CN107964047A - Chimeric peptide and its synthetic method and application based on Tyr-Pro-Trp-Phe-NH2 and neurotensin (8-13) - Google Patents

Abstract

Chimeric peptide and its synthetic method and application based on endomorphin 1 and neurotensin (8 13), are related to a kind of chimeric peptide and its synthetic method and application.Be to solve existing endomorphin 1 analgesia duration it is shorter, peripherally administered analgesic activities are relatively low, and have the problem of analgesia tolerance and gastrointestinal side effect.The amino acid sequence of the chimeric peptide is Tyr Pro Trp Phe Gly Gly Arg Arg Pro Tyr Ile Leu.Method：First, the Wang resins pretreatment of " Fmoc " protection；2nd, " Fmoc " blocking group is removed；3rd, amino acid condensation reacts；4th, the extension of peptide chain；5th, peptide chain is from the cutting on resin；6th, the desalination and purifying of thick peptide.Chimeric peptide analgesia duration length of the present invention, and have the advantages that no analgesia tolerance is low with to gastrointestinal side effect.Chimeric peptide of the present invention is used to prepare polypeptide analgesic.

Description

Chimeric peptide based on endomorphin-1 and neurotensin (8-13) and its synthesis method and application

technical field

The invention relates to a chimeric peptide and its synthesis method and application.

Background technique

The World Association for the Study of Pain (IASP) defines neuropathic pain as a pain syndrome caused by sensory pathway damage or dysfunction in the central or peripheral nervous system, characterized by spontaneous pain, hyperalgesia, hyperalgesia, or evoked pain. feature. Neuropathic pain lasts for a long time, recovers slowly, and its pain mechanism is not completely clear. There is a lack of satisfactory treatment methods in clinical practice, which seriously affects the quality of life of human beings. The study of neuropeptides in pain perception and modulation has always been a hot spot in neuroscience research. A variety of neuropeptides have been found to play an important role in the transmission and modulation of pain information, among which opioid peptides are the most familiar.

Endomorphins are endogenous μ-opioid receptor ligands widely present in mammals, and are divided into endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2). Among all known opioids, endomorphins have extremely high affinity and selectivity for μ-opioid receptors. Endomorphins participate in the regulation of many functions such as pain perception, cardiovascular, respiratory, gastrointestinal, motor, behavior, endocrine and immune functions by binding to G protein-coupled μ-opioid receptors, but their main effects are potent Analgesic activity, especially in animal models of neuropathic pain, which is more effective than most opioid peptides. In addition, endomorphins have excellent therapeutic properties, and can produce analgesic effects that are more active than morphine and have fewer side effects, such as the separation of reward behavior from analgesic effects, the formation of tolerance is slower, and the analgesic dose is significantly lower than that of The dose that causes respiratory depression and cardiovascular effects makes it have potential clinical application value. However, endomorphin has short analgesic time, lasting only 20 minutes, poor enzymatic stability, and difficult to penetrate the blood-brain barrier, which limits its application as clinical analgesic drugs. In recent years, scholars have devoted themselves to the research of new, high-efficiency, low-toxic and side-effect peptide analgesic drugs based on the structure of endomorphin. It has been reported that glycosylation and lipidation modification of endomorphin can enter the central nervous system through peripheral administration to produce central analgesic effect.

Contents of the invention

The present invention aims to solve the problems of short analgesic duration of existing endomorphin-1, low analgesic activity of peripheral administration, analgesic tolerance and gastrointestinal side effects, and provides endomorphin-1 based 1 and neurotensin (8-13) chimeric peptides and their synthesis and application.

The amino acid sequence of the chimeric peptide based on endomorphin-1 and neurotensin (8-13) of the present invention is as follows:

Tyr-Pro-Trp-Phe-Gly-Gly-Arg-Arg-Pro-Tyr-Ile-Leu.

The sequence "Tyr-Pro-Trp-Phe" is the four amino acid residue sequence of endomorphin-1, "Gly-Gly" is the middle linker of the chimeric peptide, "Arg-Arg-Pro-Tyr-Ile- Leu" is the 8th-13th amino acid residue of neurotensin, namely the six amino acid residue sequence of neurotensin (8-13).

The present invention is based on the synthetic method of the chimeric peptide of endomorphin-1 and neurotensin (8-13), comprises the following steps:

1. Pretreatment of Wang resin protected by "Fmoc": check the air tightness of the solid phase synthesizer, put the Fmoc-Leu-Wang resin with one amino acid residue into the synthesizer, add dichloromethane and stir for 30-40min, After the resin is fully soaked and swollen, filter the solvent under reduced pressure; the mass ratio of the Fmoc-Leu-Wang resin with one amino acid residue to the volume ratio of dichloromethane is 1g: (7-12)mL;

2. Remove the "Fmoc" protecting group: wash the swelled resin with DMF for 3 to 5 minutes, dry it, repeat 3 to 5 times, and then add 20% to 25% vol. Piperidine/DMF deprotection solution, stirred for 5-10 minutes, drained, repeated 2-3 times, then added piperidine/DMF deprotection solution with volume percentage concentration of 20%-25%, stirred for 15-20 minutes, and " Fmoc" protecting group is fully removed, then the solvent is drained, and finally the deprotecting solution is washed with DMF to obtain the resin that removes the "Fmoc" protecting group; wherein the quality of the resin is the same as that of the deprotecting solution added for the first time The volume ratio is 1g: (8-12) mL; the volume ratio of the mass of the resin to the deprotection solution added for the second time is 1 g: (10-14) mL;

