Application of neostigmine methylsulfate or atropine sulfate in preparation of medicine for preventing, relieving or adjunctively treating bungarus multicinctus bites

Abstract

The invention discloses application of neostigmine methylsulfate or atropine sulfate to preparation of a medicine for preventing, relieving or assisting in treating bungarus multicinctus bites, and belongs to the field of medicine. The invention protects the application of neostigmine methylsulfate or atropine sulfate in prevention, alleviation or adjuvant treatment of bungarus multicinctus bites, and solves the problems in the prior art that the quantity of anti-snake venom serum is small, the anti-snake venom serum is not easy to preserve and transport, and the anti-snake venom serum needs to be injected in time. Experiments prove that before or after a bungarus multicinctus bites, the treatment time and the treatment window can be effectively prolonged by taking the neostigmine methylsulfate or the atropine sulfate, and the survival rate of a patient is increased; meanwhile, neostigmine methylsulfate or atropine sulfate is easy to store and convenient to carry.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to the use of neostigmine methylsulfate or atropine sulfate in the preparation of a medicament for preventing, relieving or adjuvantly treating Bungarus multicinctus bites. Background Art

[0002] Globally, more than 600 species of venomous snakes have been discovered. Bungarus multicinctus is a highly venomous snake ranked fourth in terms of global toxicity. Its single venom output is about 4.6 mg, far exceeding the lethal dose for humans, and the reported bite mortality rate is as high as 23%. The situation of Bungarus multicinctus bites is very serious. If effective treatment is not received in time after being bitten, the mortality rate can be as high as over 50%. Even if treated, some patients still have sequelae such as limb dysfunction due to excessive poisoning time and severe nerve damage. Therefore, the research on Bungarus multicinctus venom and Bungarus multicinctus bites is very necessary and has important clinical reference value.

[0003] The toxins of Bungarus multicinctus mainly include: α-BGT, γ-BGT, β-BGT, κ-BGT. α-BGT acts on the postsynaptic membrane, competitively inhibits the binding of acetylcholine to the receptor, and blocks the transmission of nerve signals. β-BGT is a presynaptic neurotoxin with an intraperitoneal injection LD50 of 0.004 μg / g. β-BGT mainly acts on the neuromuscular junction and causes damage to certain cells, changes the content of acetylcholine in the synaptic cleft, and affects the transmission of nerve signals. γ-BGT is the most toxic molecule among the three-finger toxins of Bungarus multicinctus venom. It mainly acts on the M2 type acetylcholine receptor, and the specific mechanism of action is still unclear, but it is also related to the change in the content of neurotransmitters. There is a sequence of Arg-Gly-Asp (RGD) in its primary sequence, which has various bioactivities with development potential. κ-BGT belongs to a postsynaptic neurotoxin and has relatively little research, but it also has a strong blocking effect on nerve signal conduction.

[0004] After being bitten by Bungarus multicinctus, failure to receive timely treatment can lead to an extremely high fatality rate. Some may die within minutes, and if not treated promptly, the venom may take effect within 6 - 8 hours. The poisoning symptoms usually appear within 1 - 6 hours, and in severe cases, the patient may die due to respiratory muscle paralysis or cardiac arrest within 1 - 2 hours. After being bitten, the patient may have mild or no local pain, which is easily overlooked, and systemic nerve damage will occur: symptoms such as chest tightness, difficulty swallowing, ptosis of the eyelids, and muscle tremors will appear rapidly, and eventually progress to flaccid paralysis and respiratory failure. Currently, the most effective measure after being bitten by Bungarus multicinctus is to inject antivenom serum. However, in remote mountainous areas, antivenom serum is not easy to store and carry with, which is likely to delay the treatment opportunity and cause serious consequences. The treatment effect of neurotoxic venomous snake bites highly depends on early recognition and standardized treatment. If there are drugs that are easy to store and carry with, which can prevent in advance or delay the poisoning symptoms after being bitten and extend the treatment time, it will greatly improve the survival and cure rates of neurotoxic venomous snake bites. In some remote mountainous areas or rural areas of certain countries, after being bitten by Bungarus multicinctus, there may be no antivenom serum against Bungarus multicinctus in a short time, and effective treatment cannot be obtained. Future research should focus on developing safer and more effective antivenom drugs and deeply studying the toxicity mechanism of Bungarus multicinctus venom to provide a standardized, effective, and reliable basis for the diagnosis and treatment of Bungarus multicinctus bites.

