Inhibition of hyperalgesia and tolerance to opioids by cobra neurotoxin peptides and their synergistic analgesic effects

Abstract

Opioid receptor agonists, with morphine being a representative example, are the most widely used, powerful analgesics in clinical practice. These drugs effectively relieve pain, particularly postoperative pain and pain associated with advanced cancer. However, opioid use can easily lead to tolerance and hyperalgesia, often developing within a week. Once tolerance and hyperalgesia develop, the dose must be continuously increased to maintain the analgesic effect. This means that the body requires larger doses of the drug to achieve the same analgesic effect as the initial dose. Higher doses, however, can lead to more severe hyperalgesia, tolerance, constipation, addiction, and respiratory depression. Cobra neurotoxin peptides can both inhibit the hyperalgesia and tolerance associated with opioids and enhance their efficacy. When used in combination with opioids, they enable the latter to achieve analgesia at lower doses without developing tolerance and hyperalgesia, thus resolving the clinical challenge of long-term opioid use.

Description

Technical field:

[0001] The present invention belongs to the field of biopharmaceuticals and relates to a new clinical application of cobra neurotoxin, and in particular, relates to the clinical application of inhibiting the side effects of opium drugs and synergizing the analgesic effect of opium drugs. Background technology:

[0002] Opioids are substances extracted from the poppy plant. They bind to the body's own opioid receptors and, when legally prescribed, provide pain relief or analgesia for patients with acute or chronic pain; morphine is an example of an opioid analgesic. Short-term use of these medications is considered a safe method of pain management. However, when overused and / or used frequently, they can cause a range of problems, including pain hypersensitivity, tolerance, drug dependence, constipation, respiratory depression, and fatal overdose. According to the Centers for Disease Control and Prevention, opioid overdoses resulted in more than 33,000 deaths in 2015.

[0003] Opioid receptor agonists, such as morphine, are the most widely used, potent analgesics in clinical practice. These drugs can effectively relieve pain, particularly postoperative pain and pain associated with advanced cancer. However, the use of opioids like morphine can easily lead to tolerance and hyperalgesia, often developing within a week. Once tolerance and hyperalgesia develop, the dose must be continuously increased to maintain the analgesic effect. This means the body requires larger doses of the drug to achieve the same analgesic effect as the initial dose. Higher doses, in turn, can lead to more severe hyperalgesia, tolerance, constipation, addiction, and respiratory depression. Hyperalgesia and tolerance to opioids can develop through a variety of routes of administration, at varying doses (from ultra-low to high doses), and over varying durations of administration (e.g., intermittent or continuous). Therefore, addressing opioid hyperalgesia and tolerance is a pressing issue to achieve both clinical efficacy and treatment acceptance.

[0004] Drug-induced analgesic tolerance and hyperalgesia are two closely related and easily confused, yet distinct symptoms. The former refers to a significant decrease in the analgesic effect of opioid receptor agonists after long-term use, necessitating larger doses of the drug to achieve or maintain the same initial analgesic effect. The latter refers to abnormal pain responses to non-noxious stimuli or hyperalgesia to the same noxious stimuli after long-term or inappropriate use of opioid receptor agonists, such as morphine. Thus, analgesic tolerance refers to a decrease in the ability of a drug to alleviate pain, while hyperalgesia refers to an increased reactivity to painful stimuli. Although analgesic tolerance and hyperalgesia are distinct adverse reactions induced by opioid receptor agonists, they both hinder the long-term use of morphine-like drugs in clinical practice, causing significant suffering for patients who require long-term use and representing a significant unmet clinical need.

[0005] Currently, treatments for morphine tolerance and hyperalgesia are mostly still in the experimental stage, including electroacupuncture and medications such as minocycline, pentoxifylline, statins, and resveratrol. While the combination of opioids and other medications has become an effective strategy for enhancing the analgesic effects of opioids and inhibiting analgesic tolerance and hyperalgesia, there is still no proven effective treatment available, leading to a clinically promising product that can truly satisfy both doctors and patients.

