Elapidae neurotoxin enhances opioid analgesic effect and inhibits opioid induced hyperalgesia and tolerance

Abstract

Provided herein is elapidae neurotoxin, and methods for using a pharmaceutically effective amount of said compound to produce synergistic analgesic effect with an opioid for the treatment of pain. In addition, opioid induced hyperalgesia and tolerance can also be alleviated by said compound while administrated separately, or jointly with the opioid.

Claims

1. Claims:

2. A method for treating opioids induced hyperalgesia in a mammal. Said method comprising administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of elapidae neurotoxin, and a pharmaceutically acceptable carrier base for use in inhibiting or controlling opioids induced hyperalgesia.

3. A method for treating opioids induced tolerance in a mammal. Said method comprising administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of elapidae neurotoxin, and a pharmaceutically acceptable carrier base for use in inhibiting or controlling opioids induced tolerance.

4. A method for treating pain in a mammal. Said method comprising administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of elapidae neurotoxin, and a therapeutically effective amount of opioid, and a pharmaceutically acceptable carrier base for use in producing synergistic or better analgesic effect for the patients not satisfying with an opioid as analgesia.

5. A method for treating pain in a mammal. Said method comprising administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of elapidae neurotoxin, and a therapeutically effective amount of opioid, and a pharmaceutically acceptable carrier base for use in prolonging the analgesic effect of an opioid while treating pain .

6. A method for treating pain in a mammal. Said method comprising administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of elapidae neurotoxin, and a therapeutically effective amount of opioid, and a pharmaceutically acceptable carrier base for use in controlling or alleviating the pain in patients who do not respond to an opioid mono therapy .

7. The elapidae neurotoxin according to claim (1-5), characterized in that it is a elapidae neurotoxin polypeptide having the amino acid sequence shown in SEQ ID No. 1 to SEQ ID No. 22; or elapidae neurotoxin polypeptide homologues having 70% or more homology with the elapidae neurotoxin polypeptide of SEQ ID No. 1 to SEQ ID No. 22, and the biological function of the elapidae neurotoxin polypeptide homologues is the same as or similar to that of the elapidae neurotoxin polypeptide of the amino acid sequence ID No. 1 to SEQ ID No. 22.

8. Elapidae neurotoxin polypeptides or elapidae neurotoxin polypeptides homologues according to claim (1-6), characterized in that they can be derived from natural snake venoms, or synthesized from chemical polypeptides, or can be obtained from prokaryotic or eukaryotic hosts using recombinant technology (for example, Bacteria, yeast, higher plants, insects and mammalian cells).

9. The recombinantly produced elapidae neurotoxin polypeptide or its homologues according to claim (7), based on the host used in the recombinant production scheme, the polypeptide or its homologues of the present invention may be glycosylated, or may be non-glycosylated; Disulfide-bonded or non-disulfide-bonded. The polypeptides and its homologues described in the present invention may also include or exclude the starting methionine residue.

10. The elapidae neurotoxin polypeptide according to claim (1-8), further characterized in that the polypeptide in the present invention may include fragments of the above-mentioned various elapidae neurotoxin polypeptides after hydrolysis or enzymolysis, derivatives or analogs treated by physical, chemical or biological method, they are polypeptides which basically maintain the same biological function or activity as the above-mentioned elapidae neurotoxin polypeptide. The fragments, derivatives or analogs described in the present invention may be a polypeptide in which one or more amino acid residues are substituted, or a polypeptide having a substituent group in one or more amino acid residues, or combined with a compound (such as compounds that extend the half-life of a polypeptide, such as polyethylene glycol), or a polypeptide formed by fusion of a fatty chain, or a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence. As described herein, these fragments, derivatives, and analogs are within the scope of those skilled in the art.

11. The method as described in claim (1-5), wherein the respective compounds are administered simultaneously, separately or sequentially.

12. The method as described in claims 3-5, wherein the pain is acute or chronic pain, including traumatic pain, somatic pain, visceral pain, neuropathic pain, post-operative pain, cancer pain, inflammatory pain, fibromyalgia, toothache , Dysmenorrhea, kidney pain, headache, biliary colic, arthralgia, back pain, arthroscopic pain, gynecological laparoscopic pain, and pain caused by burns, rheumatoid arthritis, intraocular hypertension, and virus infection etc.

