WO2021068432A1 - 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用 - Google Patents

眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用 Download PDF

Info

Publication number
WO2021068432A1
WO2021068432A1 PCT/CN2020/000239 CN2020000239W WO2021068432A1 WO 2021068432 A1 WO2021068432 A1 WO 2021068432A1 CN 2020000239 W CN2020000239 W CN 2020000239W WO 2021068432 A1 WO2021068432 A1 WO 2021068432A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
protein
neurotoxin
snake
postsynaptic
Prior art date
Application number
PCT/CN2020/000239
Other languages
English (en)
French (fr)
Other versions
WO2021068432A9 (zh
Inventor
祁展楷
祁·海亚特
Original Assignee
祁展楷
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201910986752.2A external-priority patent/CN111135288A/zh
Priority claimed from CN202010298240.XA external-priority patent/CN111467479A/zh
Application filed by 祁展楷 filed Critical 祁展楷
Priority to EP20873458.2A priority Critical patent/EP4046648A4/en
Priority to CN202080071876.9A priority patent/CN116997350A/zh
Publication of WO2021068432A1 publication Critical patent/WO2021068432A1/zh
Publication of WO2021068432A9 publication Critical patent/WO2021068432A9/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • A61K35/583—Snakes; Lizards, e.g. chameleons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates

Definitions

  • the present invention relates to a group of cobra snake postsynaptic neurotoxin monomer molecules which can inhibit the level of inflammatory factors in the hippocampal brain tissue of Alzheimer's rats and improve the learning and memory ability of Alzheimer's rats and a method for treating Alzheimer's. It belongs to the fields of biochemistry and biopharmaceuticals.
  • Senile dementia also known as Alzheimer's Disease (AD)
  • AD Alzheimer's Disease
  • Dementia is an acquired and persistent mental retardation syndrome caused by brain dysfunction.
  • the incidence and prevalence of dementia increase with age.
  • AD has become the fourth leading cause of death in humans.
  • AD Alzheimer's disease
  • amyloid beda protein as the core in the cerebral cortex and hippocampus
  • tau protein as the core component
  • Neuropathic diseases such as fibrous tangles.
  • One of the theories is that the neurotoxicity of the metabolites of amyloid precursor protein (APP) is due to the common pathways that cause Alzheimer’s disease.
  • APP amyloid precursor protein
  • the abnormal increase in brain tissue content may induce senile dementia.
  • the main factor of dementia up to now, clinical trial drugs aimed at removing Tau protein have not been able to prove their true effectiveness in the treatment of Alzheimer's disease.
  • the present invention discloses for the first time a group that can inhibit the increase in the levels of related inflammatory cytokines IL-1 ⁇ and TNF-a in the hippocampus of senile dementia rats, and can significantly improve the learning of senile dementia rats after treatment in the Morris water maze experiment Memory ability (shorten the escape latency) of the cobra snake postsynaptic neurotoxin monomer molecule.
  • cobra snake postsynaptic neurotoxin monomer molecules can inhibit the inflammatory cytokines in the brain tissue of Alzheimer’s rats and improve the learning and memory ability of Alzheimer’s rats, except for the cobra postsynaptic neurotoxin
  • some other snake species in the Cobra family including coral snakes, king cobras, black mamba snakes, golden snakes, etc., their postsynaptic neurotoxins can also inhibit the brain tissue of Alzheimer’s rats. This finding is the first report of the inflammatory cytokines in the inflammatory cytokines and improving the learning and memory ability of Alzheimer’s rats.
  • Synapse refers to the structure in which the impulse of one neuron is transmitted to another neuron or to another cell. Synapse is the part where neurons are connected in function, and it is also the key part of information transmission. Under an optical microscope, it can be seen that the axon end of a neuron has branched many times, and finally the end of each branch swells into a cup or ball, called a synaptosome. These synaptosomes can contact the cell bodies or dendrites of multiple neurons to form synapses. Observed under an electron microscope, it can be seen that this kind of synapse is composed of three parts: the presynaptic membrane, the synaptic cleft, and the postsynaptic membrane.
  • Acetylcholine is a neurotransmitter, and acetylcholine can specifically act on various cholinergic receptors.
  • Acetylcholine receptors include two types: muscarinic receptors and nicotinic receptors. Muscarinic receptors are involved in the excitatory effects of parasympathetic nerves, such as the contraction of bronchial gastrointestinal smooth muscles; nicotinic receptors are located in the postsynaptic membrane of the ganglion or the skeletal muscle endplate membrane, which can cause the excitement of the postganglionic neurons of the autonomic ganglion , Or cause skeletal muscle excitement.
  • Cobra snake neurotoxins can be divided into two types according to their different targets: one is the postsynaptic neurotoxin or ⁇ -neurotoxin, which compete with the nerve junctions located in the postsynaptic membrane The nicotinic acetylcholine receptor binds to block the transmission of the neurotransmitter acetylcholine [7]; the other type is presynaptic neurotoxin or ⁇ -neurotoxin, which directly acts on the presynaptic membrane of the motor nerve to block acetylcholine The release of skeletal muscle loses its contraction function and becomes paralyzed.
  • the cobra snake postsynaptic neurotoxin is an antagonist of the nicotinic acetylcholine receptor (nAChR), which binds to the nicotinic acetylcholine receptor of neurons in an antagonistic and slowly reversible manner[8,9 ].
  • nAChR nicotinic acetylcholine receptor
  • they have a common three-finger structure, so they are also called three-finger toxins.
  • the active site is close to the end of the middle finger [7].
  • This three-finger structure is a multifunctional structure that can regulate acetylcholine and The function of the receptor [10].
  • the cobra snake’s postsynaptic neurotoxin can improve the learning and memory of Alzheimer’s rats by inhibiting the inflammatory response. It may be related to their common three-finger protein structure to antagonize and regulate nicotinic acetylcholine receptors, because acetylcholine is affected by acetylcholine. The body is involved in the regulation of immune inflammatory response [11,12,13,14], which also leads to the common consistency of their therapeutic effects. Therefore, by antagonizing and regulating acetylcholine receptors, the inflammatory response can be suppressed correspondingly, thereby improving the learning and memory ability of Alzheimer's rats.
  • Another advantage of the present invention is in production. Because the cobra snake postsynaptic neurotoxin monomer molecule disclosed in the present invention has a clear amino acid sequence, it can be produced through genetic engineering, which solves the practical problem of scarcity of snake venom resources. ; Even if we continue to obtain postsynaptic neurotoxin through the separation and purification of natural snake venom, it is easier to achieve quality and purity control due to the clear amino acid sequence in the process. This is the development of medicines for the monomer components in snake venom Laid the necessary foundation.
  • Figure 1 shows the 12 protein peaks separated by TSK CM-650(M) column with ammonium acetate as the buffer and eluent of the crude venom of Chinese cobra.
  • Figure 2 is a broken line chart of the escape latency (Table 1) of the control group of Alzheimer's rats, the treatment group of two isolated postsynaptic neurotoxins, and the control group of young rats before and after administration.
  • Fig. 3 is a broken line chart of the escape latency (Table 2) of the control group of Alzheimer's rats, the treatment group of 4 recombinant postsynaptic neurotoxins, and the control group of young rats before and after administration.
  • Example A Obtaining of Cobra Neurotoxin Polypeptides SEQ ID No. 1 and SEQ ID No. 10
  • the method is to pass 12 protein peaks and nicotinic acetylcholine receptor (nAChR) affinity test.
  • nAChR nicotinic acetylcholine receptor
  • ⁇ -bungarotoxin has a high affinity with the nicotinic acetylcholine receptor (nAChR)
  • the cobra neurotoxin and ⁇ -bungarotoxin will competitively bind to the nicotinic acetylcholine receptor (nAChR), and only with smoke Alkaline acetylcholine receptors (nAChR) bound to the radionuclide 125I-labeled ⁇ -Bungarotoxin can be precipitated and detected by the ⁇ immunocounter, and the unbound ones will be eluted.
  • nAChR nicotinic acetylcholine receptor
  • the binding inhibition rate of the 12 isolated proteins to the ⁇ -Bungarotoxin and the nicotinic acetylcholine receptor (nAChR) was used to measure the affinity of the isolated proteins to the nicotinic acetylcholine receptor (nAChR). That is their activity, the activity can be measured by the gamma immunometer in counts per second (Bq) as an indicator of protein activity.
  • the specific method is to inhibit the binding of 125I radiolabeled- ⁇ -Bungarotoxin and nicotinic acetylcholine receptor. Rate (%) to reflect the activity of each protein.
  • the method for identifying the cobra neurotoxin with high affinity with nicotinic acetylcholine receptor includes the following steps:
  • 125I radiolabeled- ⁇ -Bungarotoxin and nicotinic acetylcholine receptor (125I- ⁇ Btx-nAChR)
  • C ⁇ Btx, CBSA, and C snake venom respectively refer to the positive control ⁇ Btx, the negative control BSA and the activity Bq value of each snake venom component;
  • ⁇ -Bungarotoxin ( ⁇ Btx) (final concentration 4 ⁇ g/mi) is used as the positive control (ie 100 % Inhibition);
  • Bovine serum albumin (BSA) (final concentration 4 ⁇ g/ml) was used as a negative control (ie 0% inhibition).
