Use of cobra neurotoxin and formulation thereof in preparation of drug for preventing and / or treating alzheimer's disease

Abstract

Provided is use of cobra neurotoxin and a formulation thereof in the preparation of a drug for preventing and / or treating Alzheimer's disease. The cobra neurotoxin is selected from Chinese cobrotoxin and / or Thai cobratoxin. The formulation of the cobra neurotoxin is selected from Cobratide, Cobratide injection, Nyloxin, cobraxin, peperon, or at least one of clinically acceptable non-injection dosage forms prepared from the described formulations. Intranasal administration of Cobratide has a good effect in treating Alzheimer's disease. Meanwhile, no significant toxic and side effects are found in the therapeutic dose, and Cobratide has the advantages of a small dosage and safety.

Description

Use of cobra neurotoxins and their preparations in the preparation of medicines for the prevention and / or treatment of Alzheimer's disease

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202410977393.5, filed on July 19, 2024, entitled “Use of cobra neurotoxin and its preparations in the preparation of medicaments for the prevention and / or treatment of Alzheimer’s disease”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the pharmaceutical field, specifically to the use of a cobra neurotoxin and its preparations in the preparation of medicaments for the prevention and / or treatment of Alzheimer's disease. Background Technology

[0004] Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease characterized by memory loss, cognitive decline, and behavioral disturbances. It is estimated that more than 100 million people will be affected by AD by 2050, and the disease is trending towards affecting younger people. Alzheimer's disease has become a major threat to the health of the elderly.

[0005] The pathological features of Alzheimer's disease include the deposition of amyloid plaques, abnormal phosphorylation of tau protein, and the formation of neurofibrillary tangles (NFTs). Its pathogenesis is highly complex, including genetic factors and various other pathogenic hypotheses, such as the amyloid plaque hypothesis, abnormal tau protein metabolism, neuroinflammation hypothesis, mitochondrial dysfunction, and metabolic syndrome hypothesis.

[0006] In the pathological process of Alzheimer's disease (AD), either excessive production of Aβ or reduced clearance leads to an increase in Aβ in the brain, forming Aβ plaques and hyperphosphorylation of Tau protein, which are key factors driving neuroinflammation and nerve damage in Alzheimer's disease. Aβ aggregates and fibroblasts can bind to pattern recognition receptors on microglia, stimulating Toll-like receptors and activating NLRP3 inflammasomes, producing large amounts of pro-inflammatory factors such as IL-1β, IL-6, IL-17, and TNF-α. However, most previous efforts to develop new drugs to clear Aβ have failed, so clearing Aβ alone may not be sufficient to treat AD. Neuroinflammation is an important pathological mechanism in AD, but previous treatments with anti-inflammatory drugs for AD have often been ineffective, indicating that anti-inflammatory drugs alone are insufficient to reverse the pathological state of AD. Numerous studies have shown that microglia are the main cells clearing Aβ, and excessive activation of microglia reduces their ability to clear Aβ, leading to the accumulation of extracellular Aβ and intracellular NFTs. The accumulation of Aβ and NFTs leads to the activation of inflammasomes and damage to surrounding healthy neurons, resulting in the gradual loss of neurons. During the pathological process of Alzheimer's disease, the secretion of pro-inflammatory factors increases, and neurons in areas such as the hippocampus and cortex are damaged, lose function, or die, causing a gradual decline in cognitive function. Therefore, regulating microglia to accelerate Aβ clearance, while simultaneously reducing neuroinflammation, repairing neurons, and promoting neurogenesis, may be key to treating Alzheimer's disease.

[0007] Currently, there is no effective treatment for Alzheimer's disease. Acetylcholinesterase inhibitors, which enhance central cholinergic activity, and anti-anxiety and antidepressant drugs, which control accompanying psychiatric symptoms, are the main medications used clinically to treat Alzheimer's disease. Some traditional Chinese medicines have also shown potential in treating Alzheimer's disease, but the efficacy of these drugs is uncertain and they are accompanied by varying degrees of toxic side effects. In 2023, lecanemab received full approval from the U.S. Food and Drug Administration (FDA) for the treatment of AD, marking the first "causal" treatment for AD in 20 years. Its current clinical indication is early-stage AD, primarily including patients with mild cognitive impairment due to AD and early-stage dementia. This drug has been rapidly introduced into clinical practice in China, reflecting the urgent need for AD treatment drugs in the Chinese clinical setting. However, according to the results of clinical trials, lecanemab is only suitable for mild AD and has side effects such as cerebral edema and cerebral hemorrhage. Therefore, there is an urgent need to find new targets and treatment methods for Alzheimer's disease.

[0008] Traditional Chinese medicine has a long history of using cobra neurotoxins for treating diseases, including cancer treatment, arthritis treatment, and analgesia. There are 186 species of snakes in 44 genera in the family Elapdae, which can be divided into the subfamily Bungarinae, the subfamily Elapinae, and the subfamily Hydrophiinae. There are 8 species in 4 genera distributed in China. Naja atra is one of them. Each snake contains several α-neurotoxins, so there are hundreds of natural cobra α-neurotoxins. Cobra α-neurotoxins belong to the three-finger protein family, but the number of amino acids, the number of disulfide bonds, the amino acid sequence, and the function are different. Neurotoxins are the main active components of cobra venom. According to the action site and mode, they can be divided into two categories: α and β. The cobratoxin contained in the Thai cobra is a long-chain α-neurotoxin, composed of 71 amino acids and has 5 disulfide bonds. Naja atra contains three short-chain α-neurotoxins. Among them, cobrotoxin (Chinese name: Kobopeptide) is composed of 62 amino acids and has 4 disulfide bonds. These disulfide bonds are considered necessary to maintain activity. The other two short-chain neurotoxins have similar structures and pharmacological effects. The α-neurotoxin of cobra venom (Kobopeptide injection) is already a clinically approved analgesic drug in China. In addition to the analgesic effect, in recent years, it has been found that Kobopeptide also has the effects of inhibiting inflammation, improving rheumatoid arthritis, and regulating immune responses.

