Polypeptide and application thereof in preparation of TRPV1 inhibitor

Abstract

The invention provides a polypeptide and application thereof in preparation of a TRPV1 inhibitor, and particularly belongs to the technical field of biomedicine. The invention provides a polypeptide which is named as Cath2, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1. The Cath2 can significantly inhibit activation of a TRPV1 ion channel of a mammal, inhibits TRPV1 receptors of other species including human beings in a targeted manner, and can be applied to research of TRPV1-related ion channel diseases and development of drugs. Specifically, the composition can enhance the resistance of mammals to pruritus and pain, can be used as a resource to be developed into an itching relieving product or an analgesic product and the like, and is applied to human beings and other animals to relieve pruritus and pain.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide and its application in the preparation of TRPV1 inhibitors. Background Technology

[0002] TPRV1 is a non-selective cation channel closely associated with pain and inflammation, and can be activated by a variety of naturally occurring stimuli and extracellular acids. Natural stimuli for TPRV1 include capsaicin (the active ingredient in chili peppers), DkTx (a polypeptide toxin produced by spider mites), and RTX (a resinous toxin isolated from Euphorbia milii). TPRV1 also senses temperature changes (>42°C), thus participating in the body's thermoregulation. Due to the importance of TPRV1-mediated signal transduction, it has become a potentially significant clinical therapeutic target. For example, inhibition of TPRV1 as a nociceptor in animals can dull the animal's pain response for a certain period. Early studies targeting TPRV1 inhibitors for analgesia and itch relief were based on the crucial role of TPRV1 in various pain modalities and types of pain, attempting to achieve broad-spectrum analgesia for multiple pain types through systemic administration, small molecules, and strong antagonism strategies. However, as research progressed, adverse reactions such as increased body temperature and altered thermal pain thresholds were gradually revealed, along with unstable analgesic effects and a narrow therapeutic window. Therefore, developing new TRPV1 inhibitor products is of great significance for the treatment of TRPV1-related diseases. Summary of the Invention

[0003] The purpose of this invention is to provide a polypeptide and its application in the preparation of TRPV1 inhibitors. Polypeptides, as natural products, have advantages such as high safety and large potential for modification, making them an important resource for the development of TRPV1 inhibitors. The polypeptide described in this invention can significantly inhibit the activation of mammalian TRPV1 ion channels and target and inhibit TRPV1 receptors in other species, including humans, and can be applied to the research and drug development of TRPV1-related ion channel diseases.

[0004] The present invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] The present invention also provides the application of the peptide in the preparation of TRPV1 inhibitors.

[0006] The present invention also provides the application of the polypeptide in the preparation of analgesic products.

[0007] The present invention also provides the application of the polypeptide in the preparation of antipruritic products.

[0008] The present invention also provides the use of the polypeptide in the preparation of analgesic and antipruritic products.

[0009] The present invention also provides the use of the polypeptide in the preparation of medicaments for the prevention and / or treatment of diseases related to TRPV1.

[0010] Preferably, the TRPV1-related diseases include acute and chronic pain, acute and chronic itching, and skin inflammation associated with TRPV1.

[0011] The present invention also provides an analgesic and / or antipruritic product, comprising the polypeptide and excipients described in the above technical solution.

[0012] Preferably, the dosage form of the product includes an injection or a topical application.

[0013] Preferably, the product includes pharmaceuticals or biological pesticides.

