WO2011072510A1 - 实体瘤被动靶向性抗癌前药及其制备方法 - Google Patents

实体瘤被动靶向性抗癌前药及其制备方法 Download PDF

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WO2011072510A1
WO2011072510A1 PCT/CN2010/072908 CN2010072908W WO2011072510A1 WO 2011072510 A1 WO2011072510 A1 WO 2011072510A1 CN 2010072908 W CN2010072908 W CN 2010072908W WO 2011072510 A1 WO2011072510 A1 WO 2011072510A1
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pectin
solid tumor
doxorubicin
targeted anticancer
anticancer prodrug
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PCT/CN2010/072908
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English (en)
French (fr)
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唐小海
邱宇
宋鑫
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重庆莱美药业股份有限公司
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Priority to US13/381,483 priority Critical patent/US20120244193A1/en
Priority to JP2012520893A priority patent/JP5536210B2/ja
Priority to CA2771188A priority patent/CA2771188C/en
Priority to EP10836948.9A priority patent/EP2514441B1/en
Publication of WO2011072510A1 publication Critical patent/WO2011072510A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a solid tumor passive targeted anticancer prodrug and a preparation method thereof, and belongs to the field of antitumor drugs.
  • BACKGROUND OF THE INVENTION The microvascular endothelium in normal tissues is dense and structurally intact, and macromolecules and lipid particles are not easily transmitted through the blood vessel wall.
  • solid tumor tissue is rich in blood vessels, irregular microvascular structure, lack of wall, loose endothelial cells, poor structural integrity, wide gaps in endothelial cells, and lack of lymphatic drainage, resulting in macromolecules.
  • Pectin is often a natural macromolecular polysaccharide polymer, widely present in the cell wall of plants, and is an acidic macromolecular polysaccharide composed of ⁇ _ (1 ⁇ 4) -D-galactopyranoic acid units (Hyunjo Kim, et Al. International
  • Pectin enhances host immune function by enhancing mononuclear macrophage system, activating macrophages, T cells and B cells, NK cells and complement systems, promoting cytokine secretion, enhancing red blood cell immunity, etc.
  • Cancer angiogenesis plays a direct role in anticancer (Chinese Journal of Traditional Chinese Medicine, 1999, 5: 64).
  • the inventors of the present invention began researching macromolecular anticancer prodrugs using pectin as a carrier from 2005, and applied for three Chinese invention patent applications, respectively.
  • anticancer drugs can freely pass through the blood vessel walls of normal tissues and tumor tissues, and are also easy to excrete. But because of its normal tissue and tumor tissue The drug distribution is consistent, the anticancer effect is poorly selective, the drug stays in the body for a short period of time, and the frequency of administration is high.
  • the anti-cancer prodrug prepared by using macromolecular pectin as a carrier can produce passive targeting of EPR-based tumors, has significant accumulation in tumor tissues, and multiple administrations do not affect its distribution. Because pectin is not easily degraded in the body, it can only be excreted through the kidneys.
  • pectin anticancer prodrug is passively targeted by the EPR effect-based tumor, and at the same time, after entering the solid tumor tissue, the drug is released and the carrier is decomposed into small molecules to facilitate excretion.
  • the macromolecular pectin is cut into Mw 0. 5-4.
  • pectin-doxorubicin conjugate 10 to 1 million (preferably 200,000 to 800,000, further preferably 400,000 to 600,000).
  • the pectin-doxorubicin conjugate was made into a suspension (pectin-adriamycin conjugate was added to water, and then PVP and glycerin were added to mix), and then treated by a nano-high pressure homogenizer to obtain a particle size of 100 nm. ⁇ 200nm, preferably 130nm_180nm, melting point 220 °C ⁇ 245 °C solid tumor passive targeted anticancer prodrug.
  • the pectin and the doxorubicin are linked by an amide bond, and the pectin and the pectin are linked by an ester bond of a hydroxyl group of the pectin molecule to the hydroxyl group.
  • the anti-cancer prodrug is capable of generating EPR-based tumor passive targeting after entering the solid tumor tissue, has a significant accumulation in the tumor tissue, has a long plasma half-life, and prolongs the systemic circulation time; simultaneously releases the drug and the carrier Decomposed into small molecules, which is good for excretion.
  • the molecular weights of the present invention are all weight average molecular weights Mw.
  • the solid tumor passively targeted anticancer prodrug has a water solubility of 42 mg/L.
  • the method for preparing a solid tumor passively targeted anticancer prodrug is accomplished by the following steps:
  • a Mw 0. 5 ⁇ 4.
  • 50,000 (preferably Mw 1 ⁇ 30,000) small molecule pectin dissolved in water add doxorubicin hydrochloride, mix well and react with EDC-HC1, dialysis, and dry to obtain MwlO million ⁇ 1 million pectin-doxorubicin conjugate;
  • pectin-doxorubicin conjugate added to water, then add PVP and glycerin (may also add a certain amount of lecithin or DMS0, added in no more than pectin - 2% of the doxorubicin conjugate, mixed and prepared into a suspension, and treated by a nano-Ultra-high pressure homogenizer to obtain a passive targeted anti-cancer prodrug of a solid tumor having a particle diameter of 100 n m-200 nm.
  • the pectin-doxorubicin conjugate is obtained by reacting a small molecule of pectin with doxorubicin at a pH of 5 to 7, EDC ⁇ HC1 at 40 to 60 ° C.
  • the preferred treatment method is as follows: The suspension is processed into the nano ultrahigh pressure homogenizer in 3 times, the first pressure is 120mpa, the second pressure is 180mpa, and the third pressure is 190mpa.
