), as they all reflect an abnormal condition of the human or animal body or part thereof that impairs normal functioning, usually exhibits obvious signs and symptoms, and results in a decrease in the life span or quality of life of the human or animal.

[0125] The terms "reduction", "decreasing", "attenuating" or "inhibition" as used herein generally refer to a statistically significant amount of reduction. However, for the avoidance of doubt, "reduction", "decreasing", "attenuating" or "inhibition" refers to a reduction of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80% reduction, or at least about 90% or up to 100% reduction (e.g., absent compared to a reference sample), or any reduction between 10-100% compared to a reference level.

[0126] As used herein, the term "modulation" refers to any change in a biological state, ie, an increase, a decrease, etc.

[0127] The terms "increase", "enhancement", "enhancement" or "activation" as used herein generally refer to an increase in a statically significant amount, and for the avoidance of any doubt, the terms "increase", "enhancement", "enhancement" or "activation" refer to an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80% increase, or at least about 90% or up to 100% increase compared to a reference level, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0128] The term "effective amount" or "effective dose" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent refers to the amount of any drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease, expressed as a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of damage or disability due to the affliction of the disease. A "prophylactically effective amount" or "prophylactically effective dose" of a drug refers to the amount of drug that inhibits disease progression or recurrence when administered alone or in combination with another therapeutic agent to a patient at risk of disease progression or disease recurrence. Various methods known in the art can be used to evaluate the ability of a therapeutic or prophylactic agent to promote disease regression or inhibit disease progression or recurrence, for example, by measuring the activity of the agent in human patients during clinical trials, in animal model systems predictive of human efficacy, or in in vitro measurements.

[0129] Dosage is usually relative to body weight. Thus, a dosage expressed as [g, mg or other unit] / kg (or g, mg, etc.) usually refers to "per kg (or g, mg, etc.) of body weight" [g, mg or other unit], even if the term "body weight" is not explicitly mentioned.

[0130] The terms "agent" and "drug" are used herein to refer to a chemical compound, a mixture of compounds, a biological macromolecule (e.g., a nucleic acid, an antibody, a protein or a portion thereof, e.g., a peptide), or an extract made from biological material, e.g., a bacterial, plant, fungus, or an animal (especially a mammalian) cell or tissue. The activity of these drugs may qualify them as "therapeutic agents," which are biologically, physiologically, or pharmacologically active substances (or substances) that act locally or systemically in the patient's body.

[0131] The terms "therapeutic agent," "therapeutic drug," or "therapeutic agent" may be used interchangeably and refer to a molecule or compound that confers some beneficial effect when administered to a patient. Beneficial effects include initiation of a diagnostic measure, amelioration of a disease, symptom, disorder, or pathological condition, reduction or prevention of the onset of a disease, symptom, disorder, or condition, and are typically countered by a disease, symptom, disorder, or pathological condition.

[0132] Unless the context clearly indicates otherwise, "combination" therapy as used herein is meant to include administration of two or more therapeutic agents in a coordinated manner, and includes, but is not limited to, simultaneous administration. Specifically, combination therapy includes simultaneous administration (e.g., administration of a common formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that administration of one therapeutic agent is in some way dependent on administration of another therapeutic agent. For example, one therapeutic agent is administered only after administration of a different therapeutic agent and allowing the therapeutic agent to function within a given time period. See, for example, Kohrt et al. (2011) Blood 117:2423.

[0133] "Sample", "measurement sample" and "patient sample" may be used interchangeably herein. A sample may be a sample of serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells (e.g., cells producing antibodies) or tissue. Such samples may be obtained directly from a patient and may be pre-treated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., as described herein or otherwise known in the art, to modify the properties of the sample. The terms "sample" and "biological sample" as used herein generally refer to biological material suspected of containing the analyte (e.g., antibody) being measured and / or of interest. A sample may be any tissue sample from a patient. A sample may contain proteins from a patient.

[0134] The terms "inhibit" and "antagonize" as used herein refer to decreasing the expression, stability, function, or activity of a molecule, reaction, interaction, gene, mRNA, and / or protein by a measurable amount, or preventing the same entirely. Inhibitors are compounds, e.g., antagonists, that bind, partially or completely block a stimulus, decrease, block, delay activation, inactivate, desensitize, or downregulate protein, gene, and mRNA stability, expression, function, and activity.

[0135] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0136] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound used in the present disclosure with other chemical components (e.g., carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients) that facilitate administration of the compound to a living organism.

[0137] Multiple techniques exist in the art for administering a compound, including but not limited to intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0138] As used herein, the term "pharmacologically acceptable" refers to a relatively non-toxic material, such as a carrier or diluent, that does not eliminate the biological activity or properties of a composition, and that can be administered to an individual without causing adverse biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0139] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, for carrying or transporting the disclosed compounds in or to a patient so that the compounds can perform their expected function. Usually, these compounds are carried or transported from one organ or part of the body to another. Each salt or carrier must be non-injurious to the patient and "acceptable" in the sense of being compatible with the other ingredients of the formulation. Some examples of materials that can be used as pharma- ceutically acceptable carriers include sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate, isotonic saline; excipients such as Scutellaria root powder, malt, gelatin, talc, cocoa butter and suppository wax, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, ethylene glycols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol. Polyols such as lycohol, esters such as ethyl oleate and ethyl laurate, agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, phosphate buffer solution, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emulsifying agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, antioxidants, plasticizers, gelling agents, thickening agents, hardening agents, coagulating agents, suspending agents, surfactants, humectants, transporters, stabilizers, and other non-toxic compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" further includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, etc., that are compatible with the activity of the compound and are physiologically acceptable to the patient. Additional active compounds can also be incorporated into the composition.

[0140] As used herein, the language "pharmaceutically acceptable salts" refers to salts of the administered compound prepared with pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.

[0141] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" also include the plural forms unless the context clearly dictates otherwise.

[0142] Unless otherwise stated, the words "including," "including," or "having" and variations thereof are meant to include the project(s) listed thereafter and equivalents thereof, as well as other subject matter.

[0143] The phrases "in one embodiment," "in various embodiments," "in some embodiments," and the like are used repeatedly. These phrases should not be understood to refer to the same embodiment, but may, unless context dictates otherwise.

[0144] The term "and / or" or " / " refers to any project, any combination of projects, or all of the projects associated with that term.

[0145] The word "essentially" does not exclude "completely", for example, a component that is "essentially free" of Y may be completely free of Y. Where necessary, the word "essentially" may be omitted from the definitions of this disclosure.

[0146] As used herein, the term "about" or "approximately" applied to one or more values ​​refers to a value similar to the stated reference value. In some embodiments, the term "about" or "approximately" refers to increasing or decreasing the range of values ​​within the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% based on the stated reference value, unless otherwise stated or the context dictates otherwise (except when the number exceeds 100% of the possible value). Unless otherwise stated herein, the term "about" or "approximately" is intended to include values ​​close to the stated range, such as weight percentages, which are equivalent in terms of the function of the individual components, compositions, or examples.

[0147] It should be understood that where values ​​and ranges are provided herein, all values ​​and ranges encompassed within those values ​​and ranges are intended to be included within the scope of the disclosure. Furthermore, the application also contemplates all values ​​that fall within those ranges, as well as the upper or lower limits of any series of values.