[0014] This invention provides a polypeptide with the amino acid sequence shown in SEQ ID NO.1: RFRLPFRRPPIRIHPPPFYPPFRRFLGRR. The polypeptide of this invention can significantly inhibit the activation of the TRPV1 ion channel in mammals and target and inhibit TRPV1 receptors in other species, including humans, for application in the research and drug development of TRPV1-related ion channel diseases. Specifically, it can enhance the resistance of mammals to itching and pain, and can be developed as a resource into antipruritic or analgesic products for use in humans and other animals to relieve itching and pain. Electrophysiological experiments show that Cath2 acts on the TRPV1 receptor. Specifically, by expressing the human TRPV1 receptor in HEK293T cells and recording channel currents using patch-clamp technology, it was confirmed that Cath2 has an inhibitory effect on TRPV1 channel activity, indicating its potential for cross-species application. Point mutation experiments identified key amino acid residues in TRPV1 associated with Cath2 binding, indicating that Cath2 exerts its inhibitory function by directly acting on the TRPV1 channel. Inhibition of TRPV1, as an animal nociceptor, will dull the animal's pain response for a certain period of time. This invention systematically validated the efficacy of Cath2 in relieving itching and pain using mouse acute and chronic pruritus models and pain behavior experiments. Specifically, mouse model animal experiments showed that Cath2 treatment significantly reduced acetic acid- or CFA-induced inflammatory pain and harmful heat-induced thermal pain in mice. Simultaneously, Cath2 treatment reduced histamine-induced acute pruritus and SADBE-induced chronic pruritus in mice. In summary, Cath2 reduces pain responses in mice in pain models and can be developed as a potential analgesic molecule for analgesia in humans and other animals. Furthermore, Cath2 targets the receptor TRPV1, which can be applied to research on related ion channelopathies and as a potential therapeutic agent. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The diagrams show the selectivity of Cath2 in several representative ion channels provided by this invention; wherein, A is the activation diagram of TRPA1 induced by 5 μmol / L Cath2; B is the activation diagram of TRPV1 induced by 5 μmol / L Cath2; C is the activation diagram of TRPV2 induced by 5 μmol / L Cath2; D is the activation diagram of TRPV3 induced by 5 μmol / L Cath2; E is the activation diagram of TRPV4 induced by 5 μmol / L Cath2; F is the activation diagram of TRPA1 opening induced by 500 μmol / L N-phenyl-p-phenylenediamine using 5 μmol / L Cath2. The graphs show the induction and inhibition of TRPV1 opening; G represents the opening of TRPV2 induced by 200 μmol / L 2-aminoethyl diphenylboronic acid ester and the inhibition of TRPV2 opening induced by 5 μmol / L Cath2; H represents the opening of TRPV3 induced by 100 μmol / L 2-aminoethyl diphenylboronic acid ester and the inhibition of TRPV3 opening induced by 5 μmol / L Cath2; I represents the opening of TRPV4 induced by 1 μmol / L 1-benzyl-4-[(4-chloro-2-nitrophenyl)sulfonyl]piperazine and the inhibition of TRPV4 opening induced by 5 μmol / L Cath2. Figure 2 The graphs show the current and concentration effect curves of Cath2 suppressing TRPV1; where A is a summary graph of the current of Cath2 suppressing hTRPV1; B is a representative current graph of Cath2 suppressing hTRPV1; and C is a voltage-current relationship graph of Cath2 suppressing hTRPV1. Figure 3Figure 1 shows the results of investigating the key action sites of Cath2 in inhibiting TRPV1. A shows the current curve of TRPV1 inhibition induced by mutating tyrosine at position 628 of TRPV1 to alanine and then using 5 μmol / L Cath2. B shows the current curve of TRPV1 inhibition induced by mutating asparagine at position 629 of TRPV1 to alanine and then using 5 μmol / L Cath2. C shows the current curve of TRPV1 inhibition induced by mutating serine at position 630 of TRPV1 to alanine and then using 5 μmol / L Cath2. D shows the current curve of TRPV1 inhibition induced by mutating serine at position 633 of TRPV1 to alanine and then using 5 μmol / L Cath2. E shows the current curve of TRPV1 inhibition induced by mutating serine at position 630 of TRPV1 to alanine and then using 5 μmol / L Cath2. The current curves for inhibiting open TRPV1 after mutating cysteine ​​at position 5 to alanine and inducing inhibition with 5 μmol / L Cath2 are shown in Figures F, G, H, and I. Figure 4 The graphs show the analgesic effects of Cath2 on mice. A represents the inhibition of capsaicin-induced plantar pain by Cath2; B represents the inhibition of formalin-induced phase I plantar pain by Cath2; C represents the inhibition of formalin-induced phase II plantar pain by Cath2; D represents the inhibition of mechanical pain caused by Café-Alpha (CFA)-induced plantar inflammation by Cath2-induced plantar pain induced by fibrils; E represents the inhibition of thermal pain caused by hot plate therapy by Cath2-induced plantar inflammation after CFA-induced plantar inflammation; F represents the body temperature change after injection of 100 nanomoles per kilogram of Cath2; G represents the inhibition of thermal pain caused by hot plate therapy under normal conditions by Cath2-induced plantar pain; and H represents the inhibition of thermal pain caused by hot tail immersion under normal conditions by Cath2-induced plantar pain. Figure 5 The graph shows the evaluation results of the itch-relieving effect of Cath2 on mice; where A is a schematic diagram of the chronic itch model; B is the itch-relieving effect of 10 nM and 100 nM Cath2 on chronic itch; and C is the itch-relieving effect of 10 nM and 100 nM Cath2 on acute itch. Figure 6 This is a graph showing the results of a body temperature safety assessment. Detailed Implementation

[0017] This invention provides a polypeptide whose amino acid sequence is shown in SEQ ID NO.1: RFRLPFRRPPIRIHPPPFYPPFRRFLGRR. The polypeptide of this invention is named Cath2. This invention does not impose any special limitations on the synthesis and purification of polypeptide Cath2; it was synthesized by Hangzhou Gutuo Biotechnology Co., Ltd.; and purified using conventional polypeptide purification methods.