13. The method as described in claims 1-5 comprising intravenous, intramuscular, subcutaneous, intra-articular, oral, sublingual, nasal, rectal, topical, intradermal, intraperitoneal, intrathecal administration or transdermal administration.

14. The dose of elapidae neurotoxin of the method of claims 1-5 includes from 1 g/Kg to 350 g/kg each time, and the injection frequency ranges from once a day to multiple times a day, or multiple times a year.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 is the line chart of four days average baseline pain threshold (mechanical tail pressure units (g)) test results of mice randomly divided into 4 groups, namely physiological saline group, morphine group, cobrotoxin group, and cobrotoxin+morphine group. There is no significant difference between the results of the 4 groups. Cobrotoxin of amino acid sequence ID No.1 will be used for the test.

[0010] FIG. 2 is the line chart of four days average baseline pain threshold (mechanical tail pressure units (g)) test results of mice randomly divided into 4 groups, namely physiological saline group, morphine group, cobrotoxin group, and cobrotoxin+morphine group. There is no significant difference between the results of the 4 groups. Cobrotoxin of amino acid sequence ID No.2 will be used for the test.

[0011] FIG. 3 is the average pain threshold curve (mechanical tail pressure units (g)) measured during day 5 through day 11 (total of 7 days) of 4 groups of mice to indicate the hyperalgesia induced by administration of 4 different drugs which were Physiological saline, morphine, cobrotoxin, and cobrotoxin+morphine respectively. Cobrotoxin of amino acid sequence ID No.1 was used. The ## symbols indicate a significant statistical difference between the average pain threshold of morphine group and the cobrotoxin+morphine group, P<0.05. No significant statistical differences were detected between Physiological saline group, cobrotoxin group, andcobrotoxin +morphine group.

[0012] FIG. 4 is the average pain threshold curve (mechanical tail pressure units (g)) measured during day 5 through day 11 (total of 7 days) of 4 groups of mice to indicate the hyperalgesia induced by administration of 4 different drugs which were Physiological saline, morphine, cobrotoxin, and cobrotoxin+morphine respectively. Cobrotoxin of amino acid sequence ID No.2 was used. The ## symbols indicate a significant statistical difference between the average pain threshold of morphine group and the cobrotoxin +morphine group, P<0.05. No significant statistical differences were detected between Physiological saline group, cobrotoxin group, andcobrotoxin +morphine group.

[0013] FIG. 5 is the average pain threshold column chart (mechanical pressure units (g)) measured during the fifth, eighth, and eleventh day, one hour after injections of 4 different drugs, which were morphine, physiological saline, cobrotoxin, and cobrotoxin+morphine respectively. The results of 4 groups of mice reflect the effects of analgesic tolerance. Cobrotoxin amino acid sequence ID No.1 was used. Symbols ### mean a significant statistical difference between the average pain threshold of the morphine group and the cobrotoxin+morphine group for the day five, day eight, and day eleven, P<0.01; Symbols ### also indicate a significant statistical difference of average pain threshold within the morphine group of 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.

[0014] FIG. 6 is the average pain threshold column chart (mechanical pressure units (g)) measured during the fifth, eighth, and eleventh day, one hour after injections of 4 different drug , which were morphine, physiological saline, cobrotoxin, and cobrotoxin+morphine respectively. The results of 4 groups of mice reflect the effects of analgesic tolerance. Cobrotoxin amino acid sequence ID No.2 was used. Symbols ### mean a significant statistical difference between the average pain threshold of the morphine group and the cobrotoxin+morphine group for the day five, day eight, and day eleven, P<0.01; Symbols ### also indicate a significant statistical difference of average pain threshold within the morphine group of 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.

[0015] FIG. 7 is the column charts of rat writhing numbers counted at time intervals of 60 minutes, 150 minutes, and 210 minutes after injecting 1 ml of 1.5% acetic acid solution in SD rats at each time interval. Cobrotoxin of amino acid sequence ID No.1 was used.

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.

[0016] FIG. 8 is the column charts of rat writhing numbers counted at time intervals of 60 minutes, 150 minutes, and 210 minutes after injecting 1 ml of 1.5% acetic acid solution in SD rats at each time interval. Cobrotoxin of amino acid sequence ID No.2 was used.

