Modified polypeptides and their applications in the field of analgesia

Abstract

This application relates to peptides or derivatives thereof, pharmaceutical compositions comprising such peptides or derivatives thereof, and their uses for treating pain.

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims the priority of PCT / CN2023 / 101880, an application number filed on June 21, 2023. Its prior application is regarded as part of the disclosure of this application, and the entire content is incorporated into this application.

[0002] Technical Field This application relates to the field of biopharmaceuticals. Specifically, it relates to peptides or their derivatives, and their uses in the field of analgesia.

Background Art

[0003] Neuropathic pain is pain caused by lesions or diseases of the somatic sensory nervous system. As a common chronic pain, it has a profound impact on the quality of life of patients. According to statistics, about 8% of the world's population suffers from this pain (GILRON I, BARON R, JENSEN T. Neuropathic pain: principles of diagnosis and treatment [J]. Mayo Clin Proc, 2015, 90(4): 532-545). Metabolic disorders (e.g., diabetic neuropathy), nerve entrapment disorders, tumor-related pain, post-stroke pain, Parkinson's disease-related pain, pain after spinal cord injury, trigeminal neuralgia, glossopharyngeal neuralgia, viral infections such as postherpetic neuralgia, etc. are all classified as typical neuropathic pain (SZOK D, TAJTI J, NYARI 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).

[0004] Perineuropathy, characterized by nerve damage and axonal loss, is classified as either hereditary or acquired. Acquired perineuropathy is associated with a variety of causes, including contact with toxic agents, and pain induced by antitumor compounds is called chemotherapy-induced perineuropathy (CIPN). A summary of neuroimmune interactions in several preclinical studies of CIPN has shown a link between chemotherapeutic agents and neurotoxicity. Studies have demonstrated the involvement of immune responses (innate and adaptive immunity) and the stimulation and secretion of mediators (cytokines and chemokines) as possible causes of pain symptoms. Furthermore, neuroinflammatory components have also been found in the spinal cord, and microglia and astrocytes have been shown to play important roles in CIPN (Front Immunol. 2021 Feb 4:11:626687).

[0005] Nicotinic acetylcholine receptors (nAChRs) are membrane proteins with important physiological functions that are universally present in nature and can regulate a range of physiological functions in the body, including pain, cognition, memory, and anxiety. Acetylcholine receptors are widely present in nerve cells and are closely related to pain relief, with the main subtypes being 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. Role of α7 nicotinic acetylcholine receptors in the regulation of animal inflammatory responses, Journal of Animal Nutrition, 2021, 33(11):6001-6008). Cone snails are carnivorous mollusks and are a general term for animals of the family Conidae. There are about 700 species of cone snails in the world, mainly distributed in tropical seas. Conotoxins are neurotoxic fluids secreted by cone snails, primarily composed of 10-30 amino acids and containing disulfide bonds. Professor Olivera's team at the University of Utah isolated and identified numerous conotoxin sequences and biological activities, discovering their potential as therapeutic agents for various neurological diseases. This discovery sparked a research boom among researchers both domestically and internationally, focusing on the development, structural modification, and mechanism of action of conotoxins. Among these, Vc1.1 (also known as ACV1) is a polypeptide containing 16 amino acids and two pairs of disulfide bonds, isolated from the Victoria's cone snail (Conus victoriae), and is an α-conotoxin with analgesic properties. Vc1.1 is a selective antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) and exhibits therapeutic effects against neuropathic pain in rat sciatica (CCI) and neuropathic pain (PNL) models. However, due to differences between human and rat α9α10 nAChRs, Vc1.1 had already progressed to Phase II clinical trials, but its activity against human α9α10 nAChR was 100-fold lower compared to rats, forcing the relevant institutions to discontinue the clinical study.Lorenzo Di Cesare Mannelli et al. studied the analgesic effects and mechanism of action of the conotoxin polypeptide RgIA in a rat sciatic nerve ligation model of chronic compression injury (CCI). Histological analysis of the sciatic nerve suggested that RgIA could prevent the decrease in axonal density and diameter, loss of myelin sheath, and reduction of fibers caused by CCI. Furthermore, RgIA significantly suppressed edema and inflammatory infiltration, including a decrease in CD86-positive macrophages. In the spinal cord dorsal horn, RgIA suppressed the activation of microglia and astrocytes induced by CCI. These data suggest that polypeptide RgIA may represent a new therapeutic agent for neuropathic pain, protecting surrounding nerve tissue and preventing maladaptive central nervous system plasticity by suppressing glial cell activation (PAIN:155 (2014) 1986-1995).

[0006] Ziconotide, approved by the FDA in 2004 for the treatment of severe chronic pain, was originally isolated from the venom of the magical cone (Conus magus) and consists of 25 amino acids and 3 pairs of disulfide bonds. As a selective Cav2.2 channel antagonist, ziconotide is Ca 2+ It blocks the inflow of ciconotides and suppresses the release of excitatory neurotransmitters to nerve periphery, and its analgesic effect is 1000 times stronger than that of opioid analgesics. However, due to its unique administration route of intrathecal injection and a series of central nervous system side effects, patient compliance (adherence to medication) is low, which limits the widespread application of ciconotides.

[0007] Therefore, there is a strong need for the development of improved peptides that act on nicotinic acetylcholine receptors, effectively relieve pain, and / or reduce side effects. [Overview of the Initiative]

[0008] The target of the polypeptide target in this application includes α7 nAChR and α9 nAChR, and the target is a human-derived acetylcholine receptor sequence. This avoids species-dependent activity reduction and similar risks associated with conotoxin Vc1.1. The polypeptide sequence in this application incorporates multiple non-natural amino acids and includes a pair of intramolecular disulfide bonds, thereby improving polypeptide stability, significantly increasing the half-life in the body, and greatly extending the duration of analgesic effect. Pain is a serious clinical problem that negatively impacts the quality of life (QOL) of both patients and their families. However, existing analgesics cannot adequately address various types of pain, and long-term administration also presents side effects, such as opioid analgesic dependence and the risk of drug abuse with long-term use. Polypeptides inherently possess superior safety, and the polypeptide in this application has a simple synthesis process, is easy to commercialize, and is expected to be a safe and effective novel analgesic.

[0009] As can be seen from the animal test results, the polypeptide relating to this application has a shorter onset of action than the clinically used analgesic pregabalin, and the polypeptide relating to this application has significantly improved stability in human plasma compared to the parent peptide AJ003. Animal test data also suggest that the duration of analgesic effect of the modified peptide is significantly longer than that of the parent peptide. The polypeptide relating to this application belongs to the category of novel non-opioid analgesics, and its target of action is mainly in peripheral nerve tissue, so it will not cause side effects such as drug dependence in later clinical applications. Compared to diconotide, the only conotoxin polypeptide currently on the market, the polypeptide relating to this application does not require complex intrathecal administration and contributes to improved patient medication adherence in clinical settings. Furthermore, diconotide is composed of 25 amino acids and contains 3 pairs of disulfide bonds in its sequence, making the manufacturing process complex and significantly increasing production costs. The polypeptide relating to this application is composed of 13 amino acids and contains only 1 pair of disulfide bonds. Therefore, the manufacturing process is simple, the polypeptide solid-phase synthesis technology is already very mature, and its synthesis characteristics are extremely suitable for large-scale automated production, which can significantly reduce the production cost of pharmaceuticals and alleviate the medical burden on patients. Furthermore, the polypeptides related to this application can be administered at low doses and exhibit remarkable analgesic activity even at low doses.

[0010] In a first aspect of this disclosure, a peptide or derivative thereof comprising any one sequence selected from SEQ ID NOs: 1-34 is provided.

[0011] In a second aspect of this disclosure, a peptide or derivative thereof is provided, comprising any one sequence selected from SEQ ID NOs: 1-34 having a conservative substitution.

[0012] A third aspect of this disclosure provides peptides comprising formula (I) or formula (I) having a conservative substitution in the order of N-terminus to C-terminus, or derivatives thereof.

[0013] Formula I Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8

[0014] Here, in formula (I), the Cys at position 3 and the D-Cys at position 9 form a pair of intramolecular disulfide bonds, D-Cys represents D-cysteine, the "-" between amino acid residues represents a peptide bond, the C-terminus of the peptide or its derivative is a free carboxyl group or amide, X1 is Thr or D-Thr, X5 is any one selected from Ala, Val and D-Val, and X2-X4 and X6-X8 are each any independent amino acid.