[0018] This invention also provides the application of the peptide in the preparation of TRPV1 inhibitors. TRPV1 is a capsaicin receptor, an ion channel protein activated by high temperature or capsaicin. As a non-selective cation channel, TRPV1 is widely expressed in sensory neurons and skin tissue. The peptide of this invention has an inhibitory effect on TRPV1, inhibiting the TRPV1 ion channel and inhibiting the activation of TRPV1 by agonists, thereby achieving the prevention and / or treatment of TRPV1 ion channel-related diseases. In a specific embodiment, the agonist may be capsaicin. TRPV1 activation mediates pain and inflammation, while inhibition of TRPV1 alleviates pain and itching. Experimental results show that Cath2 can significantly inhibit the activation of TRPV1 ion channels in animals and effectively inhibit the TRPV1 receptor. Cath2, by targeting and inhibiting the TRPV1 receptor, can be applied to the research of related ion channel diseases and as a potential therapeutic drug. TRPV1 is closely related to the occurrence of acute and chronic pain and itching. In a specific embodiment, the animals include mammals. In a specific embodiment, the mammals include humans.

[0019] This invention also provides the application of the aforementioned polypeptide in the preparation of analgesic products. The polypeptide of this invention, as an analgesic molecule, can relieve pain and improve the pain resistance of animals. In specific embodiments, the product can be a drug or a skin care product. This invention does not specifically limit the dosage form of the drug; it can be applied to the human body and act on the skin to exert analgesic function, such as a topical application or injection. The drug of this invention can be applied to humans or animals through local application, topical application, injection, etc., to relieve acute and chronic pain.

[0020] This invention also provides the application of the polypeptide in the preparation of antipruritic products. The polypeptide of this invention can relieve itching and improve the resistance of animals to itching. In specific embodiments, the product can be a drug or a skin care product. This invention does not specifically limit the dosage form of the drug; it can be applied to the human body and act on the skin to exert its antipruritic function, such as a topical application or injection. The drug of this invention can be applied to humans or animals through local application, topical application, injection, etc., to relieve or temporarily control acute and chronic itching.

[0021] This invention also provides the application of the aforementioned polypeptide in the preparation of analgesic and antipruritic products. Cath2 can alleviate the perception of pain and itching in animals, specifically relieving the sensation of pain and itching. Experimental results show that Cath2 enhances the resistance of animals (including mice) to itching and pain, significantly inhibits the activation of TRPV1 by agonists, and thus exhibits dual analgesic and antipruritic effects in animal models. It can be developed as a potential antipruritic or analgesic for use in humans and other animals to relieve itching and pain.

[0022] This invention also provides the use of the peptide in the preparation of medicaments for the prevention and / or treatment of TRPV1-related diseases. In specific embodiments, the TRPV1-related diseases include acute and chronic pain, acute and chronic pruritus, and skin inflammation related to TRPV1. In specific embodiments, the acute and chronic pain includes acute pain and / or chronic pain. In specific embodiments, the acute and chronic pruritus includes acute pruritus and / or chronic pruritus. In specific embodiments, the pain includes peripheral pain. Electrophysiological experiments have verified that the peptide of this invention can effectively inhibit TRPV1 receptor function in animals (including humans and mice) and is a key active peptide for regulating pain and pruritus perception. It can be used to prepare medicaments for the prevention and / or treatment of TRPV1-related diseases and improve the resistance of animals to pain and pruritus.

[0023] This invention also provides an analgesic and / or antipruritic product, comprising the polypeptide and excipients described in the above-mentioned technical solution. In specific embodiments, the dosage form of the product includes an injection or a topical application. In specific embodiments, the product comprises a drug or a biopesticide. Specifically, the product of this invention can bind to the insect's TRPV1, interfering with the pest's perception of environmental temperature or harmful stimuli, making it unable to avoid death in extreme environments; by affecting TRPV1 in the feeding-related TRP channel complex, it inhibits the pest's feeding, causing it to starve to death; it can serve as a lead framework for developing novel green biopesticides, and through further structural modifications or formulation improvements, its penetration into the pest's epidermis or its stability in the field can be enhanced to achieve pest control.

