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CN112089845B - Taxane drug-adriamycin prodrug self-assembly nanoparticles and application thereof - Google Patents

Taxane drug-adriamycin prodrug self-assembly nanoparticles and application thereof Download PDF

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CN112089845B
CN112089845B CN201910519358.8A CN201910519358A CN112089845B CN 112089845 B CN112089845 B CN 112089845B CN 201910519358 A CN201910519358 A CN 201910519358A CN 112089845 B CN112089845 B CN 112089845B
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taxane
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doxorubicin
adriamycin
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CN112089845A (en
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王永军
王颖丽
刘洪卓
何仲贵
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Shenyang Pharmaceutical University
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Abstract

The invention belongs to the field of new auxiliary materials and new dosage forms of medicinal preparations, and relates to self-assembled nanoparticles of taxane medicaments, namely adriamycin prodrugs and application thereof. In particular to the synthesis of a redox sensitive taxane drug-adriamycin prodrug with tumor tissue specific response, the preparation of self-assembled nanoparticles of the taxane drug-adriamycin prodrug, and the application of the nanoparticles in a drug delivery system. The taxane drug-adriamycin prodrug provided by the invention comprises a taxane drug-adriamycin prodrug connected with a carbon chain and a taxane drug-adriamycin prodrug connected with a redox sensitive thioether bond, and the structural general formula of the prodrug is as follows: wherein R is 1 、R 2 、X、Y、Z、n 1 And n 2 As described in the claims and specification.

Description

Taxane drug-adriamycin prodrug self-assembly nanoparticles and application thereof
Technical Field
The invention belongs to the field of new auxiliary materials and new dosage forms of medicinal preparations, and relates to self-assembled nanoparticles of taxane medicines and adriamycin prodrugs and application thereof. In particular to synthesis of a redox sensitive taxane drug-adriamycin prodrug with tumor tissue specific response, preparation of self-assembled nanoparticles of the taxane drug-adriamycin prodrug, and application of the nanoparticles in a drug delivery system.
Background
Chemotherapy is still the subject of clinical tumor treatment at present, however, clinical studies show that single chemotherapy drugs are easy to generate multi-drug resistance and have poor curative effect on solid tumors, and in addition, the single chemotherapy drugs have insignificant effect, so that the drug dosage is increased or the drug administration time is prolonged, and the toxic and side effects of the drugs are increased. Therefore, the combination is always the mainstream of clinical tumor chemotherapy. Meanwhile, the anticancer drugs with different action mechanisms can respectively act on different links in the process of cell metabolism or proliferation, can kill various tumor cells, delays the generation of multi-drug resistance of tumors, and improves the curative effect.
Taxane drugs (including paclitaxel and docetaxel) and Doxorubicin (DOX) are chemotherapy drugs which are most widely applied clinically, and the combination of the two drugs can generate a synergistic effect to improve the anti-tumor effect, so that the taxane drugs and the DOX are used as a first-line scheme for treating advanced breast cancer. Because the water solubility of the paclitaxel and the docetaxel is extremely low, a large amount of polyoxyethylene castor oil, tween and ethanol are added into a solution (taxol and taxotere) sold in the market to be used as a solubilizer, and serious toxic and side effects are easy to generate. The commercial doxorubicin is generally water-soluble doxorubicin hydrochloride, and clinically used doxorubicin injection is easy to generate serious cardiotoxicity, myelosuppression and other toxicity. The taxane drugs and the adriamycin free drugs have high toxicity, low targeting property and easy induction of multi-drug resistance, thus greatly limiting the clinical application of the drugs. The taxane medicine and the adriamycin can also be encapsulated in liposome or polymer micelle together, and can also be covalently connected on the same high molecular polymer together. However, they often have low drug loading, burst release or are difficult to release from the polymer, and the release rates of the two drugs are not consistent, resulting in suboptimal therapeutic effect, so it is necessary to invent a safe, simple and efficient new method for co-delivery of taxane drugs and doxorubicin.
Disclosure of Invention
The first purpose of the invention is to provide a prodrug capable of selectively releasing taxane drugs, namely doxorubicin, in a tumor cell redox microenvironment, and the prodrug has a remarkable cancer cell inhibition effect and low toxic and side effects.
The second purpose of the invention is to provide a method for preparing the taxane drug-adriamycin prodrug.
The third purpose of the invention is to provide a preparation method of the taxane medicine-adriamycin prodrug self-assembly nanoparticles.
The fourth purpose of the present invention is to provide and compare the effect of the taxane-doxorubicin prodrug with different connecting bonds on the drug release rate and the antitumor drug effect.
The invention realizes the aim through the following technical scheme:
in the taxane medicament-adriamycin prodrug, a taxane medicament and adriamycin are directly connected through an ester bond, an amido bond and a tumor environment sensitive bond, the tumor environment sensitive bond is a pH sensitive bond or an oxidation and reduction sensitive bond, the pH sensitive bond is a carbonate bond, an imine bond, a hydrazone bond, an acylhydrazone bond, an oxime bond, a ketal bond and a cis-aconitate glycoside bond, and the oxidation and reduction environment sensitive bond is a thioether bond, a disulfide bond or a metalloprotease sensitive bond.
