Opioid and ghrelin receptor multi-target peptide molecule as well as preparation method and application thereof

Abstract

The invention provides an opioid and ghrelin receptor multi-target peptide molecule as well as a preparation method and application thereof, and relates to the technical field of biochemistry. According to the present invention, G (1-5)-EM2 is adopted as a basic peptide, L-amino acids at the 5th site, the 7th site and the 9th site of G (1-5)-EM2 and isomer D-amino acids of G (1-5)-EM2 are modified to obtain the multi-target peptide molecule, and the multi-target peptide molecule has the multi-target agonistic activity of an opioid receptor and a ghrelin receptor, can pass through the blood brain barrier, can be used for relieving various pain symptoms such as acute pain or pathological pain, and can be used for preparing the multi-target peptide molecule, meanwhile, side effects caused by traditional opioid analgesic drugs can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry technology, and in particular to an opioid and ghrelin receptor multi-target peptide molecule, a preparation method and an application thereof. Background Art

[0002] Pain is a global public health problem and one of the common clinical symptoms. According to incomplete statistics, about 25%-30% of the people in the world are suffering from persistent or periodic pain. In view of this problem of pain, the medical community has been exploring treatment methods, including drug therapy, physical therapy, traditional Chinese medicine therapy, psychotherapy, etc. Currently, the most commonly used treatment plan is still drug therapy. Among them, opioid drugs such as morphine and fentanyl are the most commonly used analgesics in clinical practice, especially playing an important role in the treatment of moderate to severe pain. However, opioid analgesics have side effects such as tolerance, addiction, respiratory depression, and constipation, which limit their clinical application. Existing studies have shown that opioid peptides can mediate highly efficient analgesic effects, and their side effects are lower than those of traditional opioid analgesics. Therefore, opioid peptides can be used as chemical template molecules for the research and development of new opioid analgesics with high efficiency and low side effects. Research has shown that endomorphin molecules can be used as chemical template molecules for the research and development of new opioid analgesics with high efficiency and low side effects. However, so far, the disadvantages such as side effects, blood-brain barrier permeability, and poor enzymatic stability of endomorphin cannot be completely solved.

[0003] In recent years, the construction of multi-target opioid molecules has provided a new idea for the research and development of new opioid analgesics with high efficiency and low side effects. A multi-target drug refers to a single-component drug that can simultaneously and selectively act on two or more molecular targets. The multi-target synergistic effect has better effects on treating complex diseases, and at the same time can avoid the disadvantages of single-target drugs such as easy generation of side effects and poor drug resistance, and has been widely verified in the research and development of new drugs for complex diseases such as cancer, depression, and diabetes (Neurotherapeutics, 2009, 6(1): 152-162). The research strategy of traditional opioid analgesic drugs has also changed from simply improving the affinity and selectivity for μ-opioid receptors to the research of multi-target drugs. This new strategy has been successfully verified. Summary of the Invention

[0004] The purpose of the present invention is to provide an opioid and ghrelin receptor multi-target peptide molecule, a preparation method and an application thereof, which have multi-target agonist activities for opioid receptors and ghrelin receptors, can also cross the blood-brain barrier and be used to relieve various pain symptoms such as acute pain or pathological pain, and at the same time can reduce the side effects brought by traditional opioid analgesics.

[0005] In a first aspect, the present invention provides an opioid and ghrelin receptor multi-target peptide molecule, and the structure of the multi-target peptide molecule is shown in Formula I:

[0006] Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Xaa 1 -Tyr-Xaa 2 -Phe-Xaa 3 -NH2 (I);

[0007] Wherein, Xaa 1 is Leu or D-Leu, Xaa 2 is Pro or D-Pro, Xaa 3 is Phe or D-Phe, and at least one of Xaa 1 , Xaa 2 , Xaa 3 is a D-amino acid.

[0008] Optionally, the multi-target peptide molecule is selected from the following compounds:

[0009] Gly-Ser-Ser(O-CO-C7H 15 )-Phe-(D-Leu)-Tyr-Pro-Phe-Phe-NH2 (I-1);

[0010] Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Leu-Tyr-(D-Pro)-Phe-Phe-NH2 (I-2);

[0011] Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Leu-Tyr-Pro-Phe-(D-Phe)-NH2 (I-3).

[0012] In a second aspect, the present invention provides a method for preparing an opioid and ghrelin receptor multi-target peptide molecule, which is obtained by sequentially coupling amino acids on a solid-phase carrier by solid-phase synthesis and then cleaving.

[0013] Optionally, the solid-phase carrier is amino resin.

[0014] Optionally, it includes: deprotection of amino resin; condensation and extension of amino acids; cleavage and purification of polypeptide.

[0015] In a third aspect, the present invention further provides an application of an opioid and ghrelin receptor multi-target peptide molecule in the preparation of pain drugs.

[0016] Optionally, the pain medication is used for relieving and / or treating pathological pain in a subject in need thereof, and the pathological pain includes acute pain and chronic pain.

[0017] In a fourth aspect, the present invention also provides a pharmaceutical composition comprising the above-mentioned opioid and ghrelin receptor multi-target peptide molecule.

[0018] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the enzymatic stability of the multi-target peptide molecule in Examples 1 to 3 and Comparative Example 1 of the present invention; Figure 1 A is the residual rate of the multi-target peptide molecule in the cerebral plasma membrane at different time points, Figure 1 B is the residual rate of the multi-target peptide molecule in the plasma at different time points;

[0020] Figure 2 It is a graph of the fluorescence intensity of the whole body of mice during the blood-brain barrier permeability test of the multi-target peptide molecule in Examples 1 to 3 and Comparative Example 1 of the present invention;

[0021] Figure 3 During the blood-brain barrier permeability test of the multi-target peptide molecule in Examples 1 to 3 and Comparative Example 1 of the present invention, Figure 2 it is a graph of the change in fluorescence intensity of the mouse head;

[0022] Figure 4 It is a graph of the fluorescence intensity of the brain of an ex vivo mouse during the blood-brain barrier permeability test of the multi-target peptide molecule in Examples 1 to 3 and Comparative Example 1 of the present invention;

[0023] Figure 5 It is a graph of the change in analgesic effect during the warm water bath tail-flick test after the multi-target peptide molecule in Examples 1 to 3 of the present invention is injected into the lateral ventricle;

[0024] Figure 6 It is a graph of the change in analgesic effect during the warm water bath tail-flick test after the multi-target peptide molecule in Examples 1 and 3 of the present invention is injected intrathecally and intravenously; Figure 6 A, Figure 6 B is for intrathecal injection, Figure 6 C, Figure 6 D is for intravenous injection;