[0015] In one embodiment of the present disclosure, the Cys at position 3 and the D-Cys at position 9 in formula (I) form a pair of intramolecular disulfide bonds, the C-terminus of the peptide or its derivative is a free carboxyl group or amide, and X1 is Thr or D-Thr, X5 is any one selected from Ala, Val and D-Val, X2 is any one selected from Pro, D-Pro and Hyp, X3 is any one selected from Pro, D-Pro and Hyp, X4 is any one selected from Ser, D-Ser, Ala and Aib, X6 is any one selected from Ala, Leu, D-Leu, Ile, ABu and Nle, X7 is any one selected from Tyr, D-Tyr, Ala, Trp, Phe and Bip, and X8 is any one selected from Ser, D-Ser, Ala and Orn.

[0016] In one embodiment of the present disclosure, formula (I) having the above-mentioned conservative substitution has 1, 2, 3, 4, or 5 amino acid conservative substitutions. In one embodiment of the present disclosure, formula (I) having the above-mentioned conservative substitution 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 compared to formula (I) without the conservative substitution.

[0017] In one preferred embodiment of the present disclosure, the peptide or its derivative comprises any one sequence selected from: SEQ ID NOs: 1-5, SEQ ID NOs: 10-16 and SEQ ID NOs: 20-34, and SEQ ID NOs: 1-5, SEQ ID NOs: 10-16 and SEQ ID NOs: 20-34 having a conserved substitution. In one more preferred embodiment of the present disclosure, the peptide or its derivative comprises any one sequence selected from: SEQ ID NOs: 26, 28 and 32, and SEQ ID NOs: 26, 28 and 32 having a conserved substitution.

[0018] In one embodiment of the present disclosure, the sequence having the above-mentioned conservative substitutions has one, two, three, four, or five amino acid conservative substitutions, respectively. In one embodiment of the present disclosure, the sequence having the above-mentioned conservative substitutions and the sequence not having the conservative substitution formula 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.

[0019] A fourth aspect of this disclosure provides a polynucleotide, which encodes a peptide or a derivative thereof as described in this disclosure.

[0020] In a fifth aspect of this disclosure, an expression vector is provided which expresses a polynucleotide described in this disclosure.

[0021] A sixth aspect of this disclosure provides a host cell comprising a polynucleotide described in this disclosure or an expression vector described in this disclosure.

[0022] In a seventh aspect of the present disclosure, there is provided a pharmaceutical composition comprising the peptide or its derivative described in the present disclosure and a pharmaceutically acceptable carrier or salt. The present disclosure further provides a pharmaceutical composition comprising the peptide or its derivative, polynucleotide, expression vector or host cell described in the present disclosure, and a pharmaceutically acceptable carrier or salt.

[0023] In an eighth aspect of the present disclosure, there is provided the use of the peptide or its derivative described in the present disclosure or the pharmaceutical composition described in the present disclosure in the manufacture of a medicament for treating pain. The present disclosure further provides the use of the peptide or its derivative, polynucleotide, expression vector, host cell described in the present disclosure or the pharmaceutical composition described in the present disclosure in the manufacture of a medicament for treating pain.

[0024] In a ninth aspect of the present disclosure, there is provided a method for treating pain, the method comprising administering to a subject to be a subject a therapeutically effective amount of the peptide or its derivative described in the present disclosure or the pharmaceutical composition described in the present disclosure. The present disclosure further provides a method for treating pain, the method comprising administering to a subject to be a subject a therapeutically effective amount of the peptide or its derivative, polynucleotide, expression vector, host cell described in the present disclosure or the pharmaceutical composition described in the present disclosure.

Brief Description of Drawings

[0025] The following provides a brief description of the drawings. These are for explaining exemplary embodiments disclosed in the present disclosure and do not limit these embodiments. [Figure 1] It is a line graph showing the change over time of the main peak area of the polypeptide AJ003-1 in Example 3. [Figure 2] It is a line graph showing the change over time of the main peak area of the polypeptide AJ003-34 in Example 3. [Figure 3] It is a line graph showing the change over time of the main peak area of the polypeptide AJ003-35 in Example 3. [Figure 4] It is a line graph showing the change over time of the main peak area of polypeptide AJ003-36 in Example 3. [Figure 5] It is a line graph showing the change over time of the main peak area of polypeptide AJ003-37 in Example 3. [Figure 6] It is a line graph showing the change over time of the main peak area of polypeptide AJ003-38 in Example 3. [Figure 7] It is the in vitro half-life prediction result of polypeptide AJ003-39 in human plasma in Example 3. [Figure 8] It is a line graph showing the change over time of the main peak area of polypeptide AJ003-40 in Example 3. [Figure 9] It is a line graph showing the change over time of the main peak area of polypeptide AJ003-41 in Example 3. [Figure 10] It is a line graph showing the change over time of the main peak area of polypeptide AJ003-42 in Example 3. [Figure 11] It is a graph showing the mechanical pain threshold on the first day of the same dosage of polypeptide in the rat paclitaxel chronic chemotherapy-induced pain model in Example 5. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents different dosing groups. The results show the mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n = 7 - 8). Model group and control group: #p < 0.0001; dosing group and model group: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns: results not significant in statistical analysis. Pregabalin 30 mg / kg / day, once a day, intragastric administration; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ003-39 and AJ003-40 1 mg / kg / day, once a day, subcutaneous injection. [Figure 12]This figure shows the mechanical pain threshold on day 3 for identical doses of polypeptide in a rat paclitaxel chronic chemotherapy-induced pain model in Example 5. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents different dose groups. The results show the mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7-8). Model group vs. control group: ####p<0.0001; dose group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: not significant result in statistical analysis. Pregabalin 30 mg / kg / day, once daily, intragastric administration; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ003-39 and AJ003-40 1 mg / kg / day, once daily, subcutaneous injection. [Figure 13] This figure shows the mechanical pain threshold on day 5 for identical doses of polypeptide in a rat paclitaxel chronic chemotherapy-induced pain model in Example 5. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents different dose groups. The results show the mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7-8). Model group vs. control group: ####p<0.0001; Dose group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. Pregabalin 30 mg / kg / day, once daily, intragastric administration; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ003-39 and AJ003-40 1 mg / kg / day, once daily, subcutaneous injection. [Figure 14]This figure shows the mechanical pain threshold on day 12 for the same dose of polypeptide in a rat paclitaxel chronic chemotherapy-induced pain model in Example 5. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents different dose groups. The results show the mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7-8). Model group vs. control group: ####p<0.0001; dose group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. Pregabalin 30 mg / kg / day once daily, administered intragastricly; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ003-39 and AJ003-40 1 mg / kg / day once daily, administered subcutaneously. [Figure 15] This figure shows the mechanical pain threshold on day 1 for different doses of polypeptide in a rat paclitaxel chronic chemotherapy-induced pain model in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different dose 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; Dose group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. Pregabalin: 30 mg / kg / day, once daily, administered intragastricly; AJ003: 1 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously. [Figure 16]This figure shows the mechanical pain threshold on day 4 for different doses of polypeptide in a rat paclitaxel chronic chemotherapy-induced pain model in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different dose groups. The results show the mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: ####p<0.0001; Dose group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. Pregabalin: 30 mg / kg / day, once daily, administered intragastricly; AJ003: 1 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously. AJ003-40-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously. [Figure 17] This figure shows the mechanical pain threshold on day 7 for different doses of polypeptide in a rat paclitaxel chronic chemotherapy-induced pain model in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different dose groups. The results show the mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: ####p<0.0001; Dose group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. Pregabalin: 30 mg / kg / day, once daily, administered intragastricly; AJ003: 1 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously. [Figure 18]This figure shows the mechanical pain thresholds on day 12 for different doses of polypeptide in a rat paclitaxel chronic chemotherapy-induced pain model in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different dose 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; Dose group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. Pregabalin: 30 mg / kg / day, once daily, administered intragastricly; AJ003: 1 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously. [Figure 19] This figure shows the mechanical pain thresholds on day 14 for different doses of polypeptide in a rat paclitaxel chronic chemotherapy-induced pain model in Example 6. The vertical axis represents the mechanical pain threshold, and the horizontal axis represents the different dose 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; Dose group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. Pregabalin: 30 mg / kg / day, once daily, administered intragastricly; AJ003: 1 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-36-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(low dose): 0.5 mg / kg / day, once daily, administered subcutaneously; AJ003-40-(high dose): 1.5 mg / kg / day, once daily, administered subcutaneously. [Figure 20]This figure shows the time course of the mechanical pain threshold for different doses of polypeptide in a mouse oxaliplatin acute chemotherapy-induced pain model in Example 7. The results are shown as the mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=8). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. [Figure 21] This figure shows the mechanical pain threshold on day 1 for different doses of polypeptide in a rat CCI model in Example 9. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns: results not significant in statistical analysis. [Figure 22] This figure shows the mechanical pain threshold on day 4 for different doses of polypeptide in a rat CCI model in Example 9. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: #### p<0.0001; Treatment group vs. model group: **** p<0.0001; ** p<0.01; ns: results not significant in statistical analysis. [Figure 23] This figure shows the mechanical pain threshold on day 7 for different doses of polypeptide in a rat CCI model in Example 9. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; **p<0.01; *p<0.05. ns: Non-significant result in statistical analysis. [Figure 24]This figure shows the mechanical pain thresholds on day 10 for different doses of polypeptide in a rat CCI model in Example 9. The results are shown as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. ns: Non-significant result in statistical analysis. [Figure 25] The mechanical pain thresholds at day 14 for different polypeptide doses in the rat CCI model in Example 9 are shown. Results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01. ns: Non-significant result in statistical analysis. [Figure 26] This figure shows the mechanical pain threshold on day 1 for different doses of polypeptide in a mouse model of diabetic neuropathic pain in Example 10. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. ns: Non-significant result in statistical analysis. [Figure 27] This figure shows the mechanical pain threshold on day 4 for different doses of polypeptide in a mouse model of diabetic neuropathic pain in Example 10. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. ns: Non-significant result in statistical analysis. [Figure 28]This figure shows the mechanical pain threshold on day 8 for different doses of polypeptide in a mouse model of diabetic neuropathic pain in Example 10. The results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. ns: Non-significant result in statistical analysis. [Figure 29] This figure shows the mechanical pain thresholds on day 14 for different doses of polypeptide in a mouse model of diabetic neuropathic pain in Example 10. The results are shown as mechanical pain thresholds (g) (±SD). Graphpad, one-way ANOVA (n=7). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. ns: Non-significant result in statistical analysis. [Figure 30] This figure shows the time course of mechanical pain threshold for different doses of polypeptide in a mouse oxaliplatin acute chemotherapy-induced pain model in Example 8. Results are shown as mechanical pain threshold (g) (±SD). Graphpad, one-way ANOVA (n=3). Model group vs. control group: ####p<0.0001; Treatment group vs. model group: ****p<0.0001; ***p<0.001; **p<0.01. ns: Non-significant result in statistical analysis. [Figure 31] This figure shows the HPLC analysis spectrum after incubation of AJ003-40 in human plasma at 37°C for 0 hours in Example 4. [Figure 32] This figure shows the HPLC analysis spectrum after incubating AJ003-40 in human plasma at 37°C for 24 hours in Example 4. [Figure 33] This figure shows the HPLC analysis spectrum obtained after incubating AJ003-24 in human plasma at 37°C for 0 hours in Example 4. [Figure 34] This figure shows the HPLC analysis spectrum obtained after incubating AJ003-24 in human plasma at 37°C for 22 hours in Example 4. [Modes for carrying out the invention]