[0148] As used herein, the term "each," when used to refer to a collection of projects, is intended to identify a single project in the collection, but does not necessarily refer to each and every project in the collection. Exceptions may occur where expressly disclosed or otherwise specified by the context.

[0149] Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "for example" or "such as") provided herein is intended merely to better clarify the disclosure and not to limit the scope of the disclosure. Nothing in the specification should be construed as indicating that any unclaimed technical feature is essential to the practice of the disclosure. As used in this document, the word "exemplary" is intended to mean "by example" and does not imply that any particular exemplary project is preferred or required.

[0150] Unless expressly stated otherwise herein or unless clearly contradicted otherwise by context, all of the methodologies described herein may be performed in any suitable order. For any method provided, the steps of the method may be performed simultaneously or sequentially. Unless otherwise stated, when method steps are performed sequentially, the steps may be performed in any order.

[0151] Where the method includes a combination of steps, unless otherwise stated herein, every combination or subcombination of steps is included within the scope of the disclosure.

[0152] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety, but does not conflict with this disclosure. Publications disclosed herein are used solely for their disclosure prior to the filing date of this disclosure. Nothing herein should be construed as an admission that the present disclosure is not entitled to be antedated by prior invention. Also, publication dates provided may differ from the actual publication dates, which may require independent confirmation.

[0153] It should be understood that the examples and embodiments described herein are for illustrative purposes only and will suggest to those skilled in the art that various modifications or changes may be made thereto, and are to be included within the spirit and scope of the present application and the scope of the appended claims.

[0154] <Example> <Example 1> This example describes the experimental design and the materials and methods used in the following examples. The experiment was carried out as follows.

[0155] 1.1 Experiment 1 To study whether annexin A5 (A5) has a therapeutic effect on craniocerebral injury, A5 was administered at doses of 0.02 mg / kg, 0.06 mg / kg and 0.2 mg / kg immediately after the construction of the craniocerebral injury (TBI) model. The same doses of A5 were then administered 6 h, 24 h, 30 h and 48 h after the craniocerebral injury. The sham-operated and model groups were treated in parallel with the same volume of carrier. At 24 h, 48 h and 72 h after injury, the neurological outcome was evaluated using the modified neurological severity score (mNSS) and the rotarod test. The levels of cell apoptosis and glial activation were evaluated by the TUNEL method and immunofluorescence observation, respectively.

[0156] 1.2 Experiment 2 To further evaluate the time course of the neuroprotective effect of A5 on TBI animals, A5 was treated for 7 days with a single dose, 3 doses QD (once a day), QD for 7 days, and BID (twice a day) for 7 days, respectively. All A5 treatments were first performed immediately after TBI construction. mNSS assessment and rotarod test were performed at 24 hours, 48 ​​hours, 72 hours, and 168 hours after TBI.

[0157] 1.3 Experiment 3 The therapeutic time window (i.e., the time interval between the TBI insult and the start of treatment) is a key factor for the treatment of TBI drugs. Most of the drugs tested in animal TBI models lost their therapeutic effect with increasing time interval between injury and first treatment. For example, when rats were first administered N-type Ca 2+ The channel blocker ziconotide improves mitochondrial function but rapidly loses efficacy when first administered 1 to 10 hours after injury.

[0158] To determine the possible therapeutic time window of A5, TBI-treated rats were divided into four A5-treatment groups, in which 0.2 mg / kg A5 was administered immediately after TBI (0 h), 3 h, 6 h, and 12 h after TBI. The same dose was administered at 6 h, 24 h, 30 h, and 48 h after TBI. The mNSS test and rotarod test were performed at 24 h, 48 h, and 72 h after TBI. At the end of the experiment, cytokine levels in the ipsilateral hemisphere were evaluated by ELISA.

[0159] 1. Materials and Methods 1.1 Animals All animal care and experimental procedures conformed to the guidelines of the Animal Management Regulations of the Ministry of Health of the People's Republic of China (Document No. 55, 2001) and were approved by the Experimental Animal Ethics Committee of the Chinese Academy of Medical Sciences. Adult male Sprague-Dawley rats (200-220 g) were obtained from Vital River Laboratories Technology Co., Ltd. (Beijing, China). Animals were kept in a humidity-controlled room with 45-70% and temperature (21-26°C) with a 12-h light / dark cycle and were given standard rodent chow and water ad libitum.

[0160] 1.2 Surgical Procedures and Drug Administration Rats anesthetized with isoflurane were cannulated and placed on a stereotactically oriented framework (model no. 6801, RWD Life Science Co.,LTD, Shenzhen, China). A 5 mm diameter craniectomy was performed 1 mm posterior to the bregma and 2 mm lateral to the sagittal suture in the right hemisphere, and a handheld drill was used to remove the bone flap. A modified Feeney's weight-drop injury (WDI) model was used to induce TBI by advancing a 5 mm impact rod into the exposed parietal cortex, followed by dropping a 40 g body weight from a height of 25 cm. Sham-operated rats underwent surgery without the influence of vehicle treatment. The test items or vehicle (0.7% saline) were administered intravenously as described in the experimental design section. After surgery, the animals were housed under the same conditions as above.

[0161] 1.3 Neurological function assessment Neurological outcome was assessed using the modified Neurological Severity Score (mNSS), a multifunctional assessment scale consisting of sensation, balance, motor, and reflex measures (Table 2). The measurements were performed by an observer blinded to the individual's treatment. Neurological function scores ranged from 0 to 18 (normal score 0, maximum deficit score 18). The higher the score, the more severe the neurological damage.

[0162] [Table 2]

[0163] 1.4 Rotarod test The rotarod test was performed to evaluate the whole body motor function, especially the coordination and balance. Rats were placed in the rotarod cylinder, and the rotation speed was slowly increased from 5 rpm to 25 rpm within 180 s. The time each rat stayed on the rotarod was recorded as the fall latency. Fall latency longer than 180 s was recorded as 180 s. Each experiment was repeated three times, and the average value of the three measurements was used.

[0164] 1.5 Brain tissue preparation and TUNEL staining Rats anesthetized with isoflurane were perfused intracardially with 0.1 M PBS, followed by 4% paraformaldehyde (PFA) in 0.1 M PBS. Whole brains were removed, further fixed in 4% PFA for 48 h, and embedded in paraffin. Coronal sections (4 μm thick) were placed on slides and used for subsequent TUNEL staining.

[0165] Briefly, brain tissue sections were deparaffinized, washed with graded ethanol, and further hydrated. Antigen retrieval was performed by incubating sections with 20 μg / mL proteinase K without DNase for 25 min at 37°C. After three washes in PBS with 5 min intervals on a shaker, TUNEL staining solution was added in a volume of 80 μL / section and incubated for 75 min at 37°C in the dark. Hoechst33342 was added to stain cell nuclei for 15 min at room temperature. After three washes with PBS for 5 min each, sections were fixed. Images were captured using an ECLIPSE 80i (Nikon Medical Healthcare Division) fluorescent microscope. Separate images were taken from the area near the injury site, with three separate images per section. A total of three images were analyzed for the count of TUNEL-positive cells.

