A polypeptide inhibiting TRPM8 and its use

Abstract

The present invention relates to a polypeptide for inhibiting TRPM8 and its use. Specifically, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide of the present invention has an excellent inhibitory effect on TRPM8 and is used for preventing and treating diseases related to TRPM8.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a polypeptide for inhibiting TRPM8 and uses thereof. Background Art

[0002] Peripheral neuropathy is a common side effect of many platinum-based chemotherapies, which limits the dosage of anticancer drugs and reduces the quality of life of patients taking these drugs. For example, oxaliplatin is often used as a first-line chemotherapy for many tumors, such as colorectal cancer and gastric cancer. However, up to 89% of patients taking oxaliplatin experience acute neurotoxicity, which is usually manifested as patients feeling extremely cold in normally cool ambient temperatures and excruciating pain in their arms and legs. Although these neuropathic symptoms in patients, including oxaliplatin-induced cold allodynia, improve after stopping the drug, there is still no effective treatment for this type of neuropathy.

[0003] TRPM8 is a type of non-selective ion channel expressed on nociceptive 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, as well as transmembrane depolarization. In the oxaliplatin-induced cold hyperalgesia mouse model, the expression level of TRPM8 channels in nociceptive dorsal root ganglion (DRG) neurons was significantly increased. More importantly, in TRPM8 knockout mice, the administration of oxaliplatin did not cause cold hyperalgesia. Therefore, the TRPM8 ion channel is an effective drug target for peripheral neuropathy and hyperalgesia caused by platinum drugs. There is an urgent need to develop effective drugs targeting the TRPM8 ion channel for the treatment of peripheral neuropathy and hyperalgesia. Summary of the invention

[0004] The purpose of the present invention is to provide a TRPM8 inhibitor and a polypeptide thereof for preventing and treating diseases related to TRPM8.

[0005] In a first aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide or a pharmaceutically acceptable salt thereof has a structure as shown in Formula I:

[0006] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13 Formula I

[0007] In the formula,

[0008] X1 is none, any amino acid or any peptide segment;

[0009] X2 is R, K, Q, A, D or N;

[0010] X3 is R, K, Q, A, H or N;

[0011] X4 is D, A, S, R or E;

[0012] X5 is R, K, Q, A or N;

[0013] X6 is A, V, L, G, R or I;

[0014] X7 is R, K, Q, A or N;

[0015] X8 is H, N, Q, K, A or R;

[0016] X9 is Y, W, F, D, T, A, R or S;

[0017] X10 is R, K, Q, A, S, Y or N;

[0018] X11 is Q, A, R or N;

[0019] X12 is R, K, Q, A, K or N;

[0020] X13 is none, any amino acid or any peptide segment.

[0021] In another preferred embodiment, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12 and X13 are each independently an L-amino acid or a D-amino acid.

[0022] In another preferred embodiment, the polypeptide or a pharmaceutically acceptable salt thereof has the effects of (a) inhibiting TRPM8; (b) preventing and / or treating diseases related to TRPM8; (c) preventing and / or treating cold allodynia; (d) preventing and / or treating peripheral neuropathy; (e) preventing and / or treating pain; and / or (f) preventing and / or treating chronic compression injuries of neuropathic pain.

[0023] In another preferred embodiment, the polypeptide is an isolated polypeptide.

[0024] In another preferred embodiment, the polypeptide is artificially synthesized.

[0025] In another preferred embodiment, the polypeptide is a recombinant polypeptide.

[0026] In another preferred embodiment, X1 is none, C (cysteine) or M (methionine).

[0027] In another preferred embodiment, X13 is none or C (cysteine).

[0028] In another preferred embodiment, the peptide segment includes a tag protein, a leader sequence or a secretory sequence.

[0029] In another preferred embodiment, the length of X1 is 1-20aa, more preferably 1-10aa, and more preferably 1-5aa.

[0030] In another preferred embodiment, the length of X13 is 1-20aa, more preferably 1-10aa, and more preferably 1-5aa.

[0031] In another preferred embodiment, 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.

[0032] In another preferred embodiment, the X1 or X13 comprises a natural or unnatural amino acid.

[0033] In another preferred embodiment, X1 and X13 form a cyclic peptide.

[0034] In another preferred embodiment, at least one pair of disulfide bonds is formed between X1 and X13.

[0035] In another preferred embodiment, a pair of disulfide bonds is formed between X1 and X13.

[0036] In another preferred embodiment, the polypeptide has a structure shown in Formula II:

[0037] X1-RRDRARHYRQR-X13 Formula II

[0038] Wherein, the definitions of X1 and X13 are as described above.

[0039] In another preferred embodiment, the polypeptide is an N-mer.

[0040] In another preferred embodiment, the N-mer has the following structure of formula III:

[0041] -(X1-RRDRARHYRQR-X13-L1)n- Formula III

[0042] Wherein, X1 and X13 are defined as above; L1 is nothing or a connecting peptide; n is 2-10, preferably 2-7, more preferably 2-5; each "-" is independently a connecting peptide or a peptide bond.

[0043] In another preferred embodiment, the L1 is absent.

[0044] In another preferred embodiment, the length of L1 is 1-30aa, preferably, 1-20aa, and more preferably, 1-10aa.

[0045] In another preferred embodiment, n is 2, 3 or 4.

[0046] In another preferred embodiment, each "-" is independently a peptide bond.

[0047] In another preferred example, the sequence of the polypeptide is as shown in 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, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

[0048] In another preferred embodiment, the polypeptide represented by Formula I has ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥99% or 100% identity (or homology) compared with the polypeptide represented by 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 or SEQ ID NO: 19.

[0049] In another preferred embodiment, the polypeptide represented by Formula I retains at least ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, or ≥99%, for example 90-100% of the biological activity of the polypeptide represented by 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 or SEQ ID NO:19.

[0050] In another preferred embodiment, the sequence of the polypeptide is shown in SEQ ID NO.:1.

[0051] In another preferred embodiment, the polypeptide represented by formula I has ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥99% or 100% identity (or homology) compared with the polypeptide represented by SEQ ID NO.: 1.

[0052] In another preferred embodiment, the polypeptide represented by formula I retains at least ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, or ≥99%, for example 90-100% of the biological activity of the polypeptide represented by SEQ ID NO:1.

[0053] In another preferred embodiment, the polypeptide is selected from the following group:

[0054] (1) a polypeptide having an amino acid sequence as shown in 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 or SEQ ID NO: 19;

[0055] (2) The amino acid sequence 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: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 is substituted, deleted or added with 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and has the functions of (a) inhibiting TRPM8; (b) preventing and / or treating diseases related to TRPM8; (c) preventing and / or treating cold allodynia; (d) preventing and / or treating peripheral neuropathy; (e) preventing and / or treating pain; and / or (f) preventing and / or treating chronic compression injury of neuropathic pain;

[0056] (3) A polypeptide having ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥99% or 100% identity (or homology) with the polypeptide set forth in 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 or SEQ ID NO:19.

[0057] In another preferred embodiment, the polypeptide is selected from the following group:

[0058] (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1;

[0059] (2) The amino acid sequence shown in SEQ ID NO: 1 is formed by replacing, deleting or adding 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and has the effects of (a) inhibiting TRPM8; (b) preventing and / or treating diseases related to TRPM8; (c) preventing and / or treating cold allodynia; (d) preventing and / or treating peripheral neuropathy; (e) preventing and / or treating pain; and / or (f) preventing and / or treating chronic compression injuries of neuropathic pain;

[0060] (3) A polypeptide having ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95%, ≥99% or 100% identity (or homology) with the polypeptide shown in SEQ ID NO.: 1.