[0024] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes a polypeptide provided by the present invention and its application in the preparation of TRPV1 inhibitors, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1 Electrophysiological experiments on the targeting of pain-related receptors such as Cath2 and hTRPV1 The following plasmids were constructed using a double digestion method: hTRPV1 (constructed from a pcDNA3.1(+) vector with restriction endonucleases KpnI and HindIII, GeneID: 7442), hTRPV2 (constructed from a pcDNA3.1(+) vector with restriction endonucleases KpnI and HindIII, GeneID: 51393), and hTRPV3 (constructed from a pcDNA3.1(+) vector with restriction endonucleases KpnI and HindIII, GeneID: 51393). The following plasmids were transiently transfected and overexpressed in HEK293T cell lines: hTRPV4 plasmid (constructed by double digestion, based on pcDNA3.1(+) vector, with restriction endonucleases KpnI and HindIII sites, GeneID: 59341), and hTRPA1 plasmid (constructed by double digestion, based on pcDNA3.1(+) vector, with restriction endonucleases KpnI and HindIII sites, GeneID: 8989). All HEK293T cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) with 10% fetal bovine serum and 1% penicillin / streptomycin, at 37°C and 5% CO2.

[0026] Cells with relatively smooth membranes and homogeneous cytoplasm were selected under an inverted microscope for patch-clamp experiments at room temperature (20–25°C). WPI 0.86 mm thin-walled borosilicate glass capillaries were used as the glass electrode material. The glass electrodes were fabricated in five steps using a stretching apparatus (P-97, Shutter). After heat polishing, the electrode tip diameter was 1.5–3.0 μm. Intracellular fluid was then perfused into the glass electrode after fabrication. The initial resistance of the glass electrode was 1.5–2.5 MΩ. After a high-impedance king-ohm (GΩ) seal was formed between the electrode and the cell membrane, fast capacitance was applied. A short, strong negative pressure was then applied to rapidly break the cell membrane clamped in the electrode, followed by compensation for slow capacitance. Once whole-cell recording mode was established, current recording began. Drugs were perfused using a Biolab RS200, with a drug switching rate of 50 ms. The series resistance (Rs) remained constant within the range of 5–8 MΩ throughout the experiment, and the system series resistance compensation was generally between 30% and 60%. Experimental data were analyzed using Patch Master software, and further analysis was performed using Igor software. All results are expressed as average ± standard error (SEM), where n represents the number of data points in the experiment.

[0027] Cells are slowly lifted using a motorized micromanipulation system until they are flush with the lower edge of the drug delivery port on the rapid drug delivery switching system (RSC-200). The drug delivery switching system is directly connected to the amplifier, and all experimental data are recorded via the amplifier in the PatchMaster computer software. Cells are stimulated and drug-treated using PatchMaster and the switching drug delivery system, and changes in channel current are recorded to detect drug activity in the channels. To ensure the accuracy of the experimental data, stable sealing and series resistors are required throughout the recording process.

[0028] TRP channel internal and external solution: 130 mM NaCl, 3 mM HEPES and 0.2 mM EDTA, adjusted to pH 7.2 with NaOH. Drugs used in the experiment must be dissolved and prepared using the above TRP channel internal and external solution.

[0029] During the experiment, the corresponding agonists for each channel were used to verify that the channels were normally expressed in cells and could be activated to exert their functions. These agonists also served as positive controls when testing for Cath2 activation and as background for channel opening when testing for Cath2 inhibition. TRPA1 used 500 μmol / L N-phenyl-p-phenylenediamine to verify normal channel expression and activation; TRPV1 used 1 μmol / L capsaicin to verify normal channel expression and activation; TRPV2 used 1 mM / L 2-aminoethyl diphenylboronic acid ester to verify normal channel expression and activation; TRPV3 used 100 μmol / L 2-aminoethyl diphenylboronic acid ester to verify normal channel expression and activation; and TRPV4 used 1 μmol / L 1-benzyl-4-[(4-chloro-2-nitrophenyl)sulfonyl]piperazine to verify normal channel expression and activation.

