Double-target peptide molecule targeting peripheral Kappa and Mu opioid receptors as well as preparation and application of double-target peptide molecule

Abstract

The invention designs a novel double-target peptide agonist targeting peripheral Kappa and Mu opioid receptors or pharmaceutically acceptable salts of the double-target peptide agonist, namely, chimeric peptides MKOP001 and MKOP002 are newly constructed on the basis of a DN-9 and dynorphin efficient analogue [des-Arg7] Dyn A (1-11)-NH2 sequence. An in-vitro calcium mobilization experiment and an acute analgesic experiment show that the brand new peptide agonists MKOP001 and MKOP002 can simultaneously activate Kappa and Mu opioid receptors and generate efficient analgesic activity. In addition, further research shows that both the MKOP001 and the MKOP002 can generate a remarkable itching relieving effect. Therefore, the peripheral restrictive Kappa and Mu opioid receptor double-target peptide molecules MKOP001 and MKOP002 or pharmaceutically acceptable salts thereof have very high application value in the aspect of preparing high-efficiency and low-side-effect analgesic or antipruritic drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry technology, and specifically relates to dual-target analgesic peptide molecules targeting peripheral Kappa and Mu opioid receptors, as well as preparation and application thereof. Background Art

[0002] Pain is a health problem of widespread concern worldwide. More than one-third of the population suffers from pain, and more than half of these patients experience moderate or severe pain. Therefore, effective treatment of pain is crucial to improving patients' quality of life. Currently, opioids such as morphine, fentanyl, and oxycodone remain the first choice for the clinical treatment of moderate to severe pain and can significantly relieve pain. However, while these traditional opioids provide effective analgesia, they are often accompanied by side effects such as respiratory depression, constipation, and addiction. Therefore, the development of new opioid analgesics with fewer side effects has important scientific value and broad application prospects.

[0003] Opioid receptors belong to the G protein-coupled receptor family, primarily consisting of three types: Mu, Kappa, and Delta. They are widely distributed in multiple tissues, including the central nervous system, peripheral nervous system, gastrointestinal tract, and immune system. Studies have shown that Mu and Kappa opioid receptors are the primary analgesic targets, while Delta opioid receptors are primarily involved in opioid regulation. Recent studies have found that activation of peripheral opioid receptors can produce significant analgesic effects. For example, activation of peripheral Kappa opioid receptors can effectively alleviate pathological pain such as visceral pain and inflammatory pain. Furthermore, peripheral opioids do not readily penetrate the blood-brain barrier. At effective analgesic doses, they can selectively activate peripheral receptors, avoiding activation of central opioid receptors, thereby reducing side effects such as drug tolerance, addiction, respiratory depression, and anxiety and depression.

[0004] In recent years, new opioid analgesics developed using strategies such as multi-target and peripherally restricted molecules can effectively reduce the side effects of traditional opioids and have great application potential. For example, the peripherally restricted Kappa opioid receptor agonist oleicin (also called CR845) developed by Cara Therapeutics was approved in 2021 for the treatment of moderate to severe pruritus in patients with chronic kidney disease. In addition, oleicin performed well in Phase III clinical trials for the treatment of postoperative pain, with only mild central side effects. NKTR-181, a Mu opioid receptor agonist developed by Nektar Therapeutics, is an analgesic made by pegylation of oxycodone. Compared with oxycodone, NKTR-181 enters the brain at a rate of only 1 / 70 of the latter, thereby effectively avoiding the side effects caused by central Mu opioid receptor activation. Currently, NKTR-181 can significantly reduce addiction in Phase III clinical trials for the treatment of low back pain and chronic pain.

[0005] Our laboratory disclosed in patent 201610252648.7 a novel chimeric peptide, DN-9, based on opioid peptides and NPFF. This peptide can simultaneously activate Mu, Kappa, and Delta opioid receptors, as well as NPFF1 and NPFF2 receptors. Peripheral injection of DN-9 produces analgesia by activating Mu and Kappa opioid receptors independently of Delta and NPFF receptors, significantly reducing the side effects of opioids. However, the analgesic effect of DN-9 still requires further optimization. Summary of the Invention

[0006] The purpose of the present invention is to provide a more druggable dual-target peptide agonist targeting peripheral Kappa and Mu opioid receptors. The inventors used DN-9 template molecules and dynorphin high-efficiency analogs [des-Arg 7 Dyn A(1-11)-NH2 is used to construct a chimeric peptide molecule or a pharmaceutically acceptable salt thereof. Compared to the reported DN-9, its in vivo analgesic efficacy and drugability are significantly improved, and it can be used to treat various pain conditions while maintaining the advantage of no analgesic tolerance.

[0007] Another object of the present invention is to provide a method for preparing the chimeric peptide or a pharmaceutically acceptable salt thereof.

[0008] Another object of the present invention is to provide therapeutic uses of the chimeric peptide or a pharmaceutically acceptable salt thereof, which can be used to relieve various pain symptoms while reducing the side effects of traditional opioid analgesics.

[0009] Another object of the present invention is to provide therapeutic uses of the chimeric peptide or a pharmaceutically acceptable salt thereof, which can be used to relieve various itching symptoms.

[0010] In order to solve the above technical problems, the present invention discloses a novel dynorphin analogue based on DN-9 and des-Arg 7 ]Dyn A(1-11)-NH2 chimeric peptide, namely [Gln 5 ]DN-9 was used as the chemical template molecule, and its C-terminal tetrapeptide was replaced with [des-Arg 7 ]Dyn A (1-11) -NH2 6-9 position tetrapeptide structure, and Pro 9 Replaced by D.Pro 9 , its specific sequence structure is shown in Formula I:

[0011] Tyr-D.Ala-Xaa1-NMePhe-Gln-Arg-Xaa2-Arg-D.Pro-NH2(I)

[0012] In the formula, Xaa1 is Gly or D.Ala, and Xaa2 is Ile or D.Ala.

[0013] Preferably, when Xaa1 is Gly and Xaa2 is Ile, the chimeric peptide has an amino acid sequence shown in Formula II, and the chimeric peptide is labeled MKOP001.