[0166] 1.6 Frozen sections and immunofluorescence staining Rats anesthetized with isoflurane were perfused intracardially with 0.1 M PBS, followed by 4% paraformaldehyde (PFA) in 0.1 M PBS. The whole brain was removed and further fixed with 4% PFA for 48 h, then dehydrated in 10%, 20% and 30% sucrose solutions until the brains sank to the bottom of the tube in sequence. The brains were removed from the 30% sucrose solution and frozen in isopentane for 15 s. Coronal frozen sections (25 μm thick) were stored in a refrigerator at -80°C until staining. The frozen sections were removed from the refrigerator at -80°C and washed three times with PBS. The sections were then incubated with goat serum at room temperature for 1 h, then immunostained with the first antibody at 4°C overnight. After washing three times with PBS for 5 min each, the sections were incubated with the secondary antibody at room temperature for 1 h, then washed and fixed. Images were captured by fluorescence microscopy using an ECLIPSE 80i (Nikon Medical Healthcare Division).

[0167] 1.7 Measurement of serum cytokine levels Blood samples were collected at the end point of the rats by cardiac puncture. Blood samples were left to clot at room temperature for 40 min and then centrifuged at 5000 rpm for 5 min at 4°C. Serum was centrifuged again at 13000 rpm for 5 min at 4°C and then stored in a -80°C refrigerator until use. Levels of IL-1β, TNF-α and IFN-γ in serum were measured using a bead-based LEGENDplex multi-analyte flow assay kit (Biolegend, San Diego, USA) and a BD FACSVerse flow cytometer according to the manufacturer's instructions.

[0168] 1.8 Enzyme-Linked Immunosorbent Assay (ELISA) Rats anesthetized with isoflurane were perfused intracardially with 0.7% saline. Whole brains were removed and the brain tissue from the same side was weighed and homogenized in ice-cold PBS (pH 7.4) containing a protease inhibitor mixture, then centrifuged at 13000 rpm for 30 min at 4°C. The supernatant was collected and centrifuged again at 13000 rpm for 30 min at 4°C. The supernatant was collected and used to assess the levels of IL-1β, TNF-α and IFN-γ.

[0169] To measure IL-1β and IFN-γ, a Peprotech ABTS ELISA was used to develop a reagent kit (New Jersey, USA) according to the manufacturer's instructions. Cytokine concentrations were measured using an ELISA microplate reader at a wavelength of 405 nm, with wavelength calibration set at 620 nm, and quantified using a standard curve. To measure TNF-α, a Thermo uncoated ELISA reagent kit was used according to the manufacturer's instructions. Briefly, the capture antibody was diluted 1:250 in coating buffer and added to a 96-well microplate at a concentration of 100 μL / well. The microplate was incubated overnight at 4°C in the dark. After blocking the wells with dilution buffer for 2 h at room temperature and washing four times, the standards and samples were added to the wells and incubated at room temperature for 2 h. After washing four times with washing buffer, the test antibody (diluted 1:250 in coating buffer 1) was added to the wells and incubated at room temperature for 1 h. The wells were then washed and avidin-HRP was added sequentially. Cytokine concentrations were measured using an ELISA plate reader at a wavelength of 450 nm, wavelength calibration was set at 570 nm, and quantified using a standard curve.

[0170] 1.9 Statistical analysis Analysis was performed using GraphPad Prism 7.00 software. All data were expressed as mean ± SEM. A two-tailed t-test was used to determine differences between two groups, and a one-way Dunnett post-hoc test was used for three or more groups. Two-way ANOVA was performed followed by Bonferroni post-hoc test for multiple comparisons. A value of P<0.05 was considered statistically significant.

[0171] <Example 2> 2.1 Dose-dependent improvement of neurological deficits after TBI in rats with Annexin A5 A modified neurological severity score (mNSS) was used to assess neurological function after TBI and Annexin A5 treatment in rats, including motor (muscle status and abnormal movement), sensory (visual, tactile, and somatosensory), reflexes, and beam-walking tests. All measurements were performed by observers blinded to the treatment of the individual. The higher the mNSS score, the worse the sensorimotor function. At 24 hours after injury, the mNSS scores of all rats in the model group were higher than 11, indicating that the Feeney drop impact model in this study resulted in neurological function deficits. At 24 to 72 hours after injury, the mNSS scores of TBI rats treated with 0.02 to 0.2 mg / kg A5 were significantly lower than the model group in a dose-dependent manner (Figure 1A), indicating improved functional outcomes.

[0172] To evaluate motor and balance coordination, the rotarod test was performed. As shown in Figure 1B, the results of the rotarod test showed that the fall latency of the model group was significantly shorter than that of the sham-operated group at 24-72 h after TBI (p<0.001). These results indicated that the Feeney drop impact model resulted in motor and balance coordination deficits in this study. In contrast, TBI rats treated with 0.02 mg / kg-0.2 mg / kg A5 at 24 h after injury showed obviously longer fall latency in a dose-dependent manner (p<0.05 or p<0.001). At 48 h and 72 h after TBI, the fall latency of the 0.06 mg / kg A5 and 0.2 mg / kg A5 treatment groups was obviously longer than that of the model group (p<0.01 or p<0.001). However, at 48 and 72 hours after injury, the lowest dose (0.02 mg / kg) of annexin A5 did not show significantly longer fall latency compared with the model group (p>0.05) (Figure 1B). These results indicated that treatment with 0.06 mg / kg-0.2 mg / kg A5 during the acute phase could rescue the motor and balance deficits caused by TBI injury.

[0173] The above results showed that, in the acute phase, annexin A5 improved the neurological function deficit after TBI in rats in a therapeutic dose-dependent manner, and improved the neurological function deficit after TBI in rats by approximately 60% at 0.2 mg / kg, indicating the clinical therapeutic efficacy of A5 against TBI.

[0174] 2.2 Annexin A5 attenuates brain tissue cell apoptosis after TBI At 72 hours after TBI, apoptotic cells in the brain after TBI were evaluated by TUNEL measurement. TUNEL staining in the brain-operated side of the sham-operated rats was negligible. At 72 hours after TBI in the model group, a significant increase in apoptotic cells and intercellular apoptotic fragments was observed (p<0.001, compared with the sham-operated group), indicating that TBI induces significant apoptosis in brain tissue. In contrast, the number of apoptotic cells or fragments was significantly decreased in the 0.2 mg / kg Annexin A5 treatment group (p<0.05, compared with the model group). As characterized by TUNEL staining, treatment with 0.2 mg / kg Annexin A5 reduced cell apoptosis in brain tissue after TBI by about 42%. These results indicated that treatment with 0.2 mg / kg Annexin A5 could attenuate TBI-induced brain cell apoptosis (Figure 2).

[0175] 2.3 Annexin A5 inhibits microglial activation after TBI Immunofluorescence was used to detect the expression of glial fibrillary acidic protein (GFAP), an astrocyte activation marker. Only a small number of GFAP-positive cells were counted in the sham-operated group. 72 hours after TBI, the number of astrocytes GFAP-positive staining increased in the model group rats compared with the sham-operated group, and reactive astrocytes were significantly increased (p<0.001). GFAP-expressing reactive astrocytes in the model group also showed a morphology characterized by enlargement and deformation of worm-like cell bodies. However, treatment with Annexin A5 did not inhibit reactive astrocytes, and GFAP-expressing cells did not significantly decrease.