[0061] In another preferred embodiment, the polypeptide is a polypeptide represented by 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 or SEQ ID NO:19, through 1-3, preferably 1-2, more preferably 1 amino acid substitution, deletion; and / or

[0062] It is formed by adding 1-5, preferably 1-4, more preferably 1-3, and most preferably 1-2 amino acids.

[0063] In another preferred embodiment, the derived polypeptide retains ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥99%, or 100%, for example 80-100%, preferably 95-100% of the polypeptide shown in 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 or SEQ ID NO:19 (a) inhibiting TRPM8; (b) preventing and / or treating diseases related to TRPM8; (c) preventing and / or treating cold allodynia; and / or (d) preventing and / or treating peripheral neuropathy.

[0064] In another preferred embodiment, the derived polypeptide has an identity of ≥50% with 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 or SEQ ID NO:19, preferably, ≥60%, more preferably, ≥70%, more preferably, ≥80%, more preferably, ≥90%, more preferably, ≥99%.

[0065] In another preferred embodiment, the polypeptide is the polypeptide shown in SEQ ID NO.: 1 with 1-3, preferably 1-2, more preferably 1 amino acid substitutions or deletions; and / or

[0066] It is formed by adding 1-5, preferably 1-4, more preferably 1-3, and most preferably 1-2 amino acids.

[0067] In another preferred embodiment, the derived polypeptide retains ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥99%, or 100%, for example 80-100%, preferably 95-100% of the polypeptide shown in SEQ ID NO: 1 (a) inhibiting TRPM8; (b) preventing and / or treating diseases related to TRPM8; (c) preventing and / or treating cold allodynia; and / or (d) preventing and / or treating peripheral neuropathy.

[0068] In another preferred embodiment, the derivative polypeptide has an identity of ≥50% with SEQ ID NO: 1, preferably, ≥60%, more preferably, ≥70%, more preferably, ≥80%, more preferably, ≥90%, more preferably, ≥99%.

[0069] In a second aspect, the present invention provides a fusion protein, wherein the fusion protein comprises:

[0070] (a) the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof;

[0071] (b) A peptide segment fused to the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0072] In another preferred embodiment, the peptide segment includes a carrier protein.

[0073] In another preferred embodiment, the carrier protein is selected from the following group: Fc fragment, human serum albumin (HSA), CTP, transferrin, or a combination thereof.

[0074] In another preferred embodiment, the peptide segment is modified.

[0075] In another preferred embodiment, the modification includes polyethylene glycol (PEG) modification.

[0076] The third aspect of the present invention provides a polynucleotide encoding the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention.

[0077] In another preferred embodiment, the polynucleotide is an isolated polynucleotide.

[0078] The fourth aspect of the present invention provides a vector, wherein the vector comprises the polynucleotide as described in the third aspect of the present invention.

[0079] In another preferred embodiment, the vector comprises a plasmid vector.

[0080] The fifth aspect of the present invention provides a host cell, wherein the host cell comprises the vector as described in the fourth aspect of the present invention or the polynucleotide as described in the third aspect of the present invention is integrated into the chromosome of the host cell.

[0081] In a sixth aspect, the present invention provides a composition comprising:

[0082] (a) the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof; and

[0083] (b) a pharmaceutically acceptable carrier or excipient.

[0084] In another preferred embodiment, the composition is a pharmaceutical composition.

[0085] In another preferred embodiment, the composition is administered by a mode of administration selected from the group consisting of intravenous, intratumoral, intracavitary, subcutaneous or hepatic arterial administration (such as injection, drip, etc.).

[0086] In another preferred embodiment, the composition is in the form of an oral preparation, an injection preparation or an external preparation.

[0087] In another preferred embodiment, the dosage form of the composition is a solid preparation, a liquid preparation or a semisolid preparation.

[0088] In another preferred embodiment, the preparation of the composition is selected from the following group: tablets, capsules, injections, granules, sprays, and lyophilized agents.

[0089] In another preferred embodiment, the composition is in the form of an injection.

[0090] In another preferred embodiment, the injection is an intravenous injection, an intramuscular injection or a subcutaneous injection.

[0091] In another preferred embodiment, the polypeptide is administered to a mammal at a dose of 0.01-100 mg / kg body weight (each time or every day).

[0092] In the seventh aspect of the present invention, there is provided a use of the polypeptide or pharmaceutically acceptable salt thereof as described in the first aspect of the present invention, 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 vector 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 composition as described in the sixth aspect of the present invention, for preparing a composition, wherein the composition is used for one or more uses selected from the following groups: (a) inhibiting TRPM8; (b) preventing and / or treating diseases related to TRPM8; (c) preventing and / or treating cold allodynia; (d) preventing and / or treating peripheral neuropathy; (e) preventing and / or treating pain; and / or (f) preventing and / or treating chronic compression injuries of neuropathic pain.

[0093] In another preferred embodiment, the composition is a pharmaceutical composition.

[0094] In another preferred embodiment, the disease associated with TRPM8 is selected from the group consisting of peripheral neuropathy, cold allodynia, pruritus, chronic compression injury of neuropathic pain, or a combination thereof.

[0095] In another preferred embodiment, the pain is selected from the group consisting of chronic pain, cold allodynia pain, neuropathy pain of diabetic neuropathy, postoperative pain, osteoarthritis pain, rheumatoid arthritis pain, cancer pain, neuralgia, nerve injury pain, migraine, cluster headache, tension headache, fibromyalgia, neuropathy pain, static allodynia, abnormal cold pain, or a combination thereof.

[0096] In another preferred embodiment, the abnormal cold pain includes abnormal cold pain caused by chronic compression injury of neuropathic pain.

[0097] In another preferred embodiment, the static allodynia includes static allodynia caused by chronic compression injury of neuropathic pain.

[0098] In another preferred embodiment, the diseases associated with TRPM8 include diseases in which TRPM8 is upregulated.

[0099] In another preferred embodiment, the up-regulation of TRPM8 includes high expression level or activity of TRPM8.

[0100] In another preferred embodiment, the TRPM8 up-regulation refers to that the TRPM8 expression level or activity of a certain cell (such as a stimulus-receptor neuron or dorsal root ganglion) is greater than the TRPM8 expression level or activity of the same cell.

[0101] In another preferred embodiment, the TRPM8 upregulation refers to the ratio of the TRPM8 expression level or activity C1 of a certain cell (such as a stimulus-receptor 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, and more preferably ≥ 5.

[0102] In another preferred embodiment, the cells include nerve cells.

[0103] In another preferred embodiment, the stimulus-receptor neurons include nociceptive stimulus-receptor neurons.

[0104] In another preferred embodiment, the dorsal root ganglia include nociceptive dorsal root ganglia.

[0105] In another preferred embodiment, the same cell refers to cells of the same type with normal TRPM8 expression or activity.

[0106] In another preferred embodiment, the expression includes mRNA and / or protein expression.

[0107] In another preferred embodiment, the cold allodynia includes cold allodynia caused by platinum anticancer drugs.

[0108] In another preferred embodiment, the peripheral neuropathy includes peripheral neuropathy caused by platinum anticancer drugs.

[0109] In another preferred embodiment, the cold allodynia includes cold allodynia caused by platinum anticancer drugs.

[0110] In another preferred embodiment, the platinum anticancer drug is selected from the following group: carboplatin, nedaplatin, lobaplatin, oxaliplatin, or a combination thereof.