  • the solid tumor passively targeted anticancer prodrug of the invention is hydrolyzed in lysozyme, amide bond is broken, and doxorubicin is released. After hydrolysis and ultrafiltration, the filter cake is cut off. The filter cake is repeatedly washed with 95% ethanol until the liquid is red-free to remove residual doxorubicin. The solvent is evaporated, and the precipitate is dissolved by adding distilled water. The molecular weight of the gel is 1 by gel permeation chromatography. Million ⁇ 30,000.
  • the solid tumor passive targeting anticancer prodrug (pectin-doxorubicin conjugate) injection of the present invention is applied to human lung cancer cells NCI-H446 and A549.
  • the cell inhibition rates were 65.23% and 68.52%, respectively, which were comparable to doxorubicin hydrochloride (63.33% and 67.62%).
  • the tumor-bearing survival time of the pectin-doxorubicin conjugate mice was 42.3 ⁇ 12. 4 days, significantly higher than that of the doxorubicin hydrochloride group (23.1 ⁇ 10.2 days).
  • Figure 2 Infrared spectrum; a, doxorubicin; b, pectin-adriamycin conjugate; c, pectin; d, physical mixture of doxorubicin and pectin;
  • Figure 3 is a particle size distribution diagram of a passive targeted anticancer prodrug of the solid tumor of the present invention.
  • Figure 4 The lysosomal hydrolysis test of the passive tumor-targeted anticancer prodrug of the solid tumor of the present invention; the series 1 is the experimental group, the series 2 is the blank group; the abscissa time (hour, h), and the ordinate is the percentage of drug release (%) );
  • FIG. 5 Effect of passive tumor-targeted anti-cancer prodrugs of the present invention on survival time of lung cancer-bearing mice.
  • the macromolecular pectin is first cut into 5-5. 5 million small molecule pectin, preferably M w l ⁇ 30,000, and then synthesized small molecule pectin - doxorubicin (by amide bond At the same time, the carboxyl group and the hydroxyl group in the pectin molecule are condensed to form a macromolecule. Then, it is treated by a nano ultrahigh pressure homogenizer to obtain a passive targeted anticancer prodrug of 100 nm to 200 nm and a molecular weight of 100,000 to 1 million solid tumors.
  • One specific embodiment Weigh 13.3g of pectin, add 1L of steamed water and stir to dissolve, then adjust with 5M NaOH The pH was adjusted to pH 13 and reacted at 65 ° C for 10 h to stop the reaction.
  • the reaction solution was adjusted to neutral with hydrochloric acid, filtered with a milliipore having a molecular weight cut off of 30,000, and the filtrate was filtered through a millipere having a molecular weight cut off of 10,000, and a solid having a molecular weight cut off of 10,000 was collected, and concentrated under reduced pressure. Vacuum drying to obtain a small molecule pectin having a molecular weight of 1 to 30,000.
  • the measured drug loading is 15-35%, and the molecular weight is Mw10 million to 1 million, preferably 200,000 to 800,000, further preferably
  • the nano ultrahigh pressure homogenizer T-200D type Hebei Langfang General Machinery Manufacturing Co., Ltd.
  • Tumor passive targeted anticancer prodrug After the above-mentioned reddish brown solid is ground, a certain amount of PVP is added, and the nano ultrahigh pressure homogenizer (T-200D type Hebei Langfang General Machinery Manufacturing Co., Ltd.) is processed to obtain an entity having a molecular weight of 10 to 1,000,000 and a particle diameter of 100 n m to 200 nm. Tumor passive targeted anticancer prodrug.
  • Prodrug preparation ⁇ ( ⁇ ) ⁇ embodiment a passive targeting solid tumors anticancer invention embodiment
  • Determination of the drug loading of the sample Accurately weigh a certain amount of ⁇ ( ⁇ ) ⁇ , dissolved in the secondary water to prepare a solution to be tested, determine the absorbance, and measure the drug loading amount to be 21. 4%.
  • samples were prepared as prodrugs.
  • the doxorubicin, pectin, the sample of the invention, the mixture of pectin and doxorubicin were dissolved in secondary water, and the ultraviolet spectrum and the infrared spectrum are shown in Fig. 1 and Fig. 2, respectively.
  • the doxorubicin has a maximum absorption peak at 479. 5 nm, and the mixture of pectin and doxorubicin has a maximum absorption peak at 488. 5 nm.
  • ⁇ has a maximum absorption peak at 498 nm, and pectin has no absorption.
  • P (A) precede redshift at absorption at 498 nm, indicating that doxorubicin is chemically coupled to pectin.
  • the mixed absorption peak of the amide I band and the amide II band at 1620 cm- 1 of pectin-doxorubicin significantly increased the peak area ratio compared with the ester bond peak of 1750 cm" 1 . It indicates that the pectin and pectin are cross-linked by ester bond.
  • the characteristic absorption peak of doxorubicin is observed, and the absorption peak of primary amide appears at 1411.23. , indicating that pectin and doxorubicin are combined in the form of an amide bond.
  • the nano-ultra-high pressure homogenizer is treated as a suspension to obtain a passive targeted anti-cancer prodrug of a solid tumor having a molecular weight of 10 to 1 million and a particle diameter of 100 nm to 200 nm.
  • a passive targeted anti-cancer prodrug of a solid tumor having a molecular weight of 10 to 1 million and a particle diameter of 100 nm to 200 nm.
  • a solid tumor passively targeted anticancer prodrug having a molecular weight of 10 to 10 million and a particle diameter of 100 nm to 200 nm is obtained.
  • dialysis is carried out in a dialysis bag with a molecular weight cut off (Mw) of 3,500, and the distilled water is changed every 3 hours.