[0009] Currently, there are few or no relevant reports on the use of cobra neurotoxins for the treatment of Alzheimer's disease. The main reasons are as follows, which make it very difficult to obtain other cobra neurotoxins with the effect of treating Alzheimer's disease:

[0010] (1) Since the cobra family is widely distributed, with more than 10 species mainly distributed in Africa and Asia, including more than 50 other species distributed in Australia and the ocean, the functions of the cobra neurotoxin proteins secreted by these cobras are diverse;

[0011] (2) Even if the primary sequences of these snake venom proteins are similar and the spatial structures are similar, the functions are still completely different, as disclosed in the following literature:

[0012] It is pointed out in "Study on the Structure-Function Relationship of Cobra Venom Three-Finger Structure Proteins" (Huang Qingqiu et al., Proceedings of the Fourth National Conference on the Toxicology of Biological Toxins) that according to the protein structure-function relationship, it is divided into three categories. The third category is like the C-type lysozyme-α-lactalbumin family. The primary sequence homology of these two types of proteins is relatively high, and the spatial structures are extremely similar, but the functions are completely different. Some other typical representatives of the third category of proteins come from snake venom proteins, such as phospholipase A2, the snake venom C-type lectin-like protein family, and the three-finger structure protein family, and neurotoxins belong to the three-finger structure protein family;

[0013] The abstract of "Research Progress on the Trifinger Ring Toxin Family of Snakes" (Ji Xianhong et al., Journal of Yunnan University, 2019) points out that the trifinger ring toxin family is the largest group of non-enzymatic venom proteins. Although the proteins in this family are all three-ring structures composed of β-sheets, they exhibit diverse functional activities. Neurotoxins are an important component of this family. The structural differences of different functional groups within the trifinger ring toxin family lead to the diversity of their biological functions and the specificity of each functional group. Although members of this family have structural similarities, they contain diverse functional groups, each with its unique functional groups or bioactive centers, which can specifically and with high affinity act on different receptors and ion channels, thus exhibiting unique pharmacological properties. Members of this family include: neurotoxins that can act on nicotinic acetylcholine receptors; muscarinic toxins, antagonists of muscarinic acetylcholine receptors; fasciculins, toxins that inhibit acetylcholinesterase and enhance acetylcholine function; cardiotoxins / cytotoxins that exert their toxicity by forming pores at the cell membrane; and neurotoxins that can inhibit L-type Ca2+. 2+ Channel calciseptine and its associated toxins; antagonists dendroaspins involved in various cell adhesion processes, etc.

[0014] The article “Snake venomα-neurotoxins and other `three-finger` proteins” (Eur.J.Biochem.264,281±286(1999)q FEBS1999) describes long-chain and short-chain neurotoxins, as well as the differences in neurotoxin function caused by insertion or deletion in the three-finger structure.

[0015] In “Structure-function studies on Taiwan cobra long neurotoxin homolog” (Long-sen Chang, Biochimica et Biophysica Acta 1480(2000)293-301), it was verified that different cobra neurotoxins NNA1 and NNA2 showed great differences in their inhibitory effects on acetylcholine-induced muscle contraction. NNA1 and NNA2 share 66.2% sequence homology, but their inhibitory effects on neurotoxicity differ by a factor of four in the muscle contraction induced by acetylcholine.

[0016] Therefore, although the spatial structures of more than a dozen such proteins have been determined, the molecular basis for how these proteins acquire diverse functions through changes in a few key residues on such highly similar spatial structures remains unclear. Furthermore, while the mechanisms of action of neurotoxins are relatively well understood, some scholars believe that our current understanding of neurotoxins is far from comprehensive. Therefore, to date, there are no related studies or reports on the use of the cobra neurotoxin described in this application for Alzheimer's disease.

[0017] Summary of the Invention

[0018] Therefore, the technical problem to be solved by this application is to provide the use of cobra neurotoxin and its preparations in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease. This cobra neurotoxin not only significantly enhances the uptake and degradation of Aβ, but also has a strong inhibitory effect on neuroinflammation, repair of neurons and promotion of regeneration, thus comprehensively improving the main pathological changes in AD.

[0019] Therefore, this application provides the following technical solution:

[0020] Use of cobra neurotoxin and its preparations in the preparation of medicaments for the prevention and / or treatment of Alzheimer's disease, wherein the cobra neurotoxin is selected from Chinese cobra venom cobrotoxin and / or Thai cobra neurotoxin cobratoxin;

[0021] The cobra neurotoxin formulation is selected from at least one of the following: Cobraxin injection, Nyloxin, cobraxin, peperon, or a clinically acceptable non-injectable dosage form made from the above formulations.

[0022] Cobrotoxin is also known as Cobrotoxin.

[0023] Optionally, the amino acid sequence of the cobra neurotoxin is shown in SEQ ID NO.1.

[0024] Optionally, it has at least one of the following uses (1)-(4):

[0025] (1) Use in the preparation of drugs to improve cognitive abilities;

[0026] (2) Use in the preparation of drugs that reduce the accumulation of amyloid protein;

[0027] (3) Use in the preparation of drugs to inhibit neuroinflammation;

[0028] (4) Use in the preparation of drugs that increase the number of neurons in the cerebral cortex and hippocampus.

[0029] Optionally, the drug contains a therapeutically effective amount of the cobra neurotoxin.

[0030] Optionally, the content of the cobra neurotoxin in the drug is 0.01% to 1% by mass.

[0031] Optionally, the drug may also include a pharmaceutically acceptable carrier.

[0032] Optionally, the pharmaceutically acceptable carrier is selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, polymeric framework materials, and film-forming materials; more preferably, physiological saline, water, or buffer solution.

[0033] Optionally, the drug is an oral dosage form, a mouth dosage form, a nasal dosage form, a transdermal dosage form, or an injectable dosage form; preferably, it is an injectable dosage form or a nasal dosage form.

[0034] Optionally, the drug may also include an additional therapeutic agent for treating Alzheimer's disease, the additional therapeutic agent being selected from synthetic small molecule drugs, genetically engineered drugs, biologics, or gene therapy drugs.

[0035] This application also provides a method for preventing and / or treating Alzheimer's disease, characterized in that it includes administering cobra neurotoxin and its preparations to a subject in need, wherein the cobra neurotoxin is selected from Chinese cobra neurotoxin cobrotoxin and / or Thai cobra neurotoxin cobratoxin.

[0036] The cobra neurotoxin formulation is selected from at least one of the following: cobraxin injection, Nyloxin, cobraxin, and peperon, or a clinically acceptable non-injectable dosage form made from the above formulations.

[0037] In some specific embodiments, the amino acid sequence of the cobra neurotoxin is shown in SEQ ID NO.1.

[0038] In some specific embodiments, the cobra neurotoxin or its formulation has at least one of the following uses (1)-(4):

[0039] (1) Improve cognitive abilities;

[0040] (2) Reduce the accumulation of amyloid protein;

[0041] (3) Inhibits neuroinflammation;

[0042] (4) Increase the number of neurons in the cerebral cortex and hippocampus and repair neurons.

[0043] In some embodiments, the method includes administering a therapeutically effective amount of the cobra neurotoxin to a subject in need.

[0044] Optionally, the content of the cobra neurotoxin in the method is 0.01-1% by mass.

[0045] In some specific embodiments, the method of administration is oral administration, oral administration, nasal administration, transdermal administration, or injection administration.

[0046] Optionally, the method of administration may be by injection or nasal administration.

[0047] In some specific embodiments, the method may further include administration in combination with another additional therapeutic agent for treating Alzheimer's disease, said additional therapeutic agent being selected from synthetic small molecule drugs, genetically engineered drugs, biologics, or gene therapy drugs.

[0048] In some specific implementations, the dosage is 5-15 μg / kg body weight.

