WO2014190849A1 - 阿霉素前药及其制备方法和可注射的组合物 - Google Patents

阿霉素前药及其制备方法和可注射的组合物 Download PDF

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WO2014190849A1
WO2014190849A1 PCT/CN2014/077095 CN2014077095W WO2014190849A1 WO 2014190849 A1 WO2014190849 A1 WO 2014190849A1 CN 2014077095 W CN2014077095 W CN 2014077095W WO 2014190849 A1 WO2014190849 A1 WO 2014190849A1
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doxorubicin
mpeg
dox
prodrug
reaction
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欧阳钧
邢孟秋
陈骏
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南方医科大学
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    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives 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
    • A61K47/51—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
    • A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/14—Nitrogen atoms not forming part of a nitro radical

Definitions

  • the present invention relates to a prodrug and a novel medicament prepared using the prodrug, and in particular to a doxorubicin prodrug and an injectable pH sensitive hydrogel thereof.
  • Cancer is a high-risk and fatal disease. Although chemotherapeutic drugs can kill cancer cells, they often cannot distinguish between normal cells and cancer cells, and have corresponding side effects, causing normal tissues to be damaged by drugs and increasing patient suffering.
  • the methods for increasing the efficacy of the drug mainly include increasing the targeting selectivity of the drug and increasing the concentration of the locally active drug.
  • Current intravenous administration is the main route by which drugs reach solid tumors. The circulatory system and the supply of tumor arterial blood supply determine the effect of administration.
  • the local injection of a drug hydrogel has the following advantages over intravenous administration:
  • the hydrogel can release the drug for a long time
  • the hydrogel can stimulate the carrier according to the surrounding environment to achieve the desired release effect
  • Biodegradable injectable hydrogels reduce the risk of primary or secondary toxicity and immune response of nanopharmaceutical compositions
  • the application of injectable hydrogel can be applied in a non-invasive manner to achieve therapeutic effects, reducing the risk of surgery;
  • a supramolecular hydrogel is a crosslinked polymer formed by physically cross-linking a polymer component with a large amount of water. They have been extensively studied over the past 10 years as potential systems for loading molecules or cells. Due to the non-covalent nature of cross-linking, the supramolecular gel exhibits a reversible transition of the gel-sol under the action of shear. The properties of the supramolecular hydrogel "shake" make it suitable for topical, non-invasive drug injections. The injected sol locally becomes a gel in the body, and at the same time, the drug which is locally loaded is released. Many injectable hydrogels are used to load hydrophilic therapeutic drugs, such as small hydrophilic drug molecules, proteins, polypeptides, and oligonucleotides. However, only a small portion is used to make sustained-release hydrophilic anticancer drugs, including 5-fluorouracil, doxorubicin and cisplatin.
  • Li et al. used an ethylene oxide polymer to form a supramolecular gel with ⁇ -CD.
  • Zhu et al. applied cisplatin in nanoparticle as a composite structure to bind to a-CD, which has a programmed release property in the treatment of cancer.
  • a prodrug is a combination of a drug molecule and a hydrophilic complex such as (polyethylene glycol PEG, polysaccharide or acrylic polyamide). Things. When PEG is covalently bound to those poorly soluble drugs, the prodrug is imparted with long-lasting PEG removal, low immunity, and increased water solubility of the drug. The complex of PEG and drug interacts with ⁇ -CD to form a supramolecular gel with sustained release function. The prolongation of the gel decomposition time is due to the interaction of hydrophobic bonds between the low solubility drugs. Summary of the invention
  • Another object of the present invention is to provide a process for the preparation of the above doxorubicin prodrug.
  • the technical solution adopted by the present invention is:
  • a doxorubicin prodrug whose structural formula is as shown in formula (I):
  • n is an integer of from 10 to 100, preferably from 30 to 50.
  • the preparation method of the above doxorubicin prodrug comprises the following steps:
  • the PEG-hydrazide and doxorubicin were dissolved in an anhydrous polar solvent, stirred to complete the reaction, and the reaction was completed by adding an excess of triethylamine, and purified to obtain a doxorubicin prodrug, which was named MPEG-DOX.
  • the MPEG-hydrazide synthesis method comprises the following steps:
  • reaction solution A 1) taking excess barium sulfate salt dissolved in water, adjusting its P H to be alkaline, to obtain a reaction solution A;
  • the pH of the barium sulfate salt solution is adjusted to 8.5 to 10 by adding a NaOH solution.
  • the molar ratio of the methoxypolyethylene glycol succinimide acetate to the barium sulfate salt is 1 :
  • the molar ratio of MPEG-hydrazide to doxorubicin is 1: 0.2-5, preferably 1:1 to 3.
  • An injectable pH-sensitive doxorubicin prodrug composition consisting of a doxorubicin prodrug, an alpha-cyclodextrin, and an acceptable pharmaceutical excipient, wherein the doxorubicin prodrug is as described above.
  • the molar ratio of the doxorubicin prodrug to the ⁇ -cyclodextrin is 1:0.5-5, preferably 1:1.5 ⁇ 2.0.
  • the doxorubicin prodrug of the invention has simple preparation method, high yield and simple control of production cost.
  • the prodrug has low immunity and can effectively improve the water solubility of doxorubicin; in addition, the doxorubicin prodrug of the invention is cleared in the body for a long time, can effectively prolong the action time of the drug, and has pH sensitivity.
