WO2014063549A1 - 一种两亲性嵌段共聚物及其制备方法、以及该共聚物与抗肿瘤药物形成的胶束载药系统 - Google Patents

一种两亲性嵌段共聚物及其制备方法、以及该共聚物与抗肿瘤药物形成的胶束载药系统 Download PDF

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WO2014063549A1
WO2014063549A1 PCT/CN2013/083958 CN2013083958W WO2014063549A1 WO 2014063549 A1 WO2014063549 A1 WO 2014063549A1 CN 2013083958 W CN2013083958 W CN 2013083958W WO 2014063549 A1 WO2014063549 A1 WO 2014063549A1
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drug
block copolymer
micelle
group
docetaxel
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PCT/CN2013/083958
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English (en)
French (fr)
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刘珂
龚飞荣
许卉
郎跃武
范华英
韩飞
车鑫
Original Assignee
Liu Ke
Gong Feirong
Xu Hui
Lang Yuewu
Fan Huaying
Han Fei
Che Xin
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Application filed by Liu Ke, Gong Feirong, Xu Hui, Lang Yuewu, Fan Huaying, Han Fei, Che Xin filed Critical Liu Ke
Priority to US14/438,409 priority Critical patent/US9393312B2/en
Priority to AU2013334301A priority patent/AU2013334301B2/en
Priority to ES13848829.1T priority patent/ES2613876T3/es
Priority to EP13848829.1A priority patent/EP2913353B1/en
Priority to CA2889518A priority patent/CA2889518C/en
Priority to JP2015538265A priority patent/JP5893807B2/ja
Publication of WO2014063549A1 publication Critical patent/WO2014063549A1/zh

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/075Ethers or acetals
    • A61K31/085Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/7028Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61K47/42Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/107Emulsions ; Emulsion preconcentrates; Micelles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/107Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C08G63/00Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
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Definitions

  • the invention relates to an amphiphilic block copolymer, a preparation method thereof, and a stable micelle drug-loading system formed by the copolymer and the antitumor drug, and belongs to the field of nano drug preparations.
  • Tumors are a type of disease that seriously threatens the safety of human life. Studying safe and effective anti-tumor drugs is of great significance for improving the quality of life of human beings.
  • Taxanes (mainly including paclitaxel, PTX, docetaxel, DTX, cabazitaxel, and larotaxel) are a very effective and broad-spectrum anti-tumor
  • the mechanism of action of the drug is mainly to polymerize and stabilize the microtubules, which can cause the rapidly dividing tumor cells to be fixed in the mitotic stage, and the cancer cell replication is blocked and died.
  • the taxane has a significant radiosensitizing effect, allowing the cells to stop in the G2 and M phases that are sensitive to radiotherapy.
  • almost all of the taxanes are highly hydrophobic and their oral absorption is poor, and currently only the route of administration is administered.
  • Doxorubicin is an anti-tumor antibiotic. It is a cytotoxic drug like taxanes. It inhibits the synthesis of RNA and DNA. It has the strongest inhibitory effect on RA and has a wide anti-tumor spectrum. It is a non-specific drug that has a killing effect on tumor cells of various growth cycles. Mainly used for acute leukemia, for acute lymphocytic leukemia and myeloid leukemia. Conventional doxorubicin preparations have significant side effects such as cardiotoxicity and myelosuppression.
  • Epirubicin is an isomer of doxorubicin, which is equivalent or slightly higher than doxorubicin, but less toxic to the heart.
  • Curcumin is a non-cytotoxic drug with potential anti-tumor activity that has received extensive attention in recent years. Its greatest feature is that it has almost no side effects, and at the same time has anti-inflammatory, anti-oxidation and other auxiliary therapeutic effects. Its biggest drawback is water soluble Very poor performance, the preparation of stable aqueous curcumin preparation is currently a hot topic.
  • Polymer micelles are a new type of drug delivery system that has been developed in recent years.
  • the micelles usually consist of a large number of amphiphilic block copolymer molecular chains oriented, and their hydrophobic segments enclose the drug in the nucleus through weak interaction with the drug molecules, and the hydrophilic chains stabilize the micelles outward. Core-shell structure. Polymer micelles not only increase the solubility of the drug, but also increase the therapeutic dose, and the drug is wrapped therein to avoid degradation and reduce side effects.
  • the micelle size is usually below 100 nm, and the periphery is a hydrophilic PEG segment, so it can avoid the phagocytosis of the reticuloendothelial system (RES), prolong the system circulation time, and pass the EPR effect (high permeability and retention effect). And retention effect) achieves the effect of passive targeting of tumors.
  • RES reticuloendothelial system
  • EPR high permeability and retention effect
  • retention effect achieves the effect of passive targeting of tumors.
  • due to the high molecular weight of the polymer micelles renal clearance can also be prevented.
  • the polymer micelles have very low CMC values (critical micelle concentrations) and maintain the stability of the micelle structure when the drug micelles are diluted.
  • the micelle delivery system can carry up to 25% of the drug, fully meeting the needs of clinical use, while the polymer material has biodegradability and good biocompatibility.
  • polymer micelles are considered to be a promising new drug delivery system, especially for some poorly soluble antitumor drugs, their low stability in solution has been affecting this new drug delivery system.
  • the solution has a stabilization time of no more than 24 hours at room temperature (Lee SW, et al, Ionically Fixed Polymeric Nanoparticles as a Novel Drug Carrier, Pharmaceutical). Research, 2007, 24: 1508-1516).
  • the micelles can only be stabilized for about 6 hours at room temperature (Lee SW, et al, Development of docetaxel-loaded intravenous formulation, Nanoxel-PMTM using colymer-based delivery system, Journal of Controlled Release, 2011, 155: 262-271).
  • the micelles quickly disintegrate after entering the body, and the drug immediately binds to proteins in the blood (such as albumin), so the EPR effect of the micelles cannot be exerted.
  • the results of animal experiments show that the drug is no different from docetaxel injection.
  • the tolerated dose has not increased, so the advantage is not obvious.
  • the stability of its mPEG-PLA micelles is similar.
  • Taxanes are one of the greatest discoveries in the field of oncology drug discovery in the past 20 years, and will be the mainstream anti-tumor drugs for the next 20 years. Due to its dose-limiting toxicity, the full use of the drug has been a hot topic. As a promising drug delivery system for taxanes, the instability of micelles has become the biggest drawback of this drug delivery system, and the cause of this instability is still not fully understood. In order to improve the stability of the taxane drug bundle, a lot of efforts have been made. For example, patent 201010001047 discloses a method for adding amino acids to a micelle solution to improve its stability.
  • the amino acid is added during micelle formation, and the position of the amino acid in the micelle (only as a physical barrier of micelles)
  • the agent or the synergistic drug molecule together in the hydrophobic core of the micelle is not mentioned, and it is not known whether the amino acid as an auxiliary additive can maintain the stability of the micelle after being diluted by the blood after entering the body, so that it is used in vivo.
  • the effect is still unclear; it has been reported that the inclusion of paclitaxel and docetaxel in the copolymer micelle can significantly increase the drug loading and stability of the micelle, but the composite drug micelle has not been used clinically.
  • amphiphilic block copolymer of the present invention is based on a polyethylene glycol monomethyl ether (or polyethylene glycol)-polyester block copolymer having a recognized safety, and the terminal hydroxyl group of the polyester segment Modification with a hydrophobic group, introduction of a hydrophobic group having a large spatial structure such as a tert-butoxycarbonyl group or an amino acid having a benzene ring and a derivative thereof, not only improving hydrophobicity in drug molecules and block copolymers
  • the compatibility of the segments increases the interaction between them, and the introduced hydrophobic groups have a larger spatial structure, which provides more space for the drug molecules to enter the core of the micelles, making them less soluble.
  • the greatest significance of the invention is to improve the stability of the micelles in solution state, especially in vivo, thereby exerting the EPR effect of the micelles, achieving higher bioavailability and better therapeutic effects.
  • amphiphilic block copolymer characterized in that: the hydrophilic segment is polyethylene glycol (PEG) or polyethylene glycol monomethyl ether (mPEG) having a number average molecular weight between 400 and 20,000, which is hydrophobic
  • the sexual segment is selected from the group consisting of polylactide (PLA), polyglycolide (PGA), polyglycolide (PLGA), and polycaprolactone having a number average molecular weight of 500,000,000 capped with a hydrophobic group.
  • PCL polycarbonate
  • PTMC polycarbonate
  • PPDO polydioxanone
  • the amino acid derivative is preferably an amino acid derivative protected by ⁇ -benzyl glutamic acid, ⁇ -benzyl aspartic acid or amino group.
  • the amino acid derivative is further preferably an amino acid comprising a benzyl protected or a tert-butoxycarbonyl (Boc) protected.
  • the amino acid derivative is more preferably t-butoxycarbonylphenylalanine.
  • the hydrophobic segment is preferably polylactide (PLA), polyglycolide (PGA), poly(ethylene lactide) (PLGA), polycaprolactone having a number average molecular weight of 1000 50000. (PCL), polycarbonate (PTMC) or a derivative thereof, or polydioxanone (PPDO) or a derivative thereof; preferably, the hydrophilic segment is a polyethylene having a number average molecular weight of 750 5000 Glycol or polyethylene glycol monomethyl ether.
  • Another object of the present invention is to provide a process for the preparation of the above amphiphilic block copolymer.
  • the preparation method of the above amphiphilic block copolymer comprises the following steps:
  • hydrophilic segment with a number average molecular weight between 400 and 20,000 is added to the polymerization bottle, heated to 100 ° C ⁇ 130 °, vacuum dehydrated for 2 h ⁇ 4 h, and then added to form a hydrophobic segment of the polymer monomer and The monomer weight is 0.3%. _1%.
  • Another object of the present invention is to provide a micellar drug delivery system formed from the above amphiphilic block copolymer and an antitumor drug.
  • the technical solution provided by the present invention is as follows:
  • micellar drug delivery system formed by the above amphiphilic block copolymer and an antitumor drug, the micellar drug delivery system comprising at least one of the above amphiphilic block copolymers, a therapeutically effective amount of at least one antitumor drug, and A pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipient is a lyophilized excipient.
  • the lyophilized excipient is at least one of lactose, mannitol, sucrose, trehalose, fructose, glucose, sodium alginate or gelatin.
  • the pharmaceutical auxiliary material further includes an antioxidant, a metal ion complexing agent, a pH adjusting agent or an isotonic adjusting agent, etc.
  • the antioxidant is sodium sulfite, sodium hydrogen sulfite or sodium metabisulfite
  • the metal ion complexing agent is Disodium glutamate, calcium edetate or sodium cyclohexanediamine tetraacetate
  • pH regulator is citric acid, sodium bicarbonate, disodium hydrogen phosphate or sodium dihydrogen phosphate
  • isotonic regulator is chlorinated Sodium or glucose, etc.
  • the anti-tumor drug is the taxane paclitaxel (PTX), docetaxel (DTX), cabazit axe l, larotaxel, and turmeric At least one of a vegetarian, doxorubicin or epirubicin.
  • PTX taxane paclitaxel
  • DTX docetaxel
  • cabazit axe l larotaxel
  • turmeric At least one of a vegetarian, doxorubicin or epirubicin.
  • the weight ratio of the amphiphilic block copolymer to the drug in the amphiphilic block copolymer is between 99.5:0.5 and 50:50, preferably between 99:1 and 75:25.
  • the weight ratio of the lyophilized excipient to the entire system is between 0 and 99.9%, preferably between 10.0 and 80.0%.
  • the antitumor drug polymer micelle preparation prepared by the present invention can be used for the treatment of cancer, preferably for the treatment of breast cancer, prostate cancer, ovarian cancer, intestinal cancer, lung cancer, liver cancer, head and neck cancer and the like.
  • the therapeutically effective amount referred to in the present invention means that the amount of the antitumor drug contained in the above micellar drug-loading system can effectively treat cancer (specifically, it can be referred to as breast cancer, prostate cancer, ovarian cancer, intestinal cancer, lung cancer, liver cancer). , head and neck cancer, etc.).
