Application of adenosine in treatment of snake venom neurotoxicity

Abstract

The invention provides a method for treating snake venom by adenosine or a pharmaceutical composition containing adenosine. The method comprises the step of applying an effective dose of adenosine or a drug containing adenosine. The adenosine or the pharmaceutical composition containing the adenosine can be applied to snake wound first aid in primary hospitals, and a remarkable curative effect is expected to be achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method and application of adenosine in treating snake venom. Background Art

[0002] Bungarus multicinctus belongs to the animal kingdom, phylum chordata, class reptiles, order squamata, family elapidae, genus krait. The main components of Bungarus multicinctus toxins are proteins and polypeptides, among which the toxic components are mainly neurotoxins among various toxic polypeptides, including α-bungarotoxin (α-BGT), β-bungarotoxin (β-BGT), κ-bungarotoxin (κ-BGT) and some enzymes such as phospholipase A and thrombin factor. The main symptoms of being bitten by Bungarus multicinctus are wound pain, local swelling, drowsiness, motor nerve disorders, ptosis, mydriasis, local weakness, oropharyngeal paralysis, stuttering, salivation, nausea, vomiting, coma, dyspnea, respiratory failure, and death within 8 to 72 hours. If not treated in time, the mortality rate is almost 100%.

[0003] Anti-snake venom serum is currently the first choice for clinical treatment of snake bite poisoning. Its components are polyclonal immunoglobulins that are highly immunized with large animal plasma / serum. However, anti-snake venom serum still has many disadvantages. First, large-dose injection of heterologous immunoglobulins can cause a variety of adverse reactions, such as serum sickness and early adverse reactions, and even severe allergic reactions; in addition, anti-snake venom serum must be injected intravenously by professional medical personnel in medical institutions, and it relies on cold chain transportation and storage, which is expensive. Therefore, many remote areas and rural areas lack spare serum, and patients bitten by snakes in the field and in rural areas cannot receive timely treatment after being bitten, which may lead to serious complications or even death. Taken together, these limitations highlight the urgency and necessity of developing well-tolerated, accessible, affordable and sustainable snake bite treatment methods.

[0004] Pimpinella diversifolia DC. is a plant of the genus Anise of the family Apiaceae. It is also known as snake retreat. As a wild plant, it is widely distributed and rich in resources. It is one of the commonly used herbal medicines and wild vegetables in the folk. The whole plant is used as medicine, which has the effects of warming the middle and dispelling cold, clearing the lungs and removing phlegm, promoting blood circulation and relieving pain, detoxifying and reducing swelling. Pimpinella diversifolia has a long history of treating snake poisoning in folk medicine, but its efficacy has not been verified by rigorous scientific experiments, and its mechanism of action is still unclear.

[0005] Adenosine is an endogenous purine nucleoside, widely distributed in the human body. It participates in energy balance through changes in content and plays an important physiological function in the cardiovascular system and nervous system. At the same time, adenosine is also a natural estrogen, widely acts on neurotransmitters, and is an important upstream neuromodulator in the brain. As a natural estrogen, adenosine has the characteristics of mild action and few adverse reactions compared with synthetic estrogen. Adenosine injection has no allergic, hemolytic, vascular irritation and other effects. As an endogenous neuroprotectant, adenosine has neuromodulatory and protective effects on ischemia, hypoxia and stroke. Studies have reported that adenosine can reduce calcium influx and inhibit the presynaptic release of excitatory neurotransmitters (mainly glutamate) after cerebral ischemia, and prevent nerve cell damage after cerebral ischemia by reducing cell metabolism, energy consumption and brain temperature. It can also protect the nervous system by increasing cerebral blood flow and inhibiting the production of inflammatory factors by immune cells, and plays an important role in regulating inflammation, immunity and tissue repair. There are currently no public reports on adenosine and similar substances in snake venom research. Summary of the invention

[0006] The present invention finds that adenosine can reverse the toxic reaction of snake venom.

[0007] In a first aspect, the present invention provides use of adenosine or a pharmaceutical composition comprising adenosine in preparing a kit for treating snake venom or alleviating poisoning symptoms.

[0008] The present invention also provides the use of adenosine in preparing medicine for treating snake venom or alleviating poisoning symptoms.

