CN112961249B

CN112961249B - Bifunctional peptide based on opioid peptide and cannabis peptide, and preparation method and application thereof

Info

Publication number: CN112961249B
Application number: CN202110211993.7A
Authority: CN
Inventors: 方泉; 许彪; 张梦娜; 张润; 李宁
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2021-02-25
Filing date: 2021-02-25
Publication date: 2023-08-04
Anticipated expiration: 2041-02-25
Also published as: CN112961249A

Abstract

The invention discloses a bifunctional peptide based on opioid peptide and cannabis peptide, a preparation method and application thereof, and an N-terminal sequence based on opioid peptide DALDA and cannabis peptide (m) VD-Hp alpha-NH ₂ A novel bifunctional peptide constructed by the C-terminal sequence of the polypeptide. Compared with the prior art, the analgesic activity of OCP 001 and OCP 002 in the body is enhanced, the analgesic effect is more effective than that of a parent molecule, the problem of analgesic tolerance of opioid and hemp agonists and the parent molecule is solved, the analgesic effect in an intolerant form is generated, and the analgesic composition can be used for treating acute pain and pathological pain.

Description

Bifunctional peptide based on opioid peptide and cannabis peptide, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biochemistry, and in particular relates to an opioid peptide DALDA and cannabis peptide (m) VD-Hpalpha-NH-based ₂ Is prepared from the bifunctional peptide of (A) and its preparing process and application.

Background

Pain is one of the most common complaints of clinical patients, with over 10 hundred million people worldwide afflicted with chronic pain over a long period of time. Currently, opioids are the first drug to treat acute and chronic moderate to severe pain, however, their concomitant side effects during use severely affect patient compliance and quality of life. In recent years, peptide analgesic molecules have become one of the hot spots for the development of new drugs because of their higher bioactivity and lower immunogenicity. DALDA is a tetrapeptide derivative of dynorphin, and appears to be a selective agonist of MOR (J Pharmacol Exp Ther 2001, 298:57). DALDA generally does not readily cross the blood brain barrier due to its strong molecular charge, producing significant analgesia in a range of preclinical pain models. However, it still has opioid side effects such as analgesic tolerance, respiratory depression, and motor enhancement at effective analgesic doses (Eur J Pharm Sci 2016,93:11;J Pharmacol Exp Ther 2001,297:364). Therefore, development of analgesic molecules with good analgesic activity and low side effects is a key scientific problem to be solved urgently at present.

The cannabis analgesic molecules are expected to replace traditional opioid therapy due to the advantages of wide analgesic spectrum, low body dependence and the like. Cannabis has two main action targets in vivo, namely cannabis CB1 and CB2 receptors respectively. Among them, CB1 receptors are abundantly expressed in areas associated with pain conduction, such as basal ganglia, cerebral cortex, dorsal horn of spinal cord and dorsal root ganglia (Curr Mol Pharmacol 2019, 12:239). In contrast, the distribution of CB2 receptors is relatively conserved, being expressed mainly in tissues and cells associated with immune functions in the periphery, such as spleen, tonsils, thymus, macrophages and leukocytes, etc. (Curr Mol Pharmacol 2019, 12:239). Activation of CB1 and CB2 receptors in the body can significantly inhibit the pain signaling pathway.

Hemopressin (Hp) is a nonapeptide derivative derived from the alpha chain of heme and acts as an antagonist/inverse agonist of the CB1 receptor. In a later study Gomes et al isolated and identified the N-terminal extension peptides (m) VD-Hpα and (m) RVD-Hpα of heme beta chain in mouse brain homogenates, as well as the dodecapeptide derivative (m) VD-Hpβ. Wherein, (m) VD-Hpα appears to be a selective agonist of CB1 receptors (FASEB J2009, 23:3020), which produces potent analgesic activity in a range of preclinical pain models, but at efficacious analgesic doses there are still cannabis-like side effects of tolerance, hypothermia and motor inhibition (Brain Res Bull 2018,139:48;Brain Res 2018,1680:155;J Pharmacol Exp Ther 2014,348:316). Subsequently, by studying the structure-activity relationship of (m) VD-Hpα, it was found that its C-terminal amidated analogue (m) VD-Hpα -NH ₂ Has significantly enhanced analgesic activity relative to the parent molecule, and is free of stiffness, motor inhibition, constipation, sedation, ingestion promotion, and motor disturbances at effective analgesic dosesTingling-like side effects occur (Neuropharmacology 2020, 175:108178). However, as with classical cannabis agonists, the lateral ventricle was continuously injected with (m) VD-hpα -NH ₂ Still produces obvious analgesic tolerance phenomenon.