[0026] Unless otherwise stated, all numerical values ​​used in this specification and in the claims, such as “content,” “concentration,” “ratio,” “weight,” “particle size,” “percentage,” and “technical effect,” are understood to be modified in any case by the terms “approximately” or “about.” Therefore, unless otherwise indicated, the numerical parameters described in the following specification and claims are approximations.

[0027] Unless otherwise stated, terms used herein have the meanings that a person skilled in the art would ordinarily understand. A person skilled in the art should interpret each numerical parameter in accordance with significant figures and ordinary rounding methods, or in any way that a person skilled in the art would understand, based on the desired properties and effects to be achieved by this disclosure. In general, the nomenclature used herein and the experimental procedures of organic chemistry, pharmacochemistry, and biology described herein are well known and widely adopted in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meanings that a person skilled in the art of the subject of this application would ordinarily understand. If there are multiple definitions of a term used herein, the definition in this section shall prevail unless otherwise stated.

[0028] In this specification, the expression "A and / or B" includes the following three cases: (1) A; (2) B; and (3) A and B. The expression "A, B and / or C" includes the following 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 meaning of similar expressions is inferred in this way.

[0029] In this specification, the term "independently" means that there is no mutual influence between multiple events. For example, "X and Y are any one independently selected from a, b, c, d, e, f, and g" means that X may be any of a, b, c, d, e, f, and g, and Y may also be any of a, b, c, d, e, f, and g, and the selection of X and the selection of Y may be the same or different, and the two selections do not interfere with each other.

[0030] In this specification, alanine scanning (Cunningham and Wells, Science 244, 1081-1085, 1989) is used to identify amino acids that play a crucial role in the activity of the peptide of the present invention (e.g., binding affinity to the acetylcholine receptor) and to avoid substituting these amino acids. Alanine scanning identifies amino acid residues that play a critical role in the activity of the molecule by introducing mutations into each residue within the molecule and measuring the biological activity of the resulting molecule.

[0031] In this specification, the terms “includes” and “contains” indicate that the listed elements are present, but do not exclude other elements.

[0032] In this specification, the term "identity" refers to the degree to which two (nucleotide or amino acid) sequences have homologous residues at homologous positions in their alignment, and is generally expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of a sequence that are completely identical have 100% identity. Those skilled in the art will recognize that algorithms 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 ClustalW can be used to determine sequence identity using standard parameters.

[0033] Peptides or their derivatives This application provides a peptide or a derivative thereof comprising any one sequence selected from SEQ ID NOs: 1-34.

[0034] This application provides a peptide or derivative thereof comprising any one sequence selected from SEQ ID NOs: 1-34 having conservative substitutions. In some embodiments, any one of SEQ ID NOs: 1-34 having conservative substitutions has 1, 2, 3, 4, or 5 amino acid conservative substitutions. In some embodiments, any sequence that does not have a conservative substitution formula with any one of SEQ ID NOs: 1-34 having 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.

[0035] This application provides a peptide or derivative thereof comprising formula (I) or formula (I) having a conservative substitution in the order from the N-terminus to the C-terminus. Formula 1 Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8

[0036] Here, in formula (I), the Cys at position 3 and the D-Cys at position 9 form a pair of intramolecular disulfide bonds, D-Cys represents D-cysteine, the "-" between amino acid residues represents a peptide bond, the C-terminus of the peptide or its derivative is a free carboxyl group or amide, X1 is Thr or D-Thr, X5 is any one selected from Ala, Val and D-Val, and X2-X4 and X6-X8 are each any independent amino acid.

[0037] In some embodiments, X1 in formula (I) is Thr or D-Thr. In some embodiments, X5 in formula (I) is any one selected from Ala, Val and D-Val. In some embodiments, X2 in formula (I) is any one selected from Pro, D-Pro and Hyp. In some embodiments, X3 in formula (I) is any one selected from Pro, D-Pro and Hyp. In some embodiments, X4 in formula (I) is any one selected from Ser, D-Ser, Ala and Aib. In some embodiments, X6 in formula (I) is any one selected from Ala, Leu, D-Leu, Ile, ABu and Nle. In some embodiments, X7 in formula (I) is any one selected from Tyr, D-Tyr, Ala, Trp, Phe and Bip. In some embodiments, X8 in formula (I) is any one selected from Ser, D-Ser, Ala and Orn.

[0038] In some embodiments, formula (I) having the above-mentioned conservative substitution has one, two, three, four, or five amino acid conservative substitutions. In one embodiment of the present disclosure, formula (I) having the above-mentioned conservative substitution 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 compared to formula (I) without the conservative substitution.

[0039] In some embodiments, the peptide or its derivative comprises any one sequence selected from: 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 a conservative substitution. In some preferred embodiments, the peptide or its derivative comprises SEQ ID NOs: 26 or SEQ ID NOs: 26 with a conservative substitution. In some preferred embodiments, the peptide or its derivative comprises SEQ ID NOs: 28 or SEQ ID NOs: 28 with a conservative substitution. In some preferred embodiments, the peptide or its derivative comprises SEQ ID NOs: 32 or SEQ ID NOs: 32 with a conservative substitution.

[0040] In some embodiments, the sequence having the above-mentioned conservative substitution has one, two, three, four, or five amino acid conservative substitutions. In some embodiments, the sequence having the above-mentioned conservative substitution and the sequence not having the conservative substitution formula 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.

[0041] In some embodiments, the derivative includes any one or more modifications selected from amino acid modifications, conservative amino acid substitutions, and hydrogen substitutions in amino acid residues, compared to the peptide.

[0042] In this specification, the term "peptide" refers to a compound formed by the linkage of amino acids by polypeptide bonds. A polypeptide composed of three or more amino acid molecules is a polypolypeptide. In this specification, unless otherwise specified or inconsistent with the context, the term "polypeptide" includes the polypeptide itself and its pharmaceutically acceptable salts, prodrugs, and metabolites. Polypeptides in this application are produced by art-standard methods, including but not limited to solid-phase synthesis (e.g., Fmoc polypeptide synthesis) and liquid-phase synthesis.

