WO2024259977A1 - 经修饰的多肽及其在镇痛领域的应用 - Google Patents

经修饰的多肽及其在镇痛领域的应用 Download PDF

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Publication number
WO2024259977A1
WO2024259977A1 PCT/CN2024/073896 CN2024073896W WO2024259977A1 WO 2024259977 A1 WO2024259977 A1 WO 2024259977A1 CN 2024073896 W CN2024073896 W CN 2024073896W WO 2024259977 A1 WO2024259977 A1 WO 2024259977A1
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Prior art keywords
pain
peptide
derivative
administration
seq
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English (en)
French (fr)
Inventor
徐寒梅
周天雄
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Nanjing Anji Biotechnology Co Ltd
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Nanjing Anji Biotechnology Co Ltd
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Priority to AU2024314499A priority Critical patent/AU2024314499A1/en
Priority to EP24824881.7A priority patent/EP4725956A1/en
Priority to CN202480000220.6A priority patent/CN118159550B/zh
Priority to KR1020267002052A priority patent/KR20260033025A/ko
Publication of WO2024259977A1 publication Critical patent/WO2024259977A1/zh
Priority to MX2025015832A priority patent/MX2025015832A/es
Priority to IL325490A priority patent/IL325490A/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present application relates to the field of biomedicine, and specifically to a peptide or its derivatives and their use in analgesia.
  • Nicotinic acetylcholine receptor is a membrane protein with important physiological functions that is ubiquitous in nature and can regulate a series of physiological functions of organisms, such as pain, cognition, memory, anxiety, etc.
  • Acetylcholine receptors are widely present in nerve cells and are closely related to analgesia. Its subtypes are mainly It is ⁇ 7, ⁇ 9 ⁇ 10, etc. (see, for example, Hone AJ et al., Nicotinic acetylcholine receptors in neuropathic and inflammatory pain, FEBS lett.
  • Conus snails are carnivorous mollusks and are the general term for Conidae. There are about 700 species of cone snails in the world, mainly distributed in tropical waters.
  • Conotoxin is a neurotoxin secreted by cone snails, mainly composed of 10 to 30 amino acids and containing disulfide bonds.
  • Vc1.1 also known as ACV1
  • ACV1 is a 16-amino acid, two-pair disulfide-bonded polypeptide, and is an ⁇ -conotoxin isolated from Conus victoriae with analgesic effects.
  • Vc1.1 is a selective antagonist of ⁇ 9 ⁇ 10 nicotinic acetylcholine receptor (nAChR), and has therapeutic effects on neuropathic pain in rat sciatica (CCI) and neuropathic pain (PNL) models.
  • nAChR nicotinic acetylcholine receptor
  • CCI rat sciatica
  • PNL neuropathic pain
  • Vc1.1 had already entered the second phase of clinical trials, but the activity of human ⁇ 9 ⁇ 10 nAChR decreased 100 times compared with mouse ⁇ 9 ⁇ 10 nAChR, forcing the relevant institutions to suspend clinical research.
  • RgIA conotoxin peptide
  • peptide RgIA may represent a new class of drugs for the treatment of neuropathic pain, which can protect peripheral nerve tissue and prevent central maladaptive plasticity by inhibiting glial activation (PAIN:155(2014)1986–1995).
  • Ziconotide was approved by the FDA in 2004 for the treatment of severe chronic pain. It was originally isolated from the venom of Conus magus and contains 25 amino acids and three pairs of disulfide bonds. As a selective Cav2.2 channel antagonist, ziconotide blocks Ca 2+ influx and inhibits the release of excitatory transmitters from afferent nerve endings. Its analgesic efficacy is 1000 times that of opioids. However, the special administration method of intrathecal injection and a series of adverse reactions of the central nervous system make patients' compliance poor, thus limiting the widespread application of ziconotide.
  • the targets targeted by the polypeptide of the present application include ⁇ 7nAChR and ⁇ 9nAChR, and the targeted targets are acetylcholine receptors of human sequences. This can avoid the reduced activity of conotoxin Vc1.1 due to species differences and similar risks.
  • the polypeptide sequence of the present application introduces multiple non-natural amino acids and the polypeptide structure contains a pair of intramolecular disulfide bonds, which enhances the stability of the polypeptide, significantly increases the half-life of the polypeptide in the body, and significantly prolongs the duration of the analgesic effect. Pain is a very serious clinical problem that can have an adverse effect on the quality of life of patients and their families.
  • polypeptides have inherent safety advantages.
  • the polypeptide synthesis process of the present application is simple and easy to commercialize, and it is expected to be a new type of safe and effective analgesic.
  • the peptide of the present application has a faster onset time than the clinical analgesic drug pregabalin, and the stability of the peptide in the application has been significantly improved compared with the parent peptide AJ003 in human plasma. From the animal experimental data, it can also be found that the duration of the analgesic effect of the modified peptide is significantly extended compared with the parent peptide.
  • the polypeptide of the present application belongs to a new type of non-opioid analgesic drug. Since its target is mainly present in peripheral nerve tissue, it will not cause toxic and side effects such as drug addiction in the later clinical stage.
  • the peptide of the present application does not need to adopt the complex administration method of intrathecal instillation, which is conducive to improving the medication compliance of clinical patients.
  • ziconotide is composed of 25 amino acids, and the peptide sequence contains 3 pairs of disulfide bonds, the preparation process is complicated, and the production cost is greatly increased.
  • the peptide of the present application is composed of 13 amino acids, contains only 1 pair of disulfide bonds, the production and preparation process is simple, the solid phase peptide synthesis technology is currently very mature, and its synthesis characteristics are very conducive to automated large-scale production, which can greatly reduce the cost of drug production and reduce the medical burden of patients.
  • the peptide of the present application has a low administration dose and exhibits significant analgesic activity even at a low dose.
  • a peptide or a derivative thereof comprising any one sequence selected from SEQ ID NO: 1-34 is provided.
  • a peptide comprising any one sequence selected from SEQ ID NOs: 1-34 having conservative substitutions or a derivative thereof.
  • a peptide or a derivative thereof comprising the following formula I or formula I with conservative substitutions in order from N-terminus to C-terminus, Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8 Formula I
  • the 3rd Cys and the 9th D-Cys in Formula I form a pair of intramolecular disulfide bonds
  • D-Cys represents D-cysteine
  • "-" between amino acid residues represents a peptide bond
  • the C-terminus of the peptide or its derivative is a free carboxyl group or an amide
  • X1 is Thr or D-Thr
  • X5 is selected from any one of Ala
  • X2-X4 and X6-X8 are independently any one of amino acids.
  • the 3rd Cys and the 9th D-Cys in the above formula I form a pair of intramolecular disulfide bonds
  • the C-terminus of the peptide or its derivative is a free carboxyl group or an amide
  • X1 is Thr or D-Thr
  • X5 is selected from any one of Ala, Val and D-Val
  • X2 is selected from any one of Pro, D-Pro and Hyp
  • X3 is selected from any one of Pro, D-Pro and Hyp
  • X4 is selected from any one of Ser, D-Ser, Ala and Aib
  • X6 is selected from any one of Ala, Leu, D-Leu, Ile, ABu and Nle
  • X7 is selected from any one of Tyr, D-Tyr, Ala, Trp, Phe and Bip
  • X8 is selected from any one of Ser, D-Ser, Ala, and Orn.
  • the above-mentioned Formula I with conservative substitutions has 1, 2, 3, 4 or 5 amino acid conservative substitutions. In one embodiment of the present disclosure, the above-mentioned Formula I with conservative substitutions has at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity or at least 95% sequence identity with Formula I without conservative substitutions.
  • the peptide or its derivative comprises any one sequence selected from the following: SEQ ID NO: 1-5, SEQ ID NO: 10-16 and SEQ ID NO: 20-34, SEQ ID NO: 1-5, SEQ ID NO: 10-16 and SEQ ID NO: 20-34 with conservative substitutions.
  • the peptide or its derivative comprises any one sequence selected from the following: SEQ ID NO: 26, 28 and 32, SEQ ID NO: 26, 28 and 32 with conservative substitutions.
  • the above sequences with conservative substitutions have 1, 2, 3, 4 or 5 amino acid conservative substitutions, respectively. In one embodiment of the present disclosure, the above sequences with conservative substitutions have at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, having at least 84% sequence identity, having at least 85% sequence identity, having at least 90% sequence identity, having at least 92% sequence identity, or having at least 95% sequence identity.
  • a polynucleotide which encodes the peptide or its derivative according to the present disclosure.
  • an expression vector which expresses the polynucleotide according to the present disclosure.
  • a host cell which comprises the polynucleotide according to the present disclosure or the expression vector according to the present disclosure.
  • a pharmaceutical composition comprising the peptide or its derivative according to the present disclosure and a pharmaceutically acceptable carrier or salt thereof.
  • the present disclosure also provides a pharmaceutical composition, comprising the peptide or its derivative, polynucleotide, expression vector or host cell according to the present disclosure, and a pharmaceutically acceptable carrier or salt thereof.
  • the use of the peptide or its derivative according to the present disclosure or the pharmaceutical composition according to the present disclosure in the preparation of a drug for treating pain is provided.
  • the present disclosure also provides the use of the peptide or its derivative according to the present disclosure, the polynucleotide according to the present disclosure, the expression vector according to the present disclosure, the host cell according to the present disclosure or the pharmaceutical composition according to the present disclosure in the preparation of a drug for treating pain.
  • a method for treating pain comprising administering a therapeutically effective amount of a peptide or a derivative thereof according to the present disclosure or a pharmaceutical composition according to the present disclosure to a subject in need thereof.
  • the present disclosure also provides a method for treating pain, the method comprising administering a therapeutically effective amount of a peptide or a derivative thereof, a polynucleotide, an expression vector, a host cell or a pharmaceutical composition according to the present disclosure to a subject in need thereof.
  • FIG1 shows a line graph showing the change in the main peak area of polypeptide AJ003-1 in Example 3 over time.
  • FIG. 2 shows a line graph showing the change in the main peak area of polypeptide AJ003-34 over time in Example 3.
  • FIG3 shows a line graph showing the change in the main peak area of polypeptide AJ003-35 over time in Example 3.
  • FIG. 4 shows a line graph showing the change in the main peak area of polypeptide AJ003-36 over time in Example 3.
  • FIG5 shows a line graph showing the change in the main peak area of polypeptide AJ003-37 over time in Example 3.
  • FIG6 shows a line graph showing the change in the main peak area of polypeptide AJ003-38 over time in Example 3.
  • FIG. 7 shows the prediction results of the in vitro half-life of polypeptide AJ003-39 in human plasma in Example 3.
  • FIG8 shows a line graph showing the change in the main peak area of polypeptide AJ003-40 over time in Example 3.
  • FIG. 9 shows a line graph showing the change in the main peak area of polypeptide AJ003-41 over time in Example 3.
  • FIG. 10 shows a line graph showing the change in the main peak area of polypeptide AJ003-42 over time in Example 3.
  • Figure 11 shows the mechanical pain threshold on the first day of administration of the same dose of polypeptide in the paclitaxel chronic chemotherapy pain model of rats in Example 5.
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is the different groups. The results are shown as the mechanical pain threshold (g) ( ⁇ SD).
  • Graphpad, one-way analysis of variance (n 7-8).
  • Model group vs. control group ####p ⁇ 0.0001; drug administration group vs. model group: ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns: no statistical significance.
  • Pregabalin was 30 mg/kg ⁇ d ⁇ qd, gavage; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ 003-39 and AJ 003-40 were 1 mg/kg ⁇ d ⁇ qd, subcutaneous injection.
  • Figure 12 shows the mechanical pain threshold on the third day after the same dose of polypeptide was administered in the rat paclitaxel chronic chemotherapy pain model in Example 5.
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is the different groups. The results are shown as the mechanical pain threshold (g) ( ⁇ SD).
  • Graphpad, one-way analysis of variance (n 7-8).
  • Model group vs. control group ####p ⁇ 0.0001; drug administration group vs. model group: ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns: no statistical significance.
  • Pregabalin was 30 mg/kg ⁇ d ⁇ qd, gavage; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ 003-39 and AJ 003-40 were 1 mg/kg ⁇ d ⁇ qd, subcutaneous injection.
  • Figure 13 shows the mechanical pain threshold of the same dose of polypeptide administered in the rat paclitaxel chronic chemotherapy pain model in Example 5 on the 5th day.
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is the different groups. The results are shown as the mechanical pain threshold (g) ( ⁇ SD).
  • Graphpad, one-way analysis of variance (n 7-8).
  • Model group vs. control group ####p ⁇ 0.0001; drug administration group vs. model group: ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns: no statistical significance.
  • Pregabalin is 30mg/kg ⁇ d ⁇ qd, gavage; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ 003-39 and AJ 003-40 are 1mg/kg ⁇ d ⁇ qd, subcutaneous injection.
  • Figure 14 shows the mechanical pain threshold of the same dose of polypeptide administered in the rat paclitaxel chronic chemotherapy pain model on day 12 in Example 5.
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is the different groups.
  • the results are shown as mechanical pain threshold (g) ( ⁇ SD).
  • Graphpad, one-way ANOVA (n 7-8).
  • Pregabalin was 30 mg/kg ⁇ d ⁇ qd, intragastrically; AJ003, AJ003-34, AJ003-35, AJ003-36, AJ 003-39 and AJ 003-40 were 1 mg/kg ⁇ d ⁇ qd, subcutaneously.
  • Figure 15 shows the mechanical pain thresholds on day 1 of different doses of the polypeptide administered in the paclitaxel chronic chemotherapy pain model of rats in Example 6.
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is the different groups.
  • the results are shown as mechanical pain threshold (g) ( ⁇ SD).
  • Graphpad, one-way ANOVA (n 8).
  • Model group vs. control group ####p ⁇ 0.0001; drug administration group vs. model group: ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns: not statistically significant.
  • Pregabalin was 30 mg/kg ⁇ d ⁇ qd, administered orally; AJ003 was 1 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-36-low dose was 0.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-36-high dose was 1.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-40-low dose was 0.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, and AJ003-40-high dose was 1.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously.
  • Figure 16 shows the mechanical pain thresholds on the 4th day after different doses of polypeptides were administered in the paclitaxel chronic chemotherapy pain model of rats in Example 6.
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is the different groups.
  • the results are shown as mechanical pain threshold (g) ( ⁇ SD).
  • Graphpad, one-way ANOVA (n 8).
  • Model group vs. control group ####p ⁇ 0.0001; drug administration group vs. model group: ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns: not statistically significant.
  • Pregabalin was 30 mg/kg ⁇ d ⁇ qd, administered orally; AJ003 was 1 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-36-low dose was 0.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-36-high dose was 1.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-40-low dose was 0.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, and AJ003-40-high dose was 1.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously.
  • Figure 17 shows the mechanical pain thresholds on the 7th day after different doses of the polypeptides were administered in the paclitaxel chronic chemotherapy pain model of rats in Example 6.
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is the different groups.
  • the results are shown as mechanical pain thresholds (g) ( ⁇ SD).
  • Graphpad, one-way ANOVA (n 8).
  • Model group vs. control group ####p ⁇ 0.0001; drug administration group vs. model group: ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns: not statistically significant.