  • Mw molecular weight cut off
  • the solvent was evaporated to dryness and dried under vacuum for 12 h to give a red-brown solid, a macromolecularly soluble pectin-doxorubicin conjugate 1. 2 g, and a drug loading of 25.2%.
  • a solid tumor passively targeted anticancer prodrug having a molecular weight of 10 to 10 million and a particle diameter of 100 nm to 200 nm is obtained.
  • Test Example 1 The lysosomal hydrolysis test of the passive targeting anticancer prodrug of the solid tumor of the present invention
  • Lysosomal source Refer to "Cell Biology Experimental Methods and Techniques" to extract purified lysosomes.
  • PBS phosphate buffered saline
  • control group did not add lysosomes, and the other conditions were the same.
  • the experimental results are shown in Fig. 4: the maximum release of the experimental group was 35%, reaching 30% after 6h, and basically stable at 30% in the range of 6-30h, and the maximum release of the control group was 7%, which was always released during the entire release process. Very low. Description
  • the solid tumor passive targeted anticancer prodrug of the invention is hydrolyzed in lysozyme, amide bond is broken, and doxorubicin is released.
  • Chromatographic conditions Aglientl lOO series HPLC, 1362A parallax detector and G1310A unit pump; column: UltrahydrogelTM linear column (7.8 X 300mm, Waters); column temperature: 40 ° C; flow cell temperature: 35 ° C; mobile phase: 0.005M KNO 3 ; Flow rate: 0.5mL/min; Sample concentration: 5mg/mL, dissolved with 0.05% sodium azide; Injection volume: 20 ⁇ .
  • the molecular weight was measured to be 1-3 million.
  • the molecular weight of commercially available citrus high ester pectin was determined under the same conditions: the absolute molecular weight was measured to be 30,000 to 600,000.
  • Test Example 2 Using the MTT method to observe the inhibition of the growth activity of the tumor cell line in vitro by the passive targeted anticancer prodrug of the solid tumor of the present invention.
  • Doxorubicin hydrochloride was purchased from Zhejiang Haizheng Pharmaceutical Co., Ltd. and dissolved in physiological saline to prepare doxorubicin 2mg/ml.
  • the solid tumor passively targeted anticancer prodrug used in this test was prepared as in Example 1 (corresponding to doxorubicin 2 mg/ml).
  • a cell solution (concentration: 50000 / ml) grown in logarithmic growth phase cultured in RPMI 1640 medium was inoculated into a 96-well plate at 0.1 ml / well. After the incubation for 24 hours, the corresponding drug was administered. After 24 hours of drug treatment, 0.02 ml of 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) was added.
  • MTT 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide
  • DMSO dimethyl sulfoxide
  • Inhibition rate (%) (1 - absorption value absorption value / blank control group absorption value) ⁇ ⁇ %, the cell inhibition rate of each group of different drugs is shown in Table 1.
  • Doxorubicin hydrochloride was purchased from Zhejiang Haizheng Pharmaceutical Co., Ltd.;
  • the pectin-doxorubicin conjugate is a passive targeted anticancer prodrug of the solid tumor of the present invention as the sample of Example 2 (corresponding to doxorubicin 2 mg/ml).
  • mice Clean-grade inbred C57BL/6 mice were purchased from West China Experimental Animal Center, Sichuan University, female, 6-7 weeks old, weighing 20 ⁇ 3 g. Free drinking water and eating during the experiment. Daily light for 12 hours, mice (5/cage) The cages are all ventilated using a central ventilation system. Mouse melanoma cell line B16 was purchased from Shanghai Institute of Cell Biology and was preserved by our laboratory. 2. Method:
  • Cell culture routinely cultured in RPMI 1640 100mL / L fetal bovine serum plus double antibody (penicillin 100U / mL, streptomycin 100mg / L) in the culture medium, placed at 37 ° C, 50mL / L carbon dioxide incubator. After 3 and 4 generations of stable passage, the cells in logarithmic growth phase were taken, and after 1.5 g/L trypsin digestion, the cells were resuspended in serum-free medium, and the cell density was adjusted with serum-free 1640 medium for use.
  • mice C57BL/6 mice were randomly divided into several groups.
  • the tail skin of the mice was disinfected with 750 mL/L ethanol, and the B16 melanoma cell solution was injected into the tail vein of the mice.
  • the inoculation dose was 0.1 mL.
  • Fig. 5 The effect of the passive tumor-targeted anticancer prodrug of the present invention on the survival time of lung cancer-bearing mice is shown in Fig. 5. It can be seen from Figure 5 that in the efficacy study of melanoma B16 lung metastasis model mice, the tumor-bearing survival time of the pectin-doxorubicin conjugate mice was 42.3 ⁇ 12. 4 days, which was significantly higher than that of doxorubicin hydrochloride. Group (23. 1 ⁇ 10. 2 days).

