Disclosure of Invention
The present invention has found that adenosine can reverse the toxic response of snake venom.
In a first aspect, the invention provides the use of adenosine or a pharmaceutical composition comprising adenosine in the manufacture of a kit for treating snake venom or alleviating symptoms of poisoning.
The invention also provides the use of adenosine in the manufacture of a medicament for treating snake venom or alleviating symptoms of poisoning.
In one or more embodiments, the toxic symptom is a toxic symptom caused by postsynaptic neurotoxicity.
In one or more embodiments, the snake venom is selected from the group consisting of gold, silver, cobra, and taipan venom. Preferably, the snake venom is selected from the group consisting of silver cyclic venom, cobra venom, taipan venom.
In one or more embodiments, the toxic symptom is a snake venom symptom. Preferably, the toxic symptom is a snake venom symptom caused by postsynaptic neurotoxicity.
In one or more embodiments, the toxic symptoms include one or more selected from the group consisting of wound pain, localized swelling, somnolence, motor disorders, eyelid prolapse, mydriasis, localized weakness, oropharyngeal paralysis, stuttering, salivation, nausea, vomiting, coma, dyspnea, respiratory failure, quadriplegia. Preferably, the toxic symptoms include dyspnea and quadriplegia.
In one or more embodiments, the adenosine has a formula as shown below:
in a second aspect, the invention provides a kit for treating snake venom, comprising instructions for treating snake venom, and (1) adenosine or (2) a pharmaceutical composition comprising adenosine.
In one or more embodiments, the instructions for use comprise a method of administering an adenosine to treat snake venom and a dosage of administration.
In one or more embodiments, the method of administration is selected from the group consisting of intravenous drip, intramuscular injection, intraperitoneal injection. Preferably, the method of administration is intraperitoneal injection.
In one or more embodiments, the administered dose is a dose of adenosine of 6-25mg/kg, preferably 12.5-25mg/kg, more preferably 25mg/kg.
In one or more embodiments, the snake venom is selected from the group consisting of gold, silver, cobra, and taipan venom. Preferably, the snake venom is selected from the group consisting of silver cyclic venom, cobra venom, taipan venom.
In a third aspect, the invention provides a method of treating snake venom, the method comprising administering an effective dose of adenosine or a pharmaceutical composition comprising adenosine.
In one or more embodiments, the snake venom is selected from the group consisting of gold, silver, cobra, and taipan venom. Preferably, the snake venom is selected from the group consisting of silver cyclic venom, cobra venom, taipan venom.
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.
In one or more embodiments, the effective dose is a dose of adenosine ranging from 6 to 25mg/kg, preferably from 12.5 to 25mg/kg, more preferably 25mg/kg.
The invention also provides a method of reducing the likelihood of death or injury in a mammal from venom invasion, the method comprising administering to a patient suspected of having or known to have suffered venom invasion, an effective amount of adenosine or a pharmaceutical composition comprising adenosine after venom invasion but before injury is caused by local, regional or systemic venom invasion.
In one or more embodiments, the mammal is a mouse.
In one or more embodiments, the snake venom is selected from the group consisting of gold, silver, cobra, and taipan venom. Preferably, the snake venom is selected from the group consisting of silver cyclic venom, cobra venom, taipan venom.
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.
In one or more embodiments, the effective dose is a dose of adenosine ranging from 6 to 25mg/kg, preferably from 12.5 to 25mg/kg, more preferably 25mg/kg.
The invention has the advantages that:
1. the adenosine injection is easy to obtain and store (stored at room temperature for 24 months) and has low price;
2. As a common clinical medicine at present, the traditional Chinese medicine composition expands the original clinical indication, belongs to the new use of old medicines, and has the safety verified in the past clinical research;
3. can be applied to the first aid of snake bite in primary hospitals and is expected to obtain remarkable curative effect.
Detailed Description
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.