  • Pregabalin was 30 mg/kg ⁇ d ⁇ qd, administered orally; AJ003 was 1 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-36-low dose was 0.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-36-high dose was 1.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-40-low dose was 0.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, and AJ003-40-high dose was 1.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously.
  • Figure 18 shows the mechanical pain thresholds on the 12th day after different doses of the polypeptides were administered in the paclitaxel chronic chemotherapy pain model of rats in Example 6.
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is different groups. Results Shown is the mechanical pain threshold (g) ( ⁇ SD).
  • Graphpad, one-way ANOVA (n 8).
  • Model group vs. control group ####p ⁇ 0.0001; drug administration group vs. model group: ****p ⁇ 0.0001;***p ⁇ 0.001;**p ⁇ 0.01;*p ⁇ 0.05; ns: no statistical significance.
  • Pregabalin was 30 mg/kg ⁇ d ⁇ qd, gavage; AJ003 was 1 mg/kg ⁇ d ⁇ qd, subcutaneous injection, AJ003-36-low dose was 0.5 mg/kg ⁇ d ⁇ qd, subcutaneous injection, AJ003-36-high dose was 1.5 mg/kg ⁇ d ⁇ qd, subcutaneous injection, AJ003-40-low dose was 0.5 mg/kg ⁇ d ⁇ qd, subcutaneous injection and AJ003-40-high dose was 1.5 mg/kg ⁇ d ⁇ qd, subcutaneous injection.
  • Figure 19 shows the mechanical pain thresholds on the 14th day after different doses of the polypeptides were administered in the rat paclitaxel chronic chemotherapy pain model in Example 6. .
  • the vertical axis is the mechanical pain threshold, and the horizontal axis is the different groups. The results are shown as mechanical pain threshold (g) ( ⁇ SD).
  • Graphpad, one-way ANOVA (n 8).
  • Model group vs. control group ####p ⁇ 0.0001; drug administration group vs. model group: ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns: no statistical significance.
  • Pregabalin was 30 mg/kg ⁇ d ⁇ qd, administered orally; AJ003 was 1 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-36-low dose was 0.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-36-high dose was 1.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, AJ003-40-low dose was 0.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously, and AJ003-40-high dose was 1.5 mg/kg ⁇ d ⁇ qd, administered subcutaneously.
  • FIG. 31 shows the HPLC analysis profile of AJ003-40 in Example 4 after incubation in human plasma at 37° C. for 0 hours.
  • FIG. 32 shows the HPLC analysis profile of AJ003-40 in Example 4 after incubation in human plasma at 37° C. for 24 hours.
  • FIG. 33 shows the HPLC analysis profile of AJ003-24 in Example 4 after incubation in human plasma at 37° C. for 0 hours.
  • FIG. 34 shows the HPLC analysis profile of AJ003-24 in Example 4 after incubation in human plasma at 37° C. for 22 hours.
  • the expression "A and/or B” includes three cases: (1) A; (2) B; and (3) A and B.
  • the expression "A, B, and/or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C.
  • the meanings of similar expressions can be deduced analogously.
  • X and Y are independently selected from any one of a, b, c, d, e, f, and g" means that X can be any one of a, b, c, d, e, f, and g, and Y can also be any one of a, b, c, d, e, f, and g.
  • the selection of X and the selection of Y can be the same or different, and the two do not interfere with each other.
  • alanine scanning (Cunningham and Wells, Science 244, 1081-1085, 1989) is used to identify the activity of the peptides of the present invention (e.g., binding affinity to acetylcholine receptors). Alanine scanning introduces mutations into every residue of the molecule and tests the biological activity of the resulting molecule to determine the amino acid residues that are critical to the activity of the molecule.
  • identity refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity.
  • 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
  • ClustalW ClustalW.
  • the present application provides a peptide or its derivative comprising any one sequence selected from SEQ ID NO: 1-34.
  • any one of SEQ ID NO: 1-34 with conservative substitutions has 1, 2, 3, 4 or 5 conservative substitutions.
  • any one of SEQ ID NO: 1-34 with conservative substitutions has at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity or at least 95% sequence identity with a sequence without conservative substitutions.
  • the present application provides a peptide or a derivative thereof comprising the following formula I or a conservatively substituted formula I in order from N-terminus to C-terminus, Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8 Formula I
  • D-Cys represents D-cysteine
  • the "-" between the amino acid residues represents a peptide bond
  • the C-terminus of the peptide or its derivative is a free carboxyl group or an amide
  • X1 is Thr or D-Thr
  • X5 is selected from Ala, Any one of Val and D-Val, X2-X4 and X6-X8 are independently any amino acid.
  • X1 in Formula I is Thr or D-Thr.
  • X5 in Formula I is selected from any one of Ala, Val and D-Val.
  • X2 in Formula I is selected from any one of Pro, D-Pro and Hyp.
  • X3 in Formula I is selected from any one of Pro, D-Pro and Hyp.
  • X4 in Formula I is selected from any one of Ser, D-Ser, Ala and Aib.
  • X6 in Formula I is selected from any one of Ala, Leu, D-Leu, Ile, ABu and Nle.
  • X7 in Formula I is selected from any one of Tyr, D-Tyr, Ala, Trp, Phe and Bip.
  • X8 in Formula I is selected from any one of Ser, D-Ser, Ala, and Orn.
  • the above-mentioned Formula I with conservative substitutions has 1, 2, 3, 4 or 5 amino acid conservative substitutions. In one embodiment of the present disclosure, the above-mentioned Formula I with conservative substitutions has at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity or at least 95% sequence identity with Formula I without conservative substitutions.
  • the peptide or its derivative comprises any one of the sequences selected from the group consisting of SEQ ID NO: 1-5, SEQ ID NO: 10-16 and SEQ ID NO: 20-34, SEQ ID NO: 1-5, SEQ ID NO: 10-16 and SEQ ID NO: 20-34 with conservative substitutions.
  • the peptide or its derivative comprises SEQ ID NO: 26 or SEQ ID NO: 26 with conservative substitutions.
  • the peptide or its derivative comprises SEQ ID NO: 28 or SEQ ID NO: 28 with conservative substitutions.
  • the peptide or its derivative comprises SEQ ID NO: 32 or SEQ ID NO: 32 with conservative substitutions.
  • the above-mentioned sequence with conservative substitutions has 1, 2, 3, 4 or 5 amino acid conservative substitutions. In some embodiments, the above-mentioned sequence with conservative substitutions has at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity or at least 95% sequence identity with the sequence without conservative substitutions.
  • the derivative comprises any one or more modifications selected from the following compared to the peptide: amino acid modification, conservative amino acid substitution, and substitution of hydrogen in an amino acid residue.
  • peptide refers to a compound formed by amino acids linked together with peptide bonds.
  • a peptide composed of three or more amino acid molecules is a polypeptide.
  • peptide covers the peptide itself and its pharmaceutically acceptable salts, prodrugs and metabolites mentioned.
  • the peptides of the present application are generated by conventional technical means in the art, including but not limited to solid phase synthesis (such as Fmoc peptide synthesis) and liquid phase synthesis.
  • the position numbering of a polypeptide or its derivative is carried out in order from the N-terminus to the C-terminus, for example, the first amino acid starting from the N-terminus of the peptide is the first amino acid, the second amino acid starting from the N-terminus of the peptide is the second amino acid, the third amino acid starting from the N-terminus of the peptide is the third amino acid, and so on.
  • prodrug refers to all molecules that are converted into the peptides of the present disclosure or their derivatives by reactions such as oxidation, reduction, hydrolysis, etc., carried out in vivo under physiological conditions, such as by the action of enzymes, gastric acid, etc.
  • the term "metabolites” refers to all molecules in a cell or organism (preferably a human) derived from a peptide of the present disclosure or a derivative thereof.
  • amino acid and “amino acid residue” have the same meaning, which means that when amino acids are connected by chemical bonds, part of their groups are lost due to participation in the formation of the connecting bonds, and the remaining amino acid part is the amino acid residue.
  • amino acids As used herein, the names and abbreviations of amino acids correspond to the following:
  • Natural amino acids except glycine, all contain chiral carbon atoms. Unless otherwise specified, the optically active amino acids described in this application are all in the L-configuration. As usual, the structure of the peptides herein is written with the amino terminus (N-terminus) on the left and the carboxyl terminus (C-terminus) on the right.
  • amino acid as used herein includes the natural amino acids, as well as other "non-protein" ⁇ -amino acids/unnatural amino acids commonly used in the art of peptide chemistry to prepare analogs of natural peptides.
  • Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, ornithine and lysine.
  • non-protein ⁇ -amino acids/unnatural amino acids are norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline (Hyp), homoserine, cyclohexylglycine (Chg), ⁇ -amino-n-butyric acid (Aba), cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanine substituted in the phenyl moiety by alkyl, alkoxy, halogen or nitro, O-alkylated derivatives of serine, threonine and tyrosine, S-alkylated cysteine, O-sulfate esters of tyrosine, and the D-isomers of the natural amino acids.
  • a "derivative" of a peptide refers to a product obtained by amino acid modification, conservative amino acid substitution and/or replacement of hydrogen in an amino acid residue based on the peptide.
  • amino acid modification includes, but is not limited to, N-terminal modification, C-terminal modification, and side chain modification.
  • the methods of "amino acid modification” include, but are not limited to, hydroxylation, carboxylation, alkylation, acylation, phosphorylation, sulfonation, amidation, aldehyde modification, alcoholization, mercaptoethylation, esterification, glycosylation, and the like.
  • conservative amino acid substitution can generally be described as an amino acid residue being replaced by another amino acid having a similar chemical structure and/or similar chemical properties, and having little effect on the function, activity or other biological properties of the peptide. Such conservative amino acid substitutions are well known in the art.
  • Such conservative substitutions can be, for example, one amino acid in the following groups (a)-(e) being replaced by another amino acid in the same group: (a) small aliphatic, non-polar or weakly polar amino acid residues: Ala, Ser, Thr, Pro and Gly; (b) negatively charged polar amino acid residues and their amides: Asp, Asn, Glu and Gln; (c) positively charged polar amino acid residues: His, Arg and Lys; (d) large aliphatic, non-polar amino acid residues: Met, Leu, Ile, Val and Cys; (e) aromatic amino acid residues: Phe, Tyr and Trp.
  • the replacement of hydrogen in an amino acid residue may be that the hydrogen in the amino acid residue is replaced by any conventional substituent known in the art, including but not limited to alkyl, alkenyl, alkynyl, aromatic, alkoxy, carboxyl, aldehyde, carbonyl, hydroxyl, halogen, cyano, acyl, sulfonic acid, amino, thiol or nitro groups and the like.
  • the present application provides a polynucleotide encoding the peptide or its derivatives described in the present disclosure.
  • polynucleotide refers to a polymer of nucleotides (nucleotides or deoxynucleotides) of any length.
  • the term refers to the primary structure of the molecule. It thus includes both double-stranded and single-stranded RNA, and double-stranded and single-stranded DNA. It also includes polynucleotides modified, such as by methylation and/or end-capping, and unmodified forms of polypeptide nucleotides.
  • the polynucleotides described herein are not necessarily obtained by physical methods, but may be produced in any manner, including, for example, chemical synthesis or DNA replication, reverse transcription or transcription.
  • the present application provides an expression vector, which can express the polynucleotide described in the present disclosure.
  • the present application provides a host cell, which comprises the polynucleotide described in the present disclosure or the expression vector described in the present application.
  • host cells are cells that serve as recipients for expression vectors or other transformed genetic segments. They include the original cell that has been transfected and its progeny. Host cells include prokaryotes, yeast or other eukaryotic cells.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the peptide or its derivative described in the present disclosure, and a pharmaceutically acceptable carrier or salt.
  • the present application also provides a pharmaceutical composition, which comprises the peptide or its derivative, polynucleotide, expression vector or host cell according to the present disclosure, and a pharmaceutically acceptable carrier or salt thereof.
  • the term "pharmaceutical composition” refers to a mixture of the peptides of the present disclosure and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients.
  • Pharmaceutical compositions facilitate administration of the peptides to an organism.
  • ways to administer peptides in the art including, but not limited to, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral, transdermal, pulmonary, ocular and topical administration.
  • the pharmaceutical composition can be configured as a dosage form suitable for administration to a subject via a desired route of administration.
  • the dosage form includes, but is not limited to, tablets, capsules, caplets, pills, lozenges, powders, syrups, brews, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, lotions, pastes, sprays, freeze-dried, injections, and gels.
  • the term "pharmaceutically acceptable salts” includes acid addition salts and base addition salts. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include, but are not limited to, acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, cyclaminate, edisylate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, 2-(4-hydroxybenzyl)benzoate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesul
  • Suitable base addition salts are formed from bases which form non-toxic salts. Examples include, but are not limited to, aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemi-salts of acids and bases, such as hemi-sulfate and hemi-calcium salts, can also be formed.
  • bases include, but are not limited to, aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts.
  • Hemi-salts of acids and bases such as hemi-sulfate and hemi-calcium salts, can also be formed.
  • suitable salts see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (W
  • pharmaceutically acceptable carrier includes pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which are involved in carrying or delivering the peptides of the present disclosure in a subject or carrying or delivering the peptides of the present disclosure to a subject so that it can perform its intended function.
  • Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject.
  • Materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; Agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer
  • the present application provides use of the peptide or its derivative described in the present disclosure or the pharmaceutical composition described in the present disclosure in the preparation of a drug for treating pain.
  • the present application also provides use of the peptide or derivatives thereof described in the present disclosure, the polynucleotide described in the present disclosure, the expression vector described in the present disclosure, the host cell described in the present disclosure or the pharmaceutical composition described in the present disclosure in the preparation of a drug for treating pain.
  • the medicine for treating pain prepared is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal administration, pulmonary administration, ocular administration or topical administration.
  • the medicine for treating pain prepared is administered by subcutaneous injection.
  • the pain includes pain caused by acting on nicotinic acetylcholine receptors.
  • the pain includes neuralgia and inflammatory pain.
  • the pain includes pain caused by chemotherapy, diabetic peripheral neuropathy, sciatica, bone and joint pain, postherpetic neuralgia, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcoholism pain, hypothyroidism pain, autoimmune disease pain, Guillain-Barre syndrome pain, poisoning pain, drug-induced pain, tumor lesions pain, genetic disease pain and contusion pain.
  • the present application further provides the peptide or derivative thereof described in the present disclosure, the polynucleotide described in the present disclosure, the expression vector described in the present disclosure, the host cell described in the present disclosure or the pharmaceutical composition described in the present disclosure, for use in treating pain.
  • the pain includes pain caused by acting on nicotinic acetylcholine receptors.
  • the pain includes neuralgia and inflammatory pain.
  • the pain includes pain caused by chemotherapy, diabetic peripheral neuropathy, sciatica, bone and joint pain, postherpetic neuralgia, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcoholism pain, hypothyroidism pain, autoimmune disease pain, Guillain-Barre syndrome pain, poisoning pain, drug-induced pain, tumor lesions pain, genetic disease pain and contusion pain.
  • 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, pulmonary administration, ocular administration or topical administration.
  • the peptide or its derivative or pharmaceutical composition is administered by subcutaneous injection.
