Use of annexin a5

Abstract

The present application relates to the field of biomedicine and provides the use of annexin A5 for preventing and / or treating a disease or condition, wherein the disease or condition is selected from the group consisting of stroke, neurobehavioral deficit, neurodegenerative disease, nerve injury, over-activation of immune cells in the brain, blood-brain barrier injury, brain edema, increased calcium ion content, excitotoxicity, over-activation of NOS, and a disease or condition caused by at least one of the above diseases or conditions.

Description

[0001] This application claims priority to an earlier application filed on August 30, 2019, with patent application number 201910815496.0 entitled "Use of Annexin A5 in the Treatment of Stroke", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine, specifically to the use of annexin A5. Background Technology

[0003] Annexins are a family of calcium-dependent phospholipid-binding proteins, widely expressed and possessing a variety of important functions. Some annexins, such as annexin A1, are closely related to tumors, showing high expression in pancreatic cancer, liver cancer, and head and neck tumors. Other annexins, such as annexin A5 (or A5), have anti-inflammatory and anticoagulant functions. Annexin A5 was first discovered in the human placenta in 1970; its gene is located on chromosome 4q26-q28 and has 13 exons and 12 introns. As a calcium-dependent channel protein that binds to the surface of negatively charged phospholipids, annexin A5 has a molecular weight of approximately 34 kDa and is mainly found on the cell membrane and endoplasmic reticulum membrane. Its large molecular weight generally prevents it from entering brain tissue, resulting in very limited research on its use in the nervous system.

[0004] Cerebral stroke, also known as apoplexy or cerebrovascular accident (CVA), is an acute cerebrovascular disease. It primarily results from the sudden rupture or blockage of blood vessels in the brain, preventing blood flow to the brain and surrounding tissues, leading to brain damage. This includes ischemic stroke and hemorrhagic stroke. Ischemic stroke has a higher incidence than hemorrhagic stroke, accounting for 60%–70% of all strokes. Clinical studies show that multiple factors can trigger stroke. Stroke is characterized by high incidence, high disability rate, high recurrence rate, and high mortality rate. Therefore, the development of new therapeutic agents is needed to further improve treatment outcomes for stroke.

[0005] In addition, treatment methods for diseases or conditions such as neurobehavioral defects, neurodegenerative diseases, nerve damage, excessive activation of immune cells in the brain, blood-brain barrier damage, increased calcium ion content, excitotoxicity, and excessive NOS activation need further improvement, and new therapeutic agents urgently need to be developed.

[0006] Although the structure of annexin A5 has become increasingly clear, its function and applications still require further exploration by researchers, and need to be developed and confirmed experimentally. At the same time, the safety of using this protein for disease prevention and / or treatment also needs to be considered. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention provides the use of annexin A5 for the prevention and / or treatment of diseases or conditions, wherein the diseases or conditions are selected from stroke, neurobehavioral deficits, neurodegenerative diseases, nerve damage, excessive activation of immune cells in the brain, blood-brain barrier damage, cerebral edema, increased calcium ion content, excitotoxicity, excessive NOS activation, and diseases or conditions caused by at least one of the aforementioned diseases or conditions.

[0008] According to embodiments of the present invention, the present invention provides the use of annexin A5 in the preparation of a medicament for the prevention and / or treatment of the aforementioned diseases or conditions and diseases or conditions caused by at least one of the aforementioned diseases or conditions.

[0009] According to embodiments of the present invention, the neurobehavioral defects, neurodegenerative diseases, nerve damage, excessive activation of immune cells in the brain, blood-brain barrier damage, cerebral edema, increased calcium ion content, excitotoxicity, and excessive NOS activation can be selected from the diseases or conditions caused by stroke or other reasons.

[0010] According to an embodiment of the present invention, the stroke is selected from ischemic stroke or hemorrhagic stroke, for example, ischemic stroke in the acute and / or recovery phase, and hemorrhagic stroke in the acute and / or recovery phase, especially acute ischemic stroke or ischemic stroke in the acute phase.

[0011] According to embodiments of the present invention, the neurobehavioral defects include, but are not limited to, flaccid paralysis and / or weakness of the limbs.

[0012] According to embodiments of the present invention, the neurodegenerative disease includes acute neurodegenerative diseases and / or chronic neurodegenerative diseases, such as acute neurodegenerative diseases selected from the following: cerebral ischemia (CI), brain injury (BI), epilepsy; and chronic neurodegenerative diseases selected from the following: Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, etc.

[0013] According to an embodiment of the present invention, the nerve injury is selected from brain nerve injury, such as brain nerve injury caused by cerebrovascular diseases such as traumatic brain injury, cerebral arteriosclerosis, encephalitis, meningitis, and demyelinating diseases, such as cognitive nerve injury, such as olfactory nerve injury, visual nerve injury, auditory nerve injury, gustatory nerve injury, and facial nerve injury.

[0014] According to an embodiment of the present invention, the nerve injury may be brain nerve injury caused by oxygen / glucose deprivation or excitotoxicity.

[0015] According to embodiments of the present invention, the neurodegenerative disease, neurological injury, or the resulting disease or condition is selected from affective disorders, depression, major depressive disorder, postpartum depression, depression associated with bipolar disorder, Alzheimer's disease, psychosis, Parkinson's disease, Huntington's disease, anxiety, generalized anxiety disorder, social anxiety disorder, obsessive-compulsive disorder, panic disorder, panic attacks, phobias, social phobia, agoraphobia, urinary incontinence, vomiting, partial response, treatment-resistant depression, cognitive impairment, memory loss, ADHD (attention deficit hyperactivity disorder), depression, or PTSD (post-traumatic stress disorder).

[0016] According to embodiments of the present invention, the term "brain immune cells" in the context of "overactivation of brain immune cells" includes immune-active cells of the central nervous system (CNS), particularly microglia.