It is appreciated that there have been a number of preclinical and clinical studies demonstrating the functional interactions between the opiate and cannabis systems. The combined use of opioid and cannabis agonists may reduce the opioid/cannabis-like side effects by lowering the drug dose (Clin Pharmacol Ther 2011,90:844;Trends Pharmacol Sci 1999,20:287). For example, cannabis can significantly reduce the dependency of chronic pain patients on opiates while improving their sleep quality (J Pharmacol Exp Ther 2003,305:812;Postgrad Med 2020,132:56). However, the absorption and metabolic properties of different drug molecules vary greatly in the body, so patients often need to follow complex dosing regimens to achieve optimal therapeutic effects of the drugs when using multiple drugs in combination. Moreover, the occurrence of potential interactions in vivo of multiple drug molecules significantly increases the incidence of adverse reactions due to the complex pharmacokinetic properties, thus limiting the scope of application of this approach in clinical therapy (Lancet 2000, 356:1255). Therefore, the invention provides a bifunctional peptide based on opioid peptide DALDA and cannabinol (m) VD-Hp-NH2 to effectively solve the technical problems.

Disclosure of Invention

The invention aims to: aiming at the defects of the prior art, the invention provides a novel opioid peptide DALDA and cannabis peptide (m) VD-Hpalpha-NH-based compound ₂ Is a bifunctional peptide of (2).

The invention also solves the technical problem of providing a preparation method of the bifunctional peptide.

The invention further aims to provide application of the bifunctional peptide.

In order to solve the first technical problem, the invention discloses a novel opioid peptide DALDA and cannabinol (m) VD-Hpα -NH-based compound ₂ Is prepared from the opioid peptide DALDA [ Tyr- (D) Arg-Phe-Lys-NH ] ₂ ]And cannabinide (m) VD-hpα -NH ₂ (Val-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-NH ₂ ) The key pharmacophore of (2) is constructed by a chemical template, the N end of the key pharmacophore is the key pharmacophore of opioid peptide DALDA, and the C end of the key pharmacophore is cannabis peptide (m) VD-Hpalpha-NH ₂ Has an amino acid sequence represented by formula I,

Tyr-(D)Arg-Phe-Lys-Xaa5-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-NH ₂ (Ⅰ)；

wherein Xaa5 is Val or absent.

Wherein when Xaa5 is Val, the bifunctional peptide has an amino acid sequence shown in formula II, and the bifunctional peptide is labeled OCP 001.

Tyr-(D)Arg-Phe-Lys-Val-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-NH ₂ (Ⅱ)；

Wherein when Xaa5 is deleted, the bifunctional peptide has an amino acid sequence shown in formula III, and the bifunctional peptide is labeled as OCP 002.

Tyr-(D)Arg-Phe-Lys-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-NH ₂ (Ⅲ)；

In order to solve the second technical problem, the invention discloses a preparation method of the bifunctional peptide, which is to prepare polypeptide by adopting Fmoc solid-phase synthesis method, and comprises the following steps:

(1) Pretreatment of resin: swelling the amino resin, pumping under reduced pressure, and washing;

(2) Removing Fmoc group protection: after deprotection, indene is detected to obtain deprotected resin;

(3) Condensation of amino acids: adding a first N-alpha-Fmoc protected amino acid (Fmoc-Aa) according to the amino acid sequence from the C end to the N end of the polypeptide, and performing condensation reaction on the deprotected resin obtained in the step (1), wherein the indene inspection confirms that the condensation is successful; then adding a deprotection reagent to remove the protecting group on the amino acid, washing, and checking indene to confirm that the deprotection is complete;

(4) Extension of peptide chain: adding a second amino acid, and repeating the condensation and deprotection reaction process in the step (3) from the C end to the N end until the polypeptide condensation is completed;

(5) Cutting the polypeptide synthesized in the step (4) from amino resin by using a cutting agent, filtering, performing reduced pressure spin drying on a filtrate, namely a cutting solution, adding glacial ethyl ether to separate out precipitate, extracting crude peptide in the ethyl ether by using a 20% acetic acid aqueous solution, performing freeze drying to obtain white crude peptide powder, and purifying to obtain the polypeptide.

In the step (1), the amino resin is any one of Rink-Amide-MBHA resin and Rink-Amide resin, preferably Rink-Amide-MBHA resin.

In step (1), the swelling is carried out by stirring the amino resin in the solvent at 60-100rpm for 10-40min, preferably at 80rpm for 30min, to fully swell the resin.

Wherein the swelling solvent is dichloromethane.

Wherein the volume-mass ratio of the solvent to the amino resin is 8-12mL/g, preferably 10mL/g.

In the step (1), the washing is to add N, N-Dimethylformamide (DMF) for 3 times, each for 3min.