[0005] In the treatment of venomous snake bites, antivenom serum remains the main treatment method. Although antivenom serum can reduce the morbidity and mortality rates, there are still many limitations. Firstly, there are only four types of antivenom serum available on the market, and all are monovalent antivenom sera. Clinically, there is often a lack of corresponding types of antivenom serum. Secondly, due to issues such as the cost of antivenom serum, the serum supply in some countries and regions is insufficient. Moreover, the side effects of antivenom serum will also have a certain impact. In addition, for some remote areas with a high incidence of snake bites, it is difficult to store and carry antivenom serum stably and effectively for a long time. The inventor's previous research showed that: antivenom serum against Bungarus multicinctus can effectively neutralize the free β - BGT in the blood, but has a weak neutralizing ability for the three - fingered neurotoxins α - BGT, γ - BGT, and κ - BGT. In clinical treatment, some patients still develop into severe cases after injecting a sufficient amount of antivenom serum against Bungarus multicinctus. Therefore, the inventor's team believes that γ - BGT is the root cause of the clinical critical type, possibly because the content of γ - BGT in the venom of some Bungarus multicinctus is too high.

[0006] After retrieval, there is no drug for early prevention, or for alleviating or assisting in the treatment of Bungarus multicinctus bites in the prior art. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the present invention provides the use of neostigmine methylsulfate or atropine sulfate in the preparation of drugs for preventing, alleviating or assisting in the treatment of Bungarus multicinctus bites or related neurotoxin poisoning.

[0008] The present invention also protects the use of neostigmine methylsulfate or atropine sulfate in combination with antivenom in the preparation of a medicament for treating Bungarus multicinctus bites or related neurotoxin poisoning.

[0009] Further, the neurotoxin is: α-BGT, β-BGT, γ-BGT and combinations thereof.

[0010] Preferably, the administration method of neostigmine methylsulfate or atropine sulfate is subcutaneous injection or intravenous injection.

[0011] Preferably, the dose of neostigmine methylsulfate for intravenous injection does not exceed 0.5 mg / kg.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention protects the use of neostigmine methylsulfate or atropine sulfate in the preparation of a medicament for preventing, alleviating or assisting in the treatment of Bungarus multicinctus bites, and solves the problems in the prior art such as the small number of antivenoms, difficult preservation and transportation, and the need for timely injection. Experiments of the present invention prove that: before or after being bitten by Bungarus multicinctus, taking neostigmine methylsulfate or atropine sulfate can effectively prevent or alleviate Bungarus multicinctus bites, extend the treatment time and treatment window, and improve the survival rate of patients. Experiments prove that: these two drugs do not affect the effect of antivenom, and are easy to preserve and carry. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a result diagram of the preventive use of several drugs on γ-BGT, wherein Figure 1 A is the influence of pre-injecting galantamine and pilocarpine on γ-BGT, Figure 1 B is the influence of pre-injecting scopolamine on γ-BGT, Figure 1 C is the influence of pre-injecting neostigmine methylsulfate on γ-BGT;

[0015] Figure 2 It is the result of the preventive use of scopolamine and neostigmine on several snake venom toxins, wherein Figure 2 A is the preventive effect of scopolamine and neostigmine methylsulfate on α-BGT toxin; Figure 2 B is the protective effect of scopolamine and neostigmine methylsulfate on β-BGT;

[0016] Figure 3 It is the preventive effect of scopolamine and neostigmine methylsulfate on the combination of α-BGT + γ-BGT;