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Description

实体瘤被动靶向性抗癌前药及其制备方法
技术领域 本发明涉及一种实体瘤被动靶向性抗癌前药及其制备方法， 属于抗肿瘤药物领域。 背景技术 正常组织中的微血管内皮间隙致密、结构完整，大分子和脂质颗粒不易透过血管壁。 与正常组织中的微血管相比，实体瘤组织血管丰富， 微血管形状构造不规则、 膨胀、 管 壁缺失、 内皮细胞排列疏松、 结构完整性差，内皮细胞连接间隙宽大， 淋巴回流缺失， 造成大分子类物质和脂质颗粒具有选择性高通透性和滞留性，这种现象被称作实体瘤组 织的高通透性禾口滞留效应， 简称 EPR效应（ en-hanced permeabi l ity and retention effect) 0 扫描电镜显示人结肠腺癌微血管内皮细胞连接间隙达 400nm， 而正常组织中微 血管内皮细胞连接间隙平均不到 100nm。 实体瘤组织的病理结构特点， 使得大分子抗癌 药对实体瘤具有被动的靶向性或者选择性的特征，全身给药后在肿瘤组织中有较多的分 布，又称为实体瘤的被动靶向性； 而小分子抗癌药能够自由通过正常组织和肿瘤组织的 血管壁，在正常组织和肿瘤组织中的药物分布一致，是造成抗癌效应选择性差、毒副作用 较强的重要原因之一，不具备被动靶向作用。 Greish K等报道， 相对分子量 >40000的大 分子物质可以克服肾脏的滤过清除， 具有较长的血浆半衰期， 大分子物质体循环时间延 长利于实现 EPR效应，同时减少给药频率。
果胶常是天然的大分子多糖聚合物， 广泛存在于植物的细胞壁中， 是由 α _ (1→ 4) -D-吡喃半乳糖醛酸单位组成的酸性大分子多糖 （Hyunjo Kim, et al. International
Journal of Pharmaceutics, 1998, 161 : 149-159) 。 果胶可通过增强单核巨噬细胞***， 激 活巨噬细胞、 T细胞和 B细胞、 NK细胞和补体***， 促进细胞因子分泌， 增强红细胞免疫 等提高宿主免疫功能； 通过改变实体癌细胞膜的生长特性、 影响实体癌细胞内信号传递 途径、 抗自由基作用、 诱导分化与凋亡、 抑制实体癌细胞的核酸与蛋白质合成、 影响实 体癌细胞超微结构、 影响癌基因、 抗突变作用、 抑制实体癌血管形成而发挥直接的抗癌 作用 （中国中医药信息杂志， 1999, 5 : 64) 。 本发明的发明人从 2005年开始研究以果胶 为载体的大分子抗癌前药， 并申请了 3件中国发明专利申请， 申请号分别为
200610020596. 7、 200710201724. 2、 200810306463. 5。 采用小分子果胶， 抗癌药能够自 由通过正常组织和肿瘤组织的血管壁，也容易***。 但是由于其在正常组织和肿瘤组织 中的药物分布一致， 抗癌效应选择性差， 药物在体内停留时间短， 需给药频率较高。 采 用大分子果胶作为载体制备得到的抗癌前药， 能够产生基于 EPR的肿瘤被动靶向性， 在 肿瘤组织有显著的蓄积作用， 而且多次给药并不影响它的分布。 由于果胶在体内不容易 降解， 只能通过肾脏***， 过多的大分子果胶聚集体内会造成有可能沉积到肺、 肾脏等 组织脏器对其功能产生影响， 具体后果目前还没见有相关报道。 发明内容 本发明所要解决的技术问题是果胶抗癌前药基于 EPR效应的肿瘤被动靶向性， 同时 能够在进入实体瘤组织后， 释放药物并将载体分解为小分子， 利于***。
为了实现本发明的目的， 本发明的技术方案是这样实现的：
先将大分子果胶切断为 Mw 0. 5-4. 5万的小分子的果胶，优选 Mw 1_3万，由 Mw 0. 5〜
4. 5万的小分子的果胶与阿霉素反应得到的 Mw为 10〜100万的果胶-阿霉素偶联物 （优 选 20万〜 80万， 进一步优选 40万 -60万）， 果胶 -阿霉素偶联物制成混悬剂 （果胶-阿 霉素偶联物加入水中， 再加入 PVP和甘油混匀）后然后通过纳米超高压均质机处理， 得 到粒径 100nm〜200nm，优选 130nm_180nm，熔点 220 °C〜245 °C实体瘤被动靶向性抗癌前 药。 其中所述果胶 -阿霉素偶联物中， 果胶与阿霉素通过酰胺键连接， 果胶与果胶之间 通过果胶分子的羧基与羟基缩合成的酯键连接。 该抗癌前药能够在进入实体瘤组织后， 能够产生基于 EPR的肿瘤被动靶向性， 在肿瘤组织有显著的蓄积作用， 具有较长的血浆 半衰期， 体循环时间延长； 同时释放药物并将载体分解为小分子， 利于***。 本发明所 述分子量为均为重均分子量 Mw。
所述实体瘤被动靶向性抗癌前药的水溶解度为 42 mg/L。
制备实体瘤被动靶向性抗癌前药的方法由以下步骤完成：
a、 Mw 0. 5〜4. 5万 （优选 Mw 1〜3万） 的小分子果胶溶解于水， 加入盐酸阿霉素， 混合均匀后与 EDC - HC1反应，透析，干燥得到 MwlO万〜 100万的果胶-阿霉素偶联物； b、 果胶 -阿霉素偶联物加入水中， 再加入 PVP和甘油 （也可以加入一定量的卵磷脂 或 DMS0, 加入量不超过果胶-阿霉素偶联物的 2%)， 混合均匀制成混悬剂， 经纳米超高 压匀质仪处理， 即得粒径 100nm-200nm的实体瘤被动靶向性抗癌前药。
