阅读说明：本技术 一种双重敏感型聚合物-药物连接物及其制备方法和应用 (Dual-sensitive polymer-drug conjugate and preparation method and application thereof ) 是由 陈立江 王惊雷 宋柯 宋立强 石金燕 褚宇琦 于 2019-10-17 设计创作，主要内容包括：本发明公开一种双重敏感型聚合物-药物连接物及其制备方法和应用,属于高分子化学领域及药物制剂领域。将酶敏感底物制成酶敏感底物中间体；再将药物与胱胺二盐酸盐制备成含二硫键的药物衍生物；然后将酶敏感底物中间体与药物衍生物连接制备成双重敏感的药物衍生物；最后将聚乙二醇单甲醚与药物衍生物缩合为谷胱甘肽还原敏感及组织蛋白酶B敏感的双重敏感型聚合物-药物连接物。其在水中能自组装成两亲性聚合物胶束,连接键为二硫键和二肽,可在肿瘤部位响应性断裂,释放出药物。本发明还公开了mPEG-VC-SS-GA共聚物的制备方法及其作为抗癌药物载体的用途。(The invention discloses a double-sensitive polymer-drug conjugate and a preparation method and application thereof, belonging to the fields of high-molecular chemistry and pharmaceutical preparations. Preparing an enzyme sensitive substrate into an enzyme sensitive substrate intermediate; then preparing the drug and cystamine dihydrochloride into a drug derivative containing disulfide bonds; then connecting the enzyme sensitive substrate intermediate with the drug derivative to prepare the dual sensitive drug derivative; finally, polyethylene glycol monomethyl ether and the drug derivative are condensed into the dual sensitive polymer-drug conjugate which is sensitive to glutathione reduction and sensitive to cathepsin B. The amphiphilic polymer micelle can be self-assembled into an amphiphilic polymer micelle in water, and a connecting bond is a disulfide bond and a dipeptide, so that the amphiphilic polymer micelle can be broken in response at a tumor part to release a medicament. The invention also discloses a preparation method of the mPEG-VC-SS-GA copolymer and application of the mPEG-VC-SS-GA copolymer as an anticancer drug carrier.)

1. A dual sensitive polymer-drug conjugate is characterized in that the dual sensitive polymer-drug conjugate is a dual sensitive polymer-drug conjugate mPEG-Y-SS-R sensitive to glutathione reduction and sensitive to cathepsin B,

wherein Y is valine-citrulline, forms a double sensitive polymer-drug linker mPEG-VC-SS-R, and has a structural formula shown as (I):

or Y is phenylalanine-arginine, forms a double sensitive polymer-drug connector mPEG-PA-SS-R, and has a structural formula shown as (II):

wherein R is a pharmaceutical compound with carboxyl.

2. The dual sensitive polymer-drug conjugate of claim 1, wherein the drug compound having a carboxyl group is selected from gambogic acid, rhein, valsartan, methotrexate, exenatide acetate, IDN-6556, AGI-1067, azaserine, chlorophenylalanine, N-acetyl-L-phenylalanine, and N-acetyl-L-valine.

3. The dual sensitive polymer-drug conjugate of claim 2, wherein Y is valine-citrulline and the drug compound having a carboxyl group is gambogic acid, and the dual sensitive polymer-drug conjugate mPEG-VC-SS-GA that is sensitive to glutathione reduction and sensitive to cathepsin B has the structural formula shown in (iii):

4. the dual sensitive polymer-drug conjugate of claim 1,2 or 3, wherein mPEG specification is polyethylene glycol monomethyl ether mPEG 5000.