[0006] Cobra neurotoxin peptides are nicotinic acetylcholine receptor (nAChR) antagonists. They bind to muscle and neuronal nicotinic acetylcholine receptors (nAChR) in an antagonistic and slowly reversible manner. These cobra neurotoxin peptides are called postsynaptic neurotoxins or α-neurotoxins because they can block the function of nicotinic acetylcholine receptors (nAChR). [1, 2] Nicotinic acetylcholine receptors (nAChR) are involved in sensation, cognition, pain, neuronal protection, and neurotransmitter transmission. [3] Therefore, when nicotinic acetylcholine receptors (nAChR) are blocked, cognition, pain, neuronal protection, and neurotransmitter transmission will be affected to varying degrees. Summary of the invention:

[0007] The analgesic effects of cobra neurotoxin peptides have been reported, but this is the first time they have been shown to both inhibit the development of opioid hyperalgesia and tolerance and enhance the analgesic efficacy of opioids. Cobra neurotoxin peptides do not suffer from the drug dependency or dosage increase issues associated with opioids during analgesia. When combined with opioids, this combination allows opioids (such as morphine) to achieve the same or even better analgesic effects at lower doses than high-dose morphine, while avoiding and inhibiting the development of opioid hyperalgesia and tolerance, allowing for long-term use. This addresses the clinical challenges of opioid tolerance and hyperalgesia.

[0008] Cobra neurotoxin peptides, when combined with morphine, exhibit synergistic analgesic effects and significantly prolong the duration of morphine's analgesic effect. In clinical pain treatment, this combination can reduce the dose of morphine and extend the dosing interval. As a non-morphine analgesic, cobra neurotoxin peptides have the potential to be developed as an effective adjuvant for opioid analgesia due to their ability to antagonize morphine analgesic tolerance and hyperalgesia, thereby reducing the serious adverse reactions of opioids such as morphine.

[0009] The significance of the present invention is that it may allow patients to use a drug that can antagonize analgesic tolerance and hyperalgesia caused by opioids; another significance of the present invention is that it may be possible to develop an adjuvant that, when used in combination with opioids, can enable the latter to still effectively relieve pain at lower doses or even better, thereby avoiding serious side effects such as respiratory depression, constipation, and addiction caused by the need to continuously increase the dose of morphine for long-term analgesia, and at the same time, due to this effect, the purpose of long-term use of opioids is met; the last significance of the present invention may be that it can allow patients to use this adjuvant in combination to enhance the analgesic effect of opioids when the analgesic effect of a certain dose of opioids is not ideal. This will be used to treat patients who are clinically ineffective or have poor analgesic effects with higher doses of morphine, but the combined use of the two still has a good analgesic effect without the need to increase the dose of morphine again.

[0010] Implementation method:

[0011] The present invention establishes a morphine-induced mouse pain hypersensitivity and analgesia tolerance model, and conducts systematic experiments on the time-effect relationship and dose-effect relationship of the analgesic effect after the cobra neurotoxin polypeptide and morphine are used in combination, including the effects of superposition enhancement, synergistic enhancement, and prolonged action time of morphine, to observe the effects of the cobra neurotoxin polypeptide in the above-mentioned aspects through the model.

[0012] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0013] Implementation steps:

[0014] A. Measurement of basal pain values in mice. From the first to the fourth day, 100 Kunming mice were subjected to a 4-day tail pressure test to measure the mechanical pain values of the mice, which were used as basal pain values.

[0015] 1. Gently place the mouse in a restrainer and place its tail under the conical tip of the analgesic instrument. Press the foot switch and evenly increase the pressure on the proximal end of the tail until the first pain response (struggle, squeak) occurs. When the animal responds, record the pressure (in grams) that causes pain as the pain value. In the absence of any response, use a cutoff value of 700 g to avoid tissue damage. Repeat this measurement at the middle and distal ends of the tail of the same mouse, with an interval of at least 30 seconds.

[0016] 2. Place the tested mouse back into its cage and test the next mouse until all mice in the group have been tested. The average of the three measurements (i.e., the proximal, mid, and distal ends of each mouse's tail) is used as the pain value (in grams) for each mouse. Continue until all mice have undergone the tail pressure test.