  • the present application provides a method for treating pain, which comprises administering a therapeutically effective amount of the peptide or derivative thereof according to the present disclosure or the pharmaceutical composition according to the present disclosure to a subject in need thereof.
  • the present application also provides a method for treating pain, which comprises administering a therapeutically effective amount of the peptide or its derivative, polynucleotide, expression vector, host cell according to the present disclosure or the pharmaceutical composition according to the present disclosure to a subject in need thereof.
  • the pain includes pain caused by acting on nicotinic acetylcholine receptors.
  • the pain includes neuralgia and inflammatory pain.
  • the pain includes pain caused by chemotherapy, diabetic peripheral neuropathy, sciatica, bone and joint pain, postherpetic neuralgia, infection pain, nutritional deficiency pain, inflammatory pain, chronic alcoholism pain, hypothyroidism pain, autoimmune disease pain, Guillain-Barre syndrome pain, poisoning pain, drug-induced pain, tumor lesions pain, genetic disease pain and contusion pain.
  • 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, pulmonary administration, ocular administration, orally administration.
  • the peptide or its derivative or pharmaceutical composition is administered by subcutaneous injection.
  • the term "subject” includes animals, such as vertebrates, preferably mammals, such as dogs, cats, pigs, cows, sheep, horses, rodents (e.g., mice, rats, or guinea pigs), or primates (e.g., gorillas, chimpanzees, and humans).
  • animals such as vertebrates, preferably mammals, such as dogs, cats, pigs, cows, sheep, horses, rodents (e.g., mice, rats, or guinea pigs), or primates (e.g., gorillas, chimpanzees, and humans).
  • treat refers to alleviating or ameliorating a disease or disorder (i.e., slowing or arresting the development of the disease or at least one clinical symptom); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder.
  • the term "therapeutically effective dose” refers to an amount that results in benefit or treatment of a disease compared to a corresponding subject that has not received the amount, but the amount is sufficiently low within the scope of reasonable medical judgment to avoid serious side effects.
  • the therapeutically effective dose of the peptide or its derivative, polynucleotide, expression vector, host cell or pharmaceutical composition described herein will vary with the selected peptide or its derivative, polynucleotide, expression vector, host cell or pharmaceutical composition; route of administration; severity of the disease being treated; age, size, weight and physical condition of the patient being treated: medical history of the patient being treated; duration of treatment; nature of concurrent treatment; desired therapeutic effect and other factors, but can still be determined by those skilled in the art in a routine manner.
  • the principle that the conventional chemical synthesis of polypeptides is to couple amino acids one by one from the C-terminus to the N-terminus was followed.
  • the corresponding protected amino acids and condensation reagents (HoBt, DIC) were weighed, and the reaction was about 1 hour. After the condensation reaction was completed, the resin was washed with DMF 3 times. From then on, a condensation cycle (deprotection-washing-condensation-washing) was carried out, that is, 20% piperidine/DMF solution was added to the reaction vessel again to carry out the deprotection reaction. After washing with DMF, the protected amino acids and the corresponding condensation reagents were added to carry out the condensation reaction.
  • a condensation cycle deprotection-washing-condensation-washing
  • the protected amino acids are coupled to the N-terminus one by one by using the condensation cycle method until all the amino acids of the polypeptide are coupled.
  • the Fmoc group at the N-terminus of the polypeptide is removed with a 20% piperidine/DMF solution, and the resin is alternately washed with dichloromethane and methanol, and finally a shrink-dried peptide resin intermediate is obtained by vacuum drying.
  • the peptide resin is weighed and a lysis solution is prepared according to the ratio of 1 gram of peptide resin to 10 ml of lysis solution.
  • the lysis solution is added to the peptide resin, and the lysis reaction is carried out for about 3 hours. Finally, the filtrate is collected and the filtrate is precipitated with frozen methyl tert-butyl ether. The amount of methyl tert-butyl ether used is 8 times that of the filtrate.
  • the precipitate is fully stirred and centrifuged in a cup to obtain a crude peptide precipitate.
  • the methyl tert-butyl ether is continued to be washed and centrifuged, and the precipitate is vacuum dried overnight to finally obtain a linear crude peptide sample. After the linear crude peptide is dissolved in water, iodine is added as an oxidant to oxidize the polypeptide into a disulfide bond.
  • the oxidation was carried out for about 30 minutes, and the degree of oxidation was monitored by HPLC. Finally, vitamin C was added to the oxidation system to terminate the oxidation reaction.
  • the oxidized polypeptide solution was filtered with a filter membrane, separated and purified by reversed-phase high-performance liquid chromatography, and the qualified fractions were combined and finally freeze-dried using a freeze dryer to obtain the pure freeze-dried polypeptide.
  • Example 2 BLI (biofilm interferometry) detection of peptide-target protein affinity
  • BLI is a label-free technology that converts the optical interference signal occurring on the surface of the BLI biosensor into a real-time response signal.
  • the system will determine the binding constant (ka or kon) and dissociation constant (kd or koff), as well as the initial binding rate, and obtain the affinity (K D ) through fitting calculation analysis.
  • the specific steps are to place the NTA sensor (Sartorius) in a pre-wet box, place the pre-wet box in a black 96-well plate, and pre-wet the NTA sensor for at least 15 minutes with 200 ⁇ L of PBS per well.
  • the affinity detection process between the peptide and the target protein ⁇ 7nAChR is basically the same as that of ⁇ 9 nAChR, except that the protein concentration of the target protein ⁇ 7 nAChR is 20 ⁇ g/mL.
  • the BLI experiment detected the affinity of the alanine scanning peptide analogue with the target protein and clarified the reconfigurable site of the parent peptide; secondly, the BLI experiment was used to detect the affinity of the single-site non-natural amino acid mutation-modified peptide with the target protein; finally, the BLI experiment was used The affinity of the modified peptides with multi-site combination mutations to the target protein was detected, so as to screen out peptides with high affinity to the target protein for subsequent screening and research on their drugability.
  • Peptides usually have a short half-life and are easily degraded by various enzymes and matrices in the blood in animals.
  • non-natural amino acids were replaced with certain inactive key site amino acids, hoping to improve the stability of the peptide and make it more conducive to drug development.
  • sampling points of peptides AJ003 and AJ003-1 were 0min, 5min, 10min, 20min, 30min, 1h, 2h, 4h, 6h, 10h, 12h, and 24h
  • the sampling points of modified peptides AJ003-34, AJ003-35, AJ003-36, AJ003-37, AJ003-38, AJ003-39, AJ003-40, AJ003-41, and AJ003-42 were 0min, 10min, 30min, 1h, 4h, 6h, 10h, 12h, and 24h.
  • AJ003-1 is an analogue of the AJ003 polypeptide in which the 5th threonine is restructured into D-threonine.
  • the peptide sequence of AJ003-1 differs from that of AJ003 by only one amino acid configuration.
  • the in vitro plasma half-life of AJ003-1 is about 10.8h
  • the in vitro plasma half-lives of the restructured peptides AJ003-34, AJ003-35, AJ003-36, AJ003-37, AJ003-38, AJ003-39, AJ003-40, AJ003-41, and AJ003-42 are 61.9h, 45.3h, 65.3h, 71.0h, 26.7h, 43.0h, 84.0h, 30.0h, and 61.4h, respectively.
  • the in vitro plasma stability of other modified peptides in this project has been significantly improved compared with AJ003-1 (which only differs from AJ003 by one amino acid).
  • Example 4 Plasma stability test of AJ003-40 and AJ003-24
  • peptide AJ003-40 300 ⁇ L of plasma containing the peptide was aspirated at 24 hours and added to 300 ⁇ L of acetonitrile, placed in a centrifuge at 4°C, 12,000 rpm for 5 minutes, aspirate the supernatant and filter it with an organic filter membrane, and aspirate 200 ⁇ L of the filtrate into a liquid phase vial as a peptide sample at 24 hours.
  • peptide AJ003-24 300 ⁇ L of plasma containing the peptide was aspirated at 22 hours and added to 300 ⁇ L of acetonitrile, placed in a centrifuge at 4°C, 12,000 rpm for 5 minutes, aspirate the supernatant and filter it with an organic filter membrane, and aspirate 200 ⁇ L of the filtrate into a liquid phase vial as a peptide sample at 22 hours.
  • the samples of the two peptides at 0 hours and 24 hours were analyzed by HPLC.
  • the main peak area of peptide AJ003-40 decreased from 15149.38 at 0 hours to 11829.51 at 24 hours, and the main peak area degradation rate was 21.9% within 24 hours; as shown in Figures 33 and 34, the main peak area of peptide AJ003-24 decreased from 746.80 at 0 hours to 267.45 at 22 hours, and the main peak area degradation rate was 64.2% within 22 hours.
  • peptide AJ003-24 has a faster degradation rate, indicating that AJ003-24 has a poorer plasma stability than AJ003-40.
  • Example 5 Evaluation of the analgesic activity of some modified peptides at the same dose in vivo during paclitaxel chemotherapy
  • this model was calculated from the beginning of modeling, and the pain threshold decreased significantly after about 15 days, indicating that a stable rat paclitaxel chronic chemotherapy pain model was successfully constructed.
  • Drug treatment was carried out according to the administration method set in the experimental protocol, as shown in Table 4 below.
  • the blank group in Table 4 represents a group without pain modeling, and the model group represents a group with pain modeling but no drug administration.
  • the rats were given drugs at fixed times on the 1st, 3rd, 5th and 12th days.
  • the model it has significant analgesic activity compared with the model group within 4 hours, and the analgesic activity after more than 4 hours is not significant compared with the model group.
  • the 5 candidate modified peptides AJ003-34, AJ003-35, AJ003-36, AJ003-39, AJ003-40
  • AJ003-34, AJ003-36, and AJ003-40 are the three best analgesic candidates. From the analysis of the results at 7 hours on the 12th day, the analgesic activity of AJ003-36 and AJ003-40 lasts longer, which can last up to 7 hours.
  • analgesic activity of this modified peptide is slightly better than that of pregabalin at certain administration time points compared with the positive drug. Overall, it is comparable to the analgesic efficacy of the positive drug pregabalin.
  • AJ003-36 and AJ003-40 can be used as candidate molecules for further study.
  • Paclitaxel is a small molecule chemotherapy drug widely used in clinical practice. While it has anti-tumor effects, it also causes neuralgia in many cancer patients due to its own damage to the patient's nervous system.
  • the chronic chemotherapy pain model of SD male rats induced by multiple low-dose intraperitoneal injections of paclitaxel simulates the analgesic effects of 5 modified peptides, the parent peptide AJ003, and the positive drug pregabalin in this model.
  • the experimental results show that the mechanical pain threshold of the plantar right hind limb of the rat was detected at the corresponding time point after each administration.
  • the overall analysis can preliminarily screen AJ003-36 and AJ003-40 as candidate molecules for further study.
  • Example 6 Evaluation of the analgesic activity of modified peptides at different doses during paclitaxel chemotherapy
  • SPF grade SD male rats weighing 180-200 g were purchased (Experimental Animal Management Department of Shanghai Institute of Family Planning Science) and fed adaptively for about a week. The rats were weighed on the day of modeling and the basic pain threshold of the rats was tested with mechanical stimulation. The animals with large differences in weight and pain threshold were eliminated, and the remaining rats were divided into the following groups according to the experimental group: The experimental scheme was used to carry out modeling. Paclitaxel powder was dissolved in olive oil to 1 mg/mL, and each rat was intraperitoneally injected with 2 mg/kg each time. A total of 4 injections were given during the entire modeling period, and intraperitoneal injections were performed on days 1, 3, 5, and 7, respectively.
  • Rats in the negative control or blank control group were injected with the above-mentioned solvent for dissolving paclitaxel. According to literature reports, the pain threshold of this model dropped significantly after about 15 days from the beginning of modeling, indicating that a stable rat paclitaxel chronic chemotherapy pain model was successfully constructed.
  • Drug treatment was carried out according to the dosing method set in the experimental scheme, as shown in Table 5 below. Starting from the 15th day of modeling, the animals were continuously treated with medication every day, and the soles of the right hind limbs of the rats were stimulated with flat-head needles at 2h, 4h, 6h, and 8h after the medication on days 1, 4, 7, 12, and 14, and the pain thresholds of the rats to mechanical stimulation at the corresponding time points were detected.
  • the mother peptide AJ003 has significant analgesic activity in this model within 4 hours compared with the model group, and the analgesic activity is not significant compared with the model group after 4 hours.
  • the two candidate modified peptides AJ003-36 and AJ003-40 have low-dose 0.5mg/kg analgesic activity comparable to the 1mg/kg analgesic effect of the mother peptide AJ003, and are superior to the mother peptide AJ003 at the corresponding time points on some days of administration.
  • the two candidate modified peptides AJ003-36 and AJ003-40 have high-dose 1.5mg/kg analgesic activity slightly better than the positive drug pregabalin, and the analgesic effect still has significant analgesic activity compared with the model group 8 hours after administration.
  • Example 7 Evaluation of the analgesic activity of candidate modified peptides at different doses in vivo for pain induced by oxaliplatin chemotherapy
  • Oxaliplatin was dissolved in 5% glucose solution to 1 mg/mL.
  • Each C57 male mouse (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) in the modeling group was injected with a single intraperitoneal injection of 10 mg/kg of oxaliplatin.
  • the negative control or blank control group was injected with 5% glucose solution.
  • the mechanical pain threshold of mice was detected about one week after modeling, and the pain threshold of each mouse was measured 3 times, the average value was calculated, and the mice were randomly divided according to the pain threshold.
  • the mice were administered according to the dosing regimen in Table 6 (a total of 1 administration), and the mechanical pain threshold was detected at 0.5, 1, 2, 4, 6, and 8 hours after administration.
  • the low dose groups of the parent peptide AJ003 and AJ003-36 had no significant difference compared with the model group, and the medium and high dose groups of the positive drug and the modified peptide still had significant analgesic activity compared with the model group.
  • Example 8 Evaluation of the analgesic activity of various peptides in vivo for pain induced by oxaliplatin chemotherapy
  • Oxaliplatin was dissolved in 5% glucose solution to 1 mg/mL.
  • Each C57 male mouse in the modeling group (Shanghai Bikekeyi Biotechnology Co., Ltd.) was injected intraperitoneally with a single 10 mg/kg dose of oxaliplatin.
  • the negative control or blank control group was injected with 5% glucose solution.
  • the mechanical pain threshold of the mice was tested about one week after modeling. The pain threshold of each mouse was measured 3 times, the average value was calculated, and the mice were randomly divided according to the pain threshold.
  • the mice were administered according to the dosing schedule in Table 7 (a total of 1 dose), and the mechanical pain threshold was tested at 0.5, 1, 2, 4, 6, and 8 h after administration.
  • Example 9 Evaluation of the analgesic activity of candidate modified peptides at different doses in rat CCI model
  • CCI chronic constriction injury model
  • SNI SNI model of selective ligation of sciatic nerve branches
  • SNL SNL model of selective ligation of spinal nerves
  • CCD using stainless steel wire to pass through the intervertebral foramen to fix and compress the dorsal root ganglion.
  • the CCI model modeling method has been widely used in the study of neuropathic pain, providing a basis for further research on the mechanisms and strategies of nerve damage repair (Wen Xiaojuan, Medical Information. Jan. 2018. Vol. 31. No. 1).