Preferably, the taxane-doxorubicin prodrug of the invention comprises a carbon chain-linked taxane-doxorubicin prodrug and a redox sensitive thioether-linked taxane-doxorubicin prodrug, and the structural general formula of the prodrug is as follows:
Figure BDA0002096128170000021
wherein,
R 1 is-CH 2 -one of-O-or-NH-;
R 2 is-CH 2 -one of-O-or-NH-;
x is S or-CH 2 -one of;
y is OH, Z is (CH) 3 ) CO or Y being CH 3 COO and Z are Ph;
n 1 and n 2 The number of all the repeating units is an integer of 0-10; preferably an integer of 0 to 5; more preferably an integer of 0,1,2.
In the most preferred embodiment of the present invention, the structure of the taxane-doxorubicin prodrug is represented by the following formula (a), (b), (c), (d):
Figure BDA0002096128170000031
Figure BDA0002096128170000041
the invention also provides a method for synthesizing the taxane medicine-adriamycin prodrug, namely, in an organic solvent, the taxane medicine is used as a raw material, and the reaction with the raw material with a corresponding group is carried out to firstly prepare a taxane medicine prodrug intermediate, and finally the intermediate is reacted with adriamycin to prepare the taxane medicine-adriamycin prodrug.
The invention provides a synthesis route and a method of a taxane medicament-adriamycin prodrug, which specifically comprise the following steps:
(1) In the presence of an esterification catalyst A, taxane drugs and thiohydroxy acetic anhydride or glutaric anhydride are mixed and reacted in an organic solvent to obtain taxane drug prodrug intermediates 1,2,3 and 4;
(2) And (2) in an organic solvent, uniformly mixing the intermediate 1,2,3 or 4 of the taxane prodrug obtained in the step (1) with adriamycin, adding organic base in the presence of a catalyst B, stirring for reaction, connecting the intermediate 1,2,3 or 4 with adriamycin, and separating and purifying to obtain the taxane prodrug-adriamycin prodrug.
In the preparation method of the invention, the organic solvent in the step (1) can be any one of tetrahydrofuran, chloroform, dichloromethane, dimethylformamide or 1, 4-dioxane; dichloromethane is preferred.
In the preparation method of the invention, the esterification catalyst A in the step (1) can be selected from any one of Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP) or carbodiimide (EDCI); 4-dimethylaminopyridine is preferred.
In the preparation method of the invention, the organic solvent in the step (2) can be any one of dichloromethane, chloroform, dimethylformamide, tetrahydrofuran, 1, 4-dioxane or dimethyl sulfoxide; dichloromethane is preferred.
In the preparation method, the organic base in the step (2) can be selected from one or a mixture of triethylamine and N, N-diisopropylethylamine; n, N-diisopropylethylamine is preferred.
In the preparation method, the catalyst B in the step (2) can be selected from any one or any two of O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU), 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), N-hydroxysuccinimide (NHS), carbodiimide (EDCI) or 1-Hydroxybenzotriazole (HOBT); an HBTU is preferred.
In a preferred embodiment of the preparation method of the present invention, a prodrug represented by formula (a) is prepared, and the synthetic route is as follows:
Figure BDA0002096128170000051
the specific synthesis method comprises the following steps:
paclitaxel (PTX) and glutaric anhydride react under the catalysis of 4-Dimethylaminopyridine (DMAP) to prepare an intermediate 1; then, the intermediate 1 and adriamycin are mixed in dichloromethane, HBTU and DIPEA are added and stirred together, and N 2 Protecting, and separating and purifying the reaction at room temperature to obtain the prodrug shown in the formula (a): PTX-C-DOX.
In another preferred embodiment of the preparation method of the present invention, a prodrug represented by formula (b) is prepared, and the synthetic route is as follows:
Figure BDA0002096128170000061
the specific synthesis method comprises the following steps:
paclitaxel (PTX) and thiohydroxy acetic anhydride react under the catalysis of 4-Dimethylaminopyridine (DMAP) to prepare an intermediate 2; then, the intermediate 2 and adriamycin are mixed in dichloromethane, HBTU and DIPEA are added and stirred together, and N 2 Protecting, and separating and purifying the reaction at room temperature to obtain the prodrug shown in the formula (b): PTX-S-DOX.
In another preferred embodiment of the preparation method of the present invention, a prodrug of formula (c) is prepared, which is synthesized as follows:
Figure BDA0002096128170000071
the specific synthesis method comprises the following steps:
docetaxel (DTX) and glutaric anhydride react under the catalysis of 4-Dimethylaminopyridine (DMAP) to prepare an intermediate 3; then, the intermediate 3 and adriamycin are mixed in dichloromethane, HBTU and DIPEA are added to be stirred together, and N 2 Protecting, and separating and purifying the reaction at room temperature to obtain the prodrug shown in the formula (c): DTX-C-DOX.
In another preferred embodiment of the preparation method of the present invention, a prodrug of formula (d) is prepared, which is synthesized as follows:
Figure BDA0002096128170000081
the specific synthesis method comprises the following steps:
docetaxel (DTX) and thiohydroxy acetic anhydride react under the catalysis of 4-Dimethylaminopyridine (DMAP) to prepare an intermediate 4; then, the intermediate 4 and adriamycin are mixed in dichloromethane, HBTU and DIPEA are added and stirred together, and N 2 Protecting, and separating and purifying the reaction at room temperature to obtain the prodrug shown in the formula (d): DTX-S-DOX.
The taxane drug-adriamycin prodrug provided by the invention can be directly used as an anticancer drug and can also be further prepared into a pharmaceutically acceptable dosage form for treating cancers. Therefore, the invention also provides prodrug self-assembly nanoparticles prepared from the taxane medicament-adriamycin prodrug.