3. Amino acid condensation reaction: amino acid protected by "Fmoc" group, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-6 Dissolve fluorophosphate in DMF, add diisopropylethylamine and mix well to obtain a mixed solution, then add the mixed solution to the resin that removes the "Fmoc" protecting group in step 2, and stir the reaction under the protection of argon 60～72min;

The degree of completion of the condensation reaction is detected by an indene detection reagent, and after the reaction is complete, it is washed repeatedly with DMF to remove unreacted residual liquid;

4. Peptide chain extension: repeat steps 2 and 3, and condense the amino acids protected by the "Fmoc" group on the resin one by one from the C-terminal to the N-terminal of the peptide according to the sequence of the amino acid sequence of the chimeric peptide until all The amino acid residues are condensed to obtain a peptide resin;

5. Cutting of the peptide chain from the resin: completely remove the "Fmoc" group of the last amino acid connected to the peptide resin, then alternately wash the peptide resin with dichloromethane and methanol, fully drain the solvent, and pour it into the peptide resin Add cleavage reagent, and cleavage reaction at room temperature for 3-5 hours;

Collect the cutting reagent and spin dry under reduced pressure. Precipitate with ice-cold ether, remove the ether supernatant after standing still, then fully dissolve the precipitate with water and acetic acid, pour into a separatory funnel, add ethyl acetate for extraction, collect the water phase, and freeze Dried to obtain white solid powder crude peptide;

6. Desalting and purification of crude peptides: using acetic acid solution with a volume concentration of 10%-20% as the mobile phase, desalting the crude peptides through a Sephadex G25 cross-linked Sephadex column, using an ultraviolet detector to collect the main peak and freeze-drying. The desalted polypeptide compound is obtained, and then separated and purified by a reverse-phase high-performance liquid chromatography column, the main peak is collected, and a white solid powder pure peptide is obtained after freeze-drying.

In step 3, the molar weight of the amino acid protected by the "Fmoc" group is 2.5-3 times that of the Fmoc-Arg(pbf)-Wang resin.

The molar weight of N-hydroxybenzotriazole in step 3 is 2.5-3 times of the molar weight of Fmoc-Arg(pbf)-Wang resin.

In step 3, the molar weight of O-benzotriazole-N, N, N', N'-tetramethylurea-hexafluorophosphate is 2.5-3 times the molar weight of Fmoc-Arg(pbf)-Wang resin .

The molar weight of diisopropylethylamine in step 3 is 5-6 times of the molar weight of Fmoc-Leu-Wang resin.

Add 10-25 mL of cleavage reagent per gram of peptide resin in Step 5.

The cleavage reagent described in step five is prepared by mixing trifluoroacetic acid, diisopropylsilane and water in a volume ratio of 95:2.5:2.5.

The specific method for completely removing the "Fmoc" group of the last amino acid connected on the peptide resin in step five is:

Wash the peptide resin with DMF for 3 to 5 minutes, drain it, repeat 3 to 5 times, then add piperidine/DMF deprotection solution with a concentration of 20% to 25% by volume in the resin, stir for 5 to 10 minutes, and then drain it , repeat 2 to 3 times, then add piperidine/DMF deprotection solution with a concentration of 20% to 25% by volume, stir for 15 to 20 minutes to fully remove the "Fmoc" protecting group, then drain the solvent, and finally The deprotection solution was washed with DMF to obtain a resin from which the "Fmoc" protecting group was removed.

Application of the chimeric peptide based on endomorphin-1 and neurotensin (8-13) in the preparation of polypeptide analgesic drugs.

Beneficial effects of the present invention:

The present invention is based on the chimeric peptide of endomorphin-1 and neurotensin (8-13), by connecting the four amino acid residue sequences "Tyr-Pro-Trp-Phe" of endomorphin-1 through the middle The sub "Gly-Gly" is coupled with the six amino acid residue sequence "Arg-Arg-Pro-Tyr-Ile-Leu" of neurotensin (8-13), detected by mass spectrometry and chromatographic analysis, and the designed The structure is consistent, indicating that the chimeric peptide EMNT was successfully synthesized.

Adding a linker in the middle of the chimeric peptide can improve the flexibility of the chimeric peptide and enhance the biological activity of the chimeric peptide. The endomorphin-1 portion of the chimeric peptide binds more readily to the μ-opioid receptor, retaining the opioid activity of this portion. The 8th-13th amino acid of neurotensin, that is, neurotensin (8-13), is the smallest active fragment of neurotensin, and the neurotensin (8-13) part of the chimeric peptide plays a role in the production of sedative At the same time, it can activate the opioid system, cause the release of endogenous opioid peptides, and then produce a synergistic analgesic effect with the endomorphin-1 part of the chimeric peptide. In addition, the chimeric peptide has two positively charged arginine residues, and the guanidine group of arginine can form divalent hydrogen bonds with anionic groups on the cell surface to enhance the biomembrane permeability of the polypeptide. For example, the permeability of the blood-brain barrier can improve the transport ability of drugs to the central nervous system, and then produce centrally mediated efficient analgesic activity through peripheral administration. Moreover, while the chimera exerts an analgesic effect, due to the participation of the neurotensin (8-13) part, its adverse side effects of opioids will also be significantly reduced.

The analgesic duration of the chimeric peptide of the present invention is longer. After injecting the chimeric peptide into the lateral ventricle of mice for analgesic experiments, the results proved that the analgesic %MPE could still reach about 10%-20% at a dose of 1-10nmol after 50 minutes of administration, indicating that the chimeric peptide has Potent, long-lasting central analgesic activity. In addition, by subcutaneous injection of 10 μmol/kg chimeric peptide, the analgesic % MPE was determined to be 17.07% at 60 minutes after administration and 16.8% at 90 minutes, indicating that the duration of chimeric peptide can reach more than 90 minutes through peripheral administration. The results prove that the chimeric peptide designed based on the structure of endomorphin-1 and neurotensin (8-13) greatly enhances the analgesic effect and duration of action of the drug, that is, the chimeric peptide has efficient central analgesia active features.