[0025] Figure 7 It is a test chart of the blood-brain barrier permeability of the multi-target peptide molecule in Examples 1 and 3 of the present invention during acute pain after intravenous injection; Figure 7 A is a graph of the change in analgesic effect after injecting the multi-target peptide molecule in Example 1,Figure 7 B is the curve graph of the analgesic effect change of the multi-target peptide molecule in Injection Example 3;

[0026] Figure 8 It is the graph of the change in the number of writhing times during the acetic acid writhing test after intrathecal injection and tail vein injection of the multi-target peptide molecule in Example 1 and Example 3 of the present invention; Figure 8 A, Figure 8 B is for intrathecal injection, Figure 8 C, Figure 8 D is for tail vein injection;

[0027] Figure 9 It is the graph of the change in licking and paw shaking time during the formalin pain test after intrathecal injection and tail vein injection of the multi-target peptide molecule in Example 1 and Example 3 of the present invention; Figure 9 A, Figure 9 B is for intrathecal injection, Figure 9 C, Figure 9 D is for tail vein injection;

[0028] Figure 10 It is the graph of the change in mechanical withdrawal threshold in carrageenan-induced inflammatory pain after intrathecal injection and tail vein injection of the multi-target peptide molecule in Example 1 and Example 3 of the present invention; Figure 10 A, Figure 10 B is for intrathecal injection, Figure 10 C, Figure 10 D is for tail vein injection;

[0029] Figure 11 It is the graph of the change in mechanical withdrawal threshold in CFA-induced inflammatory pain after intrathecal injection and tail vein injection of the multi-target peptide molecule in Example 1 and Example 3 of the present invention; Figure 11 A, Figure 11 B is for intrathecal injection, Figure 11 C, Figure 11 D is for tail vein injection;

[0030] Figure 12 It is the graph of the change in mechanical withdrawal threshold in neuropathic pain after intrathecal injection and tail vein injection of the multi-target peptide molecule in Example 1 and Example 3 of the present invention; Figure 12 A, Figure 12 B is for intrathecal injection, Figure 12 C, Figure 12 D is for tail vein injection;

[0031] Figure 13 It is the graph of the analgesic effect change during continuous intrathecal injection of the multi-target peptide molecule in Example 1 and Example 3 of the present invention for eight days;

[0032] Figure 14This is a graph showing the changes in the regulatory effects of multi-target peptide molecules on gastrointestinal motility in mice by intrathecal injection and tail vein injection in Example 1 and Example 3 of the present invention; Figure 14 A, Figure 14 B is for intrathecal injection, Figure 14 C, Figure 14 D is for tail vein injection;

[0033] Figure 15 This is a graph showing the regulatory effect of multi-target peptide molecules on the naloxone-induced withdrawal response by intrathecal injection in Example 1 and Example 3 of the present invention;

[0034] Figure 16 This is a graph showing the regulatory effect of multi-target peptide molecules on the naloxone-induced withdrawal response by tail vein injection in Example 1 and Example 3 of the present invention. Detailed implementation manners

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention pertains.

[0036] Ghrelin is a brain-gut peptide discovered in 1999 and is the natural ligand of growth hormone secretagogue receptor 1α (GHSR-1α). Ghrelin consists of 28 amino acid residues (GSS(O-CO-C7H 15 )FLSPEHQKA QQRKESKKPPAKLQPR), with Ser at the N-terminus 3Octanoylation enables it to freely cross the blood-brain barrier [Nature, 1999, 402: 656-660]. Studies have found that ghrelin mainly mediates various biological activities after binding to GHSR-1α [CNS & Neurological Disorders - Drug Targets. 2016; 15: 956-963]. Since 2006, successive studies have reported that ghrelin plays an important regulatory role in inflammatory pain, neuropathic pain, hyperalgesia, etc. [Peptides, 2013, 43: 76-82]. Ghrelin has the function of reducing side effects such as μ-opioid addiction, dependence, and constipation (Addiction Biology, 2012, 17: 613-622. Neurogastroenterology & Motility, 2017, 23: 171-179.). The research of this experimental group found that the μ-opioid / ghrelin receptor dual-target polypeptide molecule G(1-5)-EM2 formed by the ghrelin active fragment ghrelin(1-5) and EM-2 (its molecular structure is: Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Leu-Tyr-Pro-Phe-Phe-NH2) has good central and peripheral analgesic activities; compared with endomorphin, its analgesic tolerance is also reduced, and it shows good blood-brain barrier permeability (European Journal of Pharmacology, 2025, 986: 177148). At the same time, in order to improve the enzymatic stability and drugability of G(1-5)-EM2, this experimental group further modified G(1-5)-EM2 by replacing it with D-amino acids.

[0037] The present invention provides an opioid and ghrelin receptor multi-target peptide molecule, the structure of which is shown in Formula I:

[0038] Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Xaa 1 -Tyr-Xaa 2 -Phe-Xaa 3 -NH2 (I);

[0039] Wherein, Xaa 1 is Leu or D-Leu, Xaa 2 is Pro or D-Pro, Xaa 3 is Phe or D-Phe, and at least one of Xaa 1 , Xaa 2 , Xaa 3 is a D-amino acid.

[0040] Actually, the multi-target peptide molecule provided by the present invention uses the basic peptide (G(1-5)-EM2) as a template molecule, and the amino acid sequence is obtained by modifying the L-amino acids at the 5th, 7th, and 9th positions thereof with their isomeric D-amino acids. Specifically, the structural formula of the basic peptide (G(1-5)-EM2) is: Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Leu-Tyr-Pro-Phe-Phe-NH2.

[0041] Actually, the multi-target peptide molecule provided by the present invention is selected from the compounds described in Table 1 below:

[0042] Table 1 Amino acid sequences of multi-target peptide molecules

[0043]

[0044] Actually, the opioid and ghrelin receptor multi-target peptide molecule provided by the present invention is obtained by sequentially coupling amino acids on a solid-phase carrier by solid-phase synthesis and cleaving to obtain the target peptide. In some embodiments, the preparation method provided by the present invention includes the following steps:

[0045] S1. Pretreat and deprotect the solid-phase carrier;

[0046] S2. Add the first amino acid and the deprotected solid-phase carrier for condensation and deprotection;

[0047] S3. Repeat step S2 and sequentially add amino acids from the C-terminus to the N-terminus until the peptide chain condensation is completed;

[0048] S4. Wash and dry the solid-phase carrier condensed with the peptide chain, add a cleavage agent to cleave the peptide chain, and purify and separate to obtain a crude peptide;

[0049] S5. Purify the crude peptide to obtain the target peptide (multi-target peptide molecule).