The preparation method of the taxane medicine-adriamycin prodrug self-assembly nanoparticles provided by the invention comprises the following steps:
dissolving a certain amount of taxane medicament-adriamycin prodrug into a proper amount of mixed solvent of ethanol and tetrahydrofuran or acetone solution, slowly dripping the solution into water under the condition of stirring, and spontaneously forming uniform nanoparticles by the taxane medicament-adriamycin prodrug.
The taxane drug-adriamycin prodrug self-assembly nanoparticles can be non-PEG modified taxane drug-adriamycin prodrug nanoparticles, taxane drug-adriamycin prodrug nanoparticles coated with hydrophobic micromolecule drugs or hydrophobic fluorescent substances and active targeting taxane drug-adriamycin prodrug nanoparticles.
One preferred embodiment of the invention is non-pegylated modified taxane drug-doxorubicin prodrug nanoparticles, and specifically, a certain amount of the taxane drug-doxorubicin prodrug is dissolved in an organic solvent (ethanol and tetrahydrofuran mixed solution or acetone solution), and then slowly dropped into a stirring aqueous solution, and then the organic solvent is removed to prepare the prodrug nanoparticles.
Another preferred embodiment of the present invention is PEG-modified nanoparticles of taxane drugs-doxorubicin prodrugs, which are prepared by dissolving a certain amount of the taxane drug-doxorubicin prodrug and a certain amount of PEG-modifying agent in an organic solvent (ethanol and tetrahydrofuran mixed solution or acetone solution), slowly dropping the solution into a stirring aqueous solution, and removing the organic solvent. The proportion range of the taxane medicament-adriamycin prodrug and the PEG modifier is as follows: 5-50% (w/w), the PEGylation modifier is DSPE-PEG 2k Or one or two of DSPE-PEG-AA.
Yet another preferred embodiment of the present invention is to entrap small hydrophobicityThe nanometer taxane medicine-adriamycin prodrug particle of molecular medicine or hydrophobic fluorescent material is prepared through dissolving taxane medicine-adriamycin prodrug, PEG modifier, ce6 or DiR or coumarin 6 in organic solvent, dropping the solution into water solution while stirring, and eliminating the organic solvent to obtain the nanometer taxane medicine-adriamycin prodrug particle. The PEG modifier is DSPE-PEG 2k Or one or two of DSPE-PEG-AA.
The invention has the following beneficial effects: in the invention, the taxane drugs and the adriamycin are conjugated together through different connecting bonds for the first time to form a prodrug, and the taxane drugs and the adriamycin have different action mechanisms for killing tumor cells and have synergistic action; meanwhile, the taxane drugs and the adriamycin are chemically modified, so that the release of the taxane drugs and the adriamycin in blood after intravenous injection is reduced, and the toxic and side effects of the taxane drugs and the adriamycin are greatly reduced; the taxane drugs and the adriamycin can be selectively released in the tumor cells by utilizing the special high redox characteristic in the tumor cells, so that the curative effect is improved. The prodrug self-assembly nanoparticles prepared by the one-step nano precipitation method have the advantages of simple preparation process, good reproducibility and contribution to clinical transformation, and the prepared prodrug nanoparticles have small and uniform particle size, are beneficial to enriching the prodrug nanoparticles on tumor parts through an EPR (ethylene propylene rubber) effect, reduce toxic and side effects and improve the curative effect.
Drawings
FIG. 1 is a diagram showing the preparation of ester-linked paclitaxel-doxorubicin prodrug (PTX-C-DOX) according to example 1 of the present invention 1 HNMR spectrogram.
FIG. 2 is a drawing showing the preparation of thioether-linked paclitaxel-doxorubicin prodrug (PTX-S-DOX) according to example 2 of the present invention 1 HNMR spectrogram.
FIG. 3 is a drawing of ester-linked docetaxel-doxorubicin prodrug (DTX-C-DOX) of example 3 of the present invention 1 HNMR spectrogram.
FIG. 4 is a graph of thioether-linked docetaxel-doxorubicin prodrug (DTX-S-DOX) of example 4 of the present invention 1 HNMR spectrogram.
Fig. 5 is a TEM image of PEG-modified paclitaxel-doxorubicin prodrug self-assembled nanoparticles of example 6 of the present invention.
Fig. 6 is a colloidal stability chart of the PEG-modified paclitaxel-doxorubicin prodrug self-assembled nanoparticles of example 6 of the present invention.
Fig. 7 is a graph of an in vitro release assay for PEG-modified paclitaxel-doxorubicin prodrug self-assembled nanoparticles of example 6 of the present invention.
Fig. 8 is an in vitro cytotoxicity plot of PEG-modified paclitaxel-doxorubicin prodrug self-assembled nanoparticles of example 6 of the present invention.
Fig. 9 is a cell uptake map of PEG-modified paclitaxel-doxorubicin prodrug self-assembled nanoparticles of example 6 of the present invention.
Fig. 10 is a graph of blood concentration-time curve of PEG-modified paclitaxel-doxorubicin prodrug self-assembled nanoparticles of example 6 of the present invention after intravenous injection administration.
Fig. 11 is a tissue distribution diagram of PEG-modified paclitaxel-doxorubicin prodrug self-assembly nanoparticles of example 6 of the present invention.
Fig. 12 is a graph of the change of tumor volume in the in vivo antitumor experiment of PEG-modified paclitaxel-doxorubicin prodrug self-assembled nanoparticles of example 6 of the present invention.
