CN102250251A - Polyethylene glycol derivative of enkephalin analogue - Google Patents

Abstract

The invention discloses polyethylene glycol derivatives of an enkephalin analogue, a preparation method, and a composition containing the same. The polyethylene glycol derivatives of an enkephalin analogue of the invention comprises an enkephalin analogue and polyethylene glycol covalently connected to a C end of the enkephalin analogue, wherein the enkephalin analogue has the following structure: X-Try-Y1-Gly-Phe-Y2-Y3-Z, and X is H or Ac; Y1 is D-Ala or Gly; Y2 is D-Ala or Met or Leu; Y3 is Cys or high Cys or Lys or Arg or His, and is a residue modified by PEG; Z is OH or NH2. The polyethylene glycol derivatives of the enkephalin analogue provided by the invention can be used to prepare analgesic drugs or precursor compounds of the drugs.

Description

A kind of pegylated derivative of enkephalin analog

technical field

The invention relates to a pegylated derivative of an enkephalin analog, belonging to the technical field of medicine.

Background technique

Enkephalin (Enkephalin, ENK) is an endogenous analgesic substance discovered in the mid-1970s, including leucine enkephalin (Tyr-Gly-Gly-Phe-Leu, LEK) and methionine Two types of enkephalins (Tyr-Gly-Gly-Phe-Met, MEK) mainly act on δ-type opioid receptors. The analgesic activity is 2-3 times that of morphine, and the addictive effect is significantly weaker than that of morphine. The enkephalin peptide chain is short, the structure is simple, and the chemical synthesis is easy to realize. However, due to failure to solve the two major obstacles of its short half-life in vivo and difficulty in passing through the blood-brain barrier, it has not yet been developed into a drug.

In the early 2000s, a research team at the University of Arizona had some encouraging results. They linked monosaccharides to enkephalins and found that glycosylated enkephalins formed by combining sugars such as glucose, maltose, or maltotriose with neuropeptides can pass through the blood-brain barrier. The ability of glycosylated enkephalins to bind not only to pain receptors in the brain but also to mu and delta receptors, whereas morphine only binds to mu receptors, could explain the enhanced analgesic effect of these compounds s reason. The results of animal experiments show that this type of compound does not cause the euphoria after morphine use, but its analgesic effect is 2 to 3 times that of morphine (J.Pharm.Exp.Ther.2001, 299:967-972 ). Research work at the University of Arizona suggests that the introduction of unrelated compounds may have positive implications for altering the properties of enkephalins and overcoming their weaknesses.

Bioconjugate chemistry has become one of the hot spots in biopharmaceutical research in recent years. Among the commonly used modifiers such as dextran, dextran, cyclodextrin, acetylimidazole, N-ethylmaleimide, and albumin, the research on polyethylene glycol (PEG) modification technology is the most successful. The widest range of applications. The polyethylene glycol modification of drugs, namely PEGylation, is to chemically couple activated polyethylene glycol to proteins, polypeptides, small molecule organic drugs and liposomes. Due to its non-toxic properties and good biocompatibility, polyethylene glycol has become one of the few synthetic polymers approved by the FDA that can be used as a drug for intracorporeal injection. There are already many protein drugs modified with polyethylene glycol Approved by the FDA for marketing. A series of studies have shown that the modification of protein and polypeptide drugs by polyethylene glycol can improve the water solubility and stability of such drugs, reduce immunogenicity, and prolong the drug metabolism time.

In 2005, the School of Pharmacy, University of Arizona conducted research on the enkephalin analog DPDPE ([D-Pen ² , D-Pen ⁵ ]-ENK) modified by mPEG ₂₀₀₀ (The AAPS Journal. Accepted: April 19, 2005). Pharmacodynamic and pharmacokinetic experiments showed that PEG-modified DPDPE significantly improved the analgesic effect, and 58.9% of ¹²⁵ I-labeled PEG-modified DPDPE in brain tissue was not destroyed by enzymatic hydrolysis within 30 minutes.

According to the PEG modification research of known protein drugs and the experimental conclusion analysis of the School of Pharmacy, University of Arizona, enkephalins can reduce the enzymatic degradation and prolong the half-life after PEGylation; Enkephalins modified with appropriate molecular weight PEG may pass through the blood-brain barrier.

Analysis of previous research data on PEG modification of protein drugs shows that for the same modified object, the PEG modification site, the number of covalently linked PEG, and the type of PEG may all have a greater impact on the activity of the modified product. Moreover, between the modified substance and PEG, the larger the molecular weight of PEG, the more the activity of the modified product will decrease, and the smaller the molecular weight of the modified substance, the greater the possibility that its active site will be shielded.