[0014] Tyr-D.Ala-Gly-NMePhe-Gln-Arg-Ile-Arg-D.Pro-NH2(Ⅱ)

[0015]

[0016] Preferably, when Xaa1 and Xaa2 are both D.Ala, the chimeric peptide has the amino acid sequence shown in Formula II, and the chimeric peptide is labeled MKOP002.

[0017] Tyr-D.Ala-D.Ala-NMePhe-Gln-Arg-D.Ala-Arg-D.Pro-NH2(Ⅲ)

[0018]

[0019] Another aspect of the present invention is to provide a method for synthesizing the multi-target molecules MKOP001 and MKOP002, comprising the following steps:

[0020] i. Resin pretreatment: Rink-Amide-MBHA resin was fully swollen in DCM for 30 min, drained and washed three times with DMF, and tested with ninhydrin;

[0021] ii. Cyclic condensation of amino acids:

[0022] ii.i Removal of Fmoc protecting group: Add deprotection reagent (DBU:piperidine:DMF=1:1:98) to the resin obtained in step i., stir and react three times; add DMF for washing, and test with ninhydrin;

[0023] ii.ii Amino acid condensation: Fmoc-Aa-OH, HBTU, HOBT, and DIEA (1:1:1:2) were dissolved in DMF and then added to the resin obtained in step ii.i. The mixture was stirred under argon for 1 h; washed with DMF and tested with ninhydrin;

[0024] ii.iii Peptide chain extension: According to the peptide sequence, different Fmoc-protected amino acids are sequentially added, with the last amino acid being a Boc-protected amino acid. The condensation method of all amino acids is the same as described above to obtain a peptide resin;

[0025] iii. Compression resin: Wash the peptide resin alternately with DCM and MeOH, and then dry;

[0026] iv. Peptide cleavage and precipitation extraction: Add a cleavage agent (TFA: Tis: H2O = 95: 2.5: 2.5) to the peptide resin obtained in step iii, and slowly stir the reaction at room temperature for 3 h; rotary evaporation, add pre-cooled diethyl ether to precipitate, collect the precipitate by centrifugation, dissolve it in acetonitrile and water, and lyophilize to obtain a crude peptide;

[0027] v. Peptide purification and analysis: The crude peptide was isolated and purified using a semi-preparative RP-HPLC column. The main peak was collected and lyophilized to obtain the pure peptide. The molecular weight of the peptide was then determined using ESI.

[0028] Another aspect of the present invention is to provide a pharmaceutical composition containing the multi-target molecules MKOP001 and MKOP002 of the present invention, wherein the multi-target molecules MKOP001 and MKOP002 are used as active ingredients and pharmaceutically acceptable carriers and / or excipients are added to prepare the pharmaceutical composition.

[0029] Another aspect of the present invention provides the medical uses of the multi-target molecules MKOP001 and MKOP002. In vivo animal studies have shown that the multi-target molecules MKOP001 and MKOP002 exhibit significant analgesic activity. They have potential applications in the development of highly effective, low-side effect analgesics for the relief and treatment of various pain conditions, including acute and pathological pain.

[0030] Another aspect of the present invention provides medical uses of the multi-target molecule MKOP002. In vivo animal studies have shown that MKOP002 exhibits significant antipruritic activity. It has potential applications in the development of highly effective, low-side-effect antipruritic drugs, and can alleviate and treat various pruritus models, including acute and chronic itch.

[0031] Those skilled in the art will appreciate that pharmaceutical compositions containing the multi-target molecules MKOP001 and MKOP002 of the present invention can be adapted for various routes of administration, such as oral, transdermal, intrathecal, intravenous, intramuscular, topical, and nasal administration. Depending on the route of administration employed, the polypeptide pharmaceutical compositions of the present invention can be formulated into various suitable dosage forms, comprising at least one effective dose of the polypeptide of the present invention and at least one pharmaceutically acceptable carrier. Examples of suitable dosage forms include injectable sterile solutions and dry powder injections, tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, aerosols, nasal sprays, and ointments and patches for application to the skin.

[0032] The pharmaceutical composition containing the polypeptide of the present invention can be prepared into a solution or lyophilized powder for parenteral administration. Before use, an appropriate solvent or other pharmaceutically acceptable carrier can be added to reconstitute the powder. The solution formula is generally a buffer solution, an isotonic solution or an aqueous solution.

[0033] The pharmaceutical dosage of the polypeptide of the present invention can vary within a wide range, and those skilled in the art can easily determine it based on objective factors such as the type of disease, severity of the disease, patient weight, dosage form, route of administration, etc.

[0034] Compared with the prior art, the main advantages of the present invention are:

[0035] (1) The subcutaneous analgesic activity of the peptide molecules of the present invention or their pharmaceutically acceptable salts is significantly improved compared to the parent DN-9, with the analgesic effect being increased by 73-9867 times;

[0036] (2) The subcutaneous analgesic duration of the peptide molecules of the present invention or their pharmaceutically acceptable salts was extended by 30 minutes compared to the parent DN-9;

[0037] (3) The peptide molecules of the present invention or their pharmaceutically acceptable salts retain the advantages of the parent compound in terms of high efficiency and low side effects;

[0038] (4) The peptide molecules of the present invention or their pharmaceutically acceptable salts produce significant antipruritic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the time-effect curve of the dose-dependent analgesic effect of subcutaneous injection of MKOP001 in mice in acute pain;

[0040] Figure 2 This is the time-effect curve of the dose-dependent analgesic effect of subcutaneous injection of MKOP002 in mice in acute pain;

[0041] Figure 3 The time-effect curve of the dose-dependent analgesic effect of subcutaneous injection of CR845 in mice in acute pain;

[0042] Figure 4 This is the time-effect curve of the dose-dependent analgesic effect of subcutaneous injection of MKOP002 in mice with inflammatory pain;

[0043] Figure 5 The time-effect curve of the dose-dependent analgesic effect of MKOP002 in neuropathic pain in mice after subcutaneous injection (mechanical stimulation);

[0044] Figure 6 This is the time-effect curve of the dose-dependent analgesic effect of subcutaneous injection of MKOP002 in mice with neuropathic pain (photothermal stimulation);

[0045] Figure 7 This is the time-effect curve of the analgesic effect of subcutaneous injection of MKOP002 in mice with bone cancer pain (mechanical stimulation);

[0046] Figure 8 This is the time-effect curve of the analgesic effect of subcutaneous injection of MKOP002 in mice with bone cancer pain (photothermal stimulation);

[0047] Figure 9 This is the time-effect curve of the analgesic effect of subcutaneous injection of MKOP002 in mice during surgical pain.