[0043] In this specification, the positional numbers of a polypeptide or its derivative are assigned in order from the N-terminus to the C-terminus. For example, the first amino acid from the N-terminus of a polypeptide is the 1st amino acid, the second amino acid from the N-terminus is the 2nd amino acid, the third amino acid from the N-terminus is the 3rd amino acid, and so on.

[0044] Unless otherwise defined, the left end of an amino acid sequence or polypeptide sequence is the N-terminus / amino group end, as is commonly understood by those skilled in the art, and the right end of an amino acid sequence or polypeptide sequence is the C-terminus / carboxyl end. In this specification, the term “prodrug” refers to all molecules that, under physiological conditions, are converted in a living organism through reactions such as oxidation, reduction, or hydrolysis by the action of enzymes, gastric acid, etc., to the peptides or derivatives thereof of this disclosure.

[0045] In this specification, the term “metabolite” means all molecules derived from the peptides or derivatives thereof in a cell or organism (preferably human).

[0046] In this specification, with respect to the use of peptides, "amino acid" and "amino acid residue" are synonymous, and an amino acid residue refers to the remaining amino acid portion after some functional groups are lost due to their involvement in the formation of a chemical bond when amino acids are linked together.

[0047] In this specification, the names and abbreviations of amino acids are as follows:

[0048] [Table A]

[0049] In natural amino acids, all except glycine have an asymmetric carbon atom. Unless otherwise specified, all optically active amino acids described in this application are L-type. By convention, peptide structures in this specification are shown with the amino terminus (N-terminus) on the left and the carboxyl terminus (C-terminus) on the right.

[0050] As used herein, the term "amino acid" includes not only natural amino acids but also other "non-protein" α-amino acids / non-natural amino acids commonly used in the field of peptide chemistry when preparing analogs of natural peptides.

[0051] Natural amino acids include glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, ornithine, and lysine. "Non-protein" α-amino acids / non-natural amino acids include norleucine, norvaline, allo-isoleucine, homoarginine, thiaproline, didehydroproline, hydroxyproline (Hyp), homoserine, cyclohexylglycine (Chg), α-aminobutyric acid (Aba), cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanine in which the phenyl group is substituted with an alkyl group, alkoxy group, halogen or nitro group, serine, threonine and O-alkylated derivatives of tyrosine, S-alkylated cysteine, O-sulfate of tyrosine, and D-isomers of natural amino acids.

[0052] In this specification, the term "derivative" of a peptide refers to a product obtained by modifying the peptide mentioned, performing conservative substitutions, and / or hydrogen substitutions of amino acid residues.

[0053] In this specification, the term "amino acid modification" includes, but is not limited to, N-terminal modification, C-terminal modification, and side-chain modification. Methods of "amino acid modification" include, but are not limited to, modifications such as hydroxylation, carboxylation, alkylation, acylation, phosphorylation, sulfonation, amidation, aldehydeation, alkoxylation, cysteamineation, esterification, and glycosylation.

[0054] In this specification, “conservative amino acid substitution” can be described as a substitution of an amino acid residue by another amino acid having a similar chemical structure and / or similar chemical properties, and which does not substantially affect the function, activity, or other biological properties of the peptide. Such conservative amino acid substitutions are well known in the art. Such a conservative substitution may be, for example, the substitution of one amino acid from the following groups (a) to (e) by another amino acid within the same group: (a) small aliphatic, nonpolar 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, nonpolar amino acid residues: Met, Leu, Ile, Val and Cys; (e) aromatic amino acid residues: Phe, Tyr and Trp.

[0055] In this specification, hydrogen substitution in an amino acid residue may also be the substitution of a hydrogen atom in the amino acid residue with any common substituent known in the art, including but not limited to alkyl groups, alkenyl groups, alkynyl groups, aryl groups, alkoxy groups, carboxyl groups, aldehyde groups, carbonyl groups, hydroxy, halogen, cyano groups, acyl, sulfonic acid groups, amino groups, mercapto groups, or nitro groups.

[0056] Polynucleotides, expression vectors, host cells This application provides a polynucleotide, the polynucleotide comprising a peptide or a derivative thereof as described herein.

[0057] In this specification, “polynucleotide” refers to a polymer of nucleotides (nucleotides or deoxyribonucleotides) of any length. The term refers to the primary structure of the molecule. Therefore, it includes both double-stranded and single-stranded RNA and double-stranded and single-stranded DNA. It further includes polynucleotides modified by methylation and / or capping, etc., and unmodified polynucleotides. The polynucleotides described herein are not necessarily obtained by physical means and may be produced by any means, such as chemical synthesis or DNA replication, reverse transcription, or transcription.

[0058] This application provides an expression vector which expresses the polynucleotide described herein.

[0059] This application provides a host cell comprising a polynucleotide or an expression vector as described herein.

[0060] In this specification, “host cell” is used as a cell that is a receptor for an expression vector or other transformed gene fragment. It includes transfected protocellular cells and their progeny. Host cells include prokaryotic cells, yeast, or other eukaryotic cells.

[0061] Pharmaceutical composition This application provides a pharmaceutical composition comprising a peptide or derivative thereof as described herein, and a pharmaceutically acceptable carrier or salt.

[0062] This application further provides a pharmaceutical composition comprising a peptide or derivative thereof, a polynucleotide, an expression vector or host cell as described herein, and a pharmaceutically acceptable carrier or salt.

[0063] In this specification, the term “pharmaceutical composition” refers to a mixture of the peptide described herein with other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition is useful for administering the peptide to a living organism. Peptides in the art have a variety of administration methods, including, but are not limited to, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, transpulmonary administration, ocular and topical administration.

[0064] In this application, the pharmaceutical composition may be prepared into a dosage form suitable for administration to a subject via the required route of administration. The dosage form includes, but is not limited to, tablets, capsules, capsule tablets, pills, tablets, powders, syrups, elixirs, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, washes, pastes, sprays, lyophilized agents, injections, and gels.

[0065] In this specification, the term “pharmaceutically acceptable salt” includes acid addition salts and base addition salts. Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetates, adipines, aspartates, benzoates, benzenesulfons, bicarbonates / carbonates, bisulfates / sulfates, borates, camphor sulfons, citrates, cyclohexylamine sulfons, ethanedisulfons, formates, fumarates, glucoheptons, glucons, glucurons, hexafluorophosphates, 2-(4-hydroxybenzyl)benzoates, hydrochlorides / chlorides, hydrobromates / bromine, hydroiodides / iodides, and 2-hydroxybenzyl benzoates. This includes, but is not limited to, droxyethanesulfonates, lactates, malates, maleates, malons, methanesulfonates, methylsulfonates, naphthalenesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, palmitates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, glucarates, stearates, salicylates, tannates, tartrates, toluenesulfonates, and trifluoroacetates. Suitable base addition salts are formed from bases that form non-toxic salts. Examples include, but are not limited to, aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, trometamol, and zinc. Half-salts of acids and bases, such as half-sulfates and half-calcium salts, can also be formed. For an overview of appropriate salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0066] The term “pharmaceutically acceptable carrier” includes pharmaceutically acceptable materials, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, which transport or deliver the peptides described herein within a subject or to a subject, thereby enabling them to perform their intended function. Each salt or carrier must be “acceptable” in the sense of compatibility with other components in the formulation and must not be harmful to the subject. 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; excipients such as tragacanth powder, malt, gelatin, talc, cocoa butter, and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; and polyhydric compounds such as glycerin, sorbitol, mannitol, and polyethylene glycol. Examples include esters such as cellulose, ethyl oleate and ethyl laurate, buffers such as agar, magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, phosphate buffer, diluents, granulators, lubricants, binders, disintegrants, wetting agents, emulsifiers, colorants, release agents, coating agents, sweeteners, flavoring agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, curing agents, shaping agents, suspending agents, surfactants, humectants, carriers, stabilizers, other non-toxic and compatible substances used in pharmaceutical formulations, and any combination thereof.

[0067] Uses, methods of treating diseases This application provides applications for the peptide or derivative thereof described herein, or the pharmaceutical composition described herein, in the manufacture of pharmaceuticals for the treatment of pain.

[0068] This application further provides applications for the manufacture of pharmaceuticals for treating pain, using the peptides or derivatives thereof described herein, the polynucleotides described herein, the expression vectors described herein, the host cells described herein, or the pharmaceutical compositions described herein.

[0069] In some embodiments, the pharmaceutical preparation for treating pain is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, transpulmonary administration, ocular or local administration. In a preferred embodiment, the pharmaceutical preparation for treating pain is administered by subcutaneous injection.