  • the micelle drug-loading system of the present invention can be administered by an injection route, and is generally prepared as a lyophilized powder preparation. Further, those skilled in the art can determine the dose to be administered according to the dosage of the existing anti-tumor drug, and according to the individual situation. Different up and down adjustments.
  • the invention also provides a preparation method of a micellar drug-loading system formed by an amphiphilic block copolymer and an antitumor drug, comprising a dialysis method, a direct dissolution method, a film hydration method, a solid dispersion method, a high energy homogeneous emulsification method, A film hydration method and a solid dispersion method are preferred.
  • the specific steps of the film hydration method are as follows: dissolving the polymer adjuvant and the drug in an organic solvent, removing the solvent by rotary evaporation, adding the water for injection to dissolve the drug film to obtain a drug-loaded micelle solution, and lyophilizing the gel to obtain a micelle Dry powder.
  • the specific steps of the solid dispersion method are as follows: dissolving the drug in a molten polymer material after heating (this process may be appropriately added with a small amount of an organic solvent to help dissolve) to obtain a clear mixture, and then adding water for injection to dissolve the micelle solution, After filtration and sterilization, the micelles are freeze-dried to obtain a lyophilized powder.
  • the present invention has the following features:
  • the present invention modifies the terminal hydroxyl group of the polyester segment with a hydrophobic group according to the hydrophobicity of the structure of most antitumor drugs and a large spatial structure, by improving the hydrophobicity of the drug molecule and the block copolymer.
  • the compatibility of the sexual segments increases the interaction between them, and at the same time increases the space in the micelle nucleus that can accommodate the drug molecules, and limits the drug molecules to the core of the micelles, making it difficult to dissolve, thus obtaining a series of Highly stable drug-loaded micelles both in vitro and in vivo, the drug-loaded micelles can be made into a lyophilized preparation;
  • the test results prove that: the reconstituted lyophilized preparation of the antitumor drug-loaded micelle prepared by the amphiphilic block copolymer of the invention can be rapidly dispersed to form a clear solution with a blueish opalescence.
  • the solution is stable for at least 24 hours at room temperature without significant precipitation of the drug, and the EPR effect can be effectively exerted in the body after injection, and has a good industrial application prospect.
  • Figure 3 is mPEG 2 . . . -PLA 18 . . Nuclear magnetic resonance spectrum of -BP;
  • Figure 8 is a nuclear magnetic resonance spectrum of mPEGsooo-PC ooo-BP
  • Figure 9 is mPEG 5 . . . -PCL 4 . . . -BP/paclitaxel micelle particle size map
  • Figure 10 is a graph of mPEG 2 ( KKrPLA 18Q (r BP / docetaxel micelle size;
  • Figure 11 3 ⁇ 4mPEG 2 ooo-PLGA 20 oo-TB / cabazitaxel micelle particle size map;
  • Figure 12 is a result of stability test of mPEG 2GGG -PLA 18Q () -BP / docetaxel micelle solution;
  • Figure 13 is a graph showing the stability test results of the mPEG ⁇ o-PLGA ⁇ o-TB / cabazitaxel micelle solution
  • Figure 14 is a tumor suppressing effect of docetaxel injection and docetaxel micelle injection on H460;
  • Figure 15 is a tumor suppressive effect of docetaxel injection and docetaxel micelle injection on H460;
  • Figure 16 is a graph showing the inhibitory effect of docetaxel injection and docetaxel micelle injection on MDA-MB-231 tumor;
  • Figure 17 is a tumor suppressive effect of docetaxel injection and docetaxel micelle injection on MDA-MB-231;
  • Figure 18 is a plasma drug concentration of iv-administered docetaxel micelle (5 mg/kg). Time-dependent curve;
  • Figure 19 is a graph showing the plasma drug concentration over time in iv administration of cabazitaxel micelles (5 mg/kg);
  • Figure 20 is a graph showing the time-dependent change in the total amount of plasma drug and the concentration of the encapsulated drug in rats given iv to docetaxel micelles (5 mg/kg);
  • Figure 21 is a graph showing the time-dependent change in the total amount of plasma drug and the concentration of the encapsulated drug in rats by iv administration of cabazitaxel micelles (5 mg/kg);
  • Figure 22 is a graph showing the time-dependent change of docetaxel drug concentration in tumor-bearing (MX-1) nude mice (iv 10 mg/kg) ;
  • Figure 23 mPEG 2 . . . -PLA 18 . . -BP/paclitaxel micelles and benzoyl terminated mPEG 2 . . . -PLA 18 . .
  • the drug time curve of paclitaxel micelles The drug time curve of paclitaxel micelles.
  • TB is an abbreviation for tert-butanoyl.
  • the polymer structure was determined by deuterated chloroform as solvent and 400MBruker NMR.
  • the NMR data of mPEG 2Q(K )-PLGA 2()()Q -TB was as follows:
  • BP is an abbreviation for tert-butoxycarbonylphenylalanine residue.
  • the polymer structure, mPEG 2 was determined by deuterated chloroform as a solvent and 400 MBruker NMR. . . -PLA 18 . . -
  • the nuclear magnetic resonance spectrum of BP is shown in Figure 3, and its nuclear magnetic resonance spectrum data is as follows:
  • the mPEG oo-PLA ⁇ oo synthesis method is the same as in Example 1 (2).
  • the polymer structure was determined by deuterated chloroform as solvent and 400MBruker NMR.
  • the NMR data of mPEG oo-PLA ⁇ oo-Asp are as follows:
  • Example 1 (1) The synthesis method was the same as in Example 1 (1).
  • TS is an abbreviation for a tyrosine residue.
  • the polymer structure was determined by deuterated chloroform as solvent and 400MBruker NMR.
  • the nuclear magnetic resonance data of mPEG 2 ooo-PLGA 20 oo-TS are as follows:
  • Example 1 The synthesis method is referred to in Example 1 (1).
  • TS is an abbreviation for a tyrosine residue.
  • BP is an abbreviation for tert-butoxycarbonylphenylalanine residue.
  • the polymer structure, mPEG 2 was determined by deuterated chloroform as a solvent and 400 MBruker NMR. . . -PLA 18 . . -
  • the nuclear magnetic resonance spectrum of BP is shown in Figure 8, and its nuclear magnetic resonance spectrum data is as follows:
  • paclitaxel micelle lyophilized powder 150 mg of mPEG 5 prepared in Example 1 was taken. . . -PCL 4 . . . -BP and 30 mg of paclitaxel were dissolved in 2 ml of tetrahydrofuran, and 5 ml of ultrapure water was slowly added dropwise with stirring. After the addition was completed, the solution was stirred at room temperature overnight to remove the organic solvent to obtain a clear paclitaxel micelle solution with obvious blue opalescence. 120 mg of mannitol was added, and the resulting solution was filtered through a 0.22 ⁇ sterilizing membrane and lyophilized to obtain a paclitaxel micelle lyophilized powder.
  • the drug encapsulation efficiency was 98.6%, and the drug loading was greater than 11.2%, the particle size measurement result is as shown in Fig. 9, and the average particle diameter was 33.8 nm, and the dispersion coefficient PDI was 0.1.
  • the stabilization time at room temperature was more than 7 days, which was significantly higher than that of the acetoxy group and the benzoyl terminated polymer micelle.
  • the micelle lyophilized powder is reconstituted into a solution having a concentration of 5 mg/mL by physiological saline, the docetaxel content in the dissolved state is greater than 90% in the solution for 90 days at room temperature.
  • Example 1 1.9 g of mPEG 2 obtained in Example 1 was taken. . . -PLGA 2 . . . -TB is heated to 50 ° C to melt it, then add 100 mg of cabazitaxel, dissolved in the polymer with stirring to obtain a clear and transparent mixture, add 25 ml of physiological saline preheated to 50 ° C to dissolve the polymer / drug The mixture obtained a micelle solution, and then 400 mg of sucrose was added, and the solution was filtered through a 0.22 sterilizing membrane and then freeze-dried to obtain a freeze-dried powder of cabazitaxel.
  • the drug encapsulation efficiency was 96.2%, and the drug loading was more than 4.82%.
  • the particle size measurement results are shown in Fig. 11, and the average particle diameter was 24.2 nm, and the dispersion coefficient PDI was 0.02.
  • the content of the cabazitaxel in the dissolved state is more than 90% in the solution stored at room temperature for 90 days.
  • the micelle lyophilized powder is reconstituted into a solution of 5 mg/mL concentration by physiological saline, the content of curcumin in the dissolved state is more than 90% in the solution stored at room temperature for 7 days.
  • mPEG 2 prepared in Example 1 was taken.
  • . . -PLGA 2 . . . -TB and 40mg doxorubicin hydrochloride were dissolved in chloroform at 40 ° C, added with 0.1 ml of triethylamine at room temperature for 1 h, and the organic solvent was removed by rotary evaporation, and 50 ml of a concentration of 10 mM was added.
  • the HBS buffer solution dissolves the drug film, and after removing the triethylamine hydrochloride by dialysis or ultrafiltration, 250 mg of gelatin solution is added and filtered through a 0.22 ⁇ filter, and then freeze-dried to obtain a doxorubicin micelle lyophilized powder.
  • the drug encapsulation efficiency was 94.7%
  • the drug loading was more than 22.47%
  • the average particle size was 19.4 nm
  • the micelle lyophilized powder is reconstituted into a solution of 2 mg/mL concentration by physiological saline, the doxorubicin content in the dissolved state is greater than 90% in the solution stored at room temperature for 24 hours.
  • the drug content in the dissolved state changes with time as shown in Figure 12: mPEG 2 . . . -PLA 18 . . -
  • the drug in the dissolved state of BP/Docetaxel micelle solution showed a certain degree of decline in the first 15 days, and the dissolution of the drug in the late stage was very slow, even if the drug in the dissolved state remained above 90% within 90 days.
  • the drug content in the dissolved state changes with time as shown in Figure 13: mPEG 2 . . . -PLGA 2 . . .
  • the drug in the dissolved state of the -TB/carbamazee micelle solution showed a certain degree of decline in the first 3 days, and the dissolution of the drug in the late stage was very slow, even if the drug in the dissolved state remained above 90% within 90 days.
  • the docetaxel micelle lyophilized powder prepared in Example 2 was provided by Shandong Target Drug Research Co., Ltd., and dissolved in 0.9% physiological saline;
  • Docetaxel Injection 0.5ml per bottle: 20mg, produced by Qilu Pharmaceutical Co., Ltd. Blank solvent control (blank micelles, 10 mg/kg), supplied by Shandong Target Drug Research Co., Ltd., dissolved in 0.9% physiological saline.
  • Human lung cancer H460 cells were obtained from ATCC, cultured in vitro by the laboratory, inoculated into nude mice for tumor formation, and subcultured.
  • the H460 tumor was selected to grow well, and the tumor-bearing animals were better in systemic condition, and the cervical vertebrae were killed by dislocation. Tumors were removed under aseptic conditions, and scalpels were used to cut tumors with a diameter of 2 to 3 mm.
  • the trocars were inoculated subcutaneously into the ankle of nude mice. On the 7th to 8th day after inoculation, the average tumor volume of the tumor-bearing mice reached 110 ⁇ 120mm 3 , and the animals were divided into groups according to the size of the tumor, 8 in each group.
  • the tumor of the negative control group grew naturally.
  • the blank solvent control group used the same volume of solvent as the docetaxel micelle freeze-dried powder group, and the docetaxel injection was also diluted to the same level as the docetaxel micelle freeze-dried powder group. Volume administration, each administration group was intravenously administered simultaneously.
  • each group of animals was administered intravenously once every three days as planned, for a total of three doses. The observation was terminated when the average tumor volume of the negative control group reached about 2000 mm 3 .
  • TV is the tumor volume.