[0048] Figure 10 Subcutaneous injection of MKOP002 into mice produced a dose-dependent antipruritic effect in a chloroquine-induced acute itch model;

[0049] Figure 11 Subcutaneous injection of MKOP002 into mice produced a dose-dependent antipruritic effect in the acute itch model induced by compound 48 / 80;

[0050] Figure 12 The changes in analgesia caused by subcutaneous injection of MKOP002 in mice for 6 consecutive days;

[0051] Figure 13 The regulatory effect of MKOP002 on gastrointestinal transit in mice after subcutaneous injection;

[0052] Figure 14 The regulatory effect of MKOP002 on locomotor activity (locomotor enhancement) in mice after subcutaneous injection of MKOP002;

[0053] Figure 15 The conditioning effect of MKOP002 on the conditioned place was examined in mice injected subcutaneously;

[0054] Figure 16 The response of mice to naloxone withdrawal after subcutaneous injection of MKOP002;

[0055] Figure 17 The regulatory effect of MKOP002 on the respiratory rate of mice after subcutaneous injection of MKOP002; DETAILED DESCRIPTION

[0056] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0057] The instruments and main experimental materials used are as follows:

[0058] Experimental instruments: Solid-phase peptide synthesis syringe was purchased from Shanghai Becton Dickinson Medical Devices Co., Ltd., sieve plates were purchased from Shenzhen Comma Biotechnology Co., Ltd., rotary evaporator RE-5298A was purchased from Shanghai Yarong, freeze dryer was purchased from Ningbo Shuangjia Instrument Co., Ltd., mass spectrometer ESI-Q-TOF maXis-4G, Bruker Daltonics was purchased from Daltonics, Germany, circulating water pump SHB-Ⅲ was purchased from Zhengzhou Great Wall, LC-3050N preparative high performance liquid chromatograph was purchased from Shandong Runyang Instrument Co., Ltd., preparative column: C18 reversed phase chromatography column, 19 mm × 250 mm, Suzhou Mike Wangzhi, LC1260 analytical high performance liquid chromatograph was purchased from Agilent, analytical column: Agilent Poroshell 120EC-C18, 3.0 mm × 150 mm, 2.7 μm.

[0059] Reagents: Rink-Amide-MBHA resin (substitution value S, 0.42 mmol / g) was purchased from Tianjin Nankai Hecheng Co., Ltd. N-α-Fmoc-protected amino acids (Fmoc-Aa-OH), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and N-hydroxybenzotriazole (HOBt) were purchased from Shanghai Jier Biochemical Co., Ltd. N,N-diisopropylethylamine (DIEA) was purchased from Beijing Bailingwei, and 1,8-diazabicycloundec-7-ene (DBU) was purchased from Shanghai Mairui Company. Triisopropylsilane (Tis) was purchased from Shanghai Anaiji, and ninhydrin was a product of Shanghai Reagent Factory No. 3. Dichloromethane (DCM), N,N-dimethylformamide (DMF), hexahydropyridine (piperidine), methanol (MeOH) and pyridine were purchased from Tianjin Second Reagent Factory, and trifluoroacetic acid (TFA) and phenol were both products of Tianjin Reagent Factory; all the above organic reagents were redistilled before use.

[0060] Example 1. Synthesis of MKOP001

[0061] (1) Resin pretreatment: Weigh 1 g of Rink-Amide-MBHA resin (substitution value 0.42 mmol / g), add 10 mL of DCM, stir at 80 rpm to allow swelling, stir and react for 30 min, drain, and rinse the resin three times with DMF, each time for 3 min.

[0062] (2) Removal of the Fmoc protecting group: Add 10 mL of DBU, piperidine, and DMF in a volume ratio of 1:1:98 to the resin; stir at 80 rpm for 5 min, repeat twice. After draining, add the above mixed solution again and stir at 80 rpm for 10 min. Finally, add DMF and wash four times, each for 3 min.

[0063] (3) Ninhydrin test: The ninhydrin test reagent is a 1:2:1 volume ratio of phenol:pyridine:ninhydrin solution. The phenol solution is prepared by dissolving 20g of phenol in 5mL of anhydrous ethanol, the pyridine solution is prepared by dissolving 0.05mL of KCN (0.001M) in 2.5mL of pyridine, and the ninhydrin solution is prepared by dissolving 0.5g of ninhydrin in 10mL of anhydrous ethanol. The phenol, pyridine, and anhydrous ethanol are all redistilled. Under normal circumstances, the ninhydrin test solution is blue-purple and the resin is blue-purple.

[0064] (4) Condensation of amino acids: Weigh Fmoc-D.Pro-OH, HOBt, and HBTU in a molar ratio of 1:1:1, and dissolve them in 5 mL of DMF. DIEA is added last, stirred evenly, and then added to the resin from which the Fmoc protecting group has been removed in step (2). Under argon protection, react at room temperature at a stirring rate of 80 rpm for 60 min, and drain the solvent. After the reaction is complete, perform an indole assay according to the method of step (3). If the solution is light yellow and the resin is colorless, it indicates that the amino acid has condensed onto the resin. Then, remove the Fmoc protecting group according to step (2), and perform an indole assay again according to step (3). If both the solution and the resin are dark blue, it indicates that the Fmoc protecting group has been completely removed, and a resin peptide without the Fmoc protecting group is obtained.

[0065] (5) Peptide chain extension: The peptide resin obtained in step (4) was condensed onto the peptide resin in the same manner as in steps (2-4) by sequentially condensing Fmoc-Arg(pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NMePhe-OH, Fmoc-Gly-OH, Fmoc-D.Ala-OH, and Boc-Tyr(tBu)-OH. The peptide resin Boc-Tyr(tBu)-D-Ala-Gly-NMePhe-Gln(Trt)-Arg(Pbf)-Ile-Arg(Pbf)-D.Pro-Resin was obtained.

[0066] (6) Compression and drying of the peptide chain: The resin was washed alternately with DCM (2×3 min), MeOH (1×3 min), DCM (1×3 min), and MeOH (2×3 min), and then the stirring rod was removed and dried for 4 h.