5. The method for preparing a dual sensitive polymer-drug conjugate of claim 1,2 or 3, comprising the steps of: 1) connecting an enzyme sensitive substrate with Fmoc to prepare an enzyme sensitive substrate intermediate; 2) preparing a drug compound R with carboxyl and cystamine dihydrochloride into a drug derivative R-cystamine containing disulfide bonds; 3) connecting the enzyme sensitive substrate intermediate with a disulfide bond-containing drug derivative R-cystamine to prepare a double sensitive drug derivative; 4) polyethylene glycol monomethyl ether and a double-sensitive drug derivative are condensed into a double-sensitive polymer-drug connector mPEG-Y-SS-R sensitive to glutathione reduction and sensitive to cathepsin B.

6. The method of claim 5, wherein Y is valine-citrulline and constitutes dual susceptible polymer-drug conjugate mPEG-VC-SS-R, comprising the steps of:

1) synthesis of enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit):

1.1) dissolving Fmoc-Val, HOSu and DCC in tetrahydrofuran at 0 ℃, stirring for 24h, filtering, decompressing and rotary evaporating to obtain a white solid product Fmoc-Val-OSu;

1.2) combining Cit with NaHCO₃Dissolving in distilled water, cooling to 0 ℃, and dropwise adding DME solution of Fmoc-Val-OSu to Cit and NaHCO₃Adding tetrahydrofuran for assisting dissolution, and stirring at room temperature for 24h to obtain a reaction solution;

1.3) dropwise adding saturated potassium carbonate into the reaction liquid obtained in the step 1.2) to adjust the pH value to 8-9, extracting with ethyl acetate, collecting a water layer, adding a citric acid solution to adjust the pH value to 3-4, separating out a white gelatinous solid, filtering, dissolving the obtained white gelatinous solid in a mixed solution of tetrahydrofuran and methanol, performing rotary evaporation and concentration, adding methyl tert-butyl ether, stirring overnight at 0 ℃, filtering, and performing vacuum drying to obtain a white solid product which is an enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit);

2) disulfide bond-containing drug derivative R-cystamine (R-SS-NH)₂) The synthesis of (2):

2.1) dissolving a pharmaceutical compound R with carboxyl in a dichloromethane solution, cooling to 0 ℃, adding EDCI and HOBT, activating the obtained mixture at 0 ℃ for 1h, sequentially adding a methanol solution of cystamine dihydrochloride and triethylamine, and stirring at normal temperature for 48h to obtain a reaction solution;

2.2) reacting the reaction solution obtained in the step 2.1) with NaHCO₃Washing the solution, collecting the organic layer, drying with anhydrous magnesium sulfate, filtering, separating by column chromatography, and vacuum drying to obtain disulfide bond-containing drug derivative R-cystamine (R-SS-NH)₂)；

3) Synthesis of the double sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R):

3.1) dissolving Fmoc-Val-Cit obtained in the step 1) in a mixed solution of dichloromethane and methanol, cooling to 0 ℃, adding EDCI and HOBT, activating the obtained mixture at 0 ℃ for 1h, and adding R-SS-NH obtained in the step 2)₂Stirring the obtained mixture overnight, concentrating under reduced pressure after the reaction is finished, adding ice water, storing at 4 deg.C overnight, filtering, washing with water for three times, vacuum drying, and performing column chromatographyPurifying to obtain the double sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R);

4) synthesis of the double sensitive Polymer-drug linker mPEG-VC-SS-R:

4.1) dissolving succinic anhydride and DMAP in a pyridine solution, then dropwise adding the pyridine solution into a chloroform solution of mPEG, stirring the obtained mixture at 60 ℃ under the protection of nitrogen for 24 hours, washing the obtained mixture with physiological saline, drying the obtained product with anhydrous magnesium sulfate, filtering the obtained product to obtain an organic layer, concentrating the organic layer, washing the organic layer with ether, and drying the organic layer in vacuum to obtain a white solid product mPEG-COOH;

4.2) dissolving mPEG-COOH in dichloromethane, adding EDCI, HOBT andactivating a type molecular sieve in the dark at 0 ℃ to obtain mPEG-COOH solution;

4.3) dissolving Fmoc-VC-SS-R obtained in the step 3) in THF, adding DBU, stirring for 10 minutes, adding the obtained mixed solution into mPEG-COOH solution, and reacting for 2 days at room temperature under the protection of nitrogen;

4.4) after the reaction is finished, washing with distilled water, extracting with chloroform, combining organic layers, evaporating under reduced pressure, adding ether for washing, filtering, drying the obtained solid in vacuum, adding ultrapure water, stirring, filtering, dialyzing for 2 days, replacing release media for 5 times in 2h, 6h, 12h, 24h and 36h respectively, and freeze-drying to obtain the target product, namely the dual-sensitive polymer-drug conjugate mPEG-VC-SS-R.