In the step (2), the deprotection is to put the amino resin after treatment into a deprotection reagent, stir once, repeat 1-4 times, preferably 2 times, and pump down; then placing the mixture in a deprotection reagent, stirring the mixture for the second time, and washing the mixture with N, N-Dimethylformamide (DMF) for 4 times to remove residual piperidine, thereby obtaining the resin with Fmoc protecting groups removed.

Wherein the deprotection reagent is piperidine, 1, 8-diazabicyclo [5,4,0] undec-7-ene and N, N-dimethylformamide according to 1:1:98 by volume ratio.

Wherein the volume to mass ratio of the deprotected reagent to post-pump disposal is 8-12mL/g, preferably 10mL/g.

Wherein the rate of one stirring is 60-100rpm, preferably 80rpm; the time of one stirring is 2-6min, preferably 5min.

Wherein the rate of the secondary stirring is 60-100rpm, preferably 80rpm; the secondary stirring time is 8-12min, preferably 10min.

In the step (2), the indene test is to pick a small amount of resin into a colorless transparent glass tube, add an indene test reagent, and put into boiling water to stand for 3min. If Fmoc group removal is complete, both the resin and the solution are dark blue. If the condensation is successful, the resin is colorless and the solution is pale yellow.

Wherein, the indene detection reagent is that the volume ratio is 1:2: phenol of 1: pyridine: ninhydrin solution.

Wherein, the phenol solution is prepared by dissolving 20g of phenol in 5mL of absolute ethanol, the pyridine solution is prepared by dissolving 0.05mL of KCN (0.001M) in 2.5mL of pyridine, and the ninhydrin solution is prepared by dissolving 0.5g of ninhydrin in 10mL of absolute ethanol, wherein, phenol, pyridine and absolute ethanol are subjected to redistillation treatment.

In the step (3), the condensation reaction is to react N-alpha-Fmoc protected amino acid (Fmoc-Aa), 1-hydroxybenzotriazole (HOBt), benzotriazol-N, N, N ', N' -tetramethylurea Hexafluorophosphate (HBTU), N, N-Diisopropylethylamine (DIEA) according to 1:0.2-1.5:0.2-1.5:1.5-3 (preferably 1:1:1:2) and N, N-Dimethylformamide (DMF), and the deprotected resin obtained in step (1) under inert conditions, and pumping the solvent; washing with N, N-dimethylformamide for 3 times, and draining.

Wherein DIEA was added last.

Wherein the volume-mass ratio of the N, N-dimethylformamide to the N-alpha-Fmoc protected amino acid is 20mL/g.

Wherein the volume-mass ratio of the mixed solution to the deprotected resin obtained in the step (2) is 4-6mL/g, preferably 5mL/g.

Wherein the inert environment is preferably under argon.

Wherein the stirring rate is 60-100rpm, preferably 80rpm.

Wherein the reaction is carried out at room temperature for 40-100min, preferably 60min

In step (5), trifluoroacetic acid (TFA), triisopropylsilane and (TIS) water are used in accordance with 95:2.5:2.5 by volume ratio of the cutting agent to cut the bifunctional peptide from the resin; the volume-to-mass ratio of the cutting agent to the resin is 10-20mL/g, preferably 15mL/g.

Wherein the cleavage is carried out by placing the polypeptide chain synthesized in step (4) into a cleavage agent and reacting for 1.5-4h, preferably 3h at room temperature; preferably, stirring is carried out for 1min every 15min at a stirring rate of 50 rpm/min.

Preferably, the condensed resin is alternately washed with dichloromethane DCM (2X 3 min), meOH (1X 3 min), DCM (1X 3 min), meOH (2X 3 min) before cleavage, and the synthesizer is sealed and then drained for 4h.

In the step (5), the purification is to further separate and purify the crude peptide by reverse phase high performance liquid chromatography, and collect the main peak of the sample after separation to obtain the multi-target polypeptide product.

In order to solve the third technical problem, the invention discloses application of the bifunctional peptide in preparing analgesics, and the brand new bifunctional peptide molecule can generate remarkable analgesic effects in acute pain and pathological pain, and reduces analgesic tolerance side effects of traditional opium and hemp analgesic drugs.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

(1) With reported (r) VD-Hpα, (m) VD-Hpα -NH ₂ The analgesic activity of OCP 001 and OCP 002 was enhanced in vivo compared to DALDA, resulting in a more potent analgesic effect than the parent molecule.

(2) OCP 001 and OCP 002 solve the problem of analgesic tolerance of classical opioid and cannabis agonists and the parent molecule, produce an analgesic effect in an intolerant form, and are useful in the treatment of acute and pathological pain.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

Fig. 1 is a time course of analgesic effect produced by injection of OCP 001 and OCP 002 into the ventricles of mice in acute pain.