According to the above analysis, the selection of appropriate molecular weight PEG and its type is the primary factor to effectively prolong the half-life of the modified substance, increase the biological activity of the modified substance, and avoid more shielding of the active site of the modified substance. On the other hand, the PEG modification scheme using the amino group of the enkephalin analog DPDPE as the modification site in the School of Pharmacy, University of Arizona still has disadvantages such as difficult control of the reaction conditions, affecting the yield and purity of the modified product, and increasing the synthesis (production) cost. factor.

Contents of the invention

The object of the present invention is to provide a PEGylated derivative of an enkephalin analog, to provide a new drug molecule or precursor with simple structure, mild synthetic reaction conditions, simple and controllable for scientific research and clinical treatment Compounds also provide new methods and ideas for the transformation of peptide drugs and the study of PEGylation modification.

The technical solution of the present invention is to carry out the fixed-point modification of the C-terminus polyethylene glycol to the enkephalin analogs prepared by chemical synthesis and gene recombination methods to obtain a long half-life in vivo and improve the passage of enkephalin or analog molecules. PEGylated derivatives of enkephalin analogs with blood-brain barrier penetration into brain tissue and good analgesic activity.

The PEGylated derivatives of enkephalin analogs of the present invention are composed of enkephalin analogs and polyethylene glycol covalently linked to the C-terminus of enkephalin analogs, wherein the enkephalin analogs have the following formula structure:

X-Tyr-Y ₁ -Gly-Phe-Y ₂ -Y ₃ -Z

Wherein, X is H or Ac; Y ₁ is D-Ala or Gly; Y ₂ is D-Ala or Met or Leu; Y ₃ is Cys or homoCys or Lys or Arg or His, which is a PEG-modified residue; Z is OH or NH ₂ .

In the PEGylated derivative of the enkephalin analogue of the present invention, the C-terminal of the enkephalin analogue is connected to one end or two ends of polyethylene glycol.

The preparation method of the pegylated derivatives of enkephalin analogs of the present invention comprises the following steps:

The enkephalin analog for modification is prepared by chemical synthesis method or genetic engineering method; the C terminal of the enkephalin analog is covalently connected with polyethylene glycol.

The preparation method of the enkephalin analogs in the present invention can be prepared by any method known in the art, and the preparation method is preferably solid-phase and liquid-phase chemical synthesis. The solid-phase method includes using Fmoc or Boc-protected amino acids, using a peptide synthesizer or manual synthesis to synthesize the amino acid sequence, and then excised, separated and purified by high-performance liquid phase (HPLC), and freeze-dried. Diol-modified intermediate precursor.

Enkephalin analogs in the present invention can also be prepared by genetic engineering, and its steps include:

(1) Synthesize the gene fragment according to the amino acid sequence of the enkephalin analog.

(2) The gene fragments are connected, transformed, and screened to obtain positive strains.

(3) After the strain is fermented, the cells are collected, broken, and extracted to obtain inclusion bodies.

(4) The inclusion body is lysed, and the crude product is separated and purified by HPLC, and freeze-dried. The obtained peptide is the intermediate precursor for polyethylene glycol modification.

The PEGylated derivatives of the enkephalin analogs of the present invention, the polyethylene glycol is modified at the C-terminus of the enkephalin analogs, which is characterized in that the C-terminus of the enkephalin analogs can be connected to polyethylene glycol One or both ends of the alcohol. The molecular weight range of polyethylene glycol is 2000Da-80000Da, preferably 5000Da-40000Da.

The preparation method of the pegylated derivative of the said enkephalin analogue of the present invention comprises the following steps:

(1) Prepare or obtain polyethylene glycol containing Michael addition reaction acceptors and enkephalin analogs containing Michael addition reaction donors; or polyethylene glycol containing Michael addition reaction donors; Alcohols and enkephalin analogues containing Michael addition receptors.

(2) Carry out Michael addition reaction, react with its acceptor or donor polyethylene glycol and enkephalin analogs to form PEGylated derivatives of enkephalin analogs, such as containing maleimide Derivatized polyethylene glycol reacts with cysteine-containing enkephalin analogs to form the product.

If _Y3 is Cys or homoCys in the enkephalin analog structure as described in the present invention, polyethylene glycol is covalently bonded with a thioether bond or a disulfide bond; if _Y3 is Lys, polyethylene glycol It is covalently bonded by amide bond or secondary amine; if _Y3 is His, polyethylene glycol is covalently bonded by the imidazole ring of histidine; if _Y3 is Arg, polyethylene glycol is passed through Linked by heterocycle.

The compound of the present invention is a PEGylated derivative of an enkephalin analogue, and also includes a new intermediate produced for polyethylene glycol modification, and is a precursor compound for preparing analgesic drugs or drugs.