[0176] To observe the activation status of microglia, immunofluorescence was used to detect the microglia-specific protein IBA-1. A small number of IBA-1-positive cells were found in the sham-operated group. At 72 h after TBI, the number of activated microglia in the model group was obviously increased. The cell bodies in the model group spread in an amoeboid shape. Compared with the sham-operated group, the expression of IBA-1 in the model group was significantly increased (p<0.001), indicating TBI-induced microglial activation. Treatment with 0.2 mg / kg annexin A5 significantly decreased the expression of IBA-1, decreasing it by about 60% in a dose-dependent manner at 72 h after TBI (p<0.05, compared with the model group), indicating that treatment with annexin A5 inhibited microglial activation (Figure 3).

[0177] 2.4 Time course of treatment of neurological deficits after TBI in rats by Annexin A5 TBI produced significant early deficits in mMNS and fall latency in the rotarod test, and both tests showed a gradual decrease in TBI-induced damage over time.

[0178] For the mNSS test, there were no differences in the mNSS scores of the A5 BID 7 d, A5 QD 7 d, and A5 QD 3 d treatment groups, as shown in the top of Figure 4. Two-Way ANOVA and Bonferroni adjusted multiple comparisons showed that all three treatments had a significant group main effect (p<0.001) compared to the model group.

[0179] For the rotarod test shown in the bottom of Figure 4, two-way ANOVA and multiple comparison after Bonferroni adjustment for the fall latency showed that the expression of the A5 BID 7 d group was better than that of the model group (p<0.001). Compared with the model group, the fall latency of the A5 QD 7 d and A5 QD 3 d treatment groups did not improve significantly over time (p>0.05).

[0180] These results indicated that A5 BID treatment is a better method for treating TBI-induced neurological function deficits in rats.

[0181] 2.5 The therapeutic time window for Annexin A5 after TBI insults in rats was as long as 12 hours Most of the drugs used in TBI treatment lost their therapeutic effect with increasing time interval between injury and first administration. For example, when rats were first administered N-type Ca2+ at 15 min after moderate TBI, 2+ The channel blocker ziconotide improves mitochondrial function but rapidly loses efficacy when first administered within the first hour after injury, therefore assessment of therapeutic time frames with multiple outcome measures has become a standard component of preclinical evaluation of drugs for the treatment of TBI.

[0182] TBI rats were administered vehicle or 0.2 mg / kg A5 at 0, 3, 6, or 12 hours (n=5) after TBI, then twice daily until 48 hours after TBI, and efficacy of treatment was evaluated by mNSS test and rotarod test at 24, 48, and 72 hours. After the rotarod test, brain tissue was obtained for cytokine level measurement according to the method described above.

[0183] The model group had a significantly higher mNSS score and a significantly shorter fall latency than the sham-operated group, indicating that the experimental TBI model leads to neurological function deficits. Two-way ANOVA showed that there was a significant difference (p<0.001) between each group. Bonferroni post-hoc test showed that the mNSS scores of the 0 h, 6 h and 12 h groups were significantly decreased compared to the model group (top of Figure 5). No significant difference in the mNSS scores was observed between the 3 h group and the model group, which may be due to the limited number of animals in this experiment (n=5). There was also a significant difference in the two-way ANOVA of the fall latency of each group (p<0.001). Further Bonferroni post-hoc test also showed that the fall latency of the 0 h, 3 h, 6 h and 12 h groups was significantly prolonged compared to the model group (bottom of Figure 5).

[0184] The therapeutic time window experiment showed that 0.2 mg / kg annexin A5 treatment administered 12 hours after injury could reduce neurological function deficits after TBI in rats by about 38%. These results indicated that annexin A5 could be administered within 12 hours after TBI and maintain a certain therapeutic effect in this rat TBI model.

[0185] The above results showed that A5 could alleviate TBI-induced neurological function deficits, and administration within 12 hours after injury could exert a therapeutic effect. Therefore, the effect of A5 on the inflammatory response of TBI rats 72 hours after TBI was then studied. ELISA method was used to detect the levels of cytokines IL-1β, TNF-α, and IFN-γ in the same hemisphere. Compared with the sham-operated group, the IL-1β expression level of the model group was significantly increased (p<0.01), indicating that TBI induces neuroinflammatory response in rats. Compared with the model group, the 6-hour and 12-hour time frame groups had significantly decreased IL-1β levels. Treatment with 0.2 mg / kg annexin A5 for 12 hours after injury reduced IL-1β expression after TBI in rats by about 100%. There was no statistical difference between the two groups in terms of TNF-α and IFN-γ levels (Figure 6).

[0186] <Example 3> Annexin A5 was derived from the full-length annexin of human origin. By constructing a recombinant carrier, it was expressed in large quantities in the prokaryotic organism Escherichia coli (E. coli). The expressed A5 protein was sequenced and found to have the following amino acid sequence: AQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD (sequence number 1).

[0187] As shown in SEQ ID NO: 1, the A5 protein expressed by the E. coli expression system contains 319 amino acids, the completed cDNA sequence of which is encoded by codons for 320 amino acids, and the N-terminal methionine is cleaved during the expression process. The amino acid sequence of SEQ ID NO: 1 is identical to that of natural human annexin A5, differing only in that the N-terminal amino acid of the A5 protein expressed by the system is alanine (A), whereas the N-terminus of natural human annexin A5 is acetylated alanine.

[0188] Thereafter, the following experiment was carried out using the A5 protein obtained in Example 3.

[0189] <Example 4> Based on the contents of the literature, Behavioral deficits and recovery following transient focal cerebral ischemia in rats: glutamatergic and GABAergic receptor densities (Jolkkonen J, Gallagher NP, Zilles K, Sivenius J. Behav Brain Res 2003;138:187-200) and Reversible middle cerebral artery occlusion without craniectomy in rats. (Longa EZ, Weinstein PR, Carlson S, Cummins R. Stroke 1989;20:84-91), a middle cerebral artery occlusion (MCAO) cerebral ischemia-reperfusion model was prepared by the internal carotid artery thread occlusion method and A5 0.1 mg / kg, A5 1.0 mg / kg, and A5 10.0 mg / kg groups were set up and administered edaravone intravenously (6.0 A5 protein was administered once at the time of reperfusion and once again 1 h after reperfusion in each dose group, and once immediately after reperfusion in each other dose group. Neurobehavioral symptoms were evaluated and the area of ​​cerebral infarction was measured 24 hours after cerebral ischemia.

[0190] 1. Test materials (1) Laboratory animals The rats were Sprague Dawley (SD) rats, SPF grade, female, aged approximately 6 to 8 weeks, and weighing 250 to 280 g.

[0191] Animals were excluded if they met any of the following conditions during the experimental process: a) died during the anesthesia process, b) died during the cerebral ischemia process, c) died after reperfusion until the evaluation of the indicators, d) found to have a skull base hemorrhage after the brain was removed, or e) had no infarcted area in the right brain after TTC staining.

[0192] (2) Reagents An A5 protein solution was prepared by diluting the A5 protein prepared in Example 3 with a 0.9% aqueous sodium chloride solution.

[0193] Control: Edaravone injection (purchased from Nanjing Xiansheng Dongyuan Pharmaceutical Co., Ltd.), which was diluted by mixing Edaravone injection and 0.9% sodium chloride injection in a 1:1 ratio to serve as the positive control.

[0194] 2. Study Design (1) Dose design

[0195] [Table 3]

[0196] (2) Administration Animals in each group were dosed intravenously via the tail vein, and the rate of intravenous administration was approximately 1.5-2.5 mL / min.

