Modified polypeptide and use thereof in field of analgesia

Abstract

The present application relates to a peptide or a derivative of the peptide, a pharmaceutical composition containing the peptide or the derivative of the peptide, and the use thereof in the treatment of pain.

Description

Modified polypeptides and their applications in analgesia

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application PCT / CN2023 / 101880 filed on June 21, 2023. The prior application is deemed to be part of the disclosure of this application and is incorporated in its entirety into this application. Technical Field

[0003] The present application relates to the field of biomedicine, and specifically to a peptide or its derivatives and their use in analgesia. Background Art

[0004] Neuropathic pain refers to pain caused by lesions or diseases of the somatic sensory nervous system. It is a common chronic pain that seriously affects the quality of life of patients. According to statistics, about 8% of the world's population suffers from it (Gilron I, Baron R, Jensen T. Neuropathic pain: principles of diagnosis and treatment [J]. Mayo Clin Proc, 2015, 90 (4): 532-545). Metabolic disorders (such as diabetic neuropathy), nerve entrapment, tumor-related pain, post-stroke pain, Parkinson's disease-related pain, post-spinal cord injury pain, trigeminal neuralgia, glossopharyngeal neuralgia, viral infections such as post-herpetic neuralgia, etc. are all typical neuropathic pain (Szok D, Tajti J, A, et al. Therapeutic approaches for peripheral and central neuropathic pain[J]. Behav Neurol, 2019, 2019:8685954. and Jensen TS, Baron R, Haanp M, et al. A new definition of neuropathic pain[J]. Pain, 2011, 152(10):2204-2205).

[0005] Peripheral neuropathy is characterized by nerve damage and axonal loss and can be divided into inherited or acquired forms. Acquired peripheral neuropathy is associated with a variety of causes, including exposure to toxic agents, among which pain induced by anti-tumor compounds is called chemotherapy-induced peripheral neuropathy (CIPN). A review summarizes the neuroimmune interactions in several preclinical studies of CIPN, suggesting a link between chemotherapeutic drugs and neurotoxicity. Studies have demonstrated the involvement of immune responses (innate and adaptive) and the stimulation and secretion of mediators (cytokines and chemokines) as possible causes of pain symptoms. In addition, neuroinflammatory components have also been shown in the spinal cord, with microglia and astrocytes playing an important role in CIPN (Front Immunol. 2021 Feb 4:11:626687).

[0006] Nicotinic acetylcholine receptors (nAChRs) are membrane proteins with important physiological functions that are ubiquitous in nature and can regulate a range of physiological functions in organisms, such as pain, cognition, memory, and anxiety. Acetylcholine receptors are widely present in nerve cells and are closely related to analgesia. Their subtypes are mainly α7, α9, and α10 (see, for example, Hone AJ et al., Nicotinic acetylcholine receptors in neuropathic and inflammatory pain, FEBS lett. 2018 April; 592(7): 1045–1062 and Wu Pei et al., The role of α7 nicotinic acetylcholine receptors in regulating animal inflammatory responses, Journal of Animal Nutrition, 2021, 33(11): 6001-6008). Cone snails are carnivorous mollusks and are the general term for animals of the family Conus. There are approximately 700 species of cone snails in the world, mainly distributed in tropical waters. Conotoxin is a neurotoxin secreted by cone snails, mainly composed of 10 to 30 amino acids and containing disulfide bonds. The team of Professor Olivera at the University of Utah, USA, isolated and identified the sequences and biological activities of various cone snail toxins, and found that cone snail toxins may become potential drugs for the treatment of various neurological diseases. This has triggered a wave of research on the development, structural modification and mechanism of action of cone snail toxins by researchers at home and abroad. Among them, Vc1.1, also known as ACV1, is a polypeptide containing 16 amino acids and two pairs of disulfide bonds. It is an α-conotoxin with analgesic effects isolated from Conus victoriae. Vc1.1 is a selective antagonist of α9α10 nicotinic acetylcholine receptors (nAChR) and has a therapeutic effect on neuropathic pain in rat sciatica (CCI) and neuropathic pain (PNL) models. However, due to differences in α9α10 nAChR activity between humans and mice, Vc1.1, which had already entered Phase II clinical trials, showed a 100-fold decrease in human α9α10 nAChR activity compared to mouse α9α10 nAChR activity, forcing the relevant authorities to suspend the clinical study. Lorenzo Di Cesare Mannelli et al. investigated the analgesic effects and mechanisms of the cone snail toxin peptide RgIA in a rat model of chronic constriction injury (CCI) induced by sciatic nerve ligation. Histological analysis of sciatic nerves revealed that RgIA prevented CCI-induced reductions in axonal density and diameter, myelin loss, and fiber reduction. Furthermore, RgIA significantly reduced edema and inflammatory infiltration, including a decrease in CD86+ macrophages. In the dorsal horn of the spinal cord, RgIA prevented CCI-induced microglial and astrocyte activation.These data suggest that the peptide RgIA may represent a new class of drugs for the treatment of neuropathic pain by protecting peripheral nerve tissue and preventing central maladaptive plasticity by inhibiting glial cell activation (PAIN:155(2014)1986–1995).

[0007] Ziconotide was approved by the FDA in 2004 for the treatment of severe chronic pain. It was originally isolated from the venom of the cone snail Conus magus and contains 25 amino acids and three pairs of disulfide bonds. As a selective Cav2.2 channel antagonist, ziconotide blocks Ca 2+ Ziconotide is a drug that infuses and inhibits the release of excitatory neurotransmitters from afferent nerve endings, making it 1,000 times more effective at relieving pain than opioids. However, the unique intrathecal administration method and a series of adverse reactions to the central nervous system result in poor patient compliance, thus limiting its widespread use.

[0008] Therefore, there is an urgent need to develop an improved peptide that acts on nicotinic acetylcholine receptors and can effectively relieve analgesia and / or reduce adverse reactions.

[0009] Summary of the Invention

[0010] The targets targeted by the polypeptide of the present application include α7nAChR and α9nAChR, and the target is the acetylcholine receptor of the human sequence. This can avoid the reduced activity of cone snail toxin Vc1.1 due to species differences and similar risks. The polypeptide sequence of the present application introduces multiple non-natural amino acids and the polypeptide structure contains a pair of intramolecular disulfide bonds, which enhances the stability of the polypeptide, significantly increases the half-life of the polypeptide in the body, and significantly prolongs the duration of the analgesic effect. Pain is a very serious clinical problem that can have an adverse effect on the quality of life of patients and their families. However, existing analgesics cannot specifically solve different types of pain, and there are also adverse reactions to long-term medication, such as the addiction of opioids, and long-term use will cause adverse consequences of drug abuse. Polypeptides have inherent safety advantages. The polypeptide synthesis process of the present application is simple and easy to commercialize. It is expected to be a new type of safe and effective analgesic drug.

[0011] According to the results of animal experiments, the peptide of the present application has a faster onset time than the clinical analgesic drug pregabalin, and the stability of the peptide in the application has been significantly improved in human plasma compared to the parent peptide AJ003. From the animal experimental data, it can also be found that the duration of the analgesic effect of the modified peptide is significantly prolonged compared to the parent peptide. The peptide of the present application belongs to a new type of non-opioid analgesic drug. Since its target is mainly present in peripheral nerve tissue, it will not cause toxic side effects such as drug addiction in the later clinical stage. Compared with the only cone snail toxin peptide ziconotide currently on the market, the peptide of the present application does not need to adopt the complex administration method of intrathecal instillation, which is conducive to improving the medication compliance of clinical patients. In addition, ziconotide is composed of 25 amino acids and contains 3 pairs of disulfide bonds in the peptide sequence. The preparation process is complicated, which greatly increases the production cost. The peptide of the present application is composed of 13 amino acids and contains only 1 pair of disulfide bonds. The production and preparation process is simple. Solid-phase peptide synthesis technology is currently very mature, and its synthesis characteristics are very conducive to automated large-scale production, which can greatly reduce the cost of drug production and reduce the medical burden on patients. Furthermore, the peptide of the present application has a low administration dose and exhibits significant analgesic activity even at a low dose.

[0012] In a first aspect of the present disclosure, a peptide comprising any one sequence selected from SEQ ID NOs: 1-34 or a derivative thereof is provided.

[0013] In a second aspect of the present disclosure, provided is a peptide comprising any one sequence selected from SEQ ID NOs: 1-34 having conservative substitutions, or a derivative thereof.