  • the results show that there are 3 activity peaks in 12 protein peaks, and the inhibition rate can be as high as 20-50%, indicating that they all have neurotoxin activity.
  • the protein peak with the highest inhibition rate is named A, and amino acid sequencing is continued. .
  • the amino acid sequence determination method includes the following steps:
  • the peak A was purified and desalted with a reversed-phase high performance liquid chromatography (RP-HPLC) column (4.6 ⁇ 250mm, VYDAC RP-C8, 5 ⁇ m); the N-terminal sum was performed with the American ABI 491 protein sequence analyzer.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • Determination of the C-terminal amino acid sequence BLSAT analysis of the N-terminal amino acids obtained by sequencing, the comparison results, and the calculation of the theoretical amino acid sequence of the tested cobra neurotoxin; the analysis of the peptide coverage of the cobra neurotoxin, the experimental method used is Use trypsin, chymotrypsin and Glu-C enzymes to separately digest the protein test samples; then use LC-MS/MS (XevoG2-XS QTof waters) to analyze the peptide samples after digestion; use UNIFI (1.8.2, Waters) software analyzes the LC-MS/MS data, and determines the peptide coverage of the test product according to the algorithm results.
  • the Edman degradation method can also be used to sequence and confirm the sequence.
  • the amino acid sequence of the primary structure of peak A protein obtained by sequencing is: (Fasta format, SEQ ID No. 1)
  • Example B Obtaining of postsynaptic neurotoxin SEQ ID No. 4; SEQ ID No. 6; SEQ ID No. 12; SEQ ID No. 16
  • the gene containing the postsynaptic neurotoxin SEQ ID No.4 was amplified by PCR and cloned into the pBS-T vector, and the constructed recombinant pBS-T-postsynaptic neurotoxin SEQ ID No.4 was analyzed Identification.
  • the recombinant plasmid was transformed into the E. coli expression vector pET15b, and the recombinant expression plasmid pET15b-post-synaptic neurotoxin SEQ ID No. 4 was constructed, and the correct recombinant was analyzed and identified to transform E. coli BL21 (DE3) LysS.
  • the inclusion bodies after ultrasonic cleavage are dissolved in buffer (6mol/L guanidine hydrochloride, 20mmol/L Tris-HCL, pH8.0, 0.5mol/L NaCI, 5mmol/L imidazole); pass the nickel-NTA column affinity chromatography Purification involves equilibrating with the above-mentioned buffer before loading the column, washing to the baseline with the above-mentioned buffer containing 20mmol/L imidazole after loading the sample, and finally eluting with the above-mentioned buffer containing 300mol/L imidazole. Enterokinase cleaved to obtain the postsynaptic neurotoxin SEQ ID No. 4 protein.
  • the RP-HPLC method detects the refolding results, and determines the refolded components by comparing with the retention time of the standard sample. The refolded products are stored under refrigeration.
  • the peptide coverage of the obtained neurotoxin and the Edman degradation method were analyzed, and the measured sequence was compared with the amino acid sequence of the silver ring snake postsynaptic neurotoxin SEQ ID No. 4 in the protein library, and the sequence was confirmed to be completely consistent.
  • the next step is to use in experimental treatment of Alzheimer's rats.
  • the monomer molecule SEQ ID No. 6; SEQ ID No. 12; SEQ ID No. 16 of the cobra snake postsynaptic neurotoxin monomer molecule was obtained.
  • Example C Experiment on improving the learning and memory ability of rats with Alzheimer's disease by cobra snake postsynaptic neurotoxin monomer molecules
  • the present invention adopts an animal model of natural aging cognitive impairment (Alzheimer's disease), and obtains an animal model of Alzheimer's disease through the natural aging of the animal itself, such as aging rats, etc.
  • the neurological changes such as cognitive impairment in this type of model are naturally occurring Yes, it is closer to the true pathophysiological changes of AD.
  • Cummings et al. reported that there are A ⁇ precipitation plaques in the brain of old dogs, and there is corresponding selective behavioral impairment; Higgins et al. reported that A ⁇ deposits in the basal area of the forebrain in old mice and memory deficits [15].
  • 150-month-old rats aged 21-22 are adaptively reared for 10 days, the animals are free to take water, the room temperature is controlled at 22-25 degrees, the humidity is 50%-70%, the light is 12 hours, and the black is 12 hours.
  • Morris water maze positioning experimental training was used to screen cognitively impaired rats (Alzheimer's rats).
  • the Morris water maze experiment is an experiment to force animals to swim and find an underwater platform. It is mainly used to evaluate the space learning and memory ability of animals.
  • the experimental indicators provided are sensitive and reliable, and easy to operate. It is a classic experiment for testing and evaluating indicators of Alzheimer's disease in rats. .
  • the water depth of the water maze is 50cm, the water temperature is controlled at 22-25 degrees, and a platform is set in the center of the pool. Put milk powder in the pool and mix it thoroughly until the water is milky white, so that the rat cannot visually identify the position of the platform.
  • On the training day before the formal test put the rat into the water facing the wall of the pool and train it to find an escape platform. If the rat finds and stands on the platform for 3 seconds and does not slip off again, the training can be aborted and the time and time it takes to reach the platform are recorded. Swim the distance and let it stay on the platform for 10 seconds, so that the rat can learn and remember.
  • the cognitively impaired rats After screening the cognitively impaired rats, they were randomly divided into two parts, and one part was randomly divided into three groups: the control group of Alzheimer's rats (without administration, the same volume of normal saline) and two isolated and extracted cobra snakes Post-synaptic neurotoxin (SEQ ID No. 1 and SEQ ID No.
  • treatment group post-synaptic neurotoxin is prepared as a liquid at 45 ⁇ g/Kg, and administered continuously by intragastric administration, twice a day for 8 consecutive weeks), at the same time
  • Set up a control group of young rats no administration, equal volume of saline
  • the other part is randomly divided into 5 groups: the Alzheimer’s rat control group (no administration, equal volume of saline) and 4 A recombinant cobra snake postsynaptic neurotoxin (SEQ ID No. 4; SEQ ID No. 6; SEQ ID No. 12; SEQ ID No.
  • Table-1 is a comparison of the average escape latency of the Alzheimer's rat control group, the two isolated and purified postsynaptic neurotoxin treatment groups (SEQ ID No. 1 and SEQ ID No. 10), and the young rat control group.
  • Table-2 is the control group of Alzheimer's rats, the treatment group of 4 recombinant postsynaptic neurotoxins (SEQ ID No. 4; SEQ ID No. 6; SEQ ID No. 12; SEQ ID No. 16), young rats Comparison of the average escape latency of the control group.
  • the above data show that the cobra snake postsynaptic neurotoxin can improve the average escape latency of the Alzheimer's rat group and improve the learning and memory ability of the Alzheimer's rat group.
  • Example C After the experiment, one group (the control group of Alzheimer's rats; the control group of young rats, the treatment group of neurotoxin SEQ ID No. 1) was anesthetized intraperitoneally with 10% chloral hydrate.
  • the three groups of experimental rats were sacrificed head, and then the hippocampus tissue was quickly separated on the operating table. According to 1g hippocampus tissue sample, add 4°C pre-cooled normal saline, centrifuge at 13500r/min, low temperature homogenization time 10s/time, 30s interval, 4 consecutive times, prepare 10% tissue homogenate, then low temperature and low speed (4°C , 3000r/min) centrifuge for 15min, take the supernatant, and store at -40°C.
  • the levels of inflammatory cytokines interleukin-1 ⁇ (IL-1 ⁇ ) and tumor necrosis factor-a (TNF-a) in the hippocampus were operated by the ELISA method according to the instructions of the kit.
  • IL-1 ⁇ interleukin-1 ⁇
  • Table-3 shows the comparison of the levels of inflammatory cytokines interleukin-1 ⁇ (IL-1 ⁇ ) and tumor necrosis factor-a (TNF-a) in the brain tissues of the rat hippocampus.
  • IL-1 ⁇ interleukin-1 ⁇
  • TNF-a tumor necrosis factor-a
  • Nicotinic acetylcholine receptor a7 subunit is an essential regulator of inflammation NATURE