  • T/C The evaluation index of antitumor activity is the relative tumor growth rate T/C (%) is calculated as:
  • Docetaxel injection group (10mg/kg/time) and docetaxel micelle freeze-dried powder administration group (10mg/kg/time) had obvious tumor growth inhibition effect on H460 nude mice, and the tumor volume was obvious. Reduced. Among them, the docetaxel micelle freeze-dried powder administration group (10 mg/kg/time) gradually reduced the tumor volume during the administration period and maintained the average value less than the pre-dose average for nearly 10 days. Compared with the docetaxel injection group (10 mg/kg/time), the same dose of docetaxel micelle injection significantly improved the tumor inhibition rate, the tumor weight inhibition rate was 68.35%, and the latter 97.5%. The relative tumor growth rate is 25.25% in the former, and the latter is only 3.78%, and the curative effect is improved.
  • the inhibitory effect of docetaxel injection and docetaxel micelle freeze-dried powder on H460 tumors is shown in Figure 14 and Figure 15.
  • the tumor inhibition rate and relative tumor proliferation rate of docetaxel injection and docetaxel micelle freeze-dried powder on H460 tumors are shown in Tables 1 and 2.
  • Solvent control group 8 20.9 ⁇ 1.39 24.1 soil 1.76 1.62 soil 0.510 5.81 Docetaxel injection (10 8 8 21.3 ⁇ 0.97 23.1 ⁇ 2.20 0.545 ⁇ 0.166* 68.35 mg/kg)
  • Docetaxel micelles 10 8 8 21.6 ⁇ 1.11 21.6 ⁇ 2.34 0.04 soil 0.018* 97.5 mg/kg
  • Human breast cancer MAD-MB-231 cells were obtained from ATCC, cultured in vitro by the laboratory, inoculated into nude mice for tumor formation, and subcultured. E, test method
  • the MDA-MB-231 tumor was selected to grow well, and the tumor-bearing animals were better in systemic condition, and the cervical vertebrae were dislocated and sacrificed.
  • the tumor pieces were taken out under aseptic conditions, and the tumor pieces were cut into 2 to 3 mm in diameter with a scalpel, and the trocars were inoculated subcutaneously into the ankle of the nude mice.
  • the average tumor volume of the tumor-bearing mice reached 110 ⁇ 120mm 3 , and the animals were divided into groups according to the size of the tumor, 8-9 per group.
  • the tumor of the negative control group grew naturally.
  • the blank solvent control group used the same volume of solvent as the docetaxel micelle 10 mg/kg dose group, and the docetaxel injection was also diluted into the docetaxel micelle freeze-dried powder group. The same volume was administered, and each administration group was intravenously administered at the same time.
  • T/C The evaluation index of antitumor activity is the relative tumor growth rate T/C (%) is calculated as:
  • Body column rate (%) control group average tumor weight X 100
  • the docetaxel injection group (10 mg/kg/time) and the docetaxel micelle lyophilized powder administration group (10 mg/kg/time) were administered to the MDA-MB-231 mouse intermittent vein. After 3 injections, the drug showed a significant inhibitory effect on tumor growth in nude mice. After 3 doses, the tumor volume of mice was progressively reduced compared with before administration. Docetaxel micelle freeze-dried powder administration group
  • the tumor suppressive effect of MDA-MB-231 was better than that of the docetaxel injection group of the same dose.
  • the tumor inhibition rate and relative tumor proliferation rate of Docetaxel injection and docetaxel micelle freeze-dried powder on MDA-MB-231 tumors are shown in Table 3 and Table 4.
  • Solvent control group 8 17.3 soil 0.99 20.8 soil 1.92 2.48 soil 0.886 12.3 Docetaxel injection (10 8 15.8 ⁇ 1.16 21.0 ⁇ 1.63 1.08 soil 0.646* 61.8 mg/kg)
  • Solvent control group 107 soil 27.9 2472 soil 904.9 27.26 soil 11.779 94.41 docetaxel injection (10 mg/kg) 107 soil 30.7 1054 soil 620.5 10.62 soil 5.295* 36.78 docetaxel micelles (10 mg/kg) 101 soil 42.4 0 1.96 soil 1.48** 6.79
  • mice Male Sprague-Dawley rats weighing 240 ⁇ 20 g were randomly divided into 4 groups: I, II, III and IV, with 6 in each group.
  • Docetaxel micelle lyophilized powder (a), prepared according to the method of Example 2. (2), lot number 20120907, specification: containing docetaxel 20 mg / bottle;
  • Cabazitaxel micelle lyophilized powder (b), prepared according to the method of Example 2. (3), batch number 20120830, Specifications: containing cabazitaxel 20 mg / bottle;
  • Dopafi Docetaxel Injection, c
  • Qilu Pharmaceutical Co., Ltd. Lot 1120312TA, Specification 0.5 ml: 20 mg;
  • the experimental preparation was dissolved and diluted to a suitable concentration before use.
  • docetaxel micelles (a) and cabazitaxel micelles (b) were given to rats in groups I and II by tail vein injection at a dose of 5 mg/kg (each in docetaxel, cabazitaxel).
  • Docetaxel injection (c) and cabazitaxel lyophilized powder (d) were administered to rats in groups III and IV via tail vein at a dose of 5 mg/kg.
  • Blood samples were collected from the orbital venous plexus of the rats at different times after administration, and the plasma was separated by centrifugation, and stored in an ultra-low temperature freezer at -80 °C for testing.
  • Plasma samples were deproteinized by methanol precipitation and analyzed by LC-MS/MS. The total drug concentration of docetaxel or cabazitaxel was determined. The plasma drug concentration of each drug-administered group was plotted over time. The results are shown in Figure 18 ( Rats were given intravenous docetaxel lyophilized powder (a) and docetaxel injection (c) plasma drug time curve), Figure 19 (rat intravenous administration of cabazitaxel micelle lyophilized powder (b) ) and the plasma drug time curve of the cabazitaxel lyophilized powder (d).
  • the plasma AUC of the docetaxel micelle administration group and the cabazitaxel micelle administration group were 8.56 times and 8.91 times that of the corresponding injection administration groups, respectively.
  • both docetaxel micelles and cabazitaxel micelles were intravenously encapsulated in the form of micelles 24 hours after administration.
  • the plasma pharmacokinetic characteristics exhibited by micelle administration reflect the superior stability and unique in vivo release characteristics of the micelles prepared by the present invention.
  • mice Female nude mice were inoculated with human breast cancer MX-1 cells at a density of 5 X 10 6 under the armpits until the tumor grew to ⁇ 500 mm 3 and randomly divided into two groups (Group I: docetaxel micelles).
  • the body weight of the two groups of tumor-bearing mice were 24.9 ⁇ 1.2 g and 25.0 ⁇ 1.3 g, respectively, no significant difference (P > 0.05). Every big The group was evenly divided into 7 groups, each of which had 10 tumor-bearing mice, which were reserved.
  • Docetaxel micelle lyophilized powder prepared according to the method of Example 2. 2), batch number 20120907, specification: 20 mg / bottle; Dopafi (Docetaxel Injection), Qilu Pharmaceutical Co., Ltd., batch number 1120312TA , size 0.5 ml: 20mg.
  • Docetaxel injection and docetaxel micelle lyophilized powder were dissolved and diluted to the appropriate concentration before use, and administered to the group I and II via tail vein injection at a dose of 10 mg/kg (based on docetaxel). Animals were sacrificed at 5 min, 15 min, 30 min 1 h, 3 h, 8 h and 24 h after administration, and the tumor tissues were excised, and then weighed and stored in an ultra-low temperature freezer at -80 °C for testing. .
  • LC-MS/MS analysis was performed to determine the drug concentration of docetaxel, and the drug concentration of the tumor tissue in each administration group was plotted as shown in Fig. 22 .
  • the AUC of docetaxel in the tumor tissue of group II and micelle group was 45.528 mg/L for 24 h. h and 57.089 mg/L h.
  • the results showed that the distribution of docetaxel in the tumor tissue of the micelle group was significantly higher than that of the injection group (( ⁇ 0.01), and the difference was 25.4%.
  • Example 6 Comparison of in vitro and in vivo stability of Boc-phenylalanine-terminated mPEG 2flflfl- PLA 18flfl copolymer/paclitaxel micelles and benzoyl-terminated mPEG 2flflfl- PLA 18flfl copolymer/paclitaxel micelles
  • mPEG ⁇ o-PLA ⁇ o-BP/paclitaxel micelles had no drug precipitation for at least 48h, while benzoyl terminated mPEG 2QQQ- PLA 18()() copolymer/paclitaxel micelles showed significant drug precipitation at 17h, mPEG 2 . . . -PLA 18 . .
  • the stability of the -BP/paclitaxel micelles was significantly higher than that of the benzoyl terminated mPEG 2 . . . Paclitaxel micelles prepared by -PLA 18 (K) .