  • the CCI model is used in this embodiment.
  • the remaining SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate saline solution at a dose of 0.3mL per 100g body weight, and then surgical modeling was performed. Carefully cut the skin of the right hind limb of the rat with surgical scissors, cut the muscle, and ligate the sciatic nerve three times with catgut 4-0, with an interval of 1mm, and then suture the muscle and skin, and apply antibiotics to the wound to prevent animal surgical wound infection. According to literature reports, animals show a significant decrease in pain threshold 7-10 days after surgery, which can last for about one month.
  • the pain threshold of the plantar of the rat was detected in about 10 days of modeling, and the pain threshold of each rat was measured 3 times, and the average value was calculated, and random grouping was performed according to the pain threshold of the rat.
  • the mother peptide AJ003 had no significant difference compared with the model group, and the three low, medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • the low dose groups of the mother peptide AJ003 and AJ003-36 had no significant difference compared with the model group, and the two medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • the low dose groups of the parent peptide AJ003 and AJ003-36 were not significant compared with the model group, and the two medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • the low dose groups of the parent peptide AJ003 and AJ003-36 were not significant compared with the model group, and the two medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • the low dose groups of the parent peptide AJ003 and AJ003-36 were not significant compared with the model group, and the two medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • Example 10 Evaluation of the in vivo analgesic activity of candidate modified peptides at different doses in a mouse diabetic neuropathy model
  • mice except for the control group, the rest of the C57 male mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) were continuously induced with high-sugar and high-fat feed for about ten weeks.
  • the model group mice were fasted overnight, and streptozotocin (Saen Chemical Technology (Shanghai) Co., Ltd.) was dissolved in citric acid-sodium citrate buffer, and the model group mice were intraperitoneally injected with streptozotocin 70 mg/kg. About two weeks after the injection of streptozotocin, the mechanical pain threshold of the mice was tested.
  • the detection method was to place the mice in a porous wire mesh to adapt to the environment, and then use a flat-headed needle to vertically stimulate the sole of the right hind limb of the mouse.
  • the pain threshold data was collected and recorded using a biological signal acquisition system.
  • the pain threshold of each mouse was measured 3 times, and the average value was finally calculated.
  • the mice were randomly divided into groups and treated according to the pain threshold.
  • the drug was administered according to the dosing regimen in Table 9, and the mechanical pain threshold of the plantar of mice was tested at five time points, namely, 0h, 2h, 4h, 6h, and 8h after administration.
  • the three low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group; at the two time points of 6h and 8h, the parent peptide AJ003 had no significant difference compared with the model group, and the three low, medium, and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • the drug was administered according to the dosing regimen in Table 9, and the mechanical pain threshold of the plantar of mice was tested at five time points, namely, 0h, 2h, 4h, 6h, and 8h after administration.
  • the three low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group; at the two time points of 6h and 8h, the parent peptide AJ003 had no significance compared with the model group, and the three low, medium, and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • the drug was administered according to the dosing regimen of Table 9, and the mechanical pain threshold of the mouse plantar was detected at five time points of 0h, 2h, 4h, 6h, and 8h after administration.
  • the three low, medium and high dose groups of the positive drug pregabalin, the parent peptide AJ003 and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group; at the two time points of 6h and 8h, the low dose groups of the parent peptide AJ003 and AJ003-36 were not significant compared with the model group, and the medium and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • the drug was administered according to the dosing regimen in Table 9, and the mechanical pain threshold of the plantar of the mouse was detected at five time points, namely, 0h, 2h, 4h, 6h, and 8h after administration.
  • the three low, medium, and high dose groups of the positive drug pregabalin, the parent peptide AJ003, and the candidate modified peptide AJ003-36 all had significant analgesic activity compared with the model group; at the two time points of 6h and 8h, the parent peptide AJ003 had no significance compared with the model group, and the three low, medium, and high dose groups of the positive drug and AJ003-36 still had significant analgesic activity compared with the model group.
  • the mother peptide AJ003 has significant analgesic activity in this model within 4 hours compared with the model group, and the analgesic activity is not significant after 4 hours.
  • the medium and high doses of the candidate modified peptide AJ003-36 have significant analgesic activity compared with the model group at 2 to 8 hours of detection; the low dose group of AJ003-36 has no significant difference compared with the model group when tested on the 8th day, but has significant analgesic activity compared with the model group in the other three tests; the analgesic activity of the high dose group of AJ003-36 is slightly better than that of the positive drug pregabalin.
  • This experiment suggests that the candidate modified peptide AJ003-36 can treat diabetic induced neuropathy by subcutaneous injection.

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Abstract

本申请涉及一种肽或其衍生物，一种包含该肽或其衍生物的药物组合物、及它们用于治疗疼痛的用途。

Description

经修饰的多肽及其在镇痛领域的应用
相关申请的交叉引用
本申请要求对2023年6月21日提交的申请PCT/CN2023/101880享有优先权。在先申请视为本申请公开的一部分，并整体纳入本申请。
技术领域
本申请涉及生物医药领域，具体涉及一种肽或其衍生物及其在镇痛方面的用途。
背景技术
神经病理性疼痛是指由躯体感觉神经系统病变或疾病引起的疼痛,是一种常见的慢性疼痛,严重影响患者生活质量,据统计全球约8％的人口饱受其折磨(GILRON I,BARON R,JENSEN T.Neuropathic pain:principles of diagnosis and treatment[J].Mayo Clin Proc,2015,90(4):532-545)。代谢紊乱(例如糖尿病性神经痛)、神经卡压、肿瘤相关疼痛、脑卒中后疼痛、帕金森病相关性疼痛、脊髓损伤后性疼痛和三叉神经痛、舌咽神经痛、病毒感染如带状疱疹后神经痛等都属于典型的神经病理性疼痛(SZOK D,TAJTI J,A,et al.Therapeutic approaches for peripheral and central neuropathic pain[J].Behav Neurol,2019,2019:8685954.和Jensen TS，Baron R，Haanp M，et al.A new definition of neuropathic pain[J].Pain，2011，152(10):2204-2205)。
周围神经病变的特点是神经损伤和轴突丢失可以分为遗传形式或后天形式。后天性周围神经病变与多种原因有关，包括接触有毒制剂，其中抗肿瘤化合物诱发的疼痛被称为化疗诱导周围神经病变(CIPN)。有综述总结了几项CIPN临床前研究中的神经免疫相互作用表明化疗药物和神经毒性之间的联系。研究已经证明了免疫反应的参与(先天的和适应性)以及介质(细胞因子和趋化因子)的刺激和分泌可能是疼痛症状的原因。此外，神经炎症成分也被显示在脊髓中，小胶质细胞和星形胶质细胞在CIPN中发挥重要作用(Front Immunol.2021 Feb 4:11:626687)。