[0049] In some specific implementations, the dosage is 5 μg / kg body weight, 6 μg / kg body weight, 7 μg / kg body weight, 8 μg / kg body weight, 9 μg / kg body weight, 10 μg / kg body weight, 11 μg / kg body weight, 12 μg / kg body weight, 13 μg / kg body weight, 14 μg / kg body weight, and 15 μg / kg body weight.

[0050] In some alternative implementations, the dosage is 6 μg / kg to 12 μg / kg body weight.

[0051] In some specific implementations, when the subject is a human, the dosage is 0.66 μg / kg to 1.32 μg / kg body weight.

[0052] The technical solution of this application has the following advantages:

[0053] 1. The use of the cobra neurotoxin and its formulations provided in this application in the preparation of drugs for the prevention and / or treatment of Alzheimer's disease, wherein the cobra neurotoxin is selected from cobrotoxin from the Chinese cobra and / or cobratoxin from the Thai cobra; the formulation of the cobra neurotoxin is selected from at least one of cobrotoxin injection, Nyloxin, cobraxin, peperon, or clinically acceptable non-injectable dosage forms made from the above formulations; studies have discovered a new use of cobrotoxin in the treatment of Alzheimer's disease and a new target for the neuroprotective effect of cobrotoxin. This target is achieved by cobrotoxin through regulating the phagocytosis and degradation of Aβ by microglia, thereby inhibiting neuroinflammation, protecting neurons, and restoring neuronal function. This application shows that nasal administration of cobrotoxin has a good therapeutic effect on Alzheimer's disease, inhibiting several key pathological changes in Alzheimer's disease, restoring cognitive function, and no significant toxic side effects were found at therapeutic doses; it has the advantages of small dosage and safety. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0055] Figure 1. Intranasal administration of cobrotoxin (CTX) allows it to enter the brain and distribute to various brain regions; Figure A shows the results of small animal imaging technology, and Figure B shows the distribution of fluorescently labeled cobrotoxin in brain regions as determined after brain slices.

[0056] Figure 2. Cobrotoxin (CTX) improves the learning and memory abilities of APP / PS1 mice; Figure A shows the exploration of old objects by mice in each group, Figure B shows the exploration of new objects by mice in each group, Figure C shows the spontaneous alternation rate of mice in each group, Figure D shows the trajectory of spontaneous alternation rate in the Y maze experiment, Figure E shows the latency of each group of mice in finding the platform in the water maze experiment, Figure G shows the spatial exploration of each group of mice in the target quadrant in the water maze experiment, Figure F shows the swimming trajectory of each group of mice in the water maze experiment, and Figure H shows the swimming trajectory of spatial exploration in the water maze experiment; In the figures, compared with Alzheimer's disease mice (AD), *, **, and *** indicate statistical differences, respectively, p < 0.05, p < 0.01, and p < 0.001;

[0057] Figure 3. Cobrotoxin (CTX) improves anxiety in APP / PS1 mice; Figure A shows the total distance of activity in the open field, Figure B shows the proportion of central activity in each group to the total distance of activity in the open field, Figure C shows the movement trajectory of each group of mice in the open field experiment, Figure D shows the percentage of times each group of mice entered the open arm, Figure E shows the percentage of time each group of mice spent in the open arm, and Figure F shows the movement trajectory of each group of mice in the elevated cross experiment; In the figures, compared with Alzheimer's disease mice (AD), *, **, and *** indicate statistical differences, respectively, p < 0.05, p < 0.01, and p < 0.001;

[0058] Figure 4. Effect of Cobrotoxin (CTX) on Aβ clearance; Figure A shows the Aβ levels in the brains of mice in each group, and Figure B shows the Aβ fluorescence intensity in the brains of mice in each group; In the figures, *, **, and *** indicate statistically significant differences compared with Alzheimer's disease mice (AD), respectively, p < 0.05, p < 0.01, and p < 0.001.

[0059] Figure 5. Effect of Cobrotoxin (CTX) on exogenous Aβ clearance; Figure A shows the residual Aβ in the brains of mice in each group 48 hours after stereotactic injection of Aβ, Figure B is a statistical graph of the residual Aβ in the brains of mice in each group, Figure C is a representative graph of microglia phagocytosis in each group of mice, and Figure D is a statistical graph of microglia phagocytic capacity in each group of mice; *, **, and *** indicate statistical differences, p < 0.05, p < 0.01, and p < 0.001, respectively.

[0060] Figure 6. Effects of Cobrotoxin (CTX) on microglial function; Figure A shows the microglial status of each group of mice, Figure B shows the cell body area of ​​microglial cells in each group of mice, Figure C shows the cell body diameter of microglial cells in each group of mice, Figure D shows the expression of hippocampal IL-1β gene in each group of mice, Figure E shows the expression of hippocampal TNF-α gene in each group of mice, and Figure F shows the expression of hippocampal IL-6 gene in each group of mice; In the figures, compared with Alzheimer's disease mice (AD), *, **, and *** indicate statistically significant differences, respectively, p < 0.05, p < 0.01, and p < 0.001;

[0061] Figure 7. Cobrotoxin (CTX) inhibits LPS-induced inflammatory response in BV2 microglia. In the figure, A represents cell viability of BV2 cells treated with 10, 100, and 1000 ng / ml CTX for 12 h; B represents cell viability of BV2 cells treated with 10, 100, and 1000 ng / ml CTX for 24 h; C represents IL-1β gene expression after LPS-induced inflammation induced by CTX treatment; and D represents TNF-α gene expression after LPS-induced inflammation induced by CTX treatment. In the figures, *, **, and *** indicate statistically significant differences compared to Alzheimer's disease mice (AD), respectively (p < 0.05, p < 0.01, and p < 0.001).

[0062] Figure 8. Cobrotoin (CTX) increases the number of hippocampal neurons in APP / PS1 mice. In the figure, A is a representative Nissl staining map of the hippocampus in each group of mice; B is the normal neuronal density in the CA1 region of the hippocampus; C is the normal neuronal density in the CA3 region of the hippocampus; D is the normal neuronal density in the DG region of the hippocampus; E is a representative staining map of hippocampal neurons in each group of mice; and F is a statistical chart of the number of hippocampal neurons in each group of mice. In the figure, compared with Alzheimer's disease mice (AD), *, **, and *** indicate statistically significant differences, p < 0.05, p < 0.01, and p < 0.001, respectively.

[0063] Figure 9. Effects of Cobrotoxin (CTX) on stem cells in the brains of Alzheimer's disease mice; Figure A shows representative staining of neural stem cell markers in the hippocampus of each group of mice, and Figure B shows the number of neural stem cells in the hippocampus of each group of mice; In the figure, *, **, and *** indicate statistically significant differences compared with Alzheimer's disease mice (AD), respectively, p < 0.05, p < 0.01, and p < 0.001.