[0009] In one or more embodiments, the intoxication symptoms are intoxication symptoms caused by postsynaptic neurotoxicity.

[0010] In one or more embodiments, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom. Preferably, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Cobra venom, Taipan venom.

[0011] In one or more embodiments, the poisoning symptoms are snake envenomation symptoms. Preferably, the poisoning symptoms are snake envenomation symptoms caused by postsynaptic neurotoxicity.

[0012] In one or more embodiments, the poisoning symptoms include one or more selected from the following: wound pain, local swelling, drowsiness, motor nerve disorder, ptosis, mydriasis, local weakness, oropharyngeal paralysis, stuttering, salivation, nausea, vomiting, coma, dyspnea, respiratory failure, quadriplegia. Preferably, the poisoning symptoms include dyspnea and quadriplegia.

[0013] In one or more embodiments, the chemical formula of adenosine is as shown below:

[0014]

[0015] A second aspect of the present invention provides a kit for treating snake venom, comprising instructions for use for treating snake venom, and (1) adenosine or (2) a pharmaceutical composition comprising adenosine.

[0016] In one or more embodiments, the instructions for use include methods and dosages for administering adenosine to treat snake venom.

[0017] In one or more embodiments, the administration method is selected from: intravenous drip, intramuscular injection, intraperitoneal injection. Preferably, the administration method is intraperitoneal injection.

[0018] In one or more embodiments, the administered dose is a dose of adenosine of 6-25 mg / kg, preferably 12.5-25 mg / kg, more preferably 25 mg / kg.

[0019] In one or more embodiments, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom. Preferably, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Cobra venom, Taipan venom.

[0020] A third aspect of the present invention provides a method for treating snake venom, the method comprising administering an effective dose of adenosine or a pharmaceutical composition comprising adenosine.

[0021] In one or more embodiments, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom. Preferably, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Cobra venom, Taipan venom.

[0022] In one or more embodiments, the administration is selected from: intravenous drip, intramuscular injection, intraperitoneal injection. Preferably, the administration is intraperitoneal injection.

[0023] In one or more embodiments, the effective dose is a dose of adenosine of 6-25 mg / kg, preferably 12.5-25 mg / kg, more preferably 25 mg / kg.

[0024] The present invention also provides a method for reducing the likelihood of death or injury in a mammal from snake envenomation, the method comprising administering to a patient suspected of having or known to have suffered snake envenomation, the method comprising administering an effective amount of adenosine or a pharmaceutical composition comprising adenosine after the envenomation but before local, regional or systemic envenomation results in damage.

[0025] In one or more embodiments, the mammal is a mouse.

[0026] In one or more embodiments, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom. Preferably, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Cobra venom, Taipan venom.

[0027] In one or more embodiments, the administration is selected from: intravenous drip, intramuscular injection, intraperitoneal injection. Preferably, the administration is intraperitoneal injection.

[0028] In one or more embodiments, the effective dose is a dose of adenosine of 6-25 mg / kg, preferably 12.5-25 mg / kg, more preferably 25 mg / kg.

[0029] Advantages of the present invention:

[0030] 1. Adenosine injection is easy to obtain, easy to store (room temperature storage, 24 months), and cheap;

[0031] 2. As a commonly used drug in clinical practice, expanding its original clinical indications is a new use of an old drug, and its safety has been verified in previous clinical studies;

[0032] 3. It can be used in the emergency treatment of snake bites in primary hospitals and is expected to achieve significant therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :The marketed drug adenosine injection can significantly antagonize the toxicity of Bungarus cobra venom and improve the survival rate of mice after poisoning. A. Four groups of mice were used, each with 10 mice, and received intraperitoneal injection of 3.5 times LD50 Bungarus cobra venom (the venom was diluted with PBS solution, and the final volume was 100μL) and intraperitoneal injection of different concentrations of adenosine (diluted with PBS solution, the final system was 200μL) or 200μL of simple PBS solution. Among them, all mice injected with Bungarus cobra venom and 200μL PBS solution died within 5 hours, and the 48-hour survival rate of mice injected with Bungarus cobra venom and 25mg / kg adenosine injection reached 100%; the 48-hour survival rate of mice injected with Bungarus cobra venom and 12.5mg / kg adenosine injection reached 80%, and the 48-hour survival rate of mice injected with Bungarus cobra venom and 6.25mg / kg adenosine injection reached 70%. The above results were verified by repeated experiments more than three times. B. Mice injected with Bungarus cobra venom may experience obvious poisoning symptoms such as dyspnea and quadriplegia, and eventually die. Only a portion of mice injected with Bungarus cobra venom and 25 mg / kg adenosine injection showed poisoning symptoms corresponding to grade 1-2 severity, and these poisoning symptoms gradually alleviated over time.