[0050] Example 1

[0051] This Example 1 provides a preparation method for a multi-target peptide molecule (Ⅰ-1; [D-Leu 5 G(1-5)-EM2), including the following steps:

[0052] S1. Add 20 mL of a mixed solution (V(piperidine):V(DMF)=1:4) to Rink Amide AM Resin (2 g, substitution degree 0.3 mmol / g), react for 20 min to remove the Fmoc protecting group, and then sequentially add 20 mL of DMF, MeOH, and DCM for washing 3 times each (a total of 9 times), and then filter and drain;

[0053] S2. After using the Kaiser test to detect that the solution turns blue, calculate the feeding amount of Fmoc-Phe-OH according to the loading of 0.3 mmol / g of Rink Amide AM Resin (resin mass (2 g) × molecular weight × loading × 2 = 0.47 g), and the feeding amount of the condensing agent TBTU (molecular weight × 2 × 2 × loading = 0.39 g). Add 0.47 g of Fmoc-Phe-OH, 0.39 g of TBTU, 0.1 mL of MMN (2-mercapto-5-nitrobenzoic acid), and 20 mL of DMF to the Rink Amide AM Resin. React at 35 °C for 40 min and then filter by suction. Then add 20 mL of DMF to wash the resin (wash 6 times), filter and drain. Use the Kaiser test to detect that the solution does not turn blue, indicating that Fmoc-Phe-OH has been condensed onto the resin;

[0054] S3. Repeat steps S1-S2, and sequentially condense Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-(D-Leu)-OH, Fmoc-Phe-OH, Fmoc-Ser(n-Octanoyl)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Gly-OH onto the peptide resin to obtain the peptide resin;

[0055] S4. Add DCM to wash the resin, then add ether to wash twice. Drain and dry in a vacuum environment for 2 h. Place the dried peptide resin in a eggplant-shaped flask, add 20 mL of cleavage reagent (82.5% TFA (trifluoroacetic acid)), react for 3 h and then filter. Rotate and evaporate to concentrate the filtrate to obtain a concentrated solution. Add 10 mL of ice ether to the concentrated solution to precipitate. Centrifuge at 5000 rpm for 5 min and discard the supernatant. Repeat three times and then dry the precipitate in a vacuum to obtain 450 mg of crude peptide;

[0056] S5. Use a reverse-phase high-performance liquid chromatography (RP-HPLC) C18 column (4.6 mm × 250 mm) to separate and purify the crude peptide. Use acetonitrile (containing 0.1% TFA) and deionized water (containing 0.1% TFA) for gradient elution. After separating and collecting the target peak sample, lyophilize to obtain a solid powder. Calculate the yield of the pure peptide to be 12%.

[0057] Example 2

[0058] This Example 2 provides a multi-target peptide molecule (Ⅰ-2; [D-Pro 7The preparation method of G(1-5)-EM2) is different from that of Example 1 in that in step S3, Fmoc-Phe-OH, Fmoc-(D-Pro)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Ser(n-Octanoyl)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Gly-OH are successively condensed onto the peptide resin to obtain the peptide resin; in step S4, 465 mg of crude peptide is obtained, and in step S5, the pure peptide yield is 12%.

[0059] Example 3

[0060] This Example 3 provides a preparation method of a multi-target peptide molecule (Ⅰ-3; [D-Phe 9 G(1-5)-EM2). The difference from Example 1 is that in step S3, Fmoc-(D-Phe)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Ser(n-Octanoyl)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Gly-OH are successively condensed onto the peptide resin to obtain the peptide resin; in step S4, 480 mg of crude peptide is obtained, and in step S5, the pure peptide yield is 12%.

[0061] Comparative Example 1

[0062] This Comparative Example 1 provides a preparation method of a multi-target peptide molecule (Ⅰ-4; G(1-5)-EM2). The difference from Example 1 is that in step S3, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Ser(n-Octanoyl)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Gly-OH are successively condensed onto the peptide resin to obtain the peptide resin.

[0063] Structure detection

[0064] In a system of 42-100% acetonitrile / water (0.1% TFA) (completed in 30 min), with a flow rate of 1 mL / min, a detection wavelength of 220 nm, and an analytical chromatographic column of Sinochrom ODS-BP 5C18, 4.6 mm × 250 mm, the multi-target peptide molecules prepared in Examples 1 to 3 were subjected to chromatographic and mass spectrometric detections, as shown in Table 2 below.

[0065] Table 2 Mass spectrometry and purity analysis of multi-target peptide molecules

[0066]

[0067] Performance detection

[0068] 1. In the calcium mobilization experiment, the agonist activities of the multi-target peptide molecules in Examples 1 to 3 and Comparative Example 1 on opioid receptors and ghrelin receptors were measured: Using the CHO / Gα15 cell line stably overexpressing Mu-opioid receptor, Delta-opioid receptor, and Kappa-opioid receptor and the HEK293T cells transiently expressing ghrelin receptor, the agonist activities on opioid receptors and ghrelin receptors were detected by detecting the regulation of intracellular calcium mobilization by the multi-target peptide molecules, specifically including:

[0069] Determination of opioid receptor agonist activity: Pre-inoculate the CHO / Gα15 cell line expressing Mu-opioid receptor, Delta-opioid receptor, and Kappa-opioid receptor in a 96-well plate (30,000 - 50,000 cells per well), after culturing in a carbon dioxide incubator for 24 h, aspirate the culture medium, then add 50 μL of calcium dye stock solution containing 2.5 mM probenecid, 0.9 μM Fluo-4-AM, and F127 (0.03%), after incubating at 37 °C for 30 min, aspirate the calcium dye stock solution and add 75 μL of HBSS buffer (containing 2.5 mM probenecid), detect the calcium signal change after drug administration (concentration from 10 -11 to 10 -4 M) in the FlexStation 3 calcium flux workstation; Use DAMGO, Met-enkephalin, and U69593 as controls respectively to detect the calcium mobilization experiment on Mu-opioid receptor, Delta-opioid receptor, and Kappa-opioid receptor;

[0070] Determination of ghrelin receptor agonist activity: Pre-inoculate the HEK293T cells expressing ghrelin receptor (GHSR) in a 96-well plate (70,000 cells per well), after culturing in a carbon dioxide incubator for 24 h, aspirate the culture medium, then add 50 μL of calcium dye stock solution containing 2 mM probenecid, 2 μM Fluo-4-AM, and F127 (0.03%), after incubating at 37 °C for 40 min, aspirate the calcium dye stock solution and add 90 μL of HBSS buffer (containing 2 mM probenecid), then place the plate in a microplate reader for fluorescence calcium ion detection, measure the basal fluorescence intensity before adding the multi-target peptide molecule, and then add different concentrations (from 10 -11 to 10 -4 M) of the multi-target peptide molecule and Ghrelin to the plate at 10 μL per well, and measure the difference between the basal fluorescence intensity and the maximum fluorescence intensity before and after addition;

[0071] The calcium mobilization assay represents the increase in intracellular calcium as a percentage (% control), where % control = (measured value - basal value) ÷ basal value × 100. The data are presented as mean ± standard error of the mean (Means ± S.E.M.), and the dose-response relationship is statistically analyzed using a non-linear regression model, and the EC 50 value and E max are shown in Tables 3 and 4 as follows.