所述果胶-阿霉素偶联物是由小分子的果胶与阿霉素于 pH为 5〜7， EDC · HC1作 用下， 40〜60°C反应得到。
进一步地， b步骤中， PVP的用量为果胶-阿霉素偶联物质量的 1倍〜 6倍， 甘油的 用量为果胶-阿霉素偶联物质量的 0. 1%-0. 8%。 优选的处理方法是： 混悬剂分 3次进纳米超高压匀质仪处理， 第一次压力 120mpa， 第二次压力 180mpa， 第三次压力 190mpa。
其中， 所述小分子果胶是将果胶溶于水， 与 NaOH溶液在 pH=13条的条件下反应， 然后用浓盐酸调 pH到中性， 截留分子量得到。
本发明实体瘤被动靶向性抗癌前药在溶酶中的水解， 酰胺键断裂， 释放阿霉素。 水解、超滤后截留下的滤饼， 用 95%乙醇反复洗涤滤饼直至液体无红色以去除残留的阿 霉素， 蒸干溶剂， 加入蒸熘水溶解沉淀， 凝胶渗透色谱测定分子量为 1万〜 3万。
在相同的阿霉素浓度下 （2mg/ml) ， 本发明实体瘤被动靶向性抗癌前药 （果胶-阿 霉素偶联物）注射剂对人源性肺癌细胞 NCI-H446和 A549的细胞抑制率分别为 65.23% 和 68.52%， 与盐酸阿霉素的相当 （63.33%和 67.62%)。 在黑色素瘤 B16肺转移模型小 鼠的疗效研究中， 果胶-阿霉素偶联物组小鼠的荷瘤生存期为 42.3 ± 12. 4天， 明显高于 盐酸阿霉素组 （23.1 ± 10.2天）。 附图说明 图 1、 紫外光谱；
图 2、 红外光谱图； a、 阿霉素； b、 果胶一阿霉素轭合物； c、 果胶； d、 阿霉素和果胶 的物理混合物；
图 3、 本发明实体瘤被动靶向性抗癌前药粒径分布图；
图 4、 本发明实体瘤被动靶向性抗癌前药溶酶体水解试验； 系列 1为实验组， 系列 2为空 白组； 横坐标时间 （小时， h)， 纵坐标是药物释放百分率 （％);
图 5、 本发明实体瘤被动靶向性抗癌前药对肺癌荷瘤小鼠生存时间的影响。 具体实施方式 先将大分子果胶切断为丽 0. 5-4. 5万的小分子的果胶， 优选 Mw l〜3万， 然后合 成小分子果胶-阿霉素（通过酰胺键连接）， 同时通过果胶分子之间中的羧基与羟基缩合 联成大分子。 然后通过纳米超高压均质机处理， 得到 100nm〜200nm， 分子量为 10万〜 100万实体瘤被动靶向性抗癌前药。
具体制备方法：
1、 小分子果胶的准备： 采用果胶溶解于蒸熘水后用 NaOH反应， 然后用浓盐酸调 pH到中性， 截留得到分子量为 Mw 0. 5〜4. 5万 （优选 1〜3万） 的小分子果胶。
一个具体实施方式： 称量 13.3g果胶， 加蒸熘水 1L搅拌溶解后， 用 5M NaOH调 pH， 使 pH值 =13， 于 65°C反应 10h， 停止反应。 用盐酸将反应液调到中性， 使用截留 分子量为 3万的 millipore过滤， 滤液再通过截留分子量为 1万的 millipore过滤， 收 集没有透过的截留分子量为 1万的固体， 减压浓缩干， 真空干燥得到分子量为 1〜3万 的小分子果胶。
Figure imgf000006_0001
果胶 e
2、 阿霉素的负载及小分子果胶的交联:
2.1反应式
Figure imgf000006_0002
分子量为 1-3万的小分子果胶溶解， 与盐酸阿霉素溶液在 pH为 5〜7， EDC · HC1 ( 1-乙基 -3-(3-二甲胺基丙基)碳二亚胺盐酸盐） 作用下， 40-60°C反应， 透析， 干燥， 即 得红褐色固体 （果胶 -阿霉素偶联物）， 红褐色固体难溶于水， 溶解度为 42 mg/L, 熔点 220°C-245°C。
测得载药量为 15-35%， 分子量为 MwlO万〜 100万， 优选 20万〜 80万， 进一步优选
40万 -60万。
紫外可见分光光度反分析， 果胶无吸收， 阿霉素在 479. 5nm处有最大吸收峰， 果胶 与阿霉素混合物在 488. 5nm处有最大吸收峰， 果胶-阿霉素偶联物 P (A)„在 498nm处有最 大吸收峰，吸收发生红移， 这说明阿霉素与果胶发生化学键偶联。
红外光谱上扫描， 与果胶相比， ？(^„在 1620 cm—1 处出现酰胺 I带和酰胺 II带的混 合吸收峰， 与 1750 cm- 1的酯键峰相比峰面积比值显著增加， 说明果胶之间是以酯键交 联。 且在 1100 £；1^1和 1017 cm—1处出现明显的阿霉素的蒽环特征吸收峰， 1411. 23处出 现伯酰胺的吸收峰， 说明果胶与阿霉素是以酰胺键的形式结合。
上述红褐色固体研磨处理后加入一定量的 PVP， 纳米超高压匀质仪（T-200D型河北 廊坊通用机器制造有限公司）处理， 得到分子量 10〜100万， 粒径 100nm-200nm的实体 瘤被动靶向性抗癌前药。