[0017] 3. On the following 3 days, the tail pressure pain value test was repeated for all mice.

[0018] 4. Among the mice whose pain values were measured by the tail pressure test above, those with pain values greater than 220 grams and less than 180 grams were removed, and then the mice were randomly divided into 4 groups, namely "normal saline group", "morphine group", "cobra neurotoxin peptide group" and "cobra neurotoxin peptide + morphine group", with 20 mice in each group, to conduct pain hypersensitivity and analgesic tolerance tests on the corresponding drugs, and the excess mice were removed from the group.

[0019] 5. Finally, each group of 20 mice was divided into two groups, each containing 10 mice, and used for parallel experiments on the amino acid sequence of the cobra neurotoxin polypeptide SEQ ID NO.1 and the amino acid sequence of the cobra neurotoxin polypeptide SEQ ID NO.2, respectively.

[0020] The average values of basal pain response (mechanical pressure unit: grams) of each group of mice are shown in Figures 1 and 2:

[0021] Figure 1 This is a line graph showing the average values of basal pain responses over four days for the "normal saline group," "morphine group," "cobra neurotoxin peptide group," and "cobra neurotoxin peptide + morphine group." The amino acid sequence of the cobra neurotoxin peptide used is SEQ ID NO. 1.

[0022] Figure 2The graph is a line graph showing the average values of the four-day basal pain response for the "normal saline group," the "morphine group," the "cobra neurotoxin peptide group," and the "cobra neurotoxin peptide + morphine group." The amino acid sequence of the cobra neurotoxin peptide used is SEQ ID NO. 2.

[0023] The average value of the tail pressure pain response of each group of mice will be used as the basic pain value to provide a comparison for the pain values produced by the four drugs "normal saline", "morphine", cobra neurotoxin peptide, and cobra neurotoxin peptide + morphine in the next experiment.

[0024] B. Establishment of morphine-induced pain hypersensitivity and analgesia tolerance models in mice

[0025] Principle of the morphine-induced pain hypersensitivity and analgesia tolerance model in mice:

[0026] Mice were injected with morphine for 7 consecutive days to induce pain hypersensitivity and analgesia tolerance. Starting after the first injection, the pain sensation of the mice was measured before injection for 6 consecutive days as an indicator of morphine-induced pain hypersensitivity. The pain sensation of the mice was measured on the day of injection, the 4th day, and the 7th day after injection to measure the analgesic effect of morphine and serve as an indicator of analgesia tolerance. These indices were compared with the indices of the control mice ("saline group", "cobra neurotoxin peptide group", "cobra neurotoxin peptide + morphine group") and the basal pain values of the mice to confirm whether the mice developed pain hypersensitivity and analgesia tolerance after continuous morphine injection. Cobra neurotoxin peptide (amino acid sequence SEQ ID NO. 1) and cobra neurotoxin peptide (amino acid sequence SEQ ID NO. 2) were used in parallel for each of the following experiments.

[0027] The specific steps are as follows:

[0028] 1. On day 5, the four groups of mice were subcutaneously injected with sterile saline (0.9% NaCl 1 ml), morphine (5 mg / kg), cobra neurotoxin peptide (50 μg / kg), and cobra neurotoxin peptide (50 μg / kg) plus morphine (5 mg / kg). One hour later (as the maximum analgesic effect of morphine is expected to be 45-90 minutes), the tail pressure pain response of each mouse in the "saline group," "morphine group," "cobra neurotoxin peptide group," and "cobra neurotoxin peptide + morphine group" was measured using the tail pressure test (steps 1-2 in A above) to assess the analgesic effects of the four drugs.

[0029] 2. From the sixth to the eleventh day, the pain sensation of each mouse in the "saline group", "morphine group", "cobra neurotoxin peptide group", and "cobra neurotoxin peptide + morphine group" was measured by tail pressure test (steps 1-2 in A above) as an indicator of pain hypersensitivity before receiving their respective drug injections; then the administration method of the fifth day was repeated, and the above four groups of mice were subcutaneously injected with sterile saline (NaCl 0.9% 1 ml), morphine (5 mg / kg), cobra neurotoxin peptide (50 ug / kg), and cobra neurotoxin peptide (50 ug / kg) + morphine (5 mg / kg), respectively.