[0014] In a third aspect of the present disclosure, there is provided a peptide or a derivative thereof comprising the following formula I or a conservatively substituted formula I in order from N-terminus to C-terminus: Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8 Formula I

[0015] The Cys at position 3 and the D-Cys at position 9 in Formula I form a pair of intramolecular disulfide bonds, D-Cys represents D-cysteine, "-" between amino acid residues represents a peptide bond, the C-terminus of the peptide or its derivative is a free carboxyl group or an amide, X1 is Thr or D-Thr, X5 is selected from any one of Ala, Val and D-Val, and X2-X4 and X6-X8 are independently any amino acid.

[0016] In one embodiment of the present disclosure, the 3rd Cys and the 9th D-Cys in the above formula I form a pair of intramolecular disulfide bonds, the C-terminus of the peptide or its derivative is a free carboxyl group or an amide, and X1 is Thr or D-Thr, X5 is selected from any one of Ala, Val and D-Val, X2 is selected from any one of Pro, D-Pro and Hyp, X3 is selected from any one of Pro, D-Pro and Hyp, X4 is selected from any one of Ser, D-Ser, Ala and Aib, X6 is selected from any one of Ala, Leu, D-Leu, Ile, ABu and Nle, X7 is selected from any one of Tyr, D-Tyr, Ala, Trp, Phe and Bip, and X8 is selected from any one of Ser, D-Ser, Ala, and Orn.

[0017] In one embodiment of the present disclosure, the above-mentioned Formula I with conservative substitutions has 1, 2, 3, 4 or 5 amino acid conservative substitutions. In one embodiment of the present disclosure, the above-mentioned Formula I with conservative substitutions has at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity or at least 95% sequence identity with Formula I without conservative substitutions.

[0018] In a preferred embodiment of the present disclosure, the peptide or its derivative comprises any one sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-16, and 20-34, or SEQ ID NOs: 1-5, 10-16, and 20-34 with conservative substitutions. In a more preferred embodiment of the present disclosure, the peptide or its derivative comprises any one sequence selected from the group consisting of SEQ ID NOs: 26, 28, and 32, or SEQ ID NOs: 26, 28, and 32 with conservative substitutions.

[0019] In one embodiment of the present disclosure, the above-mentioned sequences with conservative substitutions have 1, 2, 3, 4 or 5 amino acid conservative substitutions, respectively. In one embodiment of the present disclosure, the above-mentioned sequences with conservative substitutions have at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity or at least 95% sequence identity with the sequences without conservative substitutions.

[0020] In a fourth aspect of the present disclosure, a polynucleotide is provided, which encodes the peptide or derivative thereof according to the present disclosure.

[0021] In a fifth aspect of the present disclosure, an expression vector is provided, which expresses the polynucleotide according to the present disclosure.

[0022] In a sixth aspect of the present disclosure, a host cell is provided, comprising the polynucleotide according to the present disclosure or the expression vector according to the present disclosure.

[0023] In a seventh aspect of the present disclosure, a pharmaceutical composition is provided, comprising the peptide or derivative thereof according to the present disclosure and a pharmaceutically acceptable carrier or salt thereof. The present disclosure also provides a pharmaceutical composition, comprising the peptide or derivative thereof, polynucleotide, expression vector or host cell according to the present disclosure, and a pharmaceutically acceptable carrier or salt thereof.

[0024] In an eighth aspect of the present disclosure, use of the peptide or derivative thereof according to the present disclosure, or the pharmaceutical composition according to the present disclosure, in the preparation of a medicament for treating pain is provided. The present disclosure also provides use of the peptide or derivative thereof according to the present disclosure, the polynucleotide according to the present disclosure, the expression vector according to the present disclosure, the host cell according to the present disclosure, or the pharmaceutical composition according to the present disclosure, in the preparation of a medicament for treating pain.

[0025] In a ninth aspect of the present disclosure, a method for treating pain is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide or derivative thereof according to the present disclosure, or a pharmaceutical composition according to the present disclosure. The present disclosure also provides a method for treating pain, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide or derivative thereof, a polynucleotide, an expression vector, a host cell, or a pharmaceutical composition according to the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following provides a brief description of the drawings, which are provided to illustrate exemplary embodiments disclosed herein and not to limit these embodiments.

[0027] FIG1 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-1 over time in Example 3.

[0028] FIG2 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-34 over time in Example 3.

[0029] FIG3 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-35 over time in Example 3.

[0030] FIG4 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-36 over time in Example 3.

[0031] FIG5 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-37 over time in Example 3.

[0032] FIG6 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-38 over time in Example 3.

[0033] FIG7 shows the prediction results of the in vitro half-life of polypeptide AJ003-39 in human plasma in Example 3.

[0034] FIG8 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-40 over time in Example 3.

[0035] FIG9 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-41 over time in Example 3.

[0036] FIG10 shows a line graph showing the change in the main peak area of ​​polypeptide AJ003-42 over time in Example 3.

[0037] Figure 11 shows the mechanical pain threshold on day 1 of the rat paclitaxel chronic chemotherapy pain model administered with the same dose of the polypeptide in Example 5. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are shown as the mechanical pain threshold (g) (±SD). Graphpad, one-way analysis of variance (n=7-8). Model group vs. control group: ####p<0.0001; drug administration group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: not statistically significant. Pregabalin was administered at 30 mg / kg·d·qd by gavage; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ 003-39, and AJ 003-40 were administered at 1 mg / kg·d·qd by subcutaneous injection.

[0038] Figure 12 shows the mechanical pain threshold on day 3 of the rat paclitaxel chronic chemotherapy pain model administered with the same dose of the polypeptide in Example 5. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way analysis of variance (n=7-8). Model group vs. control group: ####p<0.0001; drug administration group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: not statistically significant. Pregabalin was administered at 30 mg / kg·d·qd by gavage; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ 003-39, and AJ 003-40 were administered at 1 mg / kg·d·qd by subcutaneous injection.

[0039] Figure 13 shows the mechanical pain threshold on day 5 of the rat paclitaxel chronic chemotherapy pain model administered with the same dose of the polypeptide in Example 5. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way analysis of variance (n=7-8). Model group vs. control group: ####p<0.0001; drug administration group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: not statistically significant. Pregabalin was administered at 30 mg / kg·d·qd by gavage; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ 003-39, and AJ 003-40 were administered at 1 mg / kg·d·qd by subcutaneous injection.

[0040] Figure 14 shows the mechanical pain threshold on day 12 of the rat paclitaxel chronic chemotherapy pain model administered with the same dose of the polypeptide in Example 5. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way analysis of variance (n=7-8). Model group vs. control group: ####p<0.0001; drug administration group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: not statistically significant. Pregabalin was administered at 30 mg / kg·d·qd by gavage; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ 003-39, and AJ 003-40 were administered at 1 mg / kg·d·qd by subcutaneous injection.

[0041] Figure 15 shows the mechanical pain threshold on day 1 of the rat paclitaxel chronic chemotherapy pain model administered with different doses of the polypeptide in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: #### p < 0.0001; drug administration group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns: not statistically significant. Pregabalin was 30 mg / kg·d·qd, administered orally; AJ003 was 1 mg / kg·d·qd, administered subcutaneously; AJ003-36-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; AJ003-36-high dose was 1.5 mg / kg·d·qd, administered subcutaneously; AJ003-40-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; and AJ003-40-high dose was 1.5 mg / kg·d·qd, administered subcutaneously.

[0042] Figure 16 shows the mechanical pain threshold on day 4 of the rat paclitaxel chronic chemotherapy pain model administered with different doses of the polypeptide in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: #### p < 0.0001; drug administration group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns: not statistically significant. Pregabalin was 30 mg / kg·d·qd, administered orally; AJ003 was 1 mg / kg·d·qd, administered subcutaneously; AJ003-36-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; AJ003-36-high dose was 1.5 mg / kg·d·qd, administered subcutaneously; AJ003-40-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; and AJ003-40-high dose was 1.5 mg / kg·d·qd, administered subcutaneously.

[0043] Figure 17 shows the mechanical pain thresholds on day 7 of the rat paclitaxel chronic chemotherapy pain model administered with different doses of the polypeptide in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: #### p < 0.0001; drug administration group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns: not statistically significant. Pregabalin was 30 mg / kg·d·qd, administered orally; AJ003 was 1 mg / kg·d·qd, administered subcutaneously; AJ003-36-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; AJ003-36-high dose was 1.5 mg / kg·d·qd, administered subcutaneously; AJ003-40-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; and AJ003-40-high dose was 1.5 mg / kg·d·qd, administered subcutaneously.