Fig. 13 is a graph of the change in body weight of mice in an in vivo anti-tumor experiment using PEG-modified paclitaxel-doxorubicin prodrug self-assembled nanoparticles of example 6 of the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. The present invention is further described in the following examples, which should not be construed as limiting the scope of the invention, but rather should be construed as being modified and adapted by those skilled in the art in light of the teachings herein.
Example 1: synthesis of paclitaxel-doxorubicin prodrug PTX-C-DOX
Dissolving a certain amount of paclitaxel and glutaric anhydride in a small amount of dichloromethane, stirring and reacting for 24h under the catalysis of 4-Dimethylaminopyridine (DMAP) and under the protection of nitrogen at room temperature, washing the reaction solution with saturated saline for three times after the reaction is finished, and separating CH 2 Cl 2 The layer was dried over anhydrous sodium sulfate, filtered, concentrated to dryness on a rotary evaporator, and separated and purified to obtain intermediate 1 as a white solid. Appropriate amounts of intermediate 1 and doxorubicin were mixed in dichloromethane, added HBTU and DIPEA with stirring, and stirred under nitrogen for 1 day. After the reaction, the dichloromethane layer was washed with saturated brine for three times, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and separated and purified to obtain a dark red solid powder. Measurement by nuclear magnetic resonance 1 H-NMR spectrum was used to determine the structure of the prodrug of example 1, using d-DMSO as the solvent, and the spectrum was resolved as follows, as shown in FIG. 1:
1 H-NMR (400MHz, DMSO-d 6) spectrum: δ 9.17 (d, J =8.4Hz, 1H), 8.00-7.93 (m, 2H), 7.90 (d, J =4.8Hz, 2H), 7.83-7.68 (m, 3H), 7.64 (dd, J =9.1,6.1Hz, 3H), 7.52-7.37 (m, 8H), 7.18 (d, J =6.9Hz, 1H), 6.27 (s, 1H), 5.75 (s, 3H), 5.55-5.37 (m, 3H), 5.31 (d, J =9.0Hz, 1H), 5.23 (s, 1H), 4.97-4.82 (m, 4H), 4.74 (d, J =6.0Hz, 1H), 4.64-4.55 (m, 3H), 4.17 (d, J =6.4Hz, 1H), 4.13-4.04 (m, 1H), 4.00 (d, J =4.2hz, 2h), 3.97 (s, 3H), 3.57 (d, J =7.1hz, 1h), 3.38 (d, J =17.7hz, 2h), 3.05-2.88 (m, 2H), 2.40-2.28 (m, 2H), 2.22 (s, 3H), 2.09 (s, 4H), 1.81 (d, J =14.6hz, 2h), 1.76 (s, 3H), 1.74-1.56 (m, 2H), 1.49 (s, 3H), 1.46-1.38 (m, 1H), 1.23 (s, 2H), 1.13 (d, J =6.4hz, 3h), 1.00 (d, J =13.9hz, 6.6.4hz, 6h).
Example 2: synthesis of redox-bis-sensitive thioether-linked paclitaxel-doxorubicin prodrug PTX-S-DOX
Dissolving a proper amount of paclitaxel and thiohydroxy acetic anhydride in a small amount of dichloromethane, stirring and reacting for 24 hours at room temperature under the catalysis of 4-Dimethylaminopyridine (DMAP) and under the protection of nitrogen, washing a reaction solution for three times by using saturated salt water after the reaction is finished, separating a CH2Cl2 layer, drying by using anhydrous sodium sulfate, filtering, concentrating and evaporating by using a rotary evaporator, and separating and purifying to obtain a white solid intermediate 2. Appropriate amounts of intermediate 2 and doxorubicin were mixed in dichloromethane, added HBTU and DIPEA with stirring, and stirred under nitrogen for 1 day. After the reaction, the dichloromethane layer was washed with saturated brine for three times, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and separated and purified to obtain a dark red solid powder. Measurement by nuclear magnetic resonance 1 H-NMR Hydrogen SpectroscopyTo determine the structure of the prodrug of example 2, the chosen solvent was d-DMSO, and the results are shown in FIG. 2, with the following spectral resolution:
1 H-NMR (400MHz, DMSO-d 6) spectrum: δ 9.21 (d, J =8.4Hz, 1H), 7.97 (d, J =7.6Hz, 2H), 7.89 (d, J =5.0Hz, 2H), 7.84-7.59 (m, 6H), 7.43 (t, J =11.8Hz, 3H), 7.42 (s, 4H), 7.17 (d, J =7.6Hz, 1H), 6.27 (s, 1H), 5.78 (d, J =13.4Hz, 3H), 5.56-5.43 (m, 2H), 5.38 (dd, J =16.1,8.0Hz, 2H), 5.23 (s, 1H), 4.95-4.82 (m, 5H), 4.64-4.56 (m, 3H), 4.09 (s, 2H), 4.00 (d, J =4.3hz, 2h), 3.97 (s, 3H), 3.63-3.49 (m, 3H), 3.20 (s, 2H), 2.95 (q, J =18.3hz, 2h), 2.23 (s, 3H), 2.08 (s, 3H), 1.75 (s, 3H), 1.62 (t, J =12.3hz, 1h), 1.49 (s, 1H), 1.23 (s, 5H), 1.13 (d, J =6.4hz, 3h), 0.99 (d, J =14.7hz, 6h), 0.85 (s, 1H), 0.74 (s, 3H).