[0017] Figure 4 It is the preventive effect of scopolamine and neostigmine methylsulfate on several crude snake venoms, wherein Figure 4A is the preventive effect of scopolamine and neostigmine methylsulfate on the crude venom of Bungarus multicinctus; Figure 4 B is the preventive effect of scopolamine and neostigmine methylsulfate on the crude venom of Naja naja; Figure 4 C is the preventive effect of scopolamine and neostigmine methylsulfate on the crude venom of Ophiophagus hannah (x±s, n = 6 for each group);

[0018] Figure 5 It is the alleviating effect of scopolamine, neostigmine methylsulfate, and atropine sulfate on the poisoning caused by injecting Bungarus multicinctus toxin; Figure 5 A is the alleviating effect of scopolamine, neostigmine methylsulfate, and atropine sulfate on the poisoning caused by pre-injecting γ-BGT; Figure 5 B is the alleviating effect of scopolamine, neostigmine methylsulfate, and atropine sulfate on the poisoning caused by pre-injecting α-BGT (x±s, n = 6 for each group);

[0019] Figure 6 A is the alleviating effect of intravenous injection of neostigmine methylsulfate and atropine sulfate for 10 min on γ-BGT poisoning; Figure 6 B is the alleviating effect of intravenous injection of neostigmine methylsulfate and atropine sulfate for 20 min on γ-BGT poisoning (x±s, n = 6 for each group);

[0020] Figure 7 It is the alleviating effect of scopolamine, neostigmine methylsulfate, and atropine sulfate on the poisoning caused by injecting the α-BGT + γ-BGT combination (x±s, n = 6 for each group).

[0021] Figure 8 A is the detoxifying effect of neostigmine methylsulfate and atropine sulfate on the poisoning caused by the β-BGT + γ-BGT combination; Figure 8 B is the detoxifying effect of neostigmine methylsulfate and atropine sulfate combined with anti-Bungarus multicinctus venom serum respectively on the pre-injected β-BGT + γ-BGT combination (x±s, n = 6 for each group);

[0022] Figure 9 A is the detoxifying effect of neostigmine methylsulfate and atropine sulfate on the poisoning caused by the β-BGT + α-BGT combination; Figure 9 B is the detoxifying effect of neostigmine formate and atropine sulfate combined with anti-Bungarus multicinctus venom serum respectively on the β-BGT + α-BGT combination (x±s, n = 6 for each group);

[0023] Figure 10 It is the detoxifying effect of neostigmine methylsulfate and atropine sulfate combined with anti-Bungarus multicinctus venom serum respectively on the injection of the crude venom of Bungarus multicinctus (x±s, n = 6 for each group).

[0024] Figure 11The alleviating effect of neostigmine methylsulfate and atropine sulfate at twice the dosage by intravenous injection for 10 min on the injection of γ-BGT. Detailed implementation mode

[0025] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0026] The reagents used in the following examples are as follows:

[0027] The crude venoms of Bungarus multicinctus, Naja naja, and Ophiophagus hannah are all samples retained by the Discipline Group of Functional Proteomics of Natural Medicines, Kunming Institute of Zoology, Chinese Academy of Sciences. The anti-Bungarus multicinctus antivenin is a product of Shanghai Sailen Biotech Co., Ltd. (batch number 20210301, 1000 U / ml). Scopolamine (product number: S5873, 99.99%), neostigmine methylsulfate (product number: S5886, 99.99%), bisacodyl (product number: S2455, 99.99%), pilocarpine hydrochloride (product number: S1608, 99.99%), and atropine sulfate (product number: S5493, 99.99%) are all purchased from Selleck. α-bungarotoxin, β-bungarotoxin, and γ-bungarotoxin were separated from the crude venom of Bungarus multicinctus according to the method of Lin Bo in this laboratory and then stored (Lin B, Zhang JR, Lu HJ, et al. Immunoreactivity and neutralization studyofChinese Bungarus multicinctus antivenin and lab-prepared anti-bungarotoxinantisera towards purified bungarotoxins and snake venoms[J].PLoS NeglectedTropical Diseases,2020,

[0028] 14(11):e0008873.DOI:10.1371 / journal.pntd.0008873.)。

[0029] Example 1 The preventive effect of neostigmine methylsulfate on Bungarus multicinctus bites