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Psychiatry (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicinal Preparation (AREA)

Abstract

老年性痴呆是一种以进行性记忆力障碍、判断推理障碍、运动障碍为主要临床特征的老年性疾病。老年痴呆的病理表现是一种以弥漫性脑萎缩为特征的神经系统的退行性病变，而这种退行性病变又和脑组织的炎性反应相关，故下调炎性细胞因子及抑制炎性反应可能是改善老年痴呆的病理基础及临床症状的一个重要手段。通过对实验大鼠的Morris水迷宫实验及它们脑组织炎性细胞因子的检测，发现眼镜蛇科的蛇突触后神经毒素单体能抑制老年痴呆大鼠脑组织中的炎性细胞因子，改善老年痴呆大鼠的学习记忆能力，为眼镜蛇科的蛇突触后神经毒素单体在治疗老年痴呆上的应用提供了可能。

Description

眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用 技术领域
本发明涉及一组具有抑制老年痴呆大鼠海马区脑组织中炎性因子水平，改善老年痴呆大鼠学习记忆能力的眼镜蛇科蛇突触后神经毒素单体分子和一种治疗老年痴呆的方法，属于生化和生物制药领域。
背景技术
老年性痴呆，也称为阿尔茨海默病(Alzheimer Disease，AD)，是一种以进行性记忆力障碍、判断推理能力障碍、运动障碍为主要临床特征的老年性疾病。痴呆是一种由于脑功能障碍而产生的获得性和持续性的智能障碍综合征，痴呆的发病率和患病率随年龄增加而上升。随着人口老龄化问题日益严重，AD已经成为人类第4大死亡原因。
老年性痴呆的发病机制复杂，但目前国内外研究者普遍认同的病理基础是以大脑皮质弥漫性萎缩为特征的神经系统的退行性病变，并伴有神经元损伤和死亡。AD患者整个大脑弥散性萎缩，大脑皮质和海马区出现以淀粉样蛋白(amyloid beda protein)为核心的老年斑(senile plaque，SP)，及以异常过度磷酸化的以tau蛋白为核心成分的神经原纤维缠结等神经病理性病变。其中一种理论认为，淀粉样前体蛋白(amyloid precursor protein，APP)的代谢产物的神经毒性是因引起老年性痴呆各种原的共同通路，其在脑组织中含量的异常增加可能是诱发老年性痴呆的主要因素。但是，到目前为止，只是以清除Tau蛋白为目的的临床试验药物尚没能够证明它们在治疗老年痴呆上的真正有效性。
与此同时也有证据表明老年痴呆患者脑内存在着强烈的局灶性炎症反应，老年斑附近有激活的小胶质细胞和星形胶质细胞，研究发现这些激活的细胞可以表达白细胞介素炎性因子白细胞介素-1β(interleukin-1β，IL-1β)和肿瘤坏死因子-a(TNF-a)等多种炎性细胞因子[1，2]。动物实验研究显示，用老年斑的主要成分淀粉样肽可诱导出类AD患者脑内的炎症反应，并伴有白细胞的激活与外渗，以及炎性细胞因子的产生。另外，过度磷酸化的淀粉样蛋白也会引起神经组织的炎性反应。这些研究证实了老年痴呆患者脑内除了抗炎细胞因子(IL-1ra)表达量的降低外，患者脑内TNF-a和(IL-1β)等炎性细胞因子的表达的确显著上调[3，4]。由此有专家提出神经元退行性病变可能是脑内免疫和炎症反应不适当激活所致，超强的免疫反应可“方向错误”地攻击神经组织，造成神经元损伤和死亡。因此有人提出这样的假说：尽管患者神经元退变的始动因素不同，并由此出现不同神经病理损害的结局，但它们有可能通过启动炎性细胞因子产生的级联反应这一相似的过程而引致神经元的损伤，而患者中枢神经系统炎性细胞因子的大量释放有可能加重淀粉样肽的产生及淀粉样肽在血管内聚集以及脑白质的损害，从而引起一系列的恶性循环。近年来，在此领域的研究发现证实了位于脑组织老年斑附近炎性因子的免疫反应呈阳性，并导致淀粉样蛋白合成的增加。因此抑制过度的免疫炎性反应已成为治疗老年痴呆的另一种探索。
从公开的信息来看，国外有一些关于眼镜蛇蛇毒治疗老年痴呆的报道，但眼镜蛇毒素种类繁多，已知成分有神经毒素、细胞毒素、心脏毒素、神经生长因子、溶血素(DLP)、CVA蛋白、膜活性多肽、眼镜蛇毒因子等及其他成分，如碱性磷酸单酯酶、磷酸二酯酶、乙酰胆碱酯酶、L-氨基酸氧化酶、核糖核酸酶、 蛋白水解酶等，对于眼镜蛇蛇毒制剂，混合毒素会对生物构成生命危险，这可能是在进化过程中一种增强毒性的策略。各种蛇毒中不同类的毒素或同类毒素复合物之间也存在协同作用，主要毒素如磷脂酶A2、三指毒素(神经毒素)在协同过程中发挥着重要作用[5，6]。而正是这种协同作用导致了对生物体致命的后果；也有报道用眼镜蛇神经毒素来治疗老年痴呆，但神经毒素也有多种，以往报道中没有具体指出是哪一种神经毒素，而用眼镜蛇科蛇突触后神经毒素单体分子来治疗老年痴呆还是首次被公开。
发明内容
本发明首次公开了一组能对老年痴呆大鼠海马区相关炎性细胞因子IL-1β，TNF-a的含量增高有抑制作用，在Morris水迷宫实验中能明显改善治疗后老年痴呆大鼠学习记忆能力(缩短逃避潜伏期)的眼镜蛇科蛇突触后神经毒素单体分子。
我们的研究发现，眼镜蛇科蛇突触后神经毒素单体分子可以抑制老年痴呆大鼠的脑组织中的炎性细胞因子并能改善老年痴呆大鼠的学习记忆能力，除眼镜蛇突触后神经毒素单体分子外，眼镜蛇科的其他一些蛇种，包括银环蛇，眼镜王蛇，黑曼巴蛇，金环蛇等，它们的突触后神经毒素也有同样的能抑制老年痴呆大鼠脑组织中的炎性细胞因子并改善老年痴呆大鼠学习记忆能力的功效，这个发现还是首次被报道。
突触是指一个神经元的冲动传到另一个神经元或传到另一细胞间的相互接触的结构。突触是神经元之间在功能上发生联系的部位，也是信息传递的关键部位。在光学显微镜下，可以看到一个神经元的轴突末梢经过多次分支，最后每一小支的末端膨大呈杯状或球状，叫做突触小体。这些突触小体可以与多个神经元的细胞体或树突相接触，形成突触。从电子显微镜下观察，可以看到，这种突触是由突触前膜、突触间隙和突触后膜三部分构成。
乙酰胆碱是一种神经递质，乙酰胆碱能特异性地作用于各类胆碱受体。乙酰胆碱受体包括两种：毒蕈碱型受体和烟碱型受体。毒蕈碱型受参与副交感神经兴奋效应，如支气管胃肠平滑肌的收缩等；烟碱型受体位于神经节突触后膜或骨骼肌终板膜，可引起自主神经节的节后神经元兴奋，或导致骨骼肌兴奋。
眼镜蛇科蛇神经毒素根据它们作用靶点的不同，可分为两类：一类为突触后神经毒素或α-神经毒素，这类毒素竞争性的与位于突触后膜的神经神经接头处的烟碱型乙酰胆碱受体结合，阻断神经递质乙酰胆碱的传导[7]；另一类为突触前神经毒素或β-神经毒素，其直接作用于运动神经突触前膜，阻断乙酰胆碱的释放，使骨骼肌失去收缩功能而麻痹。
所以眼镜蛇科蛇突触后神经毒素(α-神经毒素)是烟碱乙酰胆碱受体(nAChR)的拮抗剂，以拮抗和缓慢可逆的方式与神经元的烟碱型乙酰胆碱受体结合[8，9]。在结构上，它们有共同的三指的结构，故也被称为三指毒素，活性部位靠近中指末端[7]，这种三指的结构是一种多功能的结构，具有能够调节乙酰胆碱及受体的功能[10]。
眼镜蛇科蛇突触后神经毒素能够通过抑制炎性反应来改善老年痴呆大鼠学习记忆力的共性可能和他们具有共同的三指蛋白结构对烟碱型乙酰胆碱受体的拮抗和调节有关，因为乙酰胆碱受体参与了免疫炎性反应的调节[11，12，13，14]，这也导致了它们在治疗疗效上的共同一致性。故通过拮抗和调节乙酰胆碱受体就能相应的抑制炎性反应，从而改善了老年痴呆大鼠的学习记忆能力，这可能 就是眼镜蛇科蛇突触后神经毒素能够治疗老年痴呆的共同作用机理。我们的研究发现这些突触后神经毒素单体分子包括具有以下成熟蛋白或多肽的神经毒素：它们的成熟蛋白或多肽的氨基酸序列(FASTA)分别如下：
中华眼镜蛇突触后神经毒素
SEQ ID No.1
Figure PCTCN2020000239-appb-000001
SEQ ID No.2
Figure PCTCN2020000239-appb-000002
SEQ ID No.3
Figure PCTCN2020000239-appb-000003
银环蛇突触后神经毒素
SEQ ID No.4
Figure PCTCN2020000239-appb-000004
SEQ ID No.5
Figure PCTCN2020000239-appb-000005
眼镜王蛇突触后神经毒素
SEQ ID No.6
Figure PCTCN2020000239-appb-000006
SEQ ID No.7
Figure PCTCN2020000239-appb-000007
SEQ ID No.8
Figure PCTCN2020000239-appb-000008
孟加拉眼镜蛇突触后神经毒素
SEQ ID No.9
Figure PCTCN2020000239-appb-000009
SEQ ID No.10
Figure PCTCN2020000239-appb-000010
SEQ ID No.11
Figure PCTCN2020000239-appb-000011
黑曼巴眼镜蛇突触后神经毒素
SEQ ID No.12
Figure PCTCN2020000239-appb-000012
SEQ ID No.13
Figure PCTCN2020000239-appb-000013
SEQ ID No.14
Figure PCTCN2020000239-appb-000014
金环蛇突触后神经毒素
SEQ ID No.15