Therefore, the chimeric peptide based on endomorphin-1 and neurotensin (8-13) of the present invention is a polypeptide analgesic drug with high efficiency and low side effects, and has high clinical application value.

Description of drawings

Fig. 1 is the analgesic effect-time curve of intracerebroventricular injection chimeric peptide in embodiment 1;

Fig. 2 is the analgesic effect-time curve of subcutaneous injection chimeric peptide in embodiment 1;

Fig. 3 is the analgesic tolerance dose curve of intracerebroventricular injection of endomorphin-1 in embodiment 1;

Fig. 4 is the analgesic tolerance dose curve of intracerebroventricular injection of chimeric peptide in Example 1;

Figure 5 is the effect of intracerebroventricular injection of endomorphin-1 and chimeric peptide on colonic transit in Example 1;

Fig. 6 shows the effect of intracerebroventricular injection of endomorphin-1 and chimeric peptide on the propulsion of gastrointestinal charcoal meal in Example 1.

Detailed ways

The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

Specific embodiment one: the amino acid sequence of the chimeric peptide based on endomorphin-1 and neurotensin (8-13) in this embodiment is as follows:

Tyr-Pro-Trp-Phe-Gly-Gly-Arg-Arg-Pro-Tyr-Ile-Leu.

This embodiment retains the biological activity of the two neuropeptide fragments, and solves the problem of the short analgesic duration of endomorphin-1, the low analgesic activity of peripheral administration, and the analgesic tolerance and gastrointestinal side effects The problem. Through various in vitro and in vivo biological experiments, the pharmacological activity of the chimeric peptide of this embodiment was identified. The results show that the chimeric peptide of this embodiment has higher affinity to μ-opioid receptors and opioid activity on isolated specimens, especially the activity on GPI specimens is higher than that of endomorphin-1. In addition, the chimeric peptide has high analgesic activity of central and peripheral administration, long duration of analgesia, and has the advantages of no analgesic tolerance and low side effects on the gastrointestinal tract, effectively overcoming the ubiquity of opioid analgesics Tolerance and side effects such as constipation. Therefore, the chimeric peptide of this embodiment has potential application value in the preparation of clinical polypeptide analgesic drugs.

Specific embodiment two: the present embodiment is based on the synthetic method of the chimeric peptide of endomorphin-1 and neurotensin (8-13), comprises the following steps:

1. Pretreatment of Wang resin protected by "Fmoc": check the air tightness of the solid phase synthesizer, put the Fmoc-Leu-Wang resin with one amino acid residue into the synthesizer, add dichloromethane and stir for 30-40min, After the resin is fully soaked and swollen, filter the solvent under reduced pressure; the mass ratio of the Fmoc-Leu-Wang resin with one amino acid residue to the volume ratio of dichloromethane is 1g: (7-12)mL;

2. Remove the "Fmoc" protecting group: wash the swelled resin with DMF for 3 to 5 minutes, dry it, repeat 3 to 5 times, and then add 20% to 25% vol. Piperidine/DMF deprotection solution, stirred for 5-10 minutes, drained, repeated 2-3 times, then added piperidine/DMF deprotection solution with volume percentage concentration of 20%-25%, stirred for 15-20 minutes, and " Fmoc" protecting group is fully removed, then the solvent is drained, and finally the deprotecting solution is washed with DMF to obtain the resin that removes the "Fmoc" protecting group; wherein the quality of the resin is the same as that of the deprotecting solution added for the first time The volume ratio is 1g: (8-12) mL; the volume ratio of the mass of the resin to the deprotection solution added for the second time is 1 g: (10-14) mL;

3. Amino acid condensation reaction: amino acid protected by "Fmoc" group, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-6 Dissolve fluorophosphate in DMF, add diisopropylethylamine and mix well to obtain a mixed solution, then add the mixed solution to the resin that removes the "Fmoc" protecting group in step 2, and stir the reaction under the protection of argon 60～72min;

The degree of completion of the condensation reaction is detected by an indene detection reagent, and after the reaction is complete, it is washed repeatedly with DMF to remove unreacted residual liquid;

4. Peptide chain extension: repeat steps 2 and 3, and condense the amino acids protected by the "Fmoc" group on the resin one by one from the C-terminal to the N-terminal of the peptide according to the sequence of the amino acid sequence of the chimeric peptide until all The amino acid residues are condensed to obtain a peptide resin;

5. Cutting of the peptide chain from the resin: completely remove the "Fmoc" group of the last amino acid connected to the peptide resin, then alternately wash the peptide resin with dichloromethane and methanol, fully drain the solvent, and pour it into the peptide resin Add cleavage reagent, and cleavage reaction at room temperature for 3-5 hours;

Collect the cutting reagent and spin dry under reduced pressure. Precipitate with ice-cold ether, remove the ether supernatant after standing still, then fully dissolve the precipitate with water and acetic acid, pour into a separatory funnel, add ethyl acetate for extraction, collect the water phase, and freeze Dried to obtain white solid powder crude peptide;

6. Desalting and purification of crude peptides: using acetic acid solution with a volume concentration of 10%-20% as the mobile phase, desalting the crude peptides through a Sephadex G25 cross-linked Sephadex column, using an ultraviolet detector to collect the main peak and freeze-drying. The desalted polypeptide compound is obtained, and then separated and purified by a reverse-phase high-performance liquid chromatography column, the main peak is collected, and a white solid powder pure peptide is obtained after freeze-drying.