Pharmacodynamic experiments prove that the pegylated derivatives of enkephalin analogs provided by the present invention have an analgesic drug effect better than precursor enkephalins or enkephalin analogs and analgesic drugs without polyethylene glycol modification. The maintenance time of pain drug effect is longer than that of precursor enkephalin or enkephalin analog without polyethylene glycol modification. And, in the molecular weight range of 2000Da-40000Da as the molecular weight of polyethylene glycol as acceptor or donor of Michael addition reaction of the present invention, along with the increase of molecular weight of polyethylene glycol, the pegylation derivation of enkephalin analog The analgesic efficacy and maintenance time of the drug have different degrees of prolongation. Pharmacokinetic experiments prove that the distribution of the precursor enkephalin labeled with ¹²⁵ I of the present invention is quite different from that of the PEGylated derivatives in vivo, wherein the enkephalin modified with polyethylene glycol is more stable in the blood and brain tissue. The radioactive clearance time in PEG-modified enkephalins was significantly prolonged, the half-life of PEG-modified enkephalins in blood and tissues was prolonged, and the ability of precursor enkephalin molecules to pass through the blood-brain barrier was enhanced.

The present invention has obtained following technical progress:

Compared with non-polyethylene glycol-modified enkephalin and its analog precursors, polyethylene glycol modification with appropriate molecular weight improves the in vivo half-life of enkephalin and its analog precursors, and enters brain tissue through the blood-brain barrier ability and analgesic activity.

The study of different PEG modification methods found that compared with, for example, the amino modification of the enkephalin analog DPDPE carried out by the School of Pharmacy, University of Arizona, and other undefined site polyethylene glycol modification techniques (including amino, carboxyl and sulfhydryl modifications), this The polyethylene glycol derivative of the enkephalin analog modified at the C-terminal sulfhydryl group provided by the invention is easy to control the reaction conditions, and the yield and purity of the synthesized modified product are high. In the preferred embodiment provided by the present invention, a Cys with a sulfhydryl group is added to the C-terminus of the modified enkephalin analog DADAE, and after being synthesized with Rink resin, the sulfhydryl group is modified with activated mPEG-MAL, and the N-terminal amino group is modified. In comparison, the reaction conditions are easy to control, and the yield and purity of the synthesized modified product are high. Moreover, the site-specific modification at the C-terminus provided by the present invention can overcome the disadvantage that polyethylene glycol modification at an indeterminate site may shield the active site of precursor compounds, especially peptide compounds with smaller molecular weights.

Detailed ways

Unless otherwise specified, the technologies and methods used in the present invention but not explicitly or briefly described refer to the technologies and methods commonly used in the technical field, and can generally be performed according to the technologies and methods known in the art.

Unless otherwise defined, terms used in the present invention are commonly known terms in the relevant technical field. Standard chemical symbols and abbreviated symbols can be used interchangeably with their full names.

Unless otherwise specified, the amino acids used in the present invention are L-amino acids.

Abbreviations in the present invention have the following meanings:

ENK: Enkephalin

LEK: Leucine Enkephalin, amino acid sequence: Tyr-Gly-Gly-Phe-Leu

MEK: methionine (methionine) enkephalin, amino acid sequence: Tyr-Gly-Gly-Phe-Met

DADAE: [D-Ala ² , D-Ala ⁵ ]-ENK, an enkephalin analog, amino acid sequence: Tyr-D-Ala-Gly-Phe-D-Ala

DPDPE: [D-Pen ² , D-Pen ⁵ ]-ENK, an enkephalin analog, amino acid sequence: Tyr-D-Pen-Gly-Phe-D-Pen

Fmoc: fluorenylmethoxycarbonyl

Boc: tert-butoxycarbonyl

PEG: polyethylene glycol

mPEG: Monomethoxypolyethylene glycol

PEG-MAL: Imide-based polyethylene glycol

Tyr: Tyrosine

Gly: Glycine

Phe: Phenylalanine

Ala: alanine

Pen: Dimethylcysteine

Met: Methionine (Methionine)

Leu: Leucine

Cys: cysteine

Lys: Lysine

Arg: Arginine

His: Histidine

DCC: Dicyclohexylcarbodiimide

HOBT: 1-Hydroxybenzotriazole

HBTU: 2-(1H-1-hydroxybenzotriazole)-1,1,3,3-tetramethyluronium hexafluorophosphate

NMM: N-methylmorpholine

TFA: Trifluoroacetic acid

TMBS: Trimethylbromosilane

Ts-Cl: p-toluenesulfonyl chloride

EDT: 1,2-ethanedithiol

Et ₃ N: Triethylamine

HPLC: High Performance Liquid Chromatography

RP-HPLC: Reverse Phase High Performance Liquid Chromatography

MS: mass spectrometry

MALDI-TOF-MS: Matrix-Assisted Laser Ionization Time-of-Flight Mass Spectrometry

The following examples represent illustrative embodiments of the invention, but the invention is not limited by these examples.