[0030] 3. On the eighth and eleventh days, one hour after the injection, the tail pressure test was again used to measure the pain response value of each mouse in the "normal saline group", "morphine group", "cobra neurotoxin peptide group" and "cobra neurotoxin peptide + morphine group" (steps 1-2 in A above) to understand the analgesic tolerance of the above four drugs.

[0031] From the fifth to the eleventh day (a total of 7 days), the average pain response values (i.e., pain hypersensitivity index, mechanical pressure unit: gram) of the four groups of mice measured by the tail pressure test before injection are shown in Figures 3 and 4.

[0032] Figure 3 This is a line graph showing the average pain response measured by the tail pressure test before injection for the "saline group," "morphine group," "cobra neurotoxin peptide group," and "cobra neurotoxin peptide + morphine group" from days 5 to 11. The amino acid sequence of the cobra neurotoxin peptide used is SEQ ID NO. 1.

[0033] Figure 4 This is a line graph showing the average pain response measured by the tail pressure test before injection for the "saline group," "morphine group," "cobra neurotoxin peptide group," and "cobra neurotoxin peptide + morphine group" from days 5 to 11. The amino acid sequence of the cobra neurotoxin peptide used is SEQ ID NO. 2.

[0034] ## indicates that there is a statistically significant difference between the values in the “morphine group” and the “cobra neurotoxin peptide + morphine group”, P < 0.05.

[0035] The average pain response values (i.e., analgesia tolerance, mechanical pressure unit: gram) of each group of mice in the "normal saline group", "morphine group", "cobra neurotoxin polypeptide group" and "cobra neurotoxin polypeptide + morphine group" measured by tail pressure test one hour after drug injection on the fifth, eighth and eleventh days are shown in Figures 5 and 6.

[0036] Figure 5It is a bar graph corresponding to the pain response values of the "normal saline group", "morphine group", "cobra neurotoxin polypeptide group" and "cobra neurotoxin polypeptide + morphine group" (analgesic tolerance). The amino acid sequence of the cobra neurotoxin polypeptide used is SEQ ID NO.1.

[0037] The pain response value of the "cobra neurotoxin peptide + morphine group" was higher than that of the "morphine group" and the "cobra neurotoxin peptide" group, and the difference was statistically significant. The experimental data not only suggested that cobra neurotoxin peptide can inhibit morphine-induced analgesic tolerance, but also proved that cobra neurotoxin peptide has an additive and synergistic effect on the analgesia of morphine.

[0038] Figure 6 It is a bar graph corresponding to the pain response values of the "normal saline group", "morphine group", "cobra neurotoxin polypeptide group" and "cobra neurotoxin polypeptide + morphine group" (analgesic tolerance). The amino acid sequence of the cobra neurotoxin polypeptide used is SEQ ID NO.2.

[0039] The pain response value of the "cobra neurotoxin peptide + morphine group" was higher than that of the "morphine group" and the "cobra neurotoxin peptide" group, and the difference was statistically significant. The experimental data not only suggested that cobra neurotoxin peptide can inhibit morphine-induced analgesic tolerance, but also proved that cobra neurotoxin peptide has an additive and synergistic effect on the analgesia of morphine.

[0040] ### indicates a statistically significant difference between the "morphine group" and the "cobra neurotoxin peptide + morphine group," P < 0.01; it also indicates a statistically significant difference between the values on days 5, 8, and 11 in the morphine group, P < 0.01. *** indicates a statistically significant difference between the "cobra neurotoxin peptide group" and the "cobra neurotoxin peptide + morphine group," P < 0.01.

[0041] The specific experimental steps for the synergistic analgesic effect of cobra neurotoxin peptides and opioid drugs and their prolonged analgesic duration are as follows:

[0042] C. Synergistic analgesia study of cobra neurotoxin peptides and morphine

[0043] 1. Forty SD rats were randomly divided into "normal saline group", "morphine group", "cobra neurotoxin peptide group" and "cobra neurotoxin peptide + morphine group", with 10 rats in each group.