Specific embodiment three: the difference between this embodiment and specific embodiment two is: the molar weight of the amino acid protected by the "Fmoc" group in step three is 2.5-3 times the molar weight of the Fmoc-Arg(pbf)-Wang resin. Others are the same as in the second embodiment.

Specific embodiment four: the difference between this embodiment and specific embodiment two or three is: the molar weight of N-hydroxybenzotriazole in step 3 is 2.5-3 times of the molar weight of Fmoc-Arg (pbf)-Wang resin . Others are the same as the second or third specific embodiment.

Embodiment 5: The difference between this embodiment and one of Embodiments 2 to 4 is that in step 3, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphoric acid The molar weight of the salt is 2.5-3 times that of the Fmoc-Arg(pbf)-Wang resin. Others are the same as one of the second to fourth specific embodiments.

Embodiment 6: The difference between this embodiment and Embodiment 2 to 5 is that the molar weight of diisopropylethylamine in step 3 is 5-6 times that of the Fmoc-Leu-Wang resin. Others are the same as one of the second to fifth specific embodiments.

Embodiment 7: This embodiment differs from Embodiment 2 to Embodiment 6 in that: in Step 5, 10-25 mL of cleavage reagent is added per gram of peptide resin. Others are the same as one of the second to sixth specific embodiments.

Embodiment 8: The difference between this embodiment and one of Embodiments 2 to 7 is that the cutting reagent described in step 5 is prepared by mixing trifluoroacetic acid, diisopropylsilane and water in a volume ratio of 95:2.5:2.5. to make. Others are the same as one of the second to seventh specific embodiments.

Specific embodiment nine: the difference between this embodiment and one of specific embodiments two to eight is that the specific method for completely removing the "Fmoc" group of the last amino acid connected to the peptide resin in step five is:

Wash the peptide resin with DMF for 3 to 5 minutes, drain it, repeat 3 to 5 times, then add piperidine/DMF deprotection solution with a concentration of 20% to 25% by volume in the resin, stir for 5 to 10 minutes, and then drain it , repeat 2 to 3 times, then add piperidine/DMF deprotection solution with a concentration of 20% to 25% by volume, stir for 15 to 20 minutes to fully remove the "Fmoc" protecting group, then drain the solvent, and finally The deprotection solution was washed with DMF to obtain a resin from which the "Fmoc" protecting group was removed. Others are the same as one of the second to eighth specific embodiments.

Embodiment 10: This embodiment is based on the application of the chimeric peptide of endomorphin-1 and neurotensin (8-13) in the preparation of polypeptide analgesic drugs.

The following examples of the present invention are described in detail, and the following examples are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

Example 1:

This embodiment is based on the synthesis method of the chimeric peptide of endomorphin-1 and neurotensin (8-13), comprising the following steps:

1. Pretreatment of Wang resin protected by "Fmoc": Check the airtightness of the solid-phase synthesizer, put 1g of Fmoc-Leu-Wang resin with one amino acid residue into the synthesizer, add 10mL of dichloromethane and stir for 30min, After the resin is fully soaked and swollen, the solvent is filtered under reduced pressure.

2. Removal of the "Fmoc" protecting group: wash the swelled resin with DMF for 3 minutes, then dry it, and repeat 3 times. Add 10mL of 20% piperidine/DMF deprotection solution in volume percentage to the resin, drain after stirring for 5min, repeat 2 times, then add 12mL of 20% volume percentage in piperidine/DMF deprotection solution, Stir for 15 minutes to fully remove the "Fmoc" protecting group, then drain the solvent, and finally wash with 10 mL of DMF to remove the deprotecting reagent, and obtain the resin from which the "Fmoc" protecting group has been removed.

3. Amino acid condensation reaction: the amino acid protected by the "Fmoc" group equivalent to 2.5-3 times the molar amount of Fmoc-Arg(pbf)-Wang resin, N-hydroxybenzotriazole, O-benzotriazole Dissolve azole-N,N,N',N'-tetramethylurea-hexafluorophosphate in 10mL DMF, then add diisopropylethylamine equivalent to 5-6 times the molar amount of Fmoc-Leu-Wang resin Afterwards, mix well to obtain a mixed solution, and then add the mixed solution into the deprotected resin, and stir and react for 60 min under the protection of argon. The degree of completion of the condensation reaction was detected by an indene detection reagent, and after the reaction was complete, it was repeatedly washed with DMF to remove unreacted residual liquid.

4. Peptide chain extension: repeat steps 2 and 3, and condense the amino acids protected by the "Fmoc" group on the resin one by one from the C-terminal to the N-terminal of the peptide according to the sequence of the amino acid sequence of the chimeric peptide until all Condensation of the amino acid residues is complete, resulting in a peptide resin.

5. Cutting of the peptide chain from the resin: according to the method of step 2, the "Fmoc" group of the last amino acid connected to the peptide resin is completely removed. Wash the resin alternately with dichloromethane and methanol. After the solvent is fully drained, add 18 mL of cleavage reagent per gram of peptide resin. Mixed, cut and reacted at room temperature for 3 h. Collect the cutting reagent and spin dry under reduced pressure. Precipitate with ice-cold ether, remove the ether supernatant after standing for a while, then fully dissolve the precipitate with water and a small amount of acetic acid, pour it into a separatory funnel, add ethyl acetate for extraction, and collect the water phase. After freeze-drying, a white solid powder crude peptide was obtained.

6. Desalting and purification of crude peptide: using acetic acid solution with a volume concentration of 16% as the mobile phase, desalting the crude peptide through a Sephadex G25 cross-linked Sephadex column, using an ultraviolet detector to collect the main peak and freeze-drying to obtain desalted Polypeptide compounds. Separation and purification are carried out by reversed-phase high-performance liquid chromatography column, the main peak is collected, and after freeze-drying, a white solid powder pure peptide, ie chimeric peptide, is obtained.

