Use of adenosine in the treatment of neurotoxicity from snake venom

Abstract

The application provides a method for treating snake venom by adenosine or a pharmaceutical composition containing adenosine, which comprises administering an effective dose of adenosine or a pharmaceutical composition containing adenosine. The adenosine or the pharmaceutical composition containing adenosine of the application can be applied to the first-aid treatment of snake bites in primary hospitals, and is expected to achieve remarkable curative effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a method for treating snake venom by using adenosine and application thereof. BACKGROUND

[0002] Bungarus multicinctus belongs to animal kingdom, chordata, reptilia, squamata, elapidae and bungarus. The main components of bungarus multicinctus toxin are proteins and polypeptides, and the toxic components mainly include neurotoxins in various toxic polypeptides, including alpha-bungarus multicinctus toxin (alpha-BGT), beta-bungarus multicinctus toxin (beta-BGT), kappa-bungarus multicinctus toxin (kappa-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, lethargy, motor nerve disorder, eyelid ptosis, mydriasis, local weakness, glossopharyngeal paralysis, slurred speech, drooling, nausea, vomiting, coma, dyspnea, respiratory failure, and death within 8-72 hours. If not treated in time, the mortality rate is almost 100%.

[0003] Anti-snake venom serum is currently the first choice for treating bungarus multicinctus bite poisoning in clinical treatment, and its components are polyclonal immunoglobulin highly immunized by large animal plasma / serum. However, the anti-snake venom serum still has many shortcomings. First, large doses of heterologous immunoglobulin injection can cause various adverse reactions, such as serum sickness and early adverse reactions, and even severe allergic reactions; in addition, the anti-snake venom serum must be intravenously injected by professional medical personnel in medical institutions, and depends on cold chain transportation and storage, and is expensive. Therefore, many remote areas and rural areas lack of standby serum, and patients bitten by snakes in the wild and rural areas cannot be treated in time after being bitten, which may lead to serious complications and even death. In summary, these limitations highlight the urgency and necessity of developing snake bite treatment methods with good tolerance, easy access, reasonable price and sustainability.

[0004] Pimpinella diversifolia DC. is a plant of the Apiaceae family, also known as snake retreat. As a wild plant, it is widely distributed and rich in resources, and is one of the folk herbs and wild vegetables. The whole plant is used as medicine, which has the effects of warming the middle and dispelling cold, promoting lung and expelling phlegm, promoting blood circulation and relieving pain, and detoxifying and detumescence. Pimpinella diversifolia has been used for a long time in the treatment of snake venom poisoning in folk medicine, but its efficacy has not been verified by strict scientific experiments, and its mechanism is not clear.

[0005] Adenosine is an endogenous purine nucleoside, which is widely distributed in the human body, participates in energy balance by changing content, and plays an important physiological function in the cardiovascular system and nervous system. At the same time, adenosine is also a natural estrogen, which widely acts on neurotransmitters and is an important upstream neuroregulator in the brain. As a natural estrogen, adenosine has the characteristics of mild effect and small adverse reactions compared with synthetic estrogen. Adenosine injection has no effects such as allergy, hemolysis and vascular irritation. As an endogenous neuroprotective agent, adenosine has neuroregulatory 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, and 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. Adenosine and similar substances are not reported in the research of snake venom. SUMMARY

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

[0007] The present application provides the use of adenosine or a pharmaceutical composition comprising adenosine in the preparation of a kit for treating snake venom or alleviating symptoms of poisoning.

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

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

[0010] In one or more embodiments, the snake venom is selected from the group consisting of: Bungarus candidus venom, Naja naja atra venom, Ophiophagus hannah venom, Daboia russelii venom, Lycodon rufozonatus venom. Preferably, the snake venom is selected from the group consisting of: Naja naja atra venom, Ophiophagus hannah venom, Lycodon rufozonatus venom.

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

[0012] In one or more embodiments, the symptoms of poisoning include one or more selected from the group consisting of: wound pain, local swelling, lethargy, motor nerve disorder, eyelid ptosis, mydriasis, local weakness, glossopharyngeal paralysis, stuttering, drooling, nausea, vomiting, coma, dyspnea, respiratory failure, quadriplegia. Preferably, the symptoms of poisoning include dyspnea and quadriplegia.

[0013] In one or more embodiments, the adenosine has the following formula:

[0014]

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

[0016] In one or more embodiments, the instructions comprise an administration method and an administration dose of adenosine for treating snake venom.