[0030] To explore the preventive, alleviating, and adjuvant therapeutic effects of neostigmine methylsulfate on bungarotoxin and bungarus multicinctus bites, the inventors pre-injected neostigmine methylsulfate, neostigmine, pilocarpine, scopolamine, etc. subcutaneously or intraperitoneally. After waiting for the drugs to take effect (10 min), the relevant snake venoms were then injected intraperitoneally. There were 6 mice in each group, and the death times of the mice in each group were statistically counted. The dosage of α-BGT and γ-BGT was calculated according to 2 times the LD 50 value; the dosage of β-BGT, crude bungarus multicinctus venom, and crude king cobra venom was calculated according to 3 times the LD 50 value. The LD 50 of the crude king cobra venom: 1 mg / kg; according to the actual body weight of the mice and the conversion relationship between mice and humans: mouse dosage (mg / kg) = 9.1 * human dosage (mg / kg), the drug dosage was calculated. The dosages of neostigmine methylsulfate, neostigmine, pilocarpine, scopolamine, etc. were 0.1 mg / kg.

[0031] The results are as Figures 1-4 shown, among which Figure 1 as shown in C: For γ-BGT, compared with neostigmine, pilocarpine, and scopolamine, pre-injection of neostigmine methylsulfate had a highly significant preventive effect on γ-BGT ( Figure 1 -C); for α-BGT, pre-injection of neostigmine methylsulfate also had a significant preventive effect ( Figure 2 A); however, for β-BGT and crude king cobra venom, neostigmine methylsulfate did not exert a good preventive effect ( Figure 2 B, Figure 4 C); for the combination of the three-finger toxins α-BGT and γ-BGT in bungarus multicinctus venom, neostigmine methylsulfate had a good preventive effect ( Figure 3 ); for crude bungarus multicinctus venom, pre-injection of neostigmine methylsulfate had a relatively significant preventive effect and scopolamine could increase the survival time of mice to a certain extent, but there was no statistical significance ( Figure 4 A).

[0032] Example 2 Alleviating effects of neostigmine methylsulfate and atropine sulfate on bungarus multicinctus venom or toxins

[0033] To simulate a real bungarus multicinctus bite situation, different bungarus multicinctus toxins were pre-injected intraperitoneally. After 10 min, neostigmine methylsulfate, atropine sulfate, and scopolamine were injected intraperitoneally and subcutaneously. There were 6 mice in each group, and the death times of the mice in each group were statistically counted. The injection dosages of α-BGT, γ-BGT, neostigmine methylsulfate, atropine sulfate, and scopolamine were the same as above.

[0034] The results are as Figures 5-7 shown: For pre-injected γ-BGT and α-BGT, neostigmine methylsulfate had a significant detoxifying effect (Figure 5 A, Figure 5 B). To observe whether the effect is better during intravenous injection, at 10 min, neostigmine methylsulfate, atropine sulfate, and scopolamine were intravenously injected at twice the dosage. The results showed that neostigmine methylsulfate actually promoted the death of the mice. Subsequently, through experiments, it was found that when the intravenous injection of neostigmine methylsulfate exceeded 0.1 mg / kg by a certain amount, the protective effect would decline with the increase in dosage until it promoted death. At the same time, to simulate the situation where treatment was not received for a long time and whether these two drugs still had an effect in a dying state, neostigmine methylsulfate was intravenously injected at 20 min, and it was found that neostigmine methylsulfate still had a significant alleviating effect at this time ( Figure 6 ). For the pre-injection of the three-finger toxin α-BGT + γ-BGT combination in the venom of Bungarus multicinctus, neostigmine methylsulfate still had a significant detoxifying effect ( Figure 7 ). This indicates that the action site of neostigmine methylsulfate may have a certain overlap with the action sites of α-BGT and γ-BGT, and the binding ability of neostigmine methylsulfate is stronger. For the pre-injection of γ-BGT and α-BGT, atropine sulfate had a significant detoxifying effect ( Figure 5 A, Figure 5 B); To observe whether the effect is better during intravenous injection, atropine sulfate was intravenously injected at 10 min, and it was found that compared with subcutaneous injection, atropine sulfate had a better protective effect on γ-bungarotoxin. At the same time, to simulate the situation where treatment was not received for a long time and whether these two drugs still had an effect in a dying state, atropine sulfate was intravenously injected at 20 min, and it was found that atropine sulfate still had a significant alleviating effect at this time ( Figure 6 A, Figure 6 B). For the pre-injection of the three-finger toxin α-BGT + γ-BGT combination in the venom of Bungarus multicinctus, atropine sulfate still had a significant detoxifying effect ( Figure 7 ). This indicates that the action site of atropine sulfate may have a certain overlap with the action sites of α-BGT and γ-BGT, and the binding ability of atropine sulfate is stronger.