[0044] Figure 18 shows the mechanical pain thresholds on day 12 of the rat paclitaxel chronic chemotherapy pain model administered with different doses of the polypeptide in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: #### p < 0.0001; drug administration group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns: not statistically significant. Pregabalin was 30 mg / kg·d·qd, administered orally; AJ003 was 1 mg / kg·d·qd, administered subcutaneously; AJ003-36-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; AJ003-36-high dose was 1.5 mg / kg·d·qd, administered subcutaneously; AJ003-40-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; and AJ003-40-high dose was 1.5 mg / kg·d·qd, administered subcutaneously.

[0045] Figure 19 shows the mechanical pain thresholds on day 14 of the rat paclitaxel chronic chemotherapy pain model administered with different doses of the polypeptide in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different groups. The results are presented as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: #### p < 0.0001; drug administration group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns: not statistically significant. Pregabalin was 30 mg / kg·d·qd, administered orally; AJ003 was 1 mg / kg·d·qd, administered subcutaneously; AJ003-36-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; AJ003-36-high dose was 1.5 mg / kg·d·qd, administered subcutaneously; AJ003-40-low dose was 0.5 mg / kg·d·qd, administered subcutaneously; and AJ003-40-high dose was 1.5 mg / kg·d·qd, administered subcutaneously.

[0046] Figure 20 shows the changes in mechanical pain threshold over time in mice treated with different doses of the peptide in the acute oxaliplatin chemotherapy pain model described in Example 7. Results are presented as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: #### p < 0.0001; drug group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns: not statistically significant.

[0047] Figure 21 shows the mechanical pain thresholds on day 1 in rats following administration of different doses of the polypeptides described in Example 9 in the CCI model. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns: not statistically significant.

[0048] Figure 22 shows the mechanical pain thresholds on day 4 in the rat CCI model following administration of different doses of the polypeptide described in Example 9. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; ** p < 0.01; ns: not statistically significant.

[0049] Figure 23 shows the mechanical pain thresholds on day 7 in the rat CCI model following administration of different doses of the polypeptide described in Example 9. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; ** p < 0.01; * p < 0.05. ns: not statistically significant.

[0050] Figure 24 shows the mechanical pain thresholds on day 10 in the rat CCI model following administration of different doses of the polypeptide described in Example 9. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. ns: not statistically significant.

[0051] Figure 25 shows the mechanical pain thresholds on day 14 in rats treated with different doses of the polypeptide described in Example 9 in the CCI model. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01. ns: not statistically significant.

[0052] Figure 26 shows the mechanical pain thresholds on day 1 in the diabetic neuropathic pain model of mice following administration of different doses of the polypeptide described in Example 10. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. ns: not statistically significant.

[0053] Figure 27 shows the mechanical pain thresholds on day 4 in the diabetic neuropathic pain model of mice following administration of different doses of the polypeptide described in Example 10. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. ns: not statistically significant.

[0054] Figure 28 shows the mechanical pain thresholds on day 8 in the diabetic neuropathic pain model of mice following administration of different doses of the polypeptide described in Example 10. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. ns: not statistically significant.

[0055] Figure 29 shows the mechanical pain thresholds on day 14 in the diabetic neuropathic pain model of mice following administration of different doses of the polypeptide described in Example 10. Results are presented as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05. ns: not statistically significant.

[0056] Figure 30 shows the changes in mechanical pain threshold over time in the mouse oxaliplatin acute chemotherapy pain model following administration of different peptides in Example 8. Results are presented as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=3). Model group vs. control group: #### p < 0.0001; drug-treated group vs. model group: **** p < 0.0001; *** p < 0.001; ** p < 0.01. ns: not statistically significant.

[0057] FIG31 shows the HPLC analysis profile of AJ003-40 in Example 4 after incubation in human plasma at 37° C. for 0 hours.

[0058] FIG32 shows the HPLC analysis profile of AJ003-40 in Example 4 after incubation in human plasma at 37° C. for 24 hours.

[0059] FIG33 shows the HPLC analysis profile of AJ003-24 in Example 4 after incubation in human plasma at 37° C. for 0 hours.

[0060] FIG34 shows the HPLC analysis profile of AJ003-24 in Example 4 after incubation in human plasma at 37° C. for 22 hours. DETAILED DESCRIPTION

[0061] Unless otherwise indicated, all numbers used in this specification and claims to indicate content, concentration, ratio, weight, particle size, percentage, technical effect, etc. should be understood as being modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values.

[0062] Unless otherwise stated, the terms used herein have the usual understanding meaning to those skilled in the art. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained by the application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or the manner understood by those skilled in the art. In general, the nomenclature used herein and the experimental procedures of organic chemistry, medicinal chemistry, biology described herein are well known in the art and are generally adopted in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings that are commonly understood by those of ordinary skill in the art of the application. Where there are multiple definitions for the terms used herein, unless otherwise stated, the definitions in this section shall prevail.

[0063] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

[0064] As used herein, the term "independently of one another" means that multiple events do not affect each other. For example, "X and Y are independently selected from any of a, b, c, d, e, f, and g" means that X can be any of a, b, c, d, e, f, and g, and Y can also be any of a, b, c, d, e, f, and g. The choice of X and the choice of Y can be the same or different, and the two do not interfere with each other.

[0065] As used herein, alanine scanning (Cunningham and Wells, Science 244, 1081-1085, 1989) is used to identify amino acids that are critical for the activity of the peptides of the invention (e.g., binding affinity to acetylcholine receptors) so that these amino acids are not substituted. Alanine scanning introduces mutations into every residue in the molecule and tests the biological activity of the resulting molecules to determine the amino acid residues that are critical for the activity of the molecule.

[0066] As used herein, the terms "comprises" and "includes" mean that in addition to the listed elements, other elements are not excluded.

[0067] As used herein, the term "identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is generally expressed as a percentage. Preferably, identity is determined over the entire length of the compared sequences. Therefore, two copies of the identical sequence have 100% identity. Those skilled in the art will recognize that some algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0068] Peptide or its derivatives

[0069] The present application provides a peptide or a derivative thereof comprising any one sequence selected from SEQ ID NO: 1-34.

[0070] The application provides a peptide or a derivative thereof comprising any one of the sequences selected from SEQ ID NOs: 1-34 with conservative substitutions. In some embodiments, any one of SEQ ID NOs: 1-34 with conservative substitutions has 1, 2, 3, 4, or 5 conservative substitutions. In some embodiments, any one of SEQ ID NOs: 1-34 with conservative substitutions has at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity, or at least 95% sequence identity with a sequence without conservative substitutions.

[0071] The present application provides a peptide or a derivative thereof comprising the following formula I or a conservatively substituted formula I in order from N-terminus to C-terminus: Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8 Formula I

[0072] The 3rd Cys and the 9th D-Cys in Formula I form a pair of intramolecular disulfide bonds, D-Cys represents D-cysteine, "-" between amino acid residues represents a peptide bond, the C-terminus of the peptide or its derivative is a free carboxyl group or an amide, X1 is Thr or D-Thr, X5 is selected from any one of Ala, Val and D-Val, and X2-X4 and X6-X8 are independently any one of amino acids.

[0073] In some embodiments, X1 in Formula I is Thr or D-Thr. In some embodiments, X5 in Formula I is selected from any one of Ala, Val, and D-Val. In some embodiments, X2 in Formula I is selected from any one of Pro, D-Pro, and Hyp. In some embodiments, X3 in Formula I is selected from any one of Pro, D-Pro, and Hyp. In some embodiments, X4 in Formula I is selected from any one of Ser, D-Ser, Ala, and Aib. In some embodiments, X6 in Formula I is selected from any one of Ala, Leu, D-Leu, Ile, ABu, and Nle. In some embodiments, X7 in Formula I is selected from any one of Tyr, D-Tyr, Ala, Trp, Phe, and Bip. In some embodiments, X8 in Formula I is selected from any one of Ser, D-Ser, Ala, and Orn.

[0074] In some embodiments, the above-mentioned Formula I with conservative substitutions has 1, 2, 3, 4 or 5 amino acid conservative substitutions. In one embodiment of the present disclosure, the above-mentioned Formula I with conservative substitutions has at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity or at least 95% sequence identity with Formula I without conservative substitutions.