[0030] The selectivity of Cath2 in several representative ion channels is as follows: Figure 1 As shown. The results indicate that Cath2 has no activity against multiple pain and itch-related ion channels. 5 micromoles of Cath2 could not activate TRPA1 ( Figure 1 The activation map of TRPA1 induced by Cath2 at 5 μmol / L (with N-phenyl-p-phenylenediamine at 500 μmol / L as a positive control) in Figure A shows that TRPV1 ( Figure 1 The activation map of TRPV1 induced by B, 5 μmol / L Cath2, with 1 μmol / L capsaicin as a positive control), TRPV2 ( Figure 1 The activation map of TRPV2 induced by C, 5 μmol / L Cath2, with 1 mmol / L 2-aminoethyl diphenylboronic acid ester as a positive control), TRPV3 ( Figure 1The activation map of TRPV3 induced by D, 5 μmol / L Cath2, with 100 μmol / L 2-aminoethyl diphenylboronic acid ester as a positive control) and TRPV4 ( Figure 1 The activation map of TRPV4 induced by E, 5 μmol / L Cath2, with 1 μmol / L 1-benzyl-4-[(4-chloro-2-nitrophenyl)sulfonyl]piperazine as a positive control) channel was shown. Furthermore, 5 μmol / L Cath2 could not inhibit the activation of TRPA1 by 4-A (N-phenyl-p-phenylenediamine). Figure 1 The figure shows the opening of TRPA1 induced by F at 500 μmol / L N-phenyl-p-phenylenediamine and the inhibition of TRPA1 opening induced by 5 μmol / L Cath2 (Figure 1), and the activation of TRPV2 by 2-APB (2-aminoethyl diphenylboronic acid ester). Figure 1 In the figure, G, induced TRPV2 opening with 200 μmol / L 2-aminoethyl diphenylboronic acid ester, and inhibited TRPV2 opening with 5 μmol / L Cath2, 2-APB activation of TRPV3 (Figure). Figure 1 The opening of TRPV3 was induced by 100 μmol / L 2-aminoethyl diphenylboronic acid ester, while the opening of TRPV3 was inhibited by 5 μmol / L Cath2 (Figure 1747). Activation of TRPV4 by 1-benzyl-4-(4-chloro-2-nitrophenyl)sulfonylpiperazine (RN1747) (Figure 2) Figure 1 In Figure I, TRPV4 opening was induced by 1 μmol / L 1-benzyl-4-[(4-chloro-2-nitrophenyl)sulfonyl]piperazine, and TRPV4 opening was inhibited by 5 μmol / L Cath2 (Figure).

[0031] like Figure 2 The Cath2 shown significantly inhibits the TRPV1 channel. (As indicated...) Figure 2 As shown, Cath2 inhibits capsaicin activation in the hTRPV1 channel ( Figure 2 The diagram summarizes the current of A and Cath2 inhibition of hTRPV1, namely, the opening of TRPV1 induced by 1 μmol / L capsaicin and the inhibition of TRPV1 opening induced by 5 μmol / L Cath2. Figure 2 The representative current diagram of B, Cath2 inhibition of hTRPV1; that is, the representative trace diagram of TRPV1 in three different states (i.e., baseline level), induced by 1 μmol / L capsaicin and then inhibited by 5 μmol / L Cath2. Figure 2 (The voltage-current relationship between C and Cath2 suppressing hTRPV1 is shown in the figure).

[0032] The targeting of Cath2 with other itch and pain-related receptors (TRPV2, TRPV3, TRPV4, TRPA1) (besides hTRPV1 receptor) showed that it could neither activate nor inhibit these receptors, indicating strong selectivity, meaning it could only specifically inhibit the current induced by TRPV1 activation by capsaicin. Figure 1 and Figure 2 ).

[0033] Example 2 Point mutations in the hTRPV1 channel Single-point mutations were performed on individual pore regions of the hTRPV1 channels to explore the key sites of Cath2 and hTRPV1 function. All mutated hTRPV1 channels were constructed using homologous recombination and obtained using the Vazyme Mut-Express-II-Fast-Mutagenesis Kit-V2.