[0111] In an eighth aspect, 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 allodynia; (d) preventing and / or treating peripheral neuropathy; (e) preventing and / or treating pain; and / or (f) preventing and / or treating chronic compression injuries of neuropathic pain, the method comprising the steps of administering to a subject in need thereof the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention, 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 vector 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 composition as described in the sixth aspect of the present invention.

[0112] In another preferred embodiment, the subject is a human or a non-human mammal.

[0113] In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, rabbits) and primates (such as monkeys).

[0114] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0115] In another preferred embodiment, the administration is oral administration, injection administration or external administration.

[0116] In another preferred embodiment, the injection is intravenous injection, intramuscular injection or subcutaneous injection.

[0117] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1The HPLC purification, mass spectrometry verification and electrophysiological verification of the designed peptides after synthesis are shown in Figure 2. (a) HPLC purification of DeC-1.1 peptide. (b) Mass spectrometry verification of the molecular weight of DeC-1.1 peptide. (c) HPLC purification of DeC-1.2 peptide. (d) Mass spectrometry verification of the molecular weight of DeC-1.2 peptide. (e) Representative curve of whole-cell electrophysiological recording of DeC-1.2 inhibiting menthol-activated mouse TRPM8 channels. (f) Inhibitory concentration-dependent curves of DeC-1.1 and DeC-1.2 measured in whole-cell electrophysiological recording mode.

[0119] Figure 2 It is the activation mode-specific inhibition of TRPM8 by Dec-1.2. (a) Representative electrophysiological recordings of TRPM8 from the outside to the outside at +80 mV. TRPM8 channels were activated or inhibited by perfusion of saturated menthol (1 mM) and a mixed solution of saturated menthol (1 mM) and Dec-1.2 (100 μM), respectively. These four representative single-channel recordings are from the same cell membrane. (b) The whole-point histogram corresponding to the electrophysiological recording of TRPM8 single channel shown in Figure (a). The histogram was fitted with a double Gaussian function (indicated by the solid red line), and the single-channel conductance was calculated by the difference between two adjacent peaks after fitting. (c) The open probability of TRPM8 channels activated by saturating concentrations of Menthol (1 mM) was significantly reduced in the presence of Dec-1.2 (100 μM). Data are shown as the mean ± SEM of electrophysiological recording data of five independent cells in each group. **** indicates P < 0.0001. (d) The single-channel conductance of TRPM8 channels activated by saturating concentrations of Menthol (1 mM) was significantly decreased in the presence of DeC-1.2 (100 μM). Data are shown as the mean ± SEM of electrophysiological recordings from three independent cells in each group. *** indicates P < 0.001. (e) Representative electrophysiological recordings of single channels from inside to outside when TRPM8 was cold-activated at +80 mV in the presence or absence of DeC-1.2. (f) The whole-point histogram corresponding to the TRPM8 single-channel electrophysiological recording shown in Figure (e). The histogram was fitted with a double Gaussian function. (g) At 16°C, the open probability of TRPM8 channels activated by cold was not significantly changed in the presence of DeC-1.2 (100 μM). Data are shown as the mean ± SEM of electrophysiological recordings from five independent cells in each group. NS indicates no significant difference. (h) At 16°C, the single-channel conductance of TRPM8 channels activated by cold did not change significantly in the presence of DeC-1.2 (100 μM). Data are shown as the mean ± SEM of electrophysiological recordings of three independent cells in each group. NS indicates no significant difference.

[0120] Figure 3is 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 to exclude the dependence of TRPM4 on PIP2. (f) Normalized comparison of the inhibitory activity of Dec-1.2 at 500 nM on TRP channels and Nav channels. Each channel corresponds to n = 3, **** indicates P < 0.0001.

[0121] Figure 4 It is the amino acid residue that plays a key role in the inhibition of TRPM8 by Dec-1.2. (a, b, c, d, e, f, g) Representative electrophysiological records of the inhibition of ligand activation of TRPM8 by wild-type Dec-1.2 and each mutant at 5 nM. (h, i) Normalized graphs of the inhibition of ligand activation of TRPM8 by wild-type Dec-1.2 and mutants at 5 nM, *, * and *** indicate P<0.05, P<0.01, P<0.001. (j) The amino acid residues that play a key role in the inhibition of TRPM8 marked in the structure of Dec-1.2 (mutations that completely eliminate inhibition and mutations that partially eliminate inhibition at a concentration of 5 nM are indicated in orange and orange, respectively).

[0122] Figure 5This is to verify the inhibitory effect of DeC-1.2 on TRPM8 from an in vivo perspective. (a) DeC-1.2 significantly reduced the wet dog-like whole body shivering reaction in mice induced by Icilin in a dose-dependent manner. The data are shown as the mean ± SEM of six animals in each group. *, * and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively. (b) Schematic diagram of the process of establishing oxaliplatin-induced cold hyperalgesia in mice and the animal experiment flow chart of DeC-1.2 inhibiting cold hyperalgesia. (c) Representative calcium imaging responses when DRG neurons extracted from mice treated with normal saline were perfused with extracellular fluid (Vehicle), DRG neurons extracted from mice treated with oxaliplatin were perfused with extracellular fluid (Oxaliplatin) or extracellular fluid containing 10 μM DeC-1.2 (Oxaliplatin + DeC-1.2). After perfusion of 100 μM menthol, calcium influx in DRG neurons increased as menthol activated TRPM8 channels. 1 μM capsaicin activated TRPV1 in DRG neurons and produced calcium influx cells, which served as a positive control. Neurons that responded to the agonist are indicated by white arrows. (d) Representative curves of the fluorescence ratio of DRG neurons extracted from saline-treated and oxaliplatin-treated mice in response to the corresponding reagents. (e) The ratio of DRGA neurons extracted from saline-treated and oxaliplatin-treated mice in response to menthol. The total number of neurons in each group corresponding to Vehicle, oxaliplatin and oxaliplatin+DeC-1.2 were 229, 238 and 334, respectively. *, * indicates P < 0.05 and P < 0.01, respectively. Each discrete circle represents the number of neurons in a field of view.

[0123] Figure 6Figure 1 is the inhibitory effect of DeC-1.2 on oxaliplatin-induced cold allodynia in vivo. (a) The inhibitory effect of DeC-1.2 on oxaliplatin-induced cold allodynia. The total time of paw lifting, flipping or licking (here all acetone-treated paws) was recorded within 5 min after acetone treatment, as the acetone score, to evaluate the inhibitory effect of DeC-1.2. The data are shown as the mean ± SEM of six animals in each group. * indicates p < 0.05 after two-way ANOVA analysis. (b) The inhibitory effect of DeC-1.2 on oxaliplatin-induced mechanical touch-evoked allodynia. The data are shown as the mean ± SEM of 5-8 mice in each group. (c) The effect of DeC-1.2 on body temperature. The rectal temperature of each animal was measured before (baseline, BL), 5 min, 15 min, 0.5 h, 1 h, 2 h, 6 h and 24 h after DeC-1.2 injection (30 μg / g, iv). Data are shown as mean ± SEM of six animals. After one-way ANOVA analysis, NS indicates no significant difference. (d) Schematic diagram of the working principle of activation mode-specific inhibitor DeC-1.2 on TRPM8 channel. DETAILED DESCRIPTION

[0124] After extensive and in-depth research, the inventors have prepared for the first time a polypeptide such as DeC-1.2 polypeptide that has the functions of (a) inhibiting TRPM8; (b) preventing and / or treating diseases related to TRPM8; (c) preventing and / or treating cold allodynia; (d) preventing and / or treating peripheral neuropathy; (e) preventing and / or treating pain; and / or (f) preventing and / or treating chronic compression injury of neuropathy pain, and the polypeptide of the present invention has good safety and little toxicity and side effects on biological tissues. On this basis, the inventors have completed the present invention.