[0064] Figure 10. Effect of Cobrotoxin (CTX) on the number of dendritic spines in hippocampal neurons of APP / PS1 mice; Figure A is a representative Golgi staining map of the hippocampus of each group of mice, and Figure B is the density of dendritic spines in the hippocampus of each group of mice; In the figure, compared with Alzheimer's disease mice (AD), *, **, and *** indicate statistical differences, respectively, p < 0.05, p < 0.01, and p < 0.001. Detailed Implementation

[0065] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

[0066] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0067] Experimental example: Cobra neurotoxin cobrotetide treats Alzheimer's disease by inhibiting neuroinflammation.

[0068] 1. Experimental materials and instruments:

[0069] 1.1 Experimental Reagents

[0070] The Chinese cobra neurotoxin solution (cobrotoxin, CTX) (the amino acid sequence of the cobra neurotoxin is shown in SEQ ID NO.1) (concentration of 150 μg / ml) was provided by Suzhou Renben Pharmaceutical Co., Ltd.

[0071] 1.2 Laboratory Animals

[0072] APP / PS1 double transgenic mice were developed at the Model Animal Research Center of Nanjing University. The strain was B6C3-tg(appswe,psen1de9) / Nju, and the wild type was a B6C3 mouse of the same age and genetic background.

[0073] 2. Experimental Methods

[0074] 2.1 Experimental Grouping

[0075] Except for the experiment on "3.1 CTX intranasal administration into the brain," other experiments were conducted with the following groupings: Mice were divided into wild-type (WT) (n=10) and Alzheimer's disease (APP / PS1) mice. Alzheimer's disease mice were randomly divided into three groups (n=8 per group): Alzheimer's disease control group (AD), low-dose group: Alzheimer's disease + 6 μg / kg CTX (AD + 6 μg / kg CTX solution (concentration 75 μg / ml)), and high-dose group: Alzheimer's disease + 12 μg / kg CTX (AD + 12 μg / kg CTX solution (concentration 150 μg / ml)). The blank control consisted of the same number of WT mice. Alzheimer's disease mice were given medication starting at eight months of age, administered intranasally every two days, with a solution volume of 0.8 μL / 10g, for a total of 12 weeks. WT and Alzheimer's disease control mice received the same amount of drug-free solution.

[0076] 2.2 Nasal administration of cobrotoxin (CTX) from cobras

[0077] Select a 10 μL liquid chromatography syringe and attach a section of extremely thin tubing to the tip to administer medication via nasal drops. After anesthetizing the mouse with isoflurane, hold its head and neck skin to stabilize it. Carefully insert the tubing about 7 mm into the mouse's nostril while slowly pushing the drug solution in. After completion, carefully remove the tubing. The entire process should be as gentle as possible to avoid harming the experimental animal.

[0078] 2.3 Hot Plate Experiment

[0079] The hot plate test is a method of measuring the pain threshold of experimental animals to reflect their pain perception, and it is often used to screen analgesic drugs. Female ICR mice were selected as the experimental animals. The mice's baseline pain threshold was measured, and they were placed on a 55°C hot plate. The mice licking their paws was used as an observation indicator. The mice were divided into three groups of 10 mice each, based on their pain threshold. They were given 0, 6 μg / kg CTX solution (concentration 75 μg / ml), and 12 μg / kg CTX solution (concentration 150 μg / ml), respectively. The pain threshold was measured at 0.5, 1, 2, 3, and 4 hours after administration (if there was no obvious pain response within 60 seconds, the mouse was removed and the time was counted as 60 seconds). The analgesic effect of different doses of cobra neurotoxin was observed.

[0080] 2.4 Organ Imaging

[0081] Three 9-month-old WT mice and three AD mice were selected and administered equal volumes of physiological saline, 6 μg / kg of FITC-labeled CTX solution (concentration 75 μg / ml), and 12 μg / kg of FITC-labeled cobra neurotoxin (concentration 150 μg / ml) via intranasal administration for three consecutive days. Three hours after the last day of administration, the mice underwent cardiac perfusion, and brain tissue was extracted for organ imaging.

[0082] 2.5 New Object Recognition Experiment

[0083] The novel object recognition experiment can reflect the short-term memory of mice. Before the experiment, mice freely explore an empty, sealed box for 5 minutes to adapt to the environment. During the learning phase, two identical cubes of the same shape, color, and size are evenly placed diagonally in the box, and the mice are placed in them to explore for 5 minutes, with their activities recorded by a camera. Three hours after the learning phase, one of the cubes is replaced with a triangular cube of a different shape, color, and size, while the object's position remains unchanged. The mouse is then placed in this triangular cube to explore for 5 minutes, with its activities recorded by a camera. After each mouse's experiment, the box and cubes are sprayed and wiped with 75% alcohol to remove the previous mouse's scent before placing the next mouse in, preventing odor from affecting the experimental results. After the experiment, the mouse exploration video is played back, and the time spent by the mouse sniffing, touching, or engaging in other exploratory behaviors with the two objects is recorded. Behaviors such as staying at the edge of an object or lying still on an object are not considered exploratory behaviors.

[0084] 2.6Y Maze Experiment

[0085] The Y-maze can be used to assess spatial and short-term memory in mice. The Y-maze apparatus consists of three arms with an angle of 120° between them. All experimental mice enter from the same arm and are allowed to explore freely for 8 minutes. Three consecutive entries into different arms are counted as one correct spontaneous alternation response. The total number of times the mouse enters the three arms and the number of correct spontaneous alternation responses are recorded, and the spontaneous alternation response rate is calculated.

[0086] 2.7 Morris Water Maze Experiment

[0087] The Morris water maze navigation experiment is used to assess the spatial learning and memory abilities of laboratory animals. The water maze setup consists of a circular pool with a diameter of 150 cm and a height of 55 cm. The pool is divided into four quadrants using different colored and shaped cards as markers on the pool walls. A platform is placed in the center of any quadrant. During the experiment, the water is dyed white with titanium dioxide, and the platform is hidden underwater while maintaining its position. The water temperature is kept at 22±1℃, and there is no light or shadow in the pool. A camera is installed directly above the pool to record the swimming trajectory and related indicators of the experimental animals within a specified time.

[0088] The experiment lasted seven days. Day 1 was the adaptation period, during which mice were placed facing the pool wall in any quadrant, and their swimming trajectory was recorded over 60 seconds. Days 2-5 were the learning and training period, with training conducted twice daily, once in the morning and once in the afternoon. Mice were placed into the pool from different quadrants, and their swimming trajectory and latency in finding the platform were recorded over 60 seconds. A successful escape was considered to have occurred if the mouse remained on the platform for 3 seconds. If the mouse did not find the platform within 60 seconds, its latency was recorded as 60 seconds, and it was led to the platform and remained there for 15 seconds. Days 6-7 were the testing period. In the navigation test, mice were placed in the diagonal quadrant opposite the platform, and their swimming trajectory and the time it took to find the platform were recorded. Twenty-four hours after the navigation test, a spatial exploration test was conducted. The platform was removed, and mice were placed in the diagonal quadrant, and their swimming trajectory and exploration time in the target quadrant were recorded.