[0034] The inhibitory activity of Cath2 on capsaicin activation in all point-mutated hTRPV1 channels was detected, and the inhibition rate of Cath2 in wild-type and each point-mutated channel was compared. The results are as follows: Figure 3 As shown. Figure 3 The figure shows the results of the investigation into the key action site of Cath2 inhibiting TRPV1, illustrating the effect of TRPV1 point mutations on the interaction between Cath2 and TRPV1. 5 micromoles of Cath2 do not affect the TRPV1 point mutation Y628 (…). Figure 3 In A, after mutating tyrosine at position 628 of TRPV1 to alanine, the current map of inhibited TRPV1 was induced by 5 μmol / L Cath2, and N629 ( Figure 3 In B, after mutating asparagine at position 629 of TRPV1 to alanine, the current map of inhibited TRPV1 was induced by 5 μmol / L Cath2, and S630 ( Figure 3 In C, the current map of TRPV1 inhibited by mutating serine at position 630 to alanine and then inducing inhibition of open TRPV1 with 5 μmol / L Cath2, S633 ( Figure 3 In D, after mutating serine at position 633 of TRPV1 to alanine, the current map of inhibited TRPV1 was induced by 5 μmol / L Cath2. C635 ( Figure 3 In the E, after mutating cysteine ​​at position 635 of TRPV1 to alanine and inducing the inhibition of open TRPV1 with 5 μmol / L Cath2, the current map was obtained. E637 ( Figure 3 In F, after mutating glutamate at position 637 of TRPV1 to alanine, the current map of inhibited TRPV1 was induced by 5 μmol / L Cath2, and E649 ( Figure 3In G, after mutating glutamate at position 649 of TRPV1 to alanine, the current map of inhibited TRPV1 was induced by 5 μmol / L Cath2. Figure 3 The H in the figure, after mutating threonine at position 651 of TRPV1 to alanine and inducing the inhibition of open TRPV1 with 5 μmol / L Cath2, is shown in the current map, and E652 ( Figure 3 In the diagram, after mutating glutamic acid at position 652 of TRPV1 to alanine, an electrogram was generated to inhibit the open TRPV1 channel by inducing Cath2 at 5 μmol / L. The amino acid mutation at position E652 significantly weakened the inhibition of capsaicin activation of the hTRPV1 channel by Cath2, suggesting that E652 is a key amino acid site for Cath2 to inhibit hTRPV1.

[0035] Example 3 Cath2 analgesic activity Establishment and administration of a mouse pain model: Six- to eight-week-old (19–26 g) C57BL / 6 mice were housed under specific pathogen-free conditions, with controlled temperature (23 ± 2 ℃), humidity (55 ± 5%), and a 12-hour light-dark cycle. Animals were housed according to standard protocols, with five mice per cage and free access to water and standard food. All mice were randomly assigned to different experimental groups and numbered. All data collection and testing were conducted in a double-blind manner.

[0036] The experiment was conducted under constant temperature (23±2 ℃) and humidity (55±5%) conditions and a 12-h light-dark cycle.

[0037] 1. Capsaicin Pain Model: Thirty minutes before the experiment, mice in the positive control and experimental groups were intravenously injected with morphine (5 mg / kg) and peptides (10 nM / kg and 100 nM / kg, respectively) (solvent: physiological saline, the same below), while the negative control group was injected with physiological saline. Thirty minutes later, 25 µl of 0.33 mM capsaicin (approximately 2.5 µg) was subcutaneously injected into the center of the sole of the left hind limb of the mice (to facilitate observation of foot-licking / foot-shaking behavior). The duration of foot-licking by the mice was then observed and recorded within 3 minutes.

[0038] 2. Formalin Pain Model: Thirty minutes before the experiment, mice in the positive control and experimental groups were intravenously injected with morphine (5 mg / kg) and peptides (10 nM / kg and 100 nM / kg), respectively, while the negative control group was injected with saline. Thirty minutes later, 20 µl of 2.5% formalin was subcutaneously injected into the center of the sole of the right hind limb (for easy observation of foot-licking / foot-shaking behavior). The time spent licking / biting the foot was recorded during phase 1 (0–5 min) and phase 2 (15–30 min).

[0039] 3. Acetic acid writhing test: 30 min before the experiment, the positive control and experimental mice were injected with morphine (10 mg / kg) and 5 nM / 100 μM peptide, respectively, while the negative control group was injected with 200 μL of physiological saline. 30 min later, 200 μL of 0.8% acetic acid was injected. After the injection, the mice were placed in open glass boxes and the number of writhing movements within 30 minutes was recorded by a camera.

[0040] 4. Hot plate pain model: 30 min before the experiment, mice in the positive control and experimental groups were intravenously injected with morphine (5 mg / kg) and peptides (10 nM / kg and 100 nM / kg), respectively, while the negative control group was injected with saline. 30 min later, the mice were placed on a 55 ℃ (harmful heat) hot plate, and the time when the mice exhibited their first pain response (licking their paws / shaking their paws / jumping) was recorded as the withdrawal latency. The cut-off time was set to 30 s to prevent burns.