[0034] Figure 2 :The change of chicken digastric muscle tension. Adenosine can reverse the decrease of muscle strength caused by snake venom. DETAILED DESCRIPTION

[0035] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0036] The present invention is the first to discover that intraperitoneal injection of adenosine can completely reverse the toxic reaction of Bungarus cobra venom, and can achieve 100% survival in mouse experiments, with high clinical promotion and application value.

[0037] The term "subject" or "patient" may refer to patients or other animals, especially mammals, such as humans, mice, rats, dogs, monkeys, cows, horses, etc., who receive the pharmaceutical composition of the present invention to treat, prevent, improve and / or alleviate the snake venom of the present invention.

[0038] The term "adenosine" has been used in clinical medicine for angina pectoris, myocardial infarction, myocarditis and cardiogenic shock; it has a certain effect on improving palpitations, shortness of breath, chest tightness and other symptoms of rheumatic heart disease; it can improve the efficacy of acute leukemia combined with chemotherapy, and can also be used to induce remission of acute leukemia. In addition, it also has a certain effect on chronic bronchitis in the elderly, various hepatitis and psoriasis.

[0039] Dosage

[0040] Intramuscular injection, 20 mg at a time, dissolved in 2 ml 0.9% sodium chloride injection, twice a day. Intravenous injection, 20 mg at a time, dissolved in 20 ml 0.9% sodium chloride injection, bolus injection, twice a day. Intravenous drip, 40 mg of this product dissolved in 250-500 ml 5% glucose injection, once a day. Coronary heart disease is treated for 15 days, and can be used continuously for 2-3 courses; leukemia is treated for one month; psoriasis is treated for 2-3 weeks, which can be extended to 4-7 weeks, and the daily dosage can be increased to 60-80 mg.

[0041] Pharmaceutical composition

[0042] The present invention first provides a pharmaceutical composition for treating snake venom, wherein the pharmaceutical composition comprises adenosine, and preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Usually, the pharmaceutical composition is administered for therapy.

[0043] As described herein, pharmaceutical compositions can be formulated by any method known or developed in the art of pharmacology, including but not limited to contacting the active ingredient (eg, adenosine) with the excipients or other auxiliary ingredients and dividing or packaging the product into dosage units.

[0044] The pharmaceutical composition may further comprise saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, nanoparticle mimetics, or combinations thereof.

[0045] The pharmaceutical composition according to the present invention can be prepared, packaged and / or sold in bulk in a single unit dose and / or in multiple single unit doses. The amount of the active ingredient is generally equal to the dosage of the active ingredient to be administered to the subject and / or a convenient ratio of such dosage, such as half or one-third of such dosage.

[0046] As used herein, the term "pharmaceutically acceptable excipients" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents, excipients, stabilizers, preservatives, viscosity inducing agents. Examples of excipients are found in Martin (1975) Remington's Pharm. Sci. 15th Edition (Mack Publ. Co., Easton).

[0047] The pharmaceutical compositions of the present invention may include one or more excipients, non-limiting examples of which include solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, or combinations thereof.

[0048] In some embodiments, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent that can reduce or eliminate damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose and / or mannitol.

[0049] Methods and uses

[0050] The present invention provides a method of treating snake venom comprising administering to a subject a therapeutically effective amount of adenosine or a pharmaceutical composition described herein.

[0051] The treatment method herein reverses the neurotoxicity of snake venom by intravenous drip, intramuscular injection, or intraperitoneal injection, preferably intraperitoneal injection.

[0052] In one or more embodiments, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom. Preferably, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Cobra venom, Taipan venom.