Figure 2 is a time course of analgesic effect of subcutaneous injections of OCP 001 and OCP 002 in mice in acute pain.

Fig. 3 is a time course of analgesic effect of injection of OCP 001 and OCP 002 into lateral ventricle of mice in inflammatory pain.

Fig. 4 is a time course of analgesic effect of subcutaneous injections of OCP 001 and OCP 002 in mice in inflammatory pain.

Fig. 5 shows the change in analgesic effect produced by 6 consecutive days of subcutaneous injection of OCP 001 and OCP 002 in inflammatory pain in mice.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.

Instrument: high Performance Liquid Chromatography (HPLC) was Delta 600 from Waters, wherein the column (XBRID TM BEH 130Prep C18,4.6mm X250 mm) was analyzed and the column (XBRID TM BEH 130Prep C18, 19mm X250 mm) was prepared. The mass spectrometer was PE Biosystems, mariner System 5074, dalton, germany. The solid-phase polypeptide synthesizer is designed independently by the laboratory. The freeze dryer was 6KBTEL-85 from VIRTIS, U.S.A. The rotary evaporator is RE-5298A of Shanghai Asia company of China.

Reagent: the Resin was Rink-Amide-MBHA-Resin, available from Tianjin Nanking and Chengda. N-alpha-Fmoc protected amino acid (Fmoc-Aa), N-hydroxybenzotriazole (HOBt), O-benzotriazol-N, N, N ', N' -tetramethylurea-Hexafluorophosphate (HBTU) were purchased from Gill Biochemical (Shanghai) Inc. Diisopropylethylamine (DIEA) and 1, 8-diazabicyclo undec-7-ene (DBU) were purchased from beijing carbofuran technologies limited. Dichloromethane (DCM), N-Dimethylformamide (DMF), piperidine (piperidine), methanol (MeOH), and pyridine were all purchased from the second reagent plant, trifluoroacetic acid (TFA) and phenol as the first reagent plant products; the organic reagents are subjected to re-steaming treatment before use.

EXAMPLE 1 Synthesis of OCP 001

(1) Pretreatment of resin: 600mg of Rink-Amide-MBHA resin was weighed out and swollen in 6mL of dichloromethane with stirring at 80rpm for 30min and dried for 30min after the reaction. DMF was then added to wash 3 times for 3min each.

(2) Removing Fmoc group protection: the volume fraction ratio of the resin after swelling was 1:1: piperidine of 98: DBU: a mixed solution of DMF, wherein the volume-mass ratio of the mixed solution to the swelled resin is 10mL/g; stirring was performed at 80rpm for 5min and repeated 2 times. After draining, the above mixed solution was added again and stirred at 80rpm for 10min. After that, the residue was removed by washing with DMF 4 times for 3min each. The Fmoc protecting group-removed resin was obtained.

(3) Indene detection: a small amount of resin is selected and put into a colorless transparent glass tube, indene detection reagent is added, and the mixture is placed into boiling water for standing for 3min. If Fmoc group removal is complete, both the resin and the solution are dark blue. Indene detection reagent: the volume ratio is 1:2: phenol of 1: pyridine: ninhydrin solution. Wherein the phenol solution is prepared by dissolving 20g of phenol in 5mL of absolute ethanol, the pyridine solution is prepared by dissolving 0.05mL of KCN (0.001M) in 2.5mL of pyridine, and the ninhydrin solution is prepared by dissolving 0.5g of ninhydrin in 10mL of absolute ethanol, wherein phenol, pyridine and absolute ethanol are subjected to redistillation treatment.

(4) Condensation of amino acids: N-alpha-Fmoc protected histidine Fmoc-His (Trt) -OH, HOBt, HBTU and DIEA were prepared at 1:1:1:2 in DMF, the volumetric mass ratio of DMF to Fmoc-His (Trt) -OH was 20mL/g, with DIEA added last. Adding the obtained mixed solution into the resin without Fmoc group protection obtained in the step (2), wherein the volume-mass ratio of the mixed solution to the deprotected resin is 5mL/g, stirring at 80rpm in a synthesizer under the protection of argon, reacting for 60min at room temperature, and pumping out the solvent; washed 3 times with DMF and drained. And (3) indene inspection is carried out according to the step (3), if the condensation is successful, the resin is colorless, and the solution is light yellow. After indene detection, fmoc group protection is removed according to the step (2), and then indene detection is carried out according to the step (3).