烟碱乙酰胆碱受体(nAChR)是自然界普遍存在的一种具有重要生理功能的膜蛋白，能调节生物体一系列的生理功能，如：痛觉、认知、记忆、焦虑等。乙酰胆碱受体广泛存在于神经细胞中，其与镇痛密切相关，其亚型主要 是α7、α9α10等(参见例如，Hone A.J.等人，Nicotinic acetylcholine receptors in neuropathic and inflammatory pain,FEBS lett.2018 April；592(7):1045–1062和吴培等人，α7烟碱型乙酰胆碱受体在调控动物炎症反应中的作用，动物营养学报，2021,33(11):6001-6008)。芋螺为肉食性软体动物，是芋螺科动物的总称。世界上约有700种芋螺，主要分布在热带海域。芋螺毒素是芋螺分泌的神经毒液，主要由10～30个氨基酸组成，且包含二硫键。美国犹他大学Olivera教授的团队分离鉴定了多种芋螺毒素的序列和生物活性，发现芋螺毒素可能成为治疗多种神经性疾病的潜在药物，由此引发了国内外研究者对芋螺毒素开发、结构改造和作用机理研究的热潮。其中，Vc1.1，也称为ACV1，含有16个氨基酸、两对二硫键的多肽，是一种从维多利亚圆锥体(Conus victoriae)分离的具有止痛作用的α-芋螺毒素。Vc1.1是α9α10烟碱乙酰胆碱受体(nAChR)的选择性拮抗剂，在大鼠坐骨神经痛(CCI)和神经病理性疼痛(PNL)模型中对神经性疼痛有治疗效果。但是由于人体和老鼠体内α9α10 nAChR的差异性，Vc1.1本已进入二期临床，结果在人体α9α10 nAChR上相比于老鼠α9α10 nAChR活性下降了100倍，迫使相关机构中止了临床研究。Lorenzo Di Cesare Mannelli等人通过对大鼠坐骨神经结扎在慢性压迫性损伤(CCI)模型研究了芋螺毒素多肽RgIA的镇痛效果及机制，坐骨神经的组织学分析显示RgIA可以预防CCI引起轴突致密性和直径降低，髓鞘丢失及纤维减少，此外，RgIA显著减少水肿和炎症浸润，包括减少CD86+巨噬细胞。在脊髓的背角中，RgIA阻止了CCI诱导的小胶质细胞和星形胶质细胞的活化。这些数据表明，多肽RgIA可能代表了一类新的治疗神经性疼痛的药物，可以保护外周神经组织以及通过抑制神经胶质细胞活化来防止中枢适应不良可塑性(PAIN:155(2014)1986–1995)。
齐考诺肽(Ziconotide)于2004年通过FDA批准用于治疗剧烈性慢性疼痛，它最初是由僧袍芋螺(Conus magus)的毒液分离得到的，包含25个氨基酸和三对二硫键。作为选择性Cav2.2通道拮抗剂，齐考诺肽阻断Ca2+内流并抑制传入神经末梢释放兴奋性递质，其镇痛效力是阿片类药物的1000倍。但鞘内注射的特殊给药方式及一系列中枢神经系统不良反应，使得患者依从性差，从而限制了齐考诺肽的普遍应用。
因此，迫切需要开发一种作用于烟碱乙酰胆碱受体且能有效镇痛和/或减少不良反应的改良肽。
发明内容
本申请多肽靶向的目标包括α7nAChR和α9nAChR，靶向的目标为人源序列的乙酰胆碱受体。这可避免芋螺毒素Vc1.1因种属差异而活性降低以及类似的风险。本申请多肽序列引入了多个非天然氨基酸且多肽结构中包含一对分子内二硫键，增强了多肽的稳定性，显著提高了多肽在体内的半衰期，镇痛药效持续时间有了显著延长。疼痛是一个非常严重的临床问题，对患者和家属的生活质量均会造成不良影响。但现有的止痛药并不能针对性地解决不同类型的疼痛，并且还存在着长期用药的不良反应，如阿片类药物的成瘾性，长期使用会造成药物滥用的不良后果。多肽具有先天的安全性优势，本申请的多肽合成工艺简单，易于商业化生产，可有望作为一类安全有效的新型镇痛药物。
本申请的多肽从动物实验结果看，起效时间快于临床上的镇痛药物普瑞巴林，且申请中的多肽相较于母肽AJ003在人源血浆中的稳定性有了显著提升，从动物实验数据也可以发现改构后的多肽其镇痛药效的持续时间比母肽有了显著的延长。本申请的多肽属于非阿片类新型镇痛药物，由于其作用靶点主要存在于外周神经组织，因此后期在临床上不会引起药物成瘾性等毒副作用。与目前已上市的唯一一款芋螺毒素多肽齐考诺肽相比，本申请的肽不用采取鞘内滴注的复杂给药方式，有利于提高临床患者的用药依从性。此外齐考诺肽由25个氨基酸构成，且肽序中含有3对二硫键，制备工艺复杂，大大增加了生产成本。本申请的肽由13个氨基酸构成，只含有1对二硫键，生产制备工艺简单，固相多肽合成技术目前已非常成熟，且其合成特点非常有利于自动化规模生产，能够极大地降低药物生产成本，减少患者的医疗负担。进一步，本申请的肽给药剂量低，在低剂量下也有明显的镇痛活性。
在本公开的第一个方面，提供了一种包含选自SEQ ID NO:1-34中的任意一个序列的肽或其衍生物。
在本公开的第二个方面，提供了一种包含选自具有保守取代的SEQ ID NO:1-34中的任意一个序列的肽或其衍生物。
在本公开的第三个方面，提供了一种以从N末端到C末端的顺序包含下式I或具有保守取代的式I的肽或其衍生物，
Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8   式I
其中式I中的第3位Cys和第9位D-Cys形成一对分子内二硫键，D-Cys代表D型半胱氨酸，氨基酸残基之间的“-”代表肽键，所述肽或其衍生物的C末端为游离羧基或酰胺，且X1为Thr或D-Thr，X5选自Ala、Val和D-Val中的任意一个，X2-X4和X6-X8彼此独立地为任意一种氨基酸。
在本公开的一个实施例中，上述式I中的第3位Cys和第9位D-Cys形成一对分子内二硫键，所述肽或其衍生物的C末端为游离羧基或酰胺，且所述X1为Thr或D-Thr，X5选自Ala、Val和D-Val中的任意一个，X2选自Pro、D-Pro和Hyp中的任意一个，X3选自Pro、D-Pro和Hyp中的任意一个，X4选自Ser、D-Ser、Ala和Aib中的任意一个，X6选自Ala、Leu、D-Leu、Ile、ABu和Nle中的任意一个，X7选自Tyr、D-Tyr、Ala、Trp、Phe和Bip中的任意一个，X8选自Ser、D-Ser、Ala、和Orn中的任意一个。
在本公开的一个实施例中，上述具有保守取代的式I具有1、2、3、4或5个氨基酸保守取代。在本公开的一个实施例中，上述具有保守取代的式I与没有发生保守取代的式I具有至少65％的序列同一性、具有至少70％的序列同一性、具有至少75％的序列同一性、具有至少80％的序列同一性、具有至少84％的序列同一性、具有至少85％的序列同一性、具有至少90％的序列同一性、具有至少92％的序列同一性或具有至少95％的序列同一性。
在本公开的一个优选实施例中，该肽或其衍生物包含选自以下的任意一个序列：SEQ ID NO：1-5、SEQ ID NO：10-16和SEQ ID NO：20-34，具有保守取代的SEQ ID NO：1-5、SEQ ID NO：10-16和SEQ ID NO：20-34。在本公开的一个更优选的实施例中，该肽或其衍生物包含选自以下的任意一个序列：SEQ ID NO：26、28和32，具有保守取代的SEQ ID NO：26、28和32。
在本公开的一个实施例中，上述具有保守取代的序列分别具有1、2、3、4或5个氨基酸保守取代。在本公开的一个实施例中，上述具有保守取代的序列与没有发生保守取代的序列具有至少65％的序列同一性、具有至少70％的序列同一性、具有至少75％的序列同一性、具有至少80％的序列同一性、 具有至少84％的序列同一性、具有至少85％的序列同一性、具有至少90％的序列同一性、具有至少92％的序列同一性或具有至少95％的序列同一性。
在本公开的第四个方面，提供了一种多核苷酸，其编码根据本公开所述的肽或其衍生物。
在本公开的第五个方面，提供了一种表达载体，其表达根据本公开所述的多核苷酸。
在本公开的第六个方面，提供了一种宿主细胞，其包含根据本公开所述的多核苷酸或根据本公开所述的表达载体。
在本公开的第七个方面，提供了一种药物组合物，其包含根据本公开所述的肽或其衍生物以及其药学上可接受的载体或盐。本公开还提供了一种药物组合物，其包含根据本公开所述的肽或其衍生物、多核苷酸、表达载体或根据本公开所述的宿主细胞，以及其药学上可接受的载体或盐。
在本公开的第八个方面，提供了根据本公开所述的肽或其衍生物或根据本公开所述的药物组合物在制备用于治疗疼痛的药物中的用途。本公开还提供了根据本公开所述的肽或其衍生物、根据本公开所述的多核苷酸、根据本公开所述的表达载体、根据本公开所述的宿主细胞或根据本公开所述的药物组合物在制备用于治疗疼痛的药物中的用途。
在本公开的第九个方面，提供了一种用于治疗疼痛的方法，该方法包括向有此需要的受试者施用治疗有效量的根据本公开所述的肽或其衍生物或根据本公开所述的药物组合物。本公开还提供了一种用于治疗疼痛的方法，该方法包括向有此需要的受试者施用治疗有效量的根据本公开所述的肽或其衍生物、多核苷酸、表达载体、宿主细胞或根据本公开所述的药物组合物。
附图说明
以下提供附图的简要说明，其用于说明本文所公开的示例性实施方式，而不是为了限制这些实施方式。
图1示出了实施例3中多肽AJ003-1主峰面积随时间变化的折线图。
图2示出了实施例3中多肽AJ003-34主峰面积随时间变化的折线图。
图3示出了实施例3中多肽AJ003-35主峰面积随时间变化的折线图。
图4示出了实施例3中多肽AJ003-36主峰面积随时间变化的折线图。
图5示出了实施例3中多肽AJ003-37主峰面积随时间变化的折线图。
图6示出了实施例3中多肽AJ003-38主峰面积随时间变化的折线图。
图7示出了实施例3中多肽AJ003-39在人源血浆体外半衰期预测结果。
图8示出了实施例3中多肽AJ003-40主峰面积随时间变化的折线图。
图9示出了实施例3中多肽AJ003-41主峰面积随时间变化的折线图。
图10示出了实施例3中多肽AJ003-42主峰面积随时间变化的折线图。
图11示出了实施例5中在大鼠紫杉醇慢性化疗痛模型中给药同一剂量的多肽在第1天的机械痛阈值。纵轴为机械痛阈值，横轴为不同组。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7-8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003、AJ003-34、AJ003-35、AJ003-36、AJ 003-39和AJ 003-40为1mg/kg·d·qd，皮下注射。
图12示出了实施例5中在大鼠紫杉醇慢性化疗痛模型中给药同一剂量的多肽在第3天的机械痛阈值。纵轴为机械痛阈值，横轴为不同组。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7-8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003、AJ003-34、AJ003-35、AJ003-36、AJ 003-39和AJ 003-40为1mg/kg·d·qd，皮下注射。
图13示出了实施例5中在大鼠紫杉醇慢性化疗痛模型中给药同一剂量的多肽在第5天的机械痛阈值。纵轴为机械痛阈值，横轴为不同组。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7-8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003、AJ003-34、AJ003-35、AJ003-36、AJ 003-39和AJ 003-40为1mg/kg·d·qd，皮下注射。
图14示出了实施例5中在大鼠紫杉醇慢性化疗痛模型中给药同一剂量的多肽在第12天的机械痛阈值。纵轴为机械痛阈值，横轴为不同组。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7-8)。模型组与 对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003、AJ003-34、AJ003-35、AJ003-36、AJ 003-39和AJ 003-40为1mg/kg·d·qd，皮下注射。
图15示出了实施例6中在大鼠紫杉醇慢性化疗痛模型中给药不同剂量的多肽在第1天的机械痛阈值。纵轴为机械痛阈值，横轴为不同组。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003为1mg/kg·d·qd，皮下注射、AJ003-36-低剂量为0.5mg/kg·d·qd，皮下注射、AJ003-36-高剂量为1.5mg/kg·d·qd，皮下注射、AJ003-40-低剂量为0.5mg/kg·d·qd，皮下注射和AJ003-40-高剂量为1.5mg/kg·d·qd，皮下注射。
图16示出了实施例6中在大鼠紫杉醇慢性化疗痛模型中给药不同剂量的多肽在第4天的机械痛阈值。纵轴为机械痛阈值，横轴为不同组。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003为1mg/kg·d·qd，皮下注射、AJ003-36-低剂量为0.5mg/kg·d·qd，皮下注射、AJ003-36-高剂量为1.5mg/kg·d·qd，皮下注射、AJ003-40-低剂量为0.5mg/kg·d·qd，皮下注射和AJ003-40-高剂量为1.5mg/kg·d·qd，皮下注射。
图17示出了实施例6中在大鼠紫杉醇慢性化疗痛模型中给药不同剂量的多肽在第7天的机械痛阈值。纵轴为机械痛阈值，横轴为不同组。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003为1mg/kg·d·qd，皮下注射、AJ003-36-低剂量为0.5mg/kg·d·qd，皮下注射、AJ003-36-高剂量为1.5mg/kg·d·qd，皮下注射、AJ003-40-低剂量为0.5mg/kg·d·qd，皮下注射和AJ003-40-高剂量为1.5mg/kg·d·qd，皮下注射。
图18示出了实施例6中在大鼠紫杉醇慢性化疗痛模型中给药不同剂量的多肽在第12天的机械痛阈值。。纵轴为机械痛阈值，横轴为不同组。结果 显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003为1mg/kg·d·qd，皮下注射、AJ003-36-低剂量为0.5mg/kg·d·qd，皮下注射、AJ003-36-高剂量为1.5mg/kg·d·qd，皮下注射、AJ003-40-低剂量为0.5mg/kg·d·qd，皮下注射和AJ003-40-高剂量为1.5mg/kg·d·qd，皮下注射。
图19示出了实施例6中在大鼠紫杉醇慢性化疗痛模型中给药不同剂量的多肽在第14天的机械痛阈值。。纵轴为机械痛阈值，横轴为不同组。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。普瑞巴林为30mg/kg·d·qd，灌胃；AJ003为1mg/kg·d·qd，皮下注射、AJ003-36-低剂量为0.5mg/kg·d·qd，皮下注射、AJ003-36-高剂量为1.5mg/kg·d·qd，皮下注射、AJ003-40-低剂量为0.5mg/kg·d·qd，皮下注射和AJ003-40-高剂量为1.5mg/kg·d·qd，皮下注射。
图20示出了实施例7中在小鼠奥沙利铂急性化疗痛模型中给药不同剂量的多肽的机械痛阈值随时间的变化。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝8)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。
图21示出了实施例9中在大鼠CCI模型中给药不同剂量的多肽在第1天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05；ns：无统计学意义。
图22示出了实施例9中在大鼠CCI模型中给药不同剂量的多肽在第4天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；**p＜0.01；ns：无统计学意义。
图23示出了实施例9中在大鼠CCI模型中给药不同剂量的多肽在第7天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差 分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；**p＜0.01；*p＜0.05。ns：无统计学意义。
图24示出了实施例9中在大鼠CCI模型中给药不同剂量的多肽在第10天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05。ns：无统计学意义。
图25示出了实施例9中在大鼠CCI模型中给药不同剂量的多肽在第14天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01。ns：无统计学意义。
图26示出了实施例10中在小鼠糖尿病神经痛模型中给药不同剂量多肽在第1天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05。ns：无统计学意义。