[0075] In some embodiments, the peptide or derivative thereof comprises any one of the following sequences: SEQ ID NOs: 1-5, 10-16, and 20-34, or SEQ ID NOs: 1-5, 10-16, and 20-34 with conservative substitutions. In some preferred embodiments, the peptide or derivative thereof comprises SEQ ID NO: 26 or SEQ ID NO: 26 with conservative substitutions. In some preferred embodiments, the peptide or derivative thereof comprises SEQ ID NO: 28 or SEQ ID NO: 28 with conservative substitutions. In some preferred embodiments, the peptide or derivative thereof comprises SEQ ID NO: 32 or SEQ ID NO: 32 with conservative substitutions.

[0076] In some embodiments, the above-mentioned sequences with conservative substitutions have 1, 2, 3, 4 or 5 amino acid conservative substitutions. In some embodiments, the above-mentioned sequences with conservative substitutions have at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity or at least 95% sequence identity with the sequences without conservative substitutions.

[0077] In some embodiments, the derivative comprises any one or more modifications selected from the group consisting of amino acid modification, conservative amino acid substitution, and substitution of hydrogen in an amino acid residue compared to the peptide.

[0078] As used herein, the term "peptide" refers to a compound formed by amino acids linked together with peptide bonds. A peptide composed of three or more amino acid molecules is a polypeptide. As used herein, unless otherwise specified or inconsistent with the context, the term "peptide" encompasses the peptide itself and its pharmaceutically acceptable salts, prodrugs and metabolites. The peptides of the present application are produced by conventional techniques in the art, including but not limited to solid phase synthesis (such as Fmoc peptide synthesis) and liquid phase synthesis.

[0079] As used herein, the positions of a polypeptide or its derivatives are numbered in order from the N-terminus to the C-terminus, for example, the first amino acid from the N-terminus of the peptide is the first amino acid, the second amino acid from the N-terminus of the peptide is the second amino acid, the third amino acid from the N-terminus of the peptide is the third amino acid, and so on.

[0080] Unless otherwise defined, as generally understood in the art, the left-hand end of an amino acid sequence or polypeptide sequence is the N-terminus / amino terminus, and the right-hand end of an amino acid sequence or polypeptide sequence is the C-terminus / carboxyl terminus. As used herein, the term "prodrug" refers to all molecules that are converted into the peptides of the present disclosure or their derivatives by reactions such as oxidation, reduction, and hydrolysis under physiological conditions in vivo, for example, by the action of enzymes, gastric acid, and the like.

[0081] As used herein, the term "metabolites" refers to all molecules in a cell or organism (preferably a human) that are derived from a peptide of the present disclosure or a derivative thereof.

[0082] When used in this article with respect to peptides, "amino acid" and "amino acid residue" have the same meaning, which means that when amino acids are connected by chemical bonds, part of their groups are lost due to their participation in the formation of the connecting bonds, and the remaining amino acid part is the amino acid residue.

[0083] As used herein, the names and abbreviations of amino acids correspond to the following:

[0084] Natural amino acids, except glycine, all contain chiral carbon atoms. Unless otherwise specified, the optically active amino acids described in this application are all in the L-configuration. As is customary, peptide structures herein are written with the amino terminus (N-terminus) on the left and the carboxyl terminus (C-terminus) on the right.

[0085] As used herein, the term "amino acid" includes natural amino acids, as well as other "non-proteinogenic" α-amino acids / unnatural amino acids commonly used in the art of peptide chemistry to prepare analogs of natural peptides.

[0086] Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, ornithine and lysine. Examples of "non-proteinogenic" α-amino acids / unnatural amino acids are norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline (Hyp), homoserine, cyclohexylglycine (Chg), α-amino-n-butyric acid (Aba), cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanine substituted on the phenyl moiety by alkyl, alkoxy, halogen or nitro groups, O-alkylated derivatives of serine, threonine and tyrosine, S-alkylated cysteine, O-sulfate esters of tyrosine, and the D-isomers of natural amino acids.

[0087] As used herein, a "derivative" of a peptide refers to a product obtained by modifying the peptide, making conservative amino acid substitutions, and / or replacing hydrogen atoms in amino acid residues.

[0088] As used herein, the term "amino acid modification" includes, but is not limited to, N-terminal modification, C-terminal modification, and side chain modification. Modifications include, but are not limited to, hydroxylation, carboxylation, alkylation, acylation, phosphorylation, sulfonation, amidation, aldehyde formation, alcoholization, mercaptoethylation, esterification, and glycosylation.

[0089] As used herein, "conservative amino acid substitutions" can generally be described as amino acid substitutions in which one amino acid residue is replaced by another amino acid having a similar chemical structure and / or similar chemical properties, and which has little effect on the function, activity or other biological properties of the peptide. Such conservative amino acid substitutions are well known in the art. Such conservative substitutions can be, for example, substitutions of one amino acid from the following groups (a)-(e) by another amino acid from the same group: (a) small aliphatic, non-polar or weakly polar amino acid residues: Ala, Ser, Thr, Pro and Gly; (b) negatively charged polar amino acid residues and their amides: Asp, Asn, Glu and Gln; (c) positively charged polar amino acid residues: His, Arg and Lys; (d) large aliphatic, non-polar amino acid residues: Met, Leu, Ile, Val and Cys; (e) aromatic amino acid residues: Phe, Tyr and Trp.

[0090] As used herein, the replacement of hydrogen in an amino acid residue may be that the hydrogen in the amino acid residue is replaced by any conventional substituent known in the art, including but not limited to alkyl, alkenyl, alkynyl, aromatic, alkoxy, carboxyl, aldehyde, carbonyl, hydroxyl, halogen, cyano, acyl, sulfonic acid, amino, thiol or nitro groups.

[0091] Polynucleotides, expression vectors, host cells

[0092] The present application provides a polynucleotide encoding the peptide or its derivatives described in the present disclosure.

[0093] As used herein, "polynucleotide" refers to a polymer of nucleotides (nucleotides or deoxynucleotides) of any length. The term refers to the primary structure of the molecule. Thus, it includes both double-stranded and single-stranded RNA, as well as double-stranded and single-stranded DNA. It also includes polynucleotides modified, such as by methylation and / or end-capping, as well as unmodified forms of polypeptide nucleotides. The polynucleotides described herein need not be obtained by physical methods and may be produced by any means, including, for example, chemical synthesis or DNA replication, reverse transcription, or transcription.

[0094] The present application provides an expression vector, which can express the polynucleotide described in the present disclosure.

[0095] The present application provides a host cell comprising the polynucleotide described in the present disclosure or the expression vector described in the present application.

[0096] As used herein, a "host cell" is a cell that serves as a recipient for an expression vector or other transforming genetic fragment. These include the original cell that has been transfected and its progeny. Host cells include prokaryotes, yeast, or other eukaryotic cells.

[0097] Pharmaceutical composition

[0098] The present application provides a pharmaceutical composition comprising the peptide or derivative thereof disclosed herein, and a pharmaceutically acceptable carrier or salt.

[0099] The present application also provides a pharmaceutical composition comprising the peptide or derivative thereof, the polynucleotide, the expression vector or the host cell according to the present disclosure, and a pharmaceutically acceptable carrier or salt thereof.

[0100] As used herein, the term "pharmaceutical composition" refers to a mixture of the peptides of the present disclosure and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. Pharmaceutical compositions facilitate administration of the peptides to an organism. There are many ways of administering peptides in the art, including, but not limited to, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, pulmonary administration, ocular administration, and topical administration.

[0101] In the present application, the pharmaceutical composition can be configured into a dosage form suitable for administration to a subject via a desired route of administration, including but not limited to tablets, capsules, caplets, pills, lozenges, powders, syrups, brews, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, lotions, pastes, sprays, freeze-dried solutions, injections, and gels.

[0102] As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts and base addition salts. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include, but are not limited to, acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclamates, edisylate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, 2-(4-hydroxybenzyl)benzoate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, hexadecanoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, glucarate, stearate, salicylate, tannate, tartrate, toluenesulfonate, and trifluoroacetate. Suitable base addition salts are formed with bases which form non-toxic salts. Examples include, but are not limited to, aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemi-salts of acids and bases, such as hemisulfate and hemicalcium salts, can also be formed. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0103] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which are involved in carrying or transporting the peptides of the present disclosure within a subject or carrying or transporting the peptides of the present disclosure to a subject so that it can perform its intended function. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate; Esters and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavorings; perfuming agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardening agents; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other nontoxic, compatible substances used in pharmaceutical formulations, or any combination thereof.

