WO2023061487A1 - 一种抑制trpm8的多肽及其用途 - Google Patents

一种抑制trpm8的多肽及其用途 Download PDF

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WO2023061487A1
WO2023061487A1 PCT/CN2022/125397 CN2022125397W WO2023061487A1 WO 2023061487 A1 WO2023061487 A1 WO 2023061487A1 CN 2022125397 W CN2022125397 W CN 2022125397W WO 2023061487 A1 WO2023061487 A1 WO 2023061487A1
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seq
polypeptide
trpm8
pain
dec
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French (fr)
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杨帆
阿尔孜古丽艾尔肯
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浙江大学
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of medicine, in particular to a polypeptide for inhibiting TRPM8 and its use.
  • Peripheral neuropathy is a common side effect of many platinum-based chemotherapy, which limits the dose of anticancer drugs used and reduces the quality of life of patients taking these drugs.
  • Oxaliplatin is commonly used as first-line chemotherapy in the treatment of many tumors, such as colorectal and gastric cancer] and others.
  • oxaliplatin developed acute neurotoxicity, usually manifested by extreme cold and excruciating pain in the arms and legs, in place of normally cool ambient temperatures.
  • these symptoms of neuropathy in patients, including oxaliplatin-induced cold hyperalgesia and other symptoms will improve after stopping the drug, so far, there is still no effective treatment for such neuropathy.
  • TRPM8 is a class of non-selective ion channels expressed in nociceptor neurons. This channel is activated at temperatures below 28°C, so it is a cold receptor in mammals. In addition, as a multimodal receptor, TRPM8 can also be activated by a variety of physical stimuli or chemical ligands, such as menthol and Icilin, and transmembrane depolarization. In an oxaliplatin-induced mouse model of cold allodynia, expression levels of TRPM8 channels were significantly increased in nociceptive dorsal root ganglion (DRG) neurons. More importantly, administration of oxaliplatin did not induce cold allodynia in TRPM8 knockout mice. Therefore, the TRPM8 ion channel is an effective drug target for peripheral neuropathy and platinum-based drug-induced hyperalgesia, and it is urgent to develop effective drugs targeting TRPM8 ion channels for the treatment of peripheral neuropathy and hyperalgesia.
  • DRG nociceptive dorsal root
  • the object of the present invention is to provide a TRPM8 inhibitor and its polypeptide for preventing and treating diseases related to TRPM8.
  • a polypeptide or a pharmaceutically acceptable salt thereof is provided, and the polypeptide or a pharmaceutically acceptable salt thereof has a structure represented by formula I:
  • X1 is none, any amino acid or any peptide
  • X2 is R, K, Q, A, D or N;
  • X3 is R, K, Q, A, H or N;
  • X4 is D, A, S, R or E;
  • X5 is R, K, Q, A or N;
  • X6 is A, V, L, G, R or I;
  • X7 is R, K, Q, A or N;
  • X8 is H, N, Q, K, A or R;
  • X9 is Y, W, F, D, T, A, R or S;
  • X10 is R, K, Q, A, S, Y or N;
  • X11 is Q, A, R or N;
  • X12 is R, K, Q, A, K or N;
  • X13 is none, any amino acid or any peptide.
  • X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12 and X13 are each independently L-amino acid or D-amino acid.
  • X2 is R, K, Q, D or N.
  • X6 is A, V, L, R or I.
  • X12 is R, K, Q, K or N.
  • the polypeptide or a pharmaceutically acceptable salt thereof can (a) inhibit TRPM8; (b) prevent and/or treat diseases related to TRPM8; (c) prevent and/or treat cold pain sensation hypersensitivity; (d) prevention and/or treatment of peripheral neuropathy; (e) prevention and/or treatment of pain; and/or (f) prevention and/or treatment of chronic compression injuries for neuropathic pain.
  • polypeptide is an isolated polypeptide.
  • polypeptide is artificially synthesized.
  • polypeptide is a recombinant polypeptide.
  • X1 is none, C (cysteine) or M (methionine).
  • X13 is none or C (cysteine).
  • the peptide segment includes a tag protein, a leader sequence or a secretory sequence.
  • the length of X1 is 1-20aa, more preferably 1-10aa, more preferably 1-5aa.
  • the length of X13 is 1-20aa, more preferably 1-10aa, more preferably 1-5aa.
  • the length of the polypeptide or a pharmaceutically acceptable salt thereof is ⁇ 25aa, preferably ⁇ 20aa, more preferably ⁇ 18aa, more preferably ⁇ 15aa; more preferably, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, or 20aa.
  • said X1 or X13 includes natural or unnatural amino acids.
  • a cyclic peptide is formed between X1 and X13.
  • At least one pair of disulfide bonds is formed between X1 and X13.
  • a pair of disulfide bonds is formed between X1 and X13.
  • polypeptide has the structure shown in formula II:
  • polypeptide is an N-mer.
  • N-polymer has the following structure of formula III:
  • X1 and X13 are as above; L1 is none or a connecting peptide; n is 2-10, preferably, 2-7, more preferably, 2-5; each "-" is independently a connecting peptide or peptide bond.
  • the L1 is none.
  • the length of L1 is 1-30aa, preferably 1-20aa, more preferably 1-10aa.
  • n 2, 3 or 4.
  • each "-" is independently a peptide bond.
  • sequence of the polypeptide is such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:6, SEQ ID NO: ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO :15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.
  • SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19 it has ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80% %, ⁇ 90%, ⁇ 95%, ⁇ 99% or 100% identity (or homology).
  • the polypeptide represented by the formula I retains SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO :6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 , at least ⁇ 50%, ⁇ 60%, ⁇ 70% of the biological activity of the polypeptide represented by SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, ⁇ 80%, ⁇ 90%, ⁇ 95%, or ⁇ 99%, eg 90-100%.
  • sequence of the polypeptide is shown in SEQ ID NO.:1.
  • the polypeptide represented by the formula I has ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95%, >99% or 100% identity (or homology).
  • the polypeptide represented by the formula I retains at least ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90% of the biological activity of the polypeptide represented by SEQ ID NO: 1, ⁇ 95%, or ⁇ 99%, eg 90-100%.
  • polypeptide is selected from the following group:
  • SEQ ID NO:1 SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8.
  • SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, A polypeptide of the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19;
  • SEQ ID NO:1 SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8.
  • SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,
  • the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19 undergoes 1-5 (preferably 1-3, more preferably 1-2) amino acid residue substitutions, deletions or (a) inhibit TRPM8; (b) prevent and/or treat diseases related to TRPM8; (c) prevent and/or treat cold hyperalgesia; (d) prevent and/or treat peripheral nerve (e) preventing and/or treating pain; and/or (f) preventing and/or treating chronic compression injuries for neuropathic pain;
  • SEQ ID NO:1 SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8.
  • SEQ ID NO:9 SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, Compared with the polypeptide shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, it has ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 99% % or 100% identical (or homologous) polypeptides.
  • polypeptide is not SEQ ID NO:3.
  • polypeptide is selected from the following group:
  • SEQ ID NO:1 SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11.
  • SEQ ID NO:1 SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11.
  • SEQ ID NO: 12 SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, it has Polypeptides with ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 95%, ⁇ 99% or 100% identity (or homology).
  • polypeptide is selected from the following group:
  • the amino acid sequence shown in SEQ ID NO: 1 is formed by substitution, deletion or addition of 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and has ( a) inhibit TRPM8; (b) prevent and/or treat diseases associated with TRPM8; (c) prevent and/or treat cold allodynia; (d) prevent and/or treat peripheral neuropathy; (e) prevent and/or treat or treating pain; and/or (f) preventing and/or treating chronic compression injuries for neuropathic pain;
  • the polypeptide is composed of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:6, SEQ ID NO: ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO :15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19 through 1-3 preferably 1-2, more preferably 1 amino acid substitution , missing; and/or
  • the derivatized polypeptide retains ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 99%, or 100%, such as 80-100%.
  • the derived polypeptide is related to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
  • polypeptide is the polypeptide shown in SEQ ID NO.: 1 through 1-3, preferably 1-2, more preferably 1 amino acid substitution, deletion; and/or
  • the derivatized polypeptide retains ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 99%, or 100%, such as 80-100%.
  • Preferably 95-100% of the polypeptide shown in SEQ ID NO: 1 (a) inhibits TRPM8; (b) prevents and/or treats diseases related to TRPM8; (c) prevents and/or treats cold hyperalgesia; and /or (d) preventing and/or treating peripheral neuropathy.
  • the identity of the derived polypeptide to SEQ ID NO: 1 is ⁇ 50%, preferably ⁇ 60%, more preferably ⁇ 70%, more preferably ⁇ 80%, More preferably ⁇ 90%, more preferably ⁇ 99%.
  • the present invention provides a fusion protein, said fusion protein comprising:
  • the peptide segment includes a carrier protein.
  • the carrier protein is selected from the group consisting of Fc fragment, human serum albumin (HSA), CTP, transferrin, or a combination thereof.
  • the peptide is modified.
  • the modification includes polyethylene glycol (PEG) modification.
  • PEG polyethylene glycol
  • a polynucleotide encoding the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.
  • the polynucleotide is an isolated polynucleotide.
  • the fourth aspect of the present invention provides a vector, which includes the polynucleotide according to the third aspect of the present invention.
  • the vector includes a plasmid vector.
  • the fifth aspect of the present invention provides a host cell, the host cell includes the vector according to the fourth aspect of the present invention or the polynucleotide according to the third aspect of the present invention is integrated on the chromosome of the host cell .
  • a sixth aspect of the present invention provides a composition, said composition comprising:
  • the composition is a pharmaceutical composition.
  • the composition is administered by a method selected from the following group: intravenous, intratumoral, intracavitary, subcutaneous or hepatic artery administration (such as injection, instillation, etc.).
  • the dosage form of the composition is an oral preparation, an injection preparation or an external preparation.
  • the dosage form of the composition is solid preparation, liquid preparation or semi-solid preparation.
  • the formulation of the composition is selected from the group consisting of tablets, capsules, injections, granules, sprays, and freeze-dried formulations.
  • the dosage form of the composition is injection.
  • the injection is intravenous injection, intramuscular injection or subcutaneous injection.
  • the polypeptide is administered to mammals at a dose of 0.01-100 mg/kg body weight (every time or every day).
  • the seventh aspect of the present invention provides a polypeptide as described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof, a fusion protein as described in the second aspect of the present invention, a multinuclear protein as described in the third aspect of the present invention Nucleic acid, the carrier as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, and/or the purposes of the composition as described in the sixth aspect of the present invention, for preparing the composition, the The composition is used for one or more purposes selected from the group: (a) inhibiting TRPM8; (b) preventing and/or treating diseases related to TRPM8; (c) preventing and/or treating cold hyperalgesia; (d) preventing and/or treating peripheral neuropathy; (e) preventing and/or treating pain; and/or (f) preventing and/or treating chronic compression injuries for neuropathic pain.
  • the composition is a pharmaceutical composition.
  • the TRPM8-related disease is selected from the group consisting of peripheral neuropathy, cold hyperalgesia, pruritus, chronic compression injury of neuropathic pain, or a combination thereof.
  • the pain is selected from the group consisting of chronic pain, cold hypersensitivity pain, neuropathic pain of diabetic neuropathy, postoperative pain, osteoarthritis pain, rheumatoid arthritis pain, Cancer pain, neuralgia, nerve injury pain, migraine, cluster headache, tension headache, fibromyalgia, neuropathic pain, static allodynia, cold allodynia, or combinations thereof.
  • the cold allodynia includes cold allodynia caused by chronic compression injury of neuropathic pain.
  • the static allodynia includes static allodynia caused by chronic compression injury of neuropathic pain.
  • the diseases related to TRPM8 include diseases in which TRPM8 is up-regulated.
  • the up-regulation of TRPM8 includes a high expression level or activity of TRPM8.