[0035] Example 3 Auxiliary therapeutic effects of neostigmine methylsulfate and atropine sulfate on Bungarus multicinctus bites

[0036] To better simulate the situation where there may be no anti-Bungarus multicinctus venom serum in a short time after being bitten by Bungarus multicinctus in remote mountainous areas or rural areas; to explore whether the injection of neostigmine methylsulfate or atropine sulfate also has an alleviating effect before injecting anti-Bungarus multicinctus venom serum. In this experiment, different Bungarus multicinctus toxins were pre-injected intraperitoneally, and 3 min later, neostigmine methylsulfate or atropine sulfate was injected intraperitoneally or subcutaneously. 6 min later, anti-Bungarus multicinctus venom serum was injected intravenously. There were 6 mice in each group, and the death time of each group of mice was statistically analyzed. The median effective dose (ED of anti-Bungarus multicinctus venom serum was calculated according to the methods of Spearman and Karber, etc.50 : 17.68 mg / kg), the dosage of anti-bungarus multicinctus venom serum was calculated according to 2 ED 50 value. The dosage of β-BGT was calculated according to 2 times of LD 50 value; the injection dosages of α-BGT, γ-BGT, crude bungarus multicinctus venom, neostigmine methylsulfate, and atropine sulfate were the same as above.

[0037] As Figure 8 , 9 shown: For the combinations of pre-injected β-BGT + γ-BGT and β-BGT + α-BGT, neostigmine methylsulfate had a very significant antidotal effect. For the mice injected with anti-bungarus multicinctus venom serum, the survival time of the mice pre-injected with neostigmine methylsulfate was significantly prolonged compared with that of the mice only injected with anti-bungarus multicinctus venom serum, and the protective effect was significant. For the mice pre-injected with crude bungarus multicinctus venom, it was found that after the combination of neostigmine methylsulfate and anti-bungarus multicinctus venom serum, the survival time was prolonged to a certain extent, but there was no significant protective effect ( Figure 10 ), and it was speculated that it might be related to the too high content of β-BGT in the crude bungarus multicinctus venom. Based on the above results, the use of neostigmine methylsulfate was helpful for the related snake venom treatment. For the combinations of pre-injected β-BGT + γ-BGT and β-BGT + α-BGT, atropine sulfate had a very significant antidotal effect. For the mice injected with anti-bungarus multicinctus venom serum, the survival time of the mice pre-injected with atropine sulfate was significantly prolonged compared with that of the mice only injected with anti-bungarus multicinctus venom serum, and the protective effect was significant. For the mice pre-injected with crude bungarus multicinctus venom, it was found that after the combination of atropine sulfate and anti-bungarus multicinctus venom serum, the survival time was prolonged to a certain extent, but there was no significant protective effect ( Figure 10 ), and it was speculated that it might be related to the too high content of β-BGT in the crude bungarus multicinctus venom. Based on the above results, the use of atropine sulfate was helpful for the related snake venom treatment.

[0038] Based on the above results, whether for the combinations of β-BGT + γ-BGT and β-BGT + α-BGT, or for the crude bungarus multicinctus venom, the use of neostigmine methylsulfate and atropine sulfate had no adverse effects on the subsequent injection of anti-bungarus multicinctus venom serum, and even was helpful for the related snake venom treatment.