3、 大分子难溶性果胶-阿霉素偶联物经研磨处理后加入一定量的 PVP， 纳米超高压 匀质仪处理，得到分子量 10万〜 100万，粒径 100nm-200nm的实体瘤被动靶向性抗癌前 药。
以下通过具体实施例对本发明作进一步详述， 但不应理解为是对本发明的限制。 实施例 1本发明实体瘤被动靶向性抗癌前药 Ρ (Α) Π的制备
1、 小分子果胶的准备：
称量 13.3g果胶， 加蒸熘水 1L搅拌溶解后， 用 5M NaOH调 pH， 使 pH值 =13， 于 65°C反应 10h，停止反应。用浓盐酸将反应液调到中性，使用截留分子量为 3万的 millipore 过滤， 滤液再通过截留分子量为 1万的 millipore过滤， 收集没有透过的截留分子量为 1 万的固体， 减压浓缩干， 真空干燥得到分子量为 1〜3万的小分子果胶。
2、 阿霉素的负载及小分子果胶的交联：
称量分子量为 1〜3万的小分子果胶阿霉素 lg加到反应瓶中， 加 100ml水， 搅拌溶 解， 称盐酸阿霉素 0.5g， 加 50ml蒸熘水超声溶解， 将盐酸阿霉素溶液加到反应瓶中， 另用 50ml蒸熘水洗涤瓶上附着的阿霉素。 称 lg EDC · HC1加入到反应瓶中后升温 50 °C反应 6. 5h， 反应完毕后， 装入截留分子量 （丽） 为 3500的透析袋中透析 ld， 每 3h 换一次蒸熘水。 蒸干溶剂， 真空干燥 12h， 得红褐色固体， 大分子难溶果胶 -阿霉素偶联 物 1. lg， 难溶于水， 溶解度为 42 mg/L, 熔点 220°C-245°C。
采用分光光度法测定载药量：
标准曲线的建立： 准确配制盐酸阿霉素标准溶液， 浓度分别为 10. 00、 20. 00、
30. 00、 40. 00、 50. 00 μ g/mL标准液， 于 479. 5 nm (紫外可见分光光度计光谱扫描确定） 处测定吸光度。
样品载药量的测定： 准确称取一定量的 Ρ (Α) Π， 溶于二次水中配成待测溶液，测定吸 光度， 测得载药量为 21. 4%。
结构表征
为了避免 Ρ (Α) Π中羧酸盐对酯键和酰胺键的影响， 通过调节 ρΗ值， 将 Ρ (Α) Π非盐处 理， 制备成前药样品。用二次水溶解阿霉素、果胶、本发明样品、果胶与阿霉素混合物， 紫外光谱和红外光谱分别如图 1， 图 2所示。
图 1中， 阿霉素在 479. 5nm处有最大吸收峰， 果胶与阿霉素混合物在 488. 5nm处有 最大吸收峰， ？^^在 498nm处有最大吸收峰， 果胶无吸收。 P (A)„在 498nm处吸收发生 红移， 这说明阿霉素与果胶发生化学键偶联。
图 2中， 与果胶相比， 果胶一阿霉素在 1620 cm—1 处出现酰胺 I带和酰胺 II带的混 合吸收峰与 1750 cm"1的酯键峰相比峰面积比值显著增加，说明果胶与果胶之间以酯键交 联。 且在 1100 cm—1和 1017 cm—1处出现明显的阿霉素的蒽环特征吸收峰， 1411. 23处出 现伯酰胺的吸收峰， 说明果胶与阿霉素是以酰胺键的形式结合的。
3、 实体瘤被动靶向性抗癌前药的制备：
0. 468g大分子难溶性果胶-阿霉素偶联物 （载药量 21. 4%) 加入 lg PVP, 3ml甘油 和 50ml水， 研磨处理制成混悬剂， 经纳米超高压匀质仪 （T-200D型河北廊坊通用机器 制造有限公司）处理。 分 3次分别进纳米超高压匀质仪处理， 第一次压力 120mpa， 第二 次压力 180mpa，第三次压力 190mpa。经过纳米超高压匀质仪处理后的粒径分布图如图 3。
纳米超高压匀质仪处理后是悬浮液， 得到分子量 10-100万， 粒径 100nm-200nm的 实体瘤被动靶向性抗癌前药。 实施例 2本发明实体瘤被动靶向性抗癌前药 Ρ (Α) Π的制备
1、 小分子果胶的准备： 称量 13. 3g果胶， 加蒸熘水 1L搅拌溶解后， 用 5M NaOH调 pH， 使 pH值 =13， 于 65 °C反应 10h， 停止反应。 用浓盐酸将反应液调到中性， 使用截留分子量为 1 万的 mi l l ipore过滤， 滤液再通过截留分子量为 7000的透析袋透析， 收集透析液， 减压浓缩 干， 真空干燥得到分子量为 7000-10000的小分子果胶。
2、 阿霉素的负载及小分子果胶的交联：
称量分子量为 7000〜 10000的小分子果胶阿霉素 lg加到反应瓶中， 加 100ml水， 搅拌溶解， 称盐酸阿霉素 0. 5g， 加 50ml蒸熘水超声溶解， 将盐酸阿霉素溶液加到反应 瓶中， 另用 50ml蒸熘水洗涤瓶上附着的阿霉素。 称 lg EDC * HCl加入到反应瓶中后升 温 50°C反应 6. 5h， 反应完毕后， 装入截留分子量（Mw)为 3500的透析袋中透析 ld， 每 3h换一次蒸熘水。 蒸干溶剂， 真空干燥 12h， 得红褐色固体， 大分子难溶果胶-阿霉素 偶联物 1. 2g， 载药量 24. 2%。
