CN111135288A

CN111135288A - Application of cobra neurotoxin monomer molecule in treating senile dementia

Info

Publication number: CN111135288A
Application number: CN201910986752.2A
Authority: CN
Inventors: 祁展楷; 祁·海亚特
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-10-11
Filing date: 2019-10-11
Publication date: 2020-05-12

Abstract

The present invention discloses a group of nerve degenerative disease molecules which can inhibit the increase of the content of the relevant inflammatory factor IL-1 β -a in the hippocampal region of senile dementia rats, and can obviously shorten the latent period of the lens nerve toxin of the senile dementia rats after escaping treatment in the Morris water maze experiment, so as to solve the problems.

Description

Application of cobra neurotoxin monomer molecule in treating senile dementia

Technical Field

The invention relates to a group of cobra neurotoxin monomer molecules capable of inhibiting the level of inflammatory factors in hippocampal brain tissues of rats with senile dementia and improving the learning and memory abilities of rats with senile dementia and a method for treating senile dementia, belonging to the field of biological pharmacy.

Background

Senile dementia, also known as Alzheimer Disease (AD), is an senile disease characterized mainly by progressive memory disorder, judgment and reasoning ability disorder, and movement disorder. Dementia is an acquired and persistent syndrome of intellectual impairment due to brain dysfunction, and the incidence and prevalence of dementia increases with age. With the aging problem of the population becoming more serious, AD has become the 4 th leading cause of death in humans.

The cause of senile dementia is not clear, the pathogenesis is complex, and researchers at home and abroad currently generally consider that the senile dementia is a degenerative change of a nervous system characterized by diffuse atrophy of cerebral cortex and is accompanied with neuronal damage and death. Patients with AD experience diffuse atrophy of the entire brain, and neuropathic conditions such as Senile Plaque (SP) with amyloid protein (amyloid beta protein) as the core and neurofibrillary tangle with tau protein as the core component, which is abnormally over-phosphorylated, appear in the cerebral cortex and hippocampal area. One of the theories is that neurotoxicity of a metabolite of Amyloid Precursor Protein (APP) is a common pathway for various causes of senile dementia, and abnormal increase in the amount thereof in brain tissue may be a major factor in the induction of senile dementia. However, clinical trials based on the above theory have not been successful in the last decade.

There is also evidence to suggest that there is a strong focal inflammatory response in the brain of patients with senile dementia, activated microglia and astrocytes in the vicinity of senile plaques, and studies have found that these activated cells can express a variety of inflammatory cytokines and complement molecules such as interleukin inflammatory factor interleukin-1 β (interleukin-1 β -1 β) and tumor necrosis factor-a (tumor necrosis factor, TNF-a) [1, 2] animal experiments show that the use of amyloid peptide, which is the main component of senile plaques, can induce inflammatory responses in the brain of patients with AD, accompanied by activation and extravasation of leukocytes, and production of inflammatory cytokines.

From the published information, there are some reports on the treatment of senile dementia by cobra venom, but the cobra venom is various, and known components include neurotoxin, cytotoxin, cardiotoxin, nerve growth factor, hemolysin (DLP), CVA protein, membrane active polypeptide, cobra venom factor and the like, and other components such as alkaline phosphomonoesterase, phosphodiesterase, acetylcholinesterase, L-amino acid oxidase, ribonuclease, proteolytic enzyme and the like, and for cobra venom preparations, the mixed toxin may pose a life risk to organisms, which may be a strategy for enhancing toxicity during the evolution process. Synergistic effect exists among different toxins or toxin complexes of the same type in various snake venoms, and main toxins such as phospholipase A2 and toxin III (neurotoxin) play an important role in the synergistic process. [5] It is this synergy that leads to fatal consequences.

Studies on the synergy of snake venom mixtures have found that there is synergy between their toxins in a range of snake species. Two or more toxin components interact directly or indirectly to enhance toxicity to a level that exceeds the sum of their individual toxicities.