[0053] As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition being treated (e.g., symptoms of snake venom, including but not limited to wound pain, local swelling, lethargy, motor disorders, ptosis, mydriasis, local weakness, oropharyngeal paralysis, stuttering, salivation, nausea, vomiting, coma, dyspnea, respiratory failure, quadriplegia). Exemplarily, the symptoms of snake venom in this application are preferably dyspnea and quadriplegia. Treatment may optionally include a reduction or alleviation of the symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily mean complete eradication or cure of the disease or condition or its associated symptoms. As used herein, "treatment" of a disease in a subject refers to (1) inhibiting snake venom neurotoxicity or preventing its development; or (2) ameliorating or causing regression of symptoms of snake venom poisoning. As understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. Beneficial or desired results may include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the extent of a condition (including a disease), a stable (i.e., not worsening) state of a condition (including a disease), a delay or slowing of a condition (including a disease), progression, improvement or palliation of a condition (including a disease), state, and remission (whether partial or total).

[0054] A "subject" or "patient" for diagnosis or treatment is a cell or an animal, such as a mammal or a human. The subject is not limited to a particular species and includes non-human animals receiving diagnosis or treatment and those receiving infection or animal models, including but not limited to simian, mouse, rat, canine or lagomorphic species, as well as other livestock, sports animals or pets. In some embodiments, the subject or patient is a human.

[0055] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve the desired effect. In the case of therapeutic or preventive applications, the effective amount will depend on the type and severity of the condition in question and the characteristics of the individual subject or patient, such as general health, age, sex, body weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, the effective amount is an amount sufficient to cause restoration of partial or full function of a defective gene in a subject or patient. In other embodiments, the effective amount of adenosine is an amount sufficient to cause a subject or patient to alleviate snake venom. A skilled technician will be able to determine an appropriate amount based on these and other factors.

[0056] In some embodiments, the effective amount will depend on the size and nature of the application in question. It also depends on the nature and sensitivity of the target subject and the method used. Those skilled in the art will be able to determine the effective amount based on these and other considerations. According to an embodiment, the effective amount may include, consist essentially of, or consist of one or more administrations of the composition.

[0057] As used herein, the term "administering" is intended to mean delivering a substance to a subject or patient, such as an animal or a human. Administration can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be performed, and dosage levels and patterns are selected by the treating physician, or in the case of pets and other animals, by the treating veterinarian. Herein, the administration is intraperitoneal administration.

[0058] Dosage and Administration

[0059] Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, and the subject being treated. Single or multiple administrations may be performed, and the dosage level and mode are selected by the treating physician. The dosage may be affected by the route of administration. Suitable dosage formulations and methods for administering the agents are known in the art. A non-limiting example of such a suitable dosage may be 6-25 mg / kg per administration of adenosine injection, preferably, the dosage is 12.5-25 mg / kg, and more preferably, the dosage is 25 mg / kg. Exemplarily, the main component of the adenosine injection described in the present application is adenosine, with a molecular formula of C 10 H 13 N5O4, auxiliary material is sodium chloride. Specification is 30ml, 90mg adenosine.

[0060] The administration of adenosine or the pharmaceutical composition of the present invention includes injection, such as intravenous drip, intramuscular injection, intraperitoneal injection. Preferably, the injection is intraperitoneal injection.

[0061] Administration of adenosine or pharmaceutical compositions of the invention can be achieved in one dose, continuously or intermittently throughout the course of treatment. In some embodiments, adenosine or pharmaceutical compositions of the invention are administered intraperitoneally by injection. Adenosine and pharmaceutical compositions of the invention can be administered in combination with other known treatments for the conditions being treated.

[0062] Reagent test kit

[0063] In some embodiments, the adenosine or pharmaceutical compositions described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. In some embodiments, the kits of the invention include any of the adenosine or pharmaceutical compositions described herein.

[0064] In some embodiments, the kit also includes instructions for use. Specifically, such a kit can include one or more reagents as described herein, and instructions for the intended application and correct use of these reagents. In some embodiments, the kit can include instructions for one or more components of a mixed kit and / or separation and mixed samples and applied to a subject or patient. In some embodiments, the reagent in the kit is a pharmaceutical preparation and dosage suitable for a specific application and the method of administration of the reagent. Kits for research purposes can contain components of appropriate concentration or amount for carrying out various experiments.