The invention discovers that the intraperitoneal injection of the adenosine can completely reverse the toxic reaction of the silver ring snake venom, can reach 100 percent survival in a mouse experiment, and has high clinical popularization and application value.
The term "subject" or "patient" may refer to a patient or other animal, particularly a mammal, such as a human, mouse, rat, dog, monkey, cow, horse, etc., that receives a pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate a snake venom of the invention.
The term "adenosine" has been used for clinical medication, for angina pectoris, myocardial infarction, myocarditis and cardiogenic shock, has a certain effect on improving symptoms of palpitation, shortness of breath, chest distress and the like of rheumatic heart disease, can improve the curative effect of combining chemotherapy with acute leukemia, and can also be used for inducing and relieving the acute leukemia. In addition, it has certain curative effect on senile chronic bronchitis, various hepatitis and psoriasis.
Dosage of usage
Intramuscular injection, 20mg once, was dissolved in 2ml of 0.9% sodium chloride injection 2 times a day. Intravenous injection, 20mg once, and injection into 20ml of 0.9% sodium chloride injection, 2 times daily. Intravenous drip, 40mg of the product is dissolved in 250-500ml of 5% glucose injection, 1 time a day. The coronary heart disease can be used for a treatment course of 15 days, 2-3 treatment courses can be continuously applied, one month is used as a treatment course of leukemia, 2-3 weeks is used as a treatment course of psoriasis, the administration period can be prolonged to 4-7 weeks, and the daily dosage can be increased to 60-80mg.
Pharmaceutical composition
The invention first provides a pharmaceutical composition for treating snake venom, the pharmaceutical composition comprising adenosine, preferably the pharmaceutical composition further comprising pharmaceutically acceptable excipients, typically the pharmaceutical composition is administered for therapy.
As described herein, the pharmaceutical compositions may be formulated by any method known or developed in the pharmacological arts 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.
The pharmaceutical composition may further comprise saline, lipids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, nanoparticle mimics, or combinations thereof.
Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk in single unit doses and/or in multiple single unit doses. The amount of active ingredient is typically equal to the dose of active ingredient to be administered to the subject and/or a convenient ratio of such doses, for example half or one third of such doses.
As used herein, the term "pharmaceutically acceptable excipients" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water and emulsions, such as oil/water or water/oil emulsions, as well as various types of wetting agents, excipients, stabilizers, preservatives, viscosity inducers. Examples of adjuvants are found in Martin (1975) Remington's pharm. Sci. 15 th edition (Mack public Co., easton).
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, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, or combinations thereof.
In some embodiments, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent that is capable of reducing or eliminating damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and/or mannitol.
Method and use
The invention provides methods of treating snake venom comprising administering to a subject a therapeutically effective amount of an adenosine or a pharmaceutical composition described herein.
The methods of treatment herein reverse neurotoxicity of snake venom by intravenous drip, intramuscular injection, and intraperitoneal injection, preferably intraperitoneal injection.
In one or more embodiments, the snake venom is selected from the group consisting of gold, silver, cobra, and taipan venom. Preferably, the snake venom is selected from the group consisting of silver cyclic venom, cobra venom, taipan venom.
As used herein, "treating" includes any beneficial or desired effect on symptoms or lesions of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of a disease or condition under treatment (e.g., symptoms caused by snake venom, including but not limited to wound pain, localized swelling, somnolence, motor nerve disorders, eyelid sagging, mydriasis, localized weakness, oropharyngeal paralysis, stuttering, drooling, nausea, vomiting, coma, dyspnea, respiratory failure, quadriplegia). Illustratively, the symptoms produced by snake venom in the application are preferably dyspnea and quadriplegia. Treatment may optionally include a reduction or alleviation of symptoms of the disease or disorder, or a delay in the progression of the disease or disorder. "treating" does not necessarily mean complete eradication or cure of a disease or disorder or associated symptoms thereof. As used herein, "treating" a disease in a subject refers to (1) inhibiting or preventing the development of snake venom neurotoxicity, or (2) ameliorating or causing regression of symptoms of snake venom toxicity. As understood in the art, "treatment" is a method for achieving a beneficial or desired result, including clinical results. The beneficial or desired result can include one or more, but is not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including a disease), deferral or slowing of the condition (including a disease), progression, amelioration or palliation of the condition (including a disease), state, and remission (whether partial or total).