[0017] According to embodiments of the present invention, the blood-brain barrier injury includes damage, impairment, or obstruction of the barrier between plasma and brain cells and / or cerebrospinal fluid, such as damage to the barrier between plasma and brain cells formed by capillary walls and glial cells, and / or damage to the barrier between plasma and cerebrospinal fluid formed by the choroid plexus. The blood-brain barrier injury may be caused by the aforementioned diseases or conditions, or it may be caused by traumatic brain injury (such as traumatic brain injury caused by external force).

[0018] According to an embodiment of the present invention, cerebral edema refers to a pathological phenomenon in which increased water content in the brain leads to an enlarged brain volume, which can cause intracranial hypertension and damage brain tissue. Cerebral edema can be caused by various factors, including traumatic brain injury, blood-brain barrier damage, microcirculatory disturbances, cerebral hemorrhage, cerebral ischemia, cerebral hypoxia, or other factors.

[0019] According to an embodiment of the present invention, the increased calcium ion content refers to an increase in the concentration of calcium ions in brain tissue, exceeding the normal range.

[0020] According to an embodiment of the present invention, the excitotoxicity is N-methyl-D-aspartic acid-induced excitotoxicity.

[0021] According to an embodiment of the present invention, the NOS overactivation refers to the oversynthesis of NO caused by the activation of NOS.

[0022] According to some embodiments of the present invention, the neurobehavioral deficits, neurodegenerative diseases, nerve damage, excessive activation of intracranial immune cells, blood-brain barrier damage, increased calcium ion content, excitotoxicity, and excessive NOS activation can be caused by stroke. Therefore, when the use of annexin A5 or drugs prepared therefrom relates to the prevention or treatment of stroke, the use of annexin A5 may further include the following technical features:

[0023] In some embodiments of the present invention, the stroke is ischemic stroke, particularly acute ischemic stroke or ischemic stroke in the acute phase. Annexin A5 can thus improve neurobehavioral defects caused by ischemic stroke, such as limb weakness and flaccid paralysis, and reduce the area of ​​cerebral infarction, thereby achieving the purpose of treating ischemic stroke.

[0024] In some embodiments of the present invention, the stroke involves excessive activation of immune cells in the brain. Annexin A5 can be used to inhibit excessive activation of immune cells in the brain, thereby alleviating neurological damage caused by excessive activation of immune cells and treating stroke.

[0025] In some embodiments of the invention, the stroke involves intracranial nerve damage. Annexin A5 can function as a neuroprotective agent in the brain, repairing nerve damage and thus treating stroke.

[0026] In some embodiments of the invention, the stroke involves a damaged blood-brain barrier. Annexin A5 can repair the damaged blood-brain barrier, preventing further damage to the brain, thereby treating the stroke.

[0027] In some embodiments of the present invention, the immune cells are microglia. Overactivation of microglia can release large amounts of neurotoxic factors, leading to neurodegenerative diseases. Annexin A5 can inhibit the overactivation of microglia.

[0028] In some embodiments of the present invention, the neural injury is intracranial neural injury caused by oxygen / glucose deprivation. Annexin A5 can alleviate intracranial neural injury caused by oxygen / glucose deprivation. Brain hypoxia or reduced glucose can induce neural injury; annexin A5 can alleviate neural injury caused by oxygen / glucose deprivation and treat stroke.

[0029] In some embodiments of the present invention, the nerve damage is intracranial nerve damage caused by excitotoxicity. Excitotoxicity can increase the calcium ion content in brain tissue, activate NOS in large quantities, and thus lead to a series of physiological or pathological changes such as the synthesis of large amounts of NO, causing intracranial nerve damage. Annexin A5 can repair this nerve damage and treat stroke.

[0030] In some embodiments of the present invention, the excitotoxicity is N-methyl-D-aspartic acid-induced excitotoxicity.

[0031] In some embodiments of the present invention, the annexin A5 can inhibit the excessive activation of immune cells in the brain.

[0032] In some embodiments of the present invention, the annexin A5 is capable of repairing nerve damage in the brain.

[0033] In some embodiments of the present invention, the annexin A5 is capable of repairing a damaged blood-brain barrier.

[0034] Those skilled in the art should recognize that the aforementioned neurobehavioral defects, neurodegenerative diseases, nerve damage, excessive activation of immune cells in the brain, blood-brain barrier damage, cerebral edema, increased calcium ion content, excitotoxicity, and excessive NOS activation can also be caused by factors other than stroke. For example, blood-brain barrier damage caused by traumatic brain injury.

[0035] According to embodiments of the invention, the annexin A5 may be, but is not limited to, a full-length, naturally occurring human annexin A5 polypeptide or a variant thereof. The A5 polypeptide can be provided from any source or by any method, for example, a natural isolate or a suitable combination of recombinant or synthetic sources. The polypeptide sequence of annexin A5 may be based on a complete or partial naturally occurring amino acid sequence or on variants of such complete or partial naturally occurring amino acid sequences.

[0036] In some embodiments of the present invention, the annexin A5 is the natural human annexin A5. In some embodiments of the present invention, the annexin A5 is recombinant human annexin A5 expressed by a prokaryotic expression system. Therefore, the desired recombinant human annexin A5 can be obtained rapidly and efficiently using a prokaryotic expression system at a low cost. Moreover, it has been verified to be non-immunotoxic, possessing extremely low immunogenicity, and the tolerated dose of annexin A5 reaches above 4500 μg / kg in both rats and cynomolgus monkeys, and even above 9000 μg / kg in rats, demonstrating good safety. Even at higher doses, it does not pose a risk of adverse drug reactions, making it of significant therapeutic value for acute cerebrovascular diseases such as stroke. In some embodiments of the present invention, the amino acid sequence of the annexin A5 expressed by the prokaryotic expression system is shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 is identical to that of natural human annexin A5, except that the N-terminal amino acid of this sequence is alanine (A), while the N-terminus of natural human annexin A5 is acetylated alanine. It can be obtained efficiently and rapidly, and has been proven to have excellent safety. Even at high doses, it does not pose any drug risk and is of great value for the prevention and / or treatment of the aforementioned diseases or conditions.