[0089] 2.8 Open Field Experiment

[0090] The open field test is a method used to assess the exploratory behavior and anxiety levels of mice in unfamiliar environments. Before the test, the animals are placed in the experimental room and allowed to acclimatize for at least half an hour. The experimental apparatus consists of a sealed chamber with a camera mounted on top. During the test, the animals are placed in the chamber and allowed to move freely for 5 minutes. The camera records their movement during these 5 minutes. The test must be conducted in a quiet environment. After each mouse's test, the chamber is sprayed with 75% ethanol and cleaned to avoid affecting the next mouse's activity.

[0091] 2.9 Elevated Cross Maze Experiment

[0092] The elevated cross maze consists of open and closed arms that intersect in a cross shape. The maze is 50 cm above the ground. During the experiment, the experimental animal is placed facing the open arm at the intersection of the two arms. A camera above the maze records the mouse's movement trajectory over 5 minutes, including the number of times it enters each arm and the duration of its stay. After each mouse is tested, it must be wiped with 75% ethanol to remove the odor before the next mouse can be tested.

[0093] 2.10 Brain tissue slices

[0094] 1) Anesthetize mice with 4% chloral hydrate, fix them on a foam board, cut open the thoracic cavity, and expose the heart;

[0095] 2) Make a small incision at the left atrial appendage, insert an intravenous injection needle from the apex of the heart, and slowly infuse 40 mL of pre-cooled PBS solution to drain the blood. Then slowly infuse 20 mL of pre-cooled 4% paraformaldehyde solution.

[0096] 3) Remove mouse brain tissue and fix it in 4% paraformaldehyde at 4°C for 24 hours;

[0097] 4) Soak the brain tissue in a 20% sucrose solution until it sinks to the bottom, then soak it in a 30% sucrose solution for 24 hours.

[0098] 5) Sectioning: Frozen sections: Blot dry the surface moisture of the brain tissue with filter paper, freeze at -80℃, and embed the brain tissue in embedding medium during sectioning. The section thickness is 20μm. Vibrating sections: Blot dry the surface moisture with filter paper, fix the tissue on a vibrating microtome, and the section thickness is 40μm. The brain slices are placed in antifreeze and stored at -20℃.

[0099] 2.11 Nissl staining

[0100] 1) Carefully pick out a 40μm brain slice with a brush and place it in a six-well plate. Wash it twice with PBS, attach it to an adhesive glass slide, and let it air dry.

[0101] 2) Place the brain slices in a glass jar and wash them twice with double-distilled water, 3 minutes each time;

[0102] 3) Pour Nissl stain into a glass jar and slowly shake on a shaker for 30 minutes;

[0103] 4) Recover Nissl stain solution and wash twice with double-distilled water for 3 minutes each time;

[0104] 5) Decolorize with 75%, 95%, and anhydrous ethanol in sequence, 2 minutes each time, then air dry;

[0105] 6) Permeate in xylene for 10 minutes, then air dry;

[0106] 7) Add a drop of neutral resin to the brain slice, carefully cover with a coverslip, and seal the slice.

[0107] 2.12 Stereotactic Injection of the Brain

[0108] Three-month-old and nine-month-old WT mice were randomly divided into a control group and a CTX-treated group (6 μg / kg) according to age. Before stereotactic injection, mice were given either a blank solution or a drug-containing solution for five consecutive days, and the stereotactic injection experiment was performed on the fifth day.

[0109] 1) After anesthetizing the mice, cut off the hair on their heads and fix their heads steadily on the stereotaxic instrument.

[0110] 2) After disinfecting with povidone-iodine, cut open the scalp to expose the skull. Use a cotton swab to apply a small amount of H2O2 solution to the surface of the skull to expose the anterior fontanelle.

[0111] 3) Mark the anterior fontanelle with a marker pen, and use it as the origin to reset the X and Y axis coordinates to zero.

[0112] 4) Move the injection needle 1.3 mm to the right and 2.0 mm down, and mark the position with a marker to mark the needle insertion position.

[0113] 5) Gently drill through the skull at the needle insertion point with a skull drill, and slowly insert the needle until the needle tip enters below the surface of the skull, then reset the Z-axis coordinate to zero.

[0114] 6) Move the injection needle downwards slowly and uniformly for 2.7 mm, and inject 2 μL of Aβ oligomer at a constant speed of 200 nL / min.

[0115] 7) After the injection, leave the needle in place for 5 minutes, then slowly and evenly remove the needle, remove the mouse and suture it. Disinfect the suture site with iodine solution, then place the mouse on a warming blanket. After it wakes up, put it back in the cage for normal feeding.

[0116] 8) After stereotactic injection, the drug was administered normally. The victim was sacrificed 48 hours later to collect samples for subsequent experiments.

[0117] 2.13 Golgi-Cox staining

[0118] 2.13.1 Sample Preparation

[0119] 1) Mix equal volumes of solutions A and B 24 hours in advance and store at room temperature away from light;

[0120] 2) After deeply anesthetizing the mice, quickly remove the brain tissue and rinse off the surface blood with PBS;

[0121] 3) Soak the brain tissue in the pre-mixed solutions A and B at room temperature in the dark. Change the solutions A and B after 24 hours and soak for two weeks at room temperature in the dark.

[0122] 4) Transfer the brain tissue to solution C and soak it at room temperature in the dark for one week. Change the solution C once every 24 hours after the brain tissue is transferred.

[0123] 5) Remove the brain tissue from solution C, remove the surface liquid, and store at -80°C.

[0124] 2.13.2 Frozen sections

[0125] 1) After the prepared brain tissue was encapsulated with embedding agent, it was fixed on a tray and quick-frozen in a -80℃ freezer;

[0126] 2) Set the cryostat temperature to -20℃, slice to a thickness of 100μm, and fix the anti-roll plate before slicing;

[0127] 3) Place a drop of solution C on an adhesive slide, carefully transfer the cut brain slice into solution C and spread it out. Use filter paper to absorb the excess liquid and allow it to air dry at room temperature.

[0128] 2.13.3 Golgi staining

[0129] 1) Rinse the brain slices twice with double-distilled water, 4 minutes each time;

[0130] 2) Mix solutions D and E with double-distilled water in a ratio of 1:1:2 to form a solution, and drop it onto the brain slice for alkalization for 10 minutes;

[0131] 3) Rinse the brain slices twice with double-distilled water, 4 minutes each time;

[0132] 4) Use a gradient of 50%, 75%, and 95% ethanol for dehydration, 4 minutes each time;

[0133] 5) Use anhydrous ethanol for dehydration, 4 minutes each time;

[0134] 6) Permeabilize the brain slices in xylene for 12 minutes;

[0135] 7) Use neutral resin to seal the film, let it dry in the dark, and then take a picture.

[0136] 2.14 Immunofluorescence

[0137] 1) Carefully pick out a 20μm brain slice with a brush and place it in a six-well plate. Wash it twice with PBS, attach it to an adhesive glass slide, draw circles around it with an immunohistochemistry pen, let it dry, and then place it in a humidified chamber.