[0044] 2. The above four groups of rats were subcutaneously injected with sterile saline (NaCl 0.9% 1 ml), morphine (3 mg / kg), cobra neurotoxin polypeptide (50 ug / kg) and cobra neurotoxin polypeptide (25 ug / kg) + morphine (1.5 mg / kg).

[0045] 3. 60 minutes after the injection, 1 ml of 1.5% acetic acid solution was injected intraperitoneally into SD rats to establish the model. The average number of writhing bodies of rats in each group within 1 hour in the "normal saline group", "morphine group", "cobra neurotoxin peptide group" and "cobra neurotoxin peptide + morphine group" was measured by the rat writhing test as the possible maximum analgesic effect of the four drugs.

[0046] Figure 7 The average number of writhings within one hour after 60 minutes of subcutaneous injection of sterile saline (0.9% NaCl 1 ml), morphine (3 mg / kg), cobra neurotoxin peptide (50 ug / kg), and cobra neurotoxin peptide (25 ug / kg) + morphine (1.5 mg / kg) into four groups of rats were injected intraperitoneally with 1 ml (1.5%) acetic acid solution. The cobra neurotoxin peptide used was the amino acid sequence of SEQ ID NO.1.

[0047] Figure 8 The average number of writhings within one hour after 60 minutes of subcutaneous injection of sterile saline (0.9% NaCl 1 ml), morphine (3 mg / kg), cobra neurotoxin peptide (50 ug / kg), and cobra neurotoxin peptide (25 ug / kg) + morphine (1.5 mg / kg) into four groups of rats were injected intraperitoneally with 1 ml (1.5%) acetic acid solution. The cobra neurotoxin peptide used had the amino acid sequence SEQ ID NO.2.

[0048] It can be seen that the analgesic effect of half a dose of cobra neurotoxin peptide (25ug / kg) + half a dose of morphine (1.5mg / kg) is superior to that of a full dose of morphine (3mg / kg) and a full dose of cobra neurotoxin peptide (50ug / kg) alone, and is significantly better. The above data show that cobra neurotoxin peptide + morphine can produce an analgesic effect of 1+1 greater than 2, which is a synergistic analgesic effect.

[0049] D. Experiment on prolonging the analgesic effect of morphine with cobra neurotoxin peptides

[0050] Continuing with Experiment C above, the four groups of SD rats were intraperitoneally injected with 1 ml of 1.5% acetic acid solution 150 minutes and 210 minutes after injection to establish the model. The SD rats in the morphine group had no analgesic effect after 150 minutes and 210 minutes. The SD rats in the cobra neurotoxin peptide group still had an analgesic effect, but the efficacy was still inferior to that of the half-dose cobra neurotoxin peptide (25 μg / kg) + half-dose morphine (1.5 mg / kg) group, and the difference was statistically significant. Therefore, it was shown that cobra neurotoxin peptide + morphine not only had a synergistic effect, but also that the half-dose cobra neurotoxin peptide + half-dose morphine significantly prolonged the analgesic duration compared to the full-dose morphine alone. It also had a stronger analgesic effect than the full-dose cobra neurotoxin peptide alone, and this synergistic analgesic effect was not weakened by the decreasing analgesic effect of morphine alone, thus demonstrating that cobra neurotoxin peptide can prolong the analgesic effect of morphine.

[0051] 1 ml (1.5%) acetic acid solution was injected intraperitoneally into the four groups of rats 60 minutes after the injection of the four drugs respectively. The number of writhings within one hour after 60, 150 and 210 minutes is shown in Figures 7 and 8.

[0052] Figure 7 is a bar graph showing the number of writhings within one hour after 60, 150, and 210 minutes after intraperitoneal injection of 1 ml (1.5%) acetic acid solution in four groups of rats, which were subcutaneously injected with sterile saline (0.9% NaCl 1 ml), morphine (3 mg / kg), cobra neurotoxin polypeptide (50 ug / kg), and cobra neurotoxin polypeptide (25 ug / kg) + morphine (1.5 mg / kg). The cobra neurotoxin polypeptide used was the amino acid sequence SEQ ID NO.1.