[0070] In some embodiments, the pain includes pain caused by action on nicotinic acetylcholine receptors. In some embodiments, the pain includes neuropathic pain and inflammatory pain. In some embodiments, the pain includes chemotherapy-induced pain, diabetic peripheral neuropathy, sciatica, osteoarthritis, postherpetic neuralgia, infection-induced pain, nutritional deficiency-induced pain, inflammatory pain, chronic alcohol-induced pain, hypothyroidism-induced pain, autoimmune disease pain, Guillain-Barré syndrome pain, toxic pain, drug-induced pain, tumor-induced pain, genetic disease-induced pain, and contusion pain.

[0071] This application further provides applications for the treatment of pain using the peptides or derivatives thereof described herein, the polynucleotides described herein, the expression vectors described herein, the host cells described herein, or the pharmaceutical compositions described herein.

[0072] In some embodiments, the pain includes pain caused by action on nicotinic acetylcholine receptors. In some embodiments, the pain includes neuropathic pain and inflammatory pain. In some embodiments, the pain includes chemotherapy-induced pain, diabetic peripheral neuropathy, sciatica, osteoarthritis, postherpetic neuralgia, infection-induced pain, nutritional deficiency-induced pain, inflammatory pain, chronic alcohol-induced pain, hypothyroidism-induced pain, autoimmune disease pain, Guillain-Barré syndrome pain, toxic pain, drug-induced pain, tumor-induced pain, genetic disease pain, and contusion pain.

[0073] In some embodiments, the peptide or its derivative or pharmaceutical composition is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, transpulmonary administration, ocular or local administration. In preferred embodiments, the peptide or its derivative or pharmaceutical composition is administered by subcutaneous injection.

[0074] This application provides a method for treating pain, the method comprising administering to a subject a therapeutically effective amount of the peptide or a derivative thereof described herein, or the pharmaceutical composition described herein.

[0075] The present application further provides a method for treating pain, which comprises administering to a subject a therapeutically effective amount of the peptide or derivative thereof, polynucleotide, expression vector, host cell, or pharmaceutical composition described herein.

[0076] In some embodiments, the pain includes pain caused by action on nicotinic acetylcholine receptors. In some embodiments, the pain includes neuropathic pain and inflammatory pain. In some embodiments, the pain includes chemotherapy-induced pain, diabetic peripheral neuropathy, sciatica, osteoarthritis, postherpetic neuralgia, infection-induced pain, nutritional deficiency-induced pain, inflammatory pain, chronic alcohol-induced pain, hypothyroidism-induced pain, autoimmune disease pain, Guillain-Barré syndrome pain, toxic pain, drug-induced pain, tumor-induced pain, genetic disease-induced pain, and contusion pain.

[0077] In some embodiments, the peptide or its derivative or pharmaceutical composition is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, transpulmonary administration, ocular or local administration. In preferred embodiments, the peptide or its derivative or pharmaceutical composition is administered by subcutaneous injection.

[0078] In this specification, the term “subject” includes animals, such as vertebrates, preferably mammals, such as dogs, cats, pigs, cattle, sheep, horses, rodents (such as mice, rats, or guinea pigs), or primates (such as gorillas, chimpanzees, and humans).

[0079] In this specification, the term “treatment” means reducing or improving a disease or disorder (i.e., delaying or preventing the progression of the disease or at least one clinical symptom), or alleviating or improving at least one physical parameter or biomarker associated with said disease or disorder.

[0080] In this specification, the term “therapeutic dose” means the amount that, compared to a corresponding subject who did not receive that amount, results in a benefit or treatment of the disease, but is within the bounds of reasonable medical judgment and sufficiently low to avoid serious side effects. The therapeutic dose of the peptide or its derivative, polynucleotide, expression vector, host cell or pharmaceutical composition described herein varies depending on factors such as the selected peptide or its derivative, polynucleotide, expression vector, host cell or pharmaceutical composition; the route of administration; the severity of the disease being treated; the age, body type, weight and health status of the patient being treated; the patient's medical history; the duration of treatment; the nature of the combination therapy; and the expected therapeutic effect, but can be determined by conventional methods by those skilled in the art.

[0081] The various embodiments of the peptides or derivatives thereof, polynucleotides, expression vectors and host cells described in the Preamble also apply to the pharmaceutical compositions, uses and methods described in the Disclosure (unless they are essentially inconsistent with each other), and the various embodiments formed by combining them are deemed to constitute part of the Disclosure. [Examples]

[0082] The following describes representative embodiments of this application with reference to the drawings, including various details of these embodiments to aid understanding. These should be understood to be merely illustrative and not intended to limit the scope of protection of this application. The scope of protection of this application is limited solely by the claims. Therefore, those skilled in the art should be aware that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and configurations are omitted in the following description.

[0083] Unless otherwise specified, the reagents and equipment used in the following examples are all commercially available, standard products. Unless otherwise specified, experiments are conducted under normal conditions or conditions recommended by the manufacturer.

[0084] Example 1: Synthesis of conotoxin polypeptide Based on previous research, a series of polypeptide compounds were initially selected by performing alanine scanning, single-site unnatural amino acid substitution, and multi-site combination modification on the parent peptide AJ003.

[0085] Synthesis of AJ003: Fmoc-Ser(tBu)-wang resin (Tianjin Nankai Synthetic Technology Co., Ltd.) was used as the starting resin, with a synthesis scale of 1 mmol. The resin was weighed and added to a polypeptide solid-phase synthesizer (Nanjing Luanyu Hua Particleboard Instruments Co., Ltd.), and after adding dichloromethane and swelling by nitrogen gas bubbling for 30 minutes, it was washed twice with DMF (dimethylformamide). A 20% piperidine / DMF solution was added twice and reacted for 5 minutes each time. After the deprotection reaction was complete, the resin was washed six times with DMF, held for approximately 3 minutes each time. According to the polypeptide sequence, and based on the principle of sequential amino acid bonding from the C-terminus to the N-terminus, which is common in chemically synthesized polypeptides, the corresponding amounts of protective amino acids and condensing agents (HOAt, DIC) were weighed and reacted for approximately 1 hour. After the condensation reaction was complete, the resin was washed three times with DMF. Subsequently, a condensation cycle (deprotection-washing-condensation-washing) was performed. Specifically, a 20% piperidine / DMF solution was added to the reaction vessel again to carry out the deprotection reaction, followed by DMF washing. After washing, the protective amino acids and a corresponding amount of condensing agent were added to carry out the condensation reaction. The condensation cycle method was employed, and protective amino acids were bonded one by one towards the N-terminus until all amino acids of the polypeptide were bonded. The Fmoc protecting group at the N-terminus of the polypeptide was removed with a 20% piperidine / DMF solution, and the resin was washed alternately with dichloromethane and methanol. A shrink-dried polypeptide resin intermediate was obtained by vacuum drying. The polypeptide resin was weighed, and a lysis buffer was prepared at a ratio of 10 ml of lysis buffer per gram of polypeptide resin. The composition of the lysis buffer was TFA:EDT:TIS:H2O = 90:5:2.5:2.5. A dissolution buffer was added to the polypeptide resin, and a decomposition reaction was carried out for approximately 3 hours. The filtrate was collected, and precipitation was performed with cooled methyl tert-butyl ether (8 times the amount of the filtrate). After thoroughly stirring the precipitate, it was dispensed and centrifuged to obtain a crude polypeptide precipitate. Next, it was washed with methyl tert-butyl ether and centrifuged, and the resulting precipitate was vacuum-dried overnight to finally obtain a linear crude polypeptide sample. After dissolving the obtained linear crude polypeptide in water, elemental iodine was added as an oxidizing agent to carry out the disulfide bond formation reaction (oxidation) of the polypeptide.The oxidation process was carried out for approximately 30 minutes, and the progress of the oxidation was monitored by HPLC. Finally, vitamin C was added dropwise to the oxidation system to stop the oxidation reaction. After filtering the polypeptide solution with the oxidation reaction completed through a membrane filter, separation and purification were performed by reverse-phase high-performance liquid chromatography. The resulting acceptable fractions were combined, and then freeze-dried using a freeze-dryer to obtain the final freeze-dried purified product of the polypeptide.

[0086] The synthesis methods for other polypeptides were similar to those for polypeptide AJ003, and in all cases, linear crude polypeptides were obtained using representative Fmoc solid-phase polypeptide synthesis technology. For polypeptide sequences ending in an amide at the C-terminus, Rink amide-MBHA resin was used. The crude polypeptide solution was iodine-oxidized to form disulfide-bonded polypeptide fractions, followed by separation and purification by reverse-phase high-performance liquid chromatography, and finally, lyophilized refined products were obtained by lyophilization technology. The polypeptide sequences involved in this example are shown in Table 1.