  • the up-regulation of TRPM8 refers to that the TRPM8 expression level or activity of a certain cell (such as a stimulus-receptive neuron or dorsal root ganglion) is greater than the TRPM8 expression level or activity of the same cell.
  • the TRPM8 up-regulation refers to the ratio of the TRPM8 expression level or activity C1 of a certain cell (such as a stimulus sensory neuron or dorsal root ganglion) to the TRPM8 expression level or activity C0 of the same cell (C1/ C0)>1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5.
  • the cells include nerve cells.
  • the stimuli receptor neurons include nociceptive stimuli receptor neurons.
  • the dorsal root ganglion includes nociceptive dorsal root ganglion.
  • the same cell refers to a cell of the same type with normal expression or activity of TRPM8.
  • the expression includes mRNA and/or protein expression.
  • the cold hyperalgesia includes cold hyperalgesia induced by platinum-based anticancer drugs.
  • the peripheral neuropathy includes peripheral neuropathy caused by platinum-based anticancer drugs.
  • the cold hyperalgesia includes cold hyperalgesia induced by platinum-based anticancer drugs.
  • the platinum-based anticancer drug is selected from the group consisting of carboplatin, nedaplatin, lobaplatin, oxaliplatin, or a combination thereof.
  • the eighth aspect of the present invention provides a method for (a) inhibiting TRPM8; (b) preventing and/or treating diseases related to TRPM8; (c) preventing and/or treating cold hyperalgesia; (d) preventing and/or treating Peripheral neuropathy; (e) prevention and/or treatment of pain; and/or (f) method of prevention and/or treatment of chronic compression injury of neuropathic pain, said method comprising the step of: administering to a subject in need
  • the polypeptide as described in the first aspect or its pharmaceutically acceptable salt, the fusion protein as described in the second aspect of the present invention, the polynucleotide as described in the third aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention A vector, a host cell as described in the fifth aspect of the present invention, and/or a composition as described in the sixth aspect of the present invention.
  • the subject is a human or a non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, rabbits), and primates (such as monkeys).
  • the method is non-diagnostic and non-therapeutic.
  • the administration is oral administration, injection administration or external administration.
  • the injection is intravenous injection, intramuscular injection or subcutaneous injection.
  • Figure 1 shows the HPLC purification, mass spectrometry verification and electrophysiological verification of the function of the designed peptide after synthesis.
  • Figure 2 is the specific inhibition of the activation mode of TRPM8 by DeC-1.2.
  • Figure 3 is the subunit selectivity of DeC-1.2.
  • (a, b, c, d, e) Representative electrophysiological recordings of DeC-1.2 inhibition of ligand activation of TRPV1, TRPV2, TRPV3, TRPM2, and TRPM4.
  • the TRPM4-K1045A mutation was used in order to rule out the dependence of TRPM4 on PIP2.
  • Figure 4 shows the amino acid residues that play a key role in the inhibition of TRPM8 by DeC-1.2.
  • Figure 5 is to verify the inhibitory effect of DeC-1.2 on TRPM8 in vivo.
  • Figure 6 shows the effect of DeC-1.2 on inhibiting oxaliplatin-induced cold hypersensitivity in vivo.
  • the inventors After extensive and in-depth research, the inventors have prepared for the first time (a) inhibiting TRPM8; (b) preventing and/or treating diseases related to TRPM8; (c) preventing and/or treating cold allodynia; (d) Prevention and/or treatment of peripheral neuropathy; (e) prevention and/or treatment of pain; and/or (f) polypeptides such as DeC-1.2 polypeptides for the prevention and/or treatment of chronic compression injuries of neuropathic pain, and the polypeptides of the present invention It is safe and has little toxic and side effects on biological tissues. On this basis, the present inventors have completed the present invention.
  • the terms “comprising”, “comprising” and “containing” are used interchangeably to include not only open definitions, but also semi-closed, and closed definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • prevention means a method of preventing the onset of a disease and/or its attendant symptoms or protecting a subject from acquiring a disease.
  • Treatment in the present invention includes delaying and terminating the progression of the disease, or eliminating the disease, and does not require 100% inhibition, eradication and reversal.
  • a polypeptide of the invention reduces, inhibits and/or reverses a TRPM8-associated disease, e.g., by at least about 10%, at least about 30%, compared to the level observed in the absence of the polypeptide of the invention. %, at least about 50%, or at least about 80%, or 100%.
  • TRPM8 Transient receptor potential melastatin 8
  • TRP transient receptor potential ion channel protein TRP family.
  • the channel is composed of 4 identical subunits, each subunit has 6 transmembrane regions, and the N-terminal and C-terminal are located inside the cell.
  • the pore region located in the center of the TRPM8 channel is composed of four subunits, which can permeate cations non-selectively.
  • TRPM8 channels are involved in the regulation of body cold sensation, pain sensation, inflammatory response, vasoconstriction and expansion, and cell growth and proliferation.
  • polypeptide of the present invention refers to a polypeptide having the structure shown in formula I or a pharmaceutically acceptable salt thereof. It should be understood that the term also includes mixtures of the aforementioned components.
  • polypeptide of the present invention also includes variant forms of the polypeptide having the structure shown in formula I. These variant forms include (but are not limited to): adding one or several (usually within 5, preferably within 3, more preferably within 2) amino acids at the N-terminus. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. Adding one or several amino acids at the N-terminus usually does not change the structure and function of the protein. Furthermore, the term also includes monomeric and multimeric forms of the polypeptides of the invention or pharmaceutically acceptable salts thereof.
  • amino acid sequence of a polypeptide is numbered from N-terminus to C-terminus.
  • the invention also includes active fragments, derivatives and analogs of the polypeptides of the invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) A polypeptide having a substituent in each amino acid residue, or (iii) a polypeptide of the invention fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid
  • the polypeptide sequence is fused to this polypeptide sequence (subsequent protein formed by fusing with leader sequence, secretory sequence or 6His and other tag sequences).
  • Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
  • One class of preferred active derivatives refers to that compared with the amino acid sequence of the polypeptide shown in formula I, there are at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid is similar or similar in nature Amino acids are substituted to form polypeptides. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 2.
  • the invention also provides analogs of the polypeptides of the invention.
  • the difference between these analogs and the natural polypeptide of the present invention may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, ⁇ , ⁇ -amino acids).
  • Cys can form a disulfide bond with non-native Hcy. It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
  • Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • glycosylation such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme
  • the polypeptide of the invention has at least one internal disulfide bond (introduced intrachain disulfide bond).
  • the presence of the internal disulfide bond not only does not affect its activity, but also helps to prolong the half-life and improve the inhibitory activity.
  • conventional methods in the art can be used to form, for example, combining cysteine or homocysteine sulfhydryl groups under oxidative conditions to form a disulfide bond.
  • a preferred polypeptide of the present invention includes polypeptide DeC-1.2, and the amino acid sequence of polypeptide DeC-1.2 is the amino acid sequence described in SEQ ID No.: 1:
  • polypeptides of the present invention also include polypeptides modified from the polypeptide shown in SEQ ID NO.:1.
  • the polypeptides of the present invention can also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases.
  • These salts include, but are not limited to, those formed with the following acids: hydrochloric, hydrobromic, sulfuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as in the form of esters, carbamates or other conventional "prodrugs".
  • the present invention also relates to polynucleotides encoding the polypeptides of the present invention.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • DNA can be either the coding strand or the non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence or a degenerate variant.
  • the full-length nucleotide sequence of the polypeptide of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • the DNA sequence encoding the polypeptide (or its fragment, or its derivative) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present invention also relates to vectors comprising the polynucleotides of the present invention, and host cells produced by genetic engineering using the vectors of the present invention or the polypeptide coding sequences of the present invention.
  • the invention also includes polyclonal and monoclonal antibodies or antibody fragments, especially monoclonal antibodies, specific for the polypeptides of the invention.
  • nucleic acids or polypeptides when comparing and aligning sequences for maximum identity, the term “substantially identical” refers to two or more sequences or subsequences that have at least about 80%, such as at least about 85%. %, about 90%, about 95%, about 98%, or about 99% of the nucleotide or amino acid residues are identical to a particular reference sequence, as determined using the following sequence comparison methods and/or by visual inspection.
  • polypeptides of the present invention may be recombinant polypeptides or synthetic polypeptides.
  • the polypeptides of the invention may be chemically synthesized, or recombinant.
  • the polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.
  • a preferred method is to use liquid-phase synthesis technology or solid-phase synthesis technology, such as Boc solid-phase method, Fmoc solid-phase method or a combination of the two methods.
  • Solid-phase synthesis can quickly obtain samples, and the appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide.
  • the preferred solid phase carrier in the Fmoc system is Wang resin connected with the C-terminal amino acid in the peptide, the Wang resin structure is polystyrene, and the arm between the amino acid is 4-alkoxybenzyl alcohol; use 25% hexahydropyridine /dimethylformamide at room temperature for 20 minutes to remove the Fmoc protecting group, and extend from the C-terminal to the N-terminal one by one according to the given amino acid sequence.
  • the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-cresol and the protective group is removed, and the resin can be filtered off and separated by ether precipitation to obtain the crude peptide. After lyophilization of the resulting product solution, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography.
  • the preferred resin is a PAM resin connected to the C-terminal amino acid in the peptide.
  • the structure of the PAM resin is polystyrene, and the arm between the amino acid is 4-hydroxymethylphenylacetamide; synthesized in Boc
  • Boc is removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane.
  • DCM TFA/dichloromethane
  • DIEA diisopropylethylamine
  • Peptide chain condensation is completed Afterwards, use hydrogen fluoride (HF) containing p-cresol (5-10%), treat at 0°C for 1 hour, cut the peptide chain from the resin, and remove the protecting group at the same time.
  • HF hydrogen fluoride
  • acetic acid containing A small amount of mercaptoethanol
  • the solution is lyophilized and further separated and purified with molecular sieve Sephadex G10 or Tsk-40f, and then purified by high-pressure liquid phase to obtain the desired peptide.
  • Various couplings known in the field of peptide chemistry can be used Reagents and coupling methods can be used to couple amino acid residues, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetrauronium hexafluorophosphate can be used (HBTU) for direct coupling.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • HBTU 1,1,3,3-tetrauronium hexafluorophosphate
  • the polypeptide of the present invention is prepared by solid-phase synthesis according to its sequence, purified by high-performance liquid chromatography to obtain a high-purity lyophilized powder of the target peptide, and stored at -20°C.
  • Another method is to use recombinant techniques to produce the polypeptides of the invention.
  • recombinant techniques By conventional recombinant DNA techniques, the polynucleotides of the invention can be used to express or produce recombinant polypeptides of the invention.
  • Recombinant polypeptides can be expressed intracellularly or on the cell membrane or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • polypeptide of the present invention is relatively short, multiple polypeptides can be concatenated together to obtain a multimeric expression product after recombinant expression, and then the desired small peptide can be formed by enzymatic digestion and other methods.
  • the present invention also provides a composition, and the composition is preferably a pharmaceutical composition.
  • composition of the present invention contains (a) the polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient.
  • the amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof is usually 10 ⁇ g-100 mg/dose, preferably 100-1000 ⁇ g/dose.
  • the polypeptide of the present invention or a pharmaceutically acceptable salt thereof is in a safe and effective amount, and the effective dosage is about 0.01 mg/kg to 50 mg for the individual /kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof.
  • the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be used alone or together with other therapeutic agents (eg formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids, such as water, saline, glycerol and ethanol.
  • liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances in these carriers such as wetting agents or emulsifying agents, pH buffering substances and the like.
  • therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.
  • compositions of the present invention may be administered by conventional routes including, but not limited to, intramuscular, intravenous, subcutaneous, intradermal, or topical administration.
  • the subject to be prevented or treated can be an animal; especially a human.
  • the dosage form of the pharmaceutical composition is oral preparation, injection preparation or external preparation.
  • the dosage form of the pharmaceutical composition is solid preparation, liquid preparation or semi-solid preparation.