图27示出了实施例10中在小鼠糖尿病神经痛模型中给药不同剂量多肽在第4天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05。ns：无统计学意义。
图28示出了实施例10中在小鼠糖尿病神经痛模型中给药不同剂量多肽在第8天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05。ns：无统计学意义。
图29示出了实施例10中在小鼠糖尿病神经痛模型中给药不同剂量多肽在第14天的机械痛阈值。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝7)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01；*p＜0.05。ns：无统计学意义。
图30示出了实施例8中在小鼠奥沙利铂急性化疗痛模型中给药不同多肽的机械痛阈值随时间的变化。结果显示为机械痛阈值(g)(±SD)。Graphpad，单因素方差分析(n＝3)。模型组与对照组：####p＜0.0001；给药组与模型组：****p<0.0001；***p＜0.001；**p＜0.01。ns：无统计学意义。
图31示出了实施例4中AJ003-40在人源血浆37℃孵育0小时HPLC分析图谱。
图32示出了实施例4中AJ003-40在人源血浆37℃孵育24小时HPLC分析图谱。
图33示出了实施例4中AJ003-24在人源血浆37℃孵育0小时HPLC分析图谱。
图34示出了实施例4中AJ003-24在人源血浆37℃孵育22小时HPLC分析图谱。
具体实施方式
除非另有说明，否则在本说明书和权利要求书中使用的表示含量、浓度、比例、重量、粒径、百分比、技术效果等的所有数字在任何情况下均应理解为由术语“约”或“大致”修饰。因此，除非有相反的指示，否则以下说明书和所附权利要求书中列出的数字参数是近似值。
除非另有说明，本文使用的术语对所属技术领域的技术人员具有通常的理解含义。对于本领域技术人员来说，其可以根据通过本申请寻求得到的期望性质和效果而变化，应根据有效数字位数和常规舍入方法或者本领域技术人员理解的方式来解释每个数值参数。一般而言，本文使用的命名和本文所描述的有机化学、药物化学、生物学的实验操作是本领域公知的，而且在本领域中普遍采用。除非另有定义，本文使用的所有技术和科学术语一般具有本申请所属领域的普通技术人员通常理解的相同含义。在本文使用的术语存在多个定义的情况下，除非另有说明，以本部分的定义为准。
在本文中使用时，表述“A和/或B”包括三种情况：(1)A；(2)B；以及(3)A和B。表述“A、B和/或C”包括七种情况：(1)A；(2)B；(3)C；(4)A和B；(5)A和C；(6)B和C；以及(7)A、B和C。类似表述的含义可以此类推。
在本文中使用时，术语“彼此独立地”表示多个事件之间彼此没有影响。例如，“X和Y彼此独立地选自a、b、c、d、e、f、g之任一”表示X可以是a、b、c、d、e、f、g之任一，Y也可以是a、b、c、d、e、f、g之任一，X的选择和Y的选择可以相同，也可以不同，二者互不干扰。
在本文中使用时，丙氨酸扫描(Cunningham和Wells,Science244,1081-1085,1989)用于鉴定对于本发明的肽的活性(例如与乙酰胆碱受体的结合亲 和力)起关键作用的氨基酸，以便不对这些氨基酸进行取代。丙氨酸扫描将突变引入该分子的每一个残基，并检验所得到的分子的生物活性以确定对该分子的活性起关键作用的氨基酸残基。
如本文所用，术语“包括”，“包含”表示除了具有所列出的元素外，其他元素也不排除。
如本文所用，术语“同一性”是指两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个拷贝具有100％同一性。本领域技术人员将认识到，一些算法可以用于使用标准参数来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25:3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215:403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147:195-197)和ClustalW。
肽或其衍生物
本申请提供了一种包含选自SEQ ID NO:1-34中的任意一个序列的肽或其衍生物。
在申请提供了一种包含选自具有保守取代的SEQ ID NO:1-34中的任意一个序列的肽或其衍生物。在一些实施方式中，具有保守取代的SEQ ID NO:1-34中的任意一个具有1、2、3、4或5个保守取代。在一些实施方式中，具有保守取代的SEQ ID NO:1-34中的任意一个与没有发生保守取代的序列具有至少65％的序列同一性、具有至少70％的序列同一性、具有至少75％的序列同一性、具有至少80％的序列同一性、具有至少84％的序列同一性、具有至少85％的序列同一性、具有至少90％的序列同一性、具有至少92％的序列同一性或具有至少95％的序列同一性。
本申请提供了一种以从N末端到C末端的顺序包含下式I或具有保守取代的式I的肽或其衍生物，
Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8   式I
其中式I中的第3位Cys和第9位D-Cys形成一对分子内二硫键，D-Cys代表D型半胱氨酸，氨基酸残基之间的“-”代表肽键，所述所述肽或其衍生物的C末端为游离羧基或酰胺，且X1为Thr或D-Thr，X5选自Ala、 Val和D-Val中的任意一个，X2-X4和X6-X8彼此独立地为任意一种氨基酸。
在一些实施方式中，所述式I中的X1为Thr或D-Thr。在一些实施方式中，所述式I中的X5选自Ala、Val和D-Val中的任意一个。在一些实施方式中，所述式I中的X2选自Pro、D-Pro和Hyp中的任意一个。在一些实施方式中，所述式I中的X3选自Pro、D-Pro和Hyp中的任意一个。在一些实施方式中，所述式I中的X4选自Ser、D-Ser、Ala和Aib中的任意一个。在一些实施方式中，所述式I中的X6选自Ala、Leu、D-Leu、Ile、ABu和Nle中的任意一个。在一些实施方式中，所述式I中的X7选自Tyr、D-Tyr、Ala、Trp、Phe和Bip中的任意一个。在一些实施方式中，所述式I中的X8选自Ser、D-Ser、Ala、和Orn中的任意一个。
在一些实施方式中，上述具有保守取代的式I具有1、2、3、4或5个氨基酸保守取代。在本公开的一个实施例中，上述具有保守取代的式I与没有发生保守取代的式I具有至少65％的序列同一性、具有至少70％的序列同一性、具有至少75％的序列同一性、具有至少80％的序列同一性、具有至少84％的序列同一性、具有至少85％的序列同一性、具有至少90％的序列同一性、具有至少92％的序列同一性或具有至少95％的序列同一性。
在一些实施方式中，所述肽或其衍生物包含选自以下的任意一个序列：SEQ ID NO：1-5、SEQ ID NO：10-16和SEQ ID NO：20-34，具有保守取代的SEQ ID NO：1-5、SEQ ID NO：10-16和SEQ ID NO：20-34。在一些优选的实施方式中，所述肽或其衍生物包含SEQ ID NO：26或具有保守取代的SEQ ID NO：26。在一些优选的实施方式中，所述肽或其衍生物包含SEQ ID NO：28或具有保守取代的SEQ ID NO：28。在一些优选的实施方式中，所述肽或其衍生物包含SEQ ID NO：32或具有保守取代的SEQ ID NO：32。
在一些实施方式中，上述具有保守取代的序列具有1、2、3、4或5个氨基酸保守取代。在一些实施方式中，上述具有保守取代的序列与没有发生保守取代的序列具有至少65％的序列同一性、具有至少70％的序列同一性、具有至少75％的序列同一性、具有至少80％的序列同一性、具有至少84％的序列同一性、具有至少85％的序列同一性、具有至少90％的序列同一性、具有至少92％的序列同一性或具有至少95％的序列同一性。
在一些实施方式中，所述衍生物与所述肽相比包含选自以下的任意一种或多种修饰：氨基酸修饰、保守氨基酸取代、氨基酸残基中的氢被取代。
在本文中使用时，术语“肽”是指氨基酸以肽键链接在一起而形成的化合物。由三个或者三个以上氨基酸分子组成的肽为多肽。在本文中使用时，除非另有说明或与上下文相矛盾，否则术语“肽”涵盖所提及的肽本身及其药学上可接受的盐、前体药和代谢物。本申请的肽以本领域常用技术手段生成，包括但不限于固相合成法(如Fmoc肽合成法)和液相合成法。
在本文中使用时，多肽或其衍生物的位点编号是按照从N末端到C末端的顺序进行的，例如从肽的N末端开始的第一个氨基酸为第一位氨基酸，从肽的N末端开始的第二个氨基酸为第二位氨基酸，从肽的N末端开始的第三个氨基酸为第三位氨基酸，以此类推。
除另有定义，如本领域通常理解的，氨基酸序列或多肽序列的左手端为N末端/氨基端，氨基酸序列或多肽序列的右手端为C末端/羧基端。在本文中使用时，术语“前体药”指的是通过在生理条件下在活体内例如通过在酶、胃酸等作用下进行的氧化、还原、水解等反应而转化为本公开的肽或其衍生物的所有分子。
在本文中使用时，术语“代谢物”指的是在细胞或有机体(优选人)中的源自式本公开的肽或其衍生物的所有分子。
在本文中关于肽使用时，“氨基酸”与“氨基酸残基”具有相同含义，指的是：当氨基酸之间通过化学键而连接时，其部分基团由于参与了连接键的形成而损失，剩余的氨基酸部分即为氨基酸残基。
在本文中使用时，氨基酸的名称和缩写对应如下:

天然氨基酸中，除了甘氨酸，均含有手性碳原子。除非另外指明，本申请中所述的光学活性氨基酸均为L-构型。如习惯一样，本文中肽的结构是按氨基末端(N末端)在左，羧基末端(C末端)在右的方式写出的。
用在本文中的术语“氨基酸”包括天然氨基酸，以及在肽化学领域中制备天然肽的类似物时所常用的其它的“非蛋白”α-氨基酸/非天然氨基酸。
天然氨基酸是甘氨酸，丙氨酸，缬氨酸，亮氨酸，异亮氮酸，丝氨酸，甲硫氨酸，苏氨酸，苯丙氨酸，酪氨酸，色氨酸，半胱氨酸，脯氨酸，组氨酸，天冬氨酸，天冬酰胺，谷氨酸，谷氨酰胺，精氨酸，鸟氨酸和赖氨酸。“非蛋白”α-氨基酸/非天然氨基酸的例子有正亮氨酸，正缬氨酸，别异亮氨酸，高精氨酸，硫脯氨酸，脱氢脯氨酸，羟基脯氨酸(Hyp)，高丝氨酸，环己基甘氨酸(Chg)，α-氨基-正丁酸(Aba)，环己基丙氨酸(Cha)，氨基苯基丁酸(Pba)，在苯基部分被烷基、烷氧基、卤素或硝基取代的苯基丙氨酸，丝氨酸，苏氨酸和酪氨酸的O-烷基化衍生物，S-烷基化的半胱氨酸，酪氨酸的O-硫酸酯，以及天然氨基酸的D-异构体。
在本文中使用时，肽的“衍生物”指的是：在所提及肽的基础上进行氨基酸修饰、保守氨基酸取代和/或氨基酸残基中的氢被取代而得到的产物。
在本文中使用时，术语“氨基酸修饰”的种类包括但不限于N-末端修饰、C-末端修饰和侧链修饰。“氨基酸修饰”的方式包括但不限羟基化、羧基化、烷基化、酰化、磷酸化、磺酸化、酰胺化、醛基化、醇基化、巯基乙胺化、酯基化、糖基化等修饰。
在本文中使用时，“保守氨基酸取代”通常可以描述为一个氨基酸残基被另一个具有相似化学结构和/或相似化学性质的氨基酸取代，并对肽的功能、活性或其他生物特性几乎没有影响的氨基酸取代。这种保守的氨基酸取代在本领域是众所周知的。这样的保守取代例如可以是以下组(a)-(e)中的一个氨基酸被同一组的另一个氨基酸取代：(a)小脂肪族、非极性或弱极性氨基酸残基：Ala,Ser,Thr,Pro和Gly；(b)带负电的极性氨基酸残基及其酰胺：Asp,Asn， Glu和Gln；(c)带正电的极性氨基酸残基：His,Arg和Lys；(d)大脂肪族、非极性氨基酸残基：Met,Leu,Ile,Val和Cys；(e)芳族氨基酸残基：Phe,Tyr和Trp。
在本文中使用时，氨基酸残基中的氢被取代可以是氨基酸残基中的氢被本领域已知的任何常规取代基取代，包括但不限于烷基、烯基、炔基、芳香基、烷氧基、羧基、醛基、羰基、羟基、卤素、氰基、酰基、磺酸基、氨基、巯基或硝基等。
多核苷酸、表达载体、宿主细胞
本申请提供了一种多核苷酸，该多核苷酸编码本公开所述的肽或其衍生物。
在本文中使用时，“多核苷酸”指任何长度的核苷酸(核苷酸或脱氧核苷酸)的多聚体。该名词指分子的初级结构。由此它既包括双链和单链的RNA，又包括双链和单链的DNA。它也包括如用甲基化作用和/或封端修饰的多核苷酸，以及未修饰形式的多肽苷酸。本文所述的多核苷酸不一定是通过物理方法获得，也可以任何方式产生，包括诸如化学合成或者DNA复制，反转录或转录。
本申请提供了一种表达载体，该表达载体能够表达本公开所述的多核苷酸。
本申请提供了一种宿主细胞，该宿主细胞包含本公开所述的多核苷酸或本申请所述的表达载体。
在本文中使用时，“宿主细胞”用作表达载体或其他转化遗传片段的受体的细胞。它们包括已被转染的原始细胞和其后代。宿主细胞包括原核、酵母或其他真核细胞。
药物组合物
本申请提供了一种药物组合物，其包含本公开所述的肽或其衍生物，以及药学上可接受的载体或盐。
本申请还提供了一种药物组合物，其包含本公开所述的肽或其衍生物、多核苷酸、表达载体或根据本公开所述的宿主细胞，以及其药学上可接受的载体或盐。
如本文所用，术语“药物组合物”是指本公开的肽与其它化学组分例如载体、稳定剂、稀释剂、分散剂、悬浮剂、增稠剂和/或赋形剂的混合物。药物组合物有助于将该肽施用至生物体。本领域中存在施用肽的多种方式，包括但不限于，皮下注射、肌肉注射、静脉注射、腹腔注射、鞘内注射、口服、经皮、经肺、眼部和局部施用。
在本申请中，所述药物组合物可以配置为适用于通过所需要的给药途径施用至受试者的剂型。所述剂型包括但不限于片剂、胶囊、囊片、药丸、锭剂、粉末、糖浆、酿剂、混悬液、溶液、乳剂、透皮贴片、栓剂、吸入剂、霜剂、膏剂、洗液、糊剂、喷雾剂、冻干、注射剂和凝胶等。
在本文中使用时，术语“药学上可接受的盐”包括酸加成盐和碱加成盐。适当的酸加成盐是由形成无毒性盐的酸所形成的。其实例包括但不限于：乙酸盐、己二酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、碳酸氢盐/碳酸盐、硫酸氢盐/硫酸盐、硼酸盐、樟脑磺酸盐、柠檬酸盐、环己胺磺酸盐、乙二磺酸盐、甲酸盐、反丁烯二酸盐、葡萄庚糖酸盐、葡萄糖酸盐、葡萄糖醛酸盐、六氟磷酸盐、2-(4-羟苄基)苯甲酸盐、氢氯化物/氯化物、氢溴化物/溴化物、氢碘化物/碘化物、2-羟乙磺酸盐、乳酸盐、苹果酸盐、顺丁烯二酸盐、丙二酸盐、甲磺酸盐、甲基硫酸盐、萘酸盐、2-萘磺酸盐、烟碱酸盐、硝酸盐、乳清酸盐、草酸盐、十六酸盐、磷酸盐/磷酸氢盐/磷酸二氢盐、焦谷氨酸盐、葡萄糖二酸盐、硬脂酸盐、水杨酸盐、单宁酸盐、酒石酸盐、甲苯磺酸盐和三氟乙酸盐。适当的碱加成盐是由形成无毒性盐的碱所形成的。其实例包括但不限于：铝、精氨酸、钙、胆碱、二乙胺、二乙醇胺、甘氨酸、赖氨酸、镁、葡甲胺、乙醇胺、钾、钠、氨丁三醇和锌盐。还可形成酸和碱的半盐，例如半硫酸盐和半钙盐。关于合适的盐的综述，参见Handbook of Pharmaceutical Salts:Properties,Selection and Use by Stahl and Wermuth(Wiley-VCH,2002)。
术语“药学上可接受的载体”包括药学上可接受的材料、组合物或载体，例如液体或固体填充剂、稀释剂、赋形剂、溶剂或封装材料，其涉及在受试者内携带或输送本公开的肽或携带或输送本公开的肽至受试者，使得其可以执行其预期功能。每种盐或载体在与制剂的其它成分相容的意义上必须是“可接受的”，并且不对受试者有害。可以用作药学上可接受的载体的材料 的一些实例包括：糖，例如乳糖、葡萄糖和蔗糖；淀粉，例如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，例如羧甲基纤维素钠、乙基纤维素和乙酸纤维素；粉末黄蓍胶；麦芽；明胶；滑石；赋形剂，例如可可脂和栓剂蜡类；油，例如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和大豆油；二醇类，例如丙二醇；多元醇，例如甘油、山梨醇、甘露醇和聚乙二醇；酯类，例如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，例如氢氧化镁和氢氧化铝；藻酸；无热原水；等渗盐水；林格氏溶液；乙醇；磷酸盐缓冲溶液；稀释剂；造粒剂；润滑剂；粘合剂；崩解剂；润湿剂；乳化剂；着色剂；脱模剂；涂层剂；甜味剂；调味剂；加香剂；防腐剂；抗氧化剂；增塑剂；胶凝剂；增稠剂；硬化剂；定型剂；悬浮剂；表面活性剂；保湿剂；载体；稳定剂；和在药物制剂中使用的其它无毒的相容性物质，或其任何组合。
用途、疾病治疗方法
本申请提供了本公开所述的肽或其衍生物或本公开所述的药物组合物在制备用于治疗疼痛的药物中的用途。
本申请还提供了本公开所述的肽或其衍生物、本公开所述的多核苷酸、本公开所述的表达载体、本公开所述的宿主细胞或本公开所述的药物组合物在制备用于治疗疼痛的药物中的用途。
