Patent Application
20210008177
The field of the present invention relates to a composition of mater of elapidae neurotoxin, which is a nicotinic acetylcholine receptors (nAChR) modulator (inhibitors or antagonists of nAChR) that can bind to nAChR to produce analgesic and anti-inflammatory effect. Elapidae neurotoxin, while combining with an opioid, they can produce synergistic analgesic effect, and on top of that, opioid induced hyperalgesia and tolerance can also be controlled by elapidea neurotoxin. Pharmaceutical formulations including the elapidae neurotoxin , independently or in combination with an opioid, and a pharmaceutically acceptable carrier base for use in the treatment of aforementioned conditions thereof.
Opium is a substance extracted from poppy plants which binds to the opioid receptors of the human body itself and provides pain relief or analgesia to patients with acute or chronic pain. Morphine is an example of an opioid analgesia. Short-term use of opioid drugs is considered as a safe pain relief method. However, when they are frequently administered and/or overused, a series of problems arise which include hyperalgesia, tolerance build-up, drug dependency, constipation, respiratory Inhibition, and fatal overdose. According to the CDC, In 2015, over 33,000 people died in US due to opioid-related overdoses.
Opioid receptor agonists such as morphine are the most widely used potent analgesic drug in clinics. The medicine can effectively relieve the pain of patients, especially post-operative and advanced cancer pain. However, the use of opioids such as morphine often leads to tolerance build-up and hyperalgesia in patients which can occur in as little as a week. And once tolerance and hyperalgesia develops, the patient requires increasing dosages to maintain the drug's initial effectiveness. In other words, the body needs a greater dose of medication to achieve the same analgesic effect as it initially provided; larger doses lead to greater hyperalgesia, the faster build-up of tolerance, constipation, addiction, and respiratory suppression. Therefore, to achieve both the clinical efficacy and the acceptance of treatment, opioid induced hyperalgesia and tolerance are urgent problems in need of a real solution.
Opioid induced analgesic tolerance and hyperalgesia are two closely related yet different symptoms. The former refers to the significant decline in analgesic effects of an opioid, whilst the latter refers to the patient's abnormal pain response to non-injurious stimuli or a highly sensitive pain response to the same injurious stimuli caused by continuous or incorrect applications of opioid receptor agonists such as morphine. Analgesic tolerance and hyperalgesia, although two different adverse reactions induced by opioid receptor agonists, both impede the long-term clinical use of opioids. Consequently, patients under the long-term prescription of such drugs are left to face severe side effects, thus a major unmet clinical need.
Presently, treatments for opioids induced tolerance and hyperalgesia mostly remain in experimental stages. Although the combination of opioids and other drugs has become an effective strategy for enhancing the analgesic effect of opioids, and to reduce the opioid dose, however, there is no singular drug with proven efficacy that could produce synergistic effect, and at same time, address analgesic tolerance and hyperalgesia. Thus clinically, there is a demand for a product that can fully satisfy the unmet needs of patients.
The analgesic effect of elapidae neurotoxins have been previously documented (US Patent Application Number 16403651). However, elapidae neurotoxin's ability to inhibit or control opioid induced hyperalgesia and tolerance has never been reported before. Elapidae neurotoxins have no dependence on the opioid system, and no hyperalgesia or tolerance were observed during analgesic process. When combined with opioids, the combination can produce a synergistic analgesic effect and prolong opioid's effective time. These unique properties were also first proved by our invention, thereby the elapidae neurotoxin is expected to be developed into a safe and effective analgesic agent.
The primary purpose of the invention is to provide a composition of matter using elapidae neurotoxin to inhibit or to control the hyperalgesia and tolerance caused by opioids.
The further purpose of the invention is to provide a composition of matter to produce a synergistic analgesic effect for the treatment of pain by combining an elapidae neurotoxin with an opioid, thus avoid to increase the dose of an opioid. Finally, the invention is to provide a composition of matter for treating the patient not responding to opioid (morphine) as an mono therapy, but satisfying with the combination of an opioid and an elapidae neurotoxin.