  • the formulation of the pharmaceutical composition is selected from the group consisting of tablets, capsules, injections, granules, sprays, and freeze-dried formulations.
  • the injection is intravenous injection, intramuscular injection or subcutaneous injection.
  • composition of the present invention When used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the usage conditions. Preferably it is an intravenous preparation or an intratumoral injection.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.
  • the preparation of eye drops can be carried out as follows: the polypeptide of the present invention or a pharmaceutically acceptable salt thereof is dissolved in sterile water (surfactant is dissolved in sterile water) together with basic substances, and the osmotic pressure is adjusted. and pH to physiological state, and optionally add suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and viscosifiers, and then completely dissolve.
  • suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and viscosifiers
  • compositions of the present invention can also be administered in the form of sustained release formulations.
  • the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be incorporated into a pill or microcapsule supported by a slow-release polymer, and then the pill or microcapsule is surgically implanted into the tissue to be treated.
  • sustained-release polymers include ethylene-vinyl acetate copolymers, polyhydroxymethacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the pharmaceutical formulation should match the mode of administration. Agents of the invention may also be used together (including before, during or after) other co-therapeutic agents.
  • a safe and effective amount of the drug is administered to a desired subject (such as a human or a non-human mammal), usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than About 8 mg/kg body weight, preferably the dose is about 10 microgram/kg body weight to about 1 mg/kg body weight.
  • a desired subject such as a human or a non-human mammal
  • the dose is about 10 microgram/kg body weight to about 1 mg/kg body weight.
  • factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the polypeptide of the present invention can effectively inhibit (a) inhibit TRPM8; (b) prevent and/or treat diseases related to TRPM8; (c) prevent and/or treat cold hypersensitivity; (d) prevent and/or treat Peripheral neuropathy; (e) prophylaxis and/or treatment of pain; and/or (f) prophylaxis and/or treatment of chronic compression injuries for neuropathic pain.
  • polypeptide of the present invention and its derived polypeptides have small molecular weight, low toxic and side effects on biological tissues, and high safety.
  • Peptides with or without disulfide bonds were chemically synthesized at Jill Biochemical (Shanghai).
  • the crude product was further purified by reverse-phase liquid chromatography.
  • the purification efficiency of the polypeptide reaches 95% or above, it can be identified and confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) or electrospray ionization mass spectrometry (ESI-MS). Afterwards, a large number of corresponding cyclic peptides were collected after purification and mass spectrometry identification for future use.
  • MALDI-TOF matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • ESI-MS electrospray ionization mass spectrometry
  • HEK293T cells were cultured in Dulbecco's modified Eagle medium at 37°C in a 5% carbon dioxide environment, under which 10% fetal bovine serum, penicillin (100u/ml) and streptomycin (100mg /ml). Transient transfection was carried out through the Lipo 2000 (Invitrogen) transfection kit according to the operating procedure in the manual.
  • the membrane potential is raised from -80mV to +10Mv.
  • a gravity-driven rapid perfusion system (RSC-200, BioLogic) was used to rapidly perfuse specific solutions onto the cells to be tested. Different solutions are delivered through separate PVC pipes to avoid cross-contamination between solutions. For electrophysiological recordings, the glass microelectrode with the membrane will be moved just below the outlet of the perfusion tube.
  • mice (6-8 weeks old) were used as experimental animals for behavioral experiments and primary culture of DRG neurons. The mice were reared under the condition of 12 hours of light/12 hours of darkness in the photoperiod, and were supplied with sufficient standard feed and water. All animal experiment protocols were approved by the Animal Care and Use Committee (IACUC) of Zhejiang University.
  • IACUC Animal Care and Use Committee
  • mice were adapted to the behavioral test environment for 1-2 hours every day.
  • a single intraperitoneal injection of oxaliplatin (6 mg/kg body weight) was used to induce the neuropathic pain model induced by chemotherapy. Behavioral tests for neuropathic pain were performed on day 10 after oxaliplatin injection.
  • mice Each of the control group and the experimental group had at least 6 adult male CD1 mice, which were injected with normal saline and gradient doses of DeC-1.2 through the tail vein respectively.
  • Icilin 2.5 ⁇ g/g body weight
  • the mice were placed on the test frame, and the video recorder recorded the wet dog-like shivering behavior of the mice within 30 min.
  • a digital display thermometer (FT3400) was used to detect the body temperature of mice.
  • body temperature testing adult CD1 male mice were placed in a constant temperature (22 ⁇ 1° C.) environment.
  • Mice were injected with DeC-1.2 (30 mg/kg body weight) through the tail vein to evaluate the effect of DeC-1.2 on the body temperature of mice.
  • Body temperature was measured before injection, 5min, 15min, 0.5h, 1h, 2h, 6h and 24h after injection. Insert the test probe of the thermometer into the anus of the mouse about 2cm, and record it as the mouse body temperature after the temperature shows a stable value.
  • the cold sensation test induced by acetone volatilization was used to evaluate oxaliplatin-induced cold hyperalgesia. Connect a plastic hose to the head of the 10ml syringe, spray 50 ⁇ l of acetone through the plastic hose to the sole of the hind paw of the mouse, and record the total time for the mouse to raise the foot, shake the foot, and lick the foot within 5 minutes.
  • the cold allodynia behavior of mice was measured before and 10 days after oxaliplatin injection. The mice were injected with normal saline or 3.5 ⁇ g/20 ⁇ L DeC-1.2 to evaluate the effect of DeC-1.2 on the cold induced by oxaliplatin. Effects of pain-sensitive behavior.
  • mice The mechanical allodynia behavior of mice was evaluated by the change of mouse paw withdrawal threshold in VonFrey test.
  • the mouse paw withdrawal threshold was performed according to the Dixon's up-down method.
  • the middle part of the rear plantar of mice was stimulated with vonFrey filaments (0.02-2g). Mice were stimulated to show behaviors such as lifting their feet, shaking their feet, and licking their feet within 3 seconds, which was considered to be responsive.
  • the Dixon’s up-down method first stimulate with 0.16g vonFrey filaments, if the mice respond, then stimulate with adjacent low-intensity vonFrey filaments, and if there is no response, then stimulate with adjacent high-intensity vonFrey filaments.
  • mice Different intensities of vonFrey silk stimulation were selected in turn, and the interval between each two stimulations was 5s, and a total of 6 stimulations were completed, and the mouse's paw withdrawal threshold (Pawwithdrawalthreshold, PWT) was evaluated against the response scale.
  • the paw withdrawal threshold of mice was measured before and 10 days after oxaliplatin injection, and normal saline or 3.5 ⁇ g/20 ⁇ L DeC-1.2 were injected into the soles of mice to evaluate the triggering effect of DeC-1.2 on oxaliplatin. The effect of pain behavior.
  • mice were deeply anesthetized the blood was discarded by decapitation, and the DRG tissue was quickly separated and placed in ice-cold PBS solution.
  • digestive enzymes Collagenase A 20mg/100ml, Dispase II 300mg/100ml dissolved in PBS
  • digest at 37°C for 1h Centrifuge at 500g for 5min to collect the digested tissue.
  • DMEM medium containing 10% FBS, 1X Pen/Strep
  • Neurobasal medium containing 2% B27, 1mM L-Glutamine, 50ng/ml NGF2.5S, 1X Pen/Strep
  • resuspend and pipette into a single cell suspension The single cell suspension was seeded on poly-D-Lys pre-coated cell slides. After the cells adhered to the wall, an appropriate amount of medium was added to the culture dish, and cultured in a 37°C, 5% CO2 incubator for 24 hours before being used for calcium imaging experiments.
  • ECS extracellular solution
  • CaCl2 calcium dye incubation solution
  • the cell image is continuously and alternately excited by a high-speed continuous monochromatic light source at the excitation light wavelength of 340/380nm, and is recorded by the visiview software under the 20X objective lens, and the intracellular fluorescence intensity is continuously recorded by the high-speed scanning camera Flash4.0LT at a speed of 0.5fps Variety.
  • the intensity of the calcium signal was expressed as the ratio of the fluorescence signal at 340nm/380nm.
  • Menthol, DeC-1.2, Capsaicin, etc. were administered through the rapid exchange perfusion system (ALA-VM8; ALA Scientific Instruments) for 30s, 90s, and 30s, respectively.
  • the effect of DeC-1.2 on TRPM8 channel activity was evaluated by comparing the intensity of calcium signal response induced by menthol and the proportion of positive responding cells in DRG neurons from different groups.
  • the entire amino acid sequence is RRDRARHYRQR.
  • a cysteine residue at its N- and C-terminal respectively to form a disulfide bond.
  • the amino acid sequence of the polypeptide DeC-1.2 is CRRDRARHYRQRC (SEQ ID NO: 1), polypeptide DeC-1.2 cyclized.
  • the amino acid sequence of the polypeptide DeC-1.1 is CRNSRAAHDSQKC (SEQ ID NO: 2), and the N- and C-terminal cysteine residues of the polypeptide DeC-1.1 form a disulfide bond, which makes the polypeptide DeC-1.1 cyclized. Meanwhile, a negative control polypeptide S-DeC-1.2 (SEQ ID NO: 3) was designed.
  • amino acids in polypeptide DeC-1.2 and polypeptide DeC-1.1 are all L-amino acids.
  • the polypeptide DeC-1.1 was chemically synthesized, purified by HPLC ( Figure 1a), identified and confirmed by mass spectrometry ( Figure 1b), and the half maximal inhibitory concentration (IC 50 ) of DeC-1.1 was 132.8 ⁇ 73.5 ⁇ M by electrophysiological test.
  • Peptide DeC-1.2 was also chemically synthesized, purified by HPLC (Fig. 1c), and identified and confirmed by mass spectrometry (Fig. 1d). After electrophysiological tests, the polypeptide DeC-1.2 can inhibit the Menthol activation of TRPM8 at nanomolar levels ( Figure 1e), showing a lower half inhibitory concentration (IC50), and the half inhibitory concentration (IC50) of the polypeptide DeC-1.2 inhibiting TRPM8 is 4.5 ⁇ 3.0nM (Fig. 1b). The negative control S-DeC-1.2 has no inhibitory effect on TRPM8.
  • the subunit selectivity of DeC-1.2 was detected (as shown in Figure 3 and Table 3).
  • the TRPM8 ion channel belongs to the TRP channel superfamily, and the member channels of this family all have similar six transmembrane domain monomers.
  • DeC-1.2 inhibited TRPM8 channel with an IC50 of 4.5 nM, however, a hundred-fold higher concentration of DeC-1.2 (5 ⁇ M) failed to inhibit ligand activation of TRPV1 or TRPV3 (Fig. 3a, Fig. 3b and 3c).
  • IC 50 half inhibitory concentration
  • TRPM2 is the closest homologue of TRPM8, but up to 100 ⁇ M DeC-1.2 failed to inhibit ligand activation of TRPM2 (Fig. 3d).
  • DeC-1.2 the structure of the closed state of TRPM4 was used as a homology modeling template for TRPM8, so DeC-1.2 may exhibit a higher affinity for TRPM4.
  • TRPM4 showed lower sensitivity to DeC-1.2 when actually tested, and 50 ⁇ M DeC-1.2 only inhibited about half of the current of TRPM4 (Fig. 3e).
  • DeC-1.2 is both a subunit-specific and an activation mode-specific inhibitor of TRPM8 ligand activation.
  • the DeC-1.2 polypeptide was mutated (alanine scanning), as shown in Table 4 and Table 5. These point mutant peptides are also chemically synthesized and purified for quality control in the same way as wild-type cyclic peptides.
  • the amino acid residues that play a key role in the inhibition of TRPM8 by DeC-1.2 are shown in Figure 4.
  • Oxaliplatin induces increased TRPM8 activity in nociceptive DRG neurons and thus sensitizes DRG neurons.
  • the oxaliplatin-induced cold allodynia mouse model was used to detect whether DeC-1.2 could affect the oxaliplatin-induced cold allodynia behavior (Fig. 5b).