在一些实施方式中，所述制备的用于治疗疼痛的药物通过包括皮下注射、肌肉注射、静脉注射、腹腔注射、鞘内注射、口服、经皮、经肺、眼部或局部给药的方式施用。在优选的实施方式中，所述制备的用于治疗疼痛的药物通过皮下注射施用。
在一些实施方式中，所述疼痛包括作用于烟碱乙酰胆碱受体而引起的疼痛。在一些实施方式中，所述疼痛包括神经疼痛和炎症性疼痛。在一些实施方式中，所述疼痛包括化疗引起的疼痛、糖尿病性周围神经疼痛、坐骨神经痛、骨关节疼痛、带状疱疹后遗神经痛、感染疼痛、营养缺乏疼痛、炎症疼痛、慢性酒精中毒疼痛、甲状腺功能减退疼痛、自身免疫病疼痛、格林巴利综合症疼痛、中毒疼痛、药物引起的疼痛、肿瘤病变疼痛、遗传疾病疼痛和挫伤疼痛。
本申请进一步提供了本公开所述的肽或其衍生物、本公开所述的多核苷酸、本公开所述的表达载体、本公开所述的宿主细胞或本公开所述的药物组合物，其用于治疗疼痛的用途。
在一些实施方式中，所述疼痛包括作用于烟碱乙酰胆碱受体而引起的疼痛。在一些实施方式中，所述疼痛包括神经疼痛和炎症性疼痛。在一些实施方式中，所述疼痛包括化疗引起的疼痛、糖尿病性周围神经疼痛、坐骨神经痛、骨关节疼痛、带状疱疹后遗神经痛、感染疼痛、营养缺乏疼痛、炎症疼痛、慢性酒精中毒疼痛、甲状腺功能减退疼痛、自身免疫病疼痛、格林巴利综合症疼痛、中毒疼痛、药物引起的疼痛、肿瘤病变疼痛、遗传疾病疼痛和挫伤疼痛。
在一些实施方式中，所述肽或其衍生物或者药物组合物通过包括皮下注射、肌肉注射、静脉注射、腹腔注射、鞘内注射、口服、经皮、经肺、眼部或局部给药的方式施用。在优选的实施方式中，所述肽或其衍生物或者药物组合物通过皮下注射施用。
本申请提供了用于治疗疼痛的方法，该方法包括向有此需要的受试者施用治疗有效量的根据本公开所述的肽或其衍生物或根据本公开所述的药物组合物。
本申请还提供了用于治疗疼痛的方法，该方法包括向有此需要的受试者施用治疗有效量的根据本公开所述的肽或其衍生物、多核苷酸、表达载体、宿主细胞或根据本公开所述的药物组合物。
在一些实施方式中，所述疼痛包括作用于烟碱乙酰胆碱受体而引起的疼痛。在一些实施方式中，所述疼痛包括神经疼痛和炎症性疼痛。在一些实施方式中，所述疼痛包括化疗引起的疼痛、糖尿病性周围神经疼痛、坐骨神经痛、骨关节疼痛、带状疱疹后遗神经痛、感染疼痛、营养缺乏疼痛、炎症疼痛、慢性酒精中毒疼痛、甲状腺功能减退疼痛、自身免疫病疼痛、格林巴利综合症疼痛、中毒疼痛、药物引起的疼痛、肿瘤病变疼痛、遗传疾病疼痛和挫伤疼痛。
在一些实施方式中，所述肽或其衍生物或者药物组合物通过包括皮下注射、肌肉注射、静脉注射、腹腔注射、鞘内注射、口服、经皮、经肺、眼部 或局部给药的方式施用。在优选的实施方式中，所述肽或其衍生物或者药物组合物通过皮下注射施用。
在本文中使用时，术语“受试者”包括动物，如脊椎动物，优选哺乳动物，如狗、猫、猪、牛、羊、马、啮齿动物(例如小鼠、大鼠或豚鼠)或灵长类动物(例如大猩猩、黑猩猩和人)。
在本文中使用时，术语“治疗”是指缓解或改善疾病或障碍(即，减慢或阻止疾病或至少一种临床症状的发展)；或缓解或改善与该疾病或障碍相关的至少一种物理参数或生物标记。
在本文中使用时，术语“治疗有效剂量”是指相比于未接受该量的相应受试者，导致从中获益或治疗疾病的量，但该量在合理医学判断的范围内足够低以避免严重副作。本文所述的肽或其衍生物、多核苷酸、表达载体、宿主细胞或药物组合物的治疗有效剂量将随着选择的肽或其衍生物、多核苷酸、表达载体、宿主细胞或药物组合物；给药途径；治疗的疾病的严重性；治疗患者的年龄、体型、体重和身体状况：治疗患者的医疗史；治疗的持续时间；并行治疗的性质；所需治疗效果等因素而改变，但仍可通过本领域技术人员以常规方式确定。
上文针对本公开的肽或其衍生物、多核苷酸、表达载体和宿主细胞所述的各种实施方式也适用于本公开的药物组合物、用途和方法(只要它们彼此之间不是内在矛盾的)，由此组合而形成的各种实施方式都视为本公开的一部分。
实施例
以下结合附图对本申请的示范性实施例做出说明，其中包括本申请实施例的各种细节以助于理解。应该理解的是，它们认为仅仅是示范性的，绝不旨在限制本申请的保护范围。本申请的保护范围仅通过权利要求来限定。因此，本领域普通技术人员应当认识到，可以对这里描述的实施例做出各种改变和修改，而不会背离本申请的范围。同样，为了清楚和简明，以下的描述中省略了对公知功能和结构的描述。
除非另有说明，否则以下实施例中所采用的试剂和仪器均为可以通过市购获得的常规产品。除非另有说明，否则按照常规条件或制造商建议的条件进行实验。
实施例1：芋螺毒素多肽的合成
在前期研究的基础上，通过对母肽AJ003进行丙氨酸扫描、单一位点非天然氨基酸替换以及多位点组合改构，初步筛选出一批多肽化合物。
AJ003的合成：使用Fmoc-Ser(tBu)-wang树脂(天津南开合成科技有限公司)为起始树脂，合成规模1mmol，称取树脂加入至多肽固相反应器(南京銮玉化玻仪器有限公司)中，加入二氯甲烷氮气鼓吹溶胀30分钟，再用DMF(二甲基甲酰胺)洗涤2次。加20％哌啶/DMF溶液两次，每次反应5分钟。脱保护反应结束后，使用DMF洗涤树脂6次，每次约3分钟。按照该多肽序列，遵循化学合成多肽常规是从C末端向N末端逐个偶联氨基酸的原则，称取相应保护氨基酸、缩合试剂(HoBt、DIC)，反应约1小时，缩合反应结束后用DMF洗涤树脂3次。自此进行缩合循环(脱保护-洗涤-缩合-洗涤)，即再向反应容器加入20％哌啶/DMF溶液，进行脱保护反应，DMF洗涤后加入保护氨基酸及相应缩合试剂进行缩合反应。采用缩合循环的方法逐个向N末端偶联保护氨基酸，直至偶联完该多肽所有氨基酸。用20％哌啶/DMF溶液脱除多肽N末端的Fmoc基团，使用二氯甲烷和甲醇对树脂进行交替洗涤，最后通过真空干燥的方式获得收缩干燥的肽树脂中间体。称取肽树脂重量，按照1克肽树脂10毫升裂解液的比例，配制裂解液。裂解液配方为TFA:EDT:TIS:H2O＝90:5:2.5:2.5。向肽树脂加入裂解液，裂解反应3小时左右，最后收集滤液，用冰冻的甲基叔丁基醚沉降滤液，甲基叔丁基醚的使用量为滤液的8倍，充分搅拌沉降液，分杯离心，即获得粗肽沉淀，继续用甲基叔丁基醚洗涤离心，将沉淀真空干燥过夜，最终获得线性粗肽样品。将线性粗肽用水溶解后，加入碘单质作为氧化剂进行该多肽氧化成二硫键的反应。氧化约30分钟，利用HPLC进行氧化程度监测，最后向氧化体系滴加维生素C以终止氧化反应。将氧化完毕的多肽液用滤膜过滤后，使用反相高效液相色谱进行分离纯化，将接得的合格馏分合并后最终使用冷冻干燥机进行冻干，最终获得该多肽冻干纯品。
其他多肽的合成方法与上述多肽AJ003技术路线一致，均采用经典的Fmoc固相多肽合成技术获得线性粗肽。多肽序列C末端以酰胺结尾的多肽，所使用树脂为Rink酰胺-MBHA树脂。将粗肽液通过碘单质氧化形成二硫 键多肽馏分，再经过反相高效液相色谱技术进行分离纯化，最后经冷冻干燥技术获得冻干纯品。本实施例中涉及的肽序列在表1中示出。
表1.多肽序列汇总表

*D-Thr:D型苏氨酸；3,9成二硫键：序列的第三位和第九位的氨基酸残基之间形成二硫键。
实施例2：BLI(生物膜层干涉技术)检测多肽与靶蛋白亲和力
BLI是一种非标记技术，它将发生在BLI生物传感器表面的光干涉信号转化为实时的响应信号。通过对分子结合过程的实时监测，系统会测定结合常数(ka或kon)和解离常数(kd或koff)，以及起始结合速率，并通过拟合计算分析得到亲和力(KD)。具体步骤是将NTA传感器(赛多利斯)放置于预湿盒，将预湿盒放置于黑色96孔板，每孔200μL的PBS，将NTA传感器预湿至少15min以上。将靶蛋白α9 nAChR用PBS稀释为5μg/mL,每孔加200μL。将待检测多肽用PBS溶解梯度3倍稀释，浓度由高到低依次为1000nM、333.3nM、111.1nM、37.04nM、12.35nM、4.115nM、1.372nM，最后再增加一个PBS孔作为零浓度对照孔。平衡仪器基线后，先利用NTA传感器固化靶蛋白α9 nAChR，固化完成再平衡基线，然后是进行结合5min、解离5min过程。按照上述过程对待测多肽逐个进行亲和力检测。多肽与靶蛋白α7nAChR的亲和力检测过程基本与α9 nAChR相同，只是靶蛋白α7 nAChR的蛋白浓度为20μg/mL。如下表2、3所示，BLI实验检测了丙氨酸扫描多肽类似物与靶蛋白的亲和力，明确了母肽可改构位点；其次再利用BLI实验检测了单一位点非天然氨基酸突变改构肽与靶蛋白亲和力；最后利用BLI实验 检测了多位点组合突变改构肽与靶蛋白亲和力，从而筛选出与靶蛋白高亲和力多肽用于后续筛选及研究其成药性。
表2.改构肽与α9 nAChR靶蛋白亲和力检测结果
表3.改构肽与α7nAChR靶蛋白亲和力检测结果
实施例3：体外血浆稳定性预测多肽半衰期
多肽通常半衰期较短，在动物体内容易被各种酶及血液中的基质等降解。本研究中的改构肽在设计时对某些非活性关键位点氨基酸进行了非天然氨基酸替换，希望能提高多肽稳定性，更加利于成药。称取多肽用去离子水溶解为2mg/mL，再用乙腈稀释为1mg/mL后用有机滤膜过滤，将滤液吸取200微升至液相小瓶作为多肽对照组。吸取300微升人源血浆(含EDTA抗凝剂)(南京森贝伽生物科技有限公司,批号SBJ-P-HU011-100mL)加入到300微升乙腈，置于离心机4℃，12000转/分钟，离心5分钟。吸取上清液用有机滤膜过滤，将滤液吸取200微升至液相小瓶作为血浆空白对照组。另称量该多肽10mg，将5mL人源血浆加入其中涡旋混匀，吸取300微升含有多肽的血浆加入到300微升乙腈，置于离心机4℃，12000转/分钟，离心5分钟，吸取上清液用有机滤膜过滤，将滤液吸取200微升至液相小瓶作为多肽0min时间点样品。将溶解多肽的血浆置于37℃水浴锅孵育，其余时间点操作同0min时间点操作。其中多肽AJ003和AJ003-1取样点为0min、5min、10min、20min、30min、1h、2h、4h、6h、10h、12h、24h，改构肽AJ003-34、AJ003-35、AJ003-36、AJ003-37、AJ003-38、AJ003-39、AJ003-40、AJ003-41和AJ003-42取样点为0min、10min、30min、1h、4h、6h、10h、12h、24h，每个多肽的系列样品均移交分析部门进行HPLC分析，记录样品主峰面积随血浆孵育时间的变化情况，在药代动力学处理软件winolin进行每个多肽体外半衰期预测。AJ003样品因存在互变异构体，峰面积随时间不易准确积分比较。AJ003-1是AJ003多肽第5位苏氨酸改构为D-型苏氨酸的类似物，AJ003-1与AJ003相比肽序仅差一个氨基酸构型的区别。如图1-10所示，AJ003-1的体外血浆半衰期约10.8h，改构肽AJ003-34、AJ003-35、AJ003-36、AJ003-37、AJ003-38、AJ003-39、AJ003-40、AJ003-41、AJ003-42体外血浆半衰期分别是61.9h、45.3h、65.3h、71.0h、26.7h、43.0h、84.0h、30.0h和61.4h。整体来看，通过非天然氨基酸的突变，本项目其他改构肽在体外血浆稳定性与AJ003-1(与AJ003只相差一个氨基酸)相比，稳定性有了显著提升。
实施例4：AJ003-40和AJ003-24的血浆稳定性实验
称量多肽AJ003-40和AJ003-24分别加入人源血浆加入其中涡旋混匀，配制为2mg/ml。各吸取300微升含有多肽的血浆加入到300微升乙腈，置 于离心机4℃，12000转/分钟，离心5分钟，吸取上清液用有机滤膜过滤，将滤液吸取200微升至液相小瓶作为多肽0小时时间点样品。将溶解多肽的血浆置于37℃水浴锅孵育，多肽AJ003-40于24小时时间点吸取300微升含有多肽的血浆加入到300微升乙腈，置于离心机4℃，12000转/分钟，离心5分钟，吸取上清液用有机滤膜过滤，将滤液吸取200微升至液相小瓶作为多肽24小时时间点样品。多肽AJ003-24于22小时时间点吸取300微升含有多肽的血浆加入到300微升乙腈，置于离心机4℃，12000转/分钟，离心5分钟，吸取上清液用有机滤膜过滤，将滤液吸取200微升至液相小瓶作为多肽22小时时间点样品。将两个多肽0小时和24小时样品进行HPLC分析，如图31和图32所示，多肽AJ003-40主峰面积由0小时的15149.38降低到24小时的11829.51，在24小时内主峰面积降解率为21.9％；如图33和图34所示，多肽AJ003-24主峰面积由0小时的746.80降低到22小时的267.45，在22小时内主峰面积降解率为64.2％。与多肽AJ003-40相比，多肽AJ003-24降解速率更快，说明AJ003-24在血浆稳定性比AJ003-40较差。
实施例5：紫杉醇化疗痛评价部分改构肽在同一剂量下的体内镇痛活性
180-200g的SPF(Specific Pathogen Free)级SD雄性大鼠(上海市计划生育科学研究所实验动物经营部)，造模当天称量大鼠体重，并用机械刺激检测大鼠基础痛阈值，剔除体重差异和痛阈值差异较大动物，余下大鼠根据实验方案开展造模。紫杉醇粉末用橄榄油溶解为1mg/mL，每只大鼠每次腹腔注射量为2mg/kg。整个造模期间共注射4次，分别在1、3、5、7天进行腹腔注射。阴性对照或空白对照组大鼠注射上述溶解紫杉醇的溶剂。根据文献报道，此模型从造模开始算，经过15天左右痛阈值发生明显下降，标志着稳定的大鼠紫杉醇慢性化疗痛模型构建成功。按照实验方案设置的给药方式进行药物治疗，具体如下表4所示。表4中的空白组代表没有进行疼痛建模的组，模型组代表有疼痛建模但没有给与药物的组。在第1、3、5、12天在固定时间对大鼠进行给药，其中在给药第1、3、5天给完药的1h、2.5h、4h、5.5h通过平头针刺激大鼠右后肢足底，检测大鼠在相应时间点对机械刺激的痛阈值。在给药第12天检测了当天给药后4h和7h大鼠对机械刺激的痛阈值数值。如图11-14所示，综合4次给药治疗的实验结果，母肽AJ003在该 模型中4h内跟模型组相比具有显著的镇痛活性，超过4h后镇痛活性与模型组比较没有显著性。5个候选改构肽(AJ003-34、AJ003-35、AJ003-36、AJ003-39、AJ003-40)中，AJ003-34、AJ003-36、AJ003-40是镇痛候选最佳的3个，从第12天7h结果分析，尤以AJ003-36和AJ003-40的镇痛活性持续时间较长，可持续至7h，且此改构肽与阳性药相比镇痛活性+在某些给药时间点略优于普瑞巴林，整体来看与阳性药普瑞巴林镇痛药效相当。AJ003-36和AJ003-40可作为候选分子后续进一步研究。紫杉醇作为临床广泛使用的小分子化疗药物，在抗肿瘤的同时，因其自身对患者神经系统的损伤，造成许多癌症患者产生神经痛。通过紫杉醇多次低剂量腹腔注射诱导的SD雄性大鼠慢性化疗痛模型，模拟了临床上癌症患者接受紫杉醇利用该模型评价了5个改构肽以及母肽AJ003和阳性药普瑞巴林在该模型的镇痛效果。试验结果表明，每次给药后在相应时间点检测大鼠右后肢足底机械痛阈值，整体分析可以初步筛选出AJ003-36和AJ003-40可作为候选分子后续进一步研究。
表4.紫杉醇化疗痛模型筛选同一剂量下部分改构肽镇痛活性给药方案表
实施例6：紫杉醇化疗痛评价不同剂量下改构肽体内镇痛活性
购买180-200g的SPF级SD雄性大鼠(上海市计划生育科学研究所实验动物经营部)，适应性喂养一周左右，造模当天称量大鼠体重，并用机械刺激检测大鼠基础痛阈值，剔除体重差异和痛阈值差异较大动物，余下大鼠根据 实验方案开展造模。紫杉醇粉末用橄榄油溶解为1mg/mL，每只大鼠每次腹腔注射量为2mg/kg。整个造模期间共注射4次，分别在1、3、5、7天进行腹腔注射。阴性对照或空白对照组大鼠注射上述溶解紫杉醇的溶剂。根据文献报道，此模型从造模开始算，经过15天左右痛阈值发生明显下降，标志着稳定的大鼠紫杉醇慢性化疗痛模型构建成功。按照实验方案设置的给药方式进行药物治疗，具体如下表5所示。从造模第15天开始，每天连续对动物进行给药治疗，并在给药第1、4、7、12、14天给完药第2h、4h、6h、8h通过平头针刺激大鼠右后肢足底，检测大鼠在相应时间点对机械刺激的痛阈值。结果见图15-19。综合5次的给药治疗的实验结果，母肽AJ003在该模型中4h内跟模型组相比具有显著的镇痛活性，超过4h后镇痛活性与模型组比较没有显著性。2个候选改构肽AJ003-36和AJ003-40，其低剂量0.5mg/kg镇痛活性与母肽AJ003的1mg/kg的镇痛效果相当，在某些给药天数的相应时间点还优于母肽AJ003。2个候选改构肽AJ003-36和AJ003-40，其高剂量1.5mg/kg镇痛活性略优于阳性药普瑞巴林，且镇痛效果在给药8h后与模型组相比仍然具有显著镇痛活性。
表5.紫杉醇化疗痛模型筛选不同剂量下部分改构肽镇痛活性给药方案表
实施例:7：奥沙利铂化疗痛评价不同剂量下候选改构肽体内镇痛活性