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, including non-human animals that are subject to diagnosis or treatment and those animals that are subject to infection or animal models, including but not limited to simian, murine, rat, canine, or rabbit species, as well as other domestic animals, sports animals, or pets. In some embodiments, the subject or patient is a human.
The term "effective amount" as used herein is intended to mean an amount sufficient to achieve the desired effect. In the case 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, weight and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, an effective amount is an amount sufficient to cause a defective gene to restore some or all of its function in a subject or patient. In other embodiments, an effective amount of adenosine is an amount sufficient to cause a subject or patient to alleviate snake venom. The skilled artisan will be able to determine the appropriate amount based on these and other factors.
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 of use. One skilled in the art will be able to determine an effective amount based on these and other considerations. According to embodiments, an effective amount may comprise, consist essentially of, or consist of one or more administrations of the composition.
As used herein, the term "administering" is intended to mean delivering a substance to a subject or patient, such as an animal or human. Administration may be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective mode and dosage of administration are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be carried out, with the dosage level and mode selected by the treating physician or, in the case of pets and other animals, by the treating physician. Herein, the administration is intraperitoneal injection.
Dosage and administration
Methods of 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 the treatment, the purpose of the treatment, and the subject being treated. Single or multiple administrations may be performed, with the dosage level and mode being selected by the treating physician. The dosage may 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 dose may be 6-25mg/kg of adenosine injection per administration, preferably 12.5-25mg/kg, more preferably 25mg/kg. The adenosine injection disclosed by the application is mainly composed of adenosine, the molecular formula is C10H13N5O4, and the auxiliary material is sodium chloride. The specification is 30ml,90mg adenosine.
Administration of adenosine or pharmaceutical compositions suitable for use in the present invention includes administration by injection, e.g. intravenous drip, intramuscular injection, intraperitoneal injection. Preferably, the injection is intraperitoneal.
Administration of the adenosine or pharmaceutical composition of the invention may be achieved in one dose, continuously or intermittently throughout the course of treatment. In some embodiments, the adenosine or pharmaceutical composition of the invention is administered by intraperitoneal injection by injection. The adenosine and pharmaceutical compositions of the invention may be administered in combination with other known treatments for the disorder being treated.
Kit for detecting a substance in a sample
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 invention comprise any of the adenosines or pharmaceutical compositions described herein.
In some embodiments, the kit further comprises instructions for use. In particular, such kits may include one or more of the reagents described herein, as well as instructions describing the intended use and proper use of such reagents. In some embodiments, the kit may include instructions for mixing one or more components of the kit and/or separating and mixing the sample and administering to the subject or patient. In some embodiments, the agents in the kit are pharmaceutical formulations and dosages suitable for the particular application and method of administration of the agents. Kits for research purposes may contain the components in the appropriate concentrations or amounts for performing the various experiments.
Kits can be designed to facilitate use of the methods described herein, and can take many forms. Where applicable, each composition of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions may be configurable or otherwise processable (e.g., processed into an active form), e.g., by addition of a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit. In some embodiments, the composition may 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 a metalloprotease inhibitor.
In some embodiments, the kit contains any one or more of the components described herein in one or more containers. Thus, in some embodiments, a kit may comprise a container containing reagents described herein. The agent may be in the form of a liquid, gel or solid (powder). The reagents may be prepared aseptically, packaged in syringes and shipped frozen. Or they may be contained in vials or other containers for storage. The second container may have other reagents prepared aseptically. Or the kit may include pre-mixed active agents and be transported in a syringe, vial, tube or other container. The kit may have one or more or all of the components required to administer the agent to the subject, such as a syringe, a topical applicator, or an IV needle tube and bag.