0、 468g大分子难溶性果胶-阿霉素偶联物， 加入 lg PVP， 3ml甘油和 2%的卵磷脂溶 液 50ml做溶剂， 研磨处理制成混悬剂， 经纳米超高压匀质仪 （T-200D型河北廊坊通用 机器制造有限公司） 处理。 分 3次分别进纳米超高压匀质仪处理， 第一次压力 120mpa， 第二次压力 lSOmpa, 第三次压力 l90mpa。
得到分子量 10-100万， 粒径 100nm-200nm的实体瘤被动靶向性抗癌前药。
实施例 3本发明实体瘤被动靶向性抗癌前药 Ρ (Α) Π的制备
1、 小分子果胶的准备：
称量 13. 3g果胶， 加蒸熘水 1L搅拌溶解后， 用 5M NaOH调 pH， 使 pH值 =13， 于 65 °C反应 10h， 停止反应。 用浓盐酸将反应液调到中性， 使用截留分子量为 5 万的 mi l l ipore过滤， 滤液使用截留分子量为 2万的透析袋透析 48h， 每 3h换一次蒸熘水。 透析液减压浓缩干， 真空干燥得到分子量为 2万 -5万的小分子果胶。
2、 阿霉素的负载及小分子果胶的交联：
称量分子量为 2-5万的小分子果胶 lg加到反应瓶中， 加 100ml水， 搅拌溶解， 称 盐酸阿霉素 0.5g，加 50ml蒸熘水超声溶解，将盐酸阿霉素溶液加到反应瓶中，另用 50ml 蒸熘水洗涤瓶上附着的阿霉素。称 lg EDC -HC1加入到反应瓶中后升温 50°C反应 6. 5h， 反应完毕后， 装入截留分子量 （Mw) 为 3500的透析袋中透析 ld， 每 3h换一次蒸熘水。 蒸干溶剂， 真空干燥 12h， 得红褐色固体， 大分子难溶果胶-阿霉素偶联物 1. 2g， 载药 量 25· 2%。
0. 468g大分子难溶性果胶-阿霉素偶联物， 加入 lg PVP, 50ml水和 DMS0的混合溶 剂 （水： DMS0=0. 75 : 0. 25)， 制成混悬剂， 经纳米超高压匀质仪 （T-200D型河北廊坊通 用机器制造有限公司）处理。分 3次分别进纳米超高压匀质仪处理，第一次压力 120mpa， 第二次压力 180mpa， 第三次压力 190mpa。
得到分子量 10-100万， 粒径 100nm-200nm的实体瘤被动靶向性抗癌前药。 试验例 1本发明实体瘤被动靶向性抗癌前药溶酶体水解试验
溶酶体来源： 参照 《细胞生物学实验方法与技术》 提取得到纯化的溶酶体。
实验组： 25mL的锥形瓶中加入 10mL磷酸盐缓冲液（PBS ) (pH=5 )溶液， lmg/mL 实施例 2 制备的实体瘤被动靶向性抗癌前药 10 mg， 再加入 0.4mL 溶酶体的蔗糖 (0.25mol/L)混悬液， 置于 37°C恒温箱中摇床避光孵育。
对照组不加入溶酶体， 其他条件相同。
分别于保温 0.25h， 0.5h， lh， 2.5h和 18h取样， 每次取样 0.5 mL, 取样后依次加 入 0.5 mL 超纯水， 0.2 mL 1 mol/L 的 Na2C03/NaHC03 (pH 9.8)缓冲液， 2.5 mL CHCl3-MeOH (3 : 1), 混合均匀， 3,500 rpm离心 20 min， 阿霉素分布在有机相。
高效液相色谱检测阿霉素含量。 色谱柱： Phenomenex Luna C18(250x4.6 mm, 5 μιη); 流动相为： 甲醇： 乙腈： 磷酸缓冲盐 =7:4:6; 检测波长 480nm; 流速： 0.8 mL/min。
实验结果如图 4: 实验组最大释放为 35%， 在 6h后达到 30%， 并且在 6-30h范围内 基本稳定在 30%， 对照组最大释放为 7%， 在整个释放过程中的始终释放很低。 说明本 发明实体瘤被动靶向性抗癌前药在溶酶中水解， 酰胺键断裂， 释放阿霉素。
碱法降脂并且超滤后的小分子果胶分子量测定：
收集超滤后截留下的滤饼， 用 95%乙醇反复洗涤滤饼直至液体无红色， 蒸干溶剂， 加入蒸熘水溶解沉淀， 凝胶渗透色谱测定分子量。
色谱条件： Aglientl lOO系列 HPLC， 1362A视差检测器和 G1310A单元泵；色谱柱： Ultrahydrogel™ linear column ( 7.8 X 300mm, Waters); 柱温： 40°C ; 流通池温度： 35 °C ; 流动相： 0.005M KNO3; 流速： 0.5mL/min; 样品浓度： 5mg/mL， 用 0.05 %叠氮化钠溶 解； 进样量： 20 μ ί 。
标准曲线的制备：用葡聚糖作用标准品 Dextrans (Mw= 5000， 25000， 50000， 80000， 270000)
测得其分子量为 1-3万。
同等条件下对市售柑橘高酯果胶分子量测定：测得其绝对分子量为 30000〜600000。 试验例 2应用 MTT法观察本发明实体瘤被动靶向性抗癌前药对体外肿瘤细胞系生 长活性的抑制
1、 受试药物
盐酸阿霉素， 购自浙江海正药业股份有限公司， 用生理盐水溶解配制成含阿霉素 2mg/ml,
本试验采用的实体瘤被动靶向性抗癌前药为实施例 1制备 (相当于阿霉素 2mg/ml)。