[0041] 5. Heat-induced tail immersion pain model: 30 min before the experiment, mice in the positive control and experimental groups were intravenously injected with morphine (5 mg / kg) and peptides (10 nM / kg and 100 nM / kg), respectively, while the negative control group was injected with saline. 30 min later, the mice were placed in 50 ml centrifuge tubes with ventilation holes drilled in the walls and a hole drilled in the center of the cap to allow the tail to pass through, thus fixing the mice in place, allowing only the tail to be exposed. The water bath was set to 55 ℃ (harmful heat), and 1 / 3 of the mouse's tail was immersed in the hot water. The latency (s) of tail shaking / flicking was recorded.

[0042] 6. CFA-induced inflammatory pain model: 20 µl of complete Freund's adjuvant (CFA) was subcutaneously injected into the center of the sole of both hind limbs (to facilitate observation of foot-licking / foot-shaking behavior) to induce inflammation. The test consisted of two parts: a hot plate test and a Von Frey fiber test.

[0043] (1) Von Frey Before the formal experiment, mice were placed in the experimental apparatus to acclimatize until they no longer exhibited obvious escape behavior when the probe approached. Thirty minutes before the experiment, positive control and experimental mice were intravenously injected with morphine (5 mg / kg) and peptides (10 nM / kg and 100 nM / kg), respectively, while the negative control group was injected with saline. After 30 minutes, the test began. Mice were placed on a metal grid covered with individual transparent chambers. Only one fiber was used for stimulation at a time, vertically upwards to stimulate the sole of the foot (middle of the foot / arch). The fiber was slightly bent and held for approximately 1-2 seconds. A positive response was indicated by rapid foot retraction, foot lifting, or licking / shaking the foot. The Dixon / Chaplan up-and-down method was used: the mechanical pain was increased from low to high, and after the first transition (from no pain to pain), six results were recorded: N, Y, N, Y, N, and Y (N represents no response, Y represents a response). k was then determined from a table using the formula: X f δ: The logarithmic label of the last used fiber (e.g., 3.61, 3.84... the number printed on the fiber, in log10 scale). δ: The distance between two adjacent fibers on the log10 coordinate system. k: A lookup table coefficient determined by the recorded Y / N sequence pattern (the result of counting 6 times from the first direction reversal). Different patterns have different values, using the table given in Chaplan 1994.

[0044] (2) Hot plate The experimental protocol was the same as before. The difference between this experiment and the previous one, which directly tested the hot plate, was that it was conducted under CFA-induced plantar inflammation conditions.

[0045] The results of the pain-related models above are as follows: Figure 4 As shown, Figure 4 This represents the pain-relieving effect of Cath2 on different types of pain in mice. Cath2's effect on capsaicin-induced plantar pain ( Figure 4 A in the figure shows the inhibition of capsaicin-induced plantar pain by Cath2, and the first phase of formalin-induced plantar pain. Figure 4 In Figure B, Cath2 is used to inhibit formalin-induced phase I plantar pain, and formalin-induced phase II plantar pain is shown. Figure 4 C in the figure, using Cath2 to inhibit formalin-induced phase II plantar pain, acetic acid writhing ( Figure 4 F in the text) and CFA-induced inflammatory pain ( Figure 4 D, CFA-induced plantar inflammation followed by Cath2 inhibition of mechanorepain induced by fibrous fibers; and Figure 4 E, CFA-induced plantar inflammation followed by Cath2 inhibition of hot plate-induced heat pain both have analgesic effects, but hot plate pain ( Figure 4 G in the image, using Cath2 to inhibit the thermal pain induced by a hot plate under normal conditions) and thermal tail irritation ( Figure 4 The H in the figure (using Cath2 to inhibit thermal pain induced by heat tail immersion under normal conditions) showed no analgesic effect, indicating that Cath2 has a strong analgesic effect on peripheral pain, but a weak analgesic effect on central pain. This suggests that Cath2's inability to cross the blood-brain barrier may prevent it from exerting its central analgesic effect. Cath2 can alleviate various types of pain, including chemically induced pain and inflammatory pain, and enhances pain resistance. It can be developed as a potential analgesic molecule for use in human and other animal analgesia.