It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and materials used in the examples are those conventional in the art, unless otherwise indicated.
Examples
Example 1:
Fresh pimpinella diversifolia was purified and extracted with water using a herbal extractor (developed by Zhiyuanling intelligent science and technology Co., ltd., and Guangdong university of Chinese medicine). 550g of freshly collected pimpinella diversifolia is washed, cut into small pieces, put into a medicine bag with proper size, put into a stainless steel pot of an extractor, and added with 300ml of double distilled water. Vacuum negative pressure suction and physical squeezing are carried out on the machine at 85 ℃ for about 35 minutes, and the extraction liquid is collected. After the extraction was completed, 300ml of double distilled water was added to perform extraction again. The two extracts were mixed together and concentrated under reduced pressure by rotary evaporator RE-201D to 40 ml.
Next, the aqueous extract of pimpinella diversifolia was subjected to metabolite identification analysis. Firstly extracting metabolites in the concentrated extract, then analyzing the processed samples through liquid chromatography-mass spectrometry to obtain a mass spectrum original RAW file, and obtaining an identification result through software MS-DIAL 4.70 analysis and data matching. The wiff file of mass spectrum acquisition is preprocessed 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, noise removal, deconvolution, peak alignment, and derivation of a three-dimensional data matrix (original data matrix) in CSV format. The extracted peak information was compared with the database, and full library search was performed for three libraries MassBank, aspect, GNPS (total 14951 records). This three-dimensional matrix includes information such as sample information, retention time, mass-to-nuclear ratio, and mass response intensity (peak area). The results of the identification found that the extracts contained adenosine, chlorogenic acid, etc., and the main components were tested (i.e., mice were first intraperitoneally injected with different types of snake venom, then immediately intraperitoneally injected with these different pharmaceutical components (both of which were injection solutions diluted with PBS), and survival of mice was observed), and the inhibitory effect of adenosine on nerve venom was found to be strongest (table 1). (Adenosine: adenosine)
TABLE 1 effects of different pimpinella diversifolia fractions on survival of mice treated with various neurostimulants.
25Mg/kg adenosine was effective against 3.5 times the lethal dose (LD 50) of Agkistrodon, cobra venom, and taipan venom, with the best effect on Agkistrodon venom.
Example 2:
The animal model used in the experiment was C57 mice (age 8 weeks, weight 20-25 g) supplied by Peking Vitrendylar Experimental animal technologies Co. 10 mice were kept per cage, ensuring adequate food and purified water supply, and the laboratory mice were kept under constant temperature and humidity (20-21 ℃ C., about 50% humidity) following a12 hour light and dark cycle. Experimental design mice were randomly assigned to different groups and experimentally observed according to the 3R-modified WHO recommendation protocol.
The silver ring nerve venom lyophilized powder is provided by Shanghai Seleno biotechnology company. The bungarus parvus venom lyophilized powder is provided by Jiangxi Agkistrodon biotechnology company, the cobra venom lyophilized powder is provided by Shanghai spectral vibration biotechnology company, the Taipan venom lyophilized powder is provided by French Latoxan biotechnology company, and the cobra venom lyophilized powder is provided by Jiangxi Agkistrodon biotechnology company.