[0197] For each treatment group, 24 hours after cerebral ischemia, neurobehavioral symptoms were evaluated and the area of ​​cerebral infarction was measured.

[0198] 3. Evaluation Methodology (1) Neurological Deficit Symptom Score The animals were evaluated for neurological deficit symptoms 24 h after ischemia using a modified Bederson 5-point test, based on the method described in the literature: Rat middle cerebral artery occlusion, evaluation of the model and development of a neurologic examination (Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. 1986. Stroke 17: 472-476).

[0199] (2) Measurement of the degree of cerebral infarction The extent of cerebral infarction was measured using the TTC staining method with reference to the contents described in the literature: Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats (Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM. Stroke 1986;17:1304-8) and Quantification of infarct size on focal cerebral ischemia model of rats using a simple and economical method (Yang Y, Shuaib A, Li Q. J Neurosci Methods 1998;84:9-16).

[0200] Calculation of cerebral infarct area: The photographs were processed using Image J software, and the corresponding area of ​​the left brain and the non-infarct area of ​​the right brain were calculated by formula to obtain the percent infarct area. Infarct volume calculation: V=t (A1+ A2+ A3+ ………+An) t is the slice thickness and A is the infarct area. %I=(V C -V L ) / V C ×100% %I is the percent infarct area, V C is the brain volume of the control side (left hemisphere), and V L was the volume of the non-infarcted area on the infarcted side (right hemisphere).

[0201] (3) Evaluation index The main evaluation indices were the extent of cerebral infarction and neurobehavioral scores, and the clinical symptoms of the animals were observed at the same time.

[0202] Quantitative data are shown as mean ± standard error. Each efficacy index was analyzed by one-way ANOVA using GraphPad Prism (6.01) software. After the variance test was significant, the differences between groups were examined using Fisher's LSD test. P<0.05 was defined as significant.

[0203] 4. Experimental Results (1) Effect of A5 protein on neurological deficit symptoms One-way analysis of variance revealed no statistically significant differences (F (4. 69) = 2.305, P = 0.0669). However, a T-test was performed for each group with the model group, and it was found that the edaravone 6.0 mg / kg group (P < 0.01) and the A5 10.0 mg / kg group (P < 0.05) could significantly improve the neurobehavioral disorder symptoms of the ischemic animals, and the A5 1.0 mg / kg group and the A5 0.1 mg / kg group had a tendency to improve the neurobehavioral disorder symptoms of the ischemic animals, but there was no statistical difference. The effect of A5 protein on the neurobehavioral disorder symptoms is shown in detail in Table 4 and Figure 7.

[0204] [Table 4]

[0205] (2) Effect of A5 protein on the extent of cerebral infarction One-way analysis of variance revealed no statistically significant differences between groups (F (4. 69) = 2.240, P = 0.0735). However, a T-test was performed for each group and the model group, and it was found that the A5 10.0 mg / kg group (P < 0.05) could significantly reduce the cerebral infarction size of the model animals, the edaravone 6.0 mg / kg group and the A5 1.0 mg / kg group had a tendency to reduce the cerebral infarction size of the model animals, but there was no statistical difference, and the A5 0.1 mg / kg group had no effect of reducing the cerebral infarction size of the model animals. The effect of A5 protein on the cerebral infarction size is shown in detail in Table 4 and Figure 8.

[0206] In this study, the intragroup difference in the extent of cerebral infarction in the model group animals was basically within 1 / 3 of the mean value (Mean ± SD, 42.53 ± 12.65), and the model usability rate was 69.81% (excluding sham-operated animals). The experimental system was highly reliable and could be used for drug efficacy evaluation.

[0207] Compared with the model group, the A5 10.0 mg / kg group could significantly reduce the cerebral infarction area of ​​the model rats (P<0.05), the edaravone 6.0 mg / kg and A5 1.0 mg / kg groups had a tendency to reduce the cerebral infarction area of ​​the model rats, but there was no statistical difference, and A5 0.1 mg / kg had no effect of reducing the cerebral infarction area of ​​the model animals. Compared with the model group, the edaravone 6.0 mg / kg and A5 10.0 mg / kg groups could significantly improve the neurobehavioral disorder symptoms of the ischemic animals (p<0.01, p<0.05), and the remaining groups did not show obvious improvement effects in the indicators.

[0208] In this experiment, the A5 10.0 mg / kg group could significantly reduce the cerebral infarction area of ​​ischemic rats and improve the neurobehavioral disorder symptoms of ischemic animals. No bleeding risk was observed in any of the experimental groups treated with Annexin A5.

[0209] <Example 5> Microglia played an important role in the progression of inflammation in the central nervous system. Moderate activation of microglia has a protective effect on neurons, but overactivated microglia release large amounts of neurotoxic factors such as carbon monoxide, which triggered the development of neurodegenerative diseases. Lipopolysaccharide (LPS) activates microglia and causes excessive activation of microglia, thus causing neuronal damage. A5 was used to observe the effects of excessive activation of microglia by LPS stimulation.

[0210] In the experimental process, primary purified microglial cells were stimulated with LPS as the control group, and A5 protein was added at the same time as the experimental group. The cell vitality was measured by MTT method and the changes in cell morphology were observed by immunocytochemistry after the above treatment. The experimental results showed that LPS can significantly activate primary microglial cells but do not affect cell vitality. A5 protein had an inhibitory effect on the excessive activation of microglial cells stimulated by LPS. Therefore, A5 protein could be used to inhibit the excessive activation of immune cells in the brain, for example, to inhibit the excessive activation of microglial cells.

[0211] <Example 6> The oxygen glucose deprivation (OGD) model simulates the ischemia / hypoxia stimulus model at the cellular level by changing the cell culture conditions, e.g., placing the cells in a hypoxic chamber or replacing the medium with glucose-free medium, to simulate the damage that cells would suffer under ischemia / hypoxia.

[0212] Rat primary cortical neurons were selected to construct an OGD model, and the test concentrations of A5 protein were 0, 41.15, 123.5, 370.4, 1111, 3333, and 10000 nM, respectively. First, the cells in the culture plate were washed twice with PBS, and sugar-free DMEM, which had been replaced with 95% N2 + 5% CO2 for 30 min in advance, was added, and the cells were immediately placed in a 37℃, 94% N2 + 1% O2 + 5% CO2 hypoxic incubator. After 8 h of hypoxic culture, the corresponding drug-containing medium (i.e., DMEM medium containing A5) was added, and the cells were placed in a 37℃, 5% CO2 cell incubator again for 24 h of reoxygenation and culture. After returning to normal conditions and continuing to culture for 24 h, the morphological changes of the cells were observed under an inverted microscope, the cell viability was detected by CCK-8 colorimetry, and the cell apoptosis rate was detected by flow cytometry. At the same time, a normal oxygen and glucose treatment group was set up as a control group. The formula for calculating the relative cell vitality is as follows:

[0213] Relative cell vitality (%) = (treated group - background group) / (control group - background group) x 100% Compared with the model group, A5 protein showed no protective effect against OGD-induced neuronal injury within the concentration range of 41.15-1111 nM (P>0.05), and significantly reduced the activity after OGD-induced neuronal injury at concentrations of 3333-10000 nM (P<0.05, Figure 9).