In a preferred embodiment of the present invention, the PEGylation reagent is a Fluka product, Wang resin, Rink resin, DCC, HOBT, HBTU, TFA, NMM, Fmoc-protected amino acid is a Shanghai Gil biochemical product, Ts-Cl, EDT, Et ₃ N is a routinely purchased analytical reagent.

Example 1 Preparation of precursors for polyethylene glycol modification-C-terminal cysteine MEK (MEK-Cys-NH ₂ ) and C-terminal cysteine DADAE (DADAE-Cys-NH ₂ ) by solid-phase chemical synthesis :

The amino acids adopted include Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Met-OH, Fmoc-D-Ala-OH, Fmoc-Cys(Trt)-OH; wherein tBu is tert-butyl, and Trt is trityl. Synthesis of polypeptide sequences was performed manually. The solid-phase polypeptide synthesis glass reactor is custom-made, and the upper and lower chambers are separated by G2 filter plates, which are treated with silane reagents before use.

Steps:

1. Synthesis of MEK-Cys-NH ₂ and DADAE-Cys-NH ₂ :

(1) Rink Amide-AM resin, 0.160g (0.1mmol), swell in DMF for 10min, 20% piperidine/DMF1×10ml, remove Fmoc protection for 30min;

(2) CH ₂ Cl ₂ 2×10ml, wash for 1min, including the bottle cap;

(3) DMF1×10ml, wash for 1min;

(4) CH ₃ OH 1×10ml, wash for 1min;

(5) DMF1×10ml, wash for 1min;

(6) CH ₃ OH1×10ml, wash for 1min;

(7) DMF1×10ml, wash for 1min;

(8) Diethyl ether 2×10ml, wash for 1min, take a small amount of resin, and test;

(9) Weigh Fmoc-Cys(Trt)-OH, 234.3mg, HOBT 54mg, dissolve in DMF, DCC 82.2mg, dissolve in DMF/CH ₂ Cl ₂ , stir and react overnight at room temperature;

(10) CH ₂ Cl ₂ 2×10ml, wash for 1min, including the bottle cap;

(11) DMF1×10ml, wash for 1min;

(12) CH ₃ OH 1×10ml, wash for 1min;

(13) DMF1×10ml, wash for 1min;

(14) CH ₃ OH 1×10ml, wash for 1min;

(15) DMF1×10ml, wash for 1min;

(16) Diethyl ether 2×10ml, wash for 1min, take a small amount of resin, and test;

(17) 20% piperidine/DMF1×10ml, remove Fmoc protection for 30min, take a small amount of resin for detection;

(18) CH ₂ Cl ₂ 2×10ml, wash for 1min, including the bottle cap;

(19) DMF1×10ml, wash for 1min;

(20) CH ₃ OH 1×10ml, wash for 1min;

(21) DMF1×10ml, wash for 1min;

(22) CH ₃ OH 1×10ml, wash for 1min;

(23) DMF1×10ml, wash for 1min;

(24) Diethyl ether 2×10ml, wash for 1min, take a small amount of resin, and test;

Fmoc-amino acids are connected sequentially. Refer to the Kziser method to check whether the reaction is complete with ninhydrin reagent. React ninhydrin reagent with amino acid or peptide on the resin (take about 10 mg of peptide-resin) at 100°C for 5 minutes, and 5 μmol of amino acid residues per gram of resin can give a slight blue color. Two short peptide sequences of Tyr-Gly-Gly-Phe-Met-Cys-NH ₂ and Tyr-D-Ala-Gly-Phe-D-Ala-Cys-NH ₂ were obtained.

2. Cutting of Rink Amide-AM resin:

Put the peptide resin from which Fmoc has been removed in a 50ml eggplant-shaped bottle, add trifluoroacetic acid (7.5ml), m-cresol (0.1ml), and 1,2-ethanedithiol at 0°C, and stir for 90min. Filter through a _G3 funnel, take the filtrate, and rotary evaporate until no liquid evaporates. Anhydrous diethyl ether was added to the bottle, and a white precipitate was seen, and the rotary evaporation was repeated three times. Diethyl ether was then added to precipitate a white substance, filtered through a _G4 funnel, the filtrate was discarded, the white substance was dissolved in water, and then freeze-dried to obtain a crude peptide.

3. Desalination:

The above synthetic product was dissolved in 2ml of water, desalted with Sephadex G-25, and water was used as the eluent at a flow rate of 1ml/min, detected by an ultraviolet detector (wavelength 214nm), separated and freeze-dried to obtain the crude synthetic product.