The final yield of the chimeric peptide prepared in the above example was 41%, and the detection results of mass spectrometry and chromatographic analysis are shown in Table 1.

Table 1. Detection results of mass spectrometry and chromatographic analysis of chimeric peptide EMNT

The results in Table 1 show that the detected value of the chimeric peptide obtained in the present invention is consistent with the theoretical value.

The biological activity experiment of the chimeric peptide based on endomorphin-1 and neurotensin (8-13) prepared in this embodiment is as follows:

1. Isotope-labeled ligand-receptor affinity experiment

Opioid receptors were obtained by preparing rat meningeal proteins. The experiment was divided into total binding tubes, competitive binding tubes and non-specific binding tubes. Each reaction tube was sequentially added with radioligand, naloxone (Naloxone, Nx)/non-radioactive drug , Tris-HCl (pH=7.4) and meningin. Among them, only [ ³ H]DAMGO (0.5nM) or [ ³ H]DPDPE (1nM) and meningin were added to the total binding tube, and 10μM Nx/different concentrations were added to the non-specific binding tube and competition binding tube respectively. analogs and meningin, and finally added Tris-HCl buffer (pH = 7.4) to a total volume of 0.5 mL. Mix the reaction solution with a shaker, react on a shaker at 37°C for 1 h, take out the centrifuge tube, collect the reaction solution through a ZT-II cell collector, and filter it with a GF/C glass cellulose membrane. After baking the glass cellulose membrane in an oil bath at 80°C for 30 min, the filter membrane was taken out, placed in a 24-well plate, 0.7 mL of scintillation fluid was added to each well, and the membrane was sealed. Radioactive intensity (CPM) was recorded overnight with a beta liquid scintillation counter. Use the Chengdu Taimeng BL-420F biological recording system to calculate the inhibition rate of the analogue to specific binding. The negative logarithm of the concentration is the abscissa, and the inhibition rate is the ordinate. Linear regression is used to obtain the IC ₅₀ value. According to the formula Ki=IC ₅₀ /(1+[L]/KD) to calculate the K _i value. Three sets of experiments were performed in parallel for each experimental data to obtain the average value.

The experimental results are shown in Table 2. The data in Table 2 show that the affinity of the chimeric peptide to the μ-opioid receptor is about 2.5 times lower than that of endomorphin-1, and the affinity K _i (δ) to the δ-opioid receptor is >10000nM, indicating that the chimeric peptide The chimeric peptide still has high affinity characteristics for μ-opioid receptors, which may be related to the linker added in the middle of the chimeric peptide. "Gly-Gly" increases the flexibility of the chimeric peptide, making it easier to bind to the μ-opioid receptor, and thus retains the opioid-affinity activity of the endomorphin-1 portion.

Table 2. Opioid receptor affinity of chimeric peptide EMNT

polypeptide K _i (μ)[nM] K _i (δ)[nM] K _i (δ)/K _i (μ) endomorphin-1 4.55±0.16 5093±660 1119 EMNT 11.2±1.6 >10000 >893

2. Determination of opioid activity in isolated biological specimens

Guinea pig ileal longitudinal muscle test (GPI test): Guinea pigs 250-300g, male or female, fasted for 12 hours before the test, and drinking water is not limited. The occipital was killed with a stick, and two 6 cm sections of ileum near the cecum were removed by laparotomy, and immersed in Kreb's solution ( _g /L: NaCl 6.9, CaCl ₂ 0.28, _KCl 0.35, KH ₂ PO ₄ 0.16, MgSO ₄ 7 H ₂ O 0.30, NaHCO ₃ 2.1, Diphenhydramine 5×10 ^-5 , Choline Chloride 2.8×10 ^-3 , Glucose 1.98), wet it and put it in a glass On the rod, a longitudinal blood vessel along the wall of the ileum separates the longitudinal muscle from the circular muscle, and then binds one end of the longitudinal muscle to the small hook of the glass electrode and the other end to the JZ-1 type muscle with a zero-gauge silk thread On the tension sensor, put it into a glass bath tube with constant temperature of 37±0.2°C filled with Kreb's solution continuously fed with gas (95% O ₂ and 5% CO ₂ ) immediately after preparation, and preload 500 mg. The nutrient solution was changed every 5 minutes for the first 30 minutes, and every 15 minutes thereafter, and the experiment was started after 2 hours of equilibrium. Give electrical stimulation, stimulation parameters: a single square wave with a wave width of 0.3-0.5ms, a frequency of 6 times/min, and a load voltage of 50V. Record the basal contraction intensity, and then give an appropriate amount of morphine to identify its activity; then select 3-4 different concentrations of the drug to be tested, add it to the bath tube, and measure the percentage of inhibition of the contraction caused by the electrical stimulation of the specimen. The degree of inhibition should be 20%- Between 80%. After each drug action, rinse with nutrient solution 5 times, and balance for 15 minutes. Inhibition percentage=(contraction height before response-contraction height after response)/contraction height before response×100%. Then use the dose-response semi-logarithmic graphing method, with the percentage of inhibition as the ordinate and the logarithm of the concentration of the drug to be tested as the abscissa, to obtain the concentration of the drug that inhibits 50% of the contraction height (IC ₅₀ ).