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Abstract

本发明涉及一种新型的两亲性嵌段共聚物及其制备方法、以及该共聚物与抗肿瘤药物形成的胶束载药系统。该两亲性嵌段共聚物包括亲水性链段和疏水性链段,其疏水性链段端基用疏水性基团封端。本发明的两亲性嵌段共聚物以具有公认安全性的聚乙二醇单甲醚(或聚乙二醇)-聚酯嵌段共聚物为基础材料,并将聚酯链段的端羟基用疏水性基团进行改性,不仅改善药物分子和嵌段共聚物中疏水性链段的相容性,增加其相互间的作用力,同时为容纳药物分子提供更大的空间,所制备的胶束能够更有效的将药物分子限制在胶束的核中使其不易溶出,从而获得具有高度稳定性的载药胶束。

Description

一种两亲性嵌段共聚物及其制备方法、 以及该共聚物
与抗肿瘤药物形成的胶束载药系统 技术领域
本发明涉及一种两亲性嵌段共聚物及其制备方法、 以及该共聚物与抗肿瘤药物形成的 稳定胶束载药系统, 属纳米药物制剂领域。
背景技术
肿瘤是一类严重威胁人类生命安全的疾病, 研究安全、有效的抗肿瘤药物对于提高人 类的生存质量具有重要意义。
紫杉类药物 (主要包括紫杉醇 (Paclitaxel、 PTX)、 多西他赛 (Docetaxel、 DTX)、 卡 巴他赛 (cabazitaxel)、 莱龙泰素 (larotaxel)) 是一类非常有效且广谱的抗肿瘤药物, 其作 用机理主要是聚合和稳定微管,可致使快速分裂的肿瘤细胞固定于有丝分裂阶段,使癌细 胞复制受阻断而死亡。体外实验证明: 紫杉类药物具有显著的放射增敏作用, 可使细胞中 止于对放疗敏感的 G2和 M期。 然而, 几乎所有的紫杉类药物均是高度疏水性的, 其口 服吸收差, 目前只有注射途径给药。 由于难以配制成水溶液, 其市售制剂一般是通过添加 表面活性剂的方法来增加药物溶解性。然而, 该类增溶方法存在诸多缺点: (1 )无论是紫 杉醇 (商品名泰素) 中所使用的增溶剂聚氧乙烯蓖麻油 (Cremophor® EL)、 或者是多西 他赛(商品名泰素帝)和卡巴他赛(商品名 JEVTANA) 中所使用的增溶剂吐温 80, 均易 引发过敏反应, 因此患者用药前需进行抗过敏治疗; (2)药物稳定性差、注射利用度不高: 上述制剂经稀释后药物易沉淀析出,在给药时需经过特殊的过滤装置,且注射液稀释过程 需缓慢进行,往往因操作人员的不同而造成药物析出的程度不一致,从而导致进入体内的 药量不准确, 继而产生疗效差异; (3 ) 血液学毒性较高: Cremophor® EL和吐温 80均可 引起血液学毒性, 成为限制治疗剂量提高的主要因素。
阿霉素是一种抗肿瘤抗生素,和紫杉类药物一样属细胞毒类药物,可抑制 RNA和 DNA 的合成, 对 R A的抑制作用最强, 抗瘤谱较广, 对多种肿瘤均有作用, 属周期非特异性 药物, 对各种生长周期的肿瘤细胞都有杀灭作用。主要适用于急性白血病, 对急性淋巴细 胞白血病及粒细胞白血病。 常规的阿霉素制剂具有显著的心脏毒性和骨髓抑制等副作用。
表阿霉素为阿霉素的同分异构体, 与阿霉素相比, 疗效相等或略高, 但对心脏的毒性 较小。
姜黄素是近年来获得广泛关注的一种具有潜在抗肿瘤活性的非细胞毒类药物,其最大 的特点是几乎没有副作用, 并同时具有抗炎、抗氧化等辅助治疗效果。其最大缺点是水溶 性极差, 制备具有稳定性的水性姜黄素制剂是目前研究的热点。
聚合物胶束是近年来发展起来的一种新型给药系统。胶束通常由大量两亲性嵌段共聚 物分子链定向排列组成, 其疏水链段通过和药物分子之间弱的相互作用将药物包裹于核 中, 亲水链向外稳定胶束, 呈现典型的核-壳结构。 聚合物胶束不仅能增加药物的溶解度, 提高治疗剂量, 而且药物包裹其中, 可避免降解失活, 减少毒副反应。 胶束粒径通常在 lOOnm 以下, 且外围为亲水性 PEG链段, 因此可以躲避网状内皮系统 (RES) 的吞噬, 延长体循环时间,通过 EPR效应(高通透性和滞留效应, enhanced permeability and retention effect) 达到对肿瘤被动靶向的效果。 另外, 由于聚合物胶束分子量大, 因此也能防止肾 清除。 同小分子表面活性剂相比, 聚合物胶束的 CMC值 (临界胶束浓度) 非常低, 在载 药胶束稀释时, 也能保持胶束结构的稳定。 胶束给药系统载药量可达 25%, 完全满足临 床用量的需要, 同时聚合物材料具有生物可降解性和良好的生物相容性。
虽然聚合物胶束被认为是一种极具潜力的新型给药系统, 特别是针对一些难溶性的抗 肿瘤药物,但是其在溶液状态下较低的稳定性一直是影响这种新型给药系统向临床研究转 化的最关键问题, 尤其是紫杉类药物胶束, 其稳定性一般均较差。 以在韩国率先上市的注 射用紫杉醇胶束 (商品名 Genexol PM) 为例, 其溶液在室温条件下的稳定时间不超过 24h ( Lee SW, et al, Ionically Fixed Polymeric Nanoparticles as a Novel Drug Carrier, Pharmaceutical Research, 2007, 24: 1508-1516)。 虽然三养公司为改进紫杉醇胶束的稳定性 做了大量努力,如专利 CN01809632.8公开了将嵌段共聚物的端羟基用乙酰氧或者苯甲酰基 封端, 来增加疏水链段和药物的亲和性, 从而提高胶束的稳定性, 但以此共聚物制备的胶 束在体外室温下也只能稳定大约 3天, 体内稳定性则更差。 作为紫杉醇的衍生物的多西他 赛和卡巴他赛胶束溶液的稳定性更低, 至今为止, 以多西他赛胶束为例, 能向临床转化的 例子非常少, 其胶束溶液的稳定性差是一个关键原因 (Gaucher G, et al, Polyester based micelles and nanoparticles for the parenteral delivery of taxanes, Journal of Controlled Release, 2010, 143: 2-12) 。 如韩国三养公司的 Nanoxel PM™胶束 (聚合物辅料为 mPEG-PLA, 药 物为多西他赛), 当胶束的载药量为 5%, 药物浓度在 0.1-2mg/ml之间, 胶束在室温下只能 稳定 6h左右 ( Lee SW, et al, Development of docetaxel-loaded intravenous formulation, Nanoxel-PM™ using colymer-based delivery system, Journal of Controlled Release, 2011, 155: 262-271 ) 。 这种胶束在进入体内以后迅速解体, 药物随即和血液中的蛋白 (如白蛋白) 结合, 因此无法发挥胶束的 EPR效应, 动物实验的结果显示药效和多西他赛注射液没有区 别, 耐受剂量也未见提高, 因此优势并不明显。 另一方面, 由于卡巴他赛结构上和多西他 赛的相似性, 因此, 其 mPEG-PLA胶束的稳定性也与之类似。 我们研究后发现, 当 mPEG-PLA/卡巴他赛胶束的药物浓度为 5mg/mL时, 其溶液在室温条件下的稳定时间不超 过 2h, 这种胶束无论对体内使用的药效, 还是安全性均未得到有效提高, 此外, 对于这样 不稳定的胶束制剂, 其规模化的制备也存在很大的难度。
紫杉类药物是作为近 20年来肿瘤药物研发领域最伟大的发现之一, 同时也将是未来 20 年的主流抗肿瘤药。 由于其剂量限制性毒性, 充分发挥药物的疗效一直是人们研究的 热点。而胶束作为一种极具潜力的紫杉类药物给药系统,其不稳定性已成为这种给药系统 的最大缺陷,而造成这种不稳定性的原因至今依然并不十分清楚。为了提高紫杉类药物胶 束的稳定性, 人们做了大量的努力。 如专利 201010001047公开了一种在胶束溶液中添加 氨基酸提高其稳定性的方法,氨基酸在胶束形成的过程中添加,而对于氨基酸在胶束中所 处的位置(仅作为胶束的物理阻隔剂或者协同药物分子一起处于胶束的疏水核中)并未提 及,同时作为辅助添加剂的氨基酸在进入体内后经过血液的稀释后是否依然能保持胶束的 稳定性不得而知, 因此体内使用的效果依然不明确;另外有报道将紫杉醇和多西他赛一起 包埋于共聚物胶束中可显著提高胶束的载药量以及稳定性,但这种复合药物胶束在临床上 使用尚未得到认可 (邵铖炜等, 紫杉醇和多西紫杉醇双药胶束体外稳定性考察, 沈阳药科 大学学报, 2010, 41: 428-434); Huh等人合成了一种 PDENA-PEG嵌段共聚物胶束, 该 胶束包埋紫杉醇后显示长期的稳定性, 但对于该聚合物辅料的安全性尚缺乏足够数据支 持, 临床使用的安全挑战较大 (Huh KM, et al . Hydmtropic polymer micelle system for delivery of paclitaxel. Journal of Controlled Release, 2005, 101(1-3): 59-68)。
发明内容
本发明的目的在于提供一种两亲性嵌段共聚物, 以解决现有技术中存在的上述问题。 本发明的两亲性嵌段共聚物以具有公认安全性的聚乙二醇单甲醚 (或聚乙二醇) -聚酯嵌 段共聚物为基础材料,并将聚酯链段的端羟基用疏水性基团进行改性,引入具有叔丁氧羰 基或者带有苯环的氨基酸及其衍生物等具有较大空间结构的疏水性基团,不仅改善药物分 子和嵌段共聚物中疏水性链段的相容性,增加其相互间的作用力,而且引入的疏水性基团 具有较大的空间结构,为药物分子进入胶束的核提供了更大的空间,从而使其更加不易溶 出,从而获得具有高度稳定性的载药胶束。该发明的最大意义在于提高胶束的在溶液状态 的稳定性, 尤其是体内的稳定性, 从而发挥胶束的 EPR效应, 达到更高的生物利用度和 更好的治疗效果。
本发明提供的技术方案如下: 一种两亲性嵌段共聚物, 其特征在于: 其亲水性链段为数均分子量在 400 20000之间 的聚乙二醇 (PEG) 或聚乙二醇单甲醚 (mPEG), 其疏水性链段选自采用疏水性基团封 端的数均分子量在 500 100000之间的聚丙交酯 (PLA)、 聚乙交酯 (PGA)、 聚乙丙交酯 (PLGA)、聚己内酯(PCL)、聚碳酸酯(PTMC)或其衍生物、或者聚二氧环己酮(PPDO) 或其衍生物中的一种, 所述的疏水性基团选自叔丁酰基、 叔丁乙酰基、 氨基酸残基、 或氨 基酸衍生物残基中的一种。
在推荐的实施例中, 所述的氨基酸衍生物优选为 γ-苄基谷氨酸、 β-苄基天冬氨酸或氨 基得到保护的氨基酸衍生物。
在推荐的实施例中,所述的氨基酸衍生物进一步优选为含有苄基保护或者叔丁氧羰基 (Boc) 保护的氨基酸。
在推荐的实施例中, 所述的氨基酸衍生物更优选为叔丁氧羰基苯丙氨酸。
本发明中,所述疏水性链段优选为数均分子量在 1000 50000之间的聚丙交酯(PLA)、 聚乙交酯 (PGA)、 聚 (乙丙交酯) (PLGA)、 聚己内酯 (PCL)、 聚碳酸酯 (PTMC) 或 其衍生物、 或者聚二氧环己酮 (PPDO) 或其衍生物; 所述亲水性链段的优选为数均分子 量在 750 5000之间的聚乙二醇或聚乙二醇单甲醚。