[0065] Kits can be designed to facilitate the use of the methods described herein, and can take many forms. Where applicable, each composition of the kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions can be constructible or otherwise processable (e.g., processed into active forms), for example, by adding suitable solvents or other substances (e.g., water or cell culture media), which may or may not be provided with the kit. In some embodiments, the composition can be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde. In some embodiments, the preservation solution contains a certain amount of metalloproteinase inhibitors.

[0066] In some embodiments, the test kit contains any one or more components described herein in one or more containers. Therefore, in some embodiments, the test kit can include a container for holding a reagent as described herein. The reagent can be in the form of a liquid, gel or solid (powder). The reagent can be aseptically prepared, packaged in a syringe and transported frozen. Alternatively, they can be contained in a vial or other container for storage. The second container can have other reagents prepared aseptically. Alternatively, the test kit can include a premixed active agent and transported in a syringe, vial, tube or other container. The test kit can have one or more or all components required for the reagent to be applied to the subject, such as a syringe, a local application device or an IV needle tube and a bag.

[0067] It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are conventional materials and methods in the art unless otherwise specified.

[0068] Example

[0069] Embodiment 1:

[0070] Fresh Pimpinella heterophylla was cleaned and water extracted using a Chinese herbal medicine extractor (developed by Zhiyuan Lingcui Intelligent Technology Co., Ltd. and Guangdong University of Chinese Medicine). 550g of freshly collected Pimpinella heterophylla was washed and cut into small pieces, put into a medicine bag of suitable size, and then placed in the stainless steel pot of the extractor, and 300ml of double distilled water was added. The machine performs vacuum negative pressure suction and physical squeezing at 85 degrees Celsius, which is completed in about 35 minutes, and the extract is collected. After the extraction is completed, 300ml of double distilled water is added for extraction again. The two extracts are mixed together and concentrated by vacuum decompression using a rotary evaporator RE-201D to concentrate the extract to 40 ml.

[0071] The next step is to identify and analyze the metabolites of the water extract of Pimpinella heterophylla. First, the metabolites in the concentrated extract are extracted, and then the processed samples are analyzed by liquid chromatography-mass spectrometry to obtain the original RAW file of the mass spectrum. After analysis by the software MS-DIAL 4.70, the data is matched to obtain the identification results. The wiff file collected by the mass spectrometry is analyzed by the software MS-DIAL 4.70 (MS-DIAL: data independent MS / MS deAvolution for comprehensive metabolome analysis. (Nature Methods, 12, 523-526, 2015) for preprocessing, including peak extraction, denoising, deconvolution, peak alignment, and export of a three-dimensional data matrix in CSV format (raw data matrix). The extracted peak information was compared with the database, and the three libraries of MassBank, Respect, and GNPS (a total of 14,951 records) were searched in their entirety. This three-dimensional matrix includes the following information: sample information, retention time, mass-to-nuclear ratio, and mass spectrometry response intensity (peak area). The identification results showed that the extract contained adenosine, chlorogenic acid and other substances. We tested the main components (first intraperitoneally injected mice with different types of snake venom, and then immediately intraperitoneally injected these different drug components (the drug components were also diluted with PBS as injections), and observed the survival of the mice), and found that adenosine had the strongest inhibitory effect on nerve snake venom (Table 1). (Adenosine: adenosine)

[0072] Table 1: Effects of different components of Pimpinella heterophylla on the survival of mice treated with various neurotoxic snake venoms.

[0073]

[0074] 25mg / kg adenosine is effective against 3.5 times the median lethal dose (LD50) of Bungarus cobra venom, Taipan venom, and Bungarus bungarus venom, among which the best effect is against Bungarus cobra venom.

[0075] Embodiment 2:

[0076] The animal model used in the experiment was C57 mice (8 weeks old, weighing 20-25 grams), provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Ten mice were kept in each cage, with sufficient food and pure water supply. The experimental mice were raised in a constant temperature and humidity environment (20-21°C, about 50% humidity) and followed a 12-hour light and dark cycle. The experimental design was based on the 3R-modified WHO recommended protocol, and the mice were randomly assigned to different groups and experimental observations were performed.