[0067] (7) Peptide chain cleavage: Add 15 mL of cleavage agent (TFA:H2O:Tis=95:2.5:2.5) to the drained peptide resin Boc-Tyr(tBu)-D.Ala-Gly-NMePhe-Gln(Trt)-Arg(Pbf)-Ile-Arg(Pbf)-D.Pro-Resin. Stir the reaction at room temperature for 3 h, stirring at 50 rpm for 1 min every 15 min. The filtrate is fully dried under reduced pressure at no higher than 40°C, and ice ether is added and thoroughly shaken to mix. The crude peptide is fully precipitated as a white precipitate. Finally, the obtained peptide aqueous solution is freeze-dried to obtain 423 mg of white crude peptide solid powder, with a crude peptide yield of 90%.

[0068] (8) Purification of Crude Peptide: The crude peptide compound was separated and purified using a reversed-phase high-performance liquid chromatography (RP-HPLC) C18 column (XBridge™ BEH 130Prep C18, 19 mm × 250 mm) with acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA). After separation, the main peak sample was collected to obtain a purified MKOP001 sample. The sample load was 50 mg and freeze-dried to obtain 35 mg of a white pure peptide solid powder, with a pure peptide yield of 70%. The results of mass spectrometry and chromatographic analysis are shown in Table 1.

[0069] Example 2. Synthesis of MKOP002

[0070] Following the same procedure as in Example 1, the linear peptide Tyr-D.Ala-D.Ala-NMePhe-Gln-Arg-D.Ala-Arg-D.Pro-NH2 was obtained as a white solid powder. The crude peptide yield was 87%, and the yield of the purified pure peptide was 72%. The results of mass spectrometry and chromatographic analysis are shown in Table 1.

[0071] Table 1 Chemical characterization of the multi-target molecules MKOP001 and MKOP002

[0072]

[0073] Note: The gradient elution system was: 10-90% acetonitrile / water (0.1% TFA) (completed in 30 min), the flow rate was: 1 mL / min, the detection wavelength was 220 nm, and the analytical column was: Agilent Poroshell 120EC-C18, 3.0 mm × 150 mm, 2.7 μm.

[0074] Example 3. In vitro functional activity assay of MKOP001 and MKOP002:

[0075] Using CHO-K1 / G cells that stably overexpress Mu opioid receptors, Kappa opioid receptors, and Delta opioid receptorsα15 Cell lines, the agonist activity of compounds on opioid receptors was detected by examining the regulation of intracellular calcium ion mobilization by MKOP001 and MKOP002.

[0076] Experimental Methods: 30,000 to 50,000 cells were seeded per well in a 96-well plate and cultured for at least 20 hours. At the start of the experiment, the culture medium was aspirated from the culture dish, followed by the addition of 50 μL of a calcium dye stock solution containing 2.5 mM probenecid, 0.9 μM Fluo-4-AM, and F127 (0.03%), and incubation at 37°C for 30 minutes. Following incubation, the dye was aspirated and added to 75 μL of HBSS buffer (containing 2.5 mM probenecid). The 96-well drug plate, 96-well cell plate, and automated dosing pipette tip were then placed in the FlexStation 3 calcium flow workstation to monitor calcium signal changes following drug administration.

[0077] Table 2 Calcium mobilization experiments of MKOP001 and MKOP002 of the present invention on opioid receptors

[0078]

[0079] The calcium mobilization effect was expressed as the percentage increase in intracellular calcium ions (% control), where % control = (test value - basal value) / basal value * 100. The relevant % control data are expressed as mean ± standard error (SEM). The dose-effect relationships of the compounds were analyzed using a nonlinear regression model. The EC values ​​for the regulation of intracellular calcium ion mobilization by compounds MKOP001 and MKOP002 were calculated using GraphPadPrism version 8.0.1. 50 The experimental results are shown in Table 2.

[0080] Experimental results showed that both MKOP001 and MKOP002 exhibited dose-dependent opioid receptor agonist activity in cells stably expressing both the Mu and Kappa opioid receptors. In cells stably expressing the Delta opioid receptor, only MKOP001 exhibited weak agonist activity, while MKOP002 showed no agonist activity. In summary, MKOP001 is a multi-targeted agonist at the Mu / Kappa / Delta opioid receptors, while MKOP002 is a dual-targeted agonist at the Mu / Kappa opioid receptors.

[0081] Example 4. Evaluation of the analgesic activity of MKOP001 and MKOP002 in the photothermal tail-flick acute pain model :

[0082] For subcutaneous administration, select the subcutaneous region of the back as the injection site. Use a 1mL sterile syringe to inject 0.1mL of drug per 10g of body weight. Lift the skin on the mouse's back with your right hand and tilt the needle to inject the drug subcutaneously. Gently shake the needle after insertion to ensure it penetrates the subcutaneous region and avoid drug leakage.

[0083] The photothermal tail-flick test employed the method outlined by D'Amour and Smith, and the experimental parameters were optimized. Male Kunming mice weighing 21 ± 2 g were used in the experiment, and the ambient temperature was controlled at 22 ± 2°C. Mice had free access to water. Before the experiment began, they were transferred from the housing room to the experimental area and allowed to acclimate for 30 minutes. The mice were then grasped with their right hands, their tails allowed to hang naturally, and placed under a radiant light source, ensuring a distance of 2–3 mm between the tail tip and the light source. The light intensity was adjusted to ensure a tail-flick duration of 3–5 seconds, which served as the baseline pain threshold. The irradiation duration did not exceed 10 seconds to avoid tail burns, which was set as the maximum latency. After drug administration, the tail-flick latency of the mice was recorded at 0, 5, 10, 15, 20, 30, 40, 45, 60, 90, 120, 180, 240, 300, and 360 minutes.

[0084] The analgesic effect of a drug is generally evaluated by the maximum analgesic effect (MPE) (%), where MPE (%) = 100 × [(pain threshold after administration - basal pain threshold) / (10s - basal pain threshold)]. 50 (50% effective dose, ED 50 ) refers to the drug dose that causes 50% of the effect. 50 The MPE (%) and the drug concentration that achieved the maximum analgesic effect (MPE) and 95% confidence interval were calculated using the statistical software GraphPadPrism 8.0.1. The difference in analgesic effect was statistically analyzed using one-way ANOVA (Dunnett test). * P<0.05, ** P < 0.01 and * ** P<0.001 indicates that the difference between the group only injected with relevant drugs and the saline group is significant.