本发明的另一目的在于提供上述两亲性嵌段共聚物的制备方法。
本发明提供的技术方案如下:
上述两亲性嵌段共聚物的制备方法, 包括如下的步骤:
1 )取数均分子量在 400 20000之间的亲水性链段加入到聚合瓶中,加热至 100°C~130 °〇真空脱水 2h~4h后加入形成疏水性链段的聚合物单体和单体重量 0.3%。_1%。的催化剂辛 酸亚锡,真空密闭反应瓶,上述反应物在 100°C~150°C反应 12h~24h后用二氯甲烷、乙醇、 四氢呋喃、 甲醇、 乙酸乙酯或丙酮溶解, 加入大量乙醚充分沉淀聚合物后经过滤, 真空干 燥得亲水性链段和疏水性链段组成的嵌段共聚物, 所述的亲水性链段聚乙二醇(PEG)或 聚乙二醇单甲醚 (mPEG);
2) 取亲水性链段和疏水性链段组成的嵌段共聚物, 用乙酸乙酯、 四氢呋喃、 二氯甲 烷、 乙酸乙酯或双蒸水溶解, 而后加入叔丁酰基、 叔丁乙酰基、 氨基酸残基、 或氨基酸衍 生物残基等进行反应,使端羟基反应形成为疏水性基团,过滤去除不溶物后加入足量乙醚 沉淀聚合物, 过滤真空干燥后得目标共聚物。
本发明的另一个目的在于提供上述两亲性嵌段共聚物与抗肿瘤药物形成的胶束载药 系统。 本发明提供的技术方案如下:
上述两亲性嵌段共聚物与抗肿瘤药物形成的胶束载药系统,该胶束载药系统包含至少 一种上述两亲性嵌段共聚物、治疗有效量的至少一种抗肿瘤药物以及药学上可接受的药用 辅料。
在推荐的实施例中, 所述的药用辅料为冻干赋型剂。
在推荐的实施例中, 所述的冻干赋型剂为乳糖、 甘露醇、 蔗糖、 海藻糖、 果糖、 葡萄 糖、 海藻酸钠或明胶中的至少一种。
所述的药用辅料还包括抗氧剂、 金属离子络合剂、 pH调节剂或等渗调节剂等, 抗氧 剂为亚硫酸钠、亚硫酸氢钠或焦亚硫酸钠等; 金属离子络合剂为依地酸二钠、依地酸钠钙 或环己二胺四醋酸钠等; pH调节剂为枸櫞酸、 碳酸氢钠、 磷酸氢二钠或磷酸二氢钠等; 等渗调节剂为氯化钠或葡萄糖等。
在推荐的实施例中,所述的抗肿瘤药物为紫杉类药物紫杉醇(PTX)、多西他赛 (DTX)、 卡巴他赛 (cabazitaxel)、 莱龙泰素 (larotaxel), 以及姜黄素、 阿霉素或表阿霉素等中的至 少一种。
在推荐的实施例中, 两亲性嵌段共聚物中两亲性嵌段共聚物和药物的重量比在 99.5:0.5-50:50之间, 优选 99: 1 -75:25之间。
在推荐的实施例中, 冻干赋型剂占整个体系的重量比为 0~99.9%之间, 优选 10.0~80.0%之间。
本发明制备得到的抗肿瘤药物聚合物胶束制剂可以用于治疗癌症,优选用于治疗乳腺 癌、 前列腺癌、 卵巢癌、 肠癌、 肺癌、 肝癌、 头颈癌等。
本发明中提及的治疗有效量指的是上述胶束载药系统含有的抗肿瘤药物的量能有效 地治疗癌症(具体地可以指乳腺癌、 前列腺癌、 卵巢癌、 肠癌、 肺癌、 肝癌、 头颈癌等)。
本发明的胶束载药系统可通过注射途径给药, 并一般制成冻干粉制剂, 另外, 本领域 技术人员可参照现有抗肿瘤药物的给药剂量确定给药剂量,并根据个体情况的不同上下调 整。
本发明还提供了两亲性嵌段共聚物与抗肿瘤药物形成的胶束载药系统的制备方法,包 括透析法、 直接溶解法、 薄膜水化法、 固体分散法、 高能均质乳化法, 优选薄膜水化法和 固体分散法。
薄膜水化法的具体步骤为: 将聚合物辅料和药物溶解于有机溶剂, 经旋转蒸发去除溶 剂后加入注射用水溶解药膜得载药胶束溶液, 经过滤除菌冻干后得胶束冻干粉。 固体分散法的具体步骤为: 将药物溶解于加热后处于熔融状态的聚合物辅料(这一过 程可适当添加少量有机溶剂帮助溶解)得到澄清的混合物,再加入注射用水溶解得胶束溶 液, 经过滤除菌后冻干得胶束冻干粉。
与现有技术相比, 本发明具有以下的特点:
1 ) 本发明根据大多数抗肿瘤药物结构上的疏水性以及较大的空间结构, 将聚酯链段 的端羟基用疏水性基团进行改性, 通过改善药物分子和嵌段共聚物中疏水性链段的相容 性, 增加其相互间的作用力, 同时增加胶束核中可容纳药物分子的空间, 将药物分子限制 在胶束的核中使其不易溶出, 从而获得了一系列在体内外均具有高度稳定性的载药胶束, 该载药胶束可制成冻干制剂;
2) 试验结果证明: 本发明的两亲性嵌段共聚物制备得到的抗肿瘤药物载药胶束的制 成的冻干制剂复溶后可迅速分散形成略带蓝色乳光的澄清溶液,该溶液在室温环境下依然 至少可稳定 24小时以上无明显药物沉淀析出, 经注射后在体内可有效发挥 EPR效应, 具 有良好的产业化应用前景。
附图说明
附图 1为 mPEG2()()(rPLGA2mw-TB凝胶渗透色谱图, PDI=1.05;
附图 2为 mPEG^Q-PLA oQ-BP凝胶渗透色谱图, PDI=1.05;
附图 3为 mPEG2。。。-PLA18。。-BP的核磁共振氢谱图;
附图 4为 mPEG¾K )-PLAim)-Asp凝胶渗透色谱图, PDI=1.05;
附图 5为 mPEG2。。。-PLGA2。。。-TS凝胶渗透色谱图, PDI=1.05;
附图 6为 PEG2ooo-PLGA2()()()-TS凝胶渗透色谱图, PDI=1.05;
附图 7为 mPEG5。。。-PCL4。。。-BP的凝胶渗透色谱图, PDI=1.07
附图 8为 mPEGsooo-PC ooo-BP的核磁共振氢谱图
附图 9为 mPEG5。。。-PCL4。。。-BP/紫杉醇胶束粒径图;
附图 10为 mPEG2(KKrPLA18Q(rBP/多西他赛胶束粒径图;
附图 11 ¾mPEG2ooo-PLGA20oo-TB /卡巴他赛胶束粒径图;
附图 12为 mPEG2GGG-PLA18Q()-BP/多西他赛胶束溶液稳定性试验结果;
附图 13为 mPEG^o-PLGA^o-TB /卡巴他赛胶束溶液稳定性试验结果;
附图 14为多西他赛注射液与多西他赛胶束注射液对 H460肿瘤抑制作用; 附图 15为多西他赛注射液与多西他赛胶束注射液对 H460肿瘤抑制作用; 附图 16为多西他赛注射液及多西他赛胶束注射液对 MDA-MB-231肿瘤抑制作用; 附图 17为多西他赛注射液及多西他赛胶束注射液对 MDA-MB-231肿瘤抑制作用; 附图 18为 iv给予多西他赛胶束 (5 mg/kg) 血浆药物浓度经时变化曲线;
附图 19为 iv给予卡巴他赛胶束 (5mg/kg) 血浆药物浓度经时变化曲线;
附图 20为大鼠 iv给予多西他赛胶束(5mg/kg)血浆药物总量及包封药物浓度的经时 变化曲线;
附图 21为大鼠 iv给予卡巴他赛胶束(5mg/kg)血浆药物总量及包封药物浓度的经时 变化曲线;
附图 22为荷瘤(MX-1 )裸鼠瘤组织内多西他赛药物浓度经时变化曲线(iv 10mg/kg); 附图 23 mPEG2。。。-PLA18。。-BP/紫杉醇胶束和苯甲酰基封端的 mPEG2。。。-PLA18。。紫杉醇 胶束大鼠药时曲线。
具体实施方式
为了便于理解本发明, 特列举实施例, 以进一步诠释本发明, 而不是对本发明的任何 方式的限制。
实施例 1 两亲性嵌段共聚物的制备
( 1 ) 合成使用叔丁酰基封端的甲氧基聚乙二醇-聚 (乙丙交酯) 嵌段共聚物 ( mPEG誦 -PLGA誦 -TB )
18.2g mPEG (数均分子量为 2000) 加入到聚合瓶中, 加热至 100°C真空脱水 3h后加 入 26mg辛酸亚锡, 11.6g 乙交酯 ( GA) 和 14.4g D, L-丙交酯 (D, L-LA), 真空密闭反应 瓶, 上述反应物在 13CTC反应 15h后用二氯甲烷溶解反应物, 加入大量乙醚充分沉淀聚合 物后经过滤, 真空干燥得 mPEG2。。。-PLGA2。。。嵌段共聚物。
取 4g mPEG2ooo-PLGA20oo¾PA 20ml二氯甲烷溶解,加入 0.5g碳酸钾,搅拌下加入 0.25g 特戊酰氯, 室温反应 24h, 过滤去除不溶物后加入大量乙醚沉淀聚合物, 过滤真空干燥后 得 mPEG誦 -PLGA誦 -TB。
注: TB为叔丁酰基的缩写。
mPEG2。。。-PLGA2。。。-TB的凝胶渗透色谱图如图 1中所示, PDI为 1.05 ;
以氘代氯仿为溶剂, 400MBruker核磁共振仪测定聚合物结构, mPEG2Q(K)-PLGA2()()Q-TB 的核磁共振氢谱数据如下:
1H NMR (CDC13) δ (1.56ppm, ( CH3 3C, CHC¾0), δ (3.55-3.75ppm, 4H, CH2CH20), δ (5.15-5.23ppm, 1H, CHCH30) 。
( 2 ) 合成使用叔丁氧羰基苯丙氨酸残基封端的甲氧基聚乙二醇-聚丙交酯嵌段共聚物 (mPEG誦 -PLA誦 -BP)
20g mPEG (数均分子量为 2000)加入到聚合瓶中, 加热至 120°C真空脱水 3h后加入 25mg辛酸亚锡和 25g D, L-丙交酯 (D, L-LA), 真空密闭反应瓶, 上述反应物在 130°C反 应 12h后用乙醇溶解反应物, 加入大量乙醚充分沉淀聚合物后经过滤, 真空干燥得 mPEG2。。。-PLA18。。嵌段共聚物。
6.65g Boc-L-苯丙氨酸溶解于 50ml无水乙酸乙酯, 加入 4.2ml三乙胺, 将上述溶液冷 却至 -10°C, 加入 3.66ml特戊酰氯, 上述反应物在 0°C搅拌反应 2h后在室温下继续反应 lh, 过滤去除不溶物, 真空去除溶剂得粘稠液体。
加入 25ml二氯甲烷溶解后将此溶液加入到含有 15g mPEG2ooo-PLA180oW 75ml二氯甲 烷溶液中, 充分混合溶解后加入 14ml吡啶和 160mg四甲氨基吡啶, 此混合物在 0°C反应 2h后在室温下继续反应 24h, 过滤去除溶剂后所得聚合物加入 100ml乙醇溶解, 溶液在 -20 °C冷冻 lh后过滤聚合物, 真空干燥后得 mPEG G-PLA18QQ-BP。
注: BP为叔丁氧羰基苯丙氨酸残基的缩写。
mPEG2。。。-PLA18。。-BP的凝胶渗透色谱图如图 2中所示, PDI为 1.05 ;
以氘代氯仿为溶剂, 400MBruker核磁共振仪测定聚合物结构, mPEG2。。。-PLA18。。- BP 的核磁共振氢谱图如附图 3中所示, 其核磁共振氢谱数据如下:
1H NMR (CDC13) δ (1.38-1.41ppm, 9H, ( CH3) 3C, ), δ (1.51-1.60ppm, 3H, CHCH?0), δ (3.38ppm, 2H, CH2C6H5) , δ (3.63-3.70ppm, 4H, CH2CH20) , δ (4.60-4.66ppm, 1H, CHCH2C6H5), δ (5.15-5.17ppm, 1H, CHCH30)。
( 3 ) 合成使用 β-苄基天冬氨酸残基封端的甲氧基聚乙二醇-聚丙交酯嵌段共聚物 (mPEG2ooo-PLAi80o-Asp)
mPEG oo-PLA^oo合成方法同实施例 1 (2)。