[0125] the term

[0126] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0127] As used herein, the terms "include", "comprises", and "comprising" are used interchangeably, and include not only open definitions, but also semi-closed and closed definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0128] In the present invention, each amino acid and its abbreviation are shown in the following Table 1:

[0129] Table 1 Amino acids and their abbreviations

[0130] name Three letter symbols Single letter symbols name Three letter symbols Single letter symbols Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamine Gln Q Serine Ser s Glutamic acid Glu E Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V

[0131] In the present invention, the term "prevention" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease.

[0132] The "treatment" of the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, the polypeptide of the present invention reduces, inhibits and / or reverses the disease associated with TRPM8 by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, or 100%, compared to the level observed in the absence of the polypeptide of the present invention.

[0133] TRPM8

[0134] TRPM8 (Transient receptor potential melastatin 8), also known as the cold and menthol receptor, is a member of the transient receptor potential ion channel protein TRP family. The channel is composed of 4 identical subunits, each with 6 transmembrane regions, and the N-terminus and C-terminus are both located inside the cell. The pore region located in the center of the TRPM8 channel is composed of 4 subunits and can non-selectively permeate cations. The TRPM8 channel is involved in the regulation of the body's cold sensation, pain, inflammatory response, vasoconstriction and dilation, cell growth and proliferation, etc.

[0135] Peptides

[0136] In the present invention, the term "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 a mixture of the above components. In addition, the 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, and more preferably within 2) amino acids to the N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. Adding one or several amino acids to the N-terminus generally does not change the structure and function of the protein. In addition, the term also includes monomeric and polymeric forms of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof.

[0137] In the present invention, the amino acid sequence of a polypeptide is numbered from the N-terminus to the C-terminus.

[0138] The present invention also includes active fragments, derivatives and analogs of the polypeptides of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of the polypeptide of the present invention with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (and then proteins formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). According to the teachings herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0139] A preferred class of active derivatives refers to polypeptides formed by replacing at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid with similar or similar properties compared to the amino acid sequence of the polypeptide shown in Formula I. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 2.

[0140] Table 2

[0141]

[0142]

[0143] The invention also provides analogs of the polypeptides of the present invention. The difference between these analogs and the natural polypeptides of the present invention may be a difference in the amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). For example, Cys can form a disulfide bond with non-natural Hcy. It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0144] Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0145] In a preferred embodiment, the polypeptide of the present invention has at least one internal disulfide bond (introduced intrachain disulfide bond). Surprisingly, 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. Generally, it can be formed by conventional methods in the art, such as allowing cysteine ​​or homocysteine ​​sulfhydryl groups to combine under oxidative conditions to form a disulfide bond.

[0146] A preferred polypeptide of the present invention includes polypeptide DeC-1.2, the amino acid sequence of polypeptide DeC-1.2 is the amino acid sequence described in SEQ ID No.: 1:

[0147] SEQ ID NO.: 1

[0148] CRRDRARHYRQRC.

[0149] In the present invention, the terms "DeC-1.2", "polypeptide DeC-1.2" or "DeC-1.2 polypeptide" are used interchangeably.

[0150] The polypeptide of the present invention also includes a polypeptide obtained by modifying the polypeptide shown in SEQ ID NO.: 1.

[0151] 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) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium), as well as in the form of esters, carbamates or other conventional "prodrugs".

[0152] Coding sequence

[0153] The present invention also relates to polynucleotides encoding the polypeptides of the present invention. The polynucleotides of the present invention may be in the form of DNA or RNA. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence or may be a degenerate variant. The full-length nucleotide sequence of the polypeptide of the present invention or its fragments may generally be obtained by PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the polypeptide of the present invention (or its fragments, or its derivatives) may be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0154] The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the polypeptide coding sequence of the present invention.

[0155] On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies or antibody fragments specific for the polypeptide of the present invention, especially monoclonal antibodies.

[0156] In the context of two nucleic acids or polypeptides, when compared and aligned for maximum correspondence, the term "substantially identical" refers to two or more sequences or subsequences that have at least about 80%, e.g., at least about 85%, about 90%, about 95%, about 98%, or about 99% of the nucleotides or amino acid residues identical to a particular reference sequence as determined using the following sequence comparison methods and / or by visual inspection.

[0157] Preparation method

[0158] The polypeptide of the present invention can be a recombinant polypeptide or a synthetic polypeptide. The polypeptide of the present invention can be chemically synthesized or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by conventional methods or produced by recombinant methods.

[0159] 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 appropriate resin carriers and synthesis systems can be selected according to the sequence characteristics of the target peptide. For example, the preferred solid phase carrier in the Fmoc system is Wang resin connected to 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; it is treated with 25% hexahydropyridine / dimethylformamide at room temperature for 20 minutes to remove the Fmoc protecting group, and extend from the C-terminus to the N-terminus one by one according to the given amino acid sequence. After the synthesis is completed, the synthesized proinsulin-related peptide is cut from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protecting group is removed. The crude peptide can be separated by ether precipitation after filtering the resin. After the solution of the obtained product is freeze-dried, the desired peptide is purified by gel filtration and reversed-phase high-pressure liquid chromatography. When using the Boc system for solid phase synthesis, the preferred resin is a PAM resin connected to the C-terminal amino acid in the peptide, the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethylphenylacetamide; in the Boc synthesis system, in the cycle of deprotection, neutralization, and coupling, TFA / dichloromethane (DCM) is used to remove the protective group Boc and diisopropylethylamine (DIEA / dichloromethane is neutralized. After the peptide chain condensation is completed, it is treated with hydrogen fluoride (HF) containing p-cresol (5-10%) at 0°C for 1 hour to cut the peptide chain from the resin and remove the protective group at the same time. The peptide is extracted with 50-80% acetic acid (containing a small amount of mercaptoethanol), and the solution is lyophilized and further filtered with molecular sieve Sephadex G10 or Tsk-40f is separated and purified, and then purified by high pressure liquid phase to obtain the desired peptide. Various coupling agents and coupling methods known in the field of peptide chemistry can be used to couple the amino acid residues, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea hexafluorophosphate (HBTU) can be used for direct coupling. For the synthesized short peptide, its purity and structure can be confirmed by reverse phase high performance liquid chromatography and mass spectrometry analysis.

[0160] In a preferred embodiment, the polypeptide of the present invention is prepared according to its sequence by solid phase synthesis, purified by high performance liquid chromatography, and a high purity target peptide lyophilized powder is obtained, which is stored at -20°C.

[0161] Another method is to use recombinant technology to produce the polypeptide of the present invention. Through conventional recombinant DNA technology, the polynucleotide of the present invention can be used to express or produce recombinant polypeptide of the present invention. Generally speaking, there are the following steps:

[0162] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a polypeptide of the present invention, or a recombinant expression vector containing the polynucleotide;

[0163] (2) Host cells cultured in a suitable culture medium;

[0164] (3) Isolate and purify proteins from culture medium or cells.

[0165] The recombinant polypeptide can be expressed in the cell or on the cell membrane or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0166] Since the polypeptide of the present invention is relatively short, it is possible to consider connecting multiple polypeptides in series, obtaining a polymeric expression product after recombinant expression, and then forming the desired small peptide by enzyme cleavage or the like.

[0167] Compositions and methods of administration

[0168] The present invention also provides a composition, which is preferably a pharmaceutical composition.

[0169] The composition of the present invention comprises (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 generally 10 μg-100 mg / dose, preferably 100-1000 μg / dose.