According to the guidelines recommended by the world health organization, the applicant adopts a mouse tail intravenous injection method to dilute 100 mu L of PBS solution into different concentrations of venom, 6 male C57 mice (weight 20-25 g) are used in each group, death conditions are observed after 24 hours, and the number of surviving mice is recorded, based on the survival percentage, the half-killing amount (LD 50) of the silver ring venom is calculated by using a probability analysis method, and 95% confidence interval thereof, the test shows that the half-killing amount (LD 50) of the silver ring venom is 1.9 microgram/20 g of mice, meanwhile, the common adenosine injection (30 ml of medium Meadon, 90mg of adenosine is contained in 30ml of specification, adenosine is the main component of adenosine in the injection, and the solvent is physiological saline (sodium chloride)) is adopted in the specification, the adenosine injection can remarkably improve the survival rate of the mice (as shown in figure 1), the control group (snake venom+solvent is injected) can remarkably improve the survival rate of the mice in the time of 5 hours, the toxicity of the mice is remarkably reduced in the time of 12.5mg of the control group, and the toxicity of the nerve is remarkably reduced in the four limbs of the mice when the nerve is completely observed in the time of 48 mg of the mice, and the toxicity of the nerve is remarkably reduced in the four limbs of the mice is completely observed after the nerve is completely reaching to 48 mg of the toxicity of the mice.
TABLE 2 scoring criteria for neurotoxic effects in mice
Example 3:
in order to observe the mechanism by which adenosine antagonizes snake venom toxicity, muscle tissue including neuromuscular junctions is used, whether adenosine acts directly on muscle, on synapses, etc.
Bungarotoxins comprise presynaptic neurotoxin (beta-bungarotoxin) and postsynaptic neurotoxin (alpha-bungarotoxin). We studied the neurotoxicity mechanism of Agkistrodon and the mechanism of action of adenosine on its therapeutic effects. Bungarotoxin (3 μg/ml) completely inhibited indirect twitches in chicken two-abdominal muscle specimens and attenuated muscle contraction responses caused by exogenous acetylcholine (ACh) and carbachol (CCh), but had no obvious effect on muscle contraction responses caused by potassium chloride (KCl). This suggests that bungarotoxin has postsynaptic neurotoxicity but no apparent myotoxicity. The pre-addition of adenosine (30 μg/ml) significantly reduced the inhibition of ACh and CCh contractile responses by silver cyclic snake venom. Our results indicate that adenosine can significantly antagonize postsynaptic neurotoxicity of silver cyclic snake venom.
Preparation of chicken two abdominal muscle specimen by taking out shell of 4-10 days old male chicken, sucking carbon dioxide, and removing blood. Removing hair on the back of the neck, making a skin incision along the midline from the head pillow, cutting downwards until reaching the base of the neck, and ensuring that two abdominal muscles of the neck are clung to the skin at two sides of the midline, wherein one end is connected to the neck bone, slightly lifting the muscles, gradually separating the two abdominal muscles of the whole chicken neck by using a glass split needle until reaching the base of the neck, and cutting off the base of the neck to obtain a sample of the two abdominal muscles of the chicken neck. The chicken neck two abdominal specimens were mounted on a rack in a 50ml tissue bath, maintaining a static tension of 1 gram. The temperature was set at 34 ℃ to ensure 95% oxygen and 5% carbon dioxide in the experimental environment, and the tissue fluid contained ：118.4mM NaCl,4.7mM KCl,1.2mM MgSO4,1.2mM KH2PO4,2.5mM CaCl2,25mM NaHCO3 and 11.1mM glucose. The motor nerve (0.1 Hz;0.2 ms) was stimulated with a stimulator at maximum voltage (10-20V) to induce indirect contraction (i.e., nerve-mediated contraction). The addition of d-TC (10. Mu.M) blocks muscle contraction, thus demonstrating that electrical stimulation is effected by selective stimulation of the nerve. Followed by repeated washout with physiological saline to restore the contractile response of the specimen to neural stimulation. In the absence of neural stimulation, the tissue was examined for contractile response to exogenous acetylcholine (Ach; 1mM for 30 seconds), carbachol (CCh; 20. Mu.M for 60 seconds) and potassium chloride (KCl; 40mM for 30 seconds). At least 30 minutes of re-initiation of nerve stimulation is required before venom or adenosine is added. The results were analyzed with GRAPHPAD PRISM by plotting the change in tension at every two minutes time points. (as shown in figure 2).