4. Purification:

Adopt _C18 chromatographic column, mobile phase is A liquid (0.05% TFA aqueous solution) and B liquid (contain 0.05% TFA and 70% acetonitrile aqueous solution), adopt linear gradient, B liquid is from 0 to 100%, mobile phase flow rate is 2ml /min, the detection wavelength is 214nm.

5. Analysis and identification of synthetic products

(1) HPLC analysis and identification

Kr100-5 C ₁₈ analytical chromatographic column is used. Detection conditions: the mobile phase is liquid A (0.05% TFA aqueous solution) and liquid B (aqueous solution containing 0.05% TFA and 70% acetonitrile), using a linear gradient, liquid B is from 0 to 100% (30min), and the flow rate of the mobile phase is 1ml /min; detection wavelength is 214nm.

Test results: the purified synthetic and modified products MEK-Cys-NH ₂ and DADAE-Cys-NH ₂ had a purity of 95.27% and 93.89%, respectively, and a yield of 70% and 74%, respectively.

(2) MS analysis and identification

Matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used, commissioned by the Department of Chemistry of Capital Normal University and ChinaTech Peptide Co., Ltd.

Test results: MEK-Cys-NH ₂ molecular weight 677, DADAE-Cys-NH ₂ molecular weight 631, consistent with expectations.

Embodiment 2 Enkephalin analogs react with polyethylene glycol to synthesize PEGylated derivatives of enkephalin analogs:

Steps:

1. Generation of mPEG ₅₀₀₀ -MAL

(1) Weigh 40.0g (8mmol) of mPEG ₅₀₀₀ -OH, dissolve it in 100ml CH ₂ Cl ₂ , stir to dissolve, add 15ml triethylamine and 19g p-toluenesulfonyl chloride, stir and react at room temperature for about 6 hours, to After the reaction was detected by TLC, the solvent was removed by rotary evaporation, and 100 ml of anhydrous ether was added to precipitate a solid to obtain mPEG ₅₀₀₀ -OTs.

(2) Purifying the obtained mPEG ₅₀₀₀ -OTs. Dissolve the crude mPEG ₅₀₀₀ -OTs in water, first use a separating funnel to extract p-toluenesulfonyl chloride with ether, discard the ether layer, then use CH ₂ Cl ₂ to extract mPEG ₅₀₀₀ -OTs in water, discard the water layer, no Dry over magnesium sulfate, remove CH ₂ Cl ₂ by rotary evaporation, add diethyl ether to precipitate mPEG ₅₀₀₀ -OTs, filter diethyl ether, and vacuum-dry phosphorus pentoxide. When the product is impure, it can be purified several times.

(3) Dissolve 18.0 g of mPEG ₅₀₀₀ -OTs in 50 ml of DMF, add 5 g (27 mol) of potassium phthalimide, and react at 120° C. for 4 h. The solvent was distilled off under reduced pressure, the residue was dissolved in 50ml of absolute ethanol, then 13.5ml of hydrazine hydrate was added, and the mixture was refluxed for 4 hours. _The solvent was removed by rotary evaporation, the residue was dissolved in _CH2Cl2 , and the solid was precipitated with anhydrous ether. Then recrystallized with absolute ethanol ether. Yield 14.2 gm PEG ₅₀₀₀ -NH ₂ .

(4) Dissolve 5.0g of mPEG ₅₀₀₀ -NH ₂ in 10ml of dioxane, add 2.0g of maleic anhydride, and stir at 80°C for 30min. Evaporate the solvent under reduced pressure, add 50ml of anhydrous diethyl ether, cool and precipitate a solid, filter off the diethyl ether, and dry the resulting solid to dissolve in 15ml of acetic anhydride, add 5.0g of sodium acetate, and stir at 100°C for 45min. Evaporate the solvent under reduced pressure, dissolve the residue with _CH2Cl2 , filter off the insoluble matter, add _an appropriate amount of activated carbon to the filtrate, _let it stand for 30 minutes, filter off the activated carbon, remove _CH2Cl2 by rotary evaporation, add anhydrous ether, and precipitate out MPEG ₅₀₀₀ -MAL was filtered to remove ether, and phosphorus pentoxide was dried in vacuum. When the product is impure, it can be purified several times. Finally, purified mPEG ₅₀₀₀ -MAL was obtained.