[0037] Studies have found that annexin A5 has a conserved sequence and high homology across different species, performing similar or identical functions. These highly homologous or sequence-conserved annexin A5s can be used to treat stroke or to prepare drugs for treating stroke. For example, in some embodiments of the present invention, the annexin A5 shows homology of 96% or more compared to natural human annexin A5 or the sequence shown in SEQ ID NO: 1, such as 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.3% or more, 98.5% or more, 98.8% or more, 99% or more, 99.3% or more, or 99.5% or more. In some embodiments of the present invention, the annexin A5, compared to SEQ ID NO: 1, undergoes one, two, three, four, or five conserved amino acid substitutions. These annexin A5s exhibiting high homology or conserved amino acid substitutions can be synthesized artificially; alternatively, they can be isolated or synthesized by referencing naturally occurring annexin A5s in nature.

[0038] In some embodiments of the invention, the medicament is in the form of a formulation, such as an injection. The content of annexin A5 in a unit dosage form of the medicament is 0.025 mg to 250 mg. Considering the daily dose of annexin A5 of approximately 0.05 mg to 500 mg, the content of annexin A5 in a unit dosage form of the medicament can be adaptively adjusted to be between 0.025 mg and 250 mg during the preparation of the medicament. For example, it can be between 0.1-50 mg, 0.1-100 mg, 0.1-200 mg, or 0.1-250 mg, or between 1-50 mg, 1-100 mg, 1-200 mg, or 1-250 mg, preferably between 1-100 mg, such as 1 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. Treatment of the aforementioned diseases or conditions can be achieved through the preparation of annexin A5 formulations, with good safety and no toxic side effects. A unit dosage form (or unit dose) of a drug refers to a single unit of a drug designed to be formulated into different dosage forms. For example, if designed as a tablet with a tablet size of 400 mg, then 400 mg is a unit dosage form of the drug. Similarly, if a drug is designed as an injection, each individually packaged injection is a unit dosage form of the drug. Typically, unit dosage forms of drugs are used as a daily dose or a half-day dose. The annexin A5 content in these unit dosage forms can fluctuate between 0.025 and 250 mg, and is combined with other pharmaceutically available carriers to prepare the drug into unit dosage forms.

[0039] These "pharmaceutically acceptable carriers" can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc. Specific examples include one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and combinations thereof. In many cases, isotonic agents may also be used, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Of course, pharmaceutically acceptable carriers may also include trace amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, to extend the shelf life or potency of antibodies.

[0040] The present invention also provides a medicament comprising annexin A5, wherein the medicament is used for the prevention and / or treatment of diseases or conditions selected from the following: stroke, neurobehavioral deficits, neurodegenerative diseases, nerve injury, excessive activation of immune cells in the brain, blood-brain barrier damage, cerebral edema, increased calcium ion content, excitotoxicity, excessive activation of NOS, and diseases or conditions caused by at least one of the above diseases or conditions.

[0041] The present invention also provides a pharmaceutical combination comprising annexin A5 and other drugs.

[0042] According to embodiments of the present invention, the other drugs are selected from drugs other than annexin A5, such as active ingredients used for the prevention and / or treatment of diseases or conditions selected from the following: stroke, neurobehavioral deficits, neurodegenerative diseases, nerve damage, excessive activation of immune cells in the brain, blood-brain barrier damage, cerebral edema, increased calcium ion content, excitotoxicity, excessive NOS activation, and diseases or conditions caused by at least one of the above-mentioned diseases or conditions.

[0043] In some embodiments of the present invention, other drugs for the prevention and / or treatment of stroke may be selected from at least one of the following: thrombolytic drugs, antiplatelet drugs, anticoagulants, fibrinolytic drugs, volume expanders, vasodilators, or other drugs that improve cerebral blood circulation and neuroprotective agents. By combining these drugs with other medications, multi-faceted and multi-level treatment of stroke can be achieved, thereby enabling rapid treatment of stroke.

[0044] The present invention also provides a method for treating, preventing and / or treating diseases or conditions, comprising administering to a patient an effective amount of annexin A5, wherein the disease or condition is selected from stroke, neurobehavioral deficits, neurodegenerative diseases, nerve injury, excessive activation of immune cells in the brain, blood-brain barrier damage, cerebral edema, increased calcium ion content, excitotoxicity, excessive NOS activation, and diseases or conditions caused by at least one of the above-mentioned diseases or conditions.

[0045] In this article, "condition" refers to an abnormal physiological state exhibited by the patient, such as disease symptoms or abnormal physiological indicators.

[0046] In this article, "effective amount" or "therapeutic effective amount" refers to the dosage of a drug that can alleviate or rehabilitate the disease or condition.

[0047] The term "patient" can refer to an individual or group that has the aforementioned disease or condition, or is at risk of having the aforementioned disease or condition, such as a subject. Patients include humans and mammals.

[0048] In some embodiments of the present invention, the dosage of annexin A5 is 0.05 mg to 500 mg per day, preferably 2 to 200 mg. It can typically be 1 mg to 500 mg / day, or 1 mg to 450 mg / day, such as 1 mg to 400 mg / day, 1 mg to 350 mg / day, 1 mg to 300 mg / day, 1 mg to 250 mg / day, 1 mg to 200 mg / day, etc. Of course, in some patients with mild stroke, the dosage of annexin A5 can be even lower, for example, 1 mg to 100 mg / day, or 1 mg to 80 mg / day, etc.

[0049] The annexin A5 or its drug can be administered as a single dose or in multiple repeated doses. The annexin A5 or its drug can be administered via intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intrathecal injection, nasal spray, or oral spray. It can also be administered rapidly in a short period, including but not limited to rapid intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intrathecal injection, nasal spray, or oral spray. It can also be administered with continuous exposure, including but not limited to continuous slow intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or intrathecal injection. Considering that annexin A5 is a protein drug, it is preferable to prepare it as an injectable formulation, such as a lyophilized powder or other injectable drugs of specific specifications, to achieve rapid treatment and high bioavailability in vivo.