[0138] 2) Punch PBST (50mL PBS + 200μL Triton-X100) at room temperature for 30min;

[0139] 3) Block with PBST solution containing 5% BSA at room temperature for 1.5 h;

[0140] 4) Dilute the primary antibody with PBST containing 1% BSA according to the instructions, add it to the brain slices, and incubate overnight at 4°C;

[0141] 5) The next day, remove the brain slices and allow them to return to room temperature. Discard the primary antibody and wash with PBST three times, 8 minutes each time.

[0142] 6) Dilute the secondary antibody with PBST containing 1% BSA according to the ratio, add it to the brain slice, and incubate at room temperature in the dark for 3 hours;

[0143] 7) Discard the secondary antibody, wash three times with PBST, 8 minutes each time;

[0144] 8) Prepare DAPI (1:10000) with PBST containing 1% BSA and incubate at room temperature in the dark for 15 min;

[0145] 9) Discard DAPI, wash with PBST 3 times, 8 minutes each time;

[0146] 10) Add two drops of anti-quenching solution to the brain slice, cover with a glass slide, and store at 4°C protected from light.

[0147] 2.15 Quantitative Real-Time PCR

[0148] 2.15.1 Tissue RNA Extraction

[0149] 1) Take mouse hippocampus into 1 mL Trizol, homogenize using a homogenizer, and let stand;

[0150] 2) Add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand for 10 minutes;

[0151] 3) Centrifuge at 4℃, 12000g for 15 minutes;

[0152] 4) Transfer the supernatant to a new EP tube, add an equal amount of isopropanol to the supernatant, gently invert to mix, and let stand for 10 minutes;

[0153] 5) Centrifuge at 4℃, 12000g for 10 minutes;

[0154] 6) Discard the supernatant, add 1 mL of 75% ethanol, gently invert and wash, centrifuge at 7500 g for 10 min at 4℃, and discard the supernatant;

[0155] 7) Repeat step 6);

[0156] 8) Open the tube cap, place it in a fume hood to dry, and add an appropriate amount of DEPC water to dissolve it;

[0157] 9) The RNA concentration was measured using a NanoDrop2000c micro-volume spectrophotometer, and the sample concentration was adjusted to 500 ng / μL.

[0158] 2.15.2 Reverse Transcription

[0159] 1) Prepare a 20 μL reverse transcription system: x μL RNA sample, 4 μL 5×TransScript All-in-One SuperMix for qPCR, 16-x μL nuclease-free water, mix well;

[0160] 2) Reverse transcription program settings: 42℃ for 15 min, 85℃ for 5 s;

[0161] 3) After completion, remove the sample, store it at 4℃, and conduct experiments as soon as possible.

[0162] 2.15.3 PCR primer sequences:

[0163] Table 1

[0164] 2.15.4 qPCR

[0165] 1) Dilute the reverse transcribed cDNA as needed to prepare a 20 μL system: prepare 10 μL of SYRB green + 1 μL of forward primer + 1 μL of reverse primer + 8 μL of diluted cDNA, and set up three replicates for each sample.

[0166] 2) Centrifuge after mixing;

[0167] 3) Place the eight-tube strip into the real-time PCR instrument and set the program: 94℃ for 30s, 94℃ for 5s, 60℃ for 30s as one cycle, for a total of 40 cycles;

[0168] 4) Perform quantitative analysis based on the measured Ct value.

[0169] 3. Experimental Results

[0170] 3.1 Brain entry of Cobrotoxin (CTX) via intranasal administration

[0171] Three 9-month-old WT mice and three AD mice were selected. Both groups were administered FITC-labeled CTX at appropriate concentrations and an equal volume of physiological saline via intranasal administration for three consecutive days. Three hours after the last day of administration, the mice were imaged using a small animal imaging system. Cardiac perfusion was then performed, and brain tissue was harvested for organ imaging and sectioning to observe the brain penetration of FITC-CTX. The results showed that the intranasal administration of CTX resulted in strong FITC fluorescence intensity in the brain, indicating that sufficient CTX entered the brain and was distributed in various brain regions (see Figures A and B in Figure 1; Figure B corresponds to a CTX intranasal administration dose of 12 μg / kg). The yellow fluorescence in Figure 1A represents the small animal imaging results, indicating that FITC-labeled cobrodin entered the brain. The green fluorescence in Figure 1B represents the brain section observed using a laser confocal microscope; the green fluorescence represents FITC-labeled cobrodin. These results form the basis for further investigation into the therapeutic effect of intranasal administration of cobra neurotoxin on Alzheimer's disease.

[0172] In addition, cobra neurotoxin has been shown to have analgesic effects. In this application, the pain threshold of mice after intranasal administration of CTX was measured by hot plate test. The results showed that the pain threshold of mice was increased to varying degrees after administration (Table 2), which further proves that cobra neurotoxin CTX can exert its efficacy through intranasal administration.

[0173] Table 2. Comparison of pain thresholds among different groups of mice

[0174] 3.2 CTX improves learning and memory abilities in APP / PS1 mice

[0175] The novel object recognition test evaluates an animal's cognitive memory ability by assessing the time spent exploring familiar objects versus new, unfamiliar objects. Alzheimer's disease mice were administered the drug for 12 weeks, and behavioral tests were performed after the treatment. The results showed no significant difference in exploration time for familiar objects among the groups. However, both the WT and Alzheimer's disease groups showed a preference for exploring new objects, while the Alzheimer's disease mice did not exhibit a preference for exploring new objects (Figures A and B in Figure 2). These results indicate that CTX improves short-term memory in Alzheimer's disease mice.

[0176] The Y-maze can be used to assess short-term memory in mice. Spontaneous alternation assesses spatial working memory by allowing mice to freely explore the three branches of the maze. Mice with good working memory remember the arms of the maze they have already traversed and tend to enter arms that have not been recently explored. Results showed that the spontaneous alternation rate was significantly lower in Alzheimer's disease mice compared to WT mice, while the spontaneous alternation rate in the drug-treated Alzheimer's disease mice rebounded (Figures C and D in Figure 2). These results indicate that CTX improves short-term memory in Alzheimer's disease mice.

[0177] The Morris water maze test is commonly used to assess animals' long-term learning and memory abilities regarding space and orientation. Results showed that as the experimental days increased, the latency for platform finding in WT mice gradually shortened, while the latency in the Alzheimer's disease group mice only shortened slightly and showed no significant progress. Compared to the Alzheimer's disease group, the latency in the drug-treated group mice was significantly shortened on the third day, eventually becoming essentially consistent with the wild type (Figure 2, Figures E and F (results from the test on day 6)). Furthermore, the test results showed that, compared to WT mice, Alzheimer's disease mice did not exhibit a preference for navigation in the target quadrant, while compared to the Alzheimer's disease group, the navigation distance in the target quadrant was significantly increased in the drug-treated group (Figure 2, Figures G and H (spatial exploration on day 7)). This indicates that CTX improved the long-term learning and memory abilities of Alzheimer's disease mice. All these results demonstrate that CTX can improve the learning and cognitive abilities of Alzheimer's disease mice.