[0087] [Table 1-1]

[0088] [Table 1-2]

[0089] Example 2: Measurement of affinity between polypeptide and target protein using BLI (Biolayer Interferometry) BLI is a label-free technology that converts optical interference signals generated on the BLI biosensor surface into real-time response signals. By monitoring the molecular binding process in real time, the system measures the binding constant (ka or kon), dissociation constant (kd or koff), and initial binding rate, and then analyzes the affinity (K) through fitting calculations. DThe following steps were taken to calculate the target protein (α9 nAChR). The specific steps were as follows: an NTA sensor (Sartorius) was placed in a pre-wetting box, the box was placed in a black 96-well plate, 200 μL of PBS was dispensed into each well, and the NTA sensor was pre-wetted for at least 15 minutes. The target protein α9 nAChR was diluted to 5 μg / mL with PBS and 200 μL was added to each well. The target polypeptide was serially diluted threefold with PBS, with concentrations set from high to low: 1000 nM, 333.3 nM, 111.1 nM, 37.04 nM, 12.35 nM, 4.115 nM, and 1.372 nM, and finally a control well with PBS only was added. After equilibrating the instrument baseline, the target protein α9 nAChR was first fixed using the NTA sensor, the baseline was equilibrated again after fixation was complete, and then a 5-minute binding reaction and a 5-minute dissociation reaction were performed. Following the procedure described above, affinity measurements were performed on each target polypeptide. The affinity measurement process between polypeptides and the target protein α7 nAChR was basically the same as for α9 nAChR, except that the concentration of the target protein α7 nAChR was set to 20 μg / mL. As shown in Tables 2 and 3, the affinity between alanine scanning polypeptide analogs and the target protein was measured by BLI experiments, thereby identifying the modifiable sites of the parent peptide. Subsequently, the affinity between single-site non-natural amino acid modified peptides and the target protein was measured by BLI experiments. Finally, the affinity between multi-site combination modified peptides and the target protein was measured by BLI experiments, thereby screening polypeptides that showed high affinity to the target protein for subsequent selection and drug potential studies.

[0090] [Table 2]

[0091] [Table 3]

[0092] Example 3: Prediction of polypeptide half-life based on in vitro plasma stability Polypeptides generally have short half-lives and are easily degraded by various enzymes and the blood matrix in animals. In this study, the modified peptides had specific inactive major amino acids substituted with non-natural amino acids during the design phase, which is expected to improve polypeptide stability and drug potential. Polypeptides were taken, dissolved in 2 mg / mL of deionized water, diluted to 1 mg / mL with acetonitrile, filtered through an organic membrane filter, and 200 microliters were taken from the resulting filtrate and transferred to an LC vial to serve as the polypeptide control group. 300 microliters of human plasma (containing EDTA anticoagulant) (Nanjing Senbei Biotechnology Co., Ltd., lot number SBJ-P-HU011-100mL) were taken and added to 300 microliters of acetonitrile, and the mixture was centrifuged at 4°C and 12000 rpm for 5 minutes. The supernatant was filtered through an organic membrane filter, and 200 microliters were taken from the resulting filtrate and transferred to an LC vial to serve as the plasma blank control group. Furthermore, 10 mg of the polypeptide was taken, 5 mL of human plasma was added to it, and the mixture was homogeneously vortexed. 300 microliters of polypeptide-containing plasma was taken and added to 300 microliters of acetonitrile. The mixture was centrifuged at 4°C and 12000 rpm for 5 minutes. The resulting supernatant was filtered through an organic membrane filter, and 200 microliters of the filtrate was dispensed into an LC vial to be used as the polypeptide 0-minute sample. The polypeptide-dissolved plasma was incubated in a 37°C water bath, and the procedures at other time points were carried out in the same manner as the 0-minute procedure.Of these, the sampling times for polypeptides AJ003 and AJ003-1 were 0 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 10 hours, 12 hours, and 24 hours. The sampling times for modified peptides AJ003-34, AJ003-35, AJ003-36, AJ003-37, AJ003-38, AJ003-39, AJ003-40, AJ003-41, and AJ003-42 were 0 minutes, 10 minutes, 30 minutes, 1 hour, 4 hours, 6 hours, 10 hours, 12 hours, and 24 hours. All series samples of each polypeptide were handed over to the analysis department for HPLC analysis, and the change in the main peak area of ​​the sample with respect to the plasma incubation time was recorded. The in vitro half-life of each polypeptide was predicted using the pharmacokinetic analysis software Winolin. Because tautomers exist in the AJ003 sample, accurate integral comparison of peak areas over time is difficult. AJ003-1 is an analog in which the threonine at position 5 of the AJ003 polypeptide is modified to D-type threonine, and there is only one difference in the amino acid sequence when compared with AJ003. As shown in Figure 1-10, the in vitro plasma half-life of AJ003-1 was approximately 10.8 hours, while 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 were 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, due to non-natural amino acid mutations, the in vitro plasma stability of the other modified peptides in this project was significantly improved compared to AJ003-1 (which differs from AJ003 by only one amino acid).

[0093] Example 4: Plasma stability test of AJ003-40 and AJ003-24 Polypeptides AJ003-40 and AJ003-24 were each taken and added to human plasma, then vortex-mixed to prepare a concentration of 2 mg / mL. 300 microliters of polypeptide-containing plasma were taken and added to 300 microliters of acetonitrile. The mixture was centrifuged at 4°C and 12000 rpm for 5 minutes. The supernatant was filtered through an organic membrane filter, and 200 microliters of the filtrate was dispensed into an LC vial to prepare the polypeptide 0-hour sample. The polypeptide-dissolved plasma was incubated in a 37°C water bath. For polypeptide AJ003-40, 300 microliters of polypeptide-containing plasma were taken at 24 hours, added to 300 microliters of acetonitrile, centrifuged at 4°C and 12000 rpm for 5 minutes. The supernatant was filtered through an organic membrane filter, and 200 microliters of the filtrate was dispensed into an LC vial to prepare the polypeptide 24-hour sample. For polypeptide AJ003-24, 300 microliters of polypeptide-containing plasma were taken at 22 hours, added to 300 microliters of acetonitrile, centrifuged at 4°C and 12000 rpm for 5 minutes, and the resulting supernatant was filtered through an organic membrane filter. 200 microliters of the filtrate was dispensed into an LC vial to obtain the polypeptide 22-hour sample. HPLC analysis was performed on the 0-hour and 24-hour samples of the two polypeptides. As shown in Figures 31 and 32, the main peak area of ​​polypeptide AJ003-40 decreased from 15149.38 at 0 hours to 11829.51 at 24 hours, with a main peak area resolution of 21.9% within 24 hours. As shown in Figures 33 and 34, the main peak area of ​​polypeptide AJ003-24 decreased from 746.80 at 0 hours to 267.45 at 22 hours, with a main peak area resolution of 64.2% within 22 hours. Compared to polypeptide AJ003-40, polypeptide AJ003-24 exhibits a faster degradation rate, indicating that AJ003-24 has inferior stability in plasma compared to AJ003-40.

[0094] Example 5: Evaluation of in vivo analgesic activity of a modified peptide in response to paclitaxel chemotherapy-induced pain at the same dosage. SPF (Specific Pathogen Free) grade SD male rats weighing 180-200g (from the Department of Experimental Animal Management, Shanghai Institute of Family Planning Science) were used. On the day of model creation, the rats' body weight was measured, and their basal pain threshold was measured by mechanical stimulation. Individuals with a large difference between body weight and pain threshold were excluded, and the remaining rats were used to create the model according to the experimental plan. Paclitaxel powder was dissolved in olive oil at a concentration of 1 mg / mL, and 2 mg / kg was administered intraperitoneally to each rat each time. A total of four administrations were performed during the model creation period, with intraperitoneal injections on days 1, 3, 5, and 7. Negative control or blank control rats were administered the paclitaxel dissolution solvent mentioned above. According to literature reports, the pain threshold decreased significantly approximately 15 days after the start of model creation, indicating the successful creation of a stable model of rat paclitaxel chronic chemotherapy-induced pain. Drug treatment was carried out according to the administration method set out in the study protocol, as shown in Table 4. In Table 4, the blank group represents the group that did not undergo pain modeling, and the model group represents the group that underwent pain modeling but did not receive any drug. Rats were administered the drug at specific times on days 1, 3, 5, and 12. On days 1, 3, and 5, the right hind limb sole of the rats was stimulated using a flat-headed needle at 1 hour, 2.5 hours, 4 hours, and 5.5 hours after administration, and the pain threshold to mechanical stimulation was measured at each time point. On day 12, the pain threshold to mechanical stimulation of the rats was measured at 4 hours and 7 hours after administration on the day of administration. As shown in Figure 11-14, when the experimental results of the four-dose treatment were combined, the parent peptide AJ003 showed significant analgesic activity in the model compared to the model group within 4 hours, but beyond 4 hours, no significant difference in analgesic activity was observed compared to the model group.Among the five candidate modified peptides (AJ003-34, AJ003-35, AJ003-36, AJ003-39, and AJ003-40), AJ003-34, AJ003-36, and AJ003-40 were identified as the three most optimal candidates for analgesia. Analysis of the results at 7 hours on day 12 revealed that AJ003-36 and AJ003-40, in particular, exhibited a longer duration of analgesic activity, lasting up to 7 hours. Furthermore, these modified peptides showed slightly superior analgesic activity compared to pregabalin at specific administration times, and overall, they demonstrated an analgesic effect equivalent to that of pregabalin as a positive drug. AJ003-36 and AJ003-40 can be considered candidate molecules for further research. Paclitaxel, a widely used low-molecular-weight anticancer chemotherapy agent, exhibits antitumor effects but also causes neuralgia in many cancer patients due to damage to the patient's nervous system. A chronic chemotherapy-induced pain model of SD male rats, induced by several low-dose intraperitoneal administrations of paclitaxel, was used to simulate clinical treatment of cancer patients with paclitaxel and to evaluate the analgesic effects of five modified peptides, the parent peptide AJ003, and the positive drug pregabalin. The results, based on measurements of the mechanical pain threshold in the right hind paw of rats at corresponding time points after each administration, showed that AJ003-36 and AJ003-40 were preliminaryly screened as candidate molecules for further study in the overall analysis.