Figure PCTCN2020000239-appb-000015
SEQ ID No.16
Figure PCTCN2020000239-appb-000016
本发明的另一个优势是在生产上，因为本发明所公开的眼镜蛇科蛇突触后神经毒素单体分子具有明确的氨基酸序列，故能够通过基因工程来生产，解决了蛇毒资源稀缺的实际问题；即使是如果继续通过天然蛇毒的分离纯化来得到突触后神经毒素，因为过程中由于有了明确的氨基酸序列而更容易达到质量和纯度上的控制，这为蛇毒中单体成分的药品开发奠定了必要的基础。
以下结合附图和具体实施例对本发明做进一步说明，但以下实施例并非对本发明的限定；同时凡依照本发明公开内容所进行的本领域等同替换，均属于本发明的保护范围。
附图描述：
图1为中华眼镜蛇粗毒以醋酸铵为缓冲液和洗脱液，经TSK CM-650(M)柱分离后的12个蛋白峰。
图2是给药前和给药后，老年痴呆大鼠对照组，2种分离提取的突触后神经毒素治疗组，和青年大鼠对照组的逃避潜伏期(表1)对应的折线图。
图3是给药前和给药后，老年痴呆大鼠对照组，4种重组的突触后神经毒素治疗组，和青年大鼠对照组的逃避潜伏期(表2)对应的折线图。
实施方法
实施例A：眼镜蛇神经毒素多肽SEQ ID No.1和SEQ ID No.10的获得
1.对中华眼镜蛇粗毒进行分离纯化
将1g中华眼镜蛇粗毒溶解在25ml 0.025摩尔PH6.0的醋酸铵缓冲液中，低温离心，取上清液；用0.025摩尔PH6.0的醋酸铵溶液平衡TSK CM-650(M)柱；上样后用0.1～0.5摩尔和0.7～1.0摩尔，pH5.9醋酸铵缓冲液进行2厢阶梯梯度洗脱，紫外检测参数：280nm；洗脱流速：48ml/h；按峰收集各种毒素组分，收集液中洗脱出12个蛋白峰。(图1)
2.鉴别出与烟碱乙酰胆碱受体(nAChR)有亲和力的中华眼镜蛇毒素
方法是通过12个蛋白峰与烟碱乙酰胆碱受体(nAChR)亲和力试验。
亲和力试验原理：
因为α-银环蛇毒素与烟碱乙酰胆碱受体(nAChR)具有高度的亲和力，所以眼镜蛇神经毒素与α-银环蛇毒素会竞争性的与烟碱乙酰胆碱受体(nAChR)结合，只有与烟碱乙酰胆碱受体(nAChR)结合的被放射性核素125I标记的α-银环蛇毒素才能沉淀后被γ免疫计数仪测到，没结合的会被洗脱。故通过对12个分离出的蛋白各自对α-银环蛇毒素与烟碱乙酰胆碱受体(nAChR)的结合抑制率来测量分离出的蛋白各自和烟碱乙酰胆碱受体(nAChR)的亲和能力即他们的活性，活性度可通过γ免疫计数仪测每秒计数值(Bq)为蛋白活性度指标，具体 方法用对125I放射标记的-α-银环蛇毒素和烟碱乙酰胆碱受体结合抑制率(％)来体现每种蛋白的活性高低。
鉴别出与烟碱乙酰胆碱受体(nAChR)有高亲和力的眼镜蛇神经毒素的方法包括下述步骤：
取上述分离的蛇毒蛋白(每次加入一种12个蛋白峰纯化的蛇毒组分)和大鼠骨骼肌nAChR提取物，1μL抗乙酰胆碱烟碱受体单克隆抗体(mAb35)5.9mg/ml，放射性核素125I标记的α-银环蛇毒素1μL(125I-nα-Btx)0.18μg/ml，混匀后4℃静置10小时以上；次日加入兔抗-大鼠IgG(4.5mg/ml)10μL，4℃静置2小时；用13，000rpm离心5min，沉淀物以Triton X-100洗液洗涤3遍；用γ免疫计数仪测蛋白活度性指标每秒计数值Bq)。
125I放射标记的-α-银环蛇毒素与烟碱乙酰胆碱受体(125I-αBtx-nAChR)
Figure PCTCN2020000239-appb-000017
其中CαBtx、CBSA、C蛇毒分别指阳性对照αBtx、阴性对照BSA及各蛇毒组分的活性度Bq值；以α-银环蛇毒素(αBtx)(终浓度4μg/mi)为阳性对照(即100％抑制)；以牛血清白蛋白(BSA)(终浓度4μg/ml)为阴性对照(即0％抑制)。
结果显示在12个蛋白峰中有3个活性峰，抑制率可高达20～50％之间，说明它们都具有神经毒素活性，把抑制率最高的蛋白峰命名为A，对之继续进行氨基酸测序。
3.峰A蛋白质的一级结构分析即氨基酸测序
氨基酸序列测定方法包括下述步骤：
对峰A用反相高效液相色谱法(RP-HPLC)柱(4.6×250mm，VYDAC RP-C8，5μm)对其蛋白进行纯化和脱盐；用美国ABI公司491蛋白序列分析仪进行N端和C端氨基酸序列测定；对测序得到的N末端氨基酸进行BLSAT分析，比对结果，推算出被测眼镜蛇神经毒素的理论氨基酸序列；对眼镜蛇神经毒素的肽段覆盖率进行分析，采用的实验方法是使用胰蛋白酶、胰凝乳蛋白酶和Glu-C酶分别对蛋白质供试品进行酶解；然后使用LC-MS/MS(XevoG2-XS QTof waters)对酶解后的肽段样品进行分析；使用UNIFI(1.8.2，Waters)软件对LC-MS/MS数据进行分析，根据算法结果确定供试品的肽段覆盖率。另外也可用Edman降解法对序列进行测序确认。
测序得到峰A蛋白质一级结构的氨基酸序列为：(Fasta形式，SEQ ID No.1)
Figure PCTCN2020000239-appb-000018
用上述同样分离纯化的方法，我们得到了孟加拉眼镜蛇突触后神经毒素SEQ ID No.10。对应的氨基酸序列Fasta形式为：
Figure PCTCN2020000239-appb-000019
实施例B：突触后神经毒素SEQ ID No.4；SEQ ID No.6；SEQ ID No.12；SEQ ID No.16的获得
以上蛋白或多肽由基因重组来获得，具体如下：
1.重组表达载体的克隆
根据Genbank上提供的突触后神经毒素SEQ ID No.4的基因合成DNA序列，对目的DNA序列进行PCR扩增，在上游引物的5’端引入编码肠激酶识别位点的序列和Nde I酶切位点，在下游引物的5’端引入终止密码子和BamHI酶切位点。
用PCR的方法扩增出含突触后神经毒素SEQ ID No.4的基因并克隆到pBS-T载体，对所构建的重组子pBS-T-突触后神经毒素SEQ ID No.4进行分析鉴定。
2.在大肠杆菌中进行蛋白表达
把重组质粒转化到大肠杆菌表达载体pET15b中，构建重组表达质粒pET15b-突触后神经毒素SEQ ID No.4，将分析鉴定正确的重组子转化大肠杆菌BL21(DE3)LysS。将单克隆接种至5mL LB培养基中，37℃培养过夜，次日按1∶100的比例接种至50mL LB培养基中，37℃振荡培养至OD600nm＝0.4～0.6。
3.表达产物的收集分析
用1mmol/L IPTG继续培养3小时，诱导转化的大肠杆菌BL21(DE3)。表达菌经超声破碎后离心，包涵体溶于缓冲液，离心收集后上清和沉淀分别进行SDS-PAGE电泳检测，目的蛋白以包涵体的形式存在。
4.表达产品的亲和和纯化
超声破菌后的包涵体溶于缓冲液(6mol/L盐酸胍、20mmol/L Tris-HCL pH8.0、0.5mol/L NaCI、5mmol/L咪唑)中；通过镍-NTA柱亲合层析纯化，具体为上柱前用上述缓冲液平衡，上样后用含20mmol/L咪唑的上述缓冲液洗至基线，最后用含300mol/L咪唑的上述缓冲液洗脱。肠激酶切割得到了突触后神经毒素SEQ ID No.4蛋白。
5.表达产物的复性
用6mol/l盐酸胍、0.1mol/L Tris-HCL pH8.0、0.01mol/LEDTA、0.1mmol/L PMSF、10mmol/L DTT缓冲液透析上述洗脱蛋白，缓冲液中的DTT和盐酸胍浓度递减，然后再用10倍体积的0.1mol/L Tris-HCL pH8.0、5μmol/L CuSo4、20％甘油的缓冲液透析。RP-HPLC法对复性结果进行检测，通过与标样保留时间的对比确定已复性的成份，复性产物冷藏保存。
6.氨基酸序列测定
对所得神经毒素用肽段覆盖率和Edman降解法进行分析，测得的序列与蛋白库中银环蛇突触后神经毒素SEQ ID No.4的氨基酸序列进行对比，确认序列完全一致后用以进行下一步对老年痴呆大鼠的治疗实验使用。
用以上同样重组的方法获得眼镜蛇科蛇突触后神经毒素单体分子SEQ ID No.6；SEQ ID No.12；SEQ ID No.16。
实施例C：眼镜蛇科蛇突触后神经毒素单体分子对老年痴呆大鼠学习记忆力改善的实验
1.动物模型的选择
本发明采用了自然衰老认知障碍(老年痴呆)动物模型，通过动物本身的自然衰老来获得老年痴呆的动物模型，如老龄大鼠等，这类模型的认知障碍等神经系统改变是自然发生的，更贴近AD的真实病理生理改变。Cummings等报道老龄犬脑中有Aβ沉淀斑块，同时有相应的选择性的行为能力损害；Higgins等报道老龄鼠可出现Aβ沉积于前脑基底区，并有记忆缺损[15]。
2.试验动物与分组
对150个月龄在21～22的大鼠适应性饲养10天，动物自由摄水，室温控制22-25度，湿度为50％-70％，光照12小时，黑12小时。通过Morris水迷宫定位 实验训练来筛选认知障碍的大鼠(老年痴呆大鼠)。Morris水迷宫实验是一种强迫动物游泳，寻找水下平台的实验，主要用于评价动物空间学习记忆能力，提供的实验指标敏感可靠，操作简便，是测试和评价大鼠老年痴呆指标的经典实验。