(5) Extension of peptide chain: condensing Fmoc-Ser (tBu) -OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Phe-OH, fmoc-Asn (Trt) -OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Asp (OtBu) -OH, fmoc-Val-OH, fmoc-Lys (Boc) -OH, fmoc-Phe-OH, fmoc- (D) Arg (pbf) -OH and Boc-Tyr (tBu) -OH on the peptide resin sequentially according to the method of the step (4). Obtaining peptide Resin Boc-Tyr (tBu) - (D) Arg (pbf) -Phe-Lys (Boc) -Val-Asp (OtBu) -Pro-Val-Asn (Trt) -Phe-Lys (Boc) -Leu-Leu-Ser (tBu) -His (Trt) -Resin;

(6) Cleavage of peptide chain from resin: the peptide resin from step (5) was alternately washed with DCM (2X 3 min), meOH (1X 3 min), DCM (1X 3 min), meOH (2X 3 min), and the synthesizer was sealed and then drained for 4h. To the drained peptide Resin Boc-Tyr (tBu) - (D) Arg (pbf) -Phe-Lys (Boc) -Val-Asp (OtBu) -Pro-Val-Asn (Trt) -Phe-Lys (Boc) -Leu-Leu-Ser (tBu) -His (Trt) -Resin was added a cleavage agent (15 mL/g peptide Resin). The reaction was carried out at room temperature for 3 hours, and stirring was carried out at 50rpm for 1 minute every 15 minutes. The cleavage agent was TFA: TIS: water at 95:2.5:2.5 by volume. After the reaction was completed, the cleavage liquid was dried by spin drying under reduced pressure, a pre-cooled diethyl ether was added to precipitate, a 20% aqueous acetic acid solution was added to extract crude peptide in diethyl ether, and 296.1mg of a white crude peptide solid powder was obtained by freeze drying, with a crude peptide yield of 63.1%.

(7) Purification of the polypeptide: the crude peptide was isolated and purified using reverse phase high performance liquid chromatography (RP-HPLC) C18 column (XBridge TM BEH 130Prep C18, 19 mm. Times.250 mm). The mobile phase was acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA), and the main peak product was collected. The loading amount was 120.0mg, and 50.0mg of a pure peptide solid powder of OCP 001 was obtained after freeze-drying, with a pure peptide yield of 41.7%. The mass spectrum and chromatographic analysis results are shown in Table 1.

EXAMPLE 2 Synthesis of OCP 002

(5) Extension of peptide chain: condensing Fmoc-Ser (tBu) -OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Phe-OH, fmoc-Asn (Trt) -OH, fmoc-Val-OH, fmoc-Pro-OH, fmoc-Asp (OtBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Phe-OH, fmoc- (D) Arg (pbf) -OH and Boc-Tyr (tBu) -OH on the peptide resin in sequence according to the method of the step (4). Obtaining peptide Resin Boc-Tyr (tBu) - (D) Arg (pbf) -Phe-Lys (Boc) -Asp (OtBu) -Pro-Val-Asn (Trt) -Phe-Lys (Boc) -Leu-Leu-Ser (tBu) -His (Trt) -Resin;

(6) Cleavage of peptide chain from resin: the peptide resin from step (5) was alternately washed with DCM (2X 3 min), meOH (1X 3 min), DCM (1X 3 min), meOH (2X 3 min), and the synthesizer was sealed and then drained for 4h. To the drained peptide Resin Boc-Tyr (tBu) - (D) Arg (pbf) -Phe-Lys (Boc) -Asp (OtBu) -Pro-Val-Asn (Trt) -Phe-Lys (Boc) -Leu-Leu-Ser (tBu) -His (Trt) -Resin was added a cleavage agent (15 mL/g peptide Resin). The reaction was carried out at room temperature for 3 hours, and stirring was carried out at 50rpm for 1 minute every 15 minutes. The cleavage agent was TFA: TIS: water at 95:2.5:2.5 by volume. After the reaction was completed, the cleavage liquid was dried by spin drying under reduced pressure, a pre-cooled diethyl ether was added to precipitate, a 20% aqueous acetic acid solution was added to extract crude peptide in diethyl ether, and 327.8mg of a white crude peptide solid powder was obtained by freeze drying, with a crude peptide yield of 70.4%.

(7) Purification of the polypeptide: the crude peptide was isolated and purified using reverse phase high performance liquid chromatography (RP-HPLC) C18 column (XBridge TM BEH 130Prep C18, 19 mm. Times.250 mm). The mobile phase was acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA), and the main peak product was collected. The loading amount was 79.79mg, and after freeze-drying, 40.4mg of pure peptide solid powder of OCP 002 was obtained, and the yield of pure peptide was 50.6%. The mass spectrum and chromatographic analysis results are shown in Table 1.