[0104] Uses, disease treatment methods

[0105] The present application provides use of the peptide or derivative thereof described in the present disclosure or the pharmaceutical composition described in the present disclosure in the preparation of a medicament for treating pain.

[0106] The present application also provides use of the peptide or derivative thereof, the polynucleotide, the expression vector, the host cell or the pharmaceutical composition described in the present disclosure in the preparation of a drug for treating pain.

[0107] In some embodiments, the medicine for treating pain of the preparation is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, pulmonary administration, ocular administration or topical administration. In a preferred embodiment, the medicine for treating pain of the preparation is administered by subcutaneous injection.

[0108] In some embodiments, the pain includes pain caused by nicotinic acetylcholine receptors. In some embodiments, the pain includes nerve pain and inflammatory pain. In some embodiments, the pain includes pain caused by chemotherapy, diabetic peripheral neuropathy, sciatica, bone and joint pain, postherpetic neuralgia, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcoholism pain, hypothyroidism pain, autoimmune disease pain, Guillain-Barré syndrome pain, poisoning pain, drug-induced pain, tumor lesion pain, genetic disease pain, and contusion pain.

[0109] The present application further provides the peptide or derivative thereof described in the present disclosure, the polynucleotide described in the present disclosure, the expression vector described in the present disclosure, the host cell described in the present disclosure or the pharmaceutical composition described in the present disclosure, for use in treating pain.

[0110] In some embodiments, the pain includes pain caused by nicotinic acetylcholine receptors. In some embodiments, the pain includes nerve pain and inflammatory pain. In some embodiments, the pain includes pain caused by chemotherapy, diabetic peripheral neuropathy, sciatica, bone and joint pain, postherpetic neuralgia, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcoholism pain, hypothyroidism pain, autoimmune disease pain, Guillain-Barré syndrome pain, poisoning pain, drug-induced pain, tumor lesion pain, genetic disease pain, and contusion pain.

[0111] In some embodiments, the peptide or its derivative or pharmaceutical composition is administered by a method including subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, pulmonary administration, ocular administration or topical administration. In a preferred embodiment, the peptide or its derivative or pharmaceutical composition is administered by subcutaneous injection.

[0112] The present application provides a method for treating pain, which comprises administering a therapeutically effective amount of the peptide or derivative thereof according to the present disclosure or the pharmaceutical composition according to the present disclosure to a subject in need thereof.

[0113] The present application also provides a method for treating pain, which comprises administering a therapeutically effective amount of the peptide or derivative thereof, polynucleotide, expression vector, host cell according to the present disclosure, or the pharmaceutical composition according to the present disclosure to a subject in need thereof.

[0114] In some embodiments, the pain includes pain caused by nicotinic acetylcholine receptors. In some embodiments, the pain includes nerve pain and inflammatory pain. In some embodiments, the pain includes pain caused by chemotherapy, diabetic peripheral neuropathy, sciatica, bone and joint pain, postherpetic neuralgia, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcoholism pain, hypothyroidism pain, autoimmune disease pain, Guillain-Barré syndrome pain, poisoning pain, drug-induced pain, tumor lesion pain, genetic disease pain, and contusion pain.

[0115] In some embodiments, the peptide or its derivative or pharmaceutical composition is administered by a method including subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, pulmonary administration, ocular administration or topical administration. In a preferred embodiment, the peptide or its derivative or pharmaceutical composition is administered by subcutaneous injection.

[0116] As used herein, the term "subject" includes animals, such as vertebrates, preferably mammals, such as dogs, cats, pigs, cows, sheep, horses, rodents (e.g., mice, rats, or guinea pigs), or primates (e.g., gorillas, chimpanzees, and humans).

[0117] As used herein, the term "treat," ...

[0118] As used herein, the term "therapeutically effective dose" refers to an amount that results in a benefit or treatment of a disease compared to a corresponding subject not receiving that amount, but is sufficiently low within the scope of sound medical judgment to avoid serious side effects. The therapeutically effective dose of the peptides or derivatives thereof, polynucleotides, expression vectors, host cells or pharmaceutical compositions described herein will vary with the selected peptides or derivatives thereof, polynucleotides, expression vectors, host cells or pharmaceutical compositions; the route of administration; the severity of the disease being treated; the age, size, weight and physical condition of the patient being treated; the medical history of the patient being treated; the duration of treatment; the nature of concurrent treatment; the desired therapeutic effect, and the like, but can still be determined in a routine manner by those skilled in the art.

[0119] The various embodiments described above for the peptides or derivatives thereof, polynucleotides, expression vectors and host cells of the present disclosure are also applicable to the pharmaceutical compositions, uses and methods of the present disclosure (as long as they are not inherently inconsistent with each other), and the various embodiments formed by such combination are considered part of the present disclosure.

[0120] Example

[0121] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0122] Unless otherwise stated, all reagents and instruments used in the following examples are commercially available conventional products. Unless otherwise stated, experiments were performed under conventional conditions or conditions recommended by the manufacturer.

[0123] Example 1: Synthesis of Conus Toxin Polypeptide

[0124] Based on previous research, a group of polypeptide compounds were preliminarily screened by performing alanine scanning, single-site non-natural amino acid substitution, and multi-site combination modification on the parent peptide AJ003.

[0125] Synthesis of AJ003: Fmoc-Ser(tBu)-wang resin (Tianjin Nankai Synthesis Technology Co., Ltd.) was used as the starting resin, with a synthesis scale of 1 mmol. The resin was weighed and added to a peptide solid phase reactor (Nanjing Luanyu Chemical Glass Instrument Co., Ltd.). Dichloromethane and nitrogen were added to swell for 30 minutes, and then washed twice with DMF (dimethylformamide). 20% piperidine / DMF solution was added twice, and the reaction was carried out for 5 minutes each time. After the deprotection reaction was completed, the resin was washed with DMF 6 times, each time for about 3 minutes. According to the peptide sequence, following the principle that the conventional chemical synthesis of peptides is to couple amino acids one by one from the C-terminus to the N-terminus, the corresponding protected amino acids and condensation reagents (HoBt, DIC) were weighed, and the reaction was carried out for about 1 hour. After the condensation reaction, the resin was washed with DMF 3 times. From then on, the condensation cycle (deprotection-washing-condensation-washing) was carried out, that is, 20% piperidine / DMF solution was added to the reaction vessel again to carry out the deprotection reaction. After washing with DMF, the protected amino acids and the corresponding condensation reagents were added to carry out the condensation reaction. Protected amino acids were coupled to the N-terminus one by one using a condensation cycle until all amino acids of the peptide were coupled. The Fmoc group at the N-terminus of the peptide was removed using a 20% piperidine / DMF solution. The resin was washed alternately with dichloromethane and methanol, and finally vacuum-dried to obtain a shrink-dried peptide resin intermediate. The peptide resin was weighed and a lysis buffer was prepared at a ratio of 1 gram of peptide resin to 10 ml of lysis buffer. The lysis buffer formulation was TFA:EDT:TIS:H2O = 90:5:2.5:2.5. The lysis buffer was added to the peptide resin and the cleavage reaction was allowed to proceed for approximately 3 hours. The filtrate was collected and precipitated with ice-cold methyl tert-butyl ether (MTBE) at an amount of 8 times the filtrate. The precipitate was thoroughly stirred and centrifuged in separate cups to obtain a crude peptide precipitate. This was then washed and centrifuged again with MTBE and vacuum-dried overnight to obtain a crude linear peptide sample. The crude linear peptide was dissolved in water and iodine was added as an oxidant to oxidize the peptide to form disulfide bonds. Oxidation was continued for approximately 30 minutes, and the degree of oxidation was monitored using HPLC. Finally, vitamin C was added dropwise to the oxidation system to terminate the oxidation reaction. The oxidized polypeptide solution was filtered through a filter membrane and separated and purified using reverse-phase high-performance liquid chromatography. The qualified fractions were combined and finally freeze-dried using a freeze dryer to obtain the pure freeze-dried polypeptide.

[0126] The synthesis methods for other peptides were consistent with the technical route for peptide AJ003 described above, all utilizing classic Fmoc solid-phase peptide synthesis technology to obtain crude linear peptides. For peptides with an amide-terminated C-terminus, the resin used was Rink amide-MBHA resin. The crude peptide solution was oxidized with iodine to form a disulfide-bonded peptide fraction, which was then separated and purified using reversed-phase high-performance liquid chromatography (RP-HPLC). Finally, the pure lyophilized product was obtained by freeze-drying. The peptide sequences used in this example are shown in Table 1.