Example 3: synthesis of docetaxel-doxorubicin prodrug DTX-C-DOX
Dissolving a certain amount of docetaxel and glutaric anhydride in a small amount of dichloromethane, reacting for 24 hours under nitrogen protection and stirring at room temperature under the catalysis of 4-Dimethylaminopyridine (DMAP), washing a reaction solution with saturated saline for three times after the reaction is finished, separating a CH2Cl2 layer, drying with anhydrous sodium sulfate, filtering, concentrating and evaporating to dryness by using a rotary evaporator, and separating and purifying to obtain a white solid intermediate 3. The appropriate amount of intermediate 3 and doxorubicin was mixed in dichloromethane, added HBTU and DIPEA with stirring and stirred under nitrogen for 1 day. After the reaction, the dichloromethane layer was washed with saturated brine for three times, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and separated and purified to obtain a dark red solid powder. Measurement by nuclear magnetic resonance 1 H-NMR spectrum was used to determine the structure of the prodrug of example 3, using d-Chloroform as the solvent, and the results are shown in FIG. 3, and the spectrum was resolved as follows:
1 h NMR (400MHz, chloroform-d) spectrum: δ 8.07 (dd, J =23.4,7.7hz, 2h), 7.79 (t, J =8.0hz, 1h), 7.62 (t, J =7.5hz, 1h), 7.51 (t, J =7.6hz, 2h), 7.38 (dd, J =8.0,5.0hz, 2h), 7.29 (d, J =7.8hz, 7h), 5.67 (d, J =7.1hz, 1h), 5.50 (s, 1H), 5.37 (s, 1H), 5.29 (s, 1H), 5.22 (s, 1H), 4.96 (d, J =9.2hz, 1h), 4.77 (d, J =1.3hz, 2h), 4.35-4.21 (m, 2H), 4.21-4.08 (m, 3H), 4.07 (s, 2H), 3.90 (d, J =7.1hz, 1h), 3.65 (s, 23H), 3.29 (d, J =18.9hz, 1h), 3.04 (d,J＝18.8Hz,1H),2.41(s,2H),2.35(d,J＝15.2Hz,2H),2.11(s,2H),1.93(s,5H),1.82(s,8H),1.74(s,6H),1.32(s,6H),1.30–1.19(m,9H),1.12(s,2H).
example 4: synthesis of redox-double-sensitive thioether-linked docetaxel-doxorubicin prodrug DTX-S-DOX
Dissolving a proper amount of docetaxel and thiohydroxy acetic anhydride in a small amount of dichloromethane, stirring and reacting for 24 hours at room temperature under the catalysis of 4-Dimethylaminopyridine (DMAP) and under the protection of nitrogen, washing a reaction solution for three times by using saturated salt water after the reaction is finished, separating a CH2Cl2 layer, drying by using anhydrous sodium sulfate, filtering, concentrating and evaporating by using a rotary evaporator, and separating and purifying to obtain a white solid intermediate 4. Appropriate amount of intermediate 4 and adriamycin were mixed in dichloromethane, added HBTU and DIPEA and stirred together, and stirred under nitrogen for 1 day. After the reaction, the dichloromethane layer was washed with saturated brine for three times, dried over anhydrous sodium sulfate, filtered, evaporated to dryness, and separated and purified to obtain a dark red solid powder. Measurement by nuclear magnetic resonance 1 The structure of the prodrug of example 4 was confirmed by H-NMR spectroscopy using d-DMSO as the solvent, and the results are shown in FIG. 4, which is resolved as follows:
1 H NMR(400MHz,DMSO-d 6 ) Spectrum: δ 7.94 (dd, J =23.5,6.2hz, 5h), 7.80 (dd, J =19.0,8.6hz, 3h), 7.74-7.60 (m, 5H), 7.36 (dd, J =22.6,8.0hz, 5h), 7.14 (s, 1H), 5.75 (s, 1H), 5.47 (s, 1H), 5.38 (d, J =7.2hz, 1h), 5.25 (s, 1H), 5.13-4.93 (m, 6H), 4.93-4.81 (m, 5H), 4.58 (d, J =5.8hz, 2h), 4.19 (d, J =7.2hz, 1h), 3.99 (d, J =8.0hz, 7h), 3.61 (d, J =7.1hz, 2h), 3.52 (d, J =12.7hz, 6h), 3.41 (s, 2H), 3.24 (d, J =4.7hz, 3h), 3.06-2.94 (m, 3H), 2.69 (s, 4H), 2.21 (s, 4H), 1.77 (s, 1H), 1.65 (d, J =15.2hz, 5h), 1.50 (s, 4H), 1.29 (s, 8H), 1.23 (s, 4H), 1.14 (d, J =6.3hz, 5h), 0.95 (s, 6H).
Example 5: preparation of non-PEG paclitaxel-adriamycin prodrug self-assembly nanoparticles
Accurately weighing 6mg of paclitaxel-adriamycin prodrug, dissolving the paclitaxel-adriamycin prodrug by using 0.6mL of ethanol-tetrahydrofuran mixed solution, then dripping the mixed solution into the deionized water solution which is stirred, and then removing the organic solvent to form uniform nanoparticles (PTX-C-DOX nanoparticles and PTX-S-DOX nanoparticles).
Example 6: preparation of PEG-paclitaxel-adriamycin prodrug self-assembly nanoparticles
Accurately weighing 6mg of taxol-adriamycin prodrug and DSPE-PEG 2000 1.2mg, dissolving with 0.6mL ethanol-tetrahydrofuran mixed solution, then dropping the mixed solution into the deionized water solution under stirring, and then removing the organic solvent to form uniform nanoparticles (PD nanoparticles, PSD nanoparticles).