  • the effects of polypeptide DeC-1.2 on oxaliplatin-induced cold allodynia and TRPM8 activity in primary sensory neurons were detected by behavioral tests and calcium imaging experiments.
  • the dorsal root ganglion was isolated from oxaliplatin-induced cold hyperalgesia model mice and normal mice for primary culture of neurons (Figure 5c, indicated by white arrows).
  • Calcium imaging experiments of primary cultured DRG neurons showed that in the control group (normal saline injection group), menthol (Menthol) could activate TRPM8 to induce calcium activity in neurons; compared with the control group, the oxaliplatin-treated group was smaller In the mouse DRG neurons, the calcium response intensity and the proportion of positive response cells caused by activation of TRPM8 by Menthol were significantly increased; while DeC-1.2 could significantly inhibit the calcium response intensity and the positive response cell ratio of the oxaliplatin group mice DRG neurons. ratio ( Figure 5d and 5e). These results indicated that DeC-1.2 polypeptide could inhibit the activity of TRPM8 channel in the oxaliplatin-induced cold allodynia model.
  • DeC-1.2 exhibited in vivo anti-cold hypersensitivity to a similar extent as knockout TRPM8 mice.
  • the mouse's foot lifting threshold in the von Frey test was significantly reduced, and the mice were injected with normal saline and DeC-1.2 to lift their feet. Neither threshold had a significant effect (Fig. 6b).
  • Fig. 5a intravenous injection of a high dose of 30 ⁇ g/g of DeC-1.2 peptide sufficient to induce a WDS response in mice (Fig. 5a) did not significantly alter the basal body temperature of the mice (Fig. 6c).
  • Fig. 6d The schematic diagram of the working principle of the activation mode-specific inhibitor DeC-1.2 on the TRPM8 channel is shown in Fig. 6d. Therefore, it can be seen that the cyclic peptide DeC-1.2 can improve the cold-sensitivity behavior of mice through the activity of TRPM8 channel, but it does not affect the body temperature of mice.
  • CCI Chronic Compression Injury
  • a cooling plate with a temperature controller (Mode 13300-0, CAL Controls Inc.) was used as described by Tanimoto-Mori Setal. (BehavPharmacol., 19:85-90, 2008) (LHP-1700CP, TECA) was evaluated for cold allodynia. Animals were acclimated to a setup consisting of a clear acrylic box (10 x 12 x 12 cm) on a stainless steel plate (15 x 33 cm). The surface of the cooling plate was maintained at 10°C and the temperature of the plate was continuously monitored with an accuracy of 0.1°C.
  • the rats were placed on a cooling plate, and the polypeptide DeC-1.2 was administered before and after administration with 120 seconds as the cutoff value, and the paw withdrawal latency (PWL) was measured.
  • the polypeptide DeC-1.2 or its vehicle is administered orally, subcutaneously or intraperitoneally.
  • the inhibition rate (%) is [PWL (polypeptide)-PWL (vehicle)]/[PWL (polypeptide)-PWL (vehicle)] ⁇ 100.
  • CCI Chronic Compression Injury
  • VFH ciliary mechanical needle stimulation
  • the lowest pressure required to elicit a response was recorded as the paw withdrawal threshold (PWT). Static allodynia was determined if the experimental animal responded below the harmless 1.4 g VFH.
  • the polypeptide DeC-1.2 or its vehicle is administered by injection, orally, subcutaneously or intraperitoneally.
  • the inhibition rate (%) was [PWL (polypeptide)-PWL (vehicle)]/[PWL (polypeptide)-PWL (vehicle)] ⁇ 100.

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Abstract

本发明涉及一种抑制TRPM8的多肽及其用途。具体地，本发明提供一种多肽或其药学上可接受的盐，本发明所述的多肽对TRPM8具有优异的抑制作用且用于预防和治疗与TRPM8相关的疾病。

Description

一种抑制TRPM8的多肽及其用途
相关申请交叉引用
本专利申请要求于2021年10月14日提交的、申请号为2021111975109、发明名称为“一种抑制TRPM8的多肽及其用途”的中国专利申请的优先权，上述申请的全文以引用的方式并入本文中。
技术领域
本发明涉及药物领域，具体地涉一种抑制TRPM8的多肽及其用途。
背景技术
外周神经病变是许多基于铂类化疗中的常见副作用，这限制了抗癌药物的使用剂量，降低了服用这些药物的患者生活质量。例如，奥沙利铂(Oxaliplatin)通常用作许多肿瘤治疗的一线化学疗法，例如结肠直肠癌和胃癌]等。然而，服用奥沙利铂的患者中高达89％会出现急性神经毒性，通常表现为患者在正常凉爽的环境温度中反而感到极度寒冷而手臂和腿等部位感到疼痛难忍。尽管患者体内的这些神经性病变症状包括奥沙利铂诱导的冷痛觉超敏等症状在停止服药后会得以改善，然而迄今为止，仍然没有针对此类神经性病变的有效治疗手段。
TRPM8是一类表达在伤害性刺激感受神经元上的非选择性离子通道。该通道在低于28℃的温度下被激活，因此它是哺乳动物的冷感受器。此外，作为多模态受体，TRPM8也能被多种物理刺激或化学配体激活，比如薄荷醇和Icilin以及跨膜去极化等。在奥沙利铂诱导的冷痛觉超敏小鼠模型中，伤害性背根神经节(DRG)神经元中TRPM8通道的表达水平显著升高。更重要的是，在TRPM8敲除鼠中，奥沙利铂的服用没有使其出现冷痛觉超敏。因此，TRPM8离子通道是外周神经病变，以及铂类药物引起的痛觉超敏的有效药物靶点，迫切需要开发有效的针对TRPM8离子通道的药物，用于治疗外周神经病变和痛觉超敏。
发明内容
本发明的目在于提供一种TRPM8抑制剂及其预防和治疗与TRPM8相关的疾病的多肽。
本发明第一方面，提供一种多肽或其药学上可接受的盐，所述多肽或其药学上可接受的盐具有式I所示的结构：
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13  式I
式中，
X1为无、任意的氨基酸或任意的肽段；
X2为R、K、Q、A、D或N；
X3为R、K、Q、A、H或N；
X4为D、A、S、R或E；
X5为R、K、Q、A或N；
X6为A、V、L、G、R或I；
X7为R、K、Q、A或N；
X8为H、N、Q、K、A或R；
X9为Y、W、F、D、T、A、R或S；
X10为R、K、Q、A、S、Y或N；
X11为Q、A、R或N；
X12为R、K、Q、A、K或N；
X13为无、任意的氨基酸或任意的肽段。
在另一优选例中，X1、X2、X3、X4、X5、X6、X7、X8、X9、X10、X11、X12和X13各自独立地为L-氨基酸或D-氨基酸。
在另一优选例中，X2为R、K、Q、D或N。
在另一优选例中，X6为A、V、L、R或I。
在另一优选例中，X12为R、K、Q、K或N。
在另一优选例中，所述的多肽或其药学上可接受的盐具有(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤。
在另一优选例中，所述的多肽为分离的多肽。
在另一优选例中，所述多肽是人工合成的。
在另一优选例中，所述多肽是重组多肽。
在另一优选例中，X1为无、C(半胱氨酸)或M(甲硫氨酸)。
在另一优选例中，X13为无或C(半胱氨酸)。
在另一优选例中，所述肽段包括标签蛋白、前导序列或分泌序列。
在另一优选例中，所述X1的长度为1-20aa，更佳地1-10aa，更佳地1-5aa。
在另一优选例中，所述X13的长度为1-20aa，更佳地1-10aa，更佳地1-5aa。
在另一优选例中，所述多肽或其药学上可接受的盐的长度≤25aa，较佳地≤20aa，更佳地≤18aa，更佳地≤15aa；更佳地，为13aa、14aa、15aa、16aa、17aa、18aa、19aa或20aa。
在另一优选例中，所述的X1或X13包括天然或非天然氨基酸。
在另一优选例中，所述X1和X13之间形成环肽。
在另一优选例中，所述X1和X13之间形成至少一对二硫键。
在另一优选例中，所述X1和X13之间形成一对二硫键。
在另一优选例中，所述的多肽具有式II所示的结构：
X1-RRDRARHYRQR-X13  式II
其中，X1、X13的定义如上所述。
在另一优选例中，所述多肽为N聚体。
在另一优选例中，所述N聚体具有如下式III结构：
-(X1-RRDRARHYRQR-X13-L1)n-   式III
其中，X1、X13的定义如上所述；L1为无或连接肽；n为2-10，较佳地，2-7，更佳地，2-5；各“-”独立地为连接肽或肽键。
在另一优选例中，所述L1为无。
在另一优选例中，所述L1的长度为1-30aa，较佳地，1-20aa，更佳地，1-10aa。
在另一优选例中，n为2、3或4。
在另一优选例中，各“-”独立地为肽键。
在另一优选例中，所述多肽的序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示。