[0085] In the photothermal tail-flick experiment in mice, maternal DN-9 analgesic ED 50 The value is 0.216μmol / kg. Subcutaneous injection of MKOP001, MKOP002 and CR845 can produce dependent analgesic effects. 50The analgesic doses of MKOP002 and MKOP002 were 0.004 μmol / kg, 0.0002 μmol / kg, and 0.005 μmol / kg, respectively, which were 54-fold and 1080-fold higher than those of the parent compound DN-9. The effective analgesic duration was extended from 90 minutes of the parent compound to 120 minutes. Therefore, we identified MKOP002 as the superior compound. The subcutaneous analgesic dose-effect curve of the compound is shown in Figure 2. Figure 1 、 Figure 2 and Figure 3 shown.

[0086] Example 5. Evaluation of the analgesic activity of MKOP002 in an inflammatory pain model:

[0087] The carrageenan-induced inflammatory pain model is used to evaluate the analgesic efficacy of drugs in inflammatory pain. Male Kunming mice weighing 22-25 g were used. 20 μL of 2% carrageenan dissolved in sterile water was injected into the digit of the mouse's right paw. Following injection, the microinjector needle was briefly held at the injection site for a few seconds before being withdrawn with a twist to prevent leakage. Behavioral testing was conducted 24 hours after carrageenan injection.

[0088] Mechanical stimulation: Before the experiment, mice were placed in the measuring apparatus (wire mesh base covered with a plexiglass box) and allowed to acclimate for 30 minutes before the experiment began. The basal threshold for mechanical stimulation was measured using a von Frey analgesia instrument, with five measurements per mouse, separated by 5 minutes. Mechanical allodynia thresholds for inflammatory pain and neuropathic pain were measured three times, with a 5-minute interval between each measurement. After the mice were injected with saline or drug, three measurements were taken at each time point, separated by 2 minutes.

[0089] The analgesic effect of a drug is generally evaluated by the maximum analgesic effect (MPE) (%), where MPE (%) = 100 × [(pain threshold after administration - basal pain threshold) / (10s - basal pain threshold)]. 50 (50% effective dose, ED 50 ) refers to the drug dose that causes 50% of the effect. 50 The MPE (%) and the drug concentration that achieved the maximum analgesic effect (MPE) and 95% confidence interval were calculated using the statistical software GraphPadPrism 8.0.1. The difference in analgesic effect was statistically analyzed using one-way ANOVA (Dunnett test). * P<0.05, ** P < 0.01 and * ** P<0.001 indicates that the difference between the group only injected with relevant drugs and the saline group is significant.

[0090] In the mouse carrageenan inflammatory pain model, maternal DN-9 showed analgesic ED 50The value is 3.08μmol / kg. Subcutaneous injection of MKOP002 can produce an analgesic effect dependent on its analgesic ED 50 The analgesic effect of the compound was 0.042 μmol / kg, which was 73 times higher than that of the parent DN-9. The effective analgesic effect time was extended from 90 minutes of the parent to 120 minutes. Figure 4 shown.

[0091] Example 6. Evaluation of the analgesic activity of MKOP002 in a neuropathic pain model:

[0092] The sciatic nerve compression injury (CCI) model is used to evaluate the analgesic effect of drugs in the neuralgia model. 16-20g Kunming male mice were used for the experiment. First, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (80mg / kg). The hair at the base of the right leg of the mouse was cleaned and fixed on a sterile surgical tray. After disinfection with iodine, a wound of about 2cm was cut along the base of the thigh, and the muscles were separated with a glass needle to expose the sciatic nerve. Then, three loops were tied on the sciatic nerve with chromium catgut from the proximal end (spinal cord) to the distal end at intervals of 1mm. The strength of the ligature was based on the slight tremor of the mouse's legs. Finally, the wound was sutured with surgical sutures, iodine was applied, and penicillin was injected subcutaneously on the back. Behavioral experiments were performed after the mice recovered for 7 days.

[0093] Mechanical stimulation: The method is the same as above.

[0094] Thermal stimulation: Mice were placed in a measuring apparatus (a transparent-bottomed resin box covered with a plexiglass box) and allowed to acclimate for 30 minutes before the experiment began. The basal threshold for thermal stimulation was measured using a PMT-100 thermal analgesia meter. To avoid potential tissue damage, the stimulation duration was limited to 20 seconds. The frequency and intervals for basal threshold, thermal allodynia threshold, and post-drug pain threshold measurements were consistent with those for mechanical stimulation.

[0095] The analgesic effect of a drug is generally evaluated by the maximum analgesic effect (MPE) (%), where MPE (%) = 100 × [(pain threshold after administration - basal pain threshold) / (10s - basal pain threshold)]. 50 (50% effective dose, ED 50 ) refers to the drug dose that causes 50% of the effect. 50 The MPE (%) and the drug concentration that achieved the maximum analgesic effect (MPE) and 95% confidence interval were calculated using the statistical software GraphPadPrism 8.0.1. The difference in analgesic effect was statistically analyzed using one-way ANOVA (Dunnett test). * P<0.05, ** P < 0.01 and * **P<0.001 indicates that the difference between the group only injected with relevant drugs and the saline group is significant.

[0096] Maternal DN-9 analgesic EDs of mechanical and photothermal stimulation in a mouse neuropathic pain model 50 The values ​​were 3.32μmol / kg and 2.96μmol / kg, respectively. Subcutaneous injection of MKOP002 can produce analgesic effects in the neuropathic pain model, and its analgesic ED of mechanical and photothermal stimulation is 50 The values ​​were 0.0062μmol / kg, and 0.0003μmol / kg, respectively. They were 535 and 9867 times higher than the parent DN-9, and the effective analgesic duration was extended from 90 minutes of the parent to 120 minutes. The subcutaneous analgesic dose-effect curve of the compound is shown in Figure 2. Figure 5 and 6 shown.