7. The method according to claim 6, wherein in step 4.4), the dialysis bag has a molecular weight of 5kDa, and the dialysis medium is distilled water.

8. The preparation method according to claim 6 or 7, wherein the pharmaceutical compound R with carboxyl is gambogic acid, and the mPEG has the specification of mPEG 5000.

9. Use of the dual sensitive polymer-drug conjugate of claim 1,2 or 3 for the preparation of an anti-tumor drug.

Technical Field

The invention relates to the field of pharmaceutical preparations and the field of polymer chemistry, in particular to a dual-sensitive polymer-drug conjugate with glutathione reduction sensitivity and cathepsin B sensitivity, and a preparation method and application thereof.

Background content

In the 70's of the 20 th century, researchers have proposed the idea of covalently bonding water-soluble polymers to chemotherapeutic drugs. With the development of synthesis and polymers, which are becoming a rapidly developing field, such conjugates began to enter the clinic in the 90 s of the 20 th century, as poly (L-glutamic acid) -paclitaxel copolymer. Most of the antitumor drugs are insoluble drugs such as paclitaxel, camptothecin, sorafenib and the like, the poor water solubility limits the bioavailability of the antitumor drugs, and the nano drug delivery system can improve the water solubility and increase the bioavailability. Other conjugates are also under development, such as peg-camptothecin in peg carriers. Polyethylene glycol is an FDA approved hydrophilic polymer with low toxicity and immunogenicity, but the fact is that at the tumor site, the polyethylene glycol linker may be difficult to break to release the drug, resulting in a significant decrease in anticancer effect. The tumor microenvironment sensitive drug delivery system which is rapidly developing at present can release drugs in response, and provides a new strategy for overcoming the obstacles of low solubility and site-specific delivery of chemotherapeutic drugs.

Gambogic Acid (GA, C)₃₈H₄₄O₈) Is an extract in the Chinese medicinal gamboge, is one of main active compounds with anti-tumor effect, and has been applied for thousands of years. The research shows that GA has anticancer effect in many cancer types, such as prostate cancer and liver cancerBreast cancer, and the like, and the toxicity thereof is considered to be acceptable by research, and the toxicity is a hot spot of natural product anti-tumor research in recent years. But the current clinical research on the anti-tumor is limited due to poor water solubility, insignificant drug effect and low selectivity.

Glutathione (GSH) is a naturally occurring tripeptide in humans, and the concentration of glutathione in tumor tissues and lysosomes is much higher (about 2-10mM) than that in extracellular fluids (about 2-20 uM). Since tumor cells often develop resistance to chemotherapy due to high levels of GSH, some researchers have consumed GSH drugs, such as buthionine-sulfoximine (BSO), in anticipation of reducing GSH levels. However, BSO has limited and no specific effects, and can reduce the content of GSH in normal cells, thereby aggravating the side effects brought by radiotherapy and chemotherapy. The high-concentration glutathione in the tumor part can reduce disulfide bonds, and the low concentration of glutathione in normal tissues and blood vessels enables the disulfide bonds to exist stably. In addition, glutathione itself is oxidized at a high concentration after reduction of disulfide bonds, and thus is consumed.