Symbols *** represent a significant statistical difference of average pain threshold between the “cobrotoxin group” and the “cobrotoxin +morphine group” for the day five, day eight, and day eleven, P<0.01.
Symbols *** represent a significant statistical difference of average pain threshold between the “cobrotoxin group” and the “cobrotoxin+morphine group” for the day five, day eight, and day eleven, P<0.01.
Symbols ### show a significant statistical difference in the number of writhing between “morphine group” and “cobrotoxin+morphine group” at 60, 150, and 210 minutes time intervals after injection, P<0.01; Symbols ### also indicate a significant statistical difference in the number of writhing within the morphine group between 60, 150, and 210 minutes time intervals after injection, P<0.01.
Symbols *** indicate significant statistical differences in the number of writhing between “cobrotoxin group” and “cobrotoxin +morphine group” at 60, 150, and 210 minutes time intervals after injection, P<0.01.
Symbols ### show a significant statistical difference in the number of writhing between “morphine group” and “cobrotoxin+morphine group” at 60, 150, and 210 minutes time intervals after injection, P<0.01; Symbols ### also indicate a significant statistical difference in the number of writhing within the morphine group between 60, 150, and 210 minutes time intervals after injection, P<0.01.
Symbols ** and *** indicate significant statistical differences in the number of writhing between “cobrotoxin group” and “cobrotoxin +morphine group” at 60, 150, and 210 minutes time intervals after injection, P<0.05 and P<0.01 respectively.
Most widespread of the snake venom neurotoxins are the post synaptically active alpha neurotoxins (αNtx), and they are found widely in Elapidae and Hydrophiid venoms [J. White et al, 1996].
Elapidae neurotoxins are antagonists of nicotinic acetylcholine receptors (nAChR) which bind to muscle and neuronal nAChR in an antagonistic and slow reversible manner. Such elapidae neurotoxins are known as postsynaptic neurotoxins or alpha-neurotoxins due to their ability to block nAChR [Naguib M et al, 2002; Abbas M et al, 2016]. Structurally they have a three-finger appearance, with the active site near the tip of the middle finger [J. White et al, 1996], and this three-finger appearance is a multifunctional structural scaffold able to modulate cholinergic functions [Pascale Marchot et al, 2017].
nAChR influences pain, senses, cognition, neuronal protection, and neurotransmitter transmission [Li Jiangbing et al, 2017]. Elapidae neurotoxins produce analgesic effects through modulating nAChR without the involvement of the opioid receptors system. When combine with an opioid, elapidae neurotoxins can synergize the analgesic effect through anti-inflammatory function.
According to published experimental data, pro-inflammatory cytokines are associated with various types of pain, one of which is pathological neuralgia. Neuropathic pain, pain caused by artificial subcutaneous formalin injection or subarachnoid injection increases the secretion of IL-1B level significantly, whilst blocking IL-1B receptors can reduce pain [Milligan et al, 2001]. IL-6 can induce mechanical pain sensitivity and hyperalgesia, knockout IL-6 gene can inhibit pain in rats with sciatic nerve ligation [Murphy et al, 1999]. Pro-inflammatory cytokines can increase pain in several ways, in the presence of a cytokine receptor on the neurons, pro-inflammatory cytokines may act directly on the neurons of the central nervous system to augment pain; pro-inflammatory cytokines can augment pain by modulating the transmission of incoming neural signals onto primary nerve fibers as well.
Pro-Inflammatory cytokines can also induce astrocytes and small glial cells to increase the synthesis and release of nitric oxide (NO) and activate nitric oxide synthase (NOS). These substances indirectly increase the magnitude of pain [Xiang hongbing et al, 2004; Haberberger et al, 2003; Rainer Viktor et al, 2002; Papadopolou. S et al, 2004; Watkins et al, 2001]. According to published experimental data, morphine-induced hyperalgesia and tolerance are accompanied by high levels of IL-1, IL-6, NOS activity, and NO content [liang huichun, 2014; Jian daolin 2005]. Experimental data also show that numerous nicotinic acetylcholine receptor (nAChR) acts as an important intermediate link in regulating pro-inflammatory cytokines, NOS activity, and NO content. nAChR antagonists either directly reduce pro-inflammatory cytokines, NOS activity or NO content, or activate certain specific nAChR (e.g., a7-nAChR, a9-nAChR), to reduce pro-inflammatory cytokines, NOS activity or NO content [Zakrzewicz A, J et al, 2017; Patel et al, 2017; Papadopolou S, et al, 2004; Thippeswamy T, et al, 2001; Richter K, et al, 2016]. Other experimental results demonstrate that nAChR antagonists are directly involved in the process of reducing neuropathic pain [Pacini A et al, 2016; Romero H K et al, 2017; Vincler M et al, 2006; Luo S, et al, 2015; Holtman J R et al, 2011; Wala E P et al, 2012].