[0127] Table 1. Summary of peptide sequences

[0128] *D-Thr: D-threonine; 3,9 disulfide bond: a disulfide bond is formed between the third and ninth amino acid residues in the sequence.

[0129] Example 2: BLI (biofilm interferometry) detection of affinity between peptide and target protein

[0130] BLI is a label-free technology that converts the optical interference signal occurring on the surface of the BLI biosensor into a real-time response signal. By real-time monitoring of the molecular binding process, the system will measure the binding constant (ka or kon) and dissociation constant (kd or koff), as well as the initial binding rate, and obtain the affinity (K D ). The specific steps are to place the NTA sensor (Sartorius) in a pre-wet box, place the pre-wet box in a black 96-well plate, add 200μL of PBS to each well, and pre-wet the NTA sensor for at least 15 minutes. Dilute the target protein α9 nAChR with PBS to 5μg / mL, and add 200μL to each well. Dissolve the peptide to be tested in PBS and dilute it 3 times in a gradient, with the concentrations from high to low being 1000nM, 333.3nM, 111.1nM, 37.04nM, 12.35nM, 4.115nM, and 1.372nM. Finally, add a PBS well as a zero concentration control well. After balancing the instrument baseline, first use the NTA sensor to solidify the target protein α9 nAChR. After solidification is completed, re-balance the baseline, and then perform a 5-minute binding and 5-minute dissociation process. According to the above process, affinity testing is performed on the peptides to be tested one by one. The affinity testing process for peptides and the target protein α7nAChR is essentially the same as that for α9 nAChR, except that the protein concentration of the target protein α7 nAChR is 20 μg / mL. As shown in Tables 2 and 3 below, BLI experiments were used to test the affinity of alanine-scanning peptide analogs for the target protein, clarifying the site of the parent peptide that can be modified. Secondly, BLI experiments were used to test the affinity of peptides modified by single-site unnatural amino acid mutations for the target protein. Finally, BLI experiments were used to test the affinity of peptides modified by multi-site combination mutations for the target protein, thereby screening peptides with high affinity for the target protein for subsequent screening and druggability research.

[0131] Table 2. Affinity test results of modified peptides and α9 nAChR target protein

[0132] Table 3. Affinity test results of modified peptides and α7nAChR target protein

[0133] Example 3: Prediction of polypeptide half-life based on in vitro plasma stability

[0134] Peptides typically have a short half-life and are easily degraded by various enzymes and substrates in the blood within the animal body. The modified peptides used in this study were designed with non-natural amino acid substitutions at certain inactive key sites, hoping to improve peptide stability and facilitate drug development. The peptide was weighed and dissolved in deionized water to 2 mg / mL, then diluted with acetonitrile to 1 mg / mL. The solution was filtered through an organic filter membrane, and 200 μL of the filtrate was transferred to a liquid phase vial as a peptide control group. 300 μL of human plasma (containing EDTA anticoagulant) (Nanjing Senbeijia Biotechnology Co., Ltd., batch number SBJ-P-HU011-100mL) was added to 300 μL of acetonitrile and centrifuged at 4°C, 12,000 rpm, for 5 minutes. The supernatant was filtered through an organic filter membrane, and 200 μL of the filtrate was transferred to a liquid phase vial as a plasma blank control group. Weigh 10 mg of the peptide and add 5 mL of human plasma to it, vortexing to mix thoroughly. Pipette 300 μL of the peptide-containing plasma into 300 μL of acetonitrile and centrifuge at 4°C, 12,000 rpm, for 5 minutes. Filter the supernatant through an organic filter membrane and transfer 200 μL of the filtrate to a liquid phase vial as the peptide sample at the 0-minute time point. Incubate the peptide-dissolved plasma in a 37°C water bath. Perform the same procedures as at the 0-minute time point for the remaining time points. The sampling points for peptides AJ003 and AJ003-1 were 0 min, 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 6 h, 10 h, 12 h, and 24 h. The sampling points for modified peptides AJ003-34, AJ003-35, AJ003-36, AJ003-37, AJ003-38, AJ003-39, AJ003-40, AJ003-41, and AJ003-42 were 0 min, 10 min, 30 min, 1 h, 4 h, 6 h, 10 h, 12 h, and 24 h. A series of samples for each peptide were transferred to the analytical department for HPLC analysis. The changes in the main peak area of ​​the sample with plasma incubation time were recorded. The in vitro half-life of each peptide was predicted using the pharmacokinetic processing software Winolin. Due to the existence of tautomers in the AJ003 sample, the peak area over time was difficult to accurately integrate and compare. AJ003-1 is an analog of the AJ003 peptide in which the threonine at position 5 is remodeled to D-threonine. The peptide sequence of AJ003-1 differs from that of AJ003 by only one amino acid configuration. As shown in Figure 1-10, the in vitro plasma half-life of AJ003-1 is approximately 10.8 hours. The in vitro plasma half-lives of the modified peptides AJ003-34, AJ003-35, AJ003-36, AJ003-37, AJ003-38, AJ003-39, AJ003-40, AJ003-41, and AJ003-42 are 61.9 hours, 45.3 hours, 65.3 hours, 71.0 hours, 26.7 hours, 43.0 hours, 84.0 hours, 30.0 hours, and 61.4 hours, respectively.Overall, through the mutation of non-natural amino acids, the in vitro plasma stability of other modified peptides in this project has been significantly improved compared with AJ003-1 (which only differs from AJ003 by one amino acid).

[0135] Example 4: Plasma stability test of AJ003-40 and AJ003-24

[0136] Weigh the peptides AJ003-40 and AJ003-24, add them to human plasma, vortex and mix them, and prepare them to 2mg / ml. Pipette 300 microliters of plasma containing peptides and add them to 300 microliters of acetonitrile. Place them in a centrifuge at 4°C, 12,000 rpm, and centrifuge for 5 minutes. Pipette the supernatant and filter it with an organic filter membrane. Pipette 200 microliters of the filtrate into a liquid phase vial as the peptide 0-hour time point sample. Place the peptide-dissolved plasma in a 37°C water bath and incubate. At the 24-hour time point, 300 microliters of plasma containing peptides for peptide AJ003-40 were added to 300 microliters of acetonitrile. Place them in a centrifuge at 4°C, 12,000 rpm, and centrifuge for 5 minutes. Pipette the supernatant and filter it with an organic filter membrane. Pipette 200 microliters of the filtrate into a liquid phase vial as the peptide 24-hour time point sample. At the 22-hour time point, 300 μl of plasma containing the peptide AJ003-24 was aspirated and added to 300 μl of acetonitrile. The mixture was centrifuged at 4°C, 12,000 rpm, and centrifuged for 5 minutes. The supernatant was aspirated and filtered through an organic filter membrane. 200 μl of the filtrate was transferred to a liquid phase vial as the 22-hour peptide sample. The 0-hour and 24-hour samples of the two peptides were analyzed by HPLC. As shown in Figures 31 and 32, the main peak area of ​​peptide AJ003-40 decreased from 15149.38 at 0 hours to 11829.51 at 24 hours, with a main peak area degradation rate of 21.9% within 24 hours. As shown in Figures 33 and 34, the main peak area of ​​peptide AJ003-24 decreased from 746.80 at 0 hours to 267.45 at 22 hours, with a main peak area degradation rate of 64.2% within 22 hours. Compared with peptide AJ003-40, peptide AJ003-24 degraded faster, indicating that AJ003-24 had poorer plasma stability than AJ003-40.