2. PEG modification of MEK-Cys-NH ₂ and DADAE-Cys-NH ₂

Purify the synthesized MEK-Cys-NH ₂ and DADAE-Cys-NH ₂ by RP-HPLC, dissolve them in water, adjust the pH to 7-8 with sodium bicarbonate, add 2-3 equivalents of mPEG ₅₀₀₀ -MAL, respectively, React at room temperature, use RP-HPLC to monitor the reaction progress and separate the products to obtain crude MEK-Cys(mPEG ₅₀₀₀ -MAL)-NH ₂ and DADAE-Cys(mPEG ₅₀₀₀ -MAL)-NH ₂ .

3. Desalting and purification

Method is the same as embodiment one.

4. Analysis and identification of modified products

(1) HPLC analysis and identification

Detection conditions are the same as in Example 1.

Test results:

The purified modified products MEK-Cys(mPEG ₅₀₀₀ -MAL)-NH ₂ and DADAE-Cys(mPEG _{5 000} -MAL)-NH ₂ had a purity of 91.73% and 90.59% respectively, and yields of 62% and 58% respectively. %.

(2) MS analysis and identification

Detection conditions are the same as in Example 1.

Detection results: MEK-Cys(mPEG ₅₀₀₀ -MAL)-NH ₂ has a group of peaks with a difference of 44 around 5708 and DADAE-Cys(mPEG ₅₀₀₀ -MAL)-NH ₂ around 5662, which is in line with expectations.

Example 3 Drug efficacy experiment - detection of analgesic pharmacological activity of synthetic peptide:

In this example, the analgesic activity of four enkephalin analogues and polyethylene glycol-modified substances was determined, including: MEK-Cys-NH ₂ , DADAE-Cys-NH ₂ , MEK-Cys(mPEG ₅₀₀₀ -MAL) -NH ₂ , DADAE-Cys(mPEG ₅₀₀₀ -MAL)-NH ₂ (prepared in Examples 1 and 2 of the present invention). When used, they were dissolved in normal saline according to the equimolar concentration of the control drug morphine, sterilized by filtration through a 0.45 μm filter membrane, and injected into the tail vein or lateral ventricle.

The control drug in this embodiment is morphine hydrochloride (Dexamethasone, Mor; produced by Northeast Sixth Pharmaceutical Factory, batch number: 001009, specification 10 mg/ml). When in use, the clinically equivalent dose (10 mg/kg) converted from animal to human body weight is diluted with sterile normal saline, and injected into the tail vein or lateral ventricle.

The experimental animals used in this example are Kunming mice (clean grade, weighing 18-22 g, provided by the breeding farm of the Institute of Experimental Animals, Chinese Academy of Medical Sciences, certificate number SCXK (Beijing) 2004-0001).

In this embodiment, three kinds of test methods are used to measure the analgesic pharmacological activity of the test substance.

Experiment 1-Effects of lateral ventricle administration of synthetic products on pain response induced by heat stimulation in mice:

For female KM mice, the skin with a diameter of 0.5-0.8 cm at the injection site (1 mm behind the bregma point and 2 mm away from the midline) was cut off under local disinfection conditions on the day before the experiment, and penicillin was injected intramuscularly once prophylactically. On the day of the experiment, the mice were placed on a hot plate (RB-200 intelligent hot plate instrument produced by Chengdu Taimeng Technology Co., Ltd.) at 55 ± 0.5 °C under room temperature conditions of 20 ± 1 ° C. The time elapsed is the pain threshold; the mice whose pain threshold is less than 3s and greater than 30s are excluded; the qualified mice are stratified and randomly divided into 6 groups, each group has more than 7-8 mice; the pain of each mouse is measured at intervals of 5 minutes before administration. Threshold 2 times, take the average value as the pre-drug (basic) pain threshold. The MEK-Cys-NH ₂ , DADAE-Cys-NH ₂ , MEK-Cys(mPEG ₅₀₀₀ -MAL)-NH ₂ and DADAE-Cys(mPEG ₅₀₀₀ -MAL)-NH ₂ groups were injected with 3.1 μmol/kg into the test subject respectively Drugs, blank and positive drug control mice were injected with normal saline or morphine 1.0 mg/kg into the lateral ventricle, with a volume of 4 μl of the test substance injected and a needle depth of 2 mm. Observe and record the pain threshold of each mouse 15-120min after administration respectively. The detection data is represented by mean ± standard deviation, and the difference of the pain threshold before and after the administration of each mouse is calculated, and the SPSS software is used to carry out the t test, and the significance of the difference of the pain threshold before and after the administration of the mice of each group and the blank control group is compared, P<0.05 was regarded as statistically significant. The experimental results are shown in Table 1.

Table 1. Effects of intracerebroventricular administration of enkephalin analogues and PEG derivatives on pain response in mice induced by heat stimulation

Note: 1. Dose: 3.1 μmol/kg, NS: 0.4 μl/head; 2. Compared with NS group ^** P≤0.01; ^* P≤0.05.