[0178] 3.3 CTX improves anxiety in APP / PS1 mice

[0179] The open field test is a method used to assess the exploratory behavior and anxiety levels of mice in unfamiliar environments. When mice are anxious, they mainly move in the peripheral areas, with reduced activity in the central area. The open field results showed no significant difference in the total distance traveled by the four groups of mice in the open field. Compared with WT mice, Alzheimer's disease mice showed significantly reduced activity in the central area, while the proportion of activity in the central area increased in the drug-treated group (Figures A, B, and C in Figure 3), indicating that the anxiety of Alzheimer's disease mice was improved after drug administration.

[0180] In the elevated cross maze experiment, abnormal behavior in the open arms of experimental mice reflected increased anxiety, mainly manifested as a significant decrease in the percentage of times mice entered the open arms and the percentage of time spent in the open arms. The results showed that compared with WT mice, Alzheimer's disease mice exhibited a decrease in the percentage of times they entered the open arms and the percentage of time spent in the open arms, indicating increased anxiety. After drug administration, the anxiety in Alzheimer's disease mice improved (Figures D, E, and F in Figure 3). These results indicate that CTX can improve anxiety in Alzheimer's disease mice.

[0181] 3.4 Effects of CTX on Aβ clearance in the brain

[0182] Aβ is an insoluble fragment produced by the degradation of APP via the β-secretase pathway. It leads to the formation and deposition of Aβ oligomers, resulting in neurotoxicity. At the same time, it triggers an inflammatory response mediated by glial cells in the brain, causing damage and death of brain nerve cells.

[0183] Immunofluorescence experiments were performed on brain tissue sections from mice. The results showed that there was significant Aβ deposition in the brains of Alzheimer's disease mice. After administration, the Aβ in the brain tissue of Alzheimer's disease mice was significantly reduced (Figure 4, Figures A and B), indicating that CTX has a clearing effect on Aβ deposited in the brain.

[0184] 3.5 Scavenging effect of CTX on exogenous Aβ

[0185] Microglia are resident immune cells in the central nervous system. They can recognize and engulf extracellular substances such as cell debris and pathogens through phagocytosis, thereby maintaining the stability of the central nervous system. To further verify the clearance effect of CTX on Aβ and to explore whether this effect is related to enhancing the phagocytic activity of microglia, FITC-labeled Aβ oligomers were injected into the hippocampus of 3-month-old and 9-month-old WT mice using stereotactic injection. Brain tissue sections were collected 48 hours later to observe the residual Aβ. Microglia were stained using immunofluorescence experiments, and 3D reconstruction of microglia was performed using IMARIS software to evaluate their phagocytic capacity. The results showed that compared with 3-month-old mice, 9-month-old mice had a reduced ability to clear Aβ, while CTX administration promoted Aβ clearance in both 3-month-old and 9-month-old mice (Figures A and B in Figure 5). Compared with 3-month-old mice, the phagocytic capacity of microglia in the brain of 9-month-old mice was reduced, while CTX administration increased the phagocytic capacity of microglia (Figures C and D in Figure 5), indicating that CTX can achieve the effect of clearing Aβ by enhancing the phagocytic capacity of microglia.

[0186] 3.6 Effects of CTX on microglial cell function

[0187] Neuroinflammation is a common pathological basis for many neurodegenerative diseases and a prominent feature of Alzheimer's disease, widely recognized as a core component of its pathogenesis. Microglia and astrocytes are key regulators of neuroinflammation. Activation of microglia and astrocytes increases the release of inflammatory and chemokine-producing factors, which helps maintain central nervous system homeostasis. While glial cell activation is a neuroprotective mechanism, excessive activation increases neurotoxicity, leading to neuroinflammation. Persistent neuroinflammation can cause neuronal functional impairment and ultimately neuronal death.

[0188] Immunofluorescence experiments were performed on brain tissue sections from mice. The results showed that microglia in the brain tissue of WT mice were mostly in a resting state, i.e., small cell bodies and many branches; microglia in the brain tissue of Alzheimer's disease mice were generally in an activated state, i.e., enlarged cell bodies and fewer branches; and after drug administration, the activation state and number of microglia in the brain tissue of Alzheimer's disease mice were improved (see Figures A, B, and C in Figure 6). qPCR results showed that compared with the Alzheimer's disease group, the levels of inflammatory factors IL-1β, TNF-α, and IL-6 in the hippocampus of Alzheimer's disease mice decreased after drug administration, indicating that CTX can improve neuroinflammation in Alzheimer's disease (see Figures D, E, and F in Figure 6).

[0189] 3.7 CTX inhibits LPS-induced inflammatory response in BV2 cells

[0190] Cobra neurotoxin CTX treatment of BV2 cells for 12 and 24 hours: Cells were seeded into 12-well plates. When the cell density reached about 60%, cells were pretreated with drug-free complete culture medium or the corresponding concentration of CTX for 12 or 24 hours. The expression of inflammatory factors was then detected by qPCR.

[0191] The experimental method for inhibiting LPS-induced inflammatory response in BV2 cells by CTX was as follows: Cells were seeded into 12-well plates. When the cell density reached about 60%, the cells were pretreated with drug-free complete culture medium or the corresponding concentration of CTX for 3 hours. Cell inflammatory response was induced with 500 μg / ml LPS. After LPS treatment for 24 hours, the cells were collected and qPCR was performed to detect the expression of inflammatory factors.

[0192] The results showed that the cobra neurotoxin CTX had no significant toxic side effects on cells (see Figures A and B in Figure 7). Next, LPS was used to induce a BV2 inflammatory response. The treatment groups were pre-treated with LPS (10 ng / ml, 100 ng / ml, 1000 ng / ml) for 3 h before being treated with LPS. After 24 h of treatment, qPCR was used to detect the expression of inflammatory factors. The results showed that the expression levels of IL-1β and TNF-α decreased after CTX administration (see Figures C and D in Figure 7), indicating that CTX has an inhibitory effect on the LPS-induced BV2 inflammatory response. In the early stages of neuroinflammatory response, glial cells play an important protective role in clearing Aβ and maintaining microenvironmental homeostasis. However, persistent neuroinflammatory inflammation will cause neuronal functional damage and ultimately lead to neuronal death. Simultaneously, persistent neuroinflammatory inflammation will also reduce the ability of microglia to clear Aβ and promote the production of neuronal Aβ, ultimately forming a vicious cycle that continuously exacerbates AD pathological damage. Therefore, in this experiment, LPS was used to induce an inflammatory response in BV2 cells in vitro. After administration of CTX, the inflammatory response was inhibited, demonstrating the anti-inflammatory effect of CTX in vitro. This suggests that CTX improves the pathology of Alzheimer's disease by inhibiting the inflammatory response of microglia.