在另一优选例中，所述式I所示的多肽与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示多肽相比，具有≥50％，≥60％，≥70％，≥80％，≥90％、≥95％、≥99％或100％的相同性(或同源性)。
在另一优选例中，所述式I所示的多肽保留了SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示多肽的生物活性的至少≥50％，≥60％，≥70％，≥80％，≥90％、≥95％，或≥99％，例90-100％。
在另一优选例中，所述多肽的序列如SEQ ID NO.:1所示。
在另一优选例中，所述式I所示的多肽与SEQ ID NO.:1所示多肽相比，具有≥50％，≥60％，≥70％，≥80％，≥90％、≥95％、≥99％或100％的相同性(或同源性)。
在另一优选例中，所述式I所示的多肽保留了SEQ ID NO:1所示多肽的生物活性的至少≥50％，≥60％，≥70％，≥80％，≥90％、≥95％，或≥99％，例90-100％。
在另一优选例中，所述多肽选自下组：
(1)具有SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示氨基酸序列的多肽；
(2)将SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示氨基酸序列经过1-5个(较佳地1-3，更佳地1-2个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相 关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤；
(3)与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示多肽相比，具有≥50％，≥60％，≥70％，≥80％，≥90％、≥95％、≥99％或100％的相同性(或同源性)的多肽。
在另一优选例中，所述多肽不为SEQ ID NO:3。
在另一优选例中，所述多肽选自下组：
(1)具有SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示氨基酸序列的多肽；
(2)将SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示氨基酸序列经过1-5个(较佳地1-3，更佳地1-2个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤；
(3)与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示多肽相比，具有≥50％，≥60％，≥70％，≥80％，≥90％、≥95％、≥99％或100％的相同性(或同源性)的多肽。
在另一优选例中，所述多肽选自下组：
(1)具有SEQ ID NO:1所示氨基酸序列的多肽；
(2)将SEQ ID NO:1所示氨基酸序列经过1-5个(较佳地1-3，更佳地1-2个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤；
(3)与SEQ ID NO.:1所示多肽相比，具有≥50％，≥60％，≥70％，≥80％，≥90％、≥95％、≥99％或100％的相同性(或同源性)的多肽。
在另一优选例中，所述的多肽是由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示的多肽经过1-3个较佳地1-2个，更佳地1个氨基酸取代、缺失；和/或
经过1-5，较佳地1-4个，更佳地1-3个，最佳地1-2个氨基酸的添加形成的。
在另一优选例中，所述的衍生的多肽保留了≥50％，≥60％，≥70％，≥80％，≥90％、≥99％、或100％，例如80-100％，较佳地95-100％的SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示多肽的(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；和/或(d)预防和/或治疗外周神经病变。
在另一优选例中，所述的衍生的多肽与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19的相同性≥50％，较佳地，≥60％，更佳地，≥70％，更佳地，≥80％，更佳地，≥90％，更佳地，≥99％。
在另一优选例中，所述的多肽是由SEQ ID NO.:1所示的多肽经过1-3个 较佳地1-2个，更佳地1个氨基酸取代、缺失；和/或
经过1-5，较佳地1-4个，更佳地1-3个，最佳地1-2个氨基酸的添加形成的。
在另一优选例中，所述的衍生的多肽保留了≥50％，≥60％，≥70％，≥80％，≥90％、≥99％、或100％，例如80-100％，较佳地95-100％的SEQ ID NO:1所示多肽的(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；和/或(d)预防和/或治疗外周神经病变。
在另一优选例中，所述的衍生的多肽与SEQ ID NO:1的相同性≥50％，较佳地，≥60％，更佳地，≥70％，更佳地，≥80％，更佳地，≥90％，更佳地，≥99％。
本发明第二方面，提供一种融合蛋白，所述的融合蛋白包括：
(a)如本发明第一方面所述的多肽或其药学上可接受的盐；
(b)与如本发明第一方面所述的多肽或其药学上可接受的盐融合的肽段。
在另一优选例中，所述肽段包括载体蛋白。
在另一优选例中，所述载体蛋白选自下组：Fc片段、人血清白蛋白(HSA)、CTP、转铁蛋白、或其组合。
在另一优选例中，所述肽段经过修饰。
在另一优选例中，所述修饰包括聚乙二醇(PEG)修饰。
本发明第三方面，提供一种多核苷酸，所述多核苷酸编码如本发明第一方面所述的多肽或其药学上可接受的盐。
在另一优选例中，所述多核苷酸为分离的多核苷酸。
本发明第四方面，提供一种载体，所述的载体包括如本发明第三方面所述的多核苷酸。
在另一优选例中，所述载体包括质粒载体。
本发明第五方面，提供一种宿主细胞，所述的宿主细胞包括如本发明第四方面所述的载体或所述的宿主细胞的染色体上整合有如本发明第三方面所述的多核苷酸。
本发明第六方面，提供一种组合物，所述的组合物包括：
(a)如本发明第一方面所述的的多肽或其药学上可接受的盐；和
(b)药学上可接受的载体或赋形剂。
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述的组合物通过选自下组的用药方式进行给药：静脉内、瘤内、腔内、皮下或肝动脉给药(如注射、滴注等)。
在另一优选例中，所述的组合物的剂型为口服制剂、注射制剂或外用制剂。
在另一优选例中，所述的组合物的剂型为固体制剂、液体制剂或半固体制剂。
在另一优选例中，所述的组合物的制剂选自下组：片剂、胶囊剂、注射剂、颗粒剂、喷雾剂、冻干剂。
在另一优选例中，所述的组合物的剂型为注射剂。
在另一优选例中，所述的注射剂为静脉注剂、肌肉注射剂或皮下注射剂。
在另一优选例中，所述的多肽以0.01-100mg/kg体重的剂量(每次或每天)施用于哺乳动物。
本发明第七方面，提供一种如本发明第一方面所述的多肽或其药学上可接受的盐、如本发明第二方面所述的融合蛋白、如本发明第三方面所述的多核苷酸、如本发明第四方面所述的载体、如本发明第五方面所述的宿主细胞、和/或如本发明第六方面所述的组合物的用途，用于制备组合物，所述组合物用于选自下组的一种或多种用途：(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤。
在另一优选例中，所述组合物为药物组合物。
在另一优选例中，所述的与TRPM8相关的疾病选自下组：外周神经病变、冷痛觉超敏、瘙痒、神经病变性疼痛的慢性压迫损伤，或其组合。
在另一优选例中，所述的疼痛选自下组：慢性疼痛、冷痛觉超敏性疼痛、糖尿病性神经病变的神经病变性疼痛、术后疼痛、骨关节炎疼痛、类风湿关节炎疼痛、癌症疼痛、神经痛、神经损伤性疼痛、偏头痛、丛集性头痛、紧 张性头痛、纤维肌痛、神经病变性疼痛、静态异常性疼痛、异常性冷疼痛，或其组合。
在另一优选例中，所述的异常性冷疼痛包括神经病变性疼痛的慢性压迫损伤导致的异常性冷疼痛。
在另一优选例中，所述的静态异常性疼痛包括神经病变性疼痛的慢性压迫损伤导致的静态异常性疼痛。
在另一优选例中，所述的与TRPM8相关的疾病包括TRPM8上调的疾病。
在另一优选例中，所述TRPM8上调包括TRPM8表达水平或活性高。
在另一优选例中，所述TRPM8上调是指某一细胞(如刺激感受神经元或背根神经节)的TRPM8表达水平或活性大于同一细胞的TRPM8表达水平或活性。
在另一优选例中，所述TRPM8上调是指某一细胞(如刺激感受神经元或背根神经节)的TRPM8表达水平或活性C1与同一细胞的TRPM8表达水平或活性C0的比值(C1/C0)＞1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5。
在另一优选例中，所述的细胞包括神经细胞。
在另一优选例中，所述的刺激感受神经元包括伤害性刺激感受神经元。
在另一优选例中，所述的背根神经节包括伤害性背根神经节。
在另一优选例中，所述的同一细胞是指同种类且TRPM8表达或活性正常的细胞。
在另一优选例中，所述的表达包括mRNA和/或蛋白表达。
在另一优选例中，所述的冷痛觉超敏包括铂类抗癌药物引起的冷痛觉超敏。
在另一优选例中，所述的外周神经病变包括铂类抗癌药物引起的外周神经病变。
在另一优选例中，所述的冷痛觉超敏包括铂类抗癌药物引起的冷痛觉超敏。
在另一优选例中，所述的铂类抗癌药物选自下组：卡铂、奈达铂、洛铂、奥沙利铂，或其组合。
本发明第八方面，提供一种(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性 压迫损伤的方法，所述的方法包括步骤：向需要的对象施用如本发明第一方面所述的多肽或其药学上可接受的盐、如本发明第二方面所述的融合蛋白、如本发明第三方面所述的多核苷酸、如本发明第四方面所述的载体、如本发明第五方面所述的宿主细胞、和/或如本发明第六方面所述的组合物。
在另一优选例中，所述的对象是人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠、兔)、灵长类动物(如猴)。
在另一优选例中，所述的方法是非诊断和非治疗性的。
在另一优选例中，所述的施用为口服施用、注射施用或外用施用。
在另一优选例中，所述的注射为静脉注射、肌肉注射或皮下注射。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1是设计多肽合成后的HPLC纯化、质谱验证及其功能的电生理验证。(a)DeC-1.1多肽的HPLC纯化。(b)DeC-1.1多肽分子量的质谱验证。(c)DeC-1.2多肽的HPLC纯化。(d)DeC-1.2多肽分子量的质谱验证。(e)DeC-1.2抑制薄荷醇激活的小鼠TRPM8通道的全细胞电生理记录代表性曲线。(f)全细胞电生理记录模式下测量的DeC-1.1和DeC-1.2的抑制性浓度依赖曲线。
图2是DeC-1.2对TRPM8的激活模式特异性抑制。(a)在+80mV时，TRPM8的由外向外单通道代表性电生理记录。分别用饱和的薄荷醇(1mM)和饱和薄荷醇(1mM)与DeC-1.2(100μM)的混合溶液的灌流来激活或抑制TRPM8通道。这四个代表性单通道记录来自同一片细胞膜。(b)在图(a)中显示的TRPM8单通道电生理记录对应的全点直方图。直方图是用双高斯函数拟合(红实线表示)，拟合后用相邻两个峰值的差值来计算单通道电导。(c)TRPM8通道被饱和浓度Menthol(1mM)激活的开放概率在DeC-1.2(100μM)存在时显著下降。数据显示为每组五个独立细胞的电生理记录数 据的平均值±SEM。****表示P<0.0001。(d)TRPM8通道被饱和浓度Menthol(1mM)激活的单通道电导在DeC-1.2(100μM)存在时显著下降。数据显示为每组三个独立细胞的电生理记录数据的平均值±SEM。***表示P<0.001。(e)在+80mV时，有无DeC-1.2存在的情况下，TRPM8被冷激活时由内向外单通道代表性电生理记录。(f)在图(e)中显示的TRPM8单通道电生理记录对应的全点直方图。直方图是用双高斯函数拟合。(g)16℃时，TRPM8通道被冷激活的开放概率在DeC-1.2(100μM)存在时没有显著改变。数据显示为每组五个独立细胞的电生理记录数据的平均值±SEM。N.S.表示没有显著性差异。(h)16℃时，TRPM8通道被被冷激活的单通道电导在DeC-1.2(100μM)存在时无显著变化。数据显示为每组三个独立细胞的电生理记录数据的平均值±SEM。N.S.表示没有显著性差异。
图3是DeC-1.2的亚基选择性。(a、b、c、d、e)DeC-1.2对TRPV1,TRPV2,TRPV3,TRPM2和TRPM4的配体激活的抑制代表性电生理记录。其中使用TRPM4-K1045A突变以便排除TRPM4对PIP2的依赖性。(f)500nM的DeC-1.2对TRP通道和Nav通道的抑制性归一化对比。每一个通道对应n＝3,****表示P<0.0001。