Cathepsin B is a cysteine protease, an endopeptidase with a molecular weight of 30kDA, which is present intracellularly, particularly in lysosomes, in various animal tissues, but is not expressed in the normal extracellular environment. In particular pathological conditions, such as rheumatoid arthritis or tumor sites, a high expression of cathepsin B is observed, especially in various tumor sites. Tumor cells over secrete cathepsin B to aid in its metastasis, invasion, e.g. breakdown of high density collagen networks. Typical substrates for cathepsin B include valine-citrulline and phenylalanine-arginine. According to related research reports, some antibody drug conjugates using enzyme sensitive small molecule peptide fragments as linking agents show effective drug release in tumor tissues.

Therefore, the research and development of various biological sensitive polymer-drug conjugates have bright prospect and practical significance in the targeted tumor treatment.

Disclosure of Invention

The invention aims to provide a glutathione reduction sensitive and cathepsin B sensitive double sensitive polymer-drug conjugate, wherein polyethylene glycol monomethyl ether and an insoluble drug are connected to a disulfide bond and a section of cathepsin B substrate through covalent bonds, so that the water solubility of the insoluble drug is improved, and meanwhile, due to the high content of the reduced glutathione and the cathepsin B in a tumor tissue and a tumor cell, the covalent bonds of the disulfide bond and the substrate can be broken, so that the effect of targeting the tumor tissue can be achieved, and the toxic and side effects on normal cells can be reduced.

The technical scheme adopted by the invention is as follows: a dual sensitive polymer-drug linker is a dual sensitive polymer-drug linker mPEG-Y-SS-R sensitive to glutathione reduction and sensitive to cathepsin B.

Wherein Y is valine-citrulline, forms a double sensitive polymer-drug linker mPEG-VC-SS-R, and has a structural formula shown as (I):

or Y is phenylalanine-arginine, forms a double sensitive polymer-drug connector mPEG-PA-SS-R, and has a structural formula shown as (II):

in the structural formula (I) and the structural formula (II), X is partially represented by a redox sensitive fragment disulfide bond; moiety Y refers to the cathepsin B substrate fragment valine-citrulline or phenylalanine-arginine; r is a pharmaceutical compound with carboxyl.

Preferably, the pharmaceutical compound having a carboxyl group is selected from gambogic acid, rhein, valsartan, methotrexate, exenatide acetate, IDN-6556, AGI-1067, azaserine, chlorophenylalanine, N-acetyl-L-phenylalanine and N-acetyl-L-valine.

More preferably, the Y is valine-citrulline, the pharmaceutical compound with carboxyl is gambogic acid, and the dual-sensitive polymer-drug conjugate mPEG-VC-SS-GA forming the glutathione reduction sensitivity and the cathepsin B sensitivity has a structural formula shown in (III):

wherein the X moiety is designated as a redox sensitive fragment disulfide bond; moiety Y refers to the cathepsin B substrate fragment valine-citrulline; the R part is gambogic acid.

Preferably, the mPEG specification is polyethylene glycol monomethyl ether mPEG 5000.

A preparation method of a dual-sensitive polymer-drug linker comprises the following steps: 1) connecting an enzyme sensitive substrate with Fmoc to prepare an enzyme sensitive substrate intermediate; 2) preparing a drug compound R with carboxyl and cystamine dihydrochloride into a drug derivative R-cystamine containing disulfide bonds; 3) connecting the enzyme sensitive substrate intermediate with a disulfide bond-containing drug derivative R-cystamine to prepare a double sensitive drug derivative; 4) polyethylene glycol monomethyl ether and a double-sensitive drug derivative are condensed into a double-sensitive polymer-drug connector mPEG-Y-SS-R sensitive to glutathione reduction and sensitive to cathepsin B.