L-天冬氨酸苄酯 22.3g, 加入去离子水 100ml, 二氧六环 100ml做混合溶剂, 搅拌, 再加入 NaOH 4N约 30ml搅拌至苄酯完全溶解, 将反应瓶置于冰水浴中, 控制温度低于 4 °C。另外取溴乙酰溴 8.7ml溶于 35ml精制的二氧六环和 4N NaOH 约 25ml,在剧烈搅拌下, 用两个恒压漏斗同时缓慢滴加两种溶液, 控制溶液 pH=8-9 (约 30分钟左右滴完)。 滴加 完毕后继续反应 5分钟, 用浓盐酸将溶液 pH值调至 2, 再用 200ml乙醚萃取, 混合物在乙 醚层中, 再用饱和 NaCl溶液洗 5次。 最后加入无水 MgS04干燥 48小时, 在真空中浓缩产 生一种黄色粘油, 在培养皿中静置, 得到 25g晶体为溴乙酰化天冬氨酸苄酯。
将 10g mPEG QQ-PLA18()()溶解于 50ml二氯甲烷, 加入 5ml三乙胺和 2.5g溴化天冬氨酸 苄酯, 室温下搅拌反应 24后将反应物用乙醚沉淀, 真空干燥得mPEG2。。。-PLA18。。-ASp。 注: Asp为苄基天冬氨酸残基的缩写。
1^502。。。-?1^18(^¾^的凝胶渗透色谱图如图 4中所示, PDI为 1.05。
以氘代氯仿为溶剂, 400MBruker核磁共振仪测定聚合物结构, mPEG oo-PLA^oo-Asp 的核磁共振氢谱数据如下:
1H NMR (CDC13) δ (1.51-1.60ppm, 3H, CHO¾0), δ (2.90-3.15ppm, 2H, OCOCH2), δ (3.63-3.70ppm, 4H, CH2CH20), δ (5.13-5.15ppm, 2H, CH2C6H5) , δ (5.15-5.17ppm, IH, CHCH30), δ (7.34ppm, 5H, OJ¾。
( 4 ) 合成使用络氨酸残基封端的甲氧基聚乙二醇-聚 (丙交酯 /乙交酯) 嵌段共聚物 (mPEG2ooo-PLGA20oo-TS )
mPEG2。。。-PLGA2。。。合成方法同实施例 1 ( 1 )。
将 lOg mPEG oo-PLGA oo溶解于 200ml双蒸水,加入 1.91g 1-乙基 -(3-二甲基氨基丙基) 碳二亚胺盐酸盐和 1.8g络氨酸, 室温反应 48小时后用 200ml二氯甲烷分三次萃取产物, 合并有机相后饱和盐水洗涤五次, 有机相用无水硫酸镁干燥后用乙醚沉淀, 真空干燥得 mPEG誦 -PLGA誦 -TS。
注: TS为络氨酸残基的缩写。
mPEG2。。。-PLGA2。。。-TS的凝胶渗透色谱图如图 5中所示, PDI为 1.05。
以氘代氯仿为溶剂, 400MBruker核磁共振仪测定聚合物结构, mPEG2ooo-PLGA20oo-TS 的核磁共振氢谱数据如下:
1H NMR (CDC13) δ (1.51-1.60ppm, 3H, CHO¾0), δ (2.94-3. OOppm, 2H, CH2C6H5), δ (3.63-3.70ppm, 4H, CH2CH20), δ (5.15-5.17ppm, IH, CHCH3O), δ (6.74-6.93ppm, 5H, Q¾)。
( 5 ) 合成使用络氨酸残基封端的聚乙二醇-聚 (丙交酯 /乙交酯) 嵌段共聚物 (PEG2ooo-PLGA20oo-TS )
PEG2。。。-PLGA2。。。合成方法参照实施例 1 ( 1 )。
将 10g PEG OO-PLGA^OO溶解于 200ml双蒸水, 加入 1.91g 1-乙基 -(3-二甲基氨基丙基) 碳二亚胺盐酸盐和 1.8g络氨酸, 室温反应 48小时后用 200ml二氯甲烷分三次萃取产物, 合并有机相后饱和盐水洗涤五次, 有机相用无水硫酸镁干燥后用乙醚沉淀, 真空干燥得 PEG2000-PLGA2000-TS。
注: TS为络氨酸残基的缩写。
PEG2。。。-PLGA2。。。-TS的凝胶渗透色谱图如图 6中所示, PDI为 1.05。 以氘代氯仿为溶剂, 400MBruker核磁共振仪测定聚合物结构, PEG2。。。-PLGA2。。。-TS 的核磁共振氢谱数据如下:
1H NMR (CDC13) δ (1.51-1.60ppm, 3H, CHO¾0), δ (2.94-3. OOppm, 2H, CH2C6H5), δ (3.63-3.70ppm, 4H, CH2CH20), δ (5.15-5.17ppm, 1H, CHCH30), δ (6.74-6.93ppm, 5H, Q¾)。
(6) 合成叔丁氧羰基苯丙氨酸残基封端的甲氧基聚乙二醇-聚己内酯嵌段共聚物 (mPEG5000-PCL棚 -BP)
20g mPEG (数均分子量为 5000)加入到聚合瓶中, 加热至 130°C真空脱水 4h后加入 20mg辛酸亚锡和 20g 己内酯(CL), 真空密闭反应瓶, 上述反应物在 130°C反应 24h后用 二氯甲烷溶解反应物, 加入大量乙醚充分沉淀聚合物后经过滤, 真空干燥得 mPEG5。。。-PCL4。。。嵌段共聚物。
6.65g Boc-L-苯丙氨酸溶解于 50ml无水乙酸乙酯, 加入 4.2ml三乙胺, 将上述溶液冷 却至 -10°C, 加入 3.66ml特戊酰氯, 上述反应物在 0°C搅拌反应 2h后在室温下继续反应 lh, 过滤去除不溶物, 真空去除溶剂得粘稠液体。
加入 25ml二氯甲烷溶解后将此溶液加入到含有 30g mPEG^o-PLA^o的 150ml二氯甲 烷溶液中, 充分混合溶解后加入 14ml吡啶和 160mg四甲氨基吡啶, 此混合物在 0°C反应 2h后在室温下继续反应 24h, 过滤后所得聚合物溶液用 -20°C乙醚沉淀, 真空干燥后得 mPEG5000-PCL棚 -BP。
注: BP为叔丁氧羰基苯丙氨酸残基的缩写。
mPEG5。。。-PCL4。。。-BP的凝胶渗透色谱图如图 7中所示, PDI为 1.08;
以氘代氯仿为溶剂, 400MBruker核磁共振仪测定聚合物结构, mPEG2。。。-PLA18。。- BP 的核磁共振氢谱图如附图 8中所示, 其核磁共振氢谱数据如下:
1H NMR (CDC13) δ (1.38-1.43ppm, 9H, ( CH3) 3C, ), δ (1.53-1.64ppm, 4H, CH2CH2CH2), δ (2.34ppm, 2H, COCH2CH2), δ (3.63-3.70ppm, 4H, CH2CH20) , δ (4.06-4.15ppm, 2H, OCH2C¾)。
实施例 2抗肿瘤药物聚合物胶束冻干制剂制备
( 1 ) mPEG^o-PC ooo-BP/紫杉醇胶束及其冻干制剂的制备
取 150mg实施例 1中制得的 mPEG5。。。-PCL4。。。-BP和 30mg紫杉醇溶解于 2ml四氢呋喃, 搅拌下缓慢滴加 5ml超纯水,滴加完毕后该溶液在室温下搅拌过夜去除有机溶剂后得带有 明显蓝色乳光的澄清紫杉醇胶束溶液, 加入 120mg甘露醇, 所得溶液经 0.22μηι除菌膜过 滤后冻干得紫杉醇胶束冻干粉。 经 LC-MS/MS分析, 药物包封率为 98.6%, 载药量大于 11.2%, 其粒径测定结果则如图 9中所示, 平均粒径 33.8nm, 分散系数 PDI=0.1。
该胶束冻干粉用生理盐水复溶成 5mg/mL浓度的溶液后, 在室温下稳定时间大于 7 天, 显著高于乙酰氧基和苯甲酰基封端聚合物胶束。
( 2 ) mPEG^o-PLA^o-BP/多西他赛胶束及其冻干制剂的制备
取 lOOmg多西他赛和 1.9g实施例 1中制得的 mPEG2(KKrPLA18Q(rBP溶解于 25ml无水乙 醇, 45 °C下通过旋转蒸发去除有机溶剂, 加入 25ml生理盐水溶解药膜, 加入 400mg乳糖 后溶液经 0.22μηι除菌膜过滤后冻干后得多西他赛胶束冻干粉。 经 LC-MS/MS分析, 药物 包封率为 95.8%, 载药量 4.76%, 其粒径测定结果则如图 10中所示, 平均粒径为 23.3nm, 分散系数 PDI=0.02。
该胶束冻干粉经生理盐水复溶成 5mg/mL浓度的溶液后, 在室温下存放 90天溶液中 溶解状态的多西他赛含量大于 90%。
( 3 ) mPEG2ooo-PLGA20oo-TB /卡巴他赛胶束及其冻干制剂的制备
取 1.9g实施例 1 中制得的 mPEG2。。。-PLGA2。。。-TB加热至 50°C使之熔融, 然后加入 lOOmg卡巴他赛, 在搅拌下使其溶解于聚合物中得到澄清透明的混合物, 加入 25ml预热 至 50°C的生理盐水溶解聚合物 /药物的混合物得胶束溶液, 再加入 400mg蔗糖后溶液经 0.22除菌膜过滤后冻干得卡巴他赛胶束冻干粉。经 LC-MS/MS分析,药物包封率为 96.2%, 载药量大于 4.82%, 其粒径测定结果则如图 11 中所示, 平均粒径为 24.2nm, 分散系数 PDI=0.02。
该胶束冻干粉经生理盐水复溶成 5mg/mL浓度的溶液后, 在室温下存放 90天溶液中 溶解状态的卡巴他赛含量大于 90%。
( 4 ) mPEG2ooo-PLA180o-BP /姜黄素胶束及其冻干制剂的制备
取 lOOmg姜黄素和 1.9g实施例 1中制得的 mPEG oo-PLA^oo-BP溶解于 25ml无水乙醇, 50°C下通过旋转蒸发去除有机溶剂, 加入 25ml生理盐水溶解药膜, 加入 400mg海藻酸钠 后溶液经 0.22μηι除菌膜过滤后冻干后得姜黄素胶束冻干粉。 经 LC-MS/MS分析, 药物包 封率为 98.9%, 载药量 4.88%, 其粒径测定结果平均粒径为 15.3nm, 分散系数 PDI=0.02。
该胶束冻干粉经生理盐水复溶成 5mg/mL浓度的溶液后, 在室温下存放 7天溶液中 溶解状态的姜黄素含量大于 90%。
(5 ) mPEG2ooo-PLGA20oo-TB /阿霉素胶束及其冻干制剂的制备
取 300mg实施例 1中制得的 mPEG2。。。-PLGA2。。。-TB和 40mg盐酸阿霉素于 40°C溶解于 氯仿, 加入 0.1ml三乙胺室温下搅拌 lh, 旋转蒸发去除有机溶剂, 加入 50ml浓度为 10mM 的 HBS缓冲溶液溶解药膜, 透析或者超滤法去除三乙胺盐酸盐后加入 250mg明胶溶液经 0.22μηι除菌膜过滤后冻干后得阿霉素胶束冻干粉。 经 LC-MS/MS分析, 药物包封率为 94.7%, 载药量大于 22.47%, 其平均粒径为 19.4nm, 分散系数 PDI=0.04。
该胶束冻干粉经生理盐水复溶成 2mg/mL浓度的溶液后, 在室温下存放 24h溶液中 溶解状态的阿霉素含量大于 90%。
实施例 3稳定性试验
( 1 ) mPEG G-PLA18QQ-BP/多西他赛胶束稳定性试验
mPEG2。。。-PLA18。。-BP/多西他赛胶束冻干粉复溶后 (浓度为 6mg/ml, 以多西他赛计) 放置于 25 °C的恒温箱中保存, 一定时间后取出, 在 lOOOOrpm下离心 lOmin后用高效液相 色谱测定上清液中的药物含量。
溶解状态的药物含量随时间变化如图 12所示: mPEG2。。。-PLA18。。- BP/多西他赛胶束 溶液中处于溶解状态的药物在开始的 15天内呈现一定程度的下降, 后期药物的溶出非常 缓慢, 即使 90天内溶解状态的药物依然保持在 90%以上。
( 2 ) mPEG2ooo-PLGA20oo-TB /卡巴他赛胶束稳定性试验
mPEG2ooo-PLGA20oo-TB /卡巴他赛胶束冻干粉复溶后 (浓度为 6mg/ml, 以卡巴他赛 计) 放置于 25 °C的恒温箱中保存, 一定时间后取出, 在 lOOOOrpm下离心 lOmin后用高效 液相色谱测定上清液中的药物含量。