[0137] Example 5: Evaluation of the analgesic activity of some modified peptides at the same dose in vivo in patients with paclitaxel chemotherapy pain

[0138] 180-200g SPF (Specific Pathogen Free) grade SD male rats (Experimental Animal Management Department of Shanghai Institute of Family Planning Science) were weighed on the day of modeling and their basal pain threshold was tested using mechanical stimulation. Animals with large differences in weight and pain threshold were eliminated, and the remaining rats were modeled according to the experimental protocol. Paclitaxel powder was dissolved in olive oil to 1mg / mL, and each rat was injected intraperitoneally at a dose of 2mg / kg each time. A total of 4 injections were given during the entire modeling period, with intraperitoneal injections performed on days 1, 3, 5, and 7. Negative control or blank control rats were injected with the solvent used to dissolve paclitaxel. According to literature reports, the pain threshold of this model decreased significantly after about 15 days from the start of modeling, indicating that a stable rat paclitaxel chronic chemotherapy pain model was successfully established. Drug treatment was carried out according to the administration method set in the experimental protocol, as shown in Table 4 below. The blank group in Table 4 represents the group without pain modeling, and the model group represents the group with pain modeling but no drug administration. Rats were dosed at fixed times on days 1, 3, 5, and 12. On days 1, 3, and 5, the plantar surface of the rats' right hind limbs was stimulated with flat-tipped needles 1 hour, 2.5 hours, 4 hours, and 5.5 hours after dosing, and the pain thresholds to mechanical stimulation were measured at the corresponding time points. On day 12, the pain thresholds to mechanical stimulation were measured 4 hours and 7 hours after dosing. As shown in Figures 11-14, the combined results of the four dosing treatments demonstrate that the parent peptide AJ003 exhibits significant analgesic activity compared to the model group within 4 hours of treatment, but no significant analgesic activity was observed beyond 4 hours. Of the five candidate modified peptides (AJ003-34, AJ003-35, AJ003-36, AJ003-39, and AJ003-40), AJ003-34, AJ003-36, and AJ003-40 were the three best analgesic candidates. Analysis of the results at 7 hours on day 12 showed that AJ003-36 and AJ003-40 exhibited particularly long-lasting analgesic activity, lasting up to 7 hours. Furthermore, these modified peptides exhibited analgesic activity compared to the active drug, and at certain dosing time points, were slightly superior to pregabalin. Overall, their analgesic efficacy was comparable to that of the active drug, pregabalin. AJ003-36 and AJ003-40 are promising candidates for further study. Paclitaxel, a widely used small molecule chemotherapy drug, has anti-tumor properties, but also causes neuropathic pain in many cancer patients due to its damage to the patient's nervous system. Using a chronic chemotherapy pain model induced by multiple low-dose intraperitoneal injections of paclitaxel in male SD rats, mimicking clinical cancer patients receiving paclitaxel, the analgesic effects of five modified peptides, along with the parent peptide AJ003 and the active agent pregabalin, were evaluated. The results showed that mechanical pain thresholds at the plantar aspect of the right hind limb of the rats were measured at the corresponding time points after each administration. Overall analysis identified AJ003-36 and AJ003-40 as candidate molecules for further investigation.

[0139] Table 4. Dosage schedule for analgesic activity of some modified peptides at the same dose in the paclitaxel chemotherapy pain model screening

[0140] Example 6: Evaluation of the analgesic activity of modified peptides at different doses in vivo for paclitaxel chemotherapy pain

[0141] Purchase 180-200g SPF grade SD male rats (Shanghai Institute of Family Planning Science, Experimental Animal Management Department), adapt to feeding for about a week, weigh rats on the same day of modeling, and detect rat basal pain threshold with mechanical stimulation, eliminate animals with large differences in body weight and pain threshold, and the remaining rats carry out modeling according to the experimental protocol. Paclitaxel powder is dissolved into 1mg / mL with olive oil, and each rat intraperitoneal injection amount is 2mg / kg. During the whole modeling period, a total of 4 injections are injected, which are intraperitoneally injected on days 1, 3, 5, and 7 respectively. Negative control or blank control group rats are injected with the solvent of the above-mentioned dissolving paclitaxel. According to literature reports, this model is calculated from the beginning of modeling, and after about 15 days, the pain threshold has significantly decreased, marking that the stable rat paclitaxel chronic chemotherapy pain model has been successfully constructed. The administration method provided according to the experimental protocol is carried out drug treatment, as shown in Table 5 below. Starting on day 15 of modeling, the animals were treated daily. On days 1, 4, 7, 12, and 14, the plantar surface of the rats' right hind limbs was stimulated with flat-tipped needles 2, 4, 6, and 8 hours after dosing. The pain thresholds to mechanical stimulation were measured at the corresponding time points. The results are shown in Figures 15-19. Based on the results of these five treatments, the parent peptide AJ003 exhibited significant analgesic activity compared to the model group within 4 hours of treatment. However, the analgesic activity was not significant beyond 4 hours. The two candidate modified peptides AJ003-36 and AJ003-40 had analgesic activity at a low dose of 0.5 mg / kg, which was comparable to the analgesic effect of the parent peptide AJ003 at 1 mg / kg, and were even superior to the parent peptide AJ003 at the corresponding time points on certain dosing days. The two candidate modified peptides AJ003-36 and AJ003-40 had analgesic activity at a high dose of 1.5 mg / kg, which was slightly superior to the positive drug pregabalin, and the analgesic effect still had significant analgesic activity compared with the model group 8 hours after administration.

[0142] Table 5. Dosage schedule for analgesic activity of some modified peptides at different doses in the paclitaxel chemotherapy pain model

[0143] Example 7: Evaluation of the analgesic activity of candidate modified peptides at different doses in vivo for pain treatment with oxaliplatin chemotherapy

[0144] Oxaliplatin was dissolved in 5% glucose solution to 1 mg / mL. Each C57 male mouse in the modeling group (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) was injected intraperitoneally with 10 mg / kg of oxaliplatin. The negative control or blank control group was injected with 5% glucose solution. The mechanical pain threshold of the mice was detected about one week after modeling. The pain threshold of each mouse was measured 3 times, the average value was calculated, and the mice were randomly divided according to the pain threshold. The mice were administered according to the dosing regimen in Table 6 (a total of 1 dose), and the mechanical pain threshold was tested at 0.5, 1, 2, 4, 6, and 8 hours after administration. As shown in Figure 20, at the four time points of 0.5h, 1h, 2h, and 4h after administration, the low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group. At 6 hours, the parent peptide AJ003 showed no significant difference compared with the model group, while the low, medium, and high dose groups of the positive drug and the modified peptide AJ003-36 still showed significant analgesic activity compared with the model group. At 8 hours, the low dose group of the parent peptide AJ003 and AJ003-36 showed no significant difference compared with the model group, while the medium and high dose groups of the positive drug and the modified peptide still showed significant analgesic activity compared with the model group.

[0145] Table 6. Oxaliplatin chemotherapy pain medication schedule

[0146] Example 8: Evaluation of the analgesic activity of various peptides in vivo for pain caused by oxaliplatin chemotherapy

[0147] Oxaliplatin was dissolved in 5% glucose solution to 1 mg / mL. Each C57 male mouse in the modeling group (Shanghai Bike Wing Biotechnology Co., Ltd.) was injected intraperitoneally with 10 mg / kg of oxaliplatin. The negative control or blank control group was injected with 5% glucose solution. The mechanical pain threshold of mice was detected about one week after modeling. The pain threshold of each mouse was measured 3 times, the average value was calculated, and the mice were randomly divided into groups according to the pain threshold. Mice were administered according to the dosing regimen of Table 7 (administered 1 time in total), and the mechanical pain threshold was detected at 0.5, 1, 2, 4, 6, and 8 hours after administration. As shown in Figure 30, at the beginning of administration 0.5h, the mechanical pain threshold of mice in the AJ003-36 administration group was significantly reduced compared with the model group, and there was a significant difference, while the mechanical pain threshold of mice in the administration groups AJ003-24 and AJ003-25 did not reduce the mechanical pain threshold of mice. This indicates that peptide AJ003-36 can effectively relieve chemotherapy pain in mice caused by oxaliplatin, while peptides AJ003-24 and AJ003-25 cannot relieve the pain.

[0148] Table 7. Dosing schedule for the mouse oxaliplatin chemotherapy pain model experiment

[0149] Example 9: Evaluation of the analgesic activity of candidate modified peptides at different doses in rat CCI model

[0150] There are many types of neuropathic pain, and animal models can be used to simulate clinical neuropathic pain. The sciatic nerve pathological pain models of SD rats mainly include CCI (chronic constriction injury model), SNI (sciatic nerve branch selective ligation SNI model), SNL (spinal nerve selective ligation SNL model), and CCD (using stainless steel wire to pass through the intervertebral foramen to fix and compress the dorsal root ganglion). Among them, the CCI model modeling method has been widely used to study neuropathic pain and provides a basis for further studying the mechanisms and strategies of nerve damage repair (Wen Xiaojuan, Medical Information. Jan. 2018. Vol. 31. No. 1). The CCI model is used in this embodiment.