[0170] For the purpose of prevention and treatment of the present invention, in the composition of the present invention, the polypeptide of the present invention or its pharmaceutically acceptable salt is a safe and effective amount, and the effective dose is about 0.01 mg / kg to 50 mg / kg, preferably 0.05 mg / kg to 10 mg / kg of the polypeptide of the present invention or its pharmaceutically acceptable salt given to the individual. In addition, the polypeptide of the present invention or its pharmaceutically acceptable salt can be used alone or together with other therapeutic agents (such as formulated in the same pharmaceutical composition).

[0171] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not induce the production of antibodies harmful to the individual receiving the composition, and there is no excessive toxicity after administration. These carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.

[0172] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers.

[0173] Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.

[0174] Once formulated, the composition of the present invention can 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.

[0175] In a preferred embodiment of the present invention, the dosage form of the pharmaceutical composition is an oral preparation, an injection preparation or an external preparation.

[0176] In a preferred embodiment of the present invention, the dosage form of the pharmaceutical composition is a solid preparation, a liquid preparation or a semisolid preparation.

[0177] In a preferred embodiment of the present invention, the preparation of the pharmaceutical composition is selected from the following group: tablets, capsules, injections, granules, sprays, and lyophilized agents.

[0178] Typically, the injection is an intravenous injection, an intramuscular injection or a subcutaneous injection.

[0179] When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the usage, preferably intravenous preparations or intratumoral injections.

[0180] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and appropriate pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers are occasionally added, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0181] For example, 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 the basic substance, the osmotic pressure and pH are adjusted to physiological state, and suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and viscosity enhancers can be added arbitrarily, and then completely dissolved.

[0182] The pharmaceutical composition of the present invention can also be administered in the form of a sustained release agent. For example, the polypeptide of the present invention or a pharmaceutically acceptable salt thereof can be incorporated into a pill or microcapsule with a sustained release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. As examples of sustained release polymers, ethylene-vinyl acetate copolymers, polyhydroxymethylacrylate (polyhydrometaacrylate), polyacrylamide, polyvinyl pyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc. can be exemplified, and preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers can be exemplified.

[0183] The pharmaceutical preparation should be matched with the mode of administration. The medicament of the present invention can also be used with other synergistic therapeutic agents (including before, during or after use). When using the pharmaceutical composition, a safe and effective amount of the drug is applied to the desired object (such as a human or non-human mammal), and the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 8 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight-about 1 milligram / kg body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0184] The main effects of the present invention include:

[0185] (1) The polypeptide of the present invention can effectively inhibit (a) TRPM8; (b) prevent and / or treat diseases related to TRPM8; (c) prevent and / or treat cold allodynia; (d) prevent and / or treat peripheral neuropathy; (e) prevent and / or treat pain; and / or (f) prevent and / or treat chronic compression injuries of neuropathic pain.

[0186] (2) The polypeptide of the present invention and its derivative polypeptides have small molecular weight, small toxicity and side effects on biological tissues, and high safety.

[0187] (3) The polypeptide of the present invention has good stability.

[0188] (4) The polypeptide of the present invention has high specificity.

[0189] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0190] Example 1

[0191] Experimental methods:

[0192] Peptide synthesis and purification

[0193] Peptides with or without disulfide bonds were chemically synthesized at Gill Biochemical (Shanghai). The crude product was further purified by reversed phase liquid chromatography. Peptide purification efficiency of 95% or more can be confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) or electrospray ionization mass spectrometry (ESI-MS). After purification, the corresponding cyclic peptides were collected in large quantities for future use.

[0194] Cell culture and transient transfection

[0195] HEK293T cells were cultured in Dulbecco's modified Eagle's medium at 37°C in a 5% carbon dioxide atmosphere, with 10% fetal bovine serum, penicillin (100u / ml) and streptomycin (100mg / ml) added to the medium. Transient transfection was performed using the Lipo 2000 (Invitrogen) transfection kit according to the instructions.

[0196] Electrophysiological recordings

[0197] Electrophysiological recordings were performed within 24-48 hours of transient transfection. Single-channel recordings were performed within 8 hours of transfection. Cellular currents were measured in whole-cell, inside-out, and outside-out recording modes using a HEKA EPC10 amplifier. For whole-cell recordings, glass microelectrodes made of borosilicate were pulled and polished to a primary electrode resistance of approximately 3-6 MΩ. Serial resistance was complemented by 60%. Whole-cell recordings were performed at ±80 mV. For single-channel recordings, glass microelectrodes were pulled and polished to 6-8 MΩ. Currents were collected at 10 kHz and filtered at 2.9 kHz. Cell membrane potential was clamped at +80 mV. All recordings were performed at room temperature at 22°C (maximum variation of 1°C).

[0198] There will be different internal and external solution formulas for the current measurement of different ion channels. For ion channels such as TRPM8, TRPV1, TRPV3 and TRPA1, the internal and external solution formulas are: 130mM NaCl, 0.2mM EDTA and 3mM HEPES, pH = 7.2. For the current measurement of TRPM4 ion channels, the external solution formula is: 130mM NaCl and 3mM HEPES, pH = 7.2, and the internal solution formula is 500μM CaCl2, 130mM NaCl and 3mM HEPES, pH = 7.2. For the current measurement of TRPM2 ion channels, the external solution formula is: 147mM NaCl, 2mM KCl, 1mM MgCl2, 10mM HEPES, 2mM CaCl2 13mM Glucose, pH = 7.4, and the internal solution is 147mM NaCl, 1mM MgCl2, 10mM HEPES, pH = 7.4. For the current measurement of NaV1.5, NaV1.7 and other ion channels, the external solution formula is: 140mM NaCl, 3mM KCl, 1mM MgCl2, 1mM CaCl2, 10mM HEPES, pH = 7.2, and the internal solution formula is: 140mM CsF, 1mM EGTA, 10mM NaCl, 10mM MgCl2, 3mM KCl, pH = 7.2. In order to stimulate the current of the sodium channel, the membrane potential is increased from -80mV to +10Mv.

[0199] A gravity-driven rapid perfusion system (RSC-200, BioLogic) was used to rapidly perfuse specific solutions to the cells to be tested. Different solutions were delivered through separate PVC tubes to avoid cross-contamination between solutions. During electrophysiological recording, the glass microelectrode with a membrane was moved just below the outlet of the perfusion tube.

[0200] Animal experiments

[0201] Adult male CD1 mice (6-8 weeks old) were used as experimental animals for behavioral experiments and primary culture of DRG neurons. Mice were housed under a 12-h light / 12-h dark light cycle and provided with sufficient standard feed and water. All animal experimental protocols were approved by the Animal Care and Use Committee (IACUC) of Zhejiang University.

[0202] Mouse models of pain

[0203] All mice were acclimated to the behavioral testing environment for 1 to 2 hours every day 2 to 3 days before the behavioral test. A single intraperitoneal injection of oxaliplatin (6 mg / kg body weight) was used to induce the chemotherapy-induced neuropathic pain model. Behavioral testing of neuropathic pain was performed on the 10th day after oxaliplatin injection.

[0204] Icilin-induced wet-dog shivering behavior test

[0205] The control group and experimental group each had at least 6 adult male CD1 mice, which were injected with physiological saline and gradient doses of DeC-1.2 through the tail vein. Icilin (2.5μg / g body weight) was injected intraperitoneally 30 minutes after the tail vein injection. After Icilin injection, the mice were placed on the test rack, and the wet dog-like shivering behavior of the mice was recorded by a video recorder within 30 minutes.