Example 7: preparation of non-PEGylated docetaxel-doxorubicin prodrug self-assembled nanoparticles
Precisely weighing 3mg of docetaxel-doxorubicin prodrug, dissolving the docetaxel-doxorubicin prodrug in 0.5mL of acetone solution, then dripping the mixed solution into deionized water solution while stirring, and then removing the organic solvent to form uniform nanoparticles (DTX-C-DOX nanoparticles, DTX-S-DOX nanoparticles).
Example 8: preparation of PEG (polyethylene glycol) docetaxel-adriamycin prodrug self-assembly nanoparticles
Accurately weighing docetaxel-adriamycin prodrug 3mg and DSPE-PEG 2000 0.6mg, dissolved with 0.5mL acetone solution, then the mixed solution is dropped into the deionized water solution while stirring, and then the organic solvent is removed to form uniform nanoparticles (DCD nanoparticles, DSD nanoparticles).
The paclitaxel-doxorubicin prodrug self-assembled nanoparticles prepared in example 6 were assayed by transmission electron microscopy
The size and morphology of the nanoparticle size results are shown in figure 5. The transmission electron microscope picture shows that the nano-particles are all in a spherical shape with uniform particle size, and the particle size is about 100-120 nm.
Example 9: preparation of Ce6, diR or coumarin 6-entrapped paclitaxel-adriamycin prodrug self-assembled nanoparticles
Precisely weighing 3mgCe6, diR or 6,6mg taxol-adriamycin prodrug and 1.2mg DSPE-PEG 2000 Dissolving the mixture with 0.6mL of ethanol-tetrahydrofuran mixed solution, then dropping the mixed solution into the stirred deionized water solution, and then removing the organic solvent to form uniform nanoparticles.
Example 10: preparation of active targeting taxol-adriamycin prodrug self-assembly nanoparticles
Accurately weighing 0.6mgDSPE-PEG 2000 0.6mg DSPE-PEG-AA and 6mg taxol-adriamycin prodrug are dissolved by 0.6mL ethanol-tetrahydrofuran mixed solution, then the mixed solution is dripped into deionized water solution in stirring, and then the organic solvent is removed to form uniform nanoparticles.
Example 11: colloidal stability test of PEG-modified paclitaxel-doxorubicin prodrug nanoparticles
The paclitaxel-doxorubicin prodrug self-assembly nanoparticle PD nanoparticles and PSD nanoparticles (2 mg/ml) prepared in example 6 were placed at 4 ℃ for 15 days, and the particle size change of the nanoparticles was measured at a predetermined time point. The result is shown in fig. 6, the particle size of the paclitaxel-doxorubicin prodrug self-assembly nanoparticles is not obviously changed within 15 days, and the paclitaxel-doxorubicin prodrug self-assembly nanoparticles show good colloidal stability.
Example 12: in vitro release test of PEG-modified paclitaxel-doxorubicin prodrug nanoparticles
Taking pH 7.4 phosphate buffer solution containing a proper amount of ethanol as a release medium, and investigating the in-vitro release condition of the prodrug self-assembled nanoparticles: the paclitaxel-doxorubicin prodrug self-assembly nanoparticles (paclitaxel content is 200 mg) prepared in example 6 were added into 15 ml of release medium, in vitro release degree examination was performed in a constant temperature oscillator at 37 ℃, samples were taken at set time points, and the concentrations of released paclitaxel and doxorubicin were determined by high performance liquid chromatography. Adding hydrogen peroxide (H) with a certain concentration into the release medium 2 O 2 ) Or Dithiothreitol (DTT), respectively, under different redox conditions.
The results are shown in FIG. 7, where the carbon chain linked PTX-C-DOX prodrug has better stability in the presence of 10mM H 2 O 2 Or 10mM DTT, only a small amount of the prodrug is hydrolyzed, and paclitaxel and doxorubicin are hardly released from the prodrug. In contrast, PTX-S-DOX prodrugs linked by thioether bonds exhibit a degree of oxidation or reduction sensitivity. Experimental results show that the taxol-adriamycin prodrug connected by thioether has the characteristic of redox-sensitive drug release, particularly takes oxidation sensitivity as the main part, and can be used for treating the drug releaseThe specific oxidation-reduction environment of the tumor tissue responds to realize the specific drug release of the tumor part.
Example 13: in vitro cytotoxicity of PEG-modified paclitaxel-doxorubicin prodrug nanoparticles
The paclitaxel-adriamycin prodrug nanoparticles are examined on two tumor cells by adopting an MTT method: cytotoxicity of human breast cancer cells (MCF-7) and mouse breast cancer cells (4T 1). Cells in logarithmic growth phase were grown at 3X 10 3 The 1640 or DMEM culture solution/well/0.1 mL is buried in a 96-well plate and placed in an incubator for 24h to adhere to the wall. Paclitaxel, doxorubicin, a mixture of paclitaxel and doxorubicin, and the paclitaxel-doxorubicin prodrug nanoparticles prepared in example 6 were added after the cells were adherent. Add 100. Mu.L of drug-containing solution to each well, 3 wells in parallel per concentration, and incubate in an incubator. After culturing for 48h and 72h, taking out the 96-well plate, adding 20 mu L of 5mg/mL MTT solution into each well, incubating for 4h in an incubator, throwing the plate, reversely buckling the 96-well plate in filter paper, fully sucking residual liquid, adding 200 mu L DMSO into each well, oscillating for 10min in an oscillator, and measuring the absorbance of each well at 570nm by an enzyme-labeling instrument. IC50 values were calculated using GraphPad Prism 5.