[0097] Example 7. Evaluation of the analgesic activity of MKOP002 in a bone cancer pain model:

[0098] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (20 mg / kg). Subsequently, the right tibial plateau was exposed and B16-F10 melanoma cells (1×10 5 , 5 μL) was injected into the tibial cavity. The injection site was sealed with sterile medical glue to prevent cancer cell leakage. Finally, the wound was sutured and erythromycin ointment was applied to prevent infection. After recovery from anesthesia, the mice were housed individually.

[0099] The analgesic effect of a drug is generally evaluated by the maximum analgesic effect (MPE) (%), where MPE (%) = 100 × [(pain threshold after administration - basal pain threshold) / (10s - basal pain threshold)]. 50 (50% effective dose, ED 50 ) refers to the drug dose that causes 50% of the effect. 50 The MPE (%) and the drug concentration that achieved the maximum analgesic effect (MPE) and 95% confidence interval were calculated using the statistical software GraphPadPrism 8.0.1. The difference in analgesic effect was statistically analyzed using one-way ANOVA (Dunnett test). * P<0.05, ** P < 0.01 and * ** P<0.001 indicates that the difference between the group only injected with relevant drugs and the saline group is significant.

[0100] In a mouse bone cancer pain model, subcutaneous injection of MKOP002 can produce an analgesic effect dependent on the analgesic effect of mechanical and photothermal stimulation. 50The values ​​were 0.62μmol / kg and 0.0005μmol / kg respectively. Its effective analgesic effect lasted for 120min. The subcutaneous analgesic dose-effect curve of the compound is shown in Figure 2. Figure 7 and 8 shown.

[0101] Example 8. Evaluation of analgesic activity of MKOP002 in a surgical pain model:

[0102] The foot incision postoperative pain model is a classic model for postoperative pain simulation, commonly used to reproduce acute postoperative pain following skin and muscle incisions. Foot incision in mice induces a variety of nociceptive behaviors that mimic the progression of postoperative pain in humans. This model is widely used to evaluate the efficacy of new and existing analgesics.

[0103] Under isoflurane anesthesia, the mice were fixed on the operating table. After the right hind paw was disinfected, a 5mm longitudinal incision was made 2mm away from the heel with a scalpel. After incising the skin and fascia, the flexor plantar brevis muscle was lifted with forceps and incised longitudinally. During the operation, damage to muscle attachment points, nerves and blood vessels should be avoided. After hemostasis, the incision was sutured with 4-0 nylon suture and erythromycin ointment was applied. The surgical process was controlled within 3-5 minutes. After surgery, the mice were placed in a quiet, warm, and light-proof environment and fed individually. Before and 6 hours after surgery, the mechanical threshold of the plantar surface on the operated side was measured using an electronic von Frey electronic analgesia, and its mechanical pain sensitivity response was continued to be measured at different time points after drug administration.

[0104] The analgesic effect of a drug is generally evaluated by the maximum analgesic effect (MPE) (%), where MPE (%) = 100 × [(pain threshold after administration - basal pain threshold) / (10s - basal pain threshold)]. 50 (50% effective dose, ED 50 ) refers to the drug dose that causes 50% of the effect. 50 The MPE (%) and the drug concentration that achieved the maximum analgesic effect (MPE) and 95% confidence interval were calculated using the statistical software GraphPadPrism 8.0.1. The difference in analgesic effect was statistically analyzed using one-way ANOVA (Dunnett test). * P<0.05, ** P < 0.01 and * ** P<0.001 indicates that the difference between the group only injected with relevant drugs and the saline group is significant.

[0105] Subcutaneous injection of MKOP002 can produce dose-dependent analgesia in the surgical pain model. 50 The value is 1.84μmol / kg. The subcutaneous analgesic dose-effect curve of the compound is as follows Figure 9shown.

[0106] Example 9. Evaluation of antipruritic activity of subcutaneous injection of MKOP002:

[0107] Male Kunming mice weighing 22 to 25 g were used for the acute itch experiment, and the experimental environment temperature was controlled at 22±2°C. Before the experiment, the mice needed to adapt to the environment for two days, with each adaptation time of 30 minutes, and the environment needed to maintain low light. One day before the experiment, the hair on the skin of the neck and back of the mice was shaved. The mice were placed in a 14×18×12 cm polypropylene glass box, and after 30 minutes of adaptation, the test drug MKOP002 was injected subcutaneously. Five minutes later, 50 μL of the itch-inducing substances chloroquine and compound 48 / 80 were injected intradermally into the back of the mice's neck, and the scratching behavior of the mice within 30 minutes was recorded by video recording in a quiet environment. The effective dose (ED) of the drug was calculated by the maximum antipruritic effect (MPE%). 50 ), MPE (%) = 100 × [(number of scratches in the saline group - number of scratches in the drug group) / number of scratches in the saline group]. GraphPad Prism 8.0.1 statistical software was used to calculate drug ED 50 The difference in antipruritic effect was analyzed using one-way ANOVA (Dunnett's test). * P<0.05, ** P < 0.01 and * ** P<0.001 indicates that the difference between the drug group and the saline group is significant.

[0108] like Figure 10 and 11 As shown in the results, subcutaneous injection of MKOP002 showed a dose-dependent antipruritic effect in the acute itch model induced by chloroquine and compound 48 / 80. 50 0.64μmol / kg and 0.51μmol / kg respectively.

[0109] Example 10. Changes in analgesic effect caused by subcutaneous injection of MKOP002 for 6 consecutive days:

[0110] Analgesic tolerance was also evaluated using the photothermal tail-flick model. Mice were injected with the drug once daily for eight consecutive days. After each injection, the tail-flick latency corresponding to the highest analgesic point was measured. The tail-flick latency was compared with the tail-flick latency after the first day of drug administration to evaluate whether the drug had caused analgesic tolerance. Fentanyl was used as a positive control. The difference in analgesic effect was statistically analyzed using one-way ANOVA (Tukey test). * P<0.05, ** P < 0.01 and * ** P<0.001 indicates that the difference between the group only injected with relevant drugs and the saline group is significant.

[0111] Subcutaneous injection of fentanyl showed a significant decrease in analgesic effect on the fourth day, while subcutaneous injection of MKOP002 did not cause analgesic tolerance. Figure 12 shown.