From a molecular point of view, synergy may generally exist in two forms (1) intermolecular synergy, causing synergistically increased toxicity when two or more toxins interact with a target on one or more (related) biological pathways (2) supramolecular synergy, creating a toxicity-enhanced complex when two or more toxins interact in a synergistic manner with the same target or when two or more toxins bind to each other [6] an example of intermolecular synergy is the binding of α -neurotoxin from the family Elapidae to other toxins, resulting in synergistically effective toxic effects, leading to flaccid paralysis and respiratory failure in victims and hunters [6] in a study of how Heiman Babyshes venom results in high mortality, scientists found that potassium channel blocking activity was only one of them, and also various pathways, the concern that the combined toxicity induced by different toxins at different organ levels was high mortality [7]

Studom and Botes (1970) showed that, 48 hours after single use, the venom fraction isolated from the relevant Oriental Green Mannheim venom was not lethal, whereas the entire venom could be lethal within 10 minutes when used at similar doses. [8]

Synergistic toxins are known to enhance the toxicity of certain toxins, and these proteins alone are less toxic, but when injected in combination into mice, they exert a strong toxic effect. These toxins are similar in amino acid sequence and number of half cysteines to neurotoxins or cytotoxins, and such synergistic toxins are referred to as synergistic toxins. [9] Cobra venom, as a mixture, has complex components, great quality control difficulty and great possibility of synergy among toxins.

In addition, the skilled artisan has attempted to treat neurological disorders with mixtures of cobra neurotoxins which may be a higher level of safety than snake venoms, because they have minimized a range of toxins such as cardiotoxin, cytotoxin, phospholipase A2, and reduced the toxic potential of mixed venoms due to synergy, but which are still mixtures of several neurotoxins and still have the potential to induce synergy, e.g., a synergy of cobra neurotoxin mixture may lead to respiratory depression. The most effective way to avoid such severe toxic synergy is to replace the mixture with monomer molecules, which are safer than the mixture because they exclude the synergistic effect.

Finally, the cobra neurotoxin polypeptide molecules with a defined amino acid sequence not only have a defined standard in terms of purity and quality control over mixtures, but they can be extracted from natural snake venom, and can also be derived from polypeptide synthesis and recombination, thus potentially offering the possibility of improving product quality and yield.

Disclosure of Invention

The invention discloses a group of cobra neurotoxin monomer molecules which have an inhibiting effect on the increase of the content of a hippocampal-related inflammatory factor IL-1 β -a of a rat with senile dementia, can obviously shorten the escape latency of the rat with senile dementia after treatment in a Morris water maze experiment, and are reported for the first time, and meanwhile, the monomer molecules can avoid the synergistic toxicity effect caused by a common snake venom mixture, and can easily achieve the control of quality and purity in production.

The amino acid sequence (FASTA) of the cobra neurotoxin molecule of the invention is as follows:

SEQ ID No.1

lechnqqssq tptttgcsgg etncykkrwr dhrgyrterg cgcpsvkngi

einccttdrc nn

SEQ ID No.2

lechnqqssq tpttktcsge tncykkwwsd hrgtiiergc gcpkvkpgvn

lnccttdrcn n

SEQ ID No.3

mktllltllv vtivcldlgy tlechnqqss qtptttgcsg getncykkrw

rdhrgyrter gcgcpsvkng ieinccttdr cnn

the invention is further described with reference to the following drawings and detailed description, but the invention is not limited thereto; and equivalents in the art may be substituted for elements thereof without departing from the scope of the invention.

Description of the drawings:

FIG. 1 shows the protein peaks of 12 toxins isolated from crude venom of Chinese cobra by cation exchange through a TSK CM-650(M) column.

FIG. 2 is a line graph showing the correspondence between the escape latencies (Table-1) of the senile dementia rat control group, the neurotoxin-treated group and the young rat control group 3 before and after administration.