[0206] Mouse body temperature measurement

[0207] A digital thermometer (FT3400) was used to detect the body temperature of mice. During the temperature test, adult CD1 male mice were placed in a constant temperature (22±1℃) environment. Mice were injected with DeC-1.2 (30mg / kg body weight) via 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. The test probe of the thermometer was inserted about 2cm into the anus of the mouse, and the temperature displayed was recorded as the body temperature of the mouse after the temperature value stabilized.

[0208] Oxaliplatin-induced cold allodynia behavior

[0209] The cold sensation test caused by acetone volatilization was used to evaluate the cold allodynia behavior induced by oxaliplatin. A plastic hose was connected to the head of a 10ml syringe, and 50μl of acetone was sprayed onto the sole of the mouse's hind paw through the plastic hose. The total time of the mouse's reaction of lifting its foot, shaking its foot, licking its foot, etc. within 5 minutes was recorded. The cold allodynia behavior of mice was measured before and 10 days after oxaliplatin injection. The mice were injected with saline or 3.5μg / 20μL DeC-1.2 in the sole of their feet to evaluate the effect of DeC-1.2 on the cold allodynia behavior induced by oxaliplatin.

[0210] Oxaliplatin-induced mechanical allodynia

[0211] The mechanical touch-induced pain behavior of mice was evaluated by the change of the mouse paw withdrawal threshold in the Von Frey test. The mouse paw withdrawal threshold in the Von Frey test was carried out according to Dixon's up-down method. The middle part of the hind foot of the mouse was stimulated with von Frey fiber filaments (0.02-2g). The mouse showed behaviors such as lifting the foot, shaking the foot, and licking the foot within 3s after being stimulated, which was considered a response. According to Dixon's up-down method, first stimulate with 0.16g von Frey filaments. If the mouse responded, stimulate with the adjacent low-intensity von Frey filaments. If there was no response, stimulate with the adjacent high-intensity von Frey filaments. Select von Frey filament stimulations of different intensities in turn, with an interval of 5s between each two stimulations, and complete 6 stimulations in total. The paw withdrawal threshold (PWT) of the mouse was evaluated against the response scale. The paw withdrawal threshold of mice was measured before and 10 days after oxaliplatin injection. The mice were intraplantarly injected with normal saline or 3.5μg / 20μL DeC-1.2 to evaluate the effect of DeC-1.2 on oxaliplatin-induced allodynia behavior.

[0212] Primary culture of DRG neurons and calcium imaging experiments

[0213] After deep anesthesia, the mouse was decapitated and blood was discarded. The DRG tissue was quickly separated and placed in an ice-cold PBS solution. After rinsing the blood stains, digestive enzymes (Collagenase A 20mg / 100ml, Dispase II 300mg / 100ml dissolved in PBS) were added and digested at 37°C for 1h. Centrifuge at 500g for 5min and collect the digested tissue. Discard the enzyme solution, add an appropriate amount of DMEM culture medium (containing 10% FBS, 1X Pen / Strep), and prepare a single cell suspension by mechanical blowing. Centrifuge at 500g for 10min. Discard the supernatant, add an appropriate volume of Neurobasal culture medium (containing 2% B27, 1mM L-Glutamine, 50ng / ml NGF2.5S, 1X Pen / Strep), resuspend and blow into a single cell suspension. The single cell suspension was inoculated on a cell slide pre-coated with poly-D-Lys. After the cells adhered to the wall, an appropriate amount of culture medium was added to the culture dish, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours before being used for calcium imaging experiments.

[0214] Primary cultured DRG neurons were rinsed with extracellular solution (ECS, 140mM NaCl, 5mM KCl, 1mM MgCl2, 1.8mM CaCl2, 10mM D-Glucoes, 15mM HEPES, pH=7.4) and incubated in calcium dye incubation solution (2μM Fluo-2 AM, 0.05% Pluronic F-127in ECS) for 30 minutes. Cell images were continuously and alternately excited by a high-speed continuous monochromatic light source at an excitation wavelength of 340 / 380nm, recorded in full field of view under a 20X objective lens by visiview software, and the changes in intracellular fluorescence intensity were continuously recorded at a speed of 0.5fps by a high-speed scanning camera Flash4.0LT. The intensity of the calcium signal was represented by the ratio of the fluorescence signal at 340nm / 380nm. Menthol, DeC-1.2, and Capsaicin were administered by a 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 and the proportion of positive response cells induced by menthol in DRG neurons from different groups.

[0215] Statistics

[0216] All experiments were repeated three times independently. All statistical data are expressed as mean ± SEM. Electrophysiological data were completed using paired or unpaired t-test. Behavioral experimental data were completed using t-test, one-way or two-way anova, respectively. NS indicates no significant difference. *, *, **, and **** indicate P < 0.05, P < 0.01, P < 0.001, and 0.0001, respectively.

[0217] 1. Chemical synthesis of peptides

[0218] The amino acid sequence of polypeptide DeC-1.2 is CRRDRARHYRQRC (SEQ ID NO: 1), and the cysteine ​​residues at the N- and C-termini of polypeptide DeC-1.2 form a disulfide bond, thereby cyclizing polypeptide DeC-1.2.

[0219] The amino acid sequence of polypeptide DeC-1.1 is CRNSRAAHDSQKC (SEQ ID NO: 2), and the cysteine ​​residues at the N- and C-termini of polypeptide DeC-1.1 form a disulfide bond, thereby cyclizing polypeptide DeC-1.1.

[0220] The amino acids in polypeptide DeC-1.2 and polypeptide DeC-1.1 are all L-amino acids.

[0221] HPLC purification, mass spectrometry verification and electrophysiological verification of peptide synthesis and its function Figure 1 shown.

[0222] The peptide DeC-1.1 was chemically synthesized and HPLC( Figure 1 a) After purification, mass spectrometry ( Figure 1 b) Identification and confirmation: the half inhibitory concentration (IC) of DeC-1.1 was 50 ) was 132.8±73.5μM.

[0223] The peptide DeC-1.2 was also chemically synthesized and HPLC ( Figure 1 c) After purification, mass spectrometry ( Figure 1 d) Identification and confirmation. Electrophysiological tests showed that peptide DeC-1.2 could inhibit the Menthol activation of TRPM8 at the nanomolar level ( Figure 1 e), showing a lower half inhibition concentration (IC50), the half inhibition concentration (IC50) of peptide DeC-1.2 for inhibiting TRPM8 was 4.5±3.0nM ( Figure 1 b).

[0224] The inhibitory concentration dependence curves of peptide DeC-1.1 and peptide DeC-1.2 measured in the whole-cell electrophysiological recording mode are shown in Figure 2. Figure 1 f.

[0225] 2. Characteristics and effects of peptides

[0226] 2.1 Activation mode specificity and subunit-specific inhibition of TRPM8 by peptide DeC-1.2

[0227] Given that the peptide DeC-1.2 inhibits the Menthol activation of TRPM8 with high affinity, we further verified whether the DeC-1.2 peptide has activation mode specificity. Figure 2 As shown. The open probability and single channel conductance of TRPM8 ion channels were measured by single channel electrophysiological recording. Under the action of saturated concentration of menthol (1mM), the maximum open probability of TRPM8 was about 70% ( Figure 2 a. Figure 2 b and Figure 2 c). When 100 μM DeC-1.2 was mixed with saturated menthol (1 mM) and acted on the TRPM8 channel, its open probability was significantly reduced to 18.83±1.92% (n=5) ( Figure 2 c). In addition, the presence of DeC-1.2 can reduce the single channel conductance of TRPM8 corresponding to menthol activation from 58.83±1.85pS (n=3) to 30.97±1.16pS (n=3) ( Figure 2 b and 2d), indicating that DeC-1.2 blocks the entry and exit of ions into the TRPM8 ion channel by blocking the pore.