The MTT results are shown in FIG. 8, and compared to paclitaxel and doxorubicin solutions, IC was determined when the two solutions were mixed at 1 50 The cytotoxicity is greatly enhanced. IC 48 hours after action 50 The synergistic coefficients of the two compounds are 0.57 and 0.22 respectively, which proves that the paclitaxel and the adriamycin have strong synergistic effect. The paclitaxel-adriamycin prodrug nano particle still has strong cytotoxicity. The cytotoxicity of thioether bonds is much higher than that of non-disulfide bonds, indicating that the cleavage of thioether bonds and the release of paclitaxel and doxorubicin are key to maintaining the drug toxicity of PSD nanoparticles.
Example 14: cellular uptake of PEG-modified paclitaxel-doxorubicin prodrug nanoparticles
The uptake condition of the PEG-modified paclitaxel-doxorubicin prodrug nanoparticles in mouse breast cancer cells (4T 1) is determined by a flow cytometer. 4T1 cells were seeded onto 12-well plates and placed in an incubator for 24 hours to allow adherence. DOX, PD nanoparticles and PSD nanoparticles prepared in example 4 were incubated with 4T1 cells, and after incubation at 37 ℃ for 2 hours or 4 hours, the cells were washed, collected and dispersed in PBS, and the uptake of the cells was examined with a flow cytometer.
The results are shown in fig. 9, the cellular uptake of the doxorubicin solution, the PD nanoparticles and the PSD nanoparticles is time-dependent, and the cellular uptake increases significantly with the increase of the incubation time. However, the fluorescence intensity of both the PD nanoparticle and the PSD nanoparticle is lower than that of the adriamycin solution, which indicates that the adriamycin exists in the form of the nanoparticle and is in a fluorescence quenching state, and the prodrug is degraded along with the prolonging of time, the nanoparticle is destroyed, the adriamycin is released, and the fluorescence is recovered. The fluorescence intensity of the PSD nanoparticle is greater than that of the PD nanoparticle group, and the degradation is slower because ester bonds are stable relative to thioether bonds.
Example 15: pharmacokinetics research of PEG modified paclitaxel-adriamycin prodrug nanoparticles
9 healthy male SD rats with a body weight of 200-250g were randomly divided into 3 groups, fasted for 12h before administration, and allowed free drinking water. Paclitaxel solution (taxol) and doxorubicin solution mixed solution and the paclitaxel-doxorubicin prodrug nanoparticles prepared in example 6 were injected intravenously, respectively. Blood is collected from the orbit at a specified time point, plasma is obtained by separation, the plasma is frozen and preserved in a refrigerator at the temperature of minus 20 ℃, and the concentration of the drug in the plasma is determined by a liquid chromatography-mass spectrometry method.
The results are shown in fig. 10, compared with the paclitaxel and adriamycin mixed solution, the prodrug nanoparticle group obviously prolongs the circulation time of the drug in blood plasma, the area under the curve of the drug administration is obviously increased, the long circulation effect is achieved, and the bioavailability is obviously improved.
Example 16: tissue distribution experiment of PEG modified paclitaxel-adriamycin prodrug nanoparticles
Mouse breast cancer cell suspension (4T1, 1x10) 6 cells/100. Mu.L) were inoculated subcutaneously on the right flank of BABL/C mice until the tumor volume had grown to 200mm 3 Tumor-bearing mice were randomly divided into 3 groups of 6 mice each, and administered by tail vein injection: the mice were sacrificed after 4h and 24h in DOX solution and PD nanoparticles and PSD nanoparticles prepared in example 6, respectively, to isolate major organs (heart, liver, spleen, lung, kidney) and tumors, which were imaged in vivoAnd (6) analyzing.
The results are shown in fig. 11, compared with the doxorubicin solution group, the fluorescence intensity of the PD nanoparticles and the PSD nanoparticles in the tumor tissue is significantly increased, and the fluorescence intensity of the PD nanoparticles in the tumor tissue is smaller than that of the PSD nanoparticle group, which indicates that, in addition to the nanoparticles being capable of prolonging the blood circulation of the drug, the cleavage of the thioether bond and the release of paclitaxel and doxorubicin are the key points of the stronger fluorescence intensity of the PSD in the tumor tissue.
Example 17: in vivo antitumor experiment of PEG modified paclitaxel-adriamycin prodrug nanoparticle
Mouse breast cancer cell suspension (4T1, 1x10) 6 cells/100. Mu.L) were inoculated subcutaneously on the right ventral side of BABL/C mice until the tumor volume had grown to 100mm 3 Left and right, tumor-bearing mice were randomly divided into 6 groups of 5 mice each: blank control group (PBS), taxol group, adriamycin solution group, mixed liquor group of taxol and adriamycin, PD nanoparticle group and PSD nanoparticle group, 1 time of administration every 1d, and 5 times of continuous administration. After the administration, the survival state of the mice was observed every day, the body weight was weighed, and the tumor volume was measured. Mice were sacrificed 10 days after dosing, organs and tumors were harvested, tumors were weighed, and important organs and tissues were further evaluated analytically.