Method of implementation

1. Separating and purifying neurotoxin

Separating and purifying the crude Chinese cobra venom, and performing cation exchange on the crude Chinese cobra venom by a TSK CM-650(M) column, wherein the method for separating various toxins comprises the following steps:

a) sample preparation-1 g of Chinese cobra venom was dissolved in 25ml of 0.025 molar ammonium acetate buffer solution with pH6.0, centrifuged at low temperature, and the supernatant was collected;

b) equilibration-the TSK CM-650(M) column was equilibrated with 0.025 molar ammonium acetate solution at PH 6.0;

c) after elution and sample loading, carrying out 2-compartment step gradient elution by using 0.1-0.5 mol and 0.7-1.0 mol of ammonium acetate buffer solution with the pH value of 5.9, and detecting parameters by ultraviolet light: 280 nm; elution flow rate: 48 ml/h;

d) collecting various toxin components according to the recorded spectrogram, and eluting 12 protein peaks (figure-1) from the collected liquid;

after elution, each peak is tested for neurotoxic activity by a rat septal nerve-septal muscle specimen, the septal muscle and the septal nerve are respectively stimulated by two electrodes, the contraction tension of the two physiological recorders is described, and the working conditions of the physiological stimulator are set to be 0.2HZ in frequency, 90V in voltage and 0.5 millisecond in square wave width.

The three peaks with the highest neurotoxicity were purified and desalted by reverse phase high performance liquid chromatography (RP-HPLC) column (4.6X 250mm, VYDAC RP-C8, 5 μm).

2. Determination of amino acid sequence

The amino acid sequence of the single peak of the purified and desalted cobra neurotoxin is determined by an Edaman degradation method and the coverage rate of peptide segments (the sample state is colorless liquid, the environmental temperature is 20 ℃, the environmental relative humidity is 45 percent), and the sequences of the three peaks are respectively determined as follows: SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3. The cobra neurotoxin polypeptide is used for the next treatment of senile dementia rats.

3. Animal model selection

The invention adopts a natural aging cognitive disorder (senile dementia) animal model, and obtains the animal model of senile dementia through natural aging of animals, including aged rats, mice, monkeys and the like, wherein the change of the nervous system of the cognitive disorder and the like of the model is naturally generated and is closer to the real pathophysiological change of AD, Cummings and the like report that A β precipitated plaques exist in the brains of the aged dogs and have corresponding selective behavior ability damage, Higgins and the like report that the aged rats can generate A β which is deposited in the basal forebrain area and have memory impairment.

4. Test animals and groups

The method comprises the steps of adaptively feeding 60 rats aged 21-22 months for 10 days, enabling the animals to take water freely, controlling the room temperature to be 22-25 ℃, controlling the humidity to be 50% -70%, illuminating for 12 hours, and keeping the animals dark for 12 hours.

Rats with cognitive impairment (senile dementia rats) were screened by training of the Morris water maze orientation experiment. The Morris water maze experiment is an experiment for forcing an animal to swim and searching an underwater platform, is mainly used for evaluating the spatial learning and memory capacity of the animal, provides an experiment index which is sensitive and reliable, is simple and convenient to operate, and is a classic experiment for testing and evaluating the senile dementia index of a rat.

The water depth of the water maze is 50cm, the water temperature is controlled at 22-25 ℃, and the center of the water pool is provided with a platform. Milk powder is put into the pool and is fully and evenly mixed until the water is milk white, so that the rat can not identify the position of the platform through vision. Before formal test, the rat is put into water facing the wall of the pool to train the rat to find an escape platform, if the rat is found and stands on the platform for 3 seconds, the rat does not slide off, the training can be stopped, the time and the traveling distance of the rat reaching the platform are recorded, and the rat stays on the platform for 10 seconds, so that the rat learns and memorizes. The person who cannot be found can be guided to the platform after continuously recording for 120s and is left for 30 seconds to train the learning and memory of the person. Training is carried out 4 times every day, the next training is carried out at intervals of 15-30 seconds, water is filled from 4 different directions, and the training is continued for 4 days. And (5) performing formal test on the day, automatically recording the activity condition of the rat by a real-time image system, calculating the average time of the rat passing through the platform (central area) for the first time, and evaluating the learning and memory conditions of the animal. Taking the upper limit value of the range of 99% normal value of the average escape latency of young (4-month old) rats as a standard, and determining the old rats with the escape latency of more than 99% as the known obstacle old rats. Each rat was labeled so that the mean escape latency before dosing for each group of rats could be calculated after completion of the final trial.