[0039] Example 4 Neostigmine methylsulfate had a significant effect of promoting death on naja naja venom in neurotoxic snake venoms.

[0040] To explore the preventive, alleviating, and adjuvant therapeutic effects of neostigmine methylsulfate on different bungarus multicinctus toxins and snakes rich in neurotoxins, neostigmine methylsulfate was pre-injected subcutaneously, and after waiting for the drug to take effect (10 min), naja naja crude venom was injected intraperitoneally. There were 6 mice in each group, and the death time of each group of mice was statistically analyzed. The dosage of naja naja crude venom was calculated according to 3 times of LD50 Value calculation, LD of crude Naja atra venom 50 : 0.5 mg / kg; According to the actual body weight of mice and the conversion relationship between mice and humans: mouse dose (mg / kg) = 9.1 * human dose (mg / kg), the drug dosage was calculated, and neostigmine methylsulfate was 0.1 mg / kg.

[0041] The results showed that: for crude Naja atra venom, neostigmine methylsulfate had a significant effect of promoting the death of mice instead. This indicates that neostigmine methylsulfate is very likely to have a certain synergistic effect of promoting death with some toxins or components in Naja atra venom, suggesting that clinical diagnosis of snake bites must be accurate.

[0042] In summary, neostigmine methylsulfate and atropine sulfate have extremely significant preventive and alleviating effects on the combination of three-finger toxins α-BGT, γ-BGT, and α-BGT + γ-BGT. Neostigmine methylsulfate also has a significant preventive effect on crude Bungarus multicinctus venom. However, neostigmine methylsulfate has a death-promoting effect on Naja atra venom. After intravenous injection of neostigmine methylsulfate and atropine sulfate, it was found that atropine sulfate had a more obvious antidote effect under the same dose. Through experiments, it was found that when the doses of neostigmine methylsulfate and atropine sulfate were doubled, neostigmine methylsulfate would accelerate the death of mice ( Figure 11 、 Figure 6 A), and when the mice were on the verge of death (20 min after injection of Bungarus multicinctus toxin), injection of a normal dose of neostigmine methylsulfate had a significant protective effect instead, which could delay the death of mice, especially when administered intravenously ( Figure 6 B). Neostigmine methylsulfate or atropine sulfate has a preventive or alleviating effect on Bungarus multicinctus bites. When neostigmine methylsulfate and atropine sulfate are used in combination with anti-Bungarus multicinctus venom serum, the survival time is prolonged, and it will not affect the effect of the anti-snake venom serum.

[0043] This invention provides a scientific basis for the treatment of Bungarus multicinctus bites, especially for the situation where there may be no anti-snake venom serum in a short time after being bitten by Bungarus multicinctus in remote mountainous areas or rural areas, and provides an auxiliary treatment measure, especially atropine sulfate has the best effect. After it is determined that it is a Bungarus multicinctus bite, injection of a certain amount of atropine sulfate can extend the treatment time to a certain extent, which is helpful for the treatment of Bungarus multicinctus bites, and has no influence on the subsequent use of anti-snake venom serum injection. At the same time, it can also provide a certain reference for the treatment of severe Bungarus multicinctus bite patients. In addition, the death-promoting effect of neostigmine methylsulfate on Naja atra venom indicates that clinical diagnosis of snake bites must be accurate.

Claims

1. Use of neostigmine methylsulfate or atropine sulfate in the preparation of a drug for preventing, alleviating or assisting in the treatment of Bungarus cobra bites or related neurotoxin poisoning.

2. Use of neostigmine methylsulfate or atropine sulfate in combination with antivenom serum for preparing a drug for treating Bungarus cobra bites or related neurotoxin poisoning.

3. The use according to claim 1 or 2, characterized in that: The neurotoxins are: α-BGT, β-BGT, γ-BGT and a combination thereof.

4. The use according to any one of claims 1 to 3, characterized in that: The administration method of the neostigmine methylsulfate or atropine sulfate is subcutaneous injection or intravenous injection.

5. The use according to claim 4, characterized in that: The dosage of the intravenous neostigmine methylsulfate does not exceed 0.5 mg / kg.

Images