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.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Most widespread of the snake venom neurotoxins are the post synaptically active alpha neurotoxins (Ntx), and they are found widely in Elapidae and Hydrophiid venoms [J. White et al, 1996].

[0018] 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].

[0019] 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.

[0020] 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.

[0021] 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].

[0022] 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.

[0023] 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.

[0024] 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.

TABLE-US-00001 Cobrotoxinofaminoacid sequenceIDNo.1 (lechnqqssqtptttgcsggetncykkrwrdhrgyrterg cgcpsvkngieinccttdrcnn) Cobrotoxinofaminoacid sequenceIDNo.2 (mktllltllvvtivcldlgytlechnqqssqtptttgcsg getncykkrwrdhrgyrtergcgcpsvkngieinccttdrcnn) Cobrotoxinofaminoacid sequenceIDNo.3 (lechnqqssqtptttgcsggetncykkrwrdhrgyrterg cgcpivkngiesnccttdrcnn) Cobrotoxinofaminoacid sequenceIDNo.4 (mechnqqssqapttktcsgetncykkwwsdhrgtiiergc gcpkvkpgvnlnccttdrcnn) Cobrotoxinofaminoacid sequenceIDNo.5 (mechnqqssqtptttgcsggetncykkwwsdhrgtiierg cgcpkvkpgvnlnccttdrcnn) Cobrotoxinofaminoacid sequenceIDNo.6 (lechnqqssqtpttktcsgetncykkwwsdhrgtiiergc gcpkvkpgvnlnccttdrcnn) Bungarotoxinsofaminoacid sequenceIDNo.7 (ivchttatspisavtcppgenlcyrkmwcdafcssrgkvv elgcaatcpskkpyeevtccstdkcnphpkqrpg) Bungarotoxinofaminoacid sequenceIDNo.8 (mktllltlvvvtivcldlgytivchttatspisavtcppg enlcyrkmwcdafcssrgkvvelgcaatcpskkpyeevtc cstdkcnphpkqrpg) BlackMambaNeurotoxinofaminoacid sequenceIDNo.9 (xicynhqsttrattksceenscykkywrdhrgtiiergcg cpkvkpgvgihccqsdkcny) BlackMambaNeurotoxinofaminoacid sequenceIDNo.10 (ricynhqsttrattksceenscykkywrdhrgtiiergcg cpkykpgvgihccqsdkcny) BlackMambaNeurotoxinofaminoacid sequenceIDNo.11 (rtcnktfsdqskicppgenicytktwcdawcsrrgkivel gcaatcpkvkagvgikccstdncnlfkfgkpr) BlackMambaNeurotoxinofaminoacid sequenceIDNo.12 (rtcnktfsdqskicppgenicytktwcdawcsqrgkrvel gcaatcpkvkagveikccstddcdkfqfgkpr) Kingcobraneurotoxinsofaminoacid sequenceIDNo.13 (mktllltlvvmtivcldlgytlicfisshdsvtcapgenv cflkswcdawcgsrgkklsfgcaatcpkvnpgidieccst dncnphpklrp) Kingcobraneurotoxinsofaminoacid sequenceIDNo.14 (tkcyktgdriiseacppgqdlcymktwcdvfcgtrgrvie lgctatcptvkpheqitccstdncdphhkmlq) Kingcobraneurotoxinsofaminoacid sequenceIDNo.15 (tkcyktgdriiseacppgqdlcymktwcdvfcgtrgrvie lgctatcptvkpheqitccstdncnphpkmkq) Kingcobraneurotoxinsofaminoacid sequenceIDNo.16 (mktllltlvvvtivcldlgytrkclntplpliyktcpigq dkcikmtikklpskydvirgcidicpkssadvevlccdtnkcnk) Kingcobraneurotoxinsofaminoacid sequenceIDNo.17 (mknllltflvvtivcldlgytlichrvhglqtcepdqkfc frkttmffpnhpvllmgctyscptekysvccstdkcnk) Kingcobraneurotoxinsofaminoacid sequenceIDNo.18 (mknllltflvvtivcldlgytlichqvhglqtcepaqkfc qirttmffpnhpvllmgctyncpterysvccstdkcnk) Kingcobraneurotoxinsofaminoacid sequenceIDNo.19 (mktllltlvvvtivcldlghtlicvkqytifgvtpeicad gqnlcyktwhmvypggydhtrgcaatcpkmknhdtvhccttdkcnl) Kingcobraneurotoxinsofaminoacid sequenceIDNo.20 (mknllltflvvtivcldlgytlicnrvhglqtcepahkfc fsktvmpfpnhpltlmgctyscpternavccstdkcn) Kingcobraneurotoxinsofaminoacid sequenceIDNo.21 (mktllltlvvvtivcldlgytrkclntplpliyttcpigq dkcvkmtikklpskydvirgcidicpkssadvevlccdtnkcnk) Kingcobraneurotoxinsofaminoacid sequenceIDNo.22 (mknllltflvvtivcldlgytlichqrhglqtcepaqkfc faqtvmpfpnhpltlmgctyscpteknavccstdkcnr)