[0050] In treatment, annexin A5 can be used for administration during the acute and recovery phases of the aforementioned diseases or conditions (such as stroke), as well as for secondary prevention after stroke, and for prophylactic administration in potential stroke patients with imaging indications but no clinical symptoms.

[0051] It should be understood that annexin A5 can be used alone or in combination with surgery or other medications for the treatment of stroke. Surgical procedures include, but are not limited to, endovascular interventional therapies (such as mechanical thrombectomy, angiogenesis, and stent implantation). Other medications include, but are not limited to, thrombolytic agents (such as recombinant tissue plasminogen activator rtPA and urokinase), antiplatelet agents (such as aspirin and clopidogrel), anticoagulants, fibrinolytic agents (such as fibrinolytic enzymes and batroxobin), volume expanders, vasodilators, other drugs that improve cerebral blood circulation, and neuroprotective agents (such as edaravone). Through combined medication, stroke can be treated from multiple angles, thus enabling rapid treatment.

[0052] In some embodiments of the present invention, after administering an effective amount of annexin A5 to a subject, it can inhibit the excessive activation of immune cells in the brain, such as the excessive activation of microglia, thereby indirectly repairing nerve damage caused by excessive immune cell activation. Simultaneously, it can also directly repair nerve damage in the brain, for example, reducing nerve damage caused by oxygen / glucose deprivation and mitigating neuronal damage caused by excitotoxicity, such as N-methyl-D-aspartate. In the treatment of stroke, annexin can cross the damaged blood-brain barrier to enter the brain and exert its therapeutic function, while simultaneously repairing the damaged blood-brain barrier to prevent further deterioration of the condition, thus achieving the therapeutic goal.

[0053] In some embodiments of the present invention, the annexin used is expressed by a prokaryotic expression system, such as an Escherichia coli expression system, and the amino acid sequence of annexin A5 is shown in SEQ ID NO: 1.

[0054] During their long-term research, the inventors discovered that annexin A5 can be used to treat stroke and repair neurological damage in the brain. Using a rat ischemia-reperfusion model, they confirmed that annexin A5 can reduce the area of ​​cerebral infarction, improve neurological deficit symptoms, and promote long-term motor recovery in rats. For example, in a rat model of focal cortical ischemia induced by light exposure, followed by tail vein administration of annexin A5, the rats showed improved neurological deficit symptoms and a significant reduction in the area of ​​cerebral infarction. Furthermore, in a rat model of hemorrhagic stroke induced by intracerebral injection of autologous arterial blood, followed by tail vein injection of annexin A5, the rats showed significant improvement in neurological deficit symptoms and a reduction in the area of ​​cerebral infarction.

[0055] The inventors also discovered that annexin A5 has a repairing and mitigating effect on stroke-related damage, and therefore can be used to treat stroke.

[0056] The inventors also discovered that in cases of acute injury (stroke, trauma, etc.), the blood-brain barrier is disrupted, allowing annexin A5 to enter the brain. Radiolabeled annexin A5, acting as a contrast agent, is distributed in the brain. After administration of annexin A5, it can significantly repair blood-brain barrier damage, as well as neurological damage or the aforementioned related lesions in the subjects. Furthermore, annexin A5 that has entered the brain can be used to treat brain diseases and repair neurological damage, such as repairing cerebral ischemia or reperfusion injury, thereby preventing or treating the diseases or conditions described above. Surprisingly, while achieving the aforementioned excellent therapeutic effects, no bleeding risk was observed in the subjects during treatment, demonstrating the excellent safety profile of the drug of this invention.

[0057] In this article, "repair" or "mitigate" refers to any method or means of reducing the aforementioned brain damage. Attached Figure Description

[0058] Figure 1 This is a graph showing the effect of the annexin A5 solution provided according to an embodiment of the present invention on symptoms of neurological defects.

[0059] Figure 2 This is a graph showing the effect of annexin A5 solution provided according to an embodiment of the present invention on the extent of cerebral infarction.

[0060] Figure 3 This is a graph showing the effect of annexin A5 on OGD damage in primary cortical neurons according to Example 4 of the present invention.

[0061] Figure 4 This is a diagram showing the protective effect of annexin A5 against NMDA-induced excitability damage in primary cortical neurons of rats, as provided in Example 5 of the present invention.

[0062] Figure 5 This is a graph showing the detection results of annexin A5 on EB content in brain tissue according to Example 6 of the present invention. Detailed Implementation

[0063] The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0064] The inventors have discovered that annexin A5, capable of repairing or mitigating damage, or exerting the preventive and / or therapeutic effects of this invention, can be the full-length or partial amino acid sequence of a naturally occurring human annexin A5 polypeptide, or a variant sequence based on the full-length or partial amino acid sequence of a naturally occurring human annexin A5 polypeptide. Of course, the preparation or acquisition of these naturally occurring annexins or variants can be based on any source or method, such as direct isolation from naturally occurring substances, direct artificial synthesis, or any combination of these methods.

[0065] Taking human natural full-length annexin A5 as an example, the available annexin A5 exhibits at least 97% homology with human natural full-length annexin A5, preferably at least 98%, for example at least 98.5%, at least 98.8%, more preferably at least 99%, for example at least 99.3%, at least 99.5%, and even more for example at least 99.6%. These homologous amino acid sequences compared to human natural full-length annexin A5 can show differences of one, two, three, four, or even five, six, or seven amino acids. These amino acid differences can be due to conserved amino acid substitutions in the sequence of human natural full-length annexin A5. "Conserved amino acid substitution" refers to the substitution of an amino acid by another amino acid with biologically, chemically, or structurally similar residues. Structural similarity refers to amino acids having side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity refers to amino acids having the same charge or being either hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine, or methionine can be substituted for each other. Alternatively, polar amino acids can be used, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, and so on.