[0193] 3.8 CTX improves the neuronal state in the hippocampus of APP / PS1 mice

[0194] Neuronal damage and loss are observed in neurodegenerative diseases. Therefore, Nissl staining was used to investigate whether cobra neurotoxin could improve the neuronal state in Alzheimer's disease mice. The results showed that most neurons in the CA1, CA3, and DG regions of the hippocampus in WT mice stained lightly, with normal, plump cell morphology, clear edges, and visible nuclei. In contrast, most neurons in the hippocampus of Alzheimer's disease mice showed damaged morphology, shrinkage, and deformation (the dark dotted neurons in the image), with a reduction in normal neurons. The neuronal state of mice in the Alzheimer's disease treatment group showed varying degrees of improvement, with a significant increase in the number of normal neurons in the brain (see Figures A, B, C, and D in Figure 8).

[0195] NeuN is a protein encoded by the RBFOX3 gene and is commonly used as a neuronal biomarker. Immunofluorescence staining of neurons in the hippocampus of mice showed that Alzheimer's disease mice had a significantly reduced number of neurons compared to WT mice, while the number of neurons in Alzheimer's disease mice increased after drug administration (see Figures E and F in Figure 8). This result is consistent with Nissl staining results, indicating that CTX can exert a therapeutic effect on Alzheimer's disease by improving neuronal status.

[0196] 3.9 CTX promotes the proliferation of neural stem cells in APP / PS1 mice

[0197] Neural stem cells (CTX) are a type of cell located in the central nervous system with self-renewal and differentiation potential, capable of differentiating into neurons, astrocytes, and oligodendrocytes. When the brain is damaged or in a disease state, neural stem cells play a crucial role in the repair process. A decrease in neural stem cells reduces neurogenesis, thereby affecting learning and memory functions. Therefore, it was hypothesized that cobra neurotoxin (CTX) could improve Alzheimer's disease symptoms by promoting the proliferation of neural stem cells. Immunofluorescence staining of neural stem cells showed that compared with WT mice, the density of neural stem cells in the brains of Alzheimer's disease mice was significantly reduced. Administration of the cobra neurotoxin CTX reversed this phenomenon, with a significant increase in the density of neural stem cells in the brains of Alzheimer's disease mice after administration (see Figures A and B in Figure 9). This suggests that CTX can improve the learning, cognition, and memory abilities of Alzheimer's disease mice by promoting the proliferation of neural stem cells.

[0198] 3.10 Effects of CTX on dendritic spines

[0199] Dendritic spines are small projections extending from the dendrites of neurons, forming synapses and closely related to learning and memory. Golgi staining was used to observe changes in dendritic spines in the hippocampus of mice. The results showed that compared to WT mice, Alzheimer's disease mice had significantly decreased dendritic spine density. However, treatment with cobra neurotoxin significantly increased dendritic spine density in Alzheimer's disease mice (see Figures A and B in Figure 10), indicating that CTX can salvage synaptic impairment in the disease and improve learning and cognitive function in Alzheimer's mice by increasing dendritic spine density.

[0200] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of cobra neurotoxin and its preparations for the manufacture of a medicament for the prevention and / or treatment of Alzheimer's disease, characterized in that, The cobra neurotoxin is selected from cobrotoxin of Chinese cobra and / or cobratoxin of Thai cobra. The preparation of the cobra neurotoxin is selected from at least one of cobrotoxin injection, Nyloxin, cobraxin and peperon, or a non-injection dosage form of the above preparations.

2. Use according to claim 1, characterized in that, The amino acid sequence of the cobra neurotoxin is shown in SEQ ID NO.

1.

3. Use according to claim 1 or 2, characterized in that, The cobra neurotoxin or the preparation thereof has at least one of the following (1) - (4) uses: (1) use in the preparation of a drug for improving cognitive ability; (2) use in the preparation of a drug for reducing accumulation of amyloid; (3) use in the preparation of a drug for inhibiting neuroinflammation; (4) use in the preparation of a drug for increasing the number of neurons in the cerebral cortex and hippocampus and repairing neurons.

4. Use according to claim 3, characterized in that, The drug contains a therapeutically effective amount of the cobra neurotoxin.

5. Use according to claim 4, characterized in that, The content of the cobra neurotoxin in the drug is 0.01-1% by mass.

6. Use according to claim 5, characterized in that, The drug further comprises a pharmaceutically acceptable carrier.

7. Use according to claim 6, characterized in that, The pharmaceutically acceptable carrier is selected from at least one of a pharmaceutically acceptable solvent, a solubilizer, a cosolvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a thickening agent, a complexing agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, a release retardant, a high molecular backbone material and a film-forming material.

8. Use according to claim 7, characterized in that, The drug is in the form of oral administration, oral cavity administration, nasal cavity administration, transdermal administration or injection.

9. Use according to claim 8, characterized in that, The drug is in the form of injection or nasal cavity administration.

10. Use according to claim 9, characterized in that, The drug can further comprise another additional therapeutic agent for treating Alzheimer's disease, which is selected from a synthetic small molecule drug, a genetically engineered drug, a biological preparation or a gene therapy drug.

11. A method for preventing and / or treating Alzheimer's disease, characterized by, The method comprises administering the cobra neurotoxin or the preparation thereof to a subject in need thereof, wherein the cobra neurotoxin is selected from cobrotoxin of Chinese cobra and / or cobratoxin of Thai cobra. The preparation of the cobra neurotoxin is selected from at least one of cobrotoxin injection, Nyloxin, cobraxin and peperon, or a non-injection dosage form of the above preparations.

12. The method of claim 11, wherein, The amino acid sequence of the cobra neurotoxin is shown in SEQ ID NO.

1.

13. The method according to claim 11 or 12, characterized in that, The cobra neurotoxin or the preparation thereof has at least one of the following (1) - (4) uses: (1) improving cognitive ability; (2) reducing accumulation of amyloid; (3) inhibiting neuroinflammation; (4) increasing the number of neurons in the cerebral cortex and hippocampus and repairing neurons.

14. The method of claim 13, wherein, The method comprises administering a therapeutically effective amount of the cobra neurotoxin to a subject in need thereof.

15. The method of claim 14, wherein, The content of the cobra neurotoxin in the method is 0.01-1% by mass.

16. The method of claim 15, wherein, The administration mode of the method is oral administration, buccal administration, nasal administration, transdermal administration or injection administration.

17. The method of claim 16, wherein, The administration mode of the method is injection administration or nasal administration.

18. The method of claim 17, wherein, The method further comprises administering in combination with another additional therapeutic agent for treating Alzheimer's disease, which is selected from synthetic small molecule drugs, genetically engineered drugs, biological agents or gene therapy drugs.

19. The method of claim 11, wherein, The administration dose is 5-15 μg / Kg body weight.

20. The method of claim 19, wherein, The administration dose is 6 μg / kg-12 μg / kg body weight.

21. The method of claim 11, wherein, When the administration subject is a human, the administration dose is 0.66 μg / Kg-1.32 μg / Kg body weight.

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