[0214] The experimental results showed that A5 protein can reduce damage to neuronal cells caused by oxygen-glucose deprivation at concentrations of 3,333 to 10,000 nM, and has a neuroprotective effect.

[0215] <Example 7> Example 7 investigated the neuroprotective effect of A5 protein against NMDA (N-methyl-D-aspartate)-induced excitotoxic injury of rat cortical neurons. NMDA activates NMDA receptors to increase Ca in brain tissue. 2+ It can cause an increase in the content of NOS, leading to a large amount of activation of NOS, which causes a series of physiological or pathological changes such as the synthesis of a large amount of NO, damaging neurons in the brain. In the experiment, neurons were pretreated with A5 protein in advance, and the neuroprotective effect of A5 protein against neuronal damage caused by excitotoxicity was observed.

[0216] In the experiment, 17-day-old SD rats were selected, and cortical neurons were inoculated and cultured. The neurons were then divided into a control group, an NMDA-intoxicated group, and an A5 pretreatment group, in which the NMDA-intoxicated group was intoxicated with NMDA, the control group was not treated, and the A5 pretreatment group was intoxicated with NMDA after incubating the neurons with A5 protein for a certain period of time. The cell vitality of the different treatment groups was evaluated by trypan blue staining, apoptotic cells were detected by TUNEL staining, and neuronal morphology was measured by immunofluorescence cytochemistry. The formula for calculating the relative cell vitality is as follows:

[0217] Relative cell vitality (%) = (treated group - background group) / (control group - background group) x 100% As shown in Figure 10, NMDA at a concentration of 100 μM could induce a decrease in the cell activity of rat primary cortical neurons (P<0.001, compared with normal control), indicating that the model was successfully constructed. A5 protein showed no significant alleviating effect on NMDA-induced primary cortical neuron excitability damage within the test concentration range of 41.15-3333 nM (P>0.05, compared with model group), and A5 protein showed a significant alleviating effect on NMDA-induced primary cortical neuron excitability damage at a test concentration of 10000 nM (P<0.05, compared with model group).

[0218] The experimental results showed that, compared with the NMDA intoxication group, the A5 pretreatment group could reduce the damage to neurons caused by excitotoxicity at a test concentration of 10,000 nM.

[0219] <Example 8> The blood-brain barrier is composed of three components: brain microvascular endothelial cells, astrocytes, and basement membrane. Damage to the blood-brain barrier is one of the important pathologies of stroke brain injury.

[0220] In the experimental process, spontaneously transformed endothelial cell line and isolated and purified rat astrocytes were co-cultured to establish an in vitro blood-brain barrier model. The in vitro blood-brain barrier (BBB) ​​model was damaged after stimulation treatment, and then the blood-brain barrier model after stimulation treatment was treated with A5 protein.

[0221] A rat craniocerebral injury model by craniocerebral percussion was used to evaluate the integrity of the blood-brain barrier, and A5 10 mg / kg, A5 30 mg / kg, and model groups were set up. Each dose group and model group of A5 protein was administered once at 1 h and 2 h after model construction, respectively, for a total of two doses, and the administration method was tail vein injection. 23 h after model construction, the rats were anesthetized and then injected with 2% EB (Evans blue) dye solution at a dose of 4 mL / kg through the tail vein, and the dye was allowed to circulate in the rat's body for 1 hour before being killed. The rats were perfused with saline through the left ventricle of the heart until a colorless perfusion was obtained. After killing, the brains were removed, weighed, homogenized in formamide (3 μL / mg), and incubated at room temperature for 48 hours. After centrifugation, the supernatant was collected to determine the relative amount of EB dye and the optical density was measured at 625 nm, and the results are shown in Table 5 and Figure 11.

[0222] [Table 5]

[0223] The results showed that compared with the model group, the EB content in brain tissue of the A5 10 mg / kg group had no significant difference (P>0.05), and the EB content in brain tissue of the A5 30 mg / kg group was significantly reduced (P<0.05). In this experiment, the A5 30 mg / kg group had a repairing effect on the blood-brain barrier damage in rats caused by skull percussion. The experimental results showed that stimulation significantly disrupted the already established blood-brain barrier, and A5 could repair the disrupted blood-brain barrier.

[0224] Example 9 Immunotoxicity and Immunogenicity Studies (with Repeated Dose Toxicity Studies in SD Rats and Cynomolgus Monkeys) 1. Toxicity Experimental Study in SD Rats SD rats were intravenously injected with A5 protein for 28 consecutive days, followed by a 4-week recovery period. The rats were observed for toxic reactions and their severity, the main toxic target organs, and the reversibility of damage. The doses were 0 (blank adjuvant), 30, 150, and 750 μg / kg, respectively. Blood samples were collected to evaluate immunotoxicity (CD4 + , CD8 + T cell) and immunogenicity tests were performed. The study was divided into 4 groups, with 8 rats per group, and samples were collected at a total of 4 time points, that is, serum samples were collected from each rat at 4 time points.

[0225] Among them, the immunotoxicity results showed that the percentage and ratio of peripheral blood T lymphocyte subpopulations (CD4 + , CD8 + It was found that there was no statistically significant difference in the T cell measurements.

[0226] The immunogenicity results are shown in Table 6. The sample numbers for each group in Table 6 represent 8 rats per group, serum was collected at 4 time points, and each group had a total of 32 samples, and the number of positive individuals is the number of positive samples detected from the samples in each group.

[0227] [Table 6]

[0228] As can be seen from the results shown in Table 6, after A5 protein blank supplement was administered intravenously to SD rats, anti-drug antibodies (ADA) were not detected. After different doses of A5 protein were administered intravenously, the detection rate of positive samples was 65.6%. There was no obvious difference between the dose groups, and the positive samples were mainly concentrated after the last dose and at the end of the recovery period, which was consistent with the process of drug-resistant antibodies being produced in vivo.

[0229] 2. Experimental toxicity study in cynomolgus monkeys In this study, A5 protein was intravenously injected into cynomolgus monkeys for 28 consecutive days, followed by a 4-week recovery period to observe the toxic reactions and their severity, the main toxic target organs, and the reversibility of damage caused by the cynomolgus monkeys. The doses were 0 (blank adjuvant), 15, 75, and 375 μg / kg, respectively. Immunogenicity blood sampling and immunotoxicity (CD4 + , CD8 + T cell) was examined.

[0230] Immunotoxicity results showed that the percentage and ratio of peripheral blood T lymphocyte subpopulations (CD4 + , CD8 + It was found that there was no statistically significant difference in the T cell measurements.

[0231] The immunogenicity results are shown in Table 7. The same grouping and treatment methods as those of the rats described above were adopted, except that the number of samples in each group was increased by two, i.e., the number of samples in each group was increased by two.

[0232] [Table 7]

[0233] As can be seen from the results shown in Table 7, after intravenous administration of A5 protein blank adjuvant to cynomolgus monkeys, ADA was not detected. After intravenous administration of different doses of A5 protein, the detection rate of positive samples was 25.5%, and the positive samples were concentrated 4 weeks after administration and at the end of the recovery period, which is consistent with the process of drug-resistant antibodies being produced in vivo.

[0234] The experimental results showed that no immunotoxicity was found in either rats or cynomolgus monkeys. In the immunogenicity test, the positive incidence rate was significantly reduced in cynomolgus monkeys compared to rats. Considering that A5 protein belongs to human-derived proteins, it was expected that the incidence rate of immunogenicity in the human body would be significantly reduced.