水迷宫水深50cm，水温控制在22-25度，水池中央设平台。水池中放入奶粉，充分混匀至水呈乳白色，使大鼠无法通过视觉辨认平台位置。正式试验前训练日将大鼠面向池壁放入水中，训练其寻找逃避平台，若大鼠找到并站在平台上3秒后不再滑落，可中止此次训练，记录其抵达平台所用时间及所游距离，并让其在平台上停留10秒，由此让大鼠学习记忆。不能找到者持续记录120s后将其引至平台并放置30秒，培训其学习记忆。每天4次训练，间隔15～30秒进行下一次训练，从4个不同方向入水，连续4天。第5天正式试验测试，实时图像系统自动记录大鼠的活动情况，并计算大鼠首次穿越平台(中心区域)的平均时间，评价动物的学习记忆情况。以青年(4月龄)大鼠平均逃避潜伏期99％正常值范围上限值为标准，将逃避潜伏期大于99％上限值的老年大鼠定为知障碍老年大鼠。标签每个大鼠，以便在完成最后试验后能计算出每组大鼠用药前平均逃避潜伏期。
筛选出认知障碍大鼠后随机分为二部分，一部分随机分为3组：即老年痴呆大鼠对照组(不给药，灌胃等体积的生理盐水)和2种分离提取的眼镜蛇科蛇突触后神经毒素(SEQ ID No.1和SEQ ID No.10)治疗组(突触后神经毒素按45μg/Kg配成液态，连续灌胃给药，每天2次，连续8周)，同时设青年大鼠对照组(不给药，灌胃等体积的生理盐水)；另一部分随机分为5组：即老年痴呆大鼠对照组(不给药，灌胃等体积的生理盐水)和4种重组的眼镜蛇科蛇突触后神经毒素(SEQ ID No.4；SEQ ID No.6；SEQ ID No.12；SEQ ID No.16)治疗组(突触后神经毒素按45μg/Kg配成液态，连续灌胃给药，每天2次，连续8周)，同时设青年大鼠对照组(不给药，灌胃等体积的生理盐水)在实验过程中，保持每组10个大鼠，多余的出组。
3.行为学(学习记忆能力)的测试
给药8周后，再次连续训练5天，记录每天各组动物在水中寻找平台的平均时间，第6天直接进行Morris水迷宫测试。测定各组动物在水中寻找平台的时间。在训练过程中仍继续给药。
4.实验结果
表-1为老年痴呆大鼠对照组、2种分离纯化突触后神经毒素治疗组(SEQ ID No.1和SEQ ID No.10)、青年大鼠对照组的平均逃避潜伏期的比较。
表-1
Figure PCTCN2020000239-appb-000020
给药前，老年痴呆大鼠对照组和2种突触后神经毒素治疗组之间无显著性差异，但是它们与青年大鼠对照组存在显著性差异，###表示P＜0.001。给药8周后，再次进行Morris水迷宫定位实验训练，老年痴呆大鼠对照组和2种突触后神经毒素治疗组之间第一到第五天开始出现显著性差异，**表示P＜0.01，***表示P＜0.001。第6天，老年痴呆大鼠对照组和2种突触后神经毒素治疗组之间出现显著性差异，***表示P＜0.001。###表示青年大鼠对照组和老年痴呆大鼠对照组及2种突触后神经毒素治疗组存在显著性差异，从第一到第 六天P＜0.001。以上的数据显示：眼镜蛇科蛇突触后神经毒素能改善老年痴呆大鼠组的平均逃避潜伏期，改善老年痴呆大鼠的学习记忆能力。
表-2为老年痴呆大鼠对照组、4种重组突触后神经毒素治疗组(SEQ ID No.4；SEQ ID No.6；SEQ ID No.12；SEQ ID No.16)、青年大鼠对照组的平均逃避潜伏期的比较。
表-2
Figure PCTCN2020000239-appb-000021
给药前，老年痴呆大鼠对照组和4种突触后神经毒素治疗组之间无显著性差异，但是它们与青年大鼠对照组存在显著性差异，###表示P＜0.001。给药8周后，再次进行Morris水迷宫定位实验训练，老年痴呆大鼠对照组和4种突触后神经毒素治疗组之间第一到第五天开始出现显著性差异，*表示P＜0.05，**表示P＜0.01，***表示P＜0.001。第6天，老年痴呆大鼠对照组和4种突触后神经毒素治疗组之间出现显著性差异，***表示P＜0.001。
###表示青年大鼠对照组和老年痴呆大鼠对照组及4种突触后神经毒素治疗组存在显著性差异，从第一到第六天P＜0.001。
以上的数据显示：眼镜蛇科蛇突触后神经毒素能改善老年痴呆大鼠组的平均逃避潜伏期，改善老年痴呆大鼠的学习记忆能力。
实施例D：实验大鼠脑组织炎性细胞因子的测定
1.样本的获取与测试方式
实施例C实验结束后，对其中一组(老年痴呆大鼠对照组大鼠；青年大鼠对照组，神经毒素SEQ ID No.1治疗组)用10％水合氯醛腹腔麻醉，麻醉完毕后断头处死上述3组实验大鼠，然后在操作台上迅速分离出海马区组织。根据1g海马组织样，加入4℃预冷的生理盐水，13500r/min离心，低温匀浆时间10s/次，间隔30s，连续4次，制备出10％的组织匀浆，后低温低速(4℃，3000r/min)离心15min，取上清液，-40℃保存。海马区炎性细胞因子白细胞介素-1β(interleukin-1β，IL-1β)和肿瘤坏死因子-a(TNF-a)含量水平采用ELISA法按试剂盒说明步骤进行操作。
2.实验结果
表-3为大鼠海马区脑组织中各组的炎性细胞因子白细胞介素-1β(interleukin-1β，IL-1β)和肿瘤坏死因子-a(TNF-a)的含量水平比较。
表-3
Figure PCTCN2020000239-appb-000022
青年对照组和老年痴呆对照组的IL-1β及TNF-a相比存在显著性差异，###表示P＜0.001；老年痴呆对照组和SEQ ID No.1神经毒素治疗组的IL-1β及TNF-a相比也存在显著性差异，**表示P＜0.01。以上实验数据显示，眼镜蛇科突触后神经毒素单体分子可以通过抑制老年痴呆大鼠海马区脑组织中的炎性细胞因子水平减轻海马区神经元损伤，这可能为达到改善老年痴呆大鼠的学习记忆能力提供了病理依据。
上述实施例只为说明本发明的实施思路和技术特点，其目的在于让熟悉此技术的人员能够了解本发明的内容并据以实施，并不能以此而限制本发明的保护范围。凡根据本发明精神实质所做的任何等效改变或修饰，都应涵盖在本发明的保护范围之内。
Reference：
1.Mark RE et al；Potential inflammatory biomarkers in alzheimer’s disease.J Alzheimer’s disease 2005 8(4)369-375.
2.Griffin WS.Inflammation and Neurodegenerative disease.Am J Clin Nutr，2006 83(2)：470s-474s.
3.Wang et al；Detection of interleukoin-1and tumor necrosis factor-a in serum and celebrospinal fluid in patient with Alzheimer disease Chin J Neurol，2002 35(6)339-341.
4.沈颖华，烟碱型乙酰胆碱受体的激动与阿尔茨海默病的治疗，解放军药学学报第24卷第2期.
5.Shengwei Xiong，Chunhong Huang.Synergistic strategies of predominant toxins in snake venoms.Toxicology Letters 287(2018)142-154.
6.Laustsen，A.H.Toxin synergism in snake venoms.Toxin Reviews，35(3-4)，165-170.DOI：10.1080/15569543.2016.
7.J.White et al，1996Snake Neurotoxin.Human Toxicology，1996.
8.Naguib M et al.Advances in neurobiology of the neuromuscular junction：implications for the anesthesiologist.J Am Soc Anesthesiol.96：202-31，2002.
9，Abbas M，Rahman S.Effects of α-7 nicotinic acetylcholine receptor positive allosteric modulator on lipopolysaccharide-induced neuroinflammatory pain in mice.Eur J Pharmacol.783：85-91，2016.
10.Pascale Marchot et al，The three-finger toxin fold：a multifunctional structural scaffold able to modulate cholinergic functions Journal of neurochemistry，Vol 142，issue S2，2017.
11.周国武等.α7烟碱型乙酰胆碱受体与炎症调节.现代生物医学进展 2009年14期.
12.刘志刚.α7烟碱型乙酰胆碱受体与炎症的调节作用.中国医药指南2014年03期.
13.Hong Wang et al；.Nicotinic acetylcholine receptor a7 subunit is an essential regulator of inflammation NATURE|VOL 421|23 JANUARY 2003.
14.Stéphanie St-Pierre et al；.Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival.PLOS ONE|DOI：10.1371/journal.pone.0150230 February 29，2016.
15.王廷华等，神经疾病动物模型制备理论与技术，科学出版社2005，ISBN：978-7-03-043784-6.