Mouse vas deferens test (MVD experiment): male Kunming mice with a body weight of 30-35 g were killed by cervical dislocation, and the vas deferens on both sides _were taken out by laparotomy, and immersed in Kreb's solution ( _g / L: NaCl 6.9, CaCl ₂ 0.28, KCl 0.35, KH ₂ PO ₄ 0.16, NaHCO ₃ 2.1, Glucose 1.98), peel off the fat and blood vessels attached thereto. Cut off one end of the vas deferens, use a small cotton ball to gently press out the semen in the tube from the end to the open end, and turn it into a hollow tube, then tie one end of it to the small hook of the glass electrode with a zero-gauge silk thread, and the other end to the small hook of the glass electrode. On the JZ-1 type muscle tension sensor, put it into the Kreb's solution filled with continuous gas (95% O ₂ and 5% CO ₂ ) and a glass bath tube with a constant temperature of 36.5±0.5°C immediately after preparation, preload 100mg. The nutrient solution was changed every 5 minutes for the first 30 minutes, and every 10 minutes thereafter, and the experiment was started after 2 hours of equilibrium. Give electrical stimulation, stimulation parameters: wave width 2ms, frequency 6 times/min, load voltage 50V. Record the basal contraction intensity, and then give an appropriate amount of morphine to identify its activity; then give different doses of the drug to be tested, and record the contraction intensity. After each drug action, rinse with nutrient solution 5 times, and balance for 15 minutes. _IC50 determination method is the same as GPI experiment.

The experimental results are shown in Table 3. The data in Table 3 shows that the results of the GPI and MVD specimen experiments are consistent with the trend of the isotope-labeled ligand-receptor affinity experiment results. GPI specimens mainly contained μ-opioid receptors, and MVD specimens mainly contained μ- and δ-opioid receptors. In the GPI assay, the opioid bioactivity of the chimeric peptide was comparable to that of endomorphin-1, and its _IC50 value was slightly lower than that of endomorphin-1. In the MVD experiment, the IC ₅₀ value of the chimeric peptide was slightly lower than that of endomorphin-1. The results indicated that the chimeric peptide had higher opioid agonistic activity on isolated biological samples, especially the agonistic activity on GPI samples was higher than that of endomorphin-1.

Table 3. GPI/MVD Specimen Opioid Activity of Chimeric Peptides

3. Central and peripheral analgesia experiments

Select Kunming male mice, 18-22g, ambient temperature: 20°C, water bath temperature: 50±0.5°C. Before administration, the basic pain threshold (control latency, CL) of the mice was measured, and 1/3-1/2 of the mouse tail was immersed in a water bath, and the time from when the mouse tail was just immersed in the water bath to contraction was recorded. Mice that are too sensitive (<3s) or dull (>5s) are discarded, and the cut-off time is 10s to prevent the mice from being scalded. Tail-flick latency (test latency, TL) was measured at 5, 10, 15, 20, 25, 30, 40, and 50 minutes after intracerebroventricular administration, and at 5, 10, 15, 20, 25, 30, and 50 minutes after subcutaneous administration. TL was measured once at 45, 60 and 90 min each, and 0.9% normal saline was used as a blank control. The results are expressed as maximum possible effect (%MPE): %MPE=100×(TL-CL)/(10-CL).

The results of the central and peripheral analgesic experiments are shown in Figures 1 and 2. The chimeric peptide injected into the central ventricle at a dose of 0.3-10 nmol produced a concentration-dependent high-efficiency analgesic effect (in Figure 1 , the chimeric peptide injected at a dose of 10 nmol, ● indicates injection of chimeric peptide at a dose of 3 nmol, ▲ indicates injection of chimeric peptide at a dose of 1 nmol, ▼ indicates injection of chimeric peptide at a dose of 0.3 nmol, and ◆ indicates normal saline; ■ in Figure 2 indicates subcutaneous injection of chimeric peptide at a dose of 10 μmol/kg, ● indicates subcutaneous injection of physiological saline). The maximum analgesic effect occurs 5 minutes to 10 minutes after the injection of the drug, and the analgesic %MPE can reach about 80% during this period. Moreover, the analgesic duration of the chimeric peptide is longer, and its analgesic %MPE can still reach about 10%-20% at a dose of 1-10nmol after 50min measurement after administration, indicating that the chimeric peptide has an efficient central analgesic effect. pain activity. In addition, by subcutaneous injection of 10 μmol/kg chimeric peptide, it was found that the chimeric peptide has highly effective analgesic activity for peripheral administration, and the maximum analgesic effect is produced 10 minutes after the injection of the drug, and the analgesic duration is longer, its The analgesic %MPE was 17.07% at 60 minutes after administration, and 16.8% at 90 minutes, indicating that the duration of chimeric peptide administration can reach more than 90 minutes. The results prove that the chimeric peptide designed based on the structure of endomorphin-1 and neurotensin (8-13) greatly enhances the analgesic effect and duration of action of the drug, that is, the chimeric peptide has efficient central analgesia active features.

4. Acute tolerance test

Select Kunming male mice, 18-22g, ambient temperature: 20°C. A PE-10 cannula was used to implant a cannula into the lateral ventricle of the mouse, and the drug was administered 3 days after the operation. According to the analgesic activity of the chimeric peptide, the dose of the drug was determined, and the acute analgesic tolerance was determined. The mice were injected with drugs or normal saline once in the lateral ventricle one hour in advance, and then injected with different doses of drugs to measure the changes in pain threshold, and then evaluate the analgesic tolerance of endomorphin-1 and chimeric peptides.