Preferably, in the above method for preparing the double sensitive polymer-drug conjugate, Y is valine-citrulline, and constitutes the double sensitive polymer-drug conjugate mPEG-VC-SS-R, the method comprises the following steps:

1) synthesis of enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit):

1.1) dissolving Fmoc-Val (N- (9-fluorenylmethoxycarbonyl) -L-valine), HOSu (N-hydroxysuccinimide), DCC (N, N' -dicyclohexylcarbodiimide) in tetrahydrofuran at 0 ℃, stirring for 24h, filtering, decompressing and rotary evaporating to obtain a white solid product Fmoc-Val-OSu;

1.2) combining Cit (citrulline) with NaHCO₃Dissolving in distilled water, cooling to 0 deg.C, and adding solution of Fmoc-Val-OSu in DME (1, 2-dimethoxyethane) dropwise to Cit and NaHCO₃Adding tetrahydrofuran to assist dissolving,stirring for 24h at room temperature to obtain a reaction solution;

1.3) dropwise adding saturated potassium carbonate into the reaction liquid obtained in the step 1.2) to adjust the pH value to 8-9, extracting with ethyl acetate, collecting a water layer, adding a citric acid solution to adjust the pH value to 3-4, separating out a white gelatinous solid, filtering, dissolving the obtained white gelatinous solid in a mixed solution of tetrahydrofuran and methanol, performing rotary evaporation and concentration, adding methyl tert-butyl ether, stirring overnight at 0 ℃, filtering, and performing vacuum drying to obtain a white solid product which is an enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit);

2) disulfide bond-containing drug derivative R-cystamine (R-SS-NH)₂) The synthesis of (2):

2.1) dissolving a pharmaceutical compound R with carboxyl in a dichloromethane solution, cooling to 0 ℃, adding EDCI (N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride) and HOBT (1-hydroxybenzotriazole), activating the obtained mixture at 0 ℃ for 1h, sequentially adding a methanol solution of cystamine dihydrochloride and triethylamine, and stirring at normal temperature for 48h to obtain a reaction solution;

2.2) reacting the reaction solution obtained in the step 2.1) with NaHCO₃Washing the solution, collecting the organic layer, drying with anhydrous magnesium sulfate, filtering, separating by column chromatography, and vacuum drying to obtain disulfide bond-containing drug derivative R-cystamine (R-SS-NH)₂)；

3) Synthesis of the double sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R):

3.1) dissolving Fmoc-Val-Cit obtained in the step 1) in a mixed solution of dichloromethane and methanol, cooling to 0 ℃, adding EDCI and HOBT, activating the obtained mixture at 0 ℃ for 1h, and adding R-SS-NH obtained in the step 2)₂Stirring the obtained mixture overnight, after the reaction is finished, concentrating under reduced pressure, adding ice water, storing overnight at 4 ℃, filtering, washing with water for three times, drying in vacuum, and purifying by column chromatography to obtain the dual sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R);

4) synthesis of the double sensitive Polymer-drug linker mPEG-VC-SS-R:

4.1) dissolving succinic anhydride and DMAP (4- (dimethylamino) pyridine) in a pyridine solution, then dropwise adding the solution into a chloroform solution of mPEG, stirring the obtained mixture at 60 ℃ under the protection of nitrogen for 24 hours, washing the mixture with normal saline, drying the mixture with anhydrous magnesium sulfate, filtering the mixture to obtain an organic layer, concentrating the organic layer, washing the organic layer with ether, and drying the organic layer in vacuum to obtain a white solid product mPEG-COOH;

4.2) dissolving mPEG-COOH in dichloromethane, adding EDCI, HOBT andactivating a type molecular sieve in the dark at 0 ℃ to obtain mPEG-COOH solution;

4.3) dissolving Fmoc-VC-SS-R obtained in the step 3) in THF, adding DBU (1, 8-diazabicyclo [5.4.0] undec-7-ene), stirring for 10 minutes, adding the obtained mixed solution into mPEG-COOH solution, and reacting for 2 days at room temperature under the protection of nitrogen;

4.4) after the reaction is finished, washing with distilled water, extracting with chloroform, combining organic layers, evaporating under reduced pressure, adding ether for washing, filtering, drying the obtained solid in vacuum, adding ultrapure water, stirring, filtering, dialyzing for 2 days, replacing release media for 5 times in 2h, 6h, 12h, 24h and 36h respectively, and freeze-drying to obtain the target product, namely the dual-sensitive polymer-drug conjugate mPEG-VC-SS-R.