[0046] Example 4 Cath2's antipruritic activity 1. Acute pruritus model: Three days before the start of the formal experiment, the skin on the right posterior neck of mice was removed. Thirty minutes before the experiment, the positive control and experimental groups were intravenously injected with morphine (5 mg / kg), and the positive control group was injected with morphine (10 nM / kg), and the experimental group was injected with peptide (10 nM / kg). The negative control group was injected with saline. Thirty minutes later, 100 nM histamine was injected intradermally into the shaved area. The number of scratches by the mice within 30 minutes was observed and recorded. One scratching behavior was defined as the mouse lifting its forepaw or hind paw off the ground to scratch the treated area on the right posterior neck until the paw was placed back on the ground.

[0047] 2. Chronic pruritus model: The day before modeling began, the abdominal area of ​​mice was shaved to prepare a 1.5 × 1.5 cm sample. 2 A shaved area was prepared on the abdomen. From day 1 of modeling, 25 μL of 1% SADBE solution (SDABE dissolved in acetone) was applied to the shaved area once daily for three consecutive days. On day 6, the right cheek of the mice was shaved, and a 0.8 × 0.8 cm swab was prepared. 2 A shaved area was created on the cheek. Starting from day 8, 25 μL of 1% SADBE solution was applied to the shaved area once daily for three consecutive days to induce the ACD response. The control group was treated with 25 μL of pure acetone instead of the 1% SADBE solution. Twenty-four hours after the last application, mice in the positive control and experimental groups were intravenously injected with dexamethasone (5 mg / kg) and 10 nM / kg and 100 nM / kg peptide, respectively, while the negative control group was injected with saline. Thirty minutes later, scratching and rubbing behaviors of the mice were observed and recorded over 2 hours. To assess spontaneous scratching and rubbing behaviors, mice were placed in a 9×9×13 cm... 3Two mice were placed in separate, transparent plastic containers with a small amount of bedding material inside. A camera was mounted above the containers to record the two mice simultaneously for 2 hours. Four mirrors were attached to the bottom of the containers, aligned with the four sides of a square base, allowing for omnidirectional observation. The experimental group mice received 2 hours of training daily from day 1 to day 10, and were placed in the containers 30 minutes before video recording to allow them to acclimatize. For each 2-hour video segment, a researcher unaware of the experimental design counted the number of scratching motions (similar to itching) made by the mice with their hind limbs and rubbing motions (similar to pain) made by their forelimbs.

[0048] The results are as follows Figure 5 As shown, Figure 5 The results show the evaluation of the itch-relieving effect of Cath2 on mice; where A is a schematic diagram of the chronic itch model; B is the itch-relieving effect of 10 nM and 100 nM Cath2 on chronic itch; and C is the itch-relieving effect of 10 nM and 100 nM Cath2 on acute itch. The results indicate that 10 nM and 100 nM Cath2 can dose-dependently relieve histamine-mediated acute itch and SADBE-induced chronic itch, and can be used as an antipruritic agent in humans and other mammals.

[0049] Example 5 Body temperature safety assessment Mice were divided into three groups: a Cath2 group receiving 25 µl of 0.33 mM capsaicin (approximately 2.5 µg) via tail vein injection, a Cath2 group receiving 100 nM / kg (25 µl) via tail vein injection, and a group receiving 25 µl of saline via tail vein injection. The mice's body temperature was measured 10 minutes before injection, and rectal temperature was measured at 0, 10, 20, and 30 minutes after injection.

[0050] The results are as follows Figure 6 The results showed that body temperature remained stable 30 minutes after injection of Cath2, and no side effect of elevated body temperature was observed, indicating that it has strong safety in regulating body temperature.

[0051] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The use of the polypeptide of claim 1 in the preparation of TRPV1 inhibitors.

3. The use of the polypeptide of claim 1 in the preparation of analgesic products.

4. The use of the polypeptide of claim 1 in the preparation of antipruritic products.

5. The use of the polypeptide of claim 1 in the preparation of analgesic and antipruritic products.

6. Use of the polypeptide of claim 1 in the preparation of a medicament for the prevention and / or treatment of diseases related to TRPV1.

7. The application according to claim 6, characterized in that, The diseases associated with TRPV1 include acute and chronic pain, acute and chronic itching, and skin inflammation associated with TRPV1.

8. A pain-relieving and / or antipruritic product, characterized in that, Includes the polypeptide and excipients as described in claim 1.

9. The product according to claim 8, characterized in that, The dosage forms of the product include injections or topical applications.

10. The product according to claim 9, characterized in that, The products include pharmaceuticals or biological pesticides.

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