[0025] 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.

[0026] The following examples are provided to illustrate, but not limit the invention.

EXAMPLES

Example A

[0027] Elapidae neurotoxin preparation

[0028] Separation and Purification of cobrotoxin of amino acid sequence ID No. 1

[0029] Based on lyophilized venom powder from Naja atra, a total of 12 fractions were isolated by cation-exchange chromatography on an open column (502.5 cm I.D.) packed with TSK CM-650(M). The process was performed and described in the following sequence: [0030] i. Venom powder was dissolved in 10 ml of 0.025 M ammonium acetate (pH6.0). [0031] ii. Starting buffer (20-50 mg/ml) was applied to TSK CM-650 column equilibrated with the same buffer. [0032] iii. After the column had been washed with 300 ml of the initial buffer, the proteins adsorbed were eluted with a two-stage linear gradient (0.1-0.5 M and 0.7-1.0 M ammonium acetate buffer). [0033] iv. A reverse-phase HPLC (RP-HPLC) was performed on a Hitachi' liquid chromatograph with a model L-6200 pump. The column eluates (6 ml/tube/7.5 min) were monitored for absorbance at 280 nm. [0034] v. A total of 12 fractions from the aforementioned ion-exchange chromatography were further desalted and purified by a reverse-phase HPLC (RP-HPLC) with Vydac RP-C18 (4.6250 mm, 5.0 um). [0035] vi. The amino acid sequences of 12 fractions were further analyzed using Edman degradation method. [0036] vii. Cobrotoxin of amino acid sequence ID No. 1 was identified.

Example B

In Vivo Anti-Hyperalgesia/Tolerance Model

[0037] 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.

Morphine-Induced Hyperalgesia and Analgesic Tolerance Model

[0038]

TABLE-US-00002 Mice of morphine group receiving morphine injection from day 5-day 11 for 7 consecutive days to induce hyperalgesia and tolerance Mice average pain threshold Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 baseline measurement for 4 days Measurement of pain threshold before injections from day 5 to day 11 Day 1 Day 2 Day 3 Day 4 for 7 consecutive days to test the morphine-induced hyperalgesia Measuring Measuring Measuring tolerance tolerance tolerance after after after injection injection injection in day 5 in day 8 in day 11

[0039] Cobrotoxin of amino acid sequence ID No.1 and cobrotoxin of amino acid sequence ID No.2 were used in parallel for the tests.

[0040] Detailed steps are as follows:

Step1. Establishment of mice's baseline pain threshold.

[0041] 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. [0042] i. Put the mouse gently into the restraint and put its tail under the conical tip of the pain test apparatus. Press the pedal switch, and increase the pressure on the proximal end of the tail evenly until signs of the first pain response (struggle, squeaking) occurs. Record pain-inducing pressure when pain response occurs (units: g) as the value of pain threshold. The pressure was released upon reaching 700 g with no indication of a response to avoid tissue injury. Measurements were also conducted at the middle and distal ends of the tail of the same mouse with a minimum of 30 seconds time intervals between each measurement. [0043] ii. Put the tested mice in a cage and test the next one until every mouse in the group is tested. The average of the three measurements (i.e., the proximal, mid and distal ends of each mouse tail) was used as the pain threshold (g) of each mouse when all mice were subjected to a tail pressure test. [0044] iii. In the following 3 days, all mice underwent repeated measurements of tail pressure pain threshold. [0045] iv. The pain threshold of the mice measured by the tail pressure test ranged from 180 g to 220 g. The mice were then randomly divided into four groups: namely physiological saline group, morphine group, cobrotoxin group and cobrotoxin+morphine group. Each group comprised of 20 mice, which is used for the hyperalgesia and tolerance test of the corresponding drugs in the group's name. Any surplus mice were excluded. [0046] v. Lastly, each group of 20 mice was divided randomly again into two groups, with each group comprising 10 mice, as cobrotoxin of amino acid sequence ID NO.1, and cobrotoxin of amino acid sequence ID NO. 2 will be used for parallel testing