图4是DeC-1.2对TRPM8的抑制中起关键作用的氨基酸残基。(a、b、c、d、e、f、g)5nM的野生型DeC-1.2以及各个突变体对TRPM8的配体激活的抑制代表性电生理记录。(h、i)5nM的野生型DeC-1.2以及突变体对TRPM8的配体激活的抑制性归一化图，*，**和***表示P<0.05，P<0.01，P<0.001。(j)DeC-1.2的结构中标出的其对TRPM8的抑制性起关键作用的氨基酸残基(在5nM浓度下完全消除抑制的突变和部分消除抑制的突变，分别用橘色和橙色表示)。
图5是从在体角度验证DeC-1.2对TRPM8的抑制效果。(a)DeC-1.2剂量依赖性地显著降低了Icilin引起的小鼠湿狗样全身颤抖反应。数据显示为每组六只动物的平均值±SEM。*，**和***分别表示p<0.05，p<0.01和p<0.001。(b)在小鼠中建立奥沙利铂诱导的冷痛觉超敏反应过程示意图以及DeC-1.2抑制冷痛觉超敏反应的动物试验流程图。(c)分别从生理盐水处理组小鼠中提取的DRG神经元灌流细胞外液(Vehicle)时，奥沙利铂处理组小鼠中提取的DRG神经元灌流细胞外液(Oxaliplatin)时或含10μM DeC-1.2 的细胞外液(Oxaliplatin+DeC-1.2)时钙成像反应代表性图。在灌流100μM的薄荷醇后，随着薄荷醇激活TRPM8通道使DRG神经元的钙内流量增加。1μM辣椒素使DRG神经元中的TRPV1激活而产生的钙内流细胞可作为阳性对照。用白色的箭头指出对激动剂有反应的神经元。(d)分别从生理盐水处理组和奥沙利铂处理组小鼠中提取的DRG神经元对相应试剂的反应的荧光比率代表性曲线。(e)分别从生理盐水处理组和奥沙利铂处理组小鼠中提取的DRGA神经元对薄荷醇的反应比例。Vehicle，oxaliplatin和oxaliplatin+DeC-1.2对应各组神经元细胞总数分别为229,238和334，*，**分别表示P<0.05和P<0.01。每个离散圈代表一个视野中的神经元数。
图6是DeC-1.2体内抑制奥沙利铂诱导的冷痛觉超敏反应效果。(a)DeC-1.2对奥沙利铂诱导的冷痛觉超敏反应的抑制效果。在丙酮处理后5分钟内记录抬爪，翻转或舔爪(此处都为丙酮处理的爪)的总时间，作为丙酮分数，用来评估DeC-1.2的抑制效果。数据显示为每组六只动物的平均值±SEM。经two-way ANOVA分析方法，*表示p<0.05。(b)DeC-1.2对奥沙利铂诱导的机械触诱发痛反应的抑制效果。数据显示为每组5-8只小鼠的平均值±SEM。(c)DeC-1.2对体温的影响。每一只数在DeC-1.2注射(30μg/g,i.v.)之前(baseline,BL)，注射后5min,15min,0.5h,1h,2h,6h和24h处被测量肛温。数据显示为六只动物的平均值±SEM。经one-way ANOVA分析，N.S.表示没有显著性差异。(d)激活模式特异性抑制剂DeC-1.2对TRPM8通道的工作原理示意图。
具体实施方式
本发明人经过广泛而深入的研究，首次制备了具有(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤的多肽如DeC-1.2多肽，且本发明的多肽安全性好，对生物组织毒副作用小。在此基础上，本发明人完成了本发明。
术语
除非另有定义，否则本文中所用的所有技术和科学术语的含义与本发明 所属领域普通技术人员普遍理解的含义相同。
如本文所用，术语“包括”、“包含”与“含有”可互换使用，不仅包括开放式定义，还包括半封闭式、和封闭式定义。换言之，所述术语包括了“由......构成”、“基本上由......构成”。
在本发明中，各个氨基酸及其简写如下表1所示：
表1 氨基酸及其简写
名称 三字母符号 单字母符号 名称 三字母符号 单字母符号
丙氨酸(alanine) Ala A 亮氨酸(leucine) Leu L
精氨酸(arginine) Arg R 赖氨酸(lysine) Lys K
天冬酰胺(asparagine) Asn N 甲硫氨酸(methionine) Met M
天冬氨酸(asparticacid) Asp D 笨丙氨酸(phenylalanine) Phe F
半胱氨酸(cysteine) Cys C 脯氨酸(proline) Pro P
谷氨酰胺(glutanine) Gln Q 丝胺酸(serine) Ser S
谷氨酸(glutamicacid) Glu E 苏氨酸(threonine) Thr T
甘氨酸(Glicine) Gly G 色氨酸(tryptophan) Trp W
组氨酸(histidine) His H 酪氨酸(tyrosine) Tyr Y
异亮氨酸(isoleucine) Ile I 颉氨酸(valine) Val V
在本发明中，术语“预防”表示预防疾病和/或它的附随症状的发作或者保护对象免于获得疾病的方法。
本发明所述的“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述多肽时观察到的水平相比，本发明所述多肽将与TRPM8相关的疾病减轻、抑制和/或逆转了例如至少约10％、至少约30％、至少约50％、或至少约80％、或100％。
TRPM8
TRPM8(Transient receptor potential melastatin 8)又称冷及薄荷醇感受器，是瞬时受体电位离子通道蛋白TRP家族的一员。该通道由4个相同亚基组成，每个亚基有6个跨膜区，N端与C端均位于细胞内侧。位于TRPM8通道中央的孔区由4个亚基共同组成，可以非选择性地通透阳离子。TRPM8通道参与机体冷感觉、痛觉、炎症反应、血管收缩与扩张、细胞生长增殖等调控。
多肽
在本发明中，术语“本发明多肽”指具有式I所示的结构多肽或其药学上可接受的盐。应理解，该术语还包括上述组分的混合物。此外，本发明多肽还包括具有式I所示的结构多肽的变异形式。这些变异形式包括(但并不限于)：在N末端添加一个或数个(通常为5个以内，较佳地为3个以内，更佳地为2个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。在N末端添加一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽或其药学上可接受的盐。
在本发明中，多肽的氨基酸序列从N端至C端进行编号。
本发明还包括本发明多肽的活性片段、衍生物和类似物。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)本发明多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的然后蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I所示多肽的氨基酸序列相比，有至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽优选地根据表2进行氨基酸替换而产生。
表2
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
发明还提供本发明多肽的类似物。这些类似物与天然本发明多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。例如，Cys可与非天然的Hcy形成二硫键。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在优选例中，本发明多肽具有至少一个内部的二硫键(引入的链内二硫键)。 令人意外的是，该内部二硫键的存在不仅不影响其活性，而且有助于延长半衰期和提高抑制活性。通常，可用本领域常规的方法来形成，例如使半胱氨酸或同型半胱氨酸巯基在氧化条件下结合形成二硫键。
一种优选的本发明多肽包括多肽DeC-1.2，多肽DeC-1.2的氨基酸序列为SEQ ID No.:1所述的氨基酸序列：
SEQ ID NO.:1
CRRDRARHYRQRC。
在本发明中，术语“DeC-1.2”、“多肽DeC-1.2”或“DeC-1.2多肽”可互换使用。
本发明的多肽还包括对SEQ ID NO.:1所示多肽进行改造后的多肽。
本发明多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使用。这些盐包括(但不限于)与如下酸形成的盐：氢氯酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、苯磺酸、或羟乙磺酸。其他盐包括：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以及以酯、氨基甲酸酯或其他常规的“前体药物”的形式。
编码序列
本发明还涉及编码本发明多肽的多核苷酸。本发明的多核苷酸可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与编码区序列相同或者是简并的变异体。本发明的多肽的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明多肽(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明多肽编码序列经基因工程产生的宿主细胞。
另一方面，本发明还包括对本发明多肽具有特异性的多克隆抗体和单克隆抗体或抗体片段，尤其是单克隆抗体。
在两个核酸或多肽的背景下，当进行最大相符性序列比较和比对时，术 语“基本相同”是指两个或多个序列或亚序列，其具有至少约80％，例如至少约85％、约90％、约95％、约98％或约99％的核苷酸或氨基酸残基与特定的参考序列具有同一性，如使用以下序列比较方法和/或通过肉眼检查所测定。
制备方法
本发明多肽可以是重组多肽或合成多肽。本发明的多肽可以是化学合成的，或重组的。相应地，本发明多肽可用常规方法人工合成，也可用重组方法生产。
一种优选的方法是使用液相合成技术或固相合成技术，如Boc固相法、Fmoc固相法或是两种方法联合使用。固相合成可快速获得样品，可根据目的肽的序列特征选用适当的树脂载体及合成系统。例如，Fmoc系统中优选的固相载体如连接有肽中C端氨基酸的Wang树脂，Wang树脂结构为聚苯乙烯，与氨基酸间的手臂是4-烷氧基苄醇；用25％六氢吡啶/二甲基甲酰胺室温处理20分钟，以除去Fmoc保护基团，并按照给定的氨基酸序列由C端逐个向N端延伸。合成完成后，用含4％对甲基苯酚的三氟乙酸将合成的胰岛素原相关肽从树脂上切割下来并除去保护基，可过滤除树脂后乙醚沉淀分离得到粗肽。将所得产物的溶液冻干后，用凝胶过滤和反相高压液相层析法纯化所需的肽。当使用Boc系统进行固相合成时，优选树脂为连接有肽中C端氨基酸的PAM树脂，PAM树脂结构为聚苯乙烯，与氨基酸间的手臂是4-羟甲基苯乙酰胺；在Boc合成系统中，在去保护、中和、偶联的循环中，用TFA/二氯甲烷(DCM)除去保护基团Boc并用二异丙基乙胺(DIEA/二氯甲烷中和。肽链缩合完成后，用含对甲苯酚(5-10％)的氟化氢(HF),在0℃下处理1小时，将肽链从树脂上切下，同时除去保护基团。以50-80％乙酸(含少量巯基乙醇)抽提肽，溶液冻干后进一步用分子筛Sephadex G10或Tsk-40f分离纯化，然后再经高压液相纯化得到所需的肽。可以使用肽化学领域内已知的各种偶联剂和偶联方法偶联各氨基酸残基，例如可使用二环己基碳二亚胺(DCC)，羟基苯骈三氮唑(HOBt)或1,1,3,3-四脲六氟磷酸酯(HBTU)进行直接偶联。对于合成得到的短肽，其纯度与结构可用反相高效液相和质谱分析进行确证。
在一优选例中，本发明多肽，按其序列，采用固相合成的方法制备，行高效液相色谱纯化，获得高纯度目的肽冻干粉，-20℃贮存。
另一种方法是用重组技术产生本发明多肽通过常规的重组DNA技术，可利用本发明的多核苷酸可用来表达或生产重组的本发明多肽。一般来说有以下步骤：
(1).用本发明的编码本发明多肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
重组多肽可在细胞内或在细胞膜上表达或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
由于本发明多肽较短，因此可以考虑将多个多肽串联在一起，重组表达后获得多聚体形式的表达产物，然后通过酶切等方法形成所需的小肽。
组合物和施用方法
本发明还提供一种组合物，所述的组合物优选为药物组合物。
本发明的组合物含有(a)本发明多肽或其药学上可接受的盐；以及(b)药学上可接受的载体或赋形剂。本发明多肽或其药学上可接受的盐的数量通常为10微克-100毫克/剂，较佳地为100-1000微克/剂。
为了本发明的预防和治疗目的，在本发明所述的组合物中，本发明多肽或其药学上可接受的盐是安全有效量的，有效的剂量为给予个体约0.01毫克/千克至50毫克/千克，较佳地0.05毫克/千克至10毫克/千克体重的本发明多肽或其药学上可接受的盐。此外，本发明的多肽或其药学上可接受的盐可以单用，也可与其他治疗剂一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington’s Pharmaceutical  Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：肌内、静脉内、皮下、皮内、或局部给药。待预防或治疗的对象可以是动物；尤其是人。