TABLE 1 Mass Spectrometry and chromatographic detection results of OCP 001 and OCP 002

Note that: system 1: the gradient elution system 1 is: 10-80% acetonitrile/water (0.1% tfa) (30 min complete), flow rate: 1mL/min, detection wavelength 220nm, analysis chromatographic column: XBIdge (TM) BEH 130Prep C18,4.6mm X250 mm; system 2: the gradient elution system 2 is as follows: 10-100% acetonitrile/water (0.1% tfa) (30 min complete), flow rate: 1mL/min, detection wavelength 220nm, analysis chromatographic column: XBIdge (TM) BEH 130Prep C18,4.6mm X250 mm; wherein the acetonitrile concentration in the system 1 rises from 10% to 80% in 30 min; the concentration of acetonitrile in system 2 rose from 10% to 100% over a period of 30min.

The product prepared by the method is detected by mass spectrum and chromatographic analysis and is consistent with the designed structure of the compound. Indicating successful synthesis of opiumPeptide DALDA and cannabis peptide (m) VD-Hpα -NH ₂ The purity of the purified bifunctional peptide is more than 97%.

Example 3: based on the opioid peptide DALDA and the cannabinide (m) VD-Hpα -NH ₂ Analgesic experiments on bifunctional peptides of (2)

The invention is based on the opioid peptide DALDA and the cannabinol (m) VD-Hpα -NH ₂ Is based on the opioid peptide DALDA and the cannabinoid peptide (m) VD-Hpα -NH ₂ The constructed brand new bifunctional peptide can effectively reduce the side effects of pain relieving tolerance while playing a role in high-efficiency pain relieving. The analgesic and tolerating effects of the bifunctional peptides OCP 001 and OCP 002 of the present invention are described below by pharmacological experiments.

1. Pain test

The compounds OCP 001 and OCP 002 prepared in example 1 and example 2 were tested and compared for in vivo pain modulation activity in photothermal-induced acute pain and carrageenan-induced inflammatory pain models, respectively, after injection and subcutaneous injection through the lateral ventricle, respectively.

The specific injection amounts of OCP 001 and OCP 002 are shown in FIGS. 1-5, and normal saline is used as blank control.

(1) Lateral ventricle injection in mice

Male Kunming mice weighing 18-22g were selected, anesthetized with 80mg/kg sodium pentobarbital by intraperitoneal injection, and fixed on a brain stereotactic apparatus (68001, ruiword). After hair is removed from the head operation area and disinfected, the skull is exposed, and the position of the bregma is determined. Using the bregma as the origin, 3mm backward and 1mm left/right, the upper position of the lateral ventricle was marked by punching with a needle. A self-made stainless steel tube (with the outer diameter of 0.5mm and the inner diameter of 0.25 mm) connected with a PE-10 catheter is inserted into the position 3mm below the needle hole, namely the lateral ventricle. The dental tray powder is used for fixing the stainless steel tube, and a section of stainless steel string is inserted into the stainless steel tube to prevent cerebrospinal fluid from overflowing or being infected, and the wound is sutured. Mice were recovered 4 days after surgery was completed and used for further experimental study.

For lateral ventricle injection, 4 μl of drug (OCP 001, OCP 002 and saline, respectively) was aspirated with a microinjector, and the lateral ventricle was slowly injected at a rate of 10 μl/min through the indwelling stainless steel tube, followed by flushing the stainless steel tube with 1 μl of saline to allow the drug to completely enter the lateral ventricle. After the injection is completed, the needle of the microinjector is pulled out after staying in the stainless steel tube for a plurality of seconds so as to prevent the medicine from overflowing.

(2) Subcutaneous injection in mice

Subcutaneous injections of the drug were performed on awake mice. The skin of the cervical back of the mouse was held by hand, the head was inclined downward at about 45 °, and the syringe was penetrated into the subcutaneous tissue of the mouse via the skin of the back, and the drugs (OCP 001, OCP 002, and physiological saline, respectively) were slowly injected. After the injection is completed, the needle is pulled out in a rotating way and pressed at the injection needle hole for a plurality of seconds so as to prevent the medicine from overflowing.

(3) Pain sensation detection experiment

(1) Acute pain. Before the experiment starts, the mice injected from the ventricle at the side of the step (1) and injected subcutaneously at the step (2) are respectively adapted to 30min at the room temperature of 22+/-1 ℃ during which the mice can eat and drink water freely. The basal tail flick latency (3-5 s) of mice was measured prior to dosing, mice that were too sensitive or sluggish were discarded, and the tail flick latency of mice at 5,10,15,20,30,40,50,60 and 90min post dosing was recorded.