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

[0018] In one or more embodiments, the administration dose is a dose of adenosine, which is 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 candidus venom, Naja naja atra venom, Ophiophagus hannah venom, Daboia russelii venom, and Lycodon rufozonatus venom. Preferably, the snake venom is selected from the group consisting of Naja naja atra venom, Ophiophagus hannah venom, and Lycodon rufozonatus venom.

[0020] The third aspect of the present application 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 candidus venom, Naja naja atra venom, Ophiophagus hannah venom, Daboia russelii venom, and Lycodon rufozonatus venom. Preferably, the snake venom is selected from the group consisting of Naja naja atra venom, Ophiophagus hannah venom, and Lycodon rufozonatus venom.

[0022] In one or more embodiments, the administration is selected from the group consisting of 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, which is 6-25 mg / kg, preferably 12.5-25 mg / kg, more preferably 25 mg / kg.

[0024] The present application also provides a method for reducing the likelihood of death or injury in a mammal resulting from snake venom invasion, the method comprising administering to a patient suspected of being exposed to or known to have been exposed to snake venom invasion, the method comprising administering an effective dose of adenosine or a pharmaceutical composition comprising adenosine after snake venom invasion, but before local, regional, or systemic snake venom invasion results in injury.

[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: Naja naja atra venom, Naja naja sacal venom, Naja naja atra venom, Naja naja sacal venom, Daboia russelii venom. Preferably, the snake venom is selected from the group consisting of: Naja naja sacal venom, Naja naja atra venom, Daboia russelii venom.

[0027] In one or more embodiments, the administration is selected from the group consisting of: 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, and is 6-25 mg / kg, preferably 12.5-25 mg / kg, more preferably 25 mg / kg.

[0029] Advantages of the present application:

[0030] 1. Adenosine injection is easy to obtain, easy to store (at room temperature for 24 months), and inexpensive.

[0031] 2. As a commonly used drug in current clinical practice, it can expand the original clinical indications, and its safety has been verified in previous clinical studies.

[0032] 3. It can be used in first-aid treatment of snakebite in primary hospitals, and is expected to achieve remarkable effects. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 : The marketed drug adenosine injection can significantly antagonize the toxicity of Naja naja sacal venom and improve the survival rate of mice after poisoning. A. Four groups of mice, each with 10 mice, were injected intraperitoneally with 3.5 times the LD50 of Naja naja sacal venom (diluted with PBS solution, final volume 100 μL) and different concentrations of adenosine (diluted with PBS solution, final volume 200 μL) or pure PBS solution 200 μL. Among them, the mice injected with Naja naja sacal venom and 200 μL of PBS solution all died within 5 hours, the 48-hour survival rate of mice injected with Naja naja sacal venom and 25 mg / kg of adenosine injection reached 100%; the 48-hour survival rate of mice injected with Naja naja sacal venom and 12.5 mg / kg of adenosine injection reached 80%, and the 48-hour survival rate of mice injected with Naja naja sacal venom and 6.25 mg / kg of adenosine injection reached 70%. The above results were verified by more than three repeated experiments. B. After the mice were injected with Naja naja sacal venom, they showed obvious symptoms of poisoning such as difficulty breathing and limb paralysis, and eventually died. Only a part of the mice injected with Naja naja sacal venom and 25 mg / kg of adenosine injection showed poisoning symptoms corresponding to grades 1-2 in severity, and these poisoning symptoms gradually decreased over time.

[0034] Figure 2 : Changes in chicken biventer cervicis muscle tension, adenosine can reverse the muscle strength decrease caused by snake venom. DETAILED DESCRIPTION

[0035] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (e.g., in the examples) can be combined with each other to form a preferred technical solution.

[0036] The present application first discovers that intraperitoneal injection of adenosine can completely reverse the toxic reaction of Bungarus multicinctus venom, and can achieve 100% survival in mouse experiments, which has high clinical application value.

[0037] The term "subject" or "patient" can refer to a patient or other animal, especially a mammal, such as a human, mouse, rat, dog, monkey, cow, horse, etc., who receives the pharmaceutical composition of the present application for treatment, prevention, improvement and / or alleviation of the venom of the present application.

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

[0039] Dosage and usage

[0040] Intramuscular injection, 20 mg at a time, dissolved in 2 ml of 0.9% sodium chloride injection, 2 times a day. Intravenous injection, 20 mg at a time, dissolved in 20 ml of 0.9% sodium chloride injection, bolus, 2 times a day. Intravenous infusion, 40 mg of the product is dissolved in 250-500 ml of 5% glucose injection, 1 time a day. Coronary heart disease takes 15 days as a course, and can be continuously applied for 2-3 courses; leukemia takes one month as a course; psoriasis takes 2-3 weeks as a course, and can be extended to 4-7 weeks, and the daily dosage can be increased to 60-80 mg.