After screening out the rats with cognitive impairment, the rats were randomly divided into 2 groups: the control group (non-administration, gavage equal volume of normal saline) and the treatment group (liquid prepared by 45 ug/Kg of neurotoxin SEQ ID No.1, continuous wash stomach administration, 2 times per day, continuous 8 weeks) are used for treating senile dementia; meanwhile, a control group of young rats (no drug administration, gavage of physiological saline of the same volume) is set. During the experiment, 10 rats per group were maintained. And (4) redundant grouping.

5. Behavioural (learning and memory) test

After 8 weeks of dosing, training was continued for another 5 days, recording the average time each group of animals looked for the platform in water each day, and the Morris water maze test was performed directly on day 6. The time for each group of animals to find the platform in the water was determined. The administration of the drug is continued during the training.

6. Inflammatory factor assay

After the experiment is finished, 10% chloral hydrate is selected for abdominal anesthesia, heads are cut off and killed after the anesthesia is finished according to the dosage of 0.3ml/100g, then hippocampal tissue samples are quickly separated out, precooled physiological saline at 4 ℃ is added according to 1g of hippocampal tissue samples, centrifugation is carried out at 13500 r/min, low-temperature homogenization time is 10 s/time, interval is 30s, continuous 4 times are carried out, 10% tissue homogenate is prepared, then centrifugation is carried out at low temperature (4 ℃, 3000 r/min) for 15min, supernate is taken, storage is carried out at minus 40 ℃, and the content levels of inflammatory factors interleukin-1 β (interleukin-1 β -1 β) and tumor necrosis factors-a (tumor necrosis factors, TNF-a) in the hippocampal region are operated according to the kit specification steps by an ELISA method.

7. Results of the experiment

The average escape latency of the senile dementia rat control group, the cobra neurotoxin treatment group and the young rat control group is compared and shown in the table-1.

TABLE-1

Before administration, there was no significant difference between the senile dementia rat control group and the neurotoxin treatment group, ### # indicates that they have significant difference with the young rat control group, and P is less than 0.001.

After 8 weeks of administration, performing Morris water maze positioning experiment training again, wherein from the first day to the sixth day, the senile dementia rat control group and the neurotoxin treatment group have significant difference every day, and P is less than 0.01 when the senile dementia rat control group and the neurotoxin treatment group are expressed as the first day to the second day; and P < 0.001 on days three to six.

# indicates that they are significantly different from the control group of young rats, with P < 0.001 from the first to sixth days.

The above data show that: the cobra neurotoxin can improve the average escape latency of a rat group with senile dementia and improve the learning and memory ability of the rat group with senile dementia.

The levels of interleukin-1 β (interleukin-1 β -1 β) and TNF-a (tumor necrosis factor-a) in each group are compared in Table-2

TABLE-2

The # indicates that P is less than 0.001 when comparing IL-1 β and TNF-a of the senile dementia control group and the young control group, and P is less than 0.01 when comparing IL-1 β and TNF-a of the senile dementia control group and the neurotoxin treatment group.