溶解状态的药物含量随时间变化如图 13所示: mPEG2。。。-PLGA2。。。-TB /卡巴他赛胶 束溶液中处于溶解状态的药物在开始的 3天内呈现一定程度的下降,后期药物的溶出非常 缓慢, 即使 90天内溶解状态的药物依然保持在 90%以上。
实施例 4药效学试验
( 1 ) 多西他赛注射液与多西他赛胶束冻干粉对人肺癌 H460裸鼠肿瘤抑制作用
A、 药品及试剂:
实施例 2中制得的多西他赛胶束冻干粉, 由山东靶点药物研究有限公司提供, 0.9% 生理盐水溶解;
多西他赛注射液 (Docetaxel Injection), 每瓶 0.5ml: 20mg, 齐鲁制药有限公司生产。 空白溶剂对照 (空白胶束, 10mg/kg), 由山东靶点药物研究有限公司提供, 0.9%生 理盐水溶解。
B、 实验动物:
BALB/c nu小鼠, 4〜6周龄, 雌雄兼用 (根据瘤株需要每批实验选用同一性别), 由 北京华阜康生物科技股份有限公司提供, 合格证号: SCXK (京) 2009-0004。
C、 词养设施条件:
中国医学科学院药物研究所动物实验中心屏障设施, 许可证编号: SYXK (京) 2009-0004, 有效期: 2009年 2月 25日〜 2014年 2月 25日。
D、 肿瘤瘤株:
人肺癌 H460, 细胞引自 ATCC, 由本实验室进行体外培养, 接种于裸鼠成瘤, 传代 保存。
E、 试验方法
选择 H460肿瘤生长良好, 全身状况较好荷瘤动物, 颈椎脱臼处死。 无菌条件下取出瘤 ±夬, 用手术刀切割成直径 2〜3mm的瘤块, 套管针接种于裸小鼠腋部皮下。 接种后第 7〜8 天, 荷瘤鼠平均瘤体积达 110〜120mm3许, 将动物按瘤体积大小分层分组, 每组 8只。
设阴性对照组、 空白溶剂对照组、 多西他赛注射液组(10mg/kg/次)及多西他赛胶束 冻干粉组(10mg/kg/次, 以多西他赛计) ; 其中阴性对照组动物肿瘤自然生长, 空白溶剂 对照组采用与多西他赛胶束冻干粉组相同体积溶剂,多西他赛注射液也稀释成与多西他赛 胶束冻干粉组相同体积给药, 各给药组同时静脉注射。
自分组当日开始, 每隔 3天对各组动物按计划静脉注射给药 1次, 共给药 3次。 待 阴性对照组平均肿瘤体积达 2000mm3左右时终止观察。
实验统计及疗效评价方法:
(A) 肿瘤体积计算公式: V=a X b72 (其中 a、 b分别表示长和宽)
(B)计算相对肿瘤体积 (RTV), 计算公式为: Vt/Vo
(其中 Vo为分笼给药时测量所得 TV, Vt为以后每次测量时的 TV。 )
TV为肿瘤体积。
(C)抗肿瘤活性的评价指标为相对肿瘤增殖率 T/C (%)计算公式为:
治疗 έ日 ( Τ) RTV
T/C (%) = 丑 ^ ~ X 100
阴性对照组 ( C) RTV
(D) 计算药物对肿瘤生长抑制率, 计算公式为: 體欄率 (%) =对照组平 ^ 平均瘤重 應
对^照组平均^瘤重
(E)比较各组动物肿瘤重量、肿瘤体积、 RTV等指标差别的统计学意义采用 检验法。
(F)疗¾¾^|介示准: T/C(%)〉40为无效; T/C(%) 40, 并经^ i十学处理 P〈0. 05为有效。 F、 试验结果和结论:
多西他赛注射液给药组 (10mg/kg/次) 和多西他赛胶束冻干粉给药组 (10mg/kg/次) 对 H460裸鼠肿瘤生长抑制作用非常明显, 肿瘤体积明显减小。 其中多西他赛胶束冻干粉 给药组 (10mg/kg/次) 瘤体积在给药期间逐渐缩小, 并维持近 10天低于给药前平均值。 与多西他赛注射液给药组(10mg/kg/次)相比, 相同剂量的多西他赛胶束静脉注射对肿瘤 抑制作用明显提高, 瘤重抑制率前者 68.35%, 后者 97.5%, 相对肿瘤增殖率前者 25.25%, 后者仅为 3.78%, 疗效有所提高。
多西他赛注射液与多西他赛胶束冻干粉对 H460肿瘤抑制作用如图 14、图 15中所示。 多西他赛注射液与多西他赛胶束冻干粉对 H460肿瘤的抑瘤率及相对肿瘤增殖率如表 1和表 2中所示。
表 1 多西他赛注射液与多西他赛胶束冻干粉对 H460肿瘤抑制作用
动物数 (只) 体重 (g) 瘤 重 抑瘤率 组 另 iJ 开始 结束 开始 ^¾ (g) ( %) 阴性对照组 8 8 21.0±1.00 24.0±1.41 1.72土 0.517
溶剂对照组 8 8 20.9±1.39 24.1土 1.76 1.62土 0.510 5.81 多西他赛注射液 (10 8 8 21.3±0.97 23.1±2.20 0.545±0.166* 68.35 mg/kg)
多西他赛胶束 ( 10 8 8 21.6±1.11 21.6±2.34 0.04土 0.018* 97.5 mg/kg)
*: P<0.001 , 与阴性对照组比较 表 2 多西他赛注射液与多西他赛胶束冻干粉对 H460肿瘤抑制作用
肿瘤体积 (mm3 ) T/C 组 另 J 开始 结束 RTV (%) 阴性对照组 119土 25.5 1947±748.7 16.87土 6.684
溶剂对照组 109土 20.8 1810土 481.9 16.63土 3.179 94.02 多西他赛注射液(10 mg/kg) 121土 25.3 509土 165.4 3.93土 2.838** 25.25 多西他赛胶束 (10 mg/kg) 112土 29.0 69.3土 27.2 0.64±0.243** 3.78
*: P<0.01, 与阴性对照组比较; **: P<0.001 , 与阴性对照组比较
( 2 ) 多西他赛注射液与多西他赛胶束对人乳腺癌 MDA-MB-231裸鼠肿瘤抑制作用
A、 药品及试剂: 同实施例 4 ( 1 )。
B、 实验动物: 同实施例 4 ( 1 )。
C、 词养设施条件: 同实施例 4 ( 1 )。
D、 肿瘤瘤株:
人乳腺癌 MAD-MB-231 , 细胞引自 ATCC, 由本实验室进行体外培养, 接种于裸鼠 成瘤, 传代保存。 E、 试验方法
选择 MDA-MB-231肿瘤生长良好, 全身状况较好荷瘤动物, 颈椎脱臼处死。 无菌条件 下取出瘤块, 用手术刀切割成直径 2〜3mm的瘤块, 套管针接种于裸小鼠腋部皮下。 接种后 第 11天, 荷瘤鼠平均瘤体积达 110〜120mm3许, 将动物按瘤体积大小分层分组, 每组 8_9 只。
设阴性对照组、 空白溶剂对照组、 多西他赛注射液组(10mg/kg/次); 多西他赛胶束 冻干粉组(10mg/kg/次, 以多西他赛计) ; 其中阴性对照组动物肿瘤自然生长, 空白溶剂 对照组采用与多西他赛胶束 10mg/kg剂量组相同体积溶剂,多西他赛注射液也稀释成与多 西他赛胶束冻干粉组相同体积给药, 各给药组同时静脉注射。
自分组当日开始, 每隔 3天对各组动物按计划静脉注射给药 1次, 共给药 3次。 待 阴性对照组平均肿瘤体积达 2000mm3左右时终止观察。 实验统计及疗效评价方法:
(A) 肿瘤体积计算公式: V=a X b72 (其中 a、 b分别表示长和宽)
(B)计算相对肿瘤体积 (RTV), 计算公式为: Vt/Vo
(其中 Vo为分笼给药时测量所得 TV, Vt为以后每次测量时的 TV。 )
(C)抗肿瘤活性的评价指标为相对肿瘤增殖率 T/C (%)计算公式为:
治疗 ( T) RTV
T/C (%) = 丑 X 100
阴性对照组 ( C) RTV
(D) 计算药物对肿瘤生长抑制率, 计算公式为: 體欄率 (%) =对照组平 均瘤重 X 100
对照 ^组平均^ 平
瘤重
(E) 比较各组动物肿瘤重量、 肿瘤体积、 RTV等指标差别的统计学意义采用 检验 法。
(F)疗效 iff介标准: T/C(%)〉40为无效; T/C(%) 40, 并经统计学麵 P〈0. 05为有效。
F、 试验结果和结论:
将多西他赛注射液给药组(10mg/kg/次)及多西他赛胶束冻干粉给药组(10mg/kg/次) 对荷乳腺癌 MDA-MB-231小鼠间断静脉注射 3次, 药物对裸鼠肿瘤生长表现非常明显抑制 作用, 3次给药后, 小鼠肿瘤体积较给药前进行性缩小, 多西他赛胶束冻干粉给药组
( 10mg/kg/次) 肿瘤几乎停止生长。 经比较, 同剂量的多西他赛胶束冻干粉给药组对
MDA-MB-231肿瘤抑制作用好于相同剂量的多西他赛注射液给药组。
多西他赛注射液及多西他赛胶束冻干粉对 MDA-MB-231肿瘤抑制作用如图 16、 图 17 中所示;
多西他赛注射液及多西他赛胶束冻干粉对 MDA-MB-231肿瘤的抑瘤率和及相对肿瘤 增殖率如表 3、 表 4中所示。
表 3 多西他赛注射液及多西他赛胶束冻干粉对 MDA-MB-231肿瘤抑制作用
动物数 (只) 体重 瘤 重 抑瘤率 组 别 开始 结束 开始 (g) (%) 阴性对照组 8 17.1土 1.12 21.30.51 2.83土 0.735
溶剂对照组 8 17.3土 0.99 20.8土 1.92 2.48土 0.886 12.3 多西他赛注射液 (10 8 15.8±1.16 21.0±1.63 1.08土 0.646* 61.8 mg/kg)
多西他赛胶束 ( 10 9 17.0土 0.71 20.1土 1.05 0.26土 0.191* 90.8 mg/kg)
: P<0.01, 与阴性对照组比较
表 4多西他赛注射液及多西他赛胶束冻干粉对 MDA-MB-231肿瘤抑制作用
肿瘤体积 (mm3 ) T/C 组 另 J 开始 结束 RTV (%) 阴性对照组 107土 22.0 2833土 782.3 28.89土 11.371
溶剂对照组 107土 27.9 2472土 904.9 27.26土 11.779 94.41 多西他赛注射液 (10 mg/kg) 107土 30.7 1054土 620.5 10.62土 5.295* 36.78 多西他赛胶束 (10 mg/kg) 101土 42.4 0 1.96土 1.448** 6.79
* : P<0.01, 与阴性对照组比较; * * : P<0.001, 与阴性对照组比较
实施例 5药代动力学试验
( 1 ) 大鼠血浆药动学对比研究
A、 实验动物:
雄性 SD大鼠, 体重 240±20 g, 随机分为 I、 II、 III、 IV共 4组, 每组均为 6只, 备用。
B、 实验制剂:
多西他赛胶束冻干粉 (a), 按实施例 2.(2)方法制备, 批号 20120907, 规格: 含多西 他赛 20 mg/瓶;
卡巴他赛胶束冻干粉 (b), 按实施例 2.(3)方法制备, 批号 20120830, 规格: 含卡巴 他赛 20 mg/瓶;
多帕菲 (多西他赛注射液, c), 齐鲁制药有限公司产品, 批号 1120312TA, 规格 0.5 ml: 20 mg;
卡巴他赛冻干粉 (d), 以吐温 -80为增溶剂配制。 C、 给药与样本采集:
实验制剂于临用前溶解稀释至合适浓度。 其中, 多西他赛胶束(a)和卡巴他赛胶束 (b) 分别以 5mg/kg剂量 (各自以多西他赛、 卡巴他赛计) 经尾静脉注射给予 I、 II组 大鼠; 多西他赛注射液 (c) 和卡巴他赛冻干粉 (d) 则分别以 5mg/kg剂量经尾静脉注射 给予 III、 IV组大鼠。分别于给药后不同时刻经大鼠眼眶静脉丛采集血样于肝素抗凝离心试 管中, 离心分离血浆, 置 -80°C超低温冰箱冻存, 待测。
D、 血浆药时曲线与药动学参数:
血浆样品以甲醇沉淀除蛋白后进行 LC-MS/MS分析, 测定其中多西他赛或卡巴他赛 的总药物浓度, 绘制各给药组的血浆药物浓度经时变化曲线, 结果如图 18 (大鼠静脉给 予多西他赛胶束冻干粉 (a) 和多西他赛注射液 (c) 的血浆药时曲线)、 图 19 (大鼠静脉 给予卡巴他赛胶束冻干粉 (b) 和卡巴他赛冻干粉 (d) 的血浆药时曲线) 所示。
同时针对采集的血浆样品超滤除去游离药物后再行 LC-MS/MS分析, 测定其中多西 他赛或卡巴他赛的包封药物浓度, 分别绘制多西他赛胶束和卡巴他赛胶束 iv给药的大鼠 血浆总药物浓度和包封药物浓度经时变化曲线,结果见图 20 (多西他赛胶束, iv 5mg/kg)、 图 21 (卡巴他赛胶束, iv 5mg/kg)。
E、 实验结果:
结果显示: 多西他赛胶束给药组和卡巴他赛胶束给药组均较相应的注射剂给药组有 显著高的血浆药物浓度和消除半衰期。 其中, 多西他赛胶束和多西他赛注射液(iv, 以多 西他赛计均为 5 mg/kg) 的血浆 AUC分别为 3732 ng/mL h和 436 ng/mL h, t1/2分别为 1.9 h 和 O.l h; 卡巴他赛胶束和卡巴他赛注射液 (iv, 以卡巴他赛计均为 5 mg/kg) 的血浆 AUC 分别为 4295 ng/mL h和 482 ng/mL h, t1/2分别为 2.7 h和 0.3 h。多西他赛胶束给药组和卡巴 他赛胶束给药组的血浆 AUC分别是相应注射剂给药组的 8.56倍和 8.91倍。此外,如图 20、 图 21所示, 无论是多西他赛胶束还是卡巴他赛胶束, 经静脉途径进入血浆后, 在给药后 24 h均主要以胶束包封的形式存在。胶束给药所表现的血浆药动学特征反映了本发明所制 备的胶束具有优越的稳定性和独特的体内释药特性。
(2) 荷瘤小鼠瘤组织药物分布对比研究
A、 实验动物:
雌性裸小鼠, 按 5 X 106的密度接种人源乳腺癌 MX-1 细胞于腋下, 至瘤生长至〜 500 mm3,随机分为两大组(Group I:多西他赛胶束冻干粉组, Group II:多西他赛注射液组), 两大组荷瘤小鼠的体重分别为 24.9± 1.2 g和 25.0± 1.3 g, 无显著性差异(P > 0.05 )。每大 组再均匀分为 7个小组, 每小组均有 10只荷瘤小鼠, 备用。
B、 实验制剂:
多西他赛胶束冻干粉, 按实施例 2. 2)方法制备, 批号 20120907, 规格: 20 mg /瓶; 多帕菲(多西他赛注射液), 齐鲁制药有限公司产品, 批号 1120312TA, 规格 0.5 ml: 20mg。
C、 给药与样本采集:
多西他赛注射液和多西他赛胶束冻干粉于临用前溶解稀释至合适浓度, 按 10mg/kg 剂量 (以多西他赛计) 分别经尾静脉注射给予 I组和 II组动物, 分别于给药后 5 min、 15 min、 30 min 1 h、 3 h、 8 h和 24h处死各小组动物, 剥取瘤组织, 称重后置 -80°C超低温 冰箱冻存, 待测。
D、 瘤组织药物分布:
瘤组织匀浆后进行 LC-MS/MS分析, 测定其中多西他赛的药物浓度, 绘制各给药组 的瘤组织药物浓度经时变化曲线, 如图 22所示。 在相同给药剂量下 (10 mg/kg), 注射剂 组(Group I )和胶束组(Group II )裸鼠在给药 24 h内瘤组织内多西他赛的 AUC分别为 45.528 mg/L h和 57.089 mg/L h。 结果表明, 胶束组的瘤组织多西他赛药物分布显著高于 注射剂组 ((Ρ < 0.01 ), 两者相差达 25.4%。
上述实施例为本发明较佳的实施方式, 但本发明的实施方式并不受上述实施例的限 制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化, 均应为等效的置换方式, 都包含在本发明的保护范围之内。
实施例 6 Boc-苯丙氨酸封端 mPEG2flflfl-PLA18flfl共聚物 /紫杉醇胶束和苯甲酰基封端 mPEG2flflfl-PLA18flfl共聚物 /紫杉醇胶束体内外稳定性对比研究
( 1 ) 共聚物制备: 参照专利 CN01809632.8 所述方法合成苯甲酰基封端 mPEG2Q(K)-PLA18(K^聚物, 采用本专利实施例 1 (2) 所述方法合成 mPEG oo-PLA^oo-BP, 分别以此两种共聚物为辅料, 紫杉醇为药物制备紫杉醇胶束。
(2) 胶束制备: 共聚物 150mg, 紫杉醇 30mg溶解于 5ml乙醇后, 于 45°C选转蒸发 去除溶剂, 然后加入 5ml生理盐水溶解药物, 所得溶液经 0.22μη膜过滤后于 37°C恒温保 存, 观察稳定性。
(3 ) 稳定性测试方法: 将胶束溶液经尾静脉注射到大鼠血液中, 分别于不同时间点 取血并用高效液相色谱测定血液中紫杉醇的含量。
( 4 ) 试验结果与结论: 两种胶束的血药浓度变化如图 23 所示, 其中 mPEG2Q(K)-PLA18(K)-BP/紫杉醇胶束组的血液浓度显著高于苯甲酰基封端 mPEG2Q(K)-PLA1800 共聚物 /紫杉醇胶束组, 而且有超过 80%的药物都是以胶束包封的形式存在, 显示出极佳 的体内稳定性。 mPEG^o-PLA^o-BP/紫杉醇胶束至少在 48h内无药物沉淀, 而苯甲酰基 封端 mPEG2QQQ-PLA18()()共聚物 /紫杉醇胶束在 17h时药物析出 明显, 显示 mPEG2。。。-PLA18。。-BP/紫杉醇胶束的稳定性显著高于苯甲酰基封端的 mPEG2。。。-PLA18(K)制 备的紫杉醇胶束。

Claims

权利要求书
1. 一种两亲性嵌段共聚物, 其特征在于: 包括亲水性链段和疏水性链段, 其亲水性链段为 数均分子量在 400 20000之间的聚乙二醇或聚乙二醇单甲醚,其疏水性链段选自采用疏 水性基团封端的数均分子量在 500 100000之间的聚丙交酯、 聚乙交酯、 聚乙丙交酯、 聚己内酯、 聚碳酸酯、 或者聚二氧环己酮中的一种, 所述的疏水性基团选自叔丁酰基、 叔丁乙酰基、 氨基酸残基、 或氨基酸衍生物残基中的一种。
2. 根据权利要求 1所述的两亲性嵌段共聚物, 其特征在于所述的氨基酸衍生物为 γ-苄基谷 氨酸、 β-苄基天冬氨酸或氨基得到保护的氨基酸衍生物。
3. 根据权利要求 2所述的两亲性嵌段共聚物,其特征在于所述的氨基酸衍生物为含有苄基 保护或者叔丁氧羰基保护的氨基酸。
4. 根据权利要求 3所述的两亲性嵌段共聚物,其特征在于所述的氨基酸衍生物为叔丁氧羰 基苯丙氨酸。
5. 根据权利要求 1所述的两亲性嵌段共聚物,其特征在于所述疏水性链段的数均分子量在 1000-50000之间; 所述亲水性链段的数均分子量在 750 5000之间。
6. 权利要求 1、 2、 3、 4或 5的两亲性嵌段共聚物的制备方法, 其特征在于, 包括以下步 骤:
1 ) 取数均分子量在 400 20000之间的亲水性链段加入到聚合瓶中, 加热至 100°C~130 °〇真空脱水 2h~4h后加入形成疏水性链段的聚合物单体和单体重量 0.3%。_1%。的催化剂辛酸 亚锡, 真空密闭反应瓶, 上述反应物在 100°C~150°C反应 12h~24h后用二氯甲烷、 乙醇、 四 氢呋喃、 甲醇、 乙酸乙酯或丙酮溶解, 加入乙醚充分沉淀聚合物后经过滤, 真空干燥得亲 水性链段和疏水性链段组成的嵌段共聚物, 所述的亲水性链段为聚乙二醇或聚乙二醇单甲 醚;
2)取亲水性链段和疏水性链段组成的嵌段共聚物, 用乙酸乙酯、 四氢呋喃、 二氯甲烷、 乙酸乙酯或双蒸水溶解, 而后加入叔丁酰基、 叔丁乙酰基、 氨基酸残基、 或氨基酸衍生物 残基进行反应, 使端羟基反应形成疏水性基团, 过滤去除不溶物后加入足量乙醚沉淀聚合 物, 过滤真空干燥后得目标共聚物。
7. 一种胶束载药系统, 其特征在于该胶束载药系统包含至少一种权利要求 1、 2、 3、 4或 5任一所述的两亲性嵌段共聚物、 治疗有效量的至少一种抗肿瘤药物以及药学上可接受 的药用辅料。
8. 根据权利要求 7所述的胶束载药系统,其特征在于两亲性嵌段共聚物和药物的重量比在 99.5:0.5~50:50之间。
9. 根据权利要求 8所述的胶束载药系统,其特征在于两亲性嵌段共聚物和药物的重量比在 99: 1 -75:25之间。
10. 根据权利要求 7所述的胶束载药系统, 其特征在于所述的药用辅料为冻干赋型剂。
11. 根据权利要求 10所述的胶束载药系统, 其特征在于所述的冻干赋型剂选自乳糖、 甘露 醇、 蔗糖、 海藻糖、 果糖、 葡萄糖、 海藻酸钠或明胶中的至少一种。
12. 根据权利要求 10所述的胶束载药系统, 其特征在于所述的冻干赋型剂占整个体系的重 量比为 0 99.9%之间。
13. 根据权利要求 12所述的胶束载药系统, 其特征在于所述的冻干赋型剂占整个体系的重 量比为 10.0 80.0%之间。
14. 根据权利要求 7所述的胶束载药系统, 其特征在于所述的药用辅料还包括抗氧剂、 金属 离子络合剂、 pH调节剂或等渗调节剂; 所述的抗氧剂为亚硫酸钠、 亚硫酸氢钠或焦亚 硫酸钠; 所述的金属离子络合剂为依地酸二钠、 依地酸钠钙或环己二胺四醋酸钠; 所述 的 pH调节剂为枸櫞酸、 碳酸氢钠、 磷酸氢二钠或磷酸二氢钠; 所述的等渗调节剂为氯 化钠或葡萄糖。
15. 根据权利要求 7所述的胶束载药系统, 其特征在于: 所述的抗肿瘤药物选自紫杉醇、 多 西他赛、 卡巴他赛、 莱龙泰素、 姜黄素、 阿霉素或表阿霉素中的至少一种。
16. 一种权利要求 7-15所述胶束载药系统的制备方法, 所述的制备方法为透析法、 直接溶 解法、 薄膜水化法、 固体分散法或高能均质乳化法。
17. 根据权利要求 16所述的制备方法, 其特征在于所述的薄膜水化法的具体步骤为: 将聚 合物辅料和药物溶解于有机溶剂,经旋转蒸发去除溶剂后加入注射用水溶解药膜得载药 胶束溶液, 经过滤除菌冻干后得胶束冻干粉。
18. 根据权利要求 16所述的制备方法, 其特征在于所述的固体分散法的具体步骤为: 将药 物溶解于加热后处于熔融状态的聚合物辅料得到澄清的混合物,再加入注射用水溶解得 胶束溶液, 经过滤除菌后冻干得胶束冻干粉。
19. 权利要求 1-5任一所述的两亲性嵌段共聚物和权利要求 7-15任一所述的胶束载药系统在 制备治疗抗肿瘤药物中的应用。
20. 根据权利要求 19所述的应用, 其特征在于所述的肿瘤是乳腺癌、 前列腺癌、 卵巢癌、 肠癌、 肺癌、 肝癌或头颈癌。
PCT/CN2013/083958 2012-10-26 2013-09-22 一种两亲性嵌段共聚物及其制备方法、以及该共聚物与抗肿瘤药物形成的胶束载药系统 WO2014063549A1 (zh)

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