[0041] Pharmaceutical composition

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

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

[0044] The pharmaceutical composition can further comprise saline, a lipidoid, a liposome, a lipid nanoparticle, a polymer, a lipoplex, a core-shell nanoparticle, a peptide, a protein, a nanoparticle mimic, or a combination thereof.

[0045] The pharmaceutical compositions according to the present teachings can be prepared, packaged, and / or sold in a single unit dosage and / or as a plurality of unit dosages. The quantity of active ingredient is generally equal to the dose which would be administered to a subject and / or a convenient fraction of such a dose such as, for example, one-half or one-third of such a dose.

[0046] As used herein, the term "pharmaceutically acceptable excipient" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, with or without various types of wetting agents, excipients, stabilizers, preservatives, viscosity inducers. Examples of excipients are found in Martin (1975) Remington's Pharm. Sci. 15th Ed. (Mack Publ. Co., Easton).

[0047] The pharmaceutical compositions of the present teachings can include one or more excipients, non-limiting examples of which include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, or a combination thereof.

[0048] In some embodiments, the pharmaceutical composition comprises a cryoprotective agent. The term "cryoprotective agent" refers to an agent that is capable of reducing or eliminating damage to a substance during freezing. Non-limiting examples of cryoprotective agents include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0049] Methods and uses

[0050] The present teachings provide methods of treating snake venom comprising administering to a subject a therapeutically effective amount of an adenosine or a pharmaceutical composition described herein.

[0051] The methods of treatment herein reverse the neurotoxicity of snake venom by intravenous infusion, intramuscular injection, intraperitoneal injection, preferably intraperitoneal injection.

[0052] In one or more embodiments, the snake venom is selected from the group consisting of: a krait venom, a cobralid venom, an eyespotted viper venom, a cobra venom, a taipan venom. Preferably, the snake venom is selected from the group consisting of: a cobralid venom, a cobra venom, a taipan venom.

[0053] As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and can include even a small reduction in one or more measurable markers of the disease or condition in therapy (e.g., symptoms of snake venom, including but not limited to, wound pain, local swelling, lethargy, motor neuropathy, ptosis, mydriasis, local weakness, glossopharyngeal paralysis, slurred speech, drooling, nausea, vomiting, coma, dyspnea, respiratory failure, quadriplegia). Illustratively, the symptoms of snake venom in the present application are preferably dyspnea and quadriplegia. Treatment can optionally include a reduction or alleviation of symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. As used herein, "treatment" of a disease in a subject refers to (1) inhibiting snake venom neurotoxicity or arresting its development; or (2) ameliorating or causing regression of the symptoms of snake venom intoxication. As understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, diminishment of extent of condition (including disease), stabilized (i.e., not worsening) state of condition (including disease), delay or slowing of condition (including disease), amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether partia l or total.

[0054] A "subject" or "patient" for diagnosis or treatment is a cell or an animal, such as a mammal or human. The subject is not limited to a particular species, including non-human animals that receive diagnosis or treatment and those animals that receive infection or animal models, including but not limited to, simian, murine, rat, canine, or rabbit species, as well as other livestock, sport, or pet animals. In some embodiments, the subject or patient is a human.

[0055] As used herein, the term "effective amount" is intended to mean the amount that is sufficient to achieve the desired effect. In the context of therapeutic or prophylactic 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 the amount sufficient to result in partial or full restoration of function of a defective gene in the subject or patient. In other some embodiments, the effective amount of adenosine is the amount sufficient to result in alleviation of snake venom in the subject or patient. The skilled artisan will be able to determine an appropriate amount depending on these and other factors.

[0056] In some embodiments, the effective amount will depend on the size and nature of the application under discussion. It will also depend on the nature and sensitivity of the subject and the method used. The person skilled in the art will be able to determine the effective amount on the basis of these and other considerations. According to the embodiments, the effective amount can comprise, consist essentially of, or consist of one or more administrations of the composition.

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

[0058] Dose and administration

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

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

[0061] Administration of the adenosine or pharmaceutical composition of the present application can be carried out in one dose, continuously or intermittently throughout the course of treatment. In some embodiments, the adenosine or pharmaceutical composition of the present application is administered by injection for intraperitoneal injection. The adenosine and pharmaceutical composition of the present application can be administered in combination with other known treatments for the condition being treated.

[0062] Kit

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

[0064] In some embodiments, the kits further include instructions for use. In particular, such kits can include one or more of the reagents described herein, along with instructions describing the intended use and proper use of the reagents. In some embodiments, the kits can include instructions for mixing one or more components of the kit and / or isolating and mixing a sample and administering to a subject or patient. In some embodiments, the reagents in the kit are in a pharmaceutical formulation and dosage appropriate for the particular application and method of administration of the reagents. Kits for research purposes can contain components in appropriate concentrations or amounts for conducting various experiments.