[0053] Figure 8 is a bar graph showing the number of writhings within one hour, 60, 150, and 210 minutes after intraperitoneal injection of 1 ml (1.5%) acetic acid solution 60 minutes after subcutaneous injection of sterile saline (0.9% NaCl 1 ml), morphine (3 mg / kg), cobra neurotoxin polypeptide (50 ug / kg), and cobra neurotoxin polypeptide (25 ug / kg) + morphine (1.5 mg / kg) in four groups of rats. The cobra neurotoxin polypeptide used is the amino acid sequence SEQ ID NO.2.

[0054] In Figures 7 and 8, ### indicates statistically significant differences between the values in the "morphine group" and the "cobra neurotoxin peptide + morphine group," P < 0.01; it also indicates statistically significant differences between the values at 60, 150, and 210 minutes in the morphine group, P < 0.01. ** and *** indicate statistically significant differences between the values in the "cobra neurotoxin peptide group" and the "cobra neurotoxin peptide + morphine group," P < 0.05 and P < 0.01, respectively.

[0055] Mechanism studies:

[0056] We have also conducted further research on the mechanism by which cobra neurotoxin peptides inhibit hyperalgesia and tolerance to opioids. The specific steps are as follows:

[0057] Determination of IL-1β, IL-6, NOS activity and NO content

[0058] After the morphine tolerance and hyperalgesia tests were completed, mice participating in the cobra neurotoxin peptide-induced opioid hyperalgesia and tolerance model were anesthetized with chloral hydrate and sacrificed by dislocation 2 hours after the last day of the experiment. Lumbar spinal cords were then quickly removed on an ice-cold plate and rinsed with ice water. The spinal cord tissue to be assayed for IL-1β, IL-6, and NOS activity and NO content was weighed and placed in pre-chilled saline. The tissue was centrifuged at 4000 rpm for 10 minutes to prepare a 10% homogenate. The values of the above parameters in the lumbar spinal cord tissue were determined by ELISA. The release of IL-1β, IL-6, NOS, and NO was detected according to the manufacturer's instructions. The total protein content in the homogenates of each sample was determined by Coomassie Brilliant Blue. The following parameters were measured for the morphine and cobra neurotoxin peptide-induced morphine groups:

[0059] Table-9

[0060]

[0061] The activities of IL-1B, IL-6, NO and NOS in the morphine tolerance group were significantly higher than those in the cobra neurotoxin peptide + morphine group, and the levels were statistically significant.

[0062] According to publicly reported experimental results, proinflammatory cytokines are associated with various types of pain, including pathological neuralgia. IL-1B secretion by spinal cord glial cells significantly increases in neuropathic pain, pain caused by artificial subcutaneous injection of formalin, or subarachnoid injection of analgesics, and blocking IL-1B receptors can relieve pain; [4] IL-6 can induce mechanical allodynia and hyperalgesia, and IL-6 gene knockout can inhibit the pain behavior of rats with sciatic nerve ligation. [5] At the same time, proinflammatory cytokines can indeed aggravate pain through multiple pathways. When cytokine receptors are expressed on neurons, proinflammatory cytokines may directly act on neurons in the central nervous system to enhance pain; this type of cytokine can also enhance pain by regulating the release of neurotransmitters from primary afferent nerve fibers.

[0063] Proinflammatory cytokines can also induce astrocytes and microglia to increase the synthesis and release of excitatory amino acids (EAAs), activate nitric oxide synthase (NOS), and increase the synthesis of nitric oxide (NO). These substances indirectly increase pain intensity. [6-10] According to published experimental data, morphine-induced pain hypersensitivity and tolerance are also accompanied by peak levels of IL-1, IL-6, NOS activity, and NO content. [11-12] In our experiments, we found that cobra neurotoxin peptides can reduce the levels of these inflammatory factors and related substances.