[0066] The aforementioned amino acid sequences exhibiting homology or conserved amino acid substitutions can be obtained through artificial design and synthesis, or they can exist directly in other species in nature. Studies have shown that the annexin A5 sequence is conserved and exhibits high homology across different species. Since the amino acid sequence shown in SEQ ID NO: 1 is identical to that of natural human annexin A5, the only difference is that the N-terminal amino acid in the sequence shown in SEQ ID NO: 1 is alanine (A), while the N-terminus of natural human annexin A5 is acetylated alanine. Therefore, the variant sites and variant amino acids shown in Table 1 below are compared using the SEQ ID NO: 1 sequence as an example; this also reflects the differences between the annexin A5 sequence and natural human annexin A5 in different species in nature. Compared to the SEQ ID NO: 1 sequence, a mutation occurs in Gorilla gorillas, where the third amino acid is changed from valine to isoleucine. For example, J.Mol.Biol.223(3), 683-704(1992) and J.Mol.Biol.223(3), 683-704(1992) also reported that there are variations in natural human annexin in humans, specifically, compared with natural human annexin A5, the 76th amino acid is mutated from glutamic acid to glutamine or from glutamic acid to glycine. Table 1 below lists the variant sites and corresponding variant amino acids in some species compared with the sequence of SEQ ID NO: 1. These sequences can be obtained from NCBI using NCBI Access Number or directly from the relevant references.

[0067] Table 1 shows the variant sites and variant amino acids found in different species compared to SEQ ID NO: 1.

[0068]

[0069]

[0070] The embodiments of the present invention will be explained below with reference to examples. Those skilled in the art will understand that the following embodiments are only for illustrative purposes to explain and illustrate the embodiments of the present invention, and should not be considered as limiting the scope of protection of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0071] Example 1

[0072] The annexin A5 (hereinafter referred to as A5 protein) used in this embodiment and the following embodiments is derived from the full-length human annexin. A recombinant vector was constructed, and the protein was expressed in large quantities in the prokaryotic organism *Escherichia coli*. The amino acid sequence of the expressed A5 protein was determined as follows:

[0073]

[0074] As shown in SEQ ID NO: 1, the A5 protein expressed by the E. coli expression system contains 319 amino acids, and its complete cDNA sequence is encoded by 320 amino acid codons. The N-terminal methionine is cleaved during expression. The amino acid sequence of SEQ ID NO: 1 is identical to that of natural human annexin A5, the only difference being that the N-terminal amino acid of the A5 protein expressed by this system is alanine (A), while the N-terminus of natural human annexin A5 is acetylated alanine.

[0075] Then, the A5 protein obtained in Example 1 was used to conduct the following experiments.

[0076] Example 2

[0077] Referring to 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 established using the internal carotid artery suture method. A5 levels were set at 0.1 mg / kg, 1.0 mg / kg, and 1.0 mg / kg, respectively. The 10.0 mg / kg group served as a positive control group, with edaravone administered intravenously (6.0 mg / kg). A model group and a sham-operated group were also included. The model and sham-operated groups received the same volume of 0.9% sodium chloride injection. A5 protein groups received a single dose upon reperfusion and a second dose 1 hour after reperfusion. The remaining groups received a single dose immediately after reperfusion. Neurological and behavioral deficits and cerebral infarction area were assessed 24 hours after cerebral ischemia.

[0078] 1. Test materials

[0079] (1) Laboratory animals

[0080] Sprague Dawley (SD) rats, SPF grade, male, aged 6-8 weeks, weighing 250-280g.

[0081] Animals will be excluded from the experiment if any of the following conditions occur: a) death during anesthesia; b) death during cerebral ischemia; c) death after reperfusion but before evaluation of indicators; d) discovery of basilar hemorrhage after brain harvesting; e) no infarct area in the right brain after TTC staining.

[0082] (2) Reagents

[0083] The A5 protein solution is prepared by diluting the A5 protein prepared in Example 1 with a 0.9% sodium chloride aqueous solution.

[0084] Reference standard: Edaravone injection (purchased from Nanjing Sinopharm Dongyuan Pharmaceutical Co., Ltd.), which was diluted with 0.9% sodium chloride injection at a ratio of 1:1 as a positive control.

[0085] 2. Experimental Design

[0086] (1) Dosage design

[0087] Table 2 Dosage table for A5 protein efficacy test

[0088]

[0089] (2) Administration

[0090] Animals in each group were administered the drug via tail vein intravenous injection at a rate of approximately 1.5–2.5 mL / min.

[0091] For each treatment group, neurobehavioral deficit symptoms and cerebral infarction area were evaluated 24 hours after cerebral ischemia.

[0092] 3. Evaluation Methods

[0093] (1) Neurological deficit symptom score

[0094] The method described in the reference 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) was used to evaluate the neurological deficit symptoms in animals 24 hours after ischemia using the modified Bederson 5-point scale.

[0095] (2) Determination of the degree of cerebral infarction

[0096] Based on the references "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 schemia model of rats using a simple and economical method" (Yang Y, Shuaib A, Li QJ Neurosci Methods 1998; 84: 9-16), the TTC staining method was used to determine the degree of cerebral infarction.

[0097] Calculation of cerebral infarction area: The images were processed using ImageJ software, and the corresponding area of ​​the left brain and the area of ​​the non-infarct lesion in the right brain were calculated according to the formula to determine the percentage of infarction range.

[0098] Infarct volume calculation method:

[0099] V = t(A1 + A2 + A3 + ... + An)

[0100] t is the slice thickness, and A is the infarct area.

[0101] %I = (V C -V L ) / V C ×100%

[0102] %I represents the percentage of infarct size, V C V represents the brain volume of the control side (left hemisphere). L The volume of the non-infarcted region on the infarcted side (right hemisphere).

[0103] (3) Evaluation indicators

[0104] The extent of cerebral infarction and neurobehavioral scores were used as the main evaluation indicators, while the clinical manifestations of the animals were observed.

[0105] Quantitative data are expressed as mean ± standard error. One-way ANOVA was performed on each efficacy index using GraphPad Prism (6.01) software. After the variance test was significant, Fisher's LSD test was used to test differences between groups. A p-value < 0.05 was defined as statistically significant.