[0235] Example 10: Single-dose toxicity study of A5 protein administered intravenously to SD rats and cynomolgus monkeys 1. Single-dose toxicity study of A5 protein administered intravenously to SD rats In the study, A5 protein was administered intravenously once to SD rats, and the animals were observed for their acute toxic reactions and their severity, as well as the main toxic target organs. The doses were 0 (blank adjuvant), 1000, 3000, and 9000 μg / kg, respectively. After intravenous administration, the animals were continuously observed for clinical symptoms such as posture, gait, reaction state, nervous activity, appetite, coat, eyes, ears, mouth, nose, limbs, breathing, and feces. After that, observations were continued until the 14th day after administration, and the recovery status of the toxic reactions of the animals was considered. On the 15th day after administration, the animals were dissected and macroscopic pathological examination was performed. During the test period, no abnormalities were observed in the clinical symptoms of all animals, no abnormalities were observed in the weight changes of female and male animals in the administration group, and no abnormalities were observed in any of the tissues and organs in the macroscopic pathological examination. Under these test conditions, the tolerated dose (MTD) of a single intravenous injection of A5 protein was 9000 μg / kg or more.

[0236] 2. Single-dose toxicity study of intravenous injection of A5 protein in cynomolgus monkeys In the study, A5 protein was administered intravenously once to cynomolgus monkeys, and the animals were observed for acute toxic reactions and their severity, as well as the main toxic target organs. The doses were 0 (blank adjuvant), 500, 1500, and 4500 μg / kg, respectively. The animals were administered by a single intravenous injection, and the animals were observed for their clinical symptoms (including feces, appearance, respiration, neurological response, activity, etc.) up to the 15th day after administration. After administration, food intake was measured once a day, and body weight was measured once a week. On the day of administration, the animals' body temperature, blood pressure, and electrocardiogram were measured before and after administration, respectively, and once more on the 1st, 7th, and 15th days after administration. Hematology (including blood coagulation) and serum biochemistry tests were performed on all animals on the 1st, 7th, and 15th days after administration, and urine tests were performed on the 14th day after administration. On the 15th day after administration, the animals were dissected for gross pathology and histopathology, if necessary. As can be seen from the results, no obvious changes related to the test substance were observed in the clinical symptoms and injection site observations, body weight, food intake, body temperature, blood pressure, electrocardiogram, urinalysis, serum biochemistry and pathological examinations of the animals in each group. On the 13th day of the quarantine period, no obvious changes were observed in the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) of the animals in each group from the 1st day after administration to the 15th day after administration. Under these test conditions, the animal tolerated dose (MTD) of cynomolgus monkeys was 4500 μg / kg or more.

[0237] The above experimental results show that the tolerance of A5 protein in both SD rats and cynomolgus monkeys is more than 4500 μg / kg, and the tolerance of A5 protein in SD rats is even more than 9000 μg / kg. The results show that at the equivalent dose where A5 protein exerts a protective effect against stroke, there is no obvious toxicity in rats and monkeys. This suggests that when A5 protein is used to treat stroke, it has good safety and brings no or little risk of drug use even when administered at a relatively high dose.

[0238] Although the embodiments of the present disclosure have been shown and described above, the above embodiments are illustrative and should not be understood as limiting the scope of the claims of the present disclosure. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present disclosure, and all of these changes, modifications, substitutions and variations should be included in the scope of the claims of the present disclosure. [Brief description of the drawings]

[0239] [Figure 1A] 13 shows the dose-dependent effect of Annexin A5 treatment on neurological outcome after TBI and presents the results of mNNS. [Figure 1B] FIG. 1 shows the dose-dependent effect of Annexin A5 treatment on neurological outcome after TBI, and the results of the rotarod test are shown. [Diagram 2] The results show that administration of Annexin A5 significantly reduces TUNEL-positive cells in the injured rat brain after TBI (###P<0.001, compared with the sham operation group; *P<0.05, compared with the model group, t-test, n=5). Among them, TUNEL positive cells represents the amount of positive cells, Sham represents the sham operation group, and Model represents the model group. [Diagram 3] The results show that administration of 0.2 mg / kg of Annexin A5 significantly reduces GFAP-positive cells in the injured rat brain after TBI (###P<0.001, compared with the sham operation group; *P<0.05, compared with the model group, t-test, n=5). Among them, Mean Fluorescence (arbitrary units) represents the mean fluorescence (arbitrary units), Sham represents the sham operation group, and Model represents the model group. [Figure 4]The effect of Annexin A5 treatment on neurofunctional outcomes after TBI is shown. (Top) MNS results, (Bottom) Rotarod test results. (###P<0.001, compared with sham operation group; ***P<0.001, compared with model group; n=5). Among them, mNNS score stands for mNNS score, Fall latency stands for fall latency, Sham stands for sham operation group, Model stands for model group, A5 One Dose stands for administration of only one dose of A5, and Time stands for time. [Diagram 5] The effect of Annexin A5 treatment on neurofunctional outcomes after TBI is shown. (Top) MNS results, (Bottom) Rotarod test results. (###P<0.001, compared with sham operation group; ***P<0.001, compared with model group; n=5). Among them, mNNS score stands for mNNS score, Fall latency stands for fall latency, Sham stands for sham operation group, Model stands for model group, A5 One Dose stands for one dose of A5, and post-TBI stands for post-TBI. [Figure 6] The levels of IL-1β, TNF-α and IFN-γ in brain tissue are shown (###P<0.001, compared with the sham operation group; **P<0.01, compared with the model group; n=5). In this figure, protein represents protein, Sham represents the sham operation group, and Model represents the model group. [Figure 7] FIG. 13 is a diagram showing the effect of an Annexin A5 solution provided by an embodiment of the present invention on neurological deficit symptoms. [Figure 8] FIG. 13 is a graph showing the effect of the Annexin A5 solution provided by an embodiment of the present invention on the extent of cerebral infarction. [Figure 9] FIG. 1 shows the effect of Annexin A5 provided by an embodiment of the present invention on primary cortical neuron OGD injury. [Figure 10] FIG. 1 shows the protective effect of Annexin A5 provided by an embodiment of the present invention against NMDA-induced excitatory injury of primary rat cortical neurons. [Figure 11]FIG. 1 shows the measurement results of Annexin A5 provided by an embodiment of the present invention on the EB content of brain tissue.

Claims

1. 1. A method for treating craniocerebral injury in a patient in need thereof, comprising: Identifying a patient suffering from or suspected of suffering from a craniocerebral injury; administering to the patient a therapeutically effective amount of annexin A5 or a variant thereof, or a pharmaceutical composition comprising a therapeutically effective amount of annexin A5 or a variant thereof; For example, according to the method of claim 1, the annexin A5 or its variant comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO:

1. For example, the craniocerebral injury is mild, moderate or severe craniocerebral injury; For example, the craniocerebral injury may be caused by head impact, penetrating head injury, non-penetrating head injury, a fall, a skull fracture, sudden acceleration or deceleration, a concussion or a contusion; For example, the patient is a human. method.