[0112] Example 11. Effect of subcutaneous injection of MKOP002 on gastrointestinal transit:

[0113] Constipation is a common side effect of opioids, and the effects of drugs on gastrointestinal motility are usually evaluated by the symptoms of constipation. The experiment selected 26±2g Kunming male mice. The mice were first starved and placed in a cage without bedding. Their diet was restricted but they were provided with free drinking water for 16 hours. After 16 hours, the mice were weighed and then orally administered. 15 minutes after administration, a pre-prepared activated carbon suspension (a suspension of 5% activated carbon and 10% gum arabic in physiological saline) was used for gavage at a dose of 0.1mL per 10g of body weight. 30 minutes after gavage, the mice were quickly killed and dissected. The entire length of the small intestine from the pylorus to the cecum was removed, and the total length of the small intestine and the distance moved by the carbon powder were measured.

[0114] Gastrointestinal motility data were expressed as a percentage of gastrointestinal motility, calculated by dividing the distance moved by the carbon powder by the total length of the small intestine. Data were expressed as mean ± standard error (SEM) of the percentage of gastrointestinal motility. Differences between the compound and saline were analyzed using one-way ANOVA with Dunnett's test. * P<0.05, ** P < 0.01 and * ** P<0.001 indicates that the difference between the injection of saline only and the injection of relevant compounds is significant.

[0115] exist Figure 13 In the study, the percentage of gastrointestinal inhibition in the saline group and the drug-treated groups was 87.2%, 68.3%, 55.5% and 43.0%, respectively, after subcutaneous injection of normal saline, 0.092, 0.92 and 9.16 μmol / kg of MKOP002. 50 It is 2.57μmol / kg.

[0116] Example 12. Determination of addictive side effects of subcutaneous injection of MKOP002:

[0117] The addictive potential of MKOP002 was assessed using the open field test, conditioned place preference (CPP), and naloxone withdrawal tests. Opioid injections promote dopamine release and increase locomotor activity in mice, so locomotor activity is often used to assess drug addictiveness.

[0118] The open field test was conducted using a 50×50×40cm topless black plexiglass box and a motion monitoring system. The experimental subjects were 21±2g Kunming male mice, and the laboratory temperature was maintained at 22±1°C to avoid the influence of temperature on the movement of the mice. Before the experiment, the experimental box was cleaned with alcohol to remove residual odors and prevent interference with the movement of the mice. At the beginning of the experiment, the basal locomotor activity of the mice was recorded for 30 minutes, and then physiological saline, 0.19μmol / kg fentanyl and 9.16μmol / kg MKOP002 were subcutaneously injected, and then the locomotor activity of the mice was recorded within 150 minutes. The locomotor activity of the mice was expressed as the total movement distance, and the data results were expressed as mean ± standard error (Means ± SEM). The differences between the compound group and the saline control group were statistically analyzed by one-way ANOVA (Bonferroni test). * P<0.05, ** P<0.01, *** P<0.001 indicates a significant difference. Figure 14 shown.

[0119] The conditioned place preference (CPP) test was conducted using a three-box apparatus consisting of two large boxes (20 × 20 × 20 cm) separated by a small box (5 × 20 × 20 cm). Each box had a closable 5 × 5 door at the bottom for mice to enter and exit. One box was white with a rough wire mesh bottom and a light intensity of 50 lux; the other was black with a smooth bottom and a light intensity of 20 lux. Male mice weighing 25 ± 5 g were used in the experiment, and the ambient temperature was 22 ± 1°C. On the first day of the experiment, mice were screened and allowed to freely move between the two boxes. The time they spent in each box was recorded. Mice that spent more than 9 minutes in a box were removed, thus identifying mice with no preference or aversion. Over the next three days, mice were continuously injected subcutaneously with saline, fentanyl (0.19 μmol / kg), and MKOP002 (9.16 μmol / kg). After the small door was closed, the mice were divided into two groups. One group was injected with saline in the white box in the morning and the drug in the black box in the afternoon; the other group was injected with saline in the black box in the morning and the drug in the white box in the afternoon. Each mouse adapted to the environment for 45 minutes and trained continuously for 3 days. On the 5th day, CPP performance test was performed. As on the first day, the time the mouse stayed in each grid after drug administration was recorded for 15 minutes. The differences between the drug group and the normal saline control group were compared by one-way ANOVA (Bonferroni test). * P<0.05, ** P<0.01, *** P<0.001 indicates a significant difference. Figure 15 shown.

[0120] The naloxone withdrawal test is a classic method commonly used to assess physiological drug addiction. The experimental protocol was based on the study of Venetia Zachariou (2003). During the experiment, fentanyl and MKOP002 were injected subcutaneously every 8 hours, and the drug dose was gradually increased. 50 For reference, the doses of morphine are (20, 40, 60, 80, 100, 100, 100 mg / kg), and their analgesic ED 50 is 1.68 mg / kg, so the dosage is analgesic ED 50 According to this method, the subcutaneous injection of fentanyl ED 50 The maximum analgesic ED of MKOP002 is 0.038 μmol / kg, so the subcutaneous injection doses are 0.38, 0.76, 1.13, 1.51, 1.89, 1.89, and 1.89 μmol / kg, respectively. 50 The dose of naloxone was 2 μmol / kg, and the corresponding subcutaneous injection doses were 20, 40, 60, 80, 100, 100, and 100 μmol / kg, respectively. Two hours after the last administration, 10 mg / kg of naloxone was injected intraperitoneally, and the mice were immediately placed in an opaque barrel structure with an inner diameter of 9 cm and a height of 32 cm. The number of jumps of the mice was recorded within 30 hours. The experimental results were expressed as the number of jumps ± standard error (Means ± SEM). The differences between the compound group and the saline control group were compared by one-way analysis of variance (Bonferroni test). * P<0.05, ** P<0.01, *** P<0.001 indicates that the difference between the two groups is significant. Figure 16 shown.

[0121] like Figure 14 As shown, in an open field test, mice were subcutaneously injected with saline, 0.19 μmol / kg fentanyl, and 9.16 μmol / kg MKOP002. The saline group traversed 119.4 ± 27.8 m, the fentanyl group traversed 235.4 ± 28.4 m, and the MKOP002 group traversed 74.9 ± 17.0 m, respectively. Compared to the saline control group, mice in the fentanyl group exhibited significantly increased locomotor activity, whereas MKOP002 had no effect on locomotor activity.