Claims (8)

  1. 一种治疗病人老年痴呆的方法，通过使用该方法含有的治疗有效量的眼镜蛇科蛇突触后神经毒素单体分子，及其药物可接受载体的组合物，来逆转或减轻老年痴呆症状。
  2. 一种治疗病人老年痴呆的方法，通过使用该方法含有的治疗有效量的眼镜蛇科蛇突触后神经毒素单体分子，及其药物可接受载体的组合物，来抑制或降低老年痴呆患者白细胞介素-1β(interleukin-1β，IL-1β)和肿瘤坏死因子-a(tumor necrosis factors-a，TNF-a)在脑内的表达水平。
  3. 根据权利要求(1-2)以上所述的眼镜蛇科蛇突触后神经毒素单体分子，其特征在于，它的成熟蛋白或多肽是具SEQ ID No.1至SEQ ID No.16所示的氨基酸序列中任何一个的眼镜蛇科蛇突触后神经毒素蛋白或多肽；或分别与SEQ ID No.1至SEQ ID No.16中的眼镜蛇科蛇突触后神经毒素蛋白或多肽具有70％或以上同源性的成熟蛋白或多肽，该成熟蛋白或多肽的功能与SEQ ID No.1至SEQ ID No.16所示的氨基酸序列的眼镜蛇科蛇突触后神经毒素蛋白或多肽功能相同或相似。
  4. 权利要求(1-2)以上所述眼镜蛇科蛇突触后神经毒素单体分子蛋白或多肽，其特征在于，它们可来自于从天然蛇毒中分离提取、或化学多肽合成、或是使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。
  5. 根据权利要求(4)以上所述重组生产的眼镜蛇科蛇突触后神经毒素单体分子蛋白或多肽，根据重组生产方案所用的宿主，本发明的蛋白或多肽可以是糖基化的，或可以是非糖基化的；可以是包含二硫键的，或可以是不包含二硫键的。本发明中所述的蛋白或多肽还可包括或不包括起始的甲硫氨酸残基。
  6. 权利要求(1-5)以上所述眼镜蛇科蛇突触后神经毒素单体分子蛋白或多肽，其特征还在于本发明中所述的蛋白或多肽可包括上述各种眼镜蛇科蛇突触后神经毒素分子蛋白或多肽经过水解或酶解后的片段、用物理和化学方法处理后的衍生物和类似物，他们是基本保持着与上述眼镜蛇科蛇突触后神经毒素分子蛋白或多肽相同的生物学功能或活性的多肽。本发明中所述的片段、衍生物或类似物可以是一个或多个氨基酸残基被取代的多肽或蛋白或在一个或多个氨基酸残基中具有取代基团的多肽或蛋白，或与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇、脂肪链融合所形成的多肽或蛋白)，或附加的氨基酸序列融合到此多肽或蛋白序列而形成的多肽或蛋白。根据本文的描述，这些片段、衍生物和类似物都属于本领域熟练技术人员公知的范围。
  7. 权利要求(1-2)的方法包括静脉注射、肌肉注射、皮下注射、口服、舌下、鼻腔、直肠、真皮内、腹膜内或鞘內给药或经皮给药。
  8. 权利要求(1-2)的方法的眼镜蛇科蛇突触后神经毒素单体分子蛋白或多肽剂量包括从1μg/Kg到350μg/kg每次，给药频率从每天一次到每天多次；或一年多次。
PCT/CN2020/000239 2019-10-11 2020-10-09 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用 WO2021068432A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP20873458.2A EP4046648A4 (en) 2019-10-11 2020-10-09 USE OF THE MONOMERIC MOLECULE OF POST-SYNAPTIC NEUROTOXIN FROM SNAKES OF THE FAMILY ELAPIDAE IN THE TREATMENT OF ALZHEIMER'S DISEASE
CN202080071876.9A CN116997350A (zh) 2019-10-11 2020-10-09 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201910986752.2 2019-10-11
CN201910986752.2A CN111135288A (zh) 2019-10-11 2019-10-11 眼镜蛇神经毒素单体分子在治疗老年痴呆上的应用
CN202010298240.XA CN111467479A (zh) 2020-04-12 2020-04-12 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用
CN202010298240.X 2020-04-12

Publications (2)

Publication Number Publication Date
WO2021068432A1 true WO2021068432A1 (zh) 2021-04-15
WO2021068432A9 WO2021068432A9 (zh) 2021-06-10

Family

ID=75437793

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/000239 WO2021068432A1 (zh) 2019-10-11 2020-10-09 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用

Country Status (3)

Country Link
EP (1) EP4046648A4 (zh)
CN (1) CN116997350A (zh)
WO (1) WO2021068432A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115561341A (zh) * 2022-07-27 2023-01-03 江苏亢钧生物科技有限公司 一种利用高效液相色谱制备眼镜王蛇抗菌肽oh-cath30标准品的方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1064242C (zh) * 1993-12-22 2001-04-11 中国科学院昆明动物所动物毒素蛇资源开发中心 蛇毒替代戒毒,镇痛及抗老年痴呆药物
CN102007142A (zh) * 2008-02-19 2011-04-06 麦欧赛普特股份有限公司 突触后靶向化学去神经剂及它们的使用方法
CN110090296A (zh) * 2018-09-21 2019-08-06 祁展楷 一组与烟碱乙酰胆碱受体具有高度亲和力能快速起效的眼镜蛇神经毒素分子在镇痛上的应用
CN111135288A (zh) * 2019-10-11 2020-05-12 祁展楷 眼镜蛇神经毒素单体分子在治疗老年痴呆上的应用
CN111467479A (zh) * 2020-04-12 2020-07-31 祁展楷 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1163515C (zh) * 2000-04-17 2004-08-25 中国科学院上海生物工程研究中心 新的眼镜蛇神经毒素亲和层析介质和纯化方法
CN1194011C (zh) * 2000-08-03 2005-03-23 上海中科伍佰豪生物工程有限公司 眼镜蛇短链神经毒素的制法和用途
US20040192594A1 (en) * 2002-01-28 2004-09-30 Paul Reid Modified neurotoxins as therapeutic agents for the treatment of diseases and methods of making
US20090209468A1 (en) * 2006-12-19 2009-08-20 Reid Paul F Alpha-neurotoxin proteins with anti-inflammatory properties and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1064242C (zh) * 1993-12-22 2001-04-11 中国科学院昆明动物所动物毒素蛇资源开发中心 蛇毒替代戒毒,镇痛及抗老年痴呆药物
CN102007142A (zh) * 2008-02-19 2011-04-06 麦欧赛普特股份有限公司 突触后靶向化学去神经剂及它们的使用方法
CN110090296A (zh) * 2018-09-21 2019-08-06 祁展楷 一组与烟碱乙酰胆碱受体具有高度亲和力能快速起效的眼镜蛇神经毒素分子在镇痛上的应用
CN111135288A (zh) * 2019-10-11 2020-05-12 祁展楷 眼镜蛇神经毒素单体分子在治疗老年痴呆上的应用
CN111467479A (zh) * 2020-04-12 2020-07-31 祁展楷 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用

Non-Patent Citations (21)