Elapidae neurotoxin, as the major antagonist of nicotinic acetylcholine receptor, has been shown in our experiments to be able to reduce pro-inflammatory cytokines, NOS activity and NO content, which is in line with the reported function of other nicotinic acetylcholine receptor antagonists.
Elapidae neurotoxins, on top of its independent analgesic effect, exhibit strong anti-inflammatory properties as well, and patients under opioids induced hyperalgesia and tolerance experience neuron inflammation, therefore, elapidae neurotoxins demonstrate dual mechanisms while treating opioids induced hyperalgesia and tolerance.
The main elapidae neurotoxins include cobrotoxins, bungarotoxins, neurotoxins from black mamba, and neurotoxins from king cobra, they all have the common three-finger appearance structure. The following elapidea neurotoxins were proved effective in enhancing opioid analgesic effect and in inhibiting opioids induced hyperalgesia and tolerance in our experiments.
The amino acid sequences of the above elapidea neurotoxins are submitted separately in ASCII text file in the name of “sequence listing”, created 2020-Aug.-12, with size of 16 KB.
The following examples are provided to illustrate, but not limit the invention.
Elapidae neurotoxin preparation
Separation and Purification of cobrotoxin of amino acid sequence ID No. 1
Based on lyophilized venom powder from Naja atra, a total of 12 fractions were isolated by cation-exchange chromatography on an open column (50×2.5 cm I.D.) packed with TSK CM-650(M). The process was performed and described in the following sequence:
To evaluate the therapeutic effects elapidea neurotoxins, one of the reliable morphine induced hyperalgesia/tolerance model in mice (Elhabazi, K et al) was created, and effects of the representative EXAMPLE compounds were investigated on the model.
Cobrotoxin of amino acid sequence ID No.1 and cobrotoxin of amino acid sequence ID No.2 were used in parallel for the tests.
Detailed steps are as follows:
Step1. Establishment of mice's baseline pain threshold.
100 Kunming mice were subjected to tail pressure tests for 4 days to measure the mechanical pain threshold and the average pain threshold will be set as the baseline pain threshold.
As we can see there were no significant statistical differences between the 4 groups of mice in both
Step2. Morphine-Induced hyperalgesia and analgesic tolerance in mice
After 4 days of measurement of the mice's baseline pain threshold described in step 1, from the fifth day to the eleventh day, the measurement of pain threshold was performed before the injection. Each 4 groups of mice (total 8 groups) underwent the tail pressure test first and then were injected with morphine (5 mg/kg), sterile saline (NaCl 0.9% 1 ml), cobrotoxin (50 ug/kg), or cobrotoxin (50 ug/kg)+morphine (5 mg/kg) respectively. The test results indicate that the mean pain threshold of the “morphine group” is significantly lower than that of the other 3 controlled groups. Cobrotoxin of amino acid sequence ID NO.1, and cobrotoxin of amino acid sequence ID NO. 2 were used for parallel testing. The experimental data is shown in
Parallelly, in the 5th, 8th, and 11th day, an hour after the injection of 4 different drugs, the tail pressure test was applied again to measure the pain threshold of each mouse of the “morphine group”, the “physiological saline group”, the “cobrotoxin group”, and the “cobrotoxin +morphine group” to determine the analgesic tolerance of these four drugs.