[0064] In addition, a considerable amount of experimental data has demonstrated that nicotinic acetylcholine receptors (nAChRs) are diverse and serve as an important intermediate in regulating the aforementioned proinflammatory cytokines, NOS, and NO. Nicotinic acetylcholine receptor antagonists increase the levels of nicotinic acetylcholine receptor agonists (such as nicotine and GTS21) in the body by antagonizing nAChRs. These agonists either directly reduce proinflammatory cytokines, NOS activity, or NO levels, or reduce proinflammatory cytokines, NOS activity, and NO levels by activating specific nAChRs, such as a7-nAChR and a9-nAChR. [13-17] The ultimate result achieved through these various pathways is a reduction in the levels of these related substances. Finally, other published experimental results have also confirmed that nAChR antagonists are directly involved in reducing neuropathic pain. [18-23]

[0065] Cobra neurotoxin peptide, as the most important nicotinic acetylcholine receptor antagonist, has also been shown in our experiments to reduce the activity of proinflammatory cytokines and NOS, and the content of NO, which is consistent with the regulatory effects of other nicotinic acetylcholine receptor antagonists in the literature on proinflammatory cytokines IL-1B, IL-6, NOS, and NO.

[0066] Cobra neurotoxin polypeptides are the most important nicotinic acetylcholine receptor (nAChR) antagonists. They either regulate pro-inflammatory cytokines, such as NOS and NO, which are closely related to pain, through nicotinic acetylcholine receptors (nAChRs); or through direct effects. This is all related to the common property of cobra neurotoxin polypeptides as antagonists of nicotinic acetylcholine receptors. The present invention used two different cobra neurotoxin polypeptides to conduct experiments and achieved almost consistent experimental results, which also confirmed this common property of cobra neurotoxin polypeptides.

[0067] The above examples are intended only to illustrate the implementation principles and technical features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be included in the scope of protection of the present invention.

[0068] References:

[0069] 1.Naguib M et al.Advances in neurobiology of the neuromuscularjunction: implications for the anesthesiologist.J Am Soc Anesthesiol, 2002, 96: 202-31.

[0070] 2.Abbas M, Rahman S. Effects of α α-7nicotinic acetylcholine receptorpositive allosteric modulator on lipopolysaccharide-induced neuroinflammatorypain in mice. Eur J Pharmacol, 2016, 783: 85-91.

[0071] 3. Li Jiangbing et al. Research progress on the interaction between α-neurotoxins and nicotinic acetylcholine receptors, Life Science, Vol. 29, No. 1, January 2017.

[0072] 4.Milligan et al.Intrathecal HIV-1envelop glycoprotein gp 120enhancedstates mediated by spinal code proinflammatory cytokins.J neuroscience 2001(8)2808-2819.

[0073] 5.Murphy et al. Endogenous interleukin-6contribute to hypersensitivity to cutanous stimuli and changes in neuropeptides associated with chroni nerveconstriction in mice. Eur J neurosci 1999, 11 (7), 2243-2253.

[0074] 6. Xiang Hongbing et al. Effects of ketamine on spinal cord astrocytes in morphine-tolerant mice. Chinese Journal of Anesthesiology, 2004, 24(4)290-293.

[0075] 7. Rainer Viktor et al. The role of spinal neuroimmune activation inmorphine induced tolerance / hyperplasia in neuropathic and sham operated rats. J Neurosci 2002, 22(22)9980-9989.

[0076] 8.Haberberger et al. Nicotinic receptor alpha7-subunits are coupled to the stimulation of nitric oxide synthase in rat dorsal root ganglion neurons. Histochem Cell Biol(2003)120:173-181.

[0077] 9. S. Papadopolou et al. Nicotinic receptor mediated stimulation of NO-generation in neurons of rat thoracic dorsal root ganglia. Neuroscience Letters 361 (2004) 32-35.

[0078] 10.Watkins et al. Spinal cord glia new player in pain, Pain 2001, 93(3): 201-205.

[0079] 11. Liang Huichun. Evaluation of the efficacy of Guiyuan in treating morphine analgesia tolerance and hyperalgesia and its mechanism. Academy of Military Science, PLA, 2014.

[0080] 12. Jian Dowling et al. Effects of morphine tolerance on IL-1B and IL-6 levels in the spinal cord of rats. Journal of Practical Medicine, Vol. 21, No. 20, 2005.