[0065] Kits can be designed to facilitate use of the methods described herein, and can take many forms. Each composition of a kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder), as applicable. In certain instances, some compositions can be constitutable or otherwise processable (e.g., into an active form), for example, by addition of a suitable solvent or other substance (e.g., water or cell culture media), which can or can not be provided with the kit. In some embodiments, compositions 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 an amount of metalloprotease inhibitor.

[0066] In some embodiments, the kits contain any one or more of the components described herein in one or more containers. Thus, in some embodiments, the kits can include containers holding the reagents described herein. The reagents can be in the form of a liquid, gel, or solid (powder). The reagents can be prepared aseptically, packaged in syringes, and shipped frozen. Alternatively, they can be contained in vials or other containers for storage. A second container can have other reagents prepared aseptically. Alternatively, the kit can include pre-mixed active agents and shipped in syringes, vials, tubes, or other containers. The kits can have one or more or all of the components needed to administer the reagents to a subject, such as syringes, topical application devices, or IV needles and bags.

[0067] It is to be understood that the examples are merely illustrative of the present application and do not limit the scope of the application. Methods and materials similar or equivalent to those described herein can be used in the practice of the present application unless otherwise stated.

[0068] Examples

[0069] Example 1:

[0070] Fresh Eshrhuzhizhuye was impurity-removed and water-extracted using a Chinese herbal medicine extraction machine (developed by Zhiyuanling Extraction Intelligence Technology Co., Ltd. and Guangdong University of Chinese Medicine). 550 g of freshly harvested Eshrhuzhizhuye was washed clean and cut into small pieces, placed in a suitable size medicine bag, and then placed in the stainless steel pot of the extraction machine, and 300 ml of double distilled water was added. The machine was operated at 85 degrees Celsius under vacuum negative pressure suction and physical pressing, and the extraction was completed in about 35 minutes, and the extraction liquid was collected. After the extraction was completed, 300 ml of double distilled water was added again for extraction. The two extraction liquids were mixed together and concentrated to 40 ml by vacuum reduction concentration using a rotary evaporator RE-201D.

[0071] Next, the Eshrhuzhizhuye water extract was subjected to metabolite identification analysis. First, the metabolites in the concentrated extract were extracted, and then the treated sample was analyzed by liquid chromatography-mass spectrometry to obtain the mass spectrum RAW file. After software MS-DIAL 4.70 analysis, matching data, the identification results were obtained. The wiff file collected by mass spectrometry was pretreated by software MS-DIAL 4.70 (MS-DIAL: data independent MS / MS deAvolution for comprehensive metabolome analysis. (Nature Methods, 12, 523-526, 2015) including peak extraction, denoising, deconvolution, peak alignment, and export of CSV format three-dimensional data matrix (original data matrix). The extracted peak information was compared with the database, and the three libraries of MassBank, Respect, and GNPS (a total of 14951 records) were searched. This three-dimensional matrix includes sample information, retention time, mass-to-charge ratio, and mass spectrometry response intensity (peak area). The identification results found 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 injected after dilution with PBS), and observed the survival of the mice), and found that adenosine had the strongest inhibitory effect on neurotoxic snake venom (Table 1). (Adenosine: adenosine)

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

[0073]

[0074] 25 mg / kg adenosine had a therapeutic effect on 3.5 times the median lethal dose (LD50) of Bungarus florens venom, Naja naja atra venom, and Ophiophagus hannah venom, with the best effect on Bungarus florens venom.

[0075] Example 2

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

[0077] The Agkistrodon laticinctus venom lyophilized powder was provided by Shanghai Sailun Biotechnology Co., Ltd. The Naja naja atra venom lyophilized powder was provided by Jiangxi Wubushen Biotechnology Co., Ltd. The Ophiophagus hannah venom lyophilized powder was provided by Shanghai Puzhen Biotechnology Co., Ltd. The Daboia russelii venom lyophilized powder was provided by Latoxan Biotechnology Co., Ltd. of France. The Ophiophagus hannah venom lyophilized powder was provided by Jiangxi Wubushen Biotechnology Co., Ltd.