[0077] The freeze-dried powder of the venom of the snake was provided by Shanghai Sailun Biotechnology Co., Ltd. The freeze-dried powder of the venom of the Bungarus venom was provided by Jiangxi Five-step Snake Biotechnology Co., Ltd.; the freeze-dried powder of the venom of the cobra was provided by Shanghai Puzhen Biotechnology Co., Ltd.; the freeze-dried powder of the venom of the Taipan snake was provided by Latoxan Biotechnology Co., Ltd. in France; and the freeze-dried powder of the venom of the king cobra was provided by Jiangxi Five-step Snake Biotechnology Co., Ltd.

[0078] The mice were intraperitoneally injected with 3.5 times the LD50 of Bungarus cobra venom (LD50 was calculated experimentally. According to the guidelines recommended by the World Health Organization, the applicant used the mouse tail vein injection method to dilute the venom with 100 μL PBS solution to obtain different concentrations. Six male C57 mice (weighing 20-25 grams) were used in each group. After 24 hours, the death was observed and the number of surviving mice was recorded. Based on the survival percentage, the median lethal dose (LD50) of Bungarus cobra venom and its 95% confidence interval were calculated using the probability analysis method. After testing, the median lethal dose (LD50) of Bungarus cobra venom toxin was 1.9 micrograms / 20 grams of mouse). At the same time, adenosine injection commonly used in clinical practice (Zhongmei Huadong, specification 30ml:90mg (30ml contains 90mg adenosine, the instructions state that the main component of the injection is adenosine, and the solvent is saline (sodium chloride)) was intraperitoneally injected. It was found that adenosine injection can significantly improve the survival rate of mice (such as Figure 1 The control group (the control group was injected with snake venom + solvent) all died within 5 hours, the 12.5mg / kg treatment group had a survival rate of 80% within 48 hours of observation, and the 25mg / kg treatment group had a survival rate of 100% within 48 hours of observation. The silver ring nerve snake venom can cause breathing difficulties, quadriplegia, and even death in mice. Adenosine can significantly reduce the neurotoxicity of mice and completely eliminate the toxicity after 13 hours.

[0079] Table 2: Scoring criteria for neurotoxic effects on mice

[0080]

[0081] Embodiment 3:

[0082] In order to observe the mechanism by which adenosine antagonizes the toxicity of snake venom, the muscle tissue used included neuromuscular junctions to observe whether adenosine acts directly on the muscle or on the synapses.

[0083] Bungarus cobra venom contains presynaptic neurotoxins (β-bungarotoxin) and postsynaptic neurotoxins (α-bungarotoxin). We investigated the neurotoxic mechanism of Bungarus cobra venom and the mechanism of action of adenosine on its therapeutic effect. Bungarus cobra venom (3 μg / ml) completely inhibited indirect twitches in chicken digastric muscle specimens and attenuated muscle contraction responses induced by exogenous acetylcholine (ACh) and carbachol (CCh), but had no significant effect on muscle contraction responses induced by potassium chloride (KCl). This suggests that Bungarus cobra venom has postsynaptic neurotoxicity but no obvious myotoxicity. The pre-addition of adenosine (30 μg / ml) significantly reduced the inhibitory effect of Bungarus cobra venom on the contraction responses of ACh and CCh. Our results show that adenosine can significantly antagonize the postsynaptic neurotoxicity of Bungarus cobra venom.

[0084] Preparation of chicken digastric muscle specimens: Male chicks aged 4-10 days after hatching were used, and they were bled to death after inhaling carbon dioxide. The hair on the back of the neck was removed, and a skin incision was made along the midline from the occipital region with surgical scissors, and the skin was cut downward to the base of the neck. It can be seen that there is a digastric muscle on both sides of the midline, which is closely attached to the skin, and one end is connected to the nuchal bone. The muscle is gently lifted, and the entire chicken digastric muscle is gradually separated to the base of the neck with a glass needle. The chicken digastric muscle specimen is cut at the base of the neck to obtain a chicken digastric muscle specimen. The chicken digastric muscle specimen is mounted on a bracket in a 50 ml tissue bath, and a static tension of 1 gram is maintained. The temperature was set at 34°C, and the experimental environment was maintained at 95% oxygen and 5% carbon dioxide. The components of the tissue fluid included: 118.4mM NaCl, 4.7mM KCl, 1.2mM MgSO4, 1.2mM KH2PO4, 2.5mM CaCl2, 25mM NaHCO3 and 11.1mM glucose. The motor nerve was stimulated (0.1Hz; 0.2ms) with a stimulator at maximum voltage (10-20V) to induce indirect contraction (i.e., nerve-mediated contraction). Addition of d-TC (10μM) blocked muscle contraction, demonstrating that electrical stimulation was effective through selective stimulation of the nerve. Subsequently, the specimens were repeatedly rinsed with saline to restore the contractile response to nerve stimulation. In the absence of nerve stimulation, the contractile response of the tissue to exogenous acetylcholine (Ach; ​​1mM 30 seconds), carbachol (CCh; 20μM 60 seconds) and potassium chloride (KCl; 40mM 30 seconds) was detected. Nerve stimulation was restarted at least 30 min before the addition of venom or adenosine. Line graphs of tension changes at 2-min time points were plotted and the results were analyzed using Graphpad Prism 10. Figure 2 shown).