The analgesic tolerance results of endomorphin-1 and chimeric peptides are shown in Figures 3 and 4 (in Figure 3 ■ represents normal saline+endomorphin-1, ● represents endomorphin-1+endomorphine at a dose of 10nmol Peptide-1; ■ in Fig. 4 indicates normal saline + chimeric peptide, ● indicates 10 nmol dose of chimeric peptide + chimeric peptide), by injecting normal saline into the mouse lateral ventricle 1 hour in advance, and then injecting 0.3-10 nmol into the lateral ventricle Doses of endomorphin-1 and chimeric peptides both produce significant analgesic effects in a concentration-dependent manner, similar to the results in Figure 1, indicating that pre-injection of normal saline does not affect endomorphin-1 and chimeric peptide analgesia role play. However, injecting 10nmol dose of endomorphin-1 into the lateral ventricle of mice one hour in advance, and then injecting different doses of endomorphin-1, the analgesic dose curve shifted to the right obviously, and the analgesic effect was significantly reduced, that is, Analgesic tolerance. However, when mice were injected with a dose of 10nmol of chimeric peptide into the lateral ventricle 1 hour earlier, and then injected with different doses of chimeric peptide, the analgesic dose curve almost coincided with the dose curve of normal saline injection in advance, and there was no significant right shift . The results indicated that the pre-injection of the chimeric peptide had no significant effect on the analgesic effect of the second injection of EMNT, proving that the chimeric peptide had high analgesic activity without tolerance side effects.

5. Colonic transit experiment

Male Kunming mice, weighing 25-30g, fasted for 1 hour before the experiment, lightly anesthetized with ether, stuffed d=3mm glass beads into the terminal colon (2cm away from the anus), the mice moved freely, and measured the basic bead row time, the mice whose bead row time was longer than 30 min were excluded. Under the same conditions, different concentrations of drugs were injected into the lateral ventricle, and the injection volume was 5 μL. Bead expulsion time was measured after 5 min, and the transport calculation formula was: %MPE=(bead expulsion time after administration−basic bead expulsion time)/basic bead expulsion time×100.

The experimental results are as shown in Figure 5 (in Figure 5, ■ represents endomorphin-1, Indicates chimeric peptide), intracerebroventricular injection of 0.5-5nmol dose of endomorphin-1 and chimeric peptide produces a concentration-dependent enhancement of the mouse colon bead discharge potential, the higher the concentration, the longer the bead discharge time. However, the effect of the chimeric peptide on colonic bead transport was significantly lower than that of endomorphin-1. The longer the bead discharge time after administration, the stronger the inhibitory effect of the drug on colonic transport in mice. This result indicated that central injection of the chimeric peptide significantly reduced the effect of intestinal transit in the colon segment of mice.

6. Gastrointestinal charcoal meal promotion experiment

Male Kunming mice, weighing 25-30 g, started the experiment after fasting for 20 hours, injected different concentrations of drugs into the lateral ventricle, and the injection volume was 5 μL. Afterwards, 0.2 mL of charcoal meal (5% gum arabic, 10% activated carbon aqueous solution) was orally administered to the mice. After 30 min, the mice were sacrificed, and the small intestine was stripped. Measure the full length of the small intestine (pyloric node to end of the cecum) and the distance the charcoal meal has advanced in the small intestine. The gastrointestinal transport activity of the drug is expressed as a percentage of the distance that the charcoal meal advances in the small intestine to the length of the entire small intestine.

The experimental results are as shown in Figure 6 (in Figure 6, ■ represents endomorphin-1, Indicates chimeric peptide), intracerebroventricular injection of 0.5-5nmol dose of endomorphin-1 and chimeric peptide produces a concentration-dependent inhibition of gastrointestinal charcoal meal propulsion, the higher the concentration, the stronger the inhibitory effect, but the chimeric peptide The inhibitory effect of char meal propulsion in the gastrointestinal tract was significantly lower than that of endomorphin-1. After administration, the lower the transport activity of the charcoal meal in the gastrointestinal tract, the stronger the inhibitory effect of the drug on the propulsion of the charcoal meal in the gastrointestinal tract of mice. This indicates that the central injection of the chimeric peptide reduces the propelling effect of the gastrointestinal carbohydrate meal, that is, it has the advantage of reducing the side effects of the gastrointestinal tract.

In summary, the present invention combines the four amino acid residue sequences "Tyr-Pro-Trp-Phe" of endomorphin-1 with neurotensin (8-13) through the intermediate linker "Gly-Gly" The six amino acid residue sequences "Arg-Arg-Pro-Tyr-Ile-Leu" are coupled to construct a chimeric peptide based on endomorphin-1 and neurotensin (8-13). Its purpose is to retain the biological activity of the two neuropeptide fragments, to solve the short duration of analgesia of endomorphin-1, the analgesic activity of peripheral administration is low, and the analgesic tolerance and gastrointestinal side effects. question. Through various in vitro and in vivo biological experiments, the pharmacological activity of the chimeric peptide synthesized by the present invention is identified. The results show that the chimeric peptide of the present invention has higher affinity to mu-opioid receptors and higher opioid agonistic activity on isolated biological samples, especially higher activity on GPI samples than endomorphin-1. The chimeric peptide has potent analgesic activity both via central ventricle and peripheral subcutaneous administration. In addition, the centrally injected chimeric peptide has no analgesic tolerance properties, and its gastrointestinal side effects are significantly reduced. Therefore, the chimeric peptide of the present invention has potential application value in the preparation of polypeptide analgesic drugs with high efficiency, no analgesic tolerance and reduced gastrointestinal side effects.