[0228] We further tested whether DeC-1.2 would inhibit the cold activation of TRPM8. When the cell membrane was cooled from 35°C to 16°C, TRPM8 was activated. At 16°C, the application of DeC-1.2 (100 μM) did not reduce the open probability of TRPM8 ( Figure 2 e. Figure 2 f and Figure 2 g). In addition, although the single-channel conductance of TRPM8 decreased to 36.16±1.74pS (n=3) upon cooling, the presence of DeC-1.2 did not further reduce the single-channel conductance of TRPM8, indicating that DeC-1.2 is an activation mode-specific inhibitor of TRPM8. The single-channel conductance of TRPM8 channels activated by cold did not change significantly in the presence of DeC-1.2 (100μM) ( Figure 2 h).

[0229] To examine the subunit selectivity of DeC-1.2 (e.g. Figure 3 and Table 3). TRPM8 ion channels belong to the TRP channel superfamily, and all members of this family have similar six transmembrane domain monomers. 50 The 4.5 nM level inhibited TRPM8 channels, whereas a concentration of DeC-1.2 (5 μM) one hundred times higher than that failed to inhibit ligand activation of TRPV1 or TRPV3 ( Figure 3 a. Figure 3 b and 3c). Although DeC-1.2 inhibited the activation of TRPV2 to a certain extent, its half inhibitory concentration (IC 50 ) is about 10 μM ( Figure 3 b and Table 3), the inhibitory effect was much lower than that on TRPM8. Among TRP channels, TRPM2 is the closest homologue of TRPM8, but up to 100 μM of DeC-1.2 failed to inhibit ligand activation of TRPM2 ( Figure 3 d). When designing DeC-1.2, the closed state structure of TRPM4 was used as a template for homology modeling of TRPM8, so DeC-1.2 may show higher affinity for TRPM4. However, in actual testing, TRPM4 showed lower sensitivity to DeC-1.2, and 50μM DeC-1.2 only inhibited about half of the TRPM4 current ( Figure 3 e). The inhibitory activity of Dec-1.2 was tested on TRPA1 and voltage-gated sodium (Nav) channels and it was found that Dec-1.2 did not inhibit them ( Figure 3f and Table 3). Therefore, DeC-1.2 is both a subunit-specific and an activation mode-specific inhibitor of TRPM8 ligand activation.

[0230] Table 3 Inhibitory effect of DeC-1.2 peptide on different ion channels

[0231]

[0232] The DeC-1.2 peptide was mutated (alanine scanning), as shown in Tables 4 and 5. These point mutation peptides were chemically synthesized and purified for quality control as the wild-type cyclic peptide. Figure 4 As shown. In the whole-cell electrophysiological recording mode, it was found that compared with the inhibitory activity of 5nM wild-type DeC-1.2, some mutants were still able to activate TRPM8, but the activity was reduced, and the inhibitory effect of some 5nM mutant cyclic peptides on TRPM8 almost disappeared ( Figure 4 a. Figure 4 b. Figure 4 c. Figure 4 d. Figure 4 e. Figure 4 f. Figure 4 g), but at high concentrations, such as 100 μM, it also has the effect of inhibiting TRPM8 channels. The key residues that can greatly change the inhibitory activity of the cyclic peptide DeC-1.2 on TRPM8 were further marked in the DeC-1.2 structure ( Figure 4 h. Figure 4 i and Figure 4 j, mutations that completely eliminated inhibition and mutations that had a great effect but did not completely eliminate inhibition are indicated in red and orange, respectively). Most of the key residues are located at the interface between DeC-1.2 and the outer side of the pore region of TRPM8, and mutations at these sites significantly affect the inhibitory activity of the cyclic peptide, indicating that DeC-1.2 binds to the outer side of the pore region of TRPM8 to exert an inhibitory effect.

[0233] Table 4 Mutant polypeptides based on DeC-1.2 polypeptide

[0234]

[0235]

[0236] Table 5 Mutant polypeptides based on DeC-1.2 polypeptide

[0237]

[0238]

[0239] 2.2 Peptides can improve peripheral neuropathy

[0240] The effect of peptide DeC-1.2 on wet dog shivers (WDS) behavior induced by icilin (an agonist of TRPM8) in mice was tested. The inhibitory effect of DeC-1.2 on TRPM8 was as follows Figure 5 The results showed that tail vein injection of low-dose DeC-1.2 (0.3 μg / g body weight) could significantly inhibit Icilin-induced WDS behavior, and its inhibitory effect was significantly dose-dependent ( Figure 5 a), The results showed that in vivo injection of DeC-1.2 could block Icilin-induced TRPM8 channel opening.

[0241] Oxaliplatin will induce increased TRPM8 activity in nociceptive DRG neurons, making them more sensitive. Using the oxaliplatin-induced cold allodynia mouse model, we tested whether DeC-1.2 could affect oxaliplatin-induced cold allodynia behavior ( Figure 5 b). Ten days after a single intraperitoneal injection of oxaliplatin (6 mg / kg body weight), mice showed obvious cold allodynia behavior. The effects of peptide DeC-1.2 on oxaliplatin-induced cold allodynia behavior and TRPM8 activity in primary sensory neurons were detected by behavioral tests and calcium imaging experiments. At the cellular level, dorsal root ganglia (DRG) were isolated from oxaliplatin-induced cold allodynia model mice and normal mice for primary culture of neurons ( Figure 5 c, 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 and cause calcium activity in neurons; compared with the control group, in the DRG neurons of mice treated with oxaliplatin, the calcium response intensity and the proportion of positive response cells caused by Menthol activating TRPM8 were significantly increased; while DeC-1.2 could significantly inhibit the calcium response intensity and the proportion of positive response cells of DRG neurons of mice treated with oxaliplatin ( Figure 5 d and 5e). These results indicate that DeC-1.2 peptide can inhibit the activity of TRPM8 channel in the oxaliplatin-induced cold hyperalgesia model.

[0242] In addition, DeC-1.2 was injected into the plantar surface to further test its effect on the cold pain response caused by acetone volatilization. The inhibitory effect of DeC-1.2 on oxaliplatin-induced cold pain hypersensitivity in vivo was as follows: Figure 6The results of behavioral tests showed that 10 days after oxaliplatin injection, the reaction time of cold pain behaviors such as lifting, licking and shaking feet caused by acetone increased significantly; the cold pain behaviors of mice in the control group (plantar injection of saline) did not change significantly before and after plantar injection; while plantar injection of 3.5μg / 20μL DeC-1.2 could significantly improve the cold pain behaviors caused by acetone volatilization ( Figure 6 a). In addition, the degree of anti-cold hypersensitivity of DeC-1.2 in vivo was similar to that of TRPM8 knockout mice. In addition, in the test of mechanical allodynia behavior, 10 days after oxaliplatin injection, the leg-lifting threshold of mice in the von Frey test was significantly reduced, and the injection of saline and DeC-1.2 had no significant effect on their leg-lifting threshold ( Figure 6 b). More importantly, we observed that the ( Figure 5 a) High-dose DeC-1.2 peptide 30 μg / g injected intravenously into mice did not significantly change the basal body temperature of mice ( Figure 6 c) Schematic diagram of the working principle of activation mode-specific inhibitor DeC-1.2 on TRPM8 channel Figure 6 Therefore, it can be seen that the cyclic peptide DeC-1.2 can improve the cold pain sensitivity behavior of mice through the activity of TRPM8 channels, but does not affect the body temperature of mice.