[0081] 13. Zakrzewicz A, J et al. (2017) Canonical and novel noncanonicalcholinergic agonists inhibit ATP-induced release of monocytic interleukin-1beta via different combinations of nicotinic acetylcholine receptor subunitsalpha7, alpha9 and alpha10. Front Cell Neurosci 11, 189.

[0082] 14.Patel et al. Anti-inflammatory effects of astroglial α7 nicotinicacetylcholine receptors are mediated by inhibition of the NF-κB pathway and activation of the Nrf2 pathway Journal of Neuroinflammation (2017) 14: 192.

[0083] 15.Papadopolou S,et al.Nicotinic receptor mediated stimulation of NO-generation in neurons of rat thoracic dorsal root ganglia.Neurosci Lett 361,32-35.77(2004).

[0084] 16.Thippeswamy T,et al.Inhibition of neuronal nitric oxide synthaseresults in neurodegenerative changes in the axotomised dorsal root ganglionneurons:evidence for a neuroprotective role of nitric oxide invivo.Neuroscience Research 2001.05.

[0085] 17.Richter K,et al.(2016)Phosphocholine-an agonist of metabotropicbut not of ionotropic functions of alpha9-containing nicotinic acetylcholinereceptors.Sci Rep 6,28660.

[0086] 18.Pacini A et al.The alpha9alpha10 nicotinic receptor antagonistalpha-conotoxin RgIA prevents neuropathic pain induced by oxaliplatintreatment.Exp Neurol 2016.282,37-48.

[0087] 19.Romero HK et al.Inhibition of a9a10 nicotinic acetylcholinereceptors prevents chemotherapy-induced neuropathic pain.Proc Natl Acad SciUSA 2017.114,1825-1832.

[0088] 20.Vincler M et al.Molecular mechanism for analgesia involvingspecific antagonism of alpha9alpha10 nicotinic acetylcholine receptors.ProcNatl Acad Sci USA 2006.103,17880-17884.

[0089] 21.Luo S,et al.Cloning,synthesis,and characterization ofalphaOconotoxin GeXIVA,a potent alpha9alpha10 nicotinic acetylcholinereceptor antagonist.Proc Natl Acad Sci USA 2015.112, E4026-E4035.

[0090] 22.Holtman JR et al.The novel small molecule alpha9alpha10 nicotinicacetylcholine receptor antagonist ZZ-204G is analgesic.Eur J Pharmacol2011.670,500-508.

[0091] 23.Wala EP et al.Novel small molecule alpha9alpha10 nicotinicreceptor antagonist prevents and reverses chemotherapy-evoked neuropathicpain in rats.Anesth Analg.2012.115,713-720.

Claims

1. Use of a cobra neurotoxin polypeptide having an amino acid sequence as shown in SEQ ID NO. 1 or 2 in the preparation of a drug for inhibiting hyperalgesia caused by opium drugs, characterized in that: The opioid drug is morphine.

2. Use of a cobra neurotoxin polypeptide having an amino acid sequence as shown in SEQ ID NO. 1 or 2 in the preparation of a drug for inhibiting analgesic tolerance caused by opioid drugs, characterized in that: The opioid drug is morphine.

3. The use according to any one of claims 1 to 2, characterized in that: The cobra neurotoxin polypeptide is separated and extracted from natural snake venom, or synthesized by chemical polypeptide, or produced from a prokaryotic or eukaryotic host using recombinant technology.

4. The use according to any one of claims 1 to 2, characterized in that: The administration of the drug includes intravenous injection, intramuscular injection, subcutaneous injection, intra-articular injection, oral administration, sublingual administration, nasal cavity administration, rectal administration, intradermal administration, intraperitoneal administration, intrathecal administration or transdermal administration.

5. The use according to any one of claims 1 to 2, characterized in that: The dosage of the cobra neurotoxin ranges from 1 μg / Kg to 350 μg / kg each time, the dosage of the opioid analgesic ranges from 1 mg / Kg to 200 mg / Kg each time, and the injection frequency ranges from once a day to multiple times a day; or multiple times a year.

Images