[0106] 4. Experimental Results

[0107] (1) Effects of A5 protein on neurological deficit symptoms

[0108] One-way ANOVA showed no statistically significant differences between the groups (F0). (4.69) =2.305, P=0.0669). However, when each group was compared with the model group by a T-test, the edaravone 6.0 mg / kg group (P<0.01) and the A5 10.0 mg / kg group (P<0.05) significantly improved the neurobehavioral deficit symptoms in ischemic animals; the A5 1.0 mg / kg group and the A5 0.1 mg / kg group showed a trend of improving the neurobehavioral deficit symptoms in ischemic animals, but there was no statistical difference. The effect of A5 protein on neurobehavioral deficit symptoms is detailed in Table 3 and... Figure 1 .

[0109] Table 3. Effects of A5 protein on acute cerebral ischemia-reperfusion injury in rats (Mean ± SEM)

[0110]

[0111] Compared with the model group, * P < 0.05 ** P < 0.01, # P < 0.05

[0112] (2) Effect of A5 protein on the extent of cerebral infarction

[0113] One-way ANOVA showed no statistically significant differences between the groups (F0). (4.69) =2.240, P=0.0735). However, when each group was compared with the model group using a T-test, the A5 10.0 mg / kg group (P<0.05) significantly reduced the cerebral infarction area in the model animals; the edaravone 6.0 mg / kg group and the A5 1.0 mg / kg group showed a trend of reducing the cerebral infarction area in the model animals, but there was no statistical difference; the A5 0.1 mg / kg group did not reduce the cerebral infarction area in the model animals. The effect of A5 protein on the cerebral infarction area is detailed in Table 3 and... Figure 2 .

[0114] In this experiment, the differences in the range of cerebral infarction within the model group were generally within one-third of the mean (Mean±SD, 42.53±12.65), and the model usability rate was 69.81% (excluding animals in the sham surgery group). The experimental system is reliable and can be used for drug efficacy evaluation.

[0115] Compared with the model group, the A5 10.0 mg / kg group significantly reduced the cerebral infarction area in model rats (P < 0.05); the edaravone 6.0 mg / kg and A5 1.0 mg / kg groups showed a trend of reducing the cerebral infarction area in model rats, but the difference was not statistically significant; A5 0.1 mg / kg did not reduce the cerebral infarction area in model animals. Compared with the model group, the edaravone 6.0 mg / kg and A5 10.0 mg / kg groups significantly improved the neurobehavioral deficit symptoms in ischemic animals (p < 0.01, p < 0.05); the other groups did not show significant improvement in this indicator.

[0116] In this experiment, the A5 10.0 mg / kg group significantly reduced the cerebral infarction area in ischemic rats and improved the neurological and behavioral deficits in ischemic animals. No bleeding risk was observed in any of the experimental groups treated with annexin A5.

[0117] Example 3

[0118] Microglia play a crucial role in the inflammatory process of the central nervous system. Moderate activation of microglia has a protective effect on neurons, but over-activated microglia release large amounts of neurotoxic factors, such as carbon monoxide, which can lead to neurodegenerative diseases. Lipopolysaccharide (LPS) activates microglia, leading to overactivation and consequently neuronal damage. The effect of A5 stimulation on LPS-induced microglia overactivation was observed.

[0119] In the experiment, primary purified microglia stimulated with LPS served as the control group, while A5 protein was added to serve as the experimental group. Cell viability was assessed using the MTT assay, and cell morphology changes were observed using immunocytochemistry.

[0120] Experimental results show that LPS can significantly activate primary microglia without affecting cell viability. The A5 protein inhibits LPS-induced microglia overactivation. Therefore, the A5 protein can be used to inhibit the overactivation of immune cells in the brain, such as microglia.

[0121] Example 4

[0122] Oxygen-glucose deprivation (OGD) models are stimulation models that simulate ischemia / hypoxia at the cellular level. By changing cell culture conditions, such as placing cells in a hypoxia chamber or replacing the culture medium with a glucose-free medium, the damage to cells under ischemic / hypoxic conditions is simulated.

[0123] A rat primary cortical neuron OGD model was established using primary cortical neurons. A5 protein concentrations were measured at 0, 41.15, 123.5, 370.4, 1111, 3333, and 10000 nM. Cells in the culture plate were washed twice with PBS and then placed in glucose-free DMEM that had been pre-purified with 95% N2 + 5% CO2 for 30 min. The plate was then rapidly placed in a hypoxic incubator at 37°C (94% N2 + 1% O2 + 5% CO2). After 8 h of hypoxic culture, the appropriate drug-containing medium (i.e., DMEM containing A5) was added, and the plate was reoxygenated for 24 h at 37°C (5% CO2). After 24 h of culture under normal conditions, cell morphology was observed under an inverted microscope. Cell viability was assessed using the CCK-8 assay, and apoptosis rate was detected by flow cytometry. A normoxic and normglycemic control group was also included. The relative cell viability was calculated using the following formula:

[0124] Relative cell viability (%) = (Drug-treated group - Baseline group) / (Control group - Baseline group) × 100%

[0125] Compared with the model group, A5 protein did not show a protective effect against OGD-induced neuronal damage in the concentration range of 41.15-1111 nM (P>0.05), and at concentrations of 3333-10000 nM, it significantly reduced the activity of neurons after OGD-induced neuronal damage (P<0.05). Figure 3 ).

[0126] Experimental results showed that A5 protein at concentrations of 3333-10000 nM can reduce the damage to neurons caused by oxygen-glucose deprivation, thus exhibiting neuroprotective effects.

[0127] Example 5

[0128] Example 5 investigated the neuroprotective effect of A5 protein against NMDA (N-methyl-D-aspartate)-induced excitotoxic damage to rat cortical neurons. NMDA can activate NMDA receptors and cause calcium in brain tissue. 2+ Increased NO levels lead to the activation of large amounts of NO, resulting in a series of physiological or pathological changes, including the synthesis of large amounts of NO, which damages neurons in the brain. This experiment pre-treated neurons with A5 protein to observe the neuroprotective effect of A5 protein against excitotoxicity-induced neuronal damage.