[0095] [Table 4]

[0096] Example 6: Evaluation of in vivo analgesic activity of modified peptides induced by paclitaxel chemotherapy at different dosages. SPF-grade SD male rats weighing 180-200g (from the Laboratory Animal Management Department of the Shanghai Institute of Family Planning Science) were purchased and reared for approximately one week to acclimate them to the environment. On the day of model creation, the rats' body weight was measured, and their basal pain threshold was measured using mechanical stimulation. Individuals with a large difference between body weight and pain threshold were excluded, and the remaining rats were used to create the model according to the experimental plan. Paclitaxel powder was dissolved in olive oil at a concentration of 1 mg / mL, and 2 mg / kg was administered intraperitoneally to each rat each time. A total of four administrations were performed during the model creation period, with intraperitoneal injections on days 1, 3, 5, and 7. Negative control or blank control rats were administered the paclitaxel dissolving solvent mentioned above. According to literature reports, the pain threshold decreased significantly approximately 15 days after the start of model creation, indicating the successful creation of a stable model of chronic paclitaxel chemotherapy-induced pain in rats. Drug treatment was carried out according to the administration method set out in the study protocol, as shown in Table 5. From day 15 of model creation, animals were administered drug therapy daily. On days 1, 4, 7, 12, and 14, the right hind limb sole of the rats was stimulated with a flat-headed needle at 2, 4, 6, and 8 hours after administration, and the pain threshold to mechanical stimulation at the corresponding times was measured. As shown in Figure 15-19, when the experimental results of the five administration treatments were combined, the parent peptide AJ003 showed significant analgesic activity compared to the model group within 4 hours in this model, but beyond 4 hours, no significant difference in analgesic activity was observed compared to the model group. The two candidate modified peptides AJ003-36 and AJ003-40 showed analgesic activity at low doses (0.5 mg / kg) comparable to the analgesic effect of the parent peptide AJ003 at 1 mg / kg, and surpassed the parent peptide AJ003 at the corresponding times on specific administration days. The two candidate modified peptides, AJ003-36 and AJ003-40, showed slightly superior analgesic activity to the positive drug pregabalin at high doses (1.5 mg / kg) and maintained significant analgesic activity compared to the model group even 8 hours after administration.

[0097] [Table 5]

[0098] Example 7: Evaluation of in vivo analgesic activity of candidate modified peptides in oxaliplatin chemotherapy-induced pain at different dosages. Oxaliplatin was dissolved in a 5% glucose solution to a concentration of 1 mg / mL. A single intraperitoneal administration of oxaliplatin was given to each C57 male mouse (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) in the model group, at a dose of 10 mg / kg. The negative control group or blank control group was administered the 5% glucose solution. Approximately one week after model creation, the mechanical pain threshold of the mice was measured, and three measurements were taken for each mouse to calculate the mean value. Based on the obtained pain threshold, the mice were randomly assigned to groups. The mice were administered according to the administration plan shown in Table 6 (total number of administrations: 1), and the mechanical pain threshold was measured at 0.5, 1, 2, 4, 6, and 8 hours after administration. As shown in Figure 20, at four time points—0.5 hours, 1 hour, 2 hours, and 4 hours after administration—all three dose groups (low, medium, and high doses) of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36 showed significantly greater analgesic activity compared to the model group. At 6 hours after administration, no significant difference was observed for parent peptide AJ003 compared to the model group, but the low, medium, and high dose groups of the positive control drug and modified peptide AJ003-36 still showed significantly greater analgesic activity compared to the model group. At 8 hours after administration, no significant difference was observed between the low-dose groups of parent peptide AJ003 and AJ003-36 and the model group, but the medium and high-dose groups of the positive control drug and modified peptide still showed significantly greater analgesic activity compared to the model group.

[0099] [Table 6]

[0100] Example 8: Evaluation of in vivo analgesic activity of multiple pyripolypeptides in oxaliplatin chemotherapy-induced pain. Oxaliplatin was dissolved in a 5% glucose solution to a concentration of 1 mg / mL. A single intraperitoneal administration of oxaliplatin was given to each C57 male mouse (Shanghai Bikaikeyi Biotechnology Co., Ltd.) in the model group, at a dose of 10 mg / kg. The negative control group or blank control group was administered the 5% glucose solution. Approximately one week after model creation, the mechanical pain threshold of the mice was measured, and three measurements were taken for each mouse to calculate the mean value. Based on the obtained pain threshold, the mice were randomly assigned to groups. The mice were administered according to the administration plan shown in Table 7 (total number of administrations: 1), and the mechanical pain threshold was measured at 0.5, 1, 2, 4, 6, and 8 hours after administration. As shown in Figure 30, from 0.5 hours after administration, the mechanical pain threshold of mice in the AJ003-36 administration group was significantly lower compared to the model group, and this difference was statistically significant. Neither the AJ003-24 nor the AJ003-25 administration groups showed any effect in lowering the mechanical pain threshold of mice. This suggests that polypeptide AJ003-36 can effectively alleviate chemotherapy pain induced by oxaliplatin in mice, while polypeptides AJ003-24 and AJ003-25 do not have the effect of alleviating this pain.

[0101] [Table 7]

[0102] Example 9: Evaluation of in vivo analgesic activity of candidate modified peptides using a rat CCI model at different dosages. Neuropathic pain is diverse, and clinical symptoms can be simulated by applying animal models. SD rat sciatic nerve pain models are mainly divided into four types: CCI (chronic compression injury model), SNI (sciatic nerve selective ligation model), SNL (spinal nerve selective ligation model), and CCD (insertion of a stainless steel rod into the intervertebral foramen to fix and compress the dorsal root ganglion). Of these, the CCI model modeling method is widely used in the study of neuropathic pain and provides a foundation for deepening research on nerve damage repair mechanisms and treatment strategies (Wen Xiaojuan, Medical Information. Jan. 2018. Vol. 31. No. 1). The CCI model was used in this example.

[0103] All SD rats, excluding the control group, were intraperitoneally administered 10% chloral hydrate saline solution at a dose of 0.3 mL per 100 g of body weight, and surgical models were created after anesthesia. The skin of the rat's right hind limb was carefully incised with surgical scissors, the muscle was separated, and the sciatic nerve was ligated at three points at 1 mm intervals using a 4-0 cutgood. The muscle and skin were then sutured, and antibiotics were applied to the wound to prevent postoperative infection. According to the literature, the animals' pain threshold significantly decreased 7-10 days post-surgery, and this condition lasted for about one month. Approximately 10 days after modeling, the plantar pain threshold of the rats was measured, with each individual measured three times. The average value was calculated, and the rats were randomly assigned to groups based on their pain threshold. The plantar pain threshold of the rats was measured at four time points after administration: 2, 4, 6, and 8 hours, according to the administration plan shown in Table 8.

[0104] On day 1 of administration, as shown in Figure 21, at 2 and 4 hours after administration, all three dose groups—low, medium, and high doses—of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36 all showed significant analgesic activity compared to the model group. At 6 hours post-administration, no significant difference was observed for parent peptide AJ003 compared to the model group, but the low, medium, and high dose groups of the positive control drug and modified peptide AJ003-36 still showed significant analgesic activity compared to the model group. At 8 hours post-administration, no significant difference was observed between the low-dose groups of parent peptide AJ003 and AJ003-36 and the model group, but the medium and high-dose groups of the positive control drug and AJ003-36 still showed significant analgesic activity compared to the model group.