在本发明一个优选例中，所述的药物组合物的剂型为口服制剂、注射制剂或外用制剂。
在本发明一个优选例中，所述的药物组合物的剂型为固体制剂、液体制剂或半固体制剂。
在本发明一个优选例中，所述的药物组合物的制剂选自下组：片剂、胶囊剂、注射剂、颗粒剂、喷雾剂、冻干剂。
代表性地，所述的注射剂为静脉注剂、肌肉注射剂或皮下注射剂。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地为静脉用药制剂或瘤内用药注射剂。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
例如，眼部滴眼液的配制可这样进行：将本发明多肽或其药学上可接受的盐与基本物质一起溶解于无菌水(在无菌水中溶解有表面活性剂)中，调节渗透压和酸碱度至生理状态，并可任意地加入合适的药物添加剂如防腐剂、稳定剂、缓冲剂、等渗剂、抗氧化剂和增粘剂，然后使其完全溶解。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明多肽或其药学上可接受的盐可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药 丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
药物制剂应与给药方式相匹配。本发明药剂还可与其他协同治疗剂一起使用(包括之前、之中或之后使用)。使用药物组合物时，是将安全有效量的药物施用于所需对象(如人或非人哺乳动物)，所述安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要效果包括：
(1)本发明多肽可有效抑(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤。
(2)本发明多肽及其衍生多肽的分子量小、对生物组织毒副作用小、安全性高。
(3)本发明多肽的稳定性好。
(4)本发明多肽的特异性高。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
实验方法：
肽的合成和纯化
带有或不带有二硫键的多肽在吉尔生化(上海)化学合成而得。粗品进一步 通过反向液相色谱进行纯化。多肽的纯化效率达到95％及以上就可通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF)或电喷雾电离质谱(ESI-MS)鉴定确认。此后经过纯化质谱鉴定大量收集对应环肽以备用。
细胞培养和瞬时转染
HEK293T细胞在Dulbecco的改性Eagle培养基中培养，在37℃，5％的二氧化碳环境中培养，其下培养基中需加入10％胎牛血清，青霉素(100u/ml)和链霉素(100mg/ml)。瞬时转染是通过Lipo 2000(Invitrogen)转染试剂盒，按照说明书中的操作流程完成的。
电生理记录
在瞬时转染24-48小时内对细胞进行电生理实验记录。单通道电生理记录是在转染后8小时内进行。使用HEKA EPC10为型号的放大器分别在全细胞记录模式，由内向外记录模式以及由外向外记录模式下进行细胞电流的测量。在全细胞电生理记录中，由硼酸硅盐材质的玻璃微电极拉制并抛光为初电极电阻大约为3-6MΩ。Serial resistance被互补60％。全细胞电生理是在±80mV下进行的。至于单通道电生理记录，玻璃微电极被拉制并抛光为6-8MΩ。在10kHZ频率上采集电流，并在2.9kHZ处过滤。细胞膜膜电位将被钳制在+80mV。所有的电生理实验记录都在22℃(最大变化幅度为1℃)室温下进行的。
对于不同的离子通道的电流的测量将会有不同的内外液配方。针对TRPM8,TRPV1,TRPV3和TRPA1等离子通道，内外液配方为：130mM NaCl,0.2mM EDTA和3mM HEPES,pH＝7.2。针对TRPM4离子通道的电流测量，外液配方为：130mM NaCl和3mM HEPES,pH＝7.2，内液配方为500μM CaCl 2，130mM NaCl和3mM HEPES,pH＝7.2。针对TRPM2离子通道的电流测量，外液配方为：147mM NaCl,2mM KCl,1mM MgCl2,10mM HEPES,2mM CaCl2 13mM Glucose,pH＝7.4，其内液为147mM NaCl,1mM MgCl 2,10mM HEPES,pH＝7.4。针对NaV1.5,NaV1.7等离子通道的电流测量，外液配方为：140mM NaCl,3mM KCl,1mM MgCl 2,1mM CaCl2,10mM HEPES,pH＝7.2，内液配方为：140mM CsF,1mM EGTA,10mM NaCl,10mM MgCl 2,3mM KCl,pH＝7.2。为了激起钠通道的电流，膜电位从-80mV提高到+10Mv。
具有重力驱动的快速灌注系统(RSC-200,BioLogic)被运用以便将特定的溶液快速灌流到待检测细胞身上。通过单独的PVC管子输送不同的溶液以免溶液间交叉污染。电生理记录中，带有膜片的玻璃微电极将被移到灌流管出口的正下方。
动物实验
成年雄性CD1鼠(6-8周龄)作为实验动物用于行为学实验和DRG神经元的原代培养。小鼠在光照周期12小时光照/12小时黑暗条件下饲养，供给充足的标准饲料和水。所有的动物实验方案都获得了浙江大学动物护理和使用委员会(IACUC)批准。
疼痛小鼠模型
在行为学测试前2～3天，所有的小鼠每天在行为学测试环境中适应1～2小时。单次腹腔注射奥沙利铂(6mg/kg体重)用于诱导化疗引起的神经病理性痛模型。神经病理性痛的行为学测试在奥沙利铂注射后第10天进行。
Icilin诱导的湿狗样颤抖行为实验
对照组和实验组各有至少6只成年雄性CD1小鼠，分别经尾静脉注射生理盐水、梯度剂量的DeC-1.2。尾静脉注射30分钟后，腹腔注射Icilin(2.5μg/g体重)。Icilin注射后，小鼠置于测试架上，录像机记录30min内小鼠的湿狗样颤抖行为。
小鼠体温测量
数显温度计(FT3400)用于小鼠的体温检测。体温测试时，成年CD1雄性小鼠置于恒温(22±1℃)环境。小鼠经尾静脉注射DeC-1.2(30mg/kg体重)，用于评估DeC-1.2对小鼠体温的影响。在注射前，注射后5min、15min、0.5h、1h、2h、6h和24h分别进行体温测量。将温度计的测试探头插入小鼠肛门2cm左右，待温度显示数值稳定后记录为小鼠体温。
奥沙利铂诱导的冷痛敏行为
丙酮挥发引起的冷觉测试用于评估奥沙利铂诱导的冷痛敏行为。在10ml注射器的头部连接一塑料软管，将50μl丙酮经塑料软管喷射至小鼠后爪足底，记录小鼠5分钟内出现抬脚、甩脚、舔脚等反应的总时间。在奥沙利铂注射前、注射后10天分别测定小鼠的冷痛敏行为，小鼠足底注射生理盐水或3.5μg/20μLDeC-1.2用于评估DeC-1.2对奥沙利铂引起的冷痛敏行为的影响。
奥沙利铂诱导的机械触诱发痛反应
以VonFrey测试中小鼠缩脚阈值的改变评估小鼠的机械触诱发痛行为。VonFrey测试中小鼠缩脚阈值按照Dixon’sup-down的方法进行。用vonFrey纤维丝(0.02-2g)对小鼠后足底中部进行刺激。小鼠受到刺激3s内表现出抬脚、甩脚、舔脚等行为，即为有反应。按照Dixon’sup-down的方法，首先用0.16gvonFrey丝刺激，若小鼠有反应则用相邻的低强度的vonFrey丝刺激，无反应则用相邻的高强度的vonFrey丝刺激。依次选择不同强度的vonFrey丝刺激，每两次刺激间隔5s，共完成6次刺激，对照反应量表评估小鼠的缩脚阈值(Pawwithdrawalthreshold，PWT)。在奥沙利铂注射前、注射后10天分别测定小鼠的缩脚阈值，小鼠足底注射生理盐水或3.5μg/20μLDeC-1.2用于评估DeC-1.2对奥沙利铂引起的触诱发痛行为的影响。
DRG神经元的原代培养和钙成像实验
小鼠深度麻醉后，断头弃血，快速分离DRG组织置于冰冷的PBS溶液中。漂洗血污后，加消化酶(Collagenase A 20mg/100ml，Dispase II 300mg/100ml溶于PBS)于37℃消化1h。500g离心5min，收集消化后的组织。弃尽酶溶液，加入适量DMEM培养基(含10％FBS、1X Pen/Strep)，通过机械吹打制备单细胞悬液。500g离心10min。弃尽上清，加适量体积的Neurobasal培养基(含2％B27、1mM L-Glutamine、50ng/ml NGF2.5S、1X Pen/Strep)，重悬并吹打成单细胞悬液。将单细胞悬液接种于poly-D-Lys预包被的细胞爬片上。待细胞贴壁后，加适量培养基至培养皿中，于37℃、5％CO2培养箱中培养24小时后用于钙成像实验。
原代培养的DRG神经元经细胞外溶液(ECS，140mM NaCl、5mM KCl、1mM MgCl2、1.8mM CaCl2、10mM D-Glucoes，15mM HEPES，pH＝7.4)漂洗后，于钙染料孵育液(2μM Fluo-2AM、0.05％Pluronic F-127in ECS)中孵育30分钟。细胞图像由高速连续的单色光源在激发光340/380nm波长下连续交替激发，由visiview软件在20X物镜下全视野记录，通过高速扫描相机Flash4.0LT以0.5fps速度连续记录细胞内荧光强度的变化。以340nm/380nm下荧光信号的比值表示钙信号的强度。通过快速交换灌注系统(ALA-VM8；ALA Scientific Instruments)给药薄荷醇、DeC-1.2、Capsaicin等分别灌注30s、90s、30s。通过比较不同组来源的DRG神经元中薄荷醇引起的钙信号反应强度、阳性反应细胞的比例，评估DeC-1.2对TRPM8通道活性的影响。
数据统计
所有实验均独立重复三次。所有统计数据都表示为平均值±SEM。电生理数据是用配对或非配对t检验完成的。行为学实验数据是分别用t检验，one-way或two-way anova完成的。N.S.表示没有显著性差异。*，**，***和****分别表示P<0.05，P<0.01，P<0.001和0.0001。
1.多肽的设计和化学合成
整个氨基酸序列为RRDRARHYRQR，为了进一步提高设计的肽链的稳定性，我们分别在其N-和C-末端引入了一个半胱氨酸残基以形成二硫键，多肽DeC-1.2的氨基酸序列为CRRDRARHYRQRC(SEQ ID NO:1)，多肽DeC-1.2环化。
多肽DeC-1.1的氨基酸序列为CRNSRAAHDSQKC(SEQ ID NO:2)，且多肽DeC-1.1的N-和C-末端的半胱氨酸残基形成二硫键，使多肽DeC-1.1环化。同时，设计了阴性对照多肽S-DeC-1.2(SEQ ID NO:3)。
多肽DeC-1.2和多肽DeC-1.1中的氨基酸均为L-氨基酸。
多肽合成后的HPLC纯化、质谱验证及其功能的电生理验证如图1所示。
多肽DeC-1.1经化学合成，HPLC(图1a)纯化后，通过质谱(图1b)鉴定确认，经电生理测试，DeC-1.1的半数抑制浓度(IC 50)为132.8±73.5μM。
多肽DeC-1.2也同样地经化学合成，HPLC(图1c)纯化后，通过质谱(图1d)鉴定确认。经电生理测试，多肽DeC-1.2能够纳摩级别抑制TRPM8的Menthol 激活(图1e)，显示出更低的半数抑制浓度(IC50)，多肽DeC-1.2抑制TRPM8的半数抑制浓度(IC50)为4.5±3.0nM(图1b)。而阴性对照S-DeC-1.2则对TRPM8无抑制作用。
全细胞电生理记录模式下测量的多肽DeC-1.1和多肽DeC-1.2的抑制性浓度依赖曲线如图1f。
2.多肽的特性和作用效果
2.1多肽DeC-1.2对TRPM8的激活方式特异性以及亚基特异性抑制
鉴于多肽DeC-1.2以高亲和力抑制TRPM8的Menthol激活，进一步验证DeC-1.2多肽是否具有激活方式特异性。DeC-1.2对TRPM8的激活模式特异性抑制如图2所示。通过单通道电生理记录测量了TRPM8离子通道的开放概率和单通道电导。在饱和浓度薄荷醇(1mM)的作用下TRPM8的最大开放概率为大约70％(图2a、图2b和图2c)。当100μM DeC-1.2与饱和的薄荷醇(1mM)混合作用于TRPM8通道时，其开放概率会明显减少至18.83±1.92％(n＝5)(图2c)。此外，DeC-1.2的存在能够使TRPM8的薄荷醇激活对应单通道电导从58.83±1.85pS(n＝3)下降至30.97±1.16pS(n＝3)(图2b和2d)，表明DeC-1.2以堵孔的方式阻碍离子进出TRPM8离子通道。
进一步测试DeC-1.2是否会抑制TRPM8的冷激活。当待测细胞膜从35℃冷却至16℃时，TRPM8被激活。16℃时，DeC-1.2(100μM)的应用并未降低TRPM8的开放概率(图2e、图2f和图2g)。此外，尽管冷却时TRPM8的单通道电导降低至36.16±1.74pS(n＝3)，DeC-1.2的存在并没有进一步降低TRPM8的单通道电导，表明DeC-1.2是TRPM8的激活模式特异性抑制剂。TRPM8通道被冷激活的单通道电导在DeC-1.2(100μM)存在时无显著变化(图2h)。