[0129] In this experiment, SD rats at 17 days of embryonic age were used, and cortical neurons were harvested and cultured. The neurons were then divided into a control group, an NMDA-treated group, and an A5 pretreatment group. In the NMDA-treated group, neurons were treated with NMDA, while the control group received no treatment. In the A5 pretreatment group, neurons were pre-incubated with A5 protein for a period of time before being treated with NMDA. For each treatment group, cell viability was assessed using trypan blue staining, apoptotic cells were detected using TUNEL staining, and neuronal morphology was determined using immunofluorescence cytochemistry. The formula for calculating relative cell viability is as follows:

[0130] Relative cell viability (%) = (Drug-treated group - Baseline group) / (Control group - Baseline group) × 100%

[0131] like Figure 4 As shown, 100 μM NMDA induced a decrease in the activity of primary cortical neurons in rats (P < 0.001, compared with the normal control group), indicating that the model was successfully established. A5 protein, within the detection concentration range of 41.15-3333 nM, did not show a significant mitigating effect on NMDA-induced excitability damage in primary cortical neurons (P > 0.05, compared with the Model group). However, A5 protein at a detection concentration of 10000 nM showed a significant mitigating effect on NMDA-induced excitability damage in primary cortical neurons (P < 0.05, compared with the Model group).

[0132] The experimental results showed that, compared with the NMDA exposure group, the A5 pretreatment group could reduce the damage of excitotoxicity to neurons at a detection concentration of 10,000 nM.

[0133] Example 6

[0134] The blood-brain barrier is composed of three components: brain microvascular endothelial cells, astrocytes, and the basement membrane. Damage to the blood-brain barrier is one of the important pathological aspects of stroke-related brain injury.

[0135] In this experiment, an in vitro blood-brain barrier model was established by co-culturing spontaneously transformed endothelial cell lines and isolated and purified rat astrocytes. The in vitro blood-brain barrier model was induced by stimulation, and then treated with A5 protein. The integrity of the blood-brain barrier was assessed using a rat model of traumatic brain injury induced by head trauma, with A5 at doses of 10 mg / kg, 30 mg / kg, and a model group. A5 protein was administered twice, once at 1 h and 2 h after modeling, via tail vein injection. The modeling process was performed 23 h after modeling. Rats were anesthetized, and then injected with 2% EB (Evans blue) dye solution at a dose of 4 ml / kg via tail vein. The dye was allowed to circulate in the rats for 1 hour before sacrifice. The heart was perfused with saline through the left ventricle until colorless perfusion was achieved. After sacrifice, the brain was removed, weighed, homogenized in formamide (3 μl / mg), and incubated at room temperature for 48 hours. After centrifugation, the supernatant was collected and the optical density was measured at 625 nm to determine the relative amount of EB dye. The results are shown in Table 4. Figure 5 :

[0136] Table 4. Effects of A5 protein on blood-brain barrier injury induced by traumatic brain injury in rats (Mean ± SD)

[0137] Model group 5 101.87±8.63 A5 10mg / kg 5 96.08±4.33 A5 30mg / kg 5 <![CDATA[89.36±5.51 * ]]>

[0138] Compared with the model group, * P < 0.05.

[0139] The results showed that, compared with the model group, there was no significant difference in EB content in the brain tissue of the A5 10 mg / kg group (P > 0.05), while the EB content in the 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 blood-brain barrier damage induced by traumatic brain injury in rats.

[0140] Experimental results show that stimulation can significantly damage the established blood-brain barrier, and A5 can repair the damaged blood-brain barrier.

[0141] Example 7: Immunotoxicity and Immunogenicity Studies (with repeated-dose toxicity studies in SD rats and cynomolgus monkeys)

[0142] 1. Toxicity experiment study in SD rats

[0143] SD rats were intravenously injected with A5 protein for 28 consecutive days, with a 4-week recovery period. The toxic reactions, their severity, major target organs, and the reversibility of the damage were observed. The dosages administered were 0 (blank excipient), 30, 150, and 750 μg / kg. Blood samples were collected for immunotoxicity (CD4+). + CD8+ T-cell and immunogenicity assays were performed. The experiment was divided into 4 groups, with 8 rats in each group. Samples were collected at 4 time points, meaning serum samples were collected from each rat at 4 time points.

[0144] Among them, the immunotoxicity results can be seen in the proportion and ratio of peripheral blood T lymphocyte subsets (CD4+) in animals of each dose group during the experiment. + No statistically significant difference was found between the CD8+ T cell assay values ​​and the control group.

[0145] The immunogenicity results are shown in Table 4. In Table 4, the number of samples in each group represents 8 rats in each group, and serum was collected at 4 time points, for a total of 32 samples in each group; the number of positive individuals is the number of positive samples detected in each group.

[0146] Table 5. Data on A5 protein antidrug antibodies in rat serum.

[0147]

[0148] As shown in Table 4, no anti-drug antibodies (ADA) were detected in SD rats after intravenous injection of A5 protein blank excipient. The positive sample detection rate was 65.6% after intravenous injection of different doses of A5 protein. There was no significant difference between the dose groups, and positive samples were mainly concentrated after the last administration and at the end of the recovery period, consistent with the process of anti-drug antibody production in vivo.

[0149] 2. Toxicity Experimental Study of Crab-Eating Mammals

[0150] This study used cynomolgus monkeys that were intravenously injected with A5 protein for 28 consecutive days, with a 4-week recovery period. The toxic reactions, their severity, major target organs, and the reversibility of the damage were observed. The dosages administered were 0 (blank excipient), 15, 75, and 375 μg / kg. Immunogenicity and immunotoxicity (CD4+) assays were performed. + CD8 + T-cell detection.

[0151] Immunotoxicity results can be seen in the proportions and ratios of peripheral blood T lymphocyte subsets (CD4+) in animals of each dose group during the experiment. + CD8 + There was no statistically significant difference in T cell measurements between the control group and the control group.