[0151] Except for the control group, all the other SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate physiological saline solution at a dosage of 0.3mL per 100g body weight, and then surgical modeling was performed. Carefully cut the skin of the right hind limb of the rat with surgical scissors, cut the muscle, and ligate the sciatic nerve three times with catgut 4-0, at intervals of 1mm. The muscle and skin were then sutured, and antibiotics could be applied to the wound to prevent infection of the animal surgical wound. According to literature reports, animals showed a significant decrease in pain threshold 7-10 days after surgery, which can be sustained for about one month. The rat foot pain threshold was tested in the model for about 10 days. Each rat measured the pain threshold 3 times, calculated the mean value, and randomly divided into groups according to the rat pain threshold. Administer the drug according to the dosage regimen of Table 8, and select four time points, 2h, 4h, 6h, and 8h after administration, to test the rat foot pain threshold.

[0152] On the first day of administration, as shown in Figure 21, at 2h and 4h, the three low, medium and high dose groups of the positive drug pregabalin, the parent peptide AJ003 and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group. At 6h, the parent peptide AJ003 was not significant compared with the model group, and the three low, medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group. At 8h, the low dose group of the parent peptide AJ003 and AJ003-36 was not significant compared with the model group, and the medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.

[0153] On day 4 of drug administration, as shown in Figure 22, at 2 and 4 hours, the low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all showed significant analgesic activity compared to the model group. At 6 and 8 hours, the low dose groups of the parent peptide AJ003 and AJ003-36 showed no significant difference compared to the model group, while the positive drug and the medium and high dose groups of AJ003-36 still showed significant analgesic activity compared to the model group.

[0154] On the 7th day of administration, as shown in Figure 23, at 2h and 4h, the three low, medium and high dose groups of the positive drug pregabalin, the parent peptide AJ003 and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group. At 6h, the parent peptide AJ003 was not significant compared with the model group, and the three low, medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group. At 8h, the low dose group of the parent peptide AJ003 and AJ003-36 was not significant compared with the model group, and the medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.

[0155] On the 10th day of administration, as shown in Figure 24, at 2h and 4h, the three low, medium and high dose groups of the positive drug pregabalin, the parent peptide AJ003 and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group. At 6h, the parent peptide AJ003 was not significant compared with the model group, and the three low, medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group. At 8h, the low dose group of the parent peptide AJ003 and AJ003-36 was not significant compared with the model group, and the medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.

[0156] On the 14th day of administration, as shown in Figure 25, at 2h and 4h, the three low, medium and high dose groups of the positive drug pregabalin, the parent peptide AJ003 and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group. At 6h, the parent peptide AJ003 was not significant compared with the model group, and the three low, medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group. At 8h, the low dose group of the parent peptide AJ003 and AJ003-36 was not significant compared with the model group, and the medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.

[0157] Table 8. Dosage schedule for rat CCI model

[0158] Example 10: Evaluation of the analgesic activity of candidate modified peptides at different doses in a mouse diabetic neuropathic pain model

[0159] Except for the control group, the remaining C57 male mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) were induced with a high-sugar and high-fat diet for about ten consecutive weeks. The model group mice were fasted overnight, and streptozotocin (Shan Chemical Technology (Shanghai) Co., Ltd.) was dissolved in citric acid-sodium citrate buffer and injected intraperitoneally with 70 mg / kg of streptozotocin. About two weeks after the injection of streptozotocin, the mechanical pain threshold of the mice was tested. The test method was to place the mice in a porous wire mesh to adapt to the environment, and then use a flat-head needle to vertically stimulate the sole of the right hind limb of the mouse. The pain threshold data was collected and recorded using a biological signal acquisition system. The pain threshold of each mouse was measured three times, and the average value was finally calculated. The mice were randomly divided into groups and treated according to their pain threshold.

[0160] On the first day of administration, drugs were administered according to the dosing regimen in Table 9, and the plantar mechanical pain threshold of mice was tested at five time points: 0 h, 2 h, 4 h, 6 h, and 8 h after administration. As shown in Figure 26, at 2 h and 4 h, the three low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group; at 6 h and 8 h, the parent peptide AJ003 had no significant difference compared with the model group, while the three low, medium, and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.

[0161] On the fourth day of administration, drugs were administered according to the dosing regimen in Table 9, and the plantar mechanical pain threshold of mice was tested at five time points: 0 h, 2 h, 4 h, 6 h, and 8 h after administration. As shown in Figure 27, at 2 h and 4 h, the three low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group; at the two time points of 6 h and 8 h, the parent peptide AJ003 had no significant difference compared with the model group, while the three low, medium, and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.

[0162] On the 8th day of administration, the drugs were administered according to the dosing regimen in Table 9, and the plantar mechanical pain threshold of the mice was tested at five time points: 0 h, 2 h, 4 h, 6 h, and 8 h after administration. As shown in Figure 28, at 2 h and 4 h, the low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group; at 6 h and 8 h, the low dose groups of the parent peptide AJ003 and AJ003-36 were not significant compared with the model group, while the medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.

[0163] On the 14th day of administration, the drugs were administered according to the dosing regimen in Table 9, and the plantar mechanical pain threshold of the mice was tested at five time points: 0 h, 2 h, 4 h, 6 h, and 8 h after administration. As shown in Figure 29, at 2 h and 4 h, the three low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group; at the two time points of 6 h and 8 h, the parent peptide AJ003 had no significant difference compared with the model group, while the three low, medium, and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.

[0164] Based on the mechanical pain threshold test results from four treatments, the parent peptide AJ003 exhibited significant analgesic activity compared to the model group within 4 hours of treatment, but the analgesic activity was not significant beyond 4 hours. The intermediate and high doses of the candidate modified peptide AJ003-36 exhibited significant analgesic activity compared to the model group from 2 to 8 hours after testing. The low dose of AJ003-36 showed no significant difference from the model group on day 8, but exhibited significant analgesic activity compared to the model group at the other three testing times. The high dose of AJ003-36 exhibited slightly superior analgesic activity to the active drug pregabalin. This study suggests that the candidate modified peptide AJ003-36 may be a potential treatment for diabetic-induced neuropathic pain via subcutaneous injection.

[0165] Table 9. Dosage schedule for the diabetic neuropathy model in mice

Claims

1. A peptide or a derivative thereof comprising the following formula I or a conservatively substituted formula I in order from N-terminus to C-terminus: Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8 Formula I in, The 3rd Cys and the 9th D-Cys in Formula I form a pair of intramolecular disulfide bonds, D-Cys represents D-cysteine, "-" between amino acid residues represents a peptide bond, the C-terminus of the peptide or its derivative is a free carboxyl group or an amide, X1 is Thr or D-Thr, X5 is selected from any one of Ala, Val and D-Val, and X2-X4 and X6-X8 are independently any one of amino acids.

2. The peptide or derivative thereof according to claim 1, wherein X2 is selected from any one of Pro, D-Pro and Hyp, X3 is selected from any one of Pro, D-Pro and Hyp, X4 is selected from any one of Ser, D-Ser, Ala and Aib, X6 is selected from any one of Ala, Leu, D-Leu, Ile, ABu and Nle, X7 is selected from any one of Tyr, D-Tyr, Ala, Trp, Phe and Bip, and X8 is selected from any one of Ser, D-Ser, Ala, and Orn.

3. The peptide or derivative thereof according to claim 1 or 2, comprising any one sequence selected from the group consisting of SEQ ID NOs: 1-5, SEQ ID NOs: 10-16 and SEQ ID NOs: 20-34, SEQ ID NOs: 1-5, SEQ ID NOs: 10-16 and SEQ ID NOs: 20-34 with conservative substitutions; preferably, the peptide or derivative thereof comprises any one sequence selected from the group consisting of SEQ ID NOs: 26, 28 and 32, SEQ ID NOs: 26, 28 and 32 with conservative substitutions.

4. The peptide or derivative thereof according to any one of claims 1 to 3, wherein the derivative comprises any one or more modifications selected from the following compared to the peptide: amino acid modification, conservative amino acid substitution, and substitution of hydrogen in an amino acid residue.

5. A pharmaceutical composition comprising the peptide or derivative thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier or salt.

6. Use of the peptide or derivative thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating pain.

7. A method for treating pain, comprising administering a therapeutically effective amount of the peptide or derivative thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 to a subject in need thereof.

8. The method according to claim 7, wherein the peptide or derivative thereof is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, pulmonary administration, ocular administration or topical administration.

9. The method according to claim 6 or claim 7, wherein the pain comprises pain caused by chemotherapy, diabetic peripheral neuropathy, sciatica, bone and joint pain, postherpetic neuralgia, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcoholism pain, hypothyroidism pain, autoimmune disease pain, Guillain-Barré syndrome pain, poisoning pain, drug-induced pain, tumor lesion pain, genetic disease pain and contusion pain.

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