[0072] Table 3 Calcium mobilization assay on opioid receptors

[0073]

[0074]

[0075] Note: The potencies are expressed as EC 50 (nM) and maximum stimulation percentage (E max ) relative to the standard agonists DAMGO (100 μM), Met-enkephalin (1 μM), or U69593 (10 μM). The data are presented as mean ± S.E.M., and "-" indicates that the multi-target peptide molecule is inactive as an agonist in the 100 μM dose range.

[0076] Table 4 Calcium mobilization assay on the Ghrelin receptor GHSR

[0077]

[0078] Note: The potencies are expressed as EC 50 (nM) and maximum stimulation percentage (E max ) relative to the standard agonist ghrelin (10 μM). The data are presented as mean ± S.E.M., and "-" indicates that the multi-target peptide molecule is inactive as an agonist in the 100 μM dose range.

[0079] As can be seen from Table 3, in the CHO / Gα15 cell line stably overexpressing the Mu-opioid receptor, Delta-opioid receptor, and Kappa-opioid receptor, the multi-target peptide molecule (Ⅰ-1; [D-Leu 5 G(1-5)-EM2) obtained in Example 1 showed only weak agonist activity against MOR compared to the positive drug DAMGO, while the multi-target peptide molecule (Ⅰ-2; [D-Pro 7 G(1-5)-EM2) obtained in Example 2 and the multi-target peptide molecule (Ⅰ-3; [D-Phe 9G(1-5)-EM2) has no agonist activity on MOR. Additionally, none of the three multi-target peptide molecules in Examples 1 to 3 exhibited agonist activity on DOR. Furthermore, compared to Comparative Example 1, the three multi-target peptide molecules in Examples 1 to 3 exhibited comparable or weaker agonist activity on KOR.

[0080] As can be seen from Table 4, in HEK293T cells transiently expressing the ghrelin receptor, the multi-target peptide molecules obtained in Examples 1 to 3 dose-dependently induced intracellular calcium mobilization, indicating that the three multi-target peptide molecules all exhibited concentration-dependent agonist activity on the ghrelin receptor. For the ghrelin receptor, compared to Comparative Example 1, the agonist activity of the multi-target peptide molecules obtained in Examples 1 to 3 was significantly increased (21-fold - 130-fold).

[0081] 2. Perform enzymatic stability tests on the multi-target peptide molecules obtained in Examples 1 to 3 and Comparative Example 1, specifically including:

[0082] Male Kunming mice weighing 25 g - 30 g were selected, sacrificed by cervical dislocation, and their brains were isolated. After blotting dry with filter paper and weighing, the mouse brains were rinsed with pre-cooled buffer Tris-HCl (1 mM, pH = 7.4) at 4°C to remove extravasated blood. 50 volumes of pre-cooled Tris-HCl (1 mM, pH = 7.4) buffer were added to the homogenizer, and the mouse brain tissue was homogenized in an ice bath and then left standing in the ice bath for 30 min to promote cell lysis; for every 10 mL of homogenate, 5 mL of Tris-HCl (50 mM, pH = 7.4) buffer was added, and then transferred to the ice for homogenization and pipetted to make it suspended. Centrifugation was performed twice at 4°C and 49000 g for 45 min, and the supernatant was discarded to obtain the precipitate; the obtained precipitate was pipetted evenly with an appropriate amount of Tris-HCl (50 mM, pH = 7.4) buffer. After determining the final concentration of brain plasma membrane in the brain homogenate to be 2.1 mg / mL using a BCA kit, it was aliquoted and stored at -80°C;

[0083] Male Kunming mice weighing 25 g - 30 g were selected, their whiskers were cut off, and blood was collected by eye puncture. The mouse blood was collected in a heparinized and ice-cold centrifuge tube, shaken well, centrifuged at 4°C and 3000 g for 20 min, and the supernatant was collected and stored at -80°C;

[0084] Quickly thaw the cerebral membrane and mouse plasma in a 37°C water bath, and stir and dissolve the multi-target peptide molecules obtained in Examples 1 to 3 and Comparative Example 1 in ultrapure water to prepare a 10 mM multi-target peptide molecule solution. Add 80 μL of the multi-target peptide molecule solution to 720 μL of the cerebral membrane solution and plasma solution respectively. Immediately take 80 μL after shaking evenly (denoted as 0 min), and incubate the remaining solution in a 37°C water bath, and take samples at 5 min, 15 min, 30 min, 60 min, 90 min, and 120 min in sequence; immediately add 80 μL of pre-cooled acetonitrile to the taken incubation solution in an ice bath, mix evenly and process on ice for 5 min, centrifuge the obtained mixture at 4°C and 13,000 g for 15 min, collect the supernatant and store it at -80°C;

[0085] Incubate the stored supernatant, filter it through a 0.22 μm filter membrane, and analyze the change in the ultraviolet absorption (220 nm) area of the sample by gradient elution. Inject 20 μL each time to identify the content change, and then calculate the half-life (t 1 / 2 ) of the multi-target peptide molecule through the first-order kinetic equation, which is expressed as the mean ± standard error (Means ± S.E.M.), as shown in Table 5 below, and plot the residual rates of different multi-target peptide molecules at different time points in the cerebral membrane and plasma as Figure 1 shown.