[0152] The immunogenicity results are shown in Table 5. The same grouping and treatment methods were used as for the rats described above, except that two more samples were taken from each group.

[0153] Table 6. Data on anti-drug antibodies against A5 protein in monkey serum.

[0154]

[0155] As can be seen from the results in Table 5, no ADA was detected in cynomolgus monkeys after intravenous injection of A5 protein blank excipient. The positive sample detection rate was 25.5% after intravenous injection of different doses of A5 protein, with positive samples concentrated at 4 weeks post-administration and at the end of the recovery period, consistent with the process of antibiotic resistance antibody production in vivo.

[0156] Experimental results showed no immunotoxicity in either rats or cynomolgus monkeys. However, in the immunogenicity test, the positive rate in cynomolgus monkeys was significantly lower than that in rats. Considering that the A5 protein is a human-derived protein, its immunogenicity in humans is expected to be significantly lower.

[0157] Example 8: Toxicity Study of Single-Dose Intravenous Injection of A5 Protein in SD Rats and Cynomolgus Monkeys

[0158] 1. Toxicity study of single-dose intravenous injection of A5 protein in SD rats

[0159] This study used SD rats to receive a single intravenous injection of A5 protein to observe the acute toxic reactions, their severity, and the main target organs. The dosages administered were 0 (blank excipient), 1000, 3000, and 9000 μg / kg. After intravenous injection, the animals' posture, gait, responsiveness, neural activity, appetite, coat, eyes, ears, mouth, nose, limbs, respiration, and feces were continuously observed. Observation continued until day 14 post-administration to assess the recovery from toxicity. On day 15 post-administration, the animals were dissected for gross pathological examination. During the experiment, no abnormalities were observed in the clinical symptoms of any animal; no abnormalities were observed in the weight changes of female and male animals in the administered group; and no abnormalities were found in any tissues or organs during gross pathological examination. Under the conditions of this experiment, the tolerated dose (MTD) of a single intravenous injection of A5 protein was above 9000 μg / kg.

[0160] 2. Toxicity Study of Single-Dose Intravenous Injection of A5 Protein in Cynomolgus Monkeys

[0161] This study used cynomolgus monkeys to receive a single intravenous injection of A5 protein, observing the acute toxic reactions, their severity, and the main target organs. The dosages administered were 0 (blank excipient), 500, 1500, and 4500 μg / kg. The animals were administered a single intravenous injection, and their clinical symptoms (including feces, appearance, respiration, neurological responses, and activity) were observed until day 15 post-administration. Food intake was measured daily, and body weight was measured weekly. Body temperature, blood pressure, and electrocardiogram were measured before and after administration on the day of administration, and again on days 1, 7, and 15. Hematological (including blood coagulation) and serum biochemical tests were performed on days 1, 7, and 15 post-administration, and urine tests were performed on day 14. On day 15 post-administration, the animals were dissected for gross pathological examination, and histopathological examination was performed when necessary. Results showed no significant changes related to the test substance in clinical symptoms, injection site observations, body weight, food intake, body temperature, blood pressure, electrocardiogram, urinalysis, serum biochemistry, and pathological examinations in any group of animals. No significant changes were observed in prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) in any group of animals from day 13 of the quarantine period to day 15 after administration. Under the conditions of this experiment, the tolerated dose (MTD) for cynomolgus monkeys was above 4500 μg / kg.

[0162] The above experimental results indicate that the tolerable dose of A5 protein in both SD rats and cynomolgus monkeys is above 4500 μg / kg, and in SD rats, the tolerable dose even exceeds 9000 μg / kg. These results demonstrate that at the equivalent dose at which A5 protein exerts its protective effect against stroke, no significant toxicity was observed in rats and monkeys. This suggests that A5 protein has a good safety profile for the treatment of stroke, and even at higher doses, it poses little to no risk.

[0163] In the description of this specification, the references to terms such as "implementation," "example," and "example" refer to specific features, structures, materials, or characteristics described in connection with that embodiment, example, or example, which are included in at least one embodiment, example, or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment, example, or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments, examples, or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments, examples, or examples described in this specification, as well as the features of different embodiments, examples, or examples.

[0164] Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention, and such changes, modifications, substitutions, and variations should be covered within the scope of protection of the present invention.

Claims

1. Use of annexin A5 in the preparation of a medicament for the prevention and / or treatment of blood-brain barrier injury, wherein the blood-brain barrier injury is selected from blood-brain barrier injuries caused by traumatic brain injury. The amino acid sequence of annexin A5 is shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein the blood-brain barrier injury is selected from damage, impairment or obstruction of the barrier between blood plasma and brain cells and / or cerebrospinal fluid.

3. The use according to claim 1, wherein the craniocerebral injury is selected from craniocerebral trauma caused by external force to the cranium.

4. The use according to any one of claims 1-3, wherein the drug is an injectable preparation.

5. The use according to any one of claims 1-3, wherein the content of annexin A5 in a unit dosage form of the drug is 0.025 mg to 250 mg.

6. The use according to claim 5, wherein the content of annexin A5 in a unit dosage form of the drug is 1-100 mg.

7. The use according to any one of claims 1-3, wherein the drug is a drug combination, the drug combination comprising: (i) Annexin A5; and (ii) Other drugs; The other drugs are selected from active ingredients other than annexin A5 for the prevention and / or treatment of diseases or conditions selected from the following: stroke, neurobehavioral deficits, neurodegenerative diseases, nerve damage, excessive activation of immune cells in the brain, blood-brain barrier damage, cerebral edema, increased calcium ion content, excitotoxicity, excessive NOS activation, and diseases or conditions caused by at least one of the above diseases or conditions.

8. The use according to claim 7, wherein the other drug is selected from at least one of the following: thrombolytic drugs, antiplatelet drugs, anticoagulants, fibrinolytic drugs, volume expanders, vasodilators, or other drugs that improve cerebral blood circulation and neuroprotective agents.

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