It can be seen from Table 1 that the positive control drug morphine showed obvious analgesic activity, the peak value was 15 minutes after the drug, and continued to 120 minutes; ENK-Cys-NH showed analgesic activity ₁₅ minutes after the drug, and the analgesic activity decreased to disappear after 30 minutes; The analgesic activity of DADAE-Cys-NH ₂ and each PEG modification lasted until 120 minutes after drug administration, most peaked at 15-60 minutes, and most of them were significantly different from the control group; the overall trend, DADAE-Cys-NH ₂ had better effect For ENK-Cys-NH ₂ , the effect of PEG-modified products was better than that of unmodified ones, and the analgesic activity of mPEG-modified DADAE-Cys-NH ₂ was stronger than that of morphine at most time points.

Experiment 2-Effects of intravenous administration of synthetic products on pain response induced by heat stimulation in mice:

Female KM mice were treated and grouped as above (Experiment 1). Each test substance was administered by tail vein injection at 31 μmol/kg, volume 0.2ml/10g body weight. Observe and record the pain threshold of each mouse 15-120min after administration respectively. The same method as above (Experiment 1) was used for statistical processing. The experimental results are shown in Table 2.

Table 2. Effects of intravenous administration of enkephalin analogues and PEG derivatives on pain response induced by heat stimulation in mice

Note: 1. Dose: 31μmol/kg, NS is 0.2ml/10g body weight; 2. Compared with NS group ^** P≤0.01; ^* P≤0.05.

It can be seen from Table 2 that the analgesic activity of the positive control drug morphine was maintained until 60 minutes after _{the drug, and the effect was obvious within 30 minutes; the obvious analgesic activity of NEK-Cys-NH 2 was} 15 minutes after the drug; The obvious analgesic activity of the compounds was observed until 30 minutes after drug administration; the effect of each test compound was weaker than that of morphine; the general trend was that the effect of DADAE-Cys-NH ₂ was better than that of ENK-Cys-NH ₂ , and the effect of PEG-modified products was better than that of unmodified products.

Experiment 3-Effects of intravenous administration of synthetic products on the pain response induced by acetic acid stimulation in mice:

KM mice, more than 8 in each group, both male and female. Grouping and administration methods are the same as experiments 1 and 2. 30 minutes after the administration, each mouse was intraperitoneally injected with 0.1mol/L glacial acetic acid (AA) 0.2ml to induce pain, and the mice showed repeated contraction of abdominal muscles, arched back, twisting of the buttocks, and stretching of the hind limbs as positive reactions of writhing, and counted Number of twists within 15 minutes. The detection data are expressed as mean ± standard deviation, and the SPSS software is used to carry out t test to compare the significance of the difference in the number of writhing times between the mice in each group and the blank control group (P<0.05 is considered statistically significant), and calculate the The percentage of pain inhibition in the drug group.

Pain suppression rate (%) = (number of times of writhing in the control group - number of times of writhing in the experimental group)/number of times of writhing in the control group × 100%

The experimental results are shown in Table 3.

Table 3. Effects of intravenous administration of enkephalin analogues and PEG derivatives on pain responses induced by chemical stimulation in mice

Note: 1. Dose: 31μmol/kg, NS is 0.2ml/10g body weight; 2. Compared with NS group ^** P≤0.01.

The results in Table 3, each test substance shows obvious analgesic activity; wherein, the pain suppression rate of morphine reaches 99.59%, and the other test substances are weaker than morphine; overall trend, mPEG ₅₀₀₀ modified product is better than unmodified product, DADAE- The effect of Cys-NH ₂ was not significantly better than that of MEK-Cys-NH ₂ .

The experimental conclusions of the above 3 kinds of test methods of the present embodiment are:

1. The substitution synthesis of D-Ala to the 2 and 5 amino acids of ENK improves the enzymatic stability of ENK and enhances the analgesic activity of the synthesized product; the experimental results are in line with expectations.

2. PEG modification of the biologically active site of the unshielded prototype compound.

3. The PEG-modified enkephalin analogs may improve the ability of the prototype compound molecules to pass through the blood-brain barrier; the experimental results are in line with expectations, but further conclusions need to be confirmed by pharmacokinetic (in vivo drug distribution) experiments.

4. Under the conditions of this experiment, PEG modification with a larger molecular weight is beneficial to improve the enzymatic stability of the modified substance, prolong its half-life in vivo, and enhance its pharmacological activity, but further conclusions need to be confirmed by pharmacokinetic experiments.

Embodiment 4 pharmacokinetic experiment:

In this example, the in vivo pharmacokinetics analysis and comparison of two synthetic products were carried out, and the specific synthetic products were MEK and mPEG ₂₀₀₀ -MEK. The purpose of the experiment is to preliminarily analyze the pharmacokinetic characteristics of PEG-modified enkephalins and modified precursor enkephalins and the differences between them.