[0086] Table 5 Enzymatic stability test of multi-target peptide molecules

[0087] <![CDATA[Brain plasma membrane (t 1 / 2 / min)]]> <![CDATA[Plasma (t 1 / 2 / min)]]> G(1-5)-EM2 213.7±7.0 107.5±14.7 <![CDATA[[D-Leu 5 G(1-5)-EM2]]> 293.3±25.9 151.5±32.9 <![CDATA[[D-Pro 7 G(1-5)-EM2]]> 132.0±0.9 160.5±13.2 <![CDATA[[D-Phe 9 G(1-5)-EM2]]> 206.7±17.2 75.5±2.3

[0088] As can be seen from Table 5, the half-lives of the multi-target peptide molecules obtained in Comparative Example 1 in the cerebral membrane and plasma are 213.7 min and 107.5 min respectively, while the enzymatic stabilities of the multi-target peptide molecules obtained in Examples 1 to 3 in the cerebral membrane and plasma have not decreased significantly. Even the half-lives of the multi-target peptide molecules obtained in Example 2 in the cerebral membrane and plasma have been extended by 80 min and 44 min respectively. It can be seen from Figure 1 that the residual rates of the multi-target peptide molecules obtained in Examples 1 and 3 in the cerebral membrane are similar to those of the multi-target peptide molecules obtained in Comparative Example 1, while the residual rate of the multi-target peptide molecules obtained in Example 2 has decreased more significantly, dropping to about 50% at 120 min. The residual rates of the multi-target peptide molecules in Example 1 in plasma at 30, 60, and 120 min are all higher than those of the multi-target peptide molecules in Comparative Example 1, and still remain above 70% at 120 min. The residual rates of the multi-target peptide molecules obtained in Examples 2 and 3 at each time point are all lower than those of the multi-target peptide molecules in Comparative Example 1.

[0089] 3. Perform blood-brain barrier permeability tests on the multi-target peptide molecules in Examples 1 to 3 and Comparative Example 1 through small animal imaging, specifically including: After pre-linking the multi-target peptide molecules obtained in Examples 1 to 3 and Comparative Example 1 with Cy7, inject them into mice (male Kunming mice weighing 18g - 22g) via the tail vein respectively, and take pictures of the fluorescence signal intensity of the whole body of the mice at the 0 min, 5 min, 10 min, 20 min, 40 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, 360 min, and 24 h time points as Figure 2 shown, and intercept the fluorescence of the head in the whole body fluorescence image of the mice for analysis as Figure 3 shown. After perfusing the heart of the mice to exclude the interference of blood vessels in the brain, take fluorescence pictures to obtain the brain fluorescence of the mice as Figure 4 shown. Among them, the fluorescence data is expressed as the mean ± standard error (Means ± S.E.M.), and the difference in fluorescence signals is tested using two-way ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001 indicate the significant difference between injecting the multi-target peptide molecules of Examples 1 to 3 and injecting the multi-target peptide molecules of Comparative Example 1.

[0090] It can be seen from Figure 2 that obvious fluorescence signals can be observed within 0 - 300 min after injecting the multi-target peptide molecules in Examples 1 to 3, and it can be seen from Figure 3 that obvious fluorescence signals can be collected in the brain for all three multi-target peptide molecules. Combining Figure 4 it can be seen that all three multi-target peptide molecules can cross the blood-brain barrier and enter the mouse brain.

[0091] 4. Determine the analgesic activity and blood-brain barrier permeability of the multi-target peptide molecules in Examples 1 to 3 in acute pain, specifically including:

[0092] (1) Warm water bath tail-flick test

[0093] Male Kunming mice weighing 18 g - 22 g were pre - adapted to a room temperature environment of 21°C - 23°C for 30 min (during which they could eat and drink freely). Then, the distal 1 / 3 of the mouse's tail was immersed in a water bath at 48°C ± 0.5°C (the mouse's basal tail - flick threshold (3 s - 5 s) was pre - measured before the experiment, and the mouse's tail should not be in the water bath for more than 10 s to prevent scalding, and the tail - flick latency was recorded as 10 s). After injecting the multi - target peptide molecules using the following methods, the tail - flick thresholds of the mice were measured and recorded at 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min after injection, and normal saline was injected as a blank control.

[0094] (1 - 1) Intracerebroventricular injection (i.c.v.) of mice

[0095] For intracerebroventricular injection of mice, the injection needle was wrapped with a plastic tube, leaving 2.5 mm of the needle tip exposed. The injection site was centered on the bregma, shifted 1 mm to the left / right and 3 - 4 mm backward (1 mm to the left / right of the intersection of the midline and the line connecting the two ears). When inserting the needle, it should be perpendicular, slow, and uniform. After injecting 4 μL of the multi - target peptide molecules obtained from Example 1 to Example 3 at 0.13 nmol, 0.4 nmol, 1.3 nmol, and 4 nmol respectively into the mouse's lateral ventricle using a syringe, the needle was withdrawn for the warm - bath tail - flick experiment; after the experiment, 4 μL of trypan blue dye was injected; the brain sections were examined. When the dye diffused in the brain cavity, it indicated that the injection position was correct and the experimental results were reliable;

[0096] (1 - 2) Intrathecal injection (i.t.) of mice

[0097] The microsyringe was vertically inserted between the 5th and 6th segments of the mouse's lumbar spine. Whether the arachnoid was successfully punctured was shown by the reflexive lateral flick of the tail or the formation of an "S" shape of the tail. After injecting 5 μL of the multi - target peptide molecules obtained from Example 1 and Example 3 at 0.04 nmol, 0.13 nmol, 0.4 nmol, and 1.3 nmol respectively, the needle was withdrawn for the warm - bath tail - flick experiment;

[0098] (1 - 3) Intravenous injection (i.v.) of mice

[0099] Using a syringe, along the parallel direction of the vein, the needle was inserted at the distal end of the mouse's tail. When inserting the needle, the bevel was upward. After tilting and inserting, the needle was advanced parallelly. After injecting 0.2 mL of the multi - target peptide molecules obtained from Example 1 and Example 3 at 6.67 nmol, 20 nmol, 66.7 nmol, and 200 nmol respectively, the needle was withdrawn for the warm - bath tail - flick experiment;

[0100] The analgesic effect of the multi - target peptide molecules was represented by the change curve of the maximum analgesic effect MPE(%), and the median effective dose ED was calculated50 (The drug dose corresponding to 50% of the maximum effect), MPE(%) = 100×[(pain threshold after drug administration - basal pain threshold)÷(10 - basal pain threshold)] (the analgesic effect data were expressed as Means±S.E.M., and the differences in analgesic effects were statistically analyzed by two-way ANOVA, * P<0.05, ** P<0.01, and *** P<0.001 indicated significant differences between the drug injection group and the saline group). The analgesic effect curves of intralateral ventricular injection of multi-target peptide molecules are as Figure 5 shown, and the analgesic effect curves of intrathecal injection of multi-target peptide molecules are as Figure 6 A, Figure 6 B shown, and the analgesic effect curves of intravenous injection of multi-target peptide molecules into the tail vein are as Figure 6 C, Figure 6 D shown.