[0078] The mice were injected intraperitoneally with 3.5 times the LD50 of Agkistrodon laticinctus venom (LD50 was calculated by experiment. According to the guidelines recommended by the World Health Organization, the applicant used the mouse tail vein injection method to dilute the venom into different concentrations with 100 μL PBS solution. Six male C57 mice (20-25 grams) were used in each group, and the survival number was recorded after 24 hours of observation. Based on the survival percentage, the probability analysis method was used to calculate the median lethal dose (LD50) and its 95% confidence interval. After testing, the median lethal dose (LD50) of Agkistrodon laticinctus venom was 1.9 μg / 20 g mice, and the intraperitoneal injection of clinically commonly used adenosine injection (East Asia, specification 30 ml:90 mg (30 ml contains 90 mg of adenosine, the main component of the injection is adenosine, and the solvent is physiological saline (sodium chloride)) was found to significantly improve the survival rate of mice (as shown in Figure 1 The control group (the control group was injected with snake venom + solvent) all died within 5 hours, the 12.5 mg / kg treatment group had a survival rate of 80% within 48 hours of observation, and the 25 mg / kg treatment group had a survival rate of 100% within 48 hours of observation. Agkistrodon laticinctus venom can cause respiratory difficulty, limb paralysis, and even death in mice, and adenosine can significantly reduce the neurotoxicity of mice, and can completely eliminate the toxicity after 13 hours.

[0079] Table 2: Scoring criteria for neurotoxicity in mice

[0080]

[0081] Example 3

[0082] To observe the mechanism of adenosine antagonizing the toxicity of the venom, the muscle tissue used included the neuromuscular junction, to observe whether adenosine acts directly on the muscle or on the synapse, etc.

[0083] The venom of Bungarus multicinctus contains presynaptic neurotoxin (beta-Bungarotoxin) and postsynaptic neurotoxin (alpha-Bungarotoxin). We studied the mechanism of neurotoxicity of Bungarus multicinctus venom and the mechanism of action of adenosine on its therapeutic effect. Bungarus multicinctus (3 μg / ml) completely inhibited the indirect twitch in chicken biventer cervicis muscle preparations and attenuated the muscle contraction response induced by exogenous acetylcholine (ACh) and carbachol (CCh), but had no significant effect on the muscle contraction response induced by potassium chloride (KCl). This indicates that Bungarus multicinctus has postsynaptic neurotoxicity, but no obvious myotoxicity. The addition of adenosine (30 μg / ml) prior to the venom significantly reduced the inhibitory effect of Bungarus multicinctus on the contraction response of ACh and CCh. Our results show that adenosine can significantly antagonize the postsynaptic neurotoxicity of Bungarus multicinctus venom.

[0084] Preparation of chicken biventer cervicis muscle preparations: Male chicks aged 4-10 days were sacrificed by exsanguination after inhalation of carbon dioxide. The skin was incised with surgical scissors on the nape of the neck along the midline, and then cut down to the base of the neck. Two biventer cervicis muscles were found on both sides of the midline, with one end attached to the atlas. The muscles were gently lifted and separated with glass dissecting needles until the base of the neck, where they were cut to obtain the biventer cervicis muscle preparations. The chicken biventer cervicis muscle preparations were mounted on a holder in a 50-ml tissue bath, maintaining a static tension of 1 g. The temperature was set to 34°C, and the experimental environment was maintained at 95% oxygen and 5% carbon dioxide. The composition of the tissue fluid included 118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 25 mM NaHCO3, and 11.1 mM glucose. The stimulator was used to stimulate the motor nerve at the maximum voltage (10-20 V) (0.1 Hz; 0.2 ms) to induce indirect contraction (i.e., nerve-mediated contraction). The addition of d-TC (10 μM) blocked the muscle contraction, proving that the electrical stimulation was effective through selective stimulation of the nerve. The preparations were then repeatedly washed with normal saline to restore their contraction response to nerve stimulation. In the absence of nerve stimulation, the tissue was tested for contraction in response to exogenous acetylcholine (Ach; 1 mM for 30 seconds), carbachol (CCh; 20 μM for 60 seconds), and potassium chloride (KCl; 40 mM for 30 seconds). At least 30 minutes of nerve stimulation was required before the venom or adenosine was added. The tension changes at each two-minute time point were plotted as a line graph, and the results were analyzed using Graphpad Prism 10. Figure 2 as shown in the figure).

Claims

1. Use of adenosine in the preparation of drugs or kits for treating snake venom or relieving symptoms of snake venom poisoning, wherein the snake venom is selected from the venom of the banded krait, cobra, and taipan.

2. The use as described in claim 1, characterized in that, The symptoms of snake venom poisoning include one or more of the following: difficulty breathing, respiratory failure, and quadriplegia.

3. The use as described in claim 1, characterized in that, The symptoms of snake venom poisoning include difficulty breathing and paralysis of the limbs.

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