2、方法：将用 RPMI 1640培养基培养的对数生长期生长的细胞溶液（浓度为 50000 个 /ml) 接种于 96孔板上， 0.1ml /孔。 孵育 24小时后给予相应药物的处理。 待药物处 理 24小时后， 加入 0.02ml的 3-(4， 5-二甲基噻唑 -2)-2， 5-二苯基四氮唑溴盐（MTT)
( 5mg/ml)处理 4小时，移去培养液加入 0.15ml的二甲亚砜（DMSO)，用酶标仪在 570nm 处测定其光吸收值。
抑制率(％)= ( 1— 吸收值吸收值 /空白对照组吸收值） χ ΐοο%， 各组不同药物的细胞 抑制率见表 1。
不同药物对各种肿瘤细胞的抑制率
Figure imgf000011_0001
试验例 3本发明实体瘤被动靶向性抗癌前药对肺转移瘤的实验研究
1. 主要材料
盐酸阿霉素， 购自浙江海正药业股份有限公司；
果胶 -阿霉素偶联物为本发明实体瘤被动靶向性抗癌前药为实施例 2 样品 （相当于 阿霉素 2mg/ml)。
细胞和动物： 清洁级近交系 C57BL/6小鼠购自四川大学华西实验动物中心， 雌性， 6-7周龄， 体重 20±3g。 实验过程中自由饮水及进食。 每日光照 12小时， 小鼠（5只 /笼) 笼均采用中央换气***通气。小鼠黑色素瘤细胞株 B16购于上海细胞生物研究所， 由本 实验室保种。 2.方法：
细胞培养： 常规培养于 RPMI 1640 100mL/L胎牛血清加双抗 （青霉素 100U/mL， 链霉素 100mg/L)的培养液内， 置于 37°C， 50mL/L二氧化碳培养箱中培养.稳定传代 3， 4代后， 取对数生长期细胞， 经 2.5g/L胰酶消化后用无血清培养液重悬收集细胞， 用无 血清的 1640培养基调整细胞密度备用。
小鼠肺部转移肿瘤模型的建立： 取 C57BL/6小鼠， 随机分为数个组， 用 750mL/L 质量浓度的乙醇消毒小鼠尾部皮肤， 取 B16黑色素瘤细胞溶液于小鼠尾静脉注射， 接种 剂量为 0.1mL。
于小鼠接种肿瘤细胞后 4〜5天开始给药，每只小鼠每周给药 2次， 40〜50μ1/只 /次。 观察小鼠的荷瘤生存期。
本发明实体瘤被动靶向性抗癌前药对肺癌荷瘤小鼠生存时间的影响见图 5。 由图 5 可见在黑色素瘤 B16肺转移模型小鼠的疗效研究中， 果胶-阿霉素偶联物组小鼠的荷瘤 生存期为 42.3 ± 12. 4天， 明显高于盐酸阿霉素组 （23. 1 ± 10. 2天）。

Claims

权利要求书
1、 实体瘤被动靶向性抗癌前药， 由果胶与阿霉素键合而成， 其特征在于： 它是由 Mw 0. 5〜4. 5万的小分子的果胶与阿霉素反应得到的 Mw为 10〜100万的果胶-阿霉素偶 联物， 制成混悬剂后通过纳米超高压均质机处理得到粒径 100nm-200nm， 熔点 220 °C〜 245 °C实体瘤被动靶向性抗癌前药； 其中， 果胶与阿霉素通过酰胺键连接， 果胶与果胶 之间通过果胶分子的羧基与羟基缩合成的酯键连接。
2、 根据权利要求 1所述的实体瘤被动靶向性抗癌前药， 其特征在于： 所述实体瘤 被动靶向性抗癌前药的粒径 130nm-180nm。
3、 根据权利要求 1或 2所述的实体瘤被动靶向性抗癌前药， 其特征在于： 所述实 体瘤被动靶向性抗癌前药的水溶解度为 42 mg/L。
4、 根据权利要求 1所述的实体瘤被动靶向性抗癌前药， 其特征在于： 所述果胶-阿 霉素偶联物是由小分子的果胶与阿霉素于 pH为 5〜7， EDC · HC1作用下， 40〜60°C反 应得到。
5、 根据权利要求 4所述的实体瘤被动靶向性抗癌前药， 其特征在于： 所述小分子 的果胶 Mw 1〜3万。
6、 根据权利要求 1〜5任一项所述的实体瘤被动靶向性抗癌前药， 其特征在于： 果 胶 -阿霉素偶联物加入水中， 再加入 PVP和甘油， 混合均匀制成混悬剂后进行纳米超高 压均质机处理。
7、 根据权利要求 6所述的制备实体瘤被动靶向性抗癌前药的方法， 其特征在于混 悬剂分 3次进纳米超高压匀质仪处理， 第一次压力 120mpa， 第二次压力 180mpa， 第三 次压力 190mpa。
8、根据权利要求 7所述的实体瘤被动靶向性抗癌前药， 其特征在于： PVP的用量为 果胶-阿霉素偶联物质量的 1 倍〜 6 倍， 甘油的用量为果胶-阿霉素偶联物质量的 0. 1%-0. 8%。
9、 制备实体瘤被动靶向性抗癌前药的方法， 其特征在于由以下步骤完成： a、 Mw 0. 5〜4. 5万的小分子果胶溶解于水，加入盐酸阿霉素，混合均匀后与 EDC HCI 反应 3〜8h， 透析， 干燥得到 MwlO万〜 100万的果胶-阿霉素偶联物；
b、 果胶 -阿霉素偶联物加入水中， 再加入 PVP和甘油， 混合均匀制成混悬剂， 经纳 米超高压匀质仪处理， 即得粒径 100nm-200nm的实体瘤被动靶向性抗癌前药。
10、 根据权利要求 9所述的制备实体瘤被动靶向性抗癌前药的方法， 其特征在于： 所述小分子果胶是将果胶溶于水， 与 NaOH溶液在 pH=13的条件下反应， 然后用浓盐 酸调 pH到中性， 截留分子量得到。
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