Claims (10)

1. Based on the chimeric peptide of endomorphin-1 and neurotensin (8-13), it is characterized in that the amino acid sequence of this chimeric peptide is as follows: Tyr-Pro-Trp-Phe-Gly-Gly-Arg-Arg-Pro-Tyr-Ile-Leu. 2. the synthetic method of the chimeric peptide based on endomorphin-1 and neurotensin (8-13) as claimed in claim 1, it is characterized in that the method comprises the following steps: 1. Pretreatment of Wang resin protected by "Fmoc": check the air tightness of the solid phase synthesizer, put the Fmoc-Leu-Wang resin with one amino acid residue into the synthesizer, add dichloromethane and stir for 30-40min, After the resin is fully soaked and swollen, filter the solvent under reduced pressure; the mass ratio of the Fmoc-Leu-Wang resin with one amino acid residue to the volume ratio of dichloromethane is 1g: (7-12)mL; 2. Remove the "Fmoc" protecting group: wash the swelled resin with DMF for 3 to 5 minutes, dry it, repeat 3 to 5 times, and then add 20% to 25% vol. Piperidine/DMF deprotection solution, stirred for 5-10 minutes, drained, repeated 2-3 times, then added piperidine/DMF deprotection solution with volume percentage concentration of 20%-25%, stirred for 15-20 minutes, and " Fmoc" protecting group is fully removed, then the solvent is drained, and finally the deprotecting solution is washed with DMF to obtain the resin that removes the "Fmoc" protecting group; wherein the quality of the resin is the same as that of the deprotecting solution added for the first time The volume ratio is 1g: (8-12) mL; the volume ratio of the mass of the resin to the deprotection solution added for the second time is 1 g: (10-14) mL; 3. Amino acid condensation reaction: amino acid protected by "Fmoc" group, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-6 Dissolve fluorophosphate in DMF, add diisopropylethylamine and mix well to obtain a mixed solution, then add the mixed solution to the resin that removes the "Fmoc" protecting group in step 2, and stir the reaction under the protection of argon 60～72min; The degree of completion of the condensation reaction is detected by an indene detection reagent, and after the reaction is complete, it is washed repeatedly with DMF to remove unreacted residual liquid; 4. Peptide chain extension: repeat steps 2 and 3, and condense the amino acids protected by the "Fmoc" group on the resin one by one from the C-terminal to the N-terminal of the peptide according to the sequence of the amino acid sequence of the chimeric peptide until all The amino acid residues are condensed to obtain a peptide resin; 5. Cutting of the peptide chain from the resin: completely remove the "Fmoc" group of the last amino acid connected to the peptide resin, then alternately wash the peptide resin with dichloromethane and methanol, fully drain the solvent, and pour it into the peptide resin Add cleavage reagent, and cleavage reaction at room temperature for 3-5 hours; Collect the cutting reagent and spin dry under reduced pressure. Precipitate with ice-cold ether, remove the ether supernatant after standing still, then fully dissolve the precipitate with water and acetic acid, pour into a separatory funnel, add ethyl acetate for extraction, collect the water phase, and freeze Dried to obtain white solid powder crude peptide; 6. Desalting and purification of crude peptides: using acetic acid solution with a volume concentration of 10%-20% as the mobile phase, desalting the crude peptides through a Sephadex G25 cross-linked Sephadex column, using an ultraviolet detector to collect the main peak and freeze-drying. The desalted polypeptide compound is obtained, and then separated and purified by a reverse-phase high-performance liquid chromatography column, the main peak is collected, and a white solid powder pure peptide is obtained after freeze-drying. 3. the synthetic method based on the chimeric peptide of endomorphin-1 and neurotensin (8-13) according to claim 2, is characterized in that the molar weight of the amino acid of " Fmoc " group protection in step 3 It is 2.5-3 times the molar weight of Fmoc-Arg(pbf)-Wang resin. 4. according to the synthetic method of the chimeric peptide based on endomorphin-1 and neurotensin (8-13) described in claim 2 or 3, it is characterized in that the N-hydroxyl benzotriazole in step 3 The molar weight is 2.5-3 times that of the Fmoc-Arg(pbf)-Wang resin. 5. the synthetic method based on the chimeric peptide of endomorphin-1 and neurotensin (8-13) according to claim 4 is characterized in that in step 3, O-benzotriazole-N,N , The molar weight of N', N'-tetramethylurea-hexafluorophosphate is 2.5-3 times that of the Fmoc-Arg(pbf)-Wang resin. 6. the synthetic method based on the chimeric peptide of endomorphin-1 and neurotensin (8-13) according to claim 5, it is characterized in that the molar weight of diisopropylethylamine in step 3 is Fmoc - 5-6 times the molar weight of Leu-Wang resin. 7. the synthetic method based on the chimeric peptide of endomorphin-1 and neurotensin (8-13) according to claim 6 is characterized in that in step 5, every gram of peptide resin adds the cleavage reagent of 10-25mL . 8. the synthetic method based on the chimeric peptide of endomorphin-1 and neurotensin (8-13) according to claim 7, it is characterized in that described in step 5 cutting reagent is by trifluoroacetic acid, diiso Propylsilane and water are mixed according to the volume ratio of 95:2.5:2.5. 9. the synthetic method based on the chimeric peptide of endomorphin-1 and neurotensin (8-13) according to claim 8, is characterized in that in step 5, " Fmoc of the last connected amino acid on the peptide resin The specific method for the complete removal of the "group is: Wash the peptide resin with DMF for 3 to 5 minutes, drain it, repeat 3 to 5 times, then add piperidine/DMF deprotection solution with a concentration of 20% to 25% by volume in the resin, stir for 5 to 10 minutes, and then drain it , repeat 2 to 3 times, then add piperidine/DMF deprotection solution with a concentration of 20% to 25% by volume, stir for 15 to 20 minutes to fully remove the "Fmoc" protecting group, then drain the solvent, and finally The deprotection solution was washed with DMF to obtain a resin from which the "Fmoc" protecting group was removed. 10. The use of chimeric peptides based on endomorphin-1 and neurotensin (8-13) in the preparation of polypeptide analgesic drugs.