Claims

1. Use of adenosine or a pharmaceutical composition comprising adenosine in the preparation of a drug or a kit for treating snake venom or alleviating poisoning symptoms.

2. The use according to claim 1, characterized in that The snake venom is selected from one or more of the following: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom, more preferably, the snake venom is selected from Bungarus bungarus venom, Cobra venom, Taipan venom.

3. The use according to claim 1, characterized in that The poisoning symptoms are poisoning symptoms caused by postsynaptic neurotoxicity, preferably, the poisoning symptoms are snake venom poisoning symptoms, more preferably, the poisoning symptoms are snake venom poisoning symptoms caused by postsynaptic neurotoxicity.

4. The use according to claim 1 or 3, characterized in that The poisoning symptoms include one or more selected from the following: wound pain, local swelling, drowsiness, motor nerve disorder, ptosis, mydriasis, local weakness, oropharyngeal paralysis, stuttering, salivation, nausea, vomiting, coma, dyspnea, respiratory failure, quadriplegia, preferably, the poisoning symptoms include dyspnea and quadriplegia.

5. The use according to claim 1, characterized in that The chemical formula of adenosine is shown below:

6. A kit for treating snake venom, comprising instructions for use for treating snake venom, and (1) adenosine or (2) a pharmaceutical composition comprising adenosine, preferably, the instructions for use comprise a method for administering adenosine for treating snake venom and an administration dosage.

7. The kit according to claim 6, characterized in that The administration method is selected from: intravenous drip, intramuscular injection, and intraperitoneal injection. Preferably, the administration method is intraperitoneal injection. The administration dose is an adenosine dose of 6-25 mg / kg, preferably 12.5-25 mg / kg, more preferably 25 mg / kg.

8. The kit according to claim 6, characterized in that The snake venom is selected from the group consisting of: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom, more preferably, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Cobra venom, Taipan venom.

9. A method for treating snake venom, the method comprising administering an effective dose of adenosine or a pharmaceutical composition comprising adenosine, preferably, further comprising one or more of the following features: The effective dose is a dose of adenosine, which is 6-25 mg / kg, preferably 12.5-25 mg / kg, and more preferably 25 mg / kg. The snake venom is selected from the group consisting of: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom, preferably, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Cobra venom, Taipan venom, The administration is selected from the group consisting of: intravenous drip, intramuscular injection, and intraperitoneal injection. Preferably, the administration is intraperitoneal injection.

10. A method of reducing the likelihood of death or injury from snake envenomation in a mammal, the method comprising administering to a patient suspected of or known to have been envenomed, the method comprising administering an effective amount of adenosine or a pharmaceutical composition comprising adenosine after envenomation but before local, regional or systemic envenomation causes damage, preferably further comprising one or more of the following features: The mammal is a mouse, The snake venom is selected from the group consisting of: Bungarus bungarus venom, Bungarus venom, Cobra venom, King cobra venom, Taipan venom, more preferably, the snake venom is selected from the group consisting of: Bungarus bungarus venom, Cobra venom, Taipan venom, The administration is selected from: intravenous drip, intramuscular injection, intraperitoneal injection, more preferably, the administration is intraperitoneal injection, The effective dose is a dose of adenosine, which is 6-25 mg / kg, more preferably, 12.5-25 mg / kg, and further preferably, 25 mg / kg.

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