[0122] like Figure 15As shown, in the CPP test, subcutaneous injection of saline and 0.19 μmol / kg fentanyl produced significant place preference, while subcutaneous injection of 9.16 μmol / kg MKOP002 did not produce significant place preference.

[0123] like Figure 16 As shown, in a naloxone withdrawal experiment, saline, fentanyl, and MKOP002 were administered subcutaneously. The saline group had 9 jumps, the fentanyl group had 40 jumps, and the MKOP002 group had 11 jumps. Compared to the saline group, the fentanyl group had a significant increase in jumps, while MKOP002 had no significant effect on jumps.

[0124] Example 13. Effect of subcutaneous injection of MKOP002 on respiratory rate in mice:

[0125] Respiratory data were obtained using a whole-body plethysmography system (TOW-INT TECH, Shanghai, China). This technology quantifies the respiratory rate of awake and unrestrained mice. Briefly, the animals were initially acclimated for 10 min in a transparent plexiglass chamber. Subsequently, 10 min of basal values ​​were measured before the injection of saline, fentanyl, and MKOP002. After drug injection, respiratory parameters were continuously monitored in unrestrained mice for 100 min. Differences between the compound and saline control groups were analyzed using one-way analysis of variance (Bonferroni test of one-way ANOVA). * P<0.05, ** P < 0.01 and * ** P<0.001 indicates that there is a significant difference between saline and the compound. Figure 17 shown.

[0126] like Figure 17 As shown, in a whole-body plethysmography experiment in mice, subcutaneous injection of 1.89 μmol / kg fentanyl produced significant respiratory depression, while subcutaneous injection of 9.16 μmol / kg MKOP002 did not produce significant respiratory depression.

[0127] The above specific description of the present invention does not limit the present invention. Those skilled in the art can make various changes and modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the appended claims of the present invention.

Claims

1. A novel high-efficiency analogue of DN-9 and dynorphin [des-Arg 7 ]Dyn A (1-11) -NH2 chimeric peptide, the structure of which is shown in Formula I: Tyr-D.Ala-Xaa1-NMePhe-Gln-Arg-Xaa2-Arg-D.Pro-NH2 (I) wherein Xaa1 is Gly or D.Ala, and Xaa2 is Ile or D.Ala.

2. According to claim 1, when Xaa1 is Gly and Xaa2 is Ile, the chimeric peptide sequence is as shown in Formula II: Tyr-D.Ala-Gly-NMePhe-Gln-Arg-Ile-Arg-D.Pro-NH2(II).

3. According to claim 1, when Xaa1 and Xaa2 are both D.Ala, the chimeric peptide sequence is as shown in Formula III Tyr-D.Ala-D.Ala-NMePhe-Gln-Arg-D.Ala-Arg-D.Pro-NH2(Ⅲ).

4. A method for preparing the chimeric peptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Including steps: i. Resin pretreatment: Place the resin in dichloromethane (DCM) solution and fully swell for 30 minutes. The resin is Rink-Amide-MBHA, the volume ratio of DCM to the mass ratio of the resin is 8-12 mL / g, and the stirring rate is 60-100 rpm. ii. Cyclic condensation of amino acids: ii.i Removal of the Fluorenylmethoxycarbonyl (Fmoc) Protecting Group: Place the resin in a volume of the deprotection solution and stir repeatedly for a specified period of time. Ninhydrin test: Under normal circumstances, the solution and the resin will be blue-purple. The deprotection reagent is a mixture of 1,8-diazabicycloundec-7-ene (DBU), piperidine and N,N-dimethylformamide (DMF) in a volume ratio of 1:1:98; the mass ratio of the deprotection mixture to the resin is 8-12 mL / g; the stirring rate is 60-100 rpm, the stirring reaction is repeated 3 times, and the reaction times are 2-6 min, 2-6 min and 8-12 min, respectively. ii.ii Amino Acid Condensation: Dissolve the amino acid and condensing agent in a small amount of DMF and add to the resin obtained in step ii.i. Stir under argon for a desired reaction time. Ninhydrin test: Normally, the solution is light yellow and the resin is colorless. Among them, N α -Fmoc or N α The molar ratio of -BOC protected amino acid, O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBT) and N,N-diisopropylethylamine (DIEA) is 1:0.2-1.5:0.2-1.5:1.5-3; DMF and N α The volume-to-mass ratio of the Fmoc-protected amino acid is 5-10 mL / g; the stirring rate is 60-100 rpm; and the reaction is carried out at room temperature for 40-100 min. ii.iii. Peptide chain extension: Based on the peptide sequence, different Fmoc-protected amino acids are sequentially attached to the peptide resin obtained in step ii.ii. The last amino acid is typically protected with a tert-butyloxycarbonyl (Boc) group, thus omitting the final Fmoc removal step. The condensation method for all amino acids is the same as described above. This yields a peptide resin. iii. Compression resin: Wash the peptide resin alternately with DCM and MeOH, seal and dry. The volume mass ratio of DCM and MeOH to the resin is 8 to 12 mL / g; and the pumping time is 3 to 5 h. iv. Peptide Cleavage and Precipitation Extraction: Add the cleavage agent mixture to the peptide resin obtained in step iii. Slowly stir the reaction at room temperature for a period of time. Afterwards, collect the cleavage solution and spin-dry it using a rotary evaporator at no more than 40°C. Pre-chilled ether is then added to the remaining cleavage solution. The precipitate is collected by centrifugation and dissolved in acetonitrile and water. The peptide solution is lyophilized to obtain the crude peptide. The cleavage agent mixture is trifluoroacetic acid (TFA), triisopropylsilane (Tis) and water (H2O) in a volume ratio of 95:2.5:2.5; the cleavage time is 1.5 to 4 hours; and the volume mass ratio of the cleavage mixture to the peptide resin is 10 to 20 mL / g. v. Purification and analysis of peptides: The crude peptide was separated and purified by preparative high performance liquid chromatography (RP-HPLC), and the purity was determined by analytical RP-HPLC to obtain pure peptides.

5. The use of the chimeric peptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Used for preparing a medicine for relieving and / or treating various types of pain, including acute pain and pathological pain.

6. The use of the chimeric peptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Used for preparing a medicament for relieving and / or treating various types of itches, including acute itches and chronic itches.

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