[0101] It can be seen from Figure 5 that the analgesic effects of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9 G(1-5)-EM2 (Example 3) are the best, while [D-Pro 7 G(1-5)-EM2 (Example 2) has no analgesic effect; in addition, the highest analgesic doses of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9 G(1-5)-EM2 (Example 3) are 1.3 nmol and 0.4 nmol respectively, the analgesic duration is 60 min, and the analgesic ED 50 are 0.52 nmol and 0.20 nmol respectively.

[0102] It can be seen from Figure 6 that further intrathecal injection and intravenous injection of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9 G(1-5)-EM2 (Example 3) produce time-dependent analgesic effects, the analgesic duration is 60 min, and the analgesic ED 50 are 0.41 nmol and 0.55 nmol respectively; the highest analgesic doses of intravenous injection of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9 G(1-5)-EM2 (Example 3) into the tail vein are 66.7 nmol and 20 nmol respectively, and the analgesic duration is 60 min.

[0103] (2) Blood-brain barrier permeability determination: Naloxone was administered by intracerebroventricular injection (i.c.v.) and Naloxone methiodide was administered by intraperitoneal injection (i.p.) to male Kunming mice weighing 18 g - 22 g. The blood-brain barrier permeability of the multi-target peptide molecules in Example 1 and Example 3 was detected by the warm water bath tail-flick test under these two treatment conditions. The antagonistic effect of the multi-target peptide molecules was compared by plotting the change in the maximum analgesic effect MPE (%) of the drug. The analgesic data was expressed as mean ± standard error (Means ± S.E.M.). The difference in analgesic effect was statistically analyzed using two-way ANOVA, * P < 0.05, ** P < 0.01 and *** P < 0.001 indicated significant differences between the drug injection group and the saline group. # P < 0.05, ## P < 0.01 and ### P < 0.001 indicated significant differences between the antagonist injection group and the group injected with the multi-target peptide molecule alone, as Figure 7 shown.

[0104] As can be seen from Figure 7 , intracerebroventricular injection of naloxone could completely antagonize the analgesic effect produced by intravenous injection of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9 G(1-5)-EM2 (Example 3). Peripherally administered Naloxone methiodide could also partially antagonize the analgesic effect produced by intravenous injection of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9 G(1-5)-EM2 (Example 3). This indicates that intravenous injection of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9 G(1-5)-EM2 (Example 3) could produce analgesic effects through the central and peripheral nervous systems, and also indicates that both analogs could penetrate the blood-brain barrier.

[0105] 5. The analgesic activity of the multi-target peptide molecules in Example 1 and Example 3 was tested for inflammatory pain, specifically including:

[0106] (1) Acetic acid writhing test

[0107] Male Kunming mice weighing 18 g - 22 g were pre - adapted to a room temperature environment of 21°C - 23°C for 30 min (during which they could freely eat and drink). Then, different doses of multi - target peptide molecules (Example 1, Example 3) were injected into the mice via intrathecal injection and tail vein injection respectively. After 5 min, 0.2 mL of 0.6% glacial acetic acid was intraperitoneally injected into each mouse. After 5 min, the number of writhing responses of the mice within 10 min was recorded (writhing response signs: the mouse's abdomen caved in, the abdominal wall contracted into an "S" shape, the hind limbs extended, the hips lifted, and the mouse crawled forward). The experimental data were expressed as the mean ± standard error (Means ± S.E.M). The inter - group differences in different doses of multi - target peptide molecule treatments were statistically analyzed by one - way analysis of variance. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicated the significant differences between the groups injected with different doses of multi - target peptide molecules and the normal saline group. The results are as Figure 8 shown.

[0108] From Figure 8 it can be seen that intrathecal injection of 0.1 pmol, 0.3 pmol, 1 pmol of [D - Leu 5 G(1 - 5)-EM2 (Example 1) produced a dose - dependent analgesic effect in the mouse acetic acid writhing model, while intrathecal injection of 0.01 nmol, 0.03 nmol, 0.1 nmol of [D - Phe 9 G(1 - 5)-EM2 (Example 3) could also produce a dose - dependent analgesic effect; after tail vein injection of 20 nmol, 66.7 nmol, 200 nmol of [D - Leu 5 G(1 - 5)-EM2 (Example 1), [D - Phe 9 G(1 - 5)-EM2 (Example 3) could also produce a dose - dependent analgesic effect. Both injection methods significantly inhibited the number of writhing responses in the mouse acetic acid writhing model, indicating that the multi - target peptide molecules provided by the present invention could exhibit obvious analgesic effects in visceral pain.

[0109] (2) Formalin test

[0110] Male Kunming mice weighing 18 g - 22 g were pre - adapted to a room temperature environment of 21°C - 23°C for 30 min (during which they could freely eat and drink). Then, different doses of multi - target peptide molecules (Example 1, Example 3) were injected into the mice intrathecally and via the tail vein respectively. After 5 min, 20 μL of 5% formalin solution was injected into the right hind paw of the mice and timing was started immediately. A stopwatch was used to record the time when the mice licked and bit the hind paw. Timing was stopped after 5 min (this is the first phase of the formalin experiment). Then there was a 10 - min rest period. From the 15th min to the 30th min was the second phase, and the time when the mice licked and bit the hind paw in the second phase was recorded; the experimental data were expressed as mean ± standard error (Means ± S.E.M). The inter - group differences in different doses of multi - target peptide molecule treatments were statistically analyzed by one - way analysis of variance. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicated the significant differences between the groups injected with different doses of multi - target peptide molecules and the saline group. The results were as Figure 9 shown.

[0111] From Figure 9 it can be seen that intrathecal injection of 0.1 nmol, 0.3 nmol, 1 nmol of [D - Leu 5 G(1 - 5)-EM2 (Example 1) and [D - Phe 9 G(1 - 5)-EM2 (Example 3) produced dose - dependent analgesic effects in the mouse formalin model, while intravenous injection of 20 nmol, 66.7 nmol, 200 nmol of [D - Leu 5 G(1 - 5)-EM2 (Example 1) and [D - Phe 9 G(1 - 5)-EM2 (Example 3) could also produce dose - dependent analgesic effects; both injection methods significantly inhibited the acute pain in the first phase and the inflammatory pain in the second phase, indicating that the multi - target peptide molecules provided by the present invention showed obvious analgesic effects in formalin pain.