The above examples are only for illustrating the technical idea and features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the contents of the present invention and to implement the present invention, and not to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Reference：

1.Mark RE et al；Potential inflammatory biomarkers in alzheimer'sdisease.J Alzheimer's disease 2005 8(4)369-375

2.Griffin WS.Inflammation and Neurodegenerative disease.Am J ClinNutr，2006 83(2)：470s-474s

3.Wang et al；Detection of interleukoin-1 and tumor necrosis factor-ain serum and celebrospinal fluid in patient with Alzheimer disease Chin JNeurol，2002 35(6)339-341

4. Shengluhua, agonism of nicotinic acetylcholine receptor and treatment of Alzheimer's disease, Release Finder pharmacopoeia Vol.24, phase 2

5.Shengwei Xiong，Chunhong Huang.Synergistic strategies of predominanttoxins 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.Anil Kumar，Varun Gupta.Neurological Implications of Dendrotoxin：AReview.EC PHARMACOLOGY AND TOXICOLOGY.May 25，2018

8.Strydom DJ，Botes DP.Snake venom toxins-I.Preliminary studies on theseparation of toxins of elapidae venoms.Toxicon 8：203-9.1970

9.Joubert，F.J.，Taljaard，N，Snake venoms.The amino-acid sequence ofprotein S2C4 from Dendroaspis jamesoni kaimosae(Jameson's mamba)venom.HoppeSeylers.Z.Physiol.Chem.360，571-580.1979

Claims

1. A method of treating Alzheimer's disease in a patient by reversing or alleviating the symptoms of Alzheimer's disease using a composition comprising a therapeutically effective amount of a pharmaceutically acceptable carrier for the cobra neurotoxin molecule.

2. A method for treating senile dementia in a patient, which comprises inhibiting or reducing the expression level of interleukin-1 β (interleukin-1 β -1 β) and tumor necrosis factor-a (TNF-a) in the body of the patient suffering senile dementia by using a composition of a pharmaceutically acceptable carrier containing a therapeutically effective amount of cobra neurotoxin molecules.

3. The cobra neurotoxin molecule according to claims (1-2) above, characterized in that it is a cobra neurotoxin polypeptide having any of the amino acid sequences shown in SEQ ID No.1 to SEQ ID No. 3; or a polypeptide having 70% or more homology with the cobra neurotoxin polypeptide of SEQ ID No.1 to SEQ ID No.3, respectively, and the function of the polypeptide is the same as or similar to the function of the cobra neurotoxin polypeptide of the amino acid sequence shown in SEQ ID No.1 to SEQ ID No. 3.

4. Cobra neurotoxin molecule polypeptides according to claims (1-2) above, characterized in that they can be derived from natural snake venom by isolation, or by chemical polypeptide synthesis, or by production using recombinant techniques from prokaryotic or eukaryotic hosts (e.g. bacteria, yeast, higher plant, insect and mammalian cells).

5. The recombinantly produced cobra neurotoxin molecule polypeptide according to claim (4) above, which may be glycosylated or may be non-glycosylated depending on the host used in the recombinant production scheme; may or may not contain disulfide bonds. The polypeptides described in the present invention may or may not also include an initial methionine residue.

6. The cobra neurotoxin molecule polypeptide of claims (1-5), further characterized in that said polypeptide of the present invention can include hydrolyzed or enzymatically hydrolyzed fragments, physically and chemically treated derivatives and analogs of the various cobra neurotoxin molecule polypeptides described above, which are polypeptides that retain substantially the same biological function or activity as the cobra neurotoxin molecule polypeptide described above. The fragment, derivative or analogue of the invention may be a polypeptide or protein in which one or more amino acid residues are substituted or a polypeptide or protein having a substituent group in one or more amino acid residues, or a polypeptide or protein fused to another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol, a polypeptide or protein formed by fusion of fatty chains), or an additional amino acid sequence to the sequence of the polypeptide or protein. Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the description herein.

7. The method of claim (1) comprising intravenous injection, intramuscular injection, subcutaneous injection, oral administration, sublingual, nasal, rectal, intradermal, intraperitoneal or intrathecal administration or transdermal administration.

8. The method of claim (1) wherein the dosage of cobra neurotoxin molecule polypeptide comprises from 1 μ g/Kg to 350 μ g/Kg per dose at a frequency of from once a day to multiple times a day; or more than once a year.