[0243] 2.3 Chronic constriction injury (CCI) model of neuropathic pain: cold allodynia

[0244] Male Sprague-Dawley rats (7 weeks at the start of the experiment, n=7-10 / treatment) were purchased from Charles River Japan, Inc. and used. CCI was established according to the method of Bennett GJ and Xie YK (Pain 1988, 33: 87-107). Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The left common sciatic nerve was exposed in the middle of the thigh with the adherent tissue removed, and four 4-0 silk threads (Ethicon Inc.) were loosely ligated around it at intervals of about 1 mm. Sham surgery was performed in the same manner except for the ligation of the sciatic nerve. After 1-2 weeks after CCI surgery, abnormal cold pain was evaluated using a cooling plate (LHP-1700CP, TECA) with a temperature controller (Model 13300-0, CAL Controls Inc.) as described by Tanimoto-Mori Setal. (Behav Pharmacol., 19: 85-90, 2008). The animals were adapted to a device consisting of a transparent acrylic box (10×12×12 cm) on a stainless steel plate (15×33 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. For the experiment, rats were placed on a cooling plate, and the polypeptide DeC-1.2 was administered before and after with a cutoff of 120 seconds, and the paw withdrawal latency (PWL) was measured. The polypeptide DeC-1.2 or its vehicle was administered orally, subcutaneously, or intraperitoneally. For example, the inhibition rate (%) is [PWL (polypeptide) - PWL (vehicle)] / [PWL (polypeptide) - PWL (vehicle)] × 100.

[0245] The results showed that the peptide DeC-1.2 showed strong activity in this model and had excellent effect in treating abnormal cold pain caused by chronic compression injury of neuropathy pain.

[0246] 2.4 Chronic constriction injury (CCI) model of neuropathic pain; static allodynia

[0247] Male Sprague Dawley rats (7 weeks old at the start of the experiment, n=7-10 / treatment) were purchased from Charles River Japan, Inc. and used. CCI was established according to the method of Bennett GJ and Xie YK (Pain 1988, 33: 87-107). Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The left common sciatic nerve was exposed in the middle of the thigh with the adherent tissue removed, and four 4-0 silk threads (Ethicon Inc.) were loosely ligated around it at intervals of about 1 mm. Sham surgery was performed in the same manner except for the ligation of the sciatic nerve. After 2-3 weeks after the CCI surgery, static allodynia was evaluated by a vibrissae mechanostimulator (VFH) as described by Field MJ et al. (Pain 1999, 83: 303-311). Before starting the experiment, the experimental animals were acclimated to a grid-bottomed cage. VFH was placed against the sole of the hind paw, and the test was performed with gradually increasing force (0.16g, 0.4g, 0.6g, 1g, 1.4g, 2g, 4g, 6g, 8g, 10g, 15g and 26g). Each VFH was pressed against the paw for 6 seconds or until a contraction reaction occurred. Once a contraction reaction occurred, the VFH was pressed again with a weaker pressure until no contraction reaction occurred. The minimum pressure required to induce a reaction was recorded as the paw withdrawal threshold (PWT). If the experimental animal reacted below the harmless 1.4g VFH, it was determined to be static allodynia. The polypeptide DeC-1.2 or its vehicle was administered by injection, oral administration, subcutaneous administration or intraperitoneal administration. The inhibition rate (%) was [PWL (polypeptide) - PWL (vehicle)] / [PWL (polypeptide) - PWL (vehicle)] × 100.

[0248] The results showed that the peptide DeC-1.2 showed strong activity in this model and had excellent static allodynia effect in treating chronic compression injury of neuropathic pain.

[0249] The above is an implementation scheme of the present invention designed for a case. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Zhejiang University <120> A polypeptide inhibiting TRPM8 and its use <130> P210635-1CNCNA1 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <400> 1 Cys Arg Arg Asp Arg Ala Arg His Tyr Arg Gln Arg Cys 1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <400> 2 Cys Arg Asn Ser Arg Ala Ala His Asp Ser Gln Lys Cys 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <400> 3 Cys Asp His Arg Arg Arg Gln Ala Arg Tyr Arg Arg Cys 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <400> 4 Cys Ala Arg Asp Arg Ala Arg His Tyr Arg Gln Arg Cys 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <400> 5 Cys Arg Ala Asp Arg Ala Arg His Tyr Arg Gln Arg Cys 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <400> 6 Cys Arg Arg Ala Arg Ala Arg His Tyr Arg Gln Arg Cys 1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <400> 7 Cys Arg Arg Asp Ala Ala Arg His Tyr Arg Gln Arg Cys 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <400> 8 Cys Arg Arg Asp Arg Gly Arg His Tyr Arg Gln Arg Cys 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <400> 9 Cys Arg Arg Asp Arg Ala Ala His Tyr Arg Gln Arg Cys 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <400> 10 Cys Arg Arg Asp Arg Ala Arg Ala Tyr Arg Gln Arg Cys 1 5 10 <210> 11 <211> 13 <212> PRT <213> Artificial Sequence <400> 11 Cys Arg Arg Asp Arg Ala Arg His Ala Arg Gln Arg Cys 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <400> 12 Cys Arg Arg Asp Arg Ala Arg His Tyr Ala Gln Arg Cys 1 5 10 <210> 13 <211> 13 <212> PRT <213> Artificial Sequence <400> 13 Cys Arg Arg Asp Arg Ala Arg His Tyr Arg Ala Arg Cys 1 5 10 <210> 14 <211> 13 <212> PRT <213> Artificial Sequence <400> 14 Cys Arg Arg Asp Arg Ala Arg His Tyr Arg Gln Ala Cys 1 5 10 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <400> 15 Cys Arg Asn Asp Arg Ala Arg His Tyr Arg Gln Arg Cys 1 5 10 <210> 16 <211> 13 <212> PRT <213> Artificial Sequence <400> 16 Cys Arg Arg Ser Arg Ala Arg His Tyr Arg Gln Arg Cys 1 5 10 <210> 17 <211> 13 <212> PRT <213> Artificial Sequence <400> 17 Cys Arg Arg Asp Arg Ala Arg His Asp Arg Gln Arg Cys 1 5 10 <210> 18 <211> 13 <212> PRT <213> Artificial Sequence <400> 18 Cys Arg Arg Asp Arg Ala Arg His Tyr Ser Gln Arg Cys 1 5 10 <210> 19 <211> 13 <212> PRT <213> Artificial Sequence <400> 19 Cys Arg Arg Asp Arg Ala Arg His Tyr Arg Gln Lys Cys 1 5 10

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide is shown in SEQ ID NO:

1.

2. A polynucleotide, characterized in that The polynucleotide encodes the polypeptide of claim 1.

3. A carrier, characterized in that The vector comprises the polynucleotide according to claim 2.

4. A host cell, characterized in that The host cell comprises the vector as claimed in claim 3 or the polynucleotide as claimed in claim 2 is integrated into the chromosome of the host cell.

5. A composition, characterized in that The composition comprises: (a) the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient.

6. Use of the polypeptide or pharmaceutically acceptable salt thereof according to claim 1, the polynucleotide according to claim 2, the vector according to claim 3, the host cell according to claim 4, and / or the composition according to claim 5, characterized in that: Used for preparing a pharmaceutical composition, the pharmaceutical composition is used for one or more uses selected from the following group: preventing and / or treating a disease associated with TRPM8, wherein the disease associated with TRPM8 is selected from the following group: peripheral neuropathy, cold allodynia, pain, itching, chronic compression injury of neuropathic pain, or a combination thereof.

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