The solid phase carrier used in the synthesis of MEK tested in this example was Wang resin, and Fmoc-amino acids were sequentially connected with reference to the method in Example 1 to prepare Tyr-Gly-Gly-Phe-Met. Prepare mPEG ₂₀₀₀ -NHCOCH ₂ CH ₂ COOH with mPEG ₂₀₀₀ -OH, modify the N-terminal of MEK with PEG, and purify to obtain mPEG ₂₀₀₀ -NHCOCH ₂ CH ₂ CO-MEK (abbreviated as mPEG ₂₀₀₀ -MEK or PEG-MEK). The MEK used in this example has a molecular weight of 573 by MS detection, and mPEG ₂₀₀₀ -NHCOCH ₂ CH ₂ CO-MEK has a group of peaks with a difference of 44 around the molecular weight of 2604 by MS detection, which is in line with expectations; the purity of the synthetic products for labeling is 90% above.

In this example, the Institute of Isotopes, China Institute of Atomic Energy was entrusted to use the chloramine T method to label MEK and mPEG ₂₀₀₀ -ENK with ¹²⁵ I. After separation and purification by PR-HPLC, the radioactive specific activities of the markers were MEK 40μCi/μg and mPEG ₂₀₀₀ -ENK 65μCi/μg. The radioactive isotope determination uses the MF-1000 gamma counter produced by Xi'an Kaipu Company.

Take Kunming mice, 4-6 in each group, and randomly divide them into 10 groups. Each mouse is injected with ¹²⁵ I-labeled MEK and mPEG ₂₀₀₀ -ENK 0.2ml/10g body weight through the tail vein, and the marker solution is MEK 150μCi/ml respectively. and mPEG ₂₀₀₀ -ENK 200 μCi/ml. At 10, 30, 60, 120, and 240 minutes after the injection, blood was collected from the eyeballs, and then the mice were sacrificed by dislocation of the cervical vertebrae. The organs (tissues) such as the heart, liver, lung, brain, kidney, and muscle were taken, weighed, and the cpm value was determined with a gamma counter. , calculate the radioactivity uptake (%ID/g) in different phases, and calculate the main pharmacokinetic parameters. The experimental results are shown in Tables 4 and 5.

Table 4. Distribution of ¹²⁵ I-labeled MEK and mPEG ₂₀₀₀ -ENK in normal mice

Note: The data in brackets in the table are examples.

Table 5. In vivo metabolic kinetic parameters of ¹²⁵ I-labeled MEK and mPEG ₂₀₀₀ -ENK in mice

The experimental results showed that there was a big difference in the distribution of ¹²⁵ I-labeled MEK and mPEG ₂₀₀₀ -ENK in vivo. Among them, mPEG ₂₀₀₀ -ENK has longer blood and brain radioactive clearance time than MEK.

The experimental conclusion of this embodiment is:

The in vivo half-life and relative bioavailability of PEG-modified enkephalins are improved, especially the PEG-modified products in the brain tissue are longer than the precursor enkephalins without PEG-modification, which shows that the PEG-modified enkephalins under the experimental conditions To a certain extent, the shortcoming that the precursor (prototype) enkephalin without PEG modification is difficult to pass through the blood-brain barrier is improved.

Claims (5)

1. A pegylated derivative of an enkephalin analogue, characterized in that: it is composed of an enkephalin analogue and polyethylene glycol covalently linked with an enkephalin analogue, wherein the enkephalin analogue is similar The substance has the following structure: X-Tyr-Y ₁ -Gly-Phe-Y ₂ -Y ₃ -Z Wherein, X is H or Ac; Y ₁ is D-Ala or Gly; Y ₂ is D-Ala or Met or Leu; Y ₃ is Cys or homoCys or Lys or Arg or His, which is a PEG-modified residue; Z is OH or NH ₂ . 2. The pegylated derivatives of enkephalin analogs according to claim 1, characterized in that polyethylene glycol is modified at the C-terminus of enkephalin analogs. 3. The pegylated derivatives of enkephalin analogues according to claim 1 or 2, characterized in that the molecular weight range of polyethylene glycol is greater than 2000Da to less than or equal to 80000Da. 4. according to claim 1 or 2 or 3 described pegylated derivatives of enkephalin analogues, it is characterized in that the C terminal of enkephalin analogues is connected at one end of polyethylene glycol or two thereof end. 5. The application of the compound according to claim 1 or 2 or 3 or 4 or the composition containing the compound, characterized in that it is used to prepare an analgesic drug or a precursor compound of the drug.