2. The annexin A5 or a variant thereof is (i) reducing diffuse axonal injury, behavioral impairment, brain tissue damage, brain atrophy, neuronal cell death or apoptosis, microglial activation, or neuronal tissue loss in a patient suffering from craniocerebral injury; or (ii) increasing cerebral blood flow or cerebral glucose uptake in patients suffering from craniocerebral injury; Alternatively, the annexin A5 reduces one or more symptoms associated with craniocerebral injury in a patient; For example, the one or more symptoms associated with the craniocerebral injury include impaired level of consciousness, cognitive impairment, impaired cognitive processing speed, impaired language, impaired motor activity, impaired memory, impaired motor skills, impaired sensory skills, cerebral ischemia, edema, intracranial pressure, hearing loss, tinnitus, headache, seizures, dizziness, nausea, vomiting, blurred vision, decreased sense of smell or taste, decreased strength, or decreased coordination. The method of claim 1.

3. administering a first drug, such as annexin A5 or a variant thereof, or the pharmaceutical composition, within a time period of up to 4 weeks from the time of the craniocerebral injury; For example, the first drug is administered within about 0 hours, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after the occurrence of craniocerebral injury. For example, a therapeutically effective amount of Annexin A5 or a variant thereof is between about 0.01 mg / kg and about 10 mg / kg, such as between about 0.01 mg / kg and about 0.5 mg / kg, such as about 0.02 mg / kg, 0.06 mg / kg or 0.2 mg / kg. The method of claim 1.

4. The method further comprises administering to the patient a second drug or treatment; The second drug or treatment is a cardioprotective drug or treatment, the cardioprotective drug or treatment including one or a combination of beta-receptor blockers, diuretics, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers, lipid-lowering therapy, statins, nitrates, antiplatelets, anticoagulants, and anticoagulants, and the therapeutic application of the drug or treatment; The method of claim 1.

5. The step of identifying the patient as suffering from a craniocerebral injury includes performing a computed tomography (CT) scan or a magnetic resonance imaging (MRI) on the patient; For example, a patient is identified as suffering from a craniocerebral injury based on the modified Neurological Severity Score (mNSS), the rotarod test, the Glasgow Coma Scale score, or the Glasgow Outcome Scale score; For example, their mNSS or Glasgow Coma Scale scores are between 3 and 8, between 9 and 12, or between 13 and 15; For example, if the mNSS or Glasgow Coma Scale score is lower than a reference level, the patient is identified as suffering from a craniocerebral injury; For example, the reference level is obtained from a reference subject that has not suffered a head injury. The method of claim 1.

6. The step of identifying a patient suffering from a craniocerebral injury includes: determining a level of a biomarker in a sample from the patient; comparing the level of the biomarker to a reference level of the biomarker; If the level of the determined biomarker is increased compared to a reference level of the biomarker, the patient is identified as suffering from craniocerebral injury. The method of claim 1.

7. the biomarker comprises a cytokine selected from IL-1β, TNF-α, IFN-γ, IL-6, IL-10, IL-8, HMGB1, TGFβ, UCH-L1, NSE, GFAP, S100B, NF proteins (L and H), MBP, Tau or phosphorylated Tau; For example, the sample is obtained after the patient's head has experienced a physical vibration, a closed or open head injury due to blunt impact of an external mechanical or other force, one or more falls, an explosion, a blast, a solid explosion, a gas explosion, or other type of blunt trauma; For example, the sample is selected from a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. The method according to claim 6.

8. 1. A method of reducing the level of a biomarker in a patient suffering from craniocerebral injury, comprising: (a) Identifying patients suffering from craniocerebral injuries; (b) administering to the patient a therapeutically effective amount of annexin A5 or a variant thereof, or a pharmaceutical composition comprising a therapeutically effective amount of annexin A5 or a variant thereof; Among these, the biomarkers include cytokines selected from IL-1β, TNF-α and IFN-γ, IL-6, IL-10, IL-8, HMGB1, TGFβ, UCH-L1, NSE, GFAP, S100B, NF proteins (L and H), MBP, Tau and phosphorylated Tau; For example, a therapeutically effective amount of annexin A5 or a variant thereof is an amount of annexin A5 or a variant thereof that increases or decreases the level of a biomarker by at least 5%, 10%, 20%, 30%, 40% or 50%. method.

9. Use of annexin A5 or a variant thereof in the manufacture of a medicament, said medicament comprising: Treatment and / or prevention of craniocerebral injuries, for example, where the craniocerebral injuries are mild, moderate or severe craniocerebral injuries; reducing diffuse axonal injury, behavioral impairment, brain tissue damage, brain atrophy, neuronal cell death or apoptosis, microglial activation, or neuronal tissue loss in patients suffering from craniocerebral injury; Increasing cerebral blood flow or cerebral glucose uptake in patients suffering from craniocerebral injury, Relief of one or more symptoms associated with craniocerebral injury in a patient, for example, where the one or more symptoms associated with the craniocerebral injury include impaired level of consciousness, cognitive impairment, impaired cognitive processing speed, impaired language, impaired motor activity, impaired memory, impaired motor skills, impaired sensory skills, cerebral ischemia, edema, intracranial pressure, hearing loss, tinnitus, headache, seizures, dizziness, nausea, vomiting, blurred vision, decreased sense of smell or taste, decreased strength, or decreased coordination; A decrease in said biomarkers in patients suffering from craniocerebral injury, for example, where the biomarkers comprise cytokines selected from IL-1β, TNF-α and IFN-γ, IL-6, IL-10, IL-8, HMGB1, TGFβ, UCH-L1, NSE, GFAP, S100B, NF proteins (L and H), MBP, Tau and phosphorylated Tau; One, two or more selected from the following are used; For example, the agent is in an amount that increases or decreases the level of a biomarker by at least 5%, 10%, 20%, 30%, 40%, or 50%; For example, annexin A5 or a variant thereof comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1, or comprises the amino acid sequence of SEQ ID NO:

1. use.

10. The craniocerebral injury is a mild, moderate or severe craniocerebral injury; For example, the craniocerebral injury may be caused by head impact, penetrating head injury, non-penetrating head injury, a fall, a skull fracture, sudden acceleration or deceleration, a concussion or a contusion; For example, the patient is a human, Preferably, the drug is in unit dosage form; For example, the amount of annexin A5 or a variant thereof in the medicament is between about 0.01 mg / kg and about 10 mg / kg of patient body weight, such as between about 0.01 mg / kg and about 0.5 mg / kg of patient body weight, such as about 0.02 mg / kg, 0.06 mg / kg or 0.2 mg / kg of patient body weight; For example, the medicament is in a unit dosage form and the amount of annexin A5 or a variant thereof in the unit dosage form is between 0.025 mg and 250 mg, such as between 0.1 and 50 mg, between 0.1 and 100 mg, between 0.1 and 200 mg or between 0.1 and 250 mg, or between 1 and 50 mg, between 1 and 100 mg, between 1 and 200 mg or between 1 and 250 mg.

10. The use according to claim 9.

11. The drug is administered within a time period from the time of the craniocerebral injury up to 4 weeks, preferably within about 0 hours, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after the occurrence of the craniocerebral injury, more preferably within about 12 hours after the craniocerebral injury, For example, the drug further comprises a cardioprotective drug, For example, the cardioprotective drug is selected from one or a combination of beta-receptor blockers, diuretics, angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers, lipid-lowering therapy, statins, nitrates, antiplatelets, anticoagulants, and anticoagulants; 11. The use according to claim 10.

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