[0047] As we can see there were no significant statistical differences between the 4 groups of mice in both FIG. 1 and FIG. 2.

Step2. Morphine-Induced hyperalgesia and analgesic tolerance in mice

[0048] 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 FIG. 3 and FIG. 4.

[0049] 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.

[0050] 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.

[0051] 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 FIG. 5 and FIG. 6.

Example C

In Vivo Synergistic Analgesic Effects and Prolongation of Morphine's Analgesia Time Model

[0052] 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.

TABLE-US-00003 Four groups of rats (10/group) were injected with sterile saline (NaCl 0.9% 1 ml), morphine (3 mg/kg), cobrotoxin (50 ug/kg), or cobrotoxin (25 ug/kg) + morphine (1.5 mg/kg) respectively. 60 minutes after 150 minutes after 210 minutes after the initial the initial the initial injection injection injection Injection of 1.5% Injection of 1.5% Injection of 1.5% acetic acid solution acetic acid solution acetic acid solution for writhing test for writhing test for writhing test To test the To test cobrotoxin's To test cobrotoxin's synergistic analgesic ability to prolong ability to prolong effect of cobrotoxin morphine's morphine's analgesia combined with morphine analgesia time time
Details as follows:

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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 FIG. 7 and FIG. 8

Step2. Prolongation of morphine's analgesia effect by cobrotoxin

[0057] 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.

[0058] 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.

[0059] 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 FIG. 7 (cobrotoxin of amino acid sequence ID NO.1 was used), and FIG. 8 (cobrotoxin of amino acid sequence ID NO.2 was used).

Example D

[0060] Test of pro-inflammatory cytokines IL-113 and IL-6, NOS activity, and NO content in tissues of mice.

[0061] Further studies were conducted on the mechanism of cobrotoxin's inhibition of hyperalgesia and tolerance, which were mainly focused on the determination of IL-1 and IL-6 blood level, NOS activity, and NO content at tissues of mice.

[0062] The specific steps were as follows: [0063] i. After completion of the aforementioned morphine tolerance and hyperalgesia tests, mice of morphine group and cobrotoxin +morphine group were set aside for 2 hours followed by anesthesia with chloral hydrate, and then dislocated. [0064] ii. The extraction of the lumbar spinal cord was performed quickly on a plate with ice, then washed with icy water. [0065] iii. The spinal tissues, weighed, then put into pre-frozen physiological saline, 4000 revolution/separation for 10 minutes, and prepared into 10% homogenate which was measured for IL-1, IL-6, NOS activity, and NO content. [0066] iv. The ELISA method was applied to determine the tested values of the lumbar spinal cord tissues. The release of IL-1, IL-6, NOS and NO was detected according to the instruction in the insert. [0067] v. Coomassie brilliant blue dye was used to determine the total protein content in the homogenate of each sample.

[0068] The levels of IL-1, 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)

TABLE-US-00004 Biomarker Group Value SD t-test IL-1 pg/mg protein Morphine Group 16.80 2.39 P < 0.01 Morphine + cobrotoxin 11.30 1.83 Group IL-6 pg/mg protein Morphine Group 19.40 2.12 P < 0.01 Morphine + cobrotoxin 14.80 1.75 Group NOS U/mg protein Morphine Group 7.56 0.14 P < 0.01 Morphine + cobrotoxin 7.15 0.12 Group NO mol/g protein Morphine Group 1.61 0.03 P < 0.01 Morphine + cobrotoxin 1.32 0.02 Group

[0069] 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.

[0070] 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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