* Cited by examiner, † Cited by third party
Title
ABBAS, MRAHMAN S: "Effects of alpha-7 Nicholic acetylcholine receptor positive allosteric modulator on lipoxycycline-induced neurodegeneration", EUR-J PHARMACOL, vol. 783, 2016, pages 85 - 91
CHUNHONG HUANGANG ET AL.: "Synergistic Strategy of Predomainant Toxins in SNAG Venoms", TOXIAECOLOGY LETTERS, vol. 287, 2018, pages 142 - 154
DATABASE PROTEIN 2 January 2001 (2001-01-02), ANONYMOUS: "short chain neurotoxin precursor [Naja atra]", XP055802051, retrieved from GENBANK Database accession no. AAG43384 *
DATABASE PROTEIN 7 May 1986 (1986-05-07), ANONYMOUS: "Chain A, ALPHA-BUNGAROTOXIN", XP055802054, retrieved from GENBANK Database accession no. PDB: 2ABX_A *
DATABASE UNIPROT 1 May 1992 (1992-05-01), ANONYMOUS: "RecName: Full=Short neurotoxin 1; AltName: Full=Toxin C-6.", XP055802059, retrieved from UNIPROTKB Database accession no. P14613 *
DATABASE UNIPROT 1 May 1992 (1992-05-01), ANONYMOUS: "UniProtKB/Swiss-Prot: P10808.1.", XP055802064, retrieved from UNIPROTKB Database accession no. P10808 *
DATABASE UNIPROT 10 July 2007 (2007-07-10), ANONYMOUS: "RecName: Full=Neurotoxin 3FTx-RI;", XP055802069, retrieved from UNIPROTKB Database accession no. P0C555 *
DATABASE UNIPROT 15 June 2001 (2001-06-15), ANONYMOUS: "Cobrotoxin precursor (CBT) (Short neurotoxin 1)", XP055802048, retrieved from UNIPROTKB Database accession no. P60770 *
GRIFFIN WWS: "FLAVANITY AND NEURODEGENERATION DISEASE", NUTR, vol. 83, no. 2, 2006, pages 470S - 474S
GUOWU ZHOU ET AL.: "ALPHA 7 NICOTINIC ACETYLCHOLINE RECEPTOR AND INFLAMMATION REGULATION", MODERN BIOMEDICAL PROGRESS, 2009
HONG WANG ET AL.: "Nicholic acetatyline receptor A 7 subunit", IS AN ESSENTIAL REGULATOR OF INTAKE NATURE, vol. 421, 23 January 2003 (2003-01-23)
J WHITE HAY ET AL.: "Snakke Neurotoxin", HUMAN NEUROLOGY, 1996
LATISEN ET AL.: "A H Toxin, Synergistic Gism in Snakke Venoms", TOXIN REVIEWS, vol. 35, no. 3-4, pages 165 - 170
MARK HERRE I ET AL.: "Potential Related Biomarkrs in Alizheim 's Disease", J ALZHEIMER'S DISEASE, vol. 8, no. 4, 2005, pages 369 - 375
NAGUIB M ET AL.: "Advances in Neurobiology of the Neuromucular Junction: Impact for the Anesthesiella", J AM, SOC., vol. 96, 2002, pages 202 - 31
PASCALE MARCHOT ET AL.: "The Three-Finger Toxin Fold: A Multifunctional Structural Scaffold Able to Modulate Cholergic Functions", JOURNAL OF NEUROCHEMISTRY, vol. 142, 2017
STINGE PHANIE ST-HERRE ET AL.: "Nicholic Acetatyline-Derived Victima-Derived Punicyte Hay-Derived Haplocyte Production and Survivale", PLOS DELTA, 29 February 2016 (2016-02-29)
TINGHUA WANG ET AL.: "Preparation of Neural Disease Animal Model Preparation Theory and Technology", 2005, SCIENCE PRESS
WANG ET AL.: "Detection of Interleukin-1 and Tumorcellsis Factor-ain Serrum and Celanese Fluid Fluid in Patient with Alzheimer Disease", CHIN J NEUOL, vol. 35, no. 6, 2002, pages 339 - 341
YINGHUA SHEN: "The activation of the nicotinic acetylcholine receptor is compared with the treatment of Alzheimer's disease. the 24th stage", PHARMACEUTICAL JOURNAL OF THE ARMY PHARMACY
ZHIGANG LIU: "Regulation of Alpha 7 nicotinic acetylcholine receptor with inflammation", CHINESE MEDICAL GUIDE, 2014

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115561341A (zh) * 2022-07-27 2023-01-03 江苏亢钧生物科技有限公司 一种利用高效液相色谱制备眼镜王蛇抗菌肽oh-cath30标准品的方法
CN115561341B (zh) * 2022-07-27 2024-04-09 江苏亢钧生物科技有限公司 一种利用高效液相色谱制备眼镜王蛇抗菌肽oh-cath30标准品的方法

Also Published As

Publication number Publication date
WO2021068432A9 (zh) 2021-06-10
CN116997350A (zh) 2023-11-03
EP4046648A1 (en) 2022-08-24
EP4046648A4 (en) 2023-10-18

Similar Documents

Publication Publication Date Title
CN107921085B (zh) 用于治疗衰老相关病症的方法和组合物
EP2066174B1 (en) Compositions containing alpha-1-antitrypsin and methods for use
US9919035B2 (en) Pharmaceutical composition comprising bee venom-phospholipase A2 (bv-PLA2) for treating or preventing diseases related to degradation of abnormal regulatory T cell activity
US10363306B2 (en) Cytokines and neuroantigens for treatment of immune disorders
JP4857396B1 (ja) 融合蛋白質
US20200093866A1 (en) Use of selected single cobrotoxin molecule as an analgesic
WO2021093134A1 (zh) 一组蛇神经生长因子及蛇神经生长因子前体在治疗老年痴呆上的应用
CN113286604A (zh) 治疗炎症性疾病的蛋白质
WO2021068432A1 (zh) 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用
EP3927817A1 (en) New recombinant diamine oxidase and its use for the treatment of diseases characterized by excess histamine
CN111135288A (zh) 眼镜蛇神经毒素单体分子在治疗老年痴呆上的应用
TW201835097A (zh) Apoc-ii擬肽
Desbiens et al. Significant contribution of mouse mast cell protease 4 in early phases of experimental autoimmune encephalomyelitis
CN110772631A (zh) 一组与烟碱乙酰胆碱受体具有高度亲和力能快速起效的眼镜蛇神经毒素分子在镇痛上的应用
US20220251155A1 (en) Composition comprising mls-stat3 for prevention or treatment of immune disease
KR20130132375A (ko) 신장 증후군 및 관련 질환을 치료하는 방법
JPH03178999A (ja) 脳由来の膜蛋白質に対する抗体
WO2021042645A1 (zh) 眼镜蛇神经毒素多肽分子在治疗肾炎蛋白尿上的应用
CN111467479A (zh) 眼镜蛇科蛇突触后神经毒素单体分子在治疗老年痴呆上的应用
US20140147441A1 (en) Compositions containing alpha-1-antitrypsin and methods for use
TW200906433A (en) Therapeutic agent for acute hepatitis or prophylactic and/or therapeutic agent for fulminant hepatitis
KR20070109347A (ko) 인간 ｃｔｒｐ1을 특이적으로 인식하는 단일클론 항체 및이를 생산하는 하이브리도마 세포
WO2023164964A1 (zh) 一种用于治疗脑损伤的基因工程药物及其制备方法
WO2023143256A1 (zh) 激肽或其衍生物在治疗vci、psci或csvd中的应用
CN116693699A (zh) 一种多肽tat-trpv1-c及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20873458

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202080071876.9

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020873458

Country of ref document: EP

Effective date: 20220510