(2) Inflammatory pain. Carrageenan was used to induce inflammatory pain in mice to evaluate the analgesic effect of the drug on inflammatory pain. Before the experiment starts, the mice injected from the ventricle at the side of the step (1) and injected subcutaneously at the step (2) are respectively adapted to 30min at the room temperature of 22+/-1 ℃ during which the mice can eat and drink water freely. The basal paw withdrawal threshold of the mice was measured using an electronic von Frey stinging machine and an inflammatory pain model was constructed by subcutaneously injecting 20 μl of 2% carrageenan solution into the sole. Paw withdrawal thresholds were recorded at 15,30,45,60 and 90min before and after administration of the mice, respectively, 24h after carrageenan injection.

(4) Calculating MPE values for drugs

The analgesic effect of the drug was evaluated with the maximum analgesic effect MPE (%); in the photo-thermal tail flick experiment, the MPE (%) calculation method comprises the following steps: MPE (%) =100× [ (pain threshold after administration-basal pain threshold)/(10 s-basal pain threshold)]. In carrageenan-induced inflammatory pain, MPE (%) was calculated as: MPE (%) =100× [ (pain threshold after administration-pain threshold before administration)/(basal pain)Threshold-pain threshold before administration]. Since the analgesic action time of the drug can last for 60min, the analgesic effect of the drug in acute pain and inflammatory pain is converted into area under the curve AUC (area under the curve) (0-60 min) to evaluate the analgesic effect of the drug. ^* P<0.05, ^** P<0.01, ^*** P<The MPE (%) value was significantly different from that of the control saline group, and the experimental results are shown in FIGS. 1 to 4. Both lateral ventricle and subcutaneous injections of OCP 001 and OCP 002 produced significant analgesia in the mice models of acute pain (fig. 1 and 2) and inflammatory pain (fig. 3 and 4).

(5) Calculating half-effective amount of the drug

ED ₅₀ Half effective amount (50%effective dose,ED) ₅₀ ) Refers to the amount of drug that causes 50% of the maximum response intensity. The relevant statistical data ED was calculated by the statistical software GraphPad Prism 5.0 using MPE (%) and the corresponding drug amounts ₅₀ And 95% confidence interval. The relevant analgesic data are shown in table 2.

Based on analgesia ED ₅₀ The values are used for comparing the strength of analgesic activity of the medicaments. In the photo-thermal tail flick experiment, the central analgesic activity of OCP 002 is improved by 44.6 times compared with (m) VD-Hpα (6.69 nmol, 201310087565.3), 45.7 times compared with (r) VD-Hpα (6.85 nmol, neuropeptides.2017, 63:83), and compared with (m) VD-Hpα -NH ₂ (1.15 nmol, neuropharmacology.2020, 175:108178) by 7.7-fold, 6.8-fold higher than morphine (1.02nmol,Br J Pharmacol.2016,173:1864), a commonly used analgesic drug in clinic; in inflammatory pain, the central analgesic activity of OCP 002 is compared to (m) VD-Hpα -NH ₂ (1.65 nmol, neuropharmacology 2020, 175:108178) was increased by a factor of 2.1.

In addition, in photothermal tail flick experiments, lateral ventricle (fig. 1) and subcutaneous (fig. 2) injection of OCP 001 required higher doses of drug to achieve the highest analgesia than OCP 002. In inflammatory pain, the amounts of analgesia required for OCP 001 and OCP 002 to achieve the same analgesic effect were comparable (FIG. 3: lateral ventricle injection; FIG. 4: subcutaneous injection).

Previous studies have shown that DALDA has relatively weak analgesic effects in preclinical acute pain, inflammatory pain and neuralgia models. For example, leatherThe injection of 5mg/kg of DALDA produced significant analgesia in mice with acute and inflammatory Pain (Reg Anesth paint Med.2020, 45:907); peripheral analgesic ED of DALDA in mouse neuralgia ₅₀ The dose of DALDA exhibiting the highest central analgesic activity in acute pain in rats at a value of 4.2mg/kg (Anesthesiology. 2016, 124:706) required 327nmol (Eur J Pharm Sci.2016, 93:11), and it is apparent from Table 2 that the present invention can greatly reduce the effective analgesic dose or the maximum analgesic dose of the drug.

TABLE 2 analgesic effect of lateral ventricle and subcutaneous horizontal injection of OCP 002

2. Pain tolerance test

Studies have shown that continuous injection of the opioid peptides DALDA and cannabinide (m) VD-Hpα -NH ₂ The obvious analgesic tolerance phenomenon can be generated, and the pharmacological activity of the bifunctional peptide in the invention in analgesic tolerance aspect is further compared. The tolerability of the mice to analgesic effect was examined in the inflammatory pain model by injecting them subcutaneously for 6 consecutive days.