奥沙利铂用5％葡萄糖溶液溶解为1mg/mL。造模组每只C57雄性小鼠(杭州子源实验动物科技有限公司)单次腹腔注射奥沙利铂量为10mg/kg。阴性对照或空白对照组注射5％葡萄糖溶液。造模一周左右检测小鼠机械痛阈值，每只小鼠测3次痛阈值，计算平均值，按照痛阈值随机分组。按照表6的给药方案对小鼠进行给药(总共给药1次)，并在给药后第0.5、1、2、4、6、8h检测机械痛阈值。如图20所示，在给药后0.5h、1h、2h、4h这4个时间点，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性。在6h时，母肽AJ003与模型组相比没有显著性，阳性药及改构肽AJ003-36的低中高不同剂量组仍与模型组比较有显著镇痛活性。在8h时，母肽AJ003、AJ003-36的低剂量组与模型组相比没有显著性，阳性药及改构肽的中、高两个剂量组仍与模型组比较有显著镇痛活性。
表6.奥沙利铂化疗痛给药表
实施例8：奥沙利铂化疗痛评价多种多肽体内镇痛活性
奥沙利铂用5％葡萄糖溶液溶解为1mg/mL。造模组每只C57雄性小鼠(上海必凯科翼生物科技有限公司)单次腹腔注射奥沙利铂量为10mg/kg。阴性对照或空白对照组注射5％葡萄糖溶液。造模一周左右检测小鼠机械痛阈值，每只小鼠测3次痛阈值，计算平均值，按照痛阈值随机分组。按照表7的给药方案对小鼠进行给药(总共给药1次)，并在给药后第0.5、1、2、4、6、8h检测机械痛阈值。如图30所示，在给药0.5h开始，AJ003-36给药组 与模型组相比，小鼠机械痛阈值明显降低，并具有显著性差异，而给药组AJ003-24和AJ003-25均没有降低小鼠的机械痛阈值。说明多肽AJ003-36可以有效缓解由奥沙利铂引起的小鼠化疗痛，而多肽AJ003-24和AJ003-25不能缓解该疼痛。
表7.小鼠奥沙利铂化疗痛模型实验给药表
实施例9：大鼠CCI模型评价不同剂量下候选改构肽体内镇痛活性
神经病理性疼痛种类众多，运用动物模型可模拟临床神经病理性疼痛。SD大鼠坐骨神经病理性疼痛模型主要有CCI(慢性压迫性损伤模型)、SNI(坐骨神经分支选择结扎SNI模型)、SNL(脊神经选择性结扎SNL模型)、CCD(利用不锈钢钢丝穿过椎间孔来固定压迫背根神经节)四大类。其中，CCI模型造模方法已经广泛运用于研究神经病理性疼痛，为进一步研究神经损伤修复的机制及策略提供基础(文晓娟，Medical Information.Jan.2018.Vol.31.No.1)。本实施例中使用的是CCI模型。
除对照组，其余SD大鼠用10％水合氯醛生理盐水溶液按照每100g体重0.3mL的剂量腹腔注射麻醉后，进行手术造模。用手术剪小心剪开大鼠右后肢皮肤，划开肌肉，用羊肠线4-0对坐骨神经进行结扎三道，间隔1mm，然后缝合肌肉与皮肤，可涂抹抗生素于伤口处防止动物手术伤口感染。按照文献报道，手术后7-10天动物出现明显痛阈值下降，可持续一个月左右。造模10天左右检测大鼠足底痛阈值，每只大鼠测3次痛阈值，计算平均值，按照大鼠痛阈值进行随机分组。按照表8的给药方案进行给药，选择给药后2h、4h、6h、8h四个时间点进行大鼠足底痛阈值检测。
给药第1天，如图21所示，在2h和4h阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇 痛活性。在6h时，母肽AJ003与模型组相比没有显著性，阳性药及AJ003-36的低中高三个剂量组均仍与模型组比较有显著镇痛活性。在8h时，母肽AJ003和AJ003-36的低剂量组与模型组相比没有显著性，阳性药及AJ003-36的中高两个剂量组均仍与模型组比较有显著镇痛活性。
给药第4天，如图22所示，在2h和4h，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性。在6h和8h两个时间点，母肽AJ003和AJ003-36的低剂量组与模型组相比没有显著性，阳性药及AJ003-36的中高两个剂量组均仍与模型组比较有显著镇痛活性。
给药第7天，如图23所示，在2h和4h，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性。在6h时，母肽AJ003与模型组相比没有显著性，阳性药及AJ003-36的低中高三个剂量组均仍与模型组比较有显著镇痛活性。在8h时，母肽AJ003和AJ003-36的低剂量组与模型组相比没有显著性，阳性药及AJ003-36的中高两个剂量组均仍与模型组比较有显著镇痛活性。
给药第10天，如图24所示，在2h和4h，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性。在6h时，母肽AJ003与模型组相比没有显著性，阳性药及AJ003-36的低中高三个剂量组均仍与模型组比较有显著镇痛活性。在8h时，母肽AJ003和AJ003-36的低剂量组与模型组相比没有显著性，阳性药及AJ003-36的中高两个剂量组均仍与模型组比较有显著镇痛活性。
给药第14天，如图25所示，在2h和4h，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性。在6h时，母肽AJ003与模型组相比没有显著性，阳性药及AJ003-36的低中高三个剂量组均仍与模型组比较有显著镇痛活性。在8h时，母肽AJ003和AJ003-36的低剂量组与模型组相比没有显著性，阳性药及AJ003-36的中高两个剂量组均仍与模型组比较有显著镇痛活性。
表8.大鼠CCI模型给药表

实施例10：小鼠糖尿病神经痛模型评价不同剂量下候选改构肽体内镇痛活性
除对照组，其余C57雄性小鼠(杭州子源实验动物科技有限公司)用高糖高脂饲料连续诱导十周左右，对造模组小鼠过夜断食空腹，用柠檬酸-柠檬酸钠缓冲液溶解链脲佐菌素(萨恩化学技术(上海)有限公司)，给造模组小鼠腹腔注射链脲佐菌素70mg/kg。注射链脲佐菌素约两周后，检测小鼠机械痛阈值，检测方法为将小鼠置于有孔隙铁丝网适应环境后，用平头针垂直刺激小鼠右后肢足底，利用生物信号采集系统收集痛阈值数据并记录，每只小鼠测3次痛阈值，最终计算平均值。按照小鼠痛阈值进行随机分组给药治疗。
给药第1天，按照表9的给药方案进行给药，选择给药后0h、2h、4h、6h、8h五个时间点进行小鼠足底机械痛阈值检测。如图26所示，在2h和4h，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性；在6h和8h两个时间点，母肽AJ003与模型组相比没有显著性，阳性药及AJ003-36的低中高三个剂量组均仍与模型组比较有显著镇痛活性。
给药第4天，按照表9的给药方案进行给药，选择给药后0h、2h、4h、6h、8h五个时间点进行小鼠足底机械痛阈值检测。如图27所示，在2h和4h，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性；在6h和8h两个时间点，母肽AJ003与模型组相比没有显著性，阳性药及AJ003-36的低中高三个剂量组均仍与模型组比较有显著镇痛活性。
给药第8天，按照表9的给药方案进行给药，选择给药后0h、2h、4h、6h、8h五个时间点进行小鼠足底机械痛阈值检测。如图28所示，在2h和4h，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性；在6h和8h两个时间点，母肽AJ003和AJ003-36的低剂量组与模型组相比没有显著性，阳性药及AJ003-36的中高两个剂量组仍与模型组比较有显著镇痛活性。
给药第14天，按照表9的给药方案进行给药，选择给药后0h、2h、4h、6h、8h五个时间点进行小鼠足底机械痛阈值检测。如图29所示，在2h和4h，阳性药普瑞巴林、母肽AJ003以及候选改构肽AJ003-36的低中高三个剂量组与模型组相比均有显著的镇痛活性；在6h和8h两个时间点，母肽AJ003与模型组相比没有显著性，阳性药及AJ003-36的低中高三个剂量组均仍与模型组比较有显著镇痛活性。
综合4次给药治疗的机械痛阈值检测结果，母肽AJ003在该模型中4h内跟模型组相比具有显著的镇痛活性，超过4h后镇痛活性与模型组比较没有显著性。候选改构肽AJ003-36的中高剂量在检测的2～8h与模型组比较均有显著镇痛活性；AJ003-36的低剂量组在第8天检测时与模型组相比没有显著性差异，而在其他3次检测时与模型组相比有显著镇痛活性；AJ003-36的高剂量组镇痛活性略优于阳性药普瑞巴林。该实验提示候选改构肽AJ003-36通过皮下注射可治疗由糖尿病诱发的神经痛。
表9.小鼠糖尿病神经痛模型给药表

Claims (9)

  1. 一种以从N末端到C末端的顺序包含下式I或具有保守取代的式I的肽或其衍生物，
    Gly-Ser-Cys-Ser-X1-X2-X3-X4-(D-Cys)-X5-X6-X7-X8     式I
    其中，
    式I中的第3位Cys和第9位D-Cys形成一对分子内二硫键，D-Cys代表D型半胱氨酸，氨基酸残基之间的“-”代表肽键，所述肽或其衍生物的C末端为游离羧基或酰胺，且X1为Thr或D-Thr，X5选自Ala、Val和D-Val中的任意一个，X2-X4和X6-X8彼此独立地为任意一种氨基酸。
  2. 根据权利要求1所述的肽或其衍生物，其中所述X2选自Pro、D-Pro和Hyp中的任意一个，X3选自Pro、D-Pro和Hyp中的任意一个，X4选自Ser、D-Ser、Ala和Aib中的任意一个，X6选自Ala、Leu、D-Leu、Ile、ABu和Nle中的任意一个，X7选自Tyr、D-Tyr、Ala、Trp、Phe和Bip中的任意一个，X8选自Ser、D-Ser、Ala、和Orn中的任意一个。
  3. 根据权利要求1或2所述的肽或其衍生物，其包含选自以下的任意一个序列：SEQ ID NO：1-5、SEQ ID NO：10-16和SEQ ID NO：20-34，具有保守取代的SEQ ID NO：1-5、SEQ ID NO：10-16和SEQ ID NO：20-34；优选地所述肽或其衍生物包含选自以下的任意一个序列：SEQ ID NO：26、28和32，具有保守取代的SEQ ID NO：26、28和32。
  4. 根据权利要求1-3中任一项所述的肽或其衍生物，其中所述衍生物与所述肽相比包含选自以下的任意一种或多种修饰：氨基酸修饰、保守氨基酸取代、氨基酸残基中的氢被取代。
  5. 一种药物组合物，其包含根据权利要求1-4中任一项所述的肽或其衍生物以及药学上可接受的载体或盐。
  6. 权利要求1-4中任一项所述的肽或其衍生物或权利要求5所述的药物组合物在制备用于治疗疼痛的药物中的用途。
  7. 一种用于治疗疼痛的方法，所述方法包括向有此需要的受试者施用治疗有效量的权利要求1-4中任一项所述的肽或其衍生物或权利要求5所述的药物组合物。
  8. 根据权利要求7所述的方法，其中所述肽或其衍生物通过皮下注射、肌肉注射、静脉注射、腹腔注射、鞘内注射、口服、经皮、经肺、眼部或局部给药的方式施用。
  9. 根据权利要求6所述的用途或根据权利要求7所述的方法，其中所述疼痛包括化疗引起的疼痛、糖尿病性周围神经疼痛、坐骨神经痛、骨关节疼痛、带状疱疹后遗神经痛、感染疼痛、营养缺乏疼痛、炎症疼痛、慢性酒精中毒疼痛、甲状腺功能减退疼痛、自身免疫病疼痛、格林巴利综合症疼痛、中毒疼痛、药物引起的疼痛、肿瘤病变疼痛、遗传疾病疼痛和挫伤疼痛。
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1793169A (zh) * 2005-12-28 2006-06-28 南京农业大学 海螺毒素变体多肽化合物、制备方法和用途
CN101307101A (zh) * 2008-03-26 2008-11-19 中山大学 一种新的具有翻译后修饰的信号芋螺p-超家族毒素及其用途
CN101381403A (zh) * 2008-09-17 2009-03-11 中国人民解放军军事医学科学院生物工程研究所 α型芋螺多肽衍生物及其应用
US20100120670A1 (en) * 2006-04-13 2010-05-13 The University Of Queensland Cyclised alpha-conotoxin peptides
CN106432422A (zh) * 2016-12-18 2017-02-22 唐琼瑶 一组具有镇痛作用的肽及其药物组合物和应用
CN107759665A (zh) * 2017-11-14 2018-03-06 中国药科大学 一种具有镇痛活性的多肽及其应用
CN109776655A (zh) * 2017-11-14 2019-05-21 南京安吉生物科技有限公司 一类具有镇痛活性的多肽及其应用
CN110167578A (zh) * 2016-12-23 2019-08-23 于利奇研究中心有限公司 用于治疗慢性和神经性疼痛的d-对映体肽

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1793169A (zh) * 2005-12-28 2006-06-28 南京农业大学 海螺毒素变体多肽化合物、制备方法和用途
US20100120670A1 (en) * 2006-04-13 2010-05-13 The University Of Queensland Cyclised alpha-conotoxin peptides
CN101307101A (zh) * 2008-03-26 2008-11-19 中山大学 一种新的具有翻译后修饰的信号芋螺p-超家族毒素及其用途
CN101381403A (zh) * 2008-09-17 2009-03-11 中国人民解放军军事医学科学院生物工程研究所 α型芋螺多肽衍生物及其应用
CN106432422A (zh) * 2016-12-18 2017-02-22 唐琼瑶 一组具有镇痛作用的肽及其药物组合物和应用
CN110167578A (zh) * 2016-12-23 2019-08-23 于利奇研究中心有限公司 用于治疗慢性和神经性疼痛的d-对映体肽
CN107759665A (zh) * 2017-11-14 2018-03-06 中国药科大学 一种具有镇痛活性的多肽及其应用
CN109776655A (zh) * 2017-11-14 2019-05-21 南京安吉生物科技有限公司 一类具有镇痛活性的多肽及其应用

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
CUNNINGHAMWELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
FRONT IMMUNOL, vol. 11, 4 February 2021 (2021-02-04), pages 626687
GILRON IBARON RJENSEN T: "Neuropathic pain: principles of diagnosis and treatment [J", MAYO CLIN PROC, vol. 90, no. 4, 2015, pages 532 - 545
HONE A.J. ET AL.: "Nicotinic acetylcholine receptors in neuropathic and inflammatory pain", FEBS LETT., vol. 592, no. 7, April 2018 (2018-04-01), pages 1045 - 1062
JENSEN TSBARON RHAANP M ET AL.: "A new definition of neuropathic pain [J", PAIN, vol. 152, no. 10, 2011, pages 2204 - 2205
PAIN, vol. 155, 2014, pages 1986 - 1995
SMITH ET AL., J. MOL. BIOL., vol. 147, 1981, pages 195 - 197
STAHLWERMUTH: "Handbook of Pharmaceutical Salts: Properties, Selection and Use", 2002, WILEY-VCH
SZOK DTAJTI JNY ÁRI A ET AL.: "Therapeutic approaches for peripheral and central neuropathic pain [J", BEHAV NEUROL, vol. 2019, 2019, pages 8685954
WEN XIAOJUN, MEDICAL INFORMATION, vol. 31, no. 1, January 2018 (2018-01-01)
WU PEI ET AL.: "Role of α7 nicotinic acetylcholine receptor in regulating inflammatory responses in animals", ACTA ZOONUTRIMENTA SINICA, vol. 33, no. 11, 2021, pages 6001 - 6008

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