[0112] (3) Carrageenan - induced inflammatory pain experiment

[0113] Male Kunming mice weighing 18 g - 22 g were pre - adapted to a room temperature environment of 21°C - 23°C for 30 min (during which they could freely eat and drink). After measuring the basal pain threshold of the right hind paw of the mice using an electronic von Frey aesthesiometer, 20 μL of 2% carrageenan was injected into the right hind paw of the mice through the sole using a microsyringe, and the pain threshold before administration was measured 24 h later. Subsequently, different doses of multi - target peptide molecules (Example 1, Example 3) were injected intrathecally and via the tail vein into the mice, and the pain thresholds at 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min after administration were tested. The experimental data were expressed as mean ± standard error (Means ± S.E.M), and the inter - group differences in different multi - target peptide molecule doses were statistically analyzed using one - way analysis of variance. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicated significant differences between the groups injected with different doses of multi - target peptide molecules and the saline group, and the results were as Figure 10 shown.

[0114] From Figure 10 it can be seen that intrathecal injection of 0.1 nmol, 0.3 nmol, 1 nmol of [D - Leu 5 G(1 - 5)-EM2 (Example 1) and [D - Phe 9 G(1 - 5)-EM2 (Example 3) produced a dose - dependent analgesic effect in the carrageenan - induced inflammatory pain model in mice, while intravenous injection of 20 nmol, 66.7 nmol, 200 nmol of [D - Leu 5 G(1 - 5)-EM2 (Example 1) and [D - Phe 9 G(1 - 5)-EM2 (Example 3) could also produce a dose - dependent analgesic effect; both injection methods significantly inhibited carrageenan - induced inflammatory pain, indicating that the multi - target peptide molecules provided by the present invention showed an obvious analgesic effect in carrageenan - induced inflammatory pain.

[0115] (4) CFA - induced inflammatory pain experiment

[0116] Male Kunming mice weighing 18 g - 22 g were pre - adapted to a room temperature environment of 21°C - 23°C for 30 min (during which they could freely eat and drink). After measuring the basal pain threshold of the right hind paw of the mice using an electronic von Frey aesthesiometer, 20 μL of CFA was injected into the right hind paw of the mice with a syringe. Four days later, the right hind paw of the mice swelled and developed pain sensitivity. The mechanical withdrawal threshold was measured, and then different doses of multi - target peptide molecules (Example 1, Example 3) were injected intrathecally and via the tail vein into the mice respectively. The mechanical pain thresholds at 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min after drug administration were tested. The experimental data were expressed as mean ± standard error (Means ± S.E.M). The inter - group differences in different doses of multi - target peptide molecule treatments were statistically analyzed using one - way analysis of variance. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicated the significant differences between the groups injected with different doses of multi - target peptide molecules and the saline group. The results were as Figure 11 shown.

[0117] From Figure 11 it can be seen that intrathecal injection and tail vein injection of different doses of [D - Leu 5 G(1 - 5)-EM2 (Example 1) and [D - Phe 9 G(1 - 5)-EM2 (Example 3) both produced significant analgesic effects, and the doses required to achieve the maximum analgesia were comparable.

[0118] 6. The analgesic activity of the multi - target peptide molecules in Example 1 and Example 3 was tested for neuropathic pain, specifically including:

[0119] Male Kunming mice weighing 18 g - 22 g were pre - adapted to a room temperature environment of 21°C - 23°C for 30 min (during which they could freely eat and drink). After measuring the basal pain threshold of the right hind paw of the mice using an electronic von Frey aesthesiometer, 0.2 mL of 6 mg / mL sodium pentobarbital solution was intraperitoneally injected to paralyze the mice. The comatose mice were fixed on their right sides under a dissecting microscope. The convexity of the right femur of the mice was found, and the skin was incised vertically in the mid - line and the fascia was cut open. The muscles were bluntly dissected using forceps (the sciatic nerve branches, the common peroneal nerve, the tibial nerve, and the sural nerve could be seen). The common peroneal nerve and the tibial nerve were lifted with a glass microprobe and ligated with a fine thread. They were cut at a position 2 mm - 3 mm from the ligation site and shortened by 1 mm (to prevent nerve re - adhesion and regeneration), and then sutured and disinfected. The mice were placed in a warm environment and waited for the anesthesia to wear off. If the wounds of the mice healed and scabbed and they survived within 14 days, it indicated that the neuropathic pain (SNI) model was successfully established.

[0120] After successful model establishment, the pain threshold before drug administration was measured. Subsequently, different doses of multi-target peptide molecules (Example 1, Example 3) were intrathecally injected and tail vein injected into the model respectively, and the pain thresholds at the 5th min, 10th min, 20th min, 30th min, 40th min, 50th min, and 60th min after drug administration were tested; the experimental data were expressed as mean ± standard error (Means ± S.E.M), and the inter-group differences in different doses of multi-target peptide molecule treatments were statistically analyzed by one-way analysis of variance. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicated the significant differences between the groups injected with different doses of multi-target peptide molecules and the normal saline group. The results were as Figure 12 shown.

[0121] From Figure 12 it can be seen that intrathecal injection and tail vein injection of different doses of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9 G(1-5)-EM2 (Example 3) both produced significant dose-dependent neuropathic pain analgesic effects, and the doses required to achieve the maximum analgesia were comparable.

[0122] 7. The analgesic tolerance phenomenon of the multi-target peptide molecules in Example 1 and Example 3 was tested. By observing the changes in the tail-flick latency in the warm water bath tail-flick experiment after continuous administration for eight days, the drug activity of the multi-target peptide molecules in analgesic tolerance was evaluated. Specifically, male Kunming mice weighing 18 g - 22 g were pre-adapted to the room temperature environment of 21°C - 23°C for 30 min (during which they could eat and drink freely), and then intrathecally injected with 1.3 nmol of multi-target peptide molecules (Example 1, Example 3) at the same time every day for eight consecutive days. The changes in the tail-flick threshold after drug injection every day were detected (the basal tail-flick threshold (3 s - 5 s) was measured on the first day and the tail-flick thresholds at the 5th min, 10th min, 20th min, 30th min, 40th min, 50th min, and 60th min after drug administration, and the tail-flick thresholds of the maximum analgesic effect were measured in the following seven days); the analgesic effect of the multi-target peptide molecules was expressed by the change in the maximum analgesic effect MPE (%), where MPE (%) = [(pain threshold after drug administration - basal pain threshold) / (10 - basal pain threshold)] × 100%, and the MPE change graph was as Figure 13 shown.

[0123] From Figure 13 it can be seen that intrathecal injection of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe 9The maximum analgesic effect of G(1-5)-EM2 (Example 3) after administration on days 2-8 showed no significant difference compared to the first day, indicating an analgesic effect without tolerance.