Preferably, in the preparation method, the pharmaceutical compound R with carboxyl is Gambogic Acid (GA), the mPEG specification is mPEG5000, and the dual-sensitive polymer-gambogic acid conjugate mPEG-VC-SS-GA is prepared.

Compared with the prior art, the invention has the following beneficial effects:

the polymer-drug copolymer improves the water solubility of insoluble drug gambogic acid, is linked with valine-citrulline through disulfide bonds, has good release response performance, enhances the targeting property of the copolymerized drug, prolongs the retention time of the anticancer drug at a tumor part, and tests on the critical micelle concentration show that the polymer-drug copolymer is easy to form micelles, and cell experiments show that the polymer-drug copolymer has good inhibition effect on liver cancer. The polymer-drug copolymer has the function of targeted intelligent drug release, the particle size is about 140nm, the accumulation of nano particles at a tumor part is facilitated, and the drug release is facilitated after GSH and enzyme responsive fracture. According to the invention, a polyethylene glycol monomethyl ether polymer targeted drug delivery technology is adopted, high-concentration glutathione and cathepsin B at a tumor part are taken as targets, and the developed polyethylene glycol monomethyl ether-valine-citrulline-S-S-gambogic acid copolymer drug delivery system is designed, so that the targeted therapeutic effect of gambogic acid is increased, the toxic and side effects are reduced, and the bioavailability is improved.

The polymer-drug copolymer has redox response and enzyme response performances, good water solubility and small toxic and side effects, and the hydrophilic section is polyethylene glycol monomethyl ether and the hydrophobic section is gambogic acid. In aqueous solution, amphiphilic polymer micelles are formed spontaneously due to the hydrophilic and hydrophobic effects, and the drug can be released at the tumor site in a response manner. The polymer-drug copolymer provided by the invention can be used as an anticancer drug carrier, and can effectively improve the water solubility of insoluble drugs.

The dual sensitive polymer-drug linker mPEG-VC-SS-GA (PVSG) sensitive to glutathione reduction and cathepsin B sensitivity designed by the invention takes the single sensitive polymer-drug linker mPE-SS-GA (PSG) reduced by glutathione as a reference, and the polymer-drug linker of the invention increases the solubility of drugs in water and evaluates the pharmacological action of the drugs in the aspect of cytotoxicity. In addition, compared with inclusion carriers such as micelle, liposome and the like, the polymer-drug conjugate has the advantages that the drug does not leak in the systemic circulation process, and the drug is a hydrophobic inner layer, so that the drug can be released more quickly when chemical bonds are broken.

Drawings

FIG. 1 shows the synthesis of dual sensitive polymer-gambogic acid linker mPEG-VC-SS-GA (PVSG).

FIG. 2 shows MALDI-TOF-MS detection of PVSG.

FIG. 3 shows MALDI-TOF-MS detection of mPEG-COOH (A) and PSG (B).

FIG. 4 is a DSC measurement of PVSG.

FIG. 5 is the measurement of the particle size and potential of gambogic acid self-assembled nanoparticles;

wherein, A: particle size of PSG nanoparticles; b: PSG nanoparticle potential; c: particle size of PVSG nanoparticles: d: PVSG nanoparticle potential.

FIG. 6 is a transmission electron microscope measurement of PVSG self-assembled nanoparticles;

wherein, A: PVSG nanoparticles; b: PSG nanoparticles.

FIG. 7 is a graph of the in vitro release of gambogic acid.

Fig. 8 is a sensitivity release diagram of gambogic acid nanoparticle glutathione.

Fig. 9 is a graph of sensitive release of gambogic acid nanoparticle cathepsin B.

FIG. 10 shows the inhibition of four cell proliferations by the double-sensitive polymer-gambogic acid conjugate (PVSG) of the present invention.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.