[0105] On day 4 of administration, as shown in Figure 22, at 2 and 4 hours after administration, all three administration groups—low, medium, and high doses of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36—showed significant analgesic activity compared to the model group. At 6 and 8 hours after administration, no significant difference was observed between the low-dose groups of parent peptide AJ003 and AJ003-36 and the model group. However, the positive control drug and the medium- and high-dose groups of AJ003-36 still showed significant analgesic activity compared to the model group.

[0106] On day 7 of administration, as shown in Figure 23, at 2 and 4 hours post-administration, all three dose groups—low, medium, and high doses—of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36 all showed significant analgesic activity compared to the model group. At 6 hours post-administration, no significant difference was observed for parent peptide AJ003 compared to the model group, but the low, medium, and high dose groups of the positive control drug and AJ003-36 still showed significant analgesic activity compared to the model group. At 8 hours post-administration, no significant difference was observed between the low-dose groups of parent peptide AJ003 and AJ003-36 and the model group, but the medium and high-dose groups of the positive control drug and AJ003-36 still showed significant analgesic activity compared to the model group.

[0107] On day 10 of administration, as shown in Figure 24, at 2 and 4 hours post-administration, all three administration groups—low, medium, and high doses of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36—showed significant analgesic activity compared to the model group. At 6 hours post-administration, no significant difference was observed for parent peptide AJ003 compared to the model group, but the low, medium, and high dose groups of the positive control drug and AJ003-36 still showed significant analgesic activity compared to the model group. At 8 hours post-administration, no significant difference was observed between the low-dose groups of parent peptide AJ003 and AJ003-36 and the model group, but the medium and high-dose groups of the positive control drug and AJ003-36 still showed significant analgesic activity compared to the model group.

[0108] On day 14 of administration, as shown in Figure 25, at 2 and 4 hours post-administration, all three administration groups—low, medium, and high doses of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36—showed significant analgesic activity compared to the model group. At 6 hours post-administration, no significant difference was observed between the parent peptide AJ003 and the model group, but the three administration groups of the positive control drug and AJ003-36 still showed significant analgesic activity compared to the model group. At 8 hours post-administration, no significant difference was observed between the low-dose groups of the parent peptide AJ003 and AJ003-36 and the model group, but the medium- and high-dose groups of the positive control drug and AJ003-36 still showed significant analgesic activity compared to the model group.

[0109] [Table 8]

[0110] Example 10: Evaluation of in vivo analgesic activity of candidate modified peptides using a mouse model of diabetic neuropathy at different dosages. All C57 male mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.), excluding the control group, were continuously induced on a high-carbohydrate, high-fat diet for approximately 10 weeks. Mice in the model group were fasted overnight and then intraperitoneally administered streptozosin (manufactured by Saen Chemical Technology (Shanghai) Co., Ltd.) dissolved in citrate-sodium citrate buffer at a dose of 70 mg / kg. Approximately two weeks after streptozosin injection, the mechanical pain threshold of the mice was measured. The measurement method was as follows: After acclimatizing the mice to the environment by placing them on a perforated wire mesh, the sole of the right hind limb was stimulated vertically using a flat-headed needle. Pain threshold data was collected and recorded using a biosignal acquisition system, and the average of three measurements for each individual was calculated as the final value. Based on the pain threshold of the mice, the administration groups were randomly divided.

[0111] On day 1 of administration, mice were administered according to the administration plan shown in Table 9, and the plantar mechanical pain threshold was measured at five time points after administration: 0, 2, 4, 6, and 8 hours. As shown in Figure 26, at 2 and 4 hours after administration, all three administration groups (low, medium, and high doses) of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36 showed significant analgesic activity compared to the model group. At 6 and 8 hours after administration, the parent peptide AJ003 did not show significant analgesic activity compared to the model group, while all three administration groups (low, medium, and high doses) of the positive control drug and AJ003-36 showed significant analgesic activity compared to the model group.

[0112] On day 4 of administration, mice were administered according to the administration plan shown in Table 9, and the plantar mechanical pain threshold was measured at five time points after administration: 0, 2, 4, 6, and 8 hours. As shown in Figure 27, at 2 and 4 hours after administration, all three administration groups (low, medium, and high doses) of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36 showed significant analgesic activity compared to the model group. At 6 and 8 hours after administration, the parent peptide AJ003 did not show significant analgesic activity compared to the model group, while all three administration groups (low, medium, and high doses) of the positive control drug and AJ003-36 showed significant analgesic activity compared to the model group.

[0113] On day 8 of administration, mice were administered according to the administration plan shown in Table 9, and the plantar mechanical pain threshold was measured at five time points after administration: 0, 2, 4, 6, and 8 hours. As shown in Figure 28, at 2 and 4 hours after administration, all three administration groups (low, medium, and high doses) of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36 showed significant analgesic activity compared to the model group. At 6 and 8 hours after administration, the low-dose groups of parent peptide AJ003 and AJ003-36 did not show significant analgesic activity compared to the model group, while the positive control drug and the medium and high-dose groups of AJ003-36 both showed significant analgesic activity compared to the model group.

[0114] On day 14 of administration, mice were administered according to the administration plan shown in Table 9, and the plantar mechanical pain threshold was measured at five time points after administration: 0, 2, 4, 6, and 8 hours. As shown in Figure 29, at 2 and 4 hours after administration, all three administration groups (low, medium, and high doses) of the positive control drug pregabalin, parent peptide AJ003, and candidate modified peptide AJ003-36 showed significant analgesic activity compared to the model group. At 6 and 8 hours after administration, the parent peptide AJ003 did not show significant analgesic activity compared to the model group, while all three administration groups (medium and high doses) of the positive control drug and AJ003-36 showed significant analgesic activity compared to the model group.

[0115] When the mechanical pain threshold measurements from the four-dose treatment were combined, the parent peptide AJ003 showed significant analgesic activity compared to the model group within 4 hours in this model, but beyond 4 hours, no significant difference in analgesic activity was observed compared to the model group. In the medium- and high-dose groups of the candidate modified peptide AJ003-36, significant analgesic activity was observed compared to the model group at measurement times of 2 to 8 hours; in the low-dose group of AJ003-36, no significant difference was observed between the group and the model group at the measurement on day 8, but significant analgesic activity was observed compared to the model group at the other three measurements; the analgesic activity of the high-dose group of AJ003-36 was slightly higher than that of the positive drug pregabalin. These experimental results suggest that the candidate modified peptide AJ003-36 can be used to treat diabetic neuropathy by subcutaneous injection.

[0116] Table 9

Claims

1. A peptide or derivative thereof comprising formula (I) or formula (I) having a conservative substitution in order from the N-terminus to the C-terminus, Formula (1) Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8 Herein, in formula (I), the Cys at position 3 and the D-Cys at position 9 form a pair of intramolecular disulfide bonds, D-Cys represents D-cysteine, the "-" between amino acid residues represents a peptide bond, the C-terminus of the peptide or its derivative is a free carboxyl group or amide group, X1 is Thr or D-Thr, X5 is any one selected from Ala, Val and D-Val, and X2-X4 and X6-X8 are each any independent amino acid, the peptide or its derivative.

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

3. The peptide or derivative thereof according to claim 1 or 2, comprising any one selected from SEQ ID NOs: 1-5, SEQ ID NOs: 10-16 and SEQ ID NOs: 20-34, and SEQ ID NOs: 1-5, SEQ ID NOs: 10-16 and SEQ ID NOs: 20-34 having a conservative substitution, preferably comprising any one sequence selected from SEQ ID NOs: 26, 28 and 32, and SEQ ID NOs: 26, 28 and 32 having a conservative substitution.

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 amino acid modification, conservative substitution of amino acids, and hydrogen atom substitution of amino acid residues, compared to the peptide.

5. A pharmaceutical composition comprising a peptide or derivative thereof as described in 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 manufacture of a pharmaceutical for treating pain.

7. A method for treating pain, the method comprising administering to a person who is to be a subject 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.

8. The method for treating pain according to claim 7, wherein the peptide or its derivative is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, transpulmonary administration, ocular administration, or local administration.

9. The method for treating pain according to claim 6 or 7, wherein the pain includes pain caused by chemotherapy, peridiabetic neuralgia, sciatica, osteoarthritis, postherpetic neuralgia, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcohol toxic pain, pain due to hypothyroidism, autoimmune disease pain, Guillain-Barré syndrome pain, toxic pain, drug-induced pain, tumor pain, genetic disease pain, and contusion pain.

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