As a result, as shown in FIG. 12, each of the administered groups showed a certain antitumor effect as compared with the control blank group. The tumor volume of the taxol group and the adriamycin solution group is rapidly increased and reaches 390-400mm on the 10 th day 3 . In contrast, the mixed solution group of taxol and adriamycin can delay the growth of tumors. However, the PD nanoparticle and the PSD nanoparticle group can obviously inhibit the tumor growth. The tumor volume of the PD nanoparticle group was about 250mm 10 days after the administration 3 Tumors in PSD group were only 110mm 3 Left and right. The anti-tumor effect of the PSD nanoparticles is consistent with the in vitro release result and the cytotoxicity result, while the anti-tumor effect of the PD nanoparticles is inconsistent with the in vitro release result and the cytotoxicity result, and the PSD nanoparticles have better anti-tumor effect probably due to the complexity of the in vivo environment. However, PSD nanoparticles show the optimal anti-tumor effect, which shows that the thioether-linked prodrug has higher sensitivity to the oxidation reduction of tumor parts, and paclitaxel and adriamycin can easily realize tumorThe medicine is released specifically, and the corresponding anti-tumor effect is good. As shown in fig. 13, there was no significant change in body weight for each group of mice. The result shows that the taxol-adriamycin prodrug nanoparticles have obvious anti-tumor effect, do not cause obvious non-specific toxicity to organisms, and are a safe and effective anti-tumor drug co-delivery system.

Claims (13)

1. The taxane drug-adriamycin prodrug is characterized by having the following general formula:
Figure 141029DEST_PATH_IMAGE001
wherein,
R 1 is-CH 2 -O-or-NH-;
R 2 is-CH 2 -one of-O-or-NH-;
x is S or-CH 2 -one of;
y is OH, Z is (CH) 3 ) CO or Y being CH 3 COO and Z are Ph;
n 1 and n 2 Are numbers of repeating units and are integers of 0-10.
2. The taxane-doxorubicin prodrug of claim 1 wherein n is n 1 And n 2 Is an integer of 0 to 5.
3. The taxane-doxorubicin prodrug of claim 1 wherein n is 1 And n 2 Is an integer of 0,1,2.
4. A taxane-doxorubicin prodrug selected from the group consisting of:
Figure 370016DEST_PATH_IMAGE003
Figure 902628DEST_PATH_IMAGE005
Figure 632687DEST_PATH_IMAGE007
Figure 957358DEST_PATH_IMAGE009
。
5. the process for preparing a taxane-doxorubicin prodrug according to claim 4,
(1) In the presence of esterification catalyst A, taxane medicine and sulfo-hydroxy acetic anhydride or glutaric anhydride
Mixing and reacting in an organic solvent to obtain a taxane prodrug intermediate 1,2,3 or 4;
(2) In an organic solvent, the taxane medicine prodrug intermediate 1,2,3 or 4 obtained in the step (1) is
Mixing with adriamycin uniformly, adding organic base under the existence of catalyst B, stirring to react, connecting the intermediate 1,2,3 or 4 with adriamycin, separating and purifying to obtain the taxane medicine-adriamycin prodrug;
Figure 481880DEST_PATH_IMAGE010
Figure DEST_PATH_IMAGE011
Figure 908314DEST_PATH_IMAGE012
Figure DEST_PATH_IMAGE013
。
6. the method for preparing a taxane-doxorubicin prodrug according to claim 5, wherein said organic solvent of the step (1) is any one selected from the group consisting of tetrahydrofuran, chloroform, dichloromethane, dimethylformamide and 1, 4-dioxane; the esterification catalyst A is selected from any one of dicyclohexylcarbodiimide and 4-dimethylaminopyridine.
7. The method for preparing a taxane-doxorubicin prodrug according to claim 5, wherein said organic solvent of step (1) is selected from the group consisting of dichloromethane; the esterification catalyst A is selected from any one of 4-dimethylamino pyridine or carbodiimide.
8. The method for preparing the taxane-doxorubicin prodrug according to claim 5, wherein the organic solvent in the step (2) is selected from the group consisting of dichloromethane, chloroform, dimethylformamide, tetrahydrofuran, 1, 4-dioxane and dimethylsulfoxide; the organic base is selected from one or a mixture of triethylamine and N, N-diisopropylethylamine; the catalyst B is selected from: any one or two of O-benzotriazole-tetramethylurea hexafluorophosphate, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N-hydroxysuccinimide, carbodiimide and 1-hydroxybenzotriazole.
9. The method for preparing a taxane-doxorubicin prodrug according to claim 5, wherein said organic solvent of step (2) is selected from the group consisting of dichloromethane; the organic base is selected from N, N-diisopropylethylamine; the catalyst B is selected from O-benzotriazole-tetramethylurea hexafluorophosphate.
10. The self-assembled nanoparticles of taxane drugs and doxorubicin prodrugs according to claim 1, comprising non-pegylated taxane drug and doxorubicin prodrug nanoparticles, taxane drug and doxorubicin prodrug nanoparticles encapsulating a hydrophobic substance, and active-targeted taxane drug and doxorubicin prodrug nanoparticles, wherein the hydrophobic substance is Ce6 or DiR or coumarin 6.
11. The taxane-doxorubicin prodrug self-assembling nanoparticle of claim 10, wherein said pegylated modifier is DSPE-PEG 2k Or DSPE-PEG-AA, the ratio range of the taxane drug-adriamycin prodrug and the PEG modifier is as follows: 5-50% w/w.
12. Use of the taxane-doxorubicin prodrug according to any one of claims 1 to 4 or the taxane-doxorubicin prodrug self-assembled nanoparticle according to claim 10 or 11 for the preparation of an antitumor drug.
13. Use of the taxane-doxorubicin prodrug of any one of claims 1-4 or the taxane-doxorubicin prodrug self-assembled nanoparticle of claim 10 or 11 in the preparation of a medicament for improving the bioavailability of the medicament.
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