检测DeC-1.2的亚基选择性(如图3和表3所示)。TRPM8离子通道属于TRP通道超家族，该家族成员通道都有相似的六个跨膜结构域单体。DeC-1.2以IC 50为4.5nM水平抑制TRPM8通道，然而，比其高一百倍浓度的DeC-1.2(5μM)未能抑制TRPV1或TRPV3的配体激活(图3a、图3b和3c)。虽然DeC-1.2在一定的程度上抑制TRPV2的激活，但是其半数抑制浓度(IC 50)为大约10μM(图3b和表3)，抑制效果远远低于TRPM8上的抑制性。在TRP通道中，TRPM2 是TRPM8最近的同源物，但高达100μM的DeC-1.2未能抑制TRPM2的配体激活(图3d)。在设计DeC-1.2时，使用TRPM4的闭合状态的结构作为TRPM8的同源建模模板，因此DeC-1.2可能对TRPM4表现出更高的亲和力。然而，实际测验时TRPM4对DeC-1.2显示出了较低的敏感性，50μM的DeC-1.2只抑制了TRPM4大约一半的电流(图3e)。在TRPA1和电压门控钠(Nav)通道上测试了DeC-1.2的抑制性，发现DeC-1.2并没有对其起到抑制作用(图3f和表3)。因此，DeC-1.2既是亚基特异性也是激活模式特异性的抑制TRPM8配体激活的抑制剂。
表3 DeC-1.2多肽对不同离子通道的抑制效果
Figure PCTCN2022125397-appb-000001
对DeC-1.2多肽进行了突变(alanine scanning)，如表4和表5所示。这些点突变多肽也和野生型环肽一样化学合成并纯化质检。DeC-1.2对TRPM8的抑制中起关键作用的氨基酸残基研究如图4所示。在全细胞电生理记录模式下，发现相对于5nM野生型DeC-1.2的抑制活性，部分突变体仍能够激活TRPM8，但活性有下降，而部分5nM突变体环肽对TRPM8的抑制效果几乎消失(图4a、图4b、图4c、图4d、图4e、图4f、图4g)，但在高浓度下，例如100μM也具有抑制TRPM8通道的效果。进一步在DeC-1.2结构中标记了能够使环肽DeC-1.2对TRPM8的抑制性有改变的那些关键残基(图4h、图4i和图4j，完全消除抑制的突变和部分抑制的突变分别用红色和橘色表示)。大多数关键残基位于DeC-1.2与TRPM8的孔区外侧界面处， 而且这些位点的突变会显著影响环肽抑制性表明DeC-1.2，会结合在TRPM8孔区外侧起到抑制作用。
表4 基于DeC-1.2多肽的突变多肽
Figure PCTCN2022125397-appb-000002
表5 基于DeC-1.2多肽的突变多肽
Figure PCTCN2022125397-appb-000003
2.2多肽能够改善外周神经病变
检测了多肽DeC-1.2对Icilin(TRPM8的一种激动剂)诱导的小鼠湿狗样颤抖(WDS)行为的影响，DeC-1.2对TRPM8的抑制效果如图5所示。结果显示：尾静脉注射低剂量的DeC-1.2(0.3μg/g体重)即能明显抑制Icilin诱导的WDS行为，并且其抑制效应具有明显的剂量依赖性(图5a)，结果表明：体内注射DeC-1.2能够阻断Icilin诱导的TRPM8通道开放。
奥沙利铂将诱导提高伤害性DRG神经元中TRPM8的活性从而使DRG神经元更敏感。通过奥沙利铂诱导的冷痛敏小鼠模型，检测DeC-1.2能否影响奥沙利铂诱导的冷痛敏行为(图5b)。单次腹腔注射奥沙利铂(6mg/kg体重)十天后，小鼠出现明显的冷痛敏行为。通过行为学测试和钙成像实验检测了多肽DeC-1.2对奥沙利铂诱导的冷痛敏行为和初级感觉神经元中TRPM8活性的影响。在细胞水平上从奥沙利铂诱导的冷痛敏模型小鼠和正常小鼠中分离背根神经节 (dorsalrootganglion,DRG)进行神经元的原代培养(图5c，白色箭头指示)。原代培养DRG神经元的钙成像实验表明：对照组(生理盐水注射组)中，薄荷醇(Menthol)能够激活TRPM8引起神经元的钙活动；与对照组相比，奥沙利铂处理组小鼠DRG神经元中，Menthol激活TRPM8引起的钙反应强度、阳性反应细胞的比例均明显增加；而DeC-1.2则能够明显抑制奥沙利铂组小鼠DRG神经元的钙反应强度、阳性反应细胞的比例(图5d和5e)。这些结果表明：DeC-1.2多肽在奥沙利铂诱导的冷痛敏模型中能够抑制TRPM8通道的活性。
此外，通过足底注射DeC-1.2，进一步检测了其对丙酮挥发所致的冷痛觉反应的影响，DeC-1.2体内抑制奥沙利铂诱导的冷痛觉超敏反应效果如图6所示。行为学测试结果表明：奥沙利铂注射后10天，丙酮所引起的抬脚、舔脚、甩脚等冷痛觉行为的反应时间明显增加；对照组(足底注射生理盐水)小鼠的冷痛觉行为在足底注射前后无明显改变；而足底注射3.5μg/20μL DeC-1.2能够明显改善丙酮挥发引起的冷痛觉行为(图6a)。另外，DeC-1.2在体内抗冷痛觉超敏反应的程度与基因敲除TRPM8小鼠类似。另外，在机械触诱发痛(mechanical allodynia)行为的测试中，奥沙利铂注射后10天，小鼠在von Frey测试中的抬脚阈值明显降低，注射生理盐水和DeC-1.2对其抬脚阈值均不产生明显影响(图6b)。更重要的是，观察到足以引起小鼠WDS反应的(图5a)高剂量DeC-1.2肽30μg/g经静脉注射进入小鼠体内后并没有显著改变小鼠的基础体温(图6c)。激活模式特异性抑制剂DeC-1.2对TRPM8通道的工作原理示意图如图6d所示。因此，可以看出，环肽DeC-1.2能够通过TRPM8通道的活性改善小鼠的冷痛敏行为，但并不影响小鼠的体温。
2.3神经病变性疼痛的慢性压迫损伤(CCI)诱导之模型：异常性冷疼痛
雄性Sprague-Dawley大鼠(购自上海斯莱克实验动物有限责任公司，实验开始时为7周，n＝7-10/一次治疗)进行使用。按照Bennett GJ和Xie YK(Pain1988,33：87-107)的方法形成CCI状态。腹膜内注射戊巴比妥钠麻醉大鼠。使左侧共同坐骨神经以去掉粘附组织的状态暴露在大腿部的中间，在其周围利用4-0丝线(Ethicon Inc.)隔着约1mm的间隔松散地结扎4道。除了坐骨神经结扎以外通过相同方式实施假手术。CCI外科手术后经过1-2周后，如Tanimoto-Mori Setal.(BehavPharmacol.,19:85-90,2008)所记载，使用带温度控制器(Mode13300-0,CAL Controls Inc.)的冷却板(LHP-1700CP，TECA)对异常性冷疼痛进行评价。使动物适应不锈钢板(15×33cm)上的由透明亚克力盒 (10×12×12cm)构成的装置。将冷却板的表面维持为10℃，以0.1℃的精确度连续监控板的温度。为了进行实验，将大鼠载置于冷却板上，以120秒为截止值，前后给药多肽DeC-1.2，测定缩足潜伏期(PWL)。将多肽DeC-1.2或其媒介物口服给药、皮下给药或腹膜内给药。如抑制率(％)为[PWL(多肽)-PWL(媒介物)]/[PWL(多肽)-PWL(媒介物)]×100。
结果表明：多肽DeC-1.2在该模型中显出较强的活性，具有优异的治疗神经病变性疼痛的慢性压迫损伤的异常性冷疼痛效果。
2.4神经病变性疼痛的慢性压迫损伤(CCI)诱导模型；静态异常性疼痛
雄性Sprague Dawley大鼠(购自上海斯莱克实验动物有限责任公司，实验开始时为7周龄，n＝7-10/一次治疗)进行使用。按照Bennett GJ和Xie YK(Pain 1988,33：87-107)的方法形成CCI状态。腹膜内注射戊巴比妥钠麻醉大鼠。使左侧共同坐骨神经以去掉粘附组织的状态暴露在大腿部的中间，在其周围利用4-0丝线(Ethicon Inc.)隔着约1mm的间隔松散地结扎4道。除了坐骨神经结扎以外通过相同方式实施假手术。CCI外科手术后经过2-3周时，如Field MJ et al.(Pain 1999,83:303-311)所记载，通过纤毛机械刺激针(VFH)对静态异常性疼痛进行评价。开始实验之前，使实验动物适应网格底笼子中。将VFH紧贴后足足底，以逐渐增加力的方式进行实验(0.16g、0.4g、0.6g、1g、1.4g、2g、4g、6g、8g、10g、15g及26g)。将各VFH按压于相侧足6秒钟，或者按压至发生收缩反应。出现一次收缩反应，则以更弱的压力按压VFH再次进行实验，直至不发生收缩反应。将诱发反应所需的最低压力记为缩足阈值(PWT)。若实验动物在无害的1.4g VFH以下作出反应，则确定为静态异常性疼痛。将多肽DeC-1.2或其媒介物注射给药、口服给药、皮下给药或腹膜内给药。抑制率(％)为[PWL(多肽)-PWL(媒介物)]/[PWL(多肽)-PWL(媒介物)]×100。
结果表明：多肽DeC-1.2在该模型中显出较强的活性，具有优异的治疗神经病变性疼痛的慢性压迫损伤的静态异常性疼痛效果。
以上所述是本发明针对一种案例设计的实施方案，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下还可以作出若干改进，这些改进也应视为本发明的保护范围。

Claims (10)

  1. 一种多肽或其药学上可接受的盐，其特征在于，所述多肽或其药学上可接受的盐具有式I所示的结构：
    X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13   式I
    式中，
    X1为无、任意的氨基酸或任意的肽段；
    X2为R、K、Q、A、D或N；
    X3为R、K、Q、A、H或N；
    X4为D、A、S、R或E；
    X5为R、K、Q、A或N；
    X6为A、V、L、G、R或I；
    X7为R、K、Q、A或N；
    X8为H、N、Q、K、A或R；
    X9为Y、W、F、D、T、A、R或S；
    X10为R、K、Q、A、S、Y或N；
    X11为Q、A、R或N；
    X12为R、K、Q、A、K或N；
    X13为无、任意的氨基酸或任意的肽段。
  2. 如权利要求1所述的多肽或其药学上可接受的盐，其特征在于，所述多肽选自下组：
    (1)具有SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示氨基酸序列的多肽；
    (2)将SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示氨基酸序列 经过1-5个(较佳地1-3，更佳地1-2个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤；
    (3)与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19所示多肽相比，具有≥50％，≥60％，≥70％，≥80％，≥90％、≥95％、≥99％或100％的相同性(或同源性)的多肽。
  3. 一种融合蛋白，其特征在于，所述的融合蛋白包括：
    (a)如权利要求1所述的多肽或其药学上可接受的盐；
    (b)与如权利要求1所述的多肽或其药学上可接受的盐融合的肽段。
  4. 一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求1所述的多肽或其药学上可接受的盐。
  5. 一种载体，其特征在于，所述的载体包括如权利要求3所述的多核苷酸。
  6. 一种宿主细胞，其特征在于，所述的宿主细胞包括如权利要求4所述的载体或所述的宿主细胞的染色体上整合有如权利要求3所述的多核苷酸。
  7. 一种组合物，其特征在于，所述的组合物包括：
    (a)如权利要求1所述的多肽或其药学上可接受的盐；和
    (b)药学上可接受的载体或赋形剂。
  8. 一种如权利要求1所述的多肽或其药学上可接受的盐、如权利要求3所述的融合蛋白、如权利要求4所述的多核苷酸、如权利要求5所述的载体、如权利要求6所述的宿主细胞、和/或如权利要求7所述的组合物的用途，其特征在于，用于制备组合物，所述组合物用于选自下组的一种或多种用途：(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤。
  9. 如权利要求8所述的用途，其特征在于，所述的与TRPM8相关的疾 病选自下组：外周神经病变、冷痛觉超敏、瘙痒、神经病变性疼痛的慢性压迫损伤，或其组合；和/或
    所述的疼痛选自下组：慢性疼痛、冷痛觉超敏性疼痛、糖尿病性神经病变的神经病变性疼痛、术后疼痛、骨关节炎疼痛、类风湿关节炎疼痛、癌症疼痛、神经痛、神经损伤性疼痛、偏头痛、丛集性头痛、紧张性头痛、纤维肌痛、神经病变性疼痛、静态异常性疼痛、异常性冷疼痛，或其组合。
  10. 一种(a)抑制TRPM8；(b)预防和/或治疗与TRPM8相关的疾病；(c)预防和/或治疗冷痛觉超敏；(d)预防和/或治疗外周神经病变；(e)预防和/或治疗疼痛；和/或(f)预防和/或治疗神经病变性疼痛的慢性压迫损伤的方法，其特征在于，所述的方法包括步骤：向需要的对象施用如权利要求1所述的多肽或其药学上可接受的盐、如权利要求3所述的融合蛋白、如权利要求4所述的多核苷酸、如权利要求5所述的载体、如权利要求6所述的宿主细胞、和/或如权利要求7所述的组合物。
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WO2020146823A1 (en) * 2019-01-10 2020-07-16 Roswell Biotechnologies Inc. Conductive synthetic peptides for molecular electronics

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