[0124] 8. Conduct constipation side effect tests on the multi-target peptide molecules in Example 1 and Example 3, specifically including:

[0125] Male Kunming mice weighing 18 g - 22 g were pre-acclimated in a room temperature environment of 21°C - 23°C for 30 min (during which they could freely eat and drink), and then fasted for 18 h (water could be freely obtained). After 5 min of intrathecal injection or tail vein injection of different doses of multi-target peptide molecules (Example 1, Example 3) into the mice, 200 μL of activated carbon suspension (physiological saline suspension containing 10% activated carbon and 5% gum arabic) was orally administered; 30 min after gavage, the mice were sacrificed by decapitation and the small intestine was dissected to measure the total length of the small intestine and the moving length of the carbon powder in the distance from the pylorus of the stomach to the cecum.

[0126] The results of gastrointestinal motility were expressed as the percentage of gastrointestinal motility. The specific calculation method was the percentage after dividing the distance traveled by the carbon powder by the total length of the small intestine. The experimental data were expressed as mean ± standard error (Means ± S.E.M.). The differences in gastrointestinal motility were statistically analyzed using one-way analysis of variance. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicated significant differences between the groups injected with different doses of multi-target peptide molecules and the saline group. The results were as Figure 14 shown.

[0127] From Figure 14 A and Figure 14 B, it can be seen that the percentages of gastrointestinal motility when intrathecally injecting [D-Leu 5 G(1-5)-EM2 (Example 1) were 71.0% (1 nmol), 74.7% (3 nmol), and 67.8% (10 nmol) respectively. The percentages of gastrointestinal motility when intrathecally injecting [D-Phe 9 G(1-5)-EM2 (Example 3) were 82.4% (1 nmol), 77.1% (3 nmol), and 72.8% (10 nmol) respectively. Compared with the percentage of gastrointestinal motility in the saline group (70.8%), the multi-target peptide molecule group did not reduce the gastrointestinal motility function, indicating that the multi-target peptide molecules had no constipation side effects.

[0128] From Figure 14 C and Figure 14 D, it can be seen that when tail vein injecting [D-Leu 5The percentages of gastrointestinal motility at [D-Phe 9 G(1-5)-EM2 (Example 1) were 76.6% (20 nmol), 70.9% (66.7 nmol), and 69.8% (200 nmol) respectively. After intravenous injection of

[0129] 9. Conduct an addiction test on the multi-target peptide molecules in Example 1 and Example 3, and use the naloxone withdrawal experiment to evaluate physiological addiction. Specifically, it includes:

[0130] Male Kunming mice weighing 18 g - 22 g were pre-adapted in a room temperature environment of 21°C - 23°C for 30 min (during which they could eat and drink freely), and then intrathecally or intravenously injected with multi-target peptide molecules (Example 1, Example 3; the dose was 1.3 nmol for intrathecal injection and 200 nmol for intravenous injection) at the same time every day for six consecutive days. 2 h after the last administration, 10 mg / kg of naloxone was intraperitoneally injected into the mice, and then the mice were immediately placed into an opaque barrel-shaped structure with an inner diameter of 9 cm and a height of 32 cm. The number of jumps, the number of paw tremors, the number of wet dog shakes, and the number of diarrhea episodes of the mice were recorded within 30 min; Diarrhea was detected for 30 min, and 1 point (maximum 6 points) was given to any diarrhea sign every 5 min; For each mouse, the overall opioid withdrawal score was calculated by summing the values obtained for each sign. Every 3 jumps and 3 paw tremors were each a characteristic point, and the other signs were the absolute values of the test data.

[0131] The data were expressed as mean ± standard error (Means ± S.E.M.), and the differences in withdrawal phenomena were statistically analyzed using one-way analysis of variance. * P < 0.05, ** P < 0.01, and *** P < 0.001 indicated significant differences between the group injected with multi-target peptide molecules and the saline group; The evaluation of physiological addiction by intrathecal injection was as Figure 15 shown, and the evaluation of physiological addiction by intravenous injection was as Figure 16 shown.

[0132] It can be seen from Figure 15 that compared with saline, intrathecal injection of [D-Leu 5 G(1-5)-EM2 (Example 1) and [D-Phe9 G(1-5)-EM2 (Example 3) did not produce obvious withdrawal reactions; from Figure 16 it can be seen that after intravenous injection of [D-Leu 5 G(1-5)-EM2 (Example 1) did not produce obvious withdrawal reactions, while after injection of [D-Phe 9 G(1-5)-EM2 (Example 3) only showed limited withdrawal reactions.

[0133] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. A multi-target peptide molecule for opioid and ghrelin receptors, characterized in that: The structure of the multi-target peptide molecule is shown in Formula I: Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Xaa 1 -Tyr-Xa 2 -Phe-Xaa 3 -NH2(Ⅰ)? Among them, Xaa 1 Leu or D-Leu, Xaa 2 For Pro or D-Pro, Xaa 3 is Phe or D-Phe, and Xaa 1 , Xaa 2 , Xaa 3 At least one of the amino acids is a D-amino acid.

2. The multi-target peptide molecule according to claim 1, characterized in that: The multi-target peptide molecules are selected from the following compounds: Gly-Ser-Ser(O-CO-C7H 15 )-Phe-(D-Leu)-Tyr-Pro-Phe-Phe-NH2(II-1); Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Leu-Tyr-(D-Pro)-Phe-Phe-NH2(II-2); Gly-Ser-Ser(O-CO-C7H 15 )-Phe-Leu-Tyr-Pro-Phe-(D-Phe)-NH2(II-3)。 3. A method for preparing the opioid and ghrelin receptor multi-target peptide molecule as claimed in claim 1, characterized in that: The solid phase synthesis method is adopted to sequentially couple amino acids to a solid phase carrier, and the target peptide is obtained after cleavage.

4. The preparation method according to claim 3, characterized in that: The solid phase carrier is an amino resin.

5. The preparation method according to claim 4, characterized in that: include: Deprotection of amino resin; Amino acid condensation and extension; polypeptide cleavage and purification.

6. Use of the opioid and ghrelin receptor multi-target peptide molecule as claimed in claim 1 in the preparation of pain medicine.

7. The use according to claim 6, characterized in that: The pain medicine is used for alleviating and / or treating pathological pain in a subject in need thereof, and the pathological pain includes acute pain and chronic pain.

8. A pharmaceutical composition comprising the opioid and ghrelin receptor multi-target peptide molecule as claimed in claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that Also included are pharmaceutically acceptable carriers and / or excipients.

Images