(1) Pain tolerance test method for mice

The highest analgesic dose of the drug was injected subcutaneously in mice for 6 consecutive days, once daily. The ambient temperature is controlled at 22+/-1 ℃, and the experimental animals can eat and drink water freely. The paw withdrawal threshold corresponding to the point in time when the drug reaches the maximum analgesic effect after the administration of the mice is recorded in the experiment.

(2) Tolerance experimental data statistics

Experimental data are expressed in terms of maximum analgesic effect MPE (%), calculated as: MPE (%) =100× [ (pain threshold after administration-pain threshold before administration)/(basal pain threshold-pain threshold before administration) ]. Differences in drug 6-day analgesic effect were counted using one-way ANOVA (Tukey HSD test for one-way ANOVA). The experimental results are shown in FIG. 5.

The normal saline group is a blank solvent control group, and the dosages of the two bifunctional peptides are respectively 10mg/kg and 6mg/kg by subcutaneous injection. Animals in each group were 6-8. Experimental data show that after mice are injected with OCP 001 and OCP 002 subcutaneously for 6 days, the MPE% value of analgesic activity of the drugs is not obviously reduced, which indicates that the two bifunctional peptides keep better intolerant analgesic activity.

The invention provides a compound based on opioid peptide DALDA and cannabis peptide (m) VD-Hpalpha-NH ₂ The above description is only of the preferred embodiments of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the scope of the present invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. Based on opioid peptide DALDA and cannabinol (m) VD-Hpα -NH ₂ Is characterized in that the amino acid sequence of the bifunctional peptide is shown as a formula I,

Tyr-(D)Arg-Phe-Lys-Xaa5-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-NH ₂ (Ⅰ)；

wherein Xaa5 is Val or absent.

2. The bifunctional peptide of claim 1, wherein the amino acid sequence of the bifunctional peptide is:

Tyr-(D)Arg-Phe-Lys-Val-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-NH ₂ 。

3. the bifunctional peptide of claim 1, wherein the amino acid sequence of the bifunctional peptide is:

Tyr-(D)Arg-Phe-Lys-Asp-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-NH ₂ 。

4. a method for producing a bifunctional peptide as claimed in any one of claims 1 to 3, comprising the steps of:

(1) Swelling and drying the amino resin;

(2) Deprotection of the resin obtained in the step (1) to obtain deprotected resin;

(3) According to the amino acid sequence from the C end to the N end of the polypeptide, adding the first oneN-condensation of the α -Fmoc protected amino acid with the deprotected resin from step (2); adding a deprotection reagent to remove a protecting group on an amino acid; condensing the second amino acid, repeating the above steps until the synthesis of the polypeptide chain is completed;

(4) And (3) cutting the polypeptide synthesized in the step (3) from amino resin by using a cutting agent to obtain the polypeptide.

5. The method according to claim 4, wherein in the step (1), the amino resin is any one of Rink-Amide-MBHA resin and Rink-Amide resin.

6. The method of claim 4, wherein in step (1), the swelling solvent is methylene chloride; the volume-mass ratio of the solvent to the amino resin is 8-12 mL/g.

7. The method of claim 4, wherein in step (2), the deprotected reagent is piperidine, 1, 8-diaza [5,4,0]Undec-7-eneN, NDimethylformamide according to 1:1:98 by volume ratio; the volume-mass ratio of the deprotected reagent to the resin after the pumping is 8-12 mL/g.

8. The process according to claim 4, wherein in the step (3), the condensation reaction is carried out byNalpha-Fmoc protected amino acid, 1-hydroxybenzotriazole and benzotriazol-N, N, N', N'-tetramethylurea hexafluorophosphate,N, NDiisopropylethylamine according to 1:0.2-1.5:0.2-1.5:1.5 to 3 molar ratioN, N-dimethylformamide to form a mixed solution, and the mixed solution is subjected to deprotection obtained in the step (2)Reacting the protected resin under inert conditions;

wherein the saidN, N-dimethylformamideN-the volume to mass ratio of the α -Fmoc protected amino acid is 20 mL/g;

wherein the volume-mass ratio of the mixed solution to the deprotected resin obtained in the step (2) is 4-6 mL/g.

9. The method of claim 4, wherein in step (4), trifluoroacetic acid, triisopropylsilane and water are used in an amount of 95:2.5:2.5 by volume ratio of the cutting agent to cut the bifunctional peptide from the resin; the volume-mass ratio of the cutting agent to the resin is 10-20 mL/g.

10. Use of a bifunctional peptide of any one of claims 1-3 for the manufacture of an analgesic.