The test results indicate that within the “morphine group”, the mean pain threshold was significantly decreased from day 5 to day 11, with highest in day 5, and lowest in day 11; between the “morphine group” and “morphine+cobrotoxin group”, the mean pain threshold was also different. The mean pain threshold of the ‘cobrotoxin+morphine group’ is higher than that of ‘cobrotoxin group’, and the difference was statistically significant for day 5, 8, and 11 as well. The experimental data suggests that the cobrotoxin can inhibit morphine-induced analgesic tolerance, and cobrotoxin+morphine can produce a stronger analgesic effect than morphine or cobrotoxin alone.
Cobrotoxin of amino acid sequence ID NO.1, and cobrotoxin of amino acid sequence ID NO. 2 were used for parallel testing. The experimental data is shown in
Synergistic analgesic effect and ability to prolong morphine's effective time of the representative EXAMPLE compounds were investigated on the model. Writhing test was applied to the rats, which is a chemical method used to induce pain of the peripheral origin by injection of irritant principles like acetic acid in rats. Analgesic effect of the test compound is inferred from the decrease in the frequency of writhe.
Details as follows:
Step1. Synergistic analgesic effect of cobrotoxin combined with morphine 80 SD rats were randomly divided into “physiological saline group”, “morphine group”, “cobrotoxin group” and “cobrotoxin+morphine group” with 20 rats in each group, and finally, each group will be divided again into two groups for 2 cobrotoxins parallel testing.
The aforementioned four groups of rats were injected with sterile saline (NaCl 0.9% 1 ml), morphine (3 mg/kg), cobrotoxin (50 ug/kg), and cobrotoxin (25 ug/kg)+morphine (1.5 mg/kg) respectively.
60 minutes after injection, 1.5% acetic acid solution was injected to SD rats (1 ml/rat). experiment results show the analgesic effect provided by half dose cobrotoxin (25ug/kg)+half dose morphine (1.5 mg/kg) is significantly higher compared to a single full dose of morphine (3 mg/kg), or a single full dose of cobrotoxin (50 ug/kg). This means that the cobrotoxin+morphine produces superior analgesic improvement rather than an additive one, indicating a synergistic analgesic effect.
Cobrotoxin of amino acid sequence ID NO.1, and cobrotoxin of amino acid sequence ID NO. 2 were used for parallel testing. The experimental data is shown in
Step2. Prolongation of morphine's analgesia effect by cobrotoxin
Following Step1, the aforementioned four groups SD rats were injected with 1.5% acetic acid solution (1 ml/rat) again 150 and 210 minutes after the initial injection of 4 different drugs respectively.
The test result indicated that SD rats of morphine group showed lower analgesic effect after 150 minutes, and almost no signs of any analgesic effect after 210 minutes; the SD rats of cobrotoxin group retained signs of analgesic effect but was inferior in comparison with the SD rats of cobrotoxin+morphine group with a significant statistical difference. The test results demonstrate the synergy formed when combining half a dose of cobrotoxin and half a dose of morphine. The combination has a stronger analgesic effect than a single full dose of cobrotoxin or morphine, and this synergistic effect did not decline with the decrease of morphine's analgesic effect at 150 and 210 minutes, which showed a prolonged analgesic effect of morphine through combination with cobrotoxin.
After the four groups of SD rats received injection of their respective drugs, the mean number of writhing per hour measured after 60, 150, and 210 minutes of initial injection was shown in
Test of pro-inflammatory cytokines IL-113 and IL-6, NOS activity, and NO content in tissues of mice.
Further studies were conducted on the mechanism of cobrotoxin's inhibition of hyperalgesia and tolerance, which were mainly focused on the determination of IL-1β and IL-6 blood level, NOS activity, and NO content at tissues of mice.
The specific steps were as follows:
The levels of IL-1β, IL-6, NOS activity, and NO content detected in the “morphine group” and “cobrotoxin+morphine group” were as follows: (cobrotoxin of amino acid sequence ID NO.1 was used for the test)
The experimental data showed that the level of IL-1B, IL-6, NOS activity, and NO content of the “morphine group” were significantly higher than that of “cobrotoxin+morphine group”.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is, therefore, to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2019106533746 | Jul 2019 | CN | national |