  • Doxorubicin can be released in situ under acidic pH conditions, while forming PEG.
  • the preparation method of the invention has low requirements on the reaction conditions, and the reaction can be completed by stirring at room temperature; the reaction process is easy to control, the safety is high, the yield is high, and the product is easy to be purified.
  • the doxorubicin prodrug composition of the invention can be dissolved in a nearly neutral aqueous solution to form a stable aqueous solution, and when the pH changes to acidic, a supramolecular hydrogel can be formed in a few minutes, which can be long-lasting. In situ release of doxorubicin can effectively prevent the spread of doxorubicin, affect the function of normal cells, and reduce the side effects of drugs.
  • Figure 1 is a nuclear magnetic resonance spectrum of MPEG-DOX of the present invention
  • Figure 2 is an FT-IR diagram of DOX, PEG, and MPEG-DOX;
  • Figure 3 is a sol-gel conversion diagram of the doxorubicin prodrug composition of the present invention.
  • Figure 4 is a rheological kinetic test chart of MPEG-DOX and ⁇ -CD supramolecular polymer gels
  • Figure 5 is a graph showing the relationship between MPEG-DOX and a-CD concentration and gel modulus
  • Figure 6 is a graph showing the in vitro DOX release of 5 wt% MPEG-DOX/8 wt% ⁇ -CD at different pH;
  • Figure 7 is a graph showing the cell viability of drug release in a supramolecular hydrogel at different DOX concentrations
  • Figure 8 is a graph showing the cytotoxicity of MPEG/a-CD hydrogel at different PEG concentrations
  • Figure 9 is a confocal micrograph of human MCF-7 cells co-cultured with drug-released samples at different time points;
  • Figure 10 is a confocal microscopy of human MCF-7 cells cultured in supramolecular hydrogels at different pHs. .
  • Adriamycin prodrug its structure (I) shows:
  • n is an integer of from 10 to 100, preferably from 30 to 50.
  • n is an integer of 10 to 100, preferably 30 to 50.
  • the preparation method of the above doxorubicin prodrug comprises the following steps:
  • the MPEG-hydrazide synthesis method comprises the following steps:
  • reaction solution A 1) taking excess barium sulfate salt dissolved in water, adjusting its P H to be alkaline, to obtain a reaction solution A;
  • the pH of the barium sulfate salt solution is adjusted to 8.5 ⁇ 10 by adding a NaOH solution, and the molecular weight of the barium sulfate salt is small, and it is easy to pass through the semipermeable membrane.
  • the barium sulfate salt during the reaction.
  • the amount is preferably excessive so that the methoxypolyethylene glycol succinimide acetate reacts as completely as possible. Therefore, as a further improvement of the present invention, the molar ratio of methoxypolyethylene glycol succinimide acetate to barium sulfate is 1:0.5 to 10, preferably 1:3 to 5.
  • the molar ratio of MPEG-hydrazide to doxorubicin is 1: 0.2-5, preferably 1: 1 ⁇
  • An injectable pH-sensitive doxorubicin prodrug composition consisting of a doxorubicin prodrug, alpha-cyclodextrin, and an acceptable pharmaceutical excipient, wherein the doxorubicin prodrug is as described above.
  • the molar ratio of the doxorubicin prodrug to the ⁇ -cyclodextrin is 1 : 0.5-5, preferably 1 : 1.5 to 2.0.
  • the barium sulfate salt (1 mmol, 130 mg) was dissolved in distilled water, and the pH was adjusted to 9.0 by adding a NaOH solution having a concentration of 1 mmol/L to obtain a reaction liquid A;
  • Methoxy polyethylene glycol succinimide acetate (0.1 mmol, 500 mg) was added to the reaction solution A, and the reaction was stirred at room temperature for 24 hours. The unreacted small molecule was removed by dialysis, and lyophilized to obtain a purified MPEG-acyl group. Hey.
  • the reaction yield was calculated to be 90%.
  • MPEG-DOX synthesis Dissolve MPEG-hydrazide (0.1 mmol, 550 mg) with doxorubicin DOX (0.2 mmol, 118 mg) in 10 ml anhydrous
  • the product was precipitated in diethyl ether, purified, and dried in vacuo to give a pink powder.
  • the reaction yield was calculated to be 95%.
  • the obtained product MPEG-DOX was subjected to nuclear magnetic resonance analysis (300 MHz), and the nuclear magnetic resonance instrument used was Bruker Avance 300, and the solvent was d6-DMSO, and its 1H-NMR chart is shown in Fig. 1.
  • the peaks at 1.18 ppm, 7.60 - 7.90 ppm show the characteristic proton peaks produced by the methyl and aryl groups in the DOX of the conjugate; the peaks at 4.20 ppm and 3.55 ppm show the methylene group attached to the carbonyl group, respectively.
  • the characteristic proton peak of the methylene group is repeated in the MPEG of the conjugate.
  • the structure of MPEG-DOX is as follows:
  • PEG is linked to DOX via a ⁇ bond.
  • the FT-IR technique is used to analyze DOX, PEG and MPEG-DOX, and the FT-IR diagram is shown in Fig. 2. The result also shows that MPEG is coupled to DOX.
  • the infrared spectrum of MPEG-DOX has a typical ether bond from PEG at 1108
  • the telescopic oscillation peak of cm" 1 is derived from the stretching and oscillating peak of the carbonyl group at 1730 cm" 1 in DOX.
  • MPEG-DOX can be dissolved in water to form a uniform aqueous solution.
  • a-CD ⁇ -cyclodextrin
  • the aqueous MPEG-DOX solution can be converted into an injectable hydrogel.
  • the formation of the gel depends on the ratio between MPEG-DOX and ⁇ -CD and can be generated under mild conditions without the need for high temperatures, emulsifiers or crosslinkers. This is because MPEG-DOX and a-CD can form inclusion complexes in a hybrid system.
  • MPEG-DOX and a-CD were each dissolved in a PBS buffer of pH 7.4 to obtain an aqueous solution thereof.
  • MPEG-DOX and a-CD will gel at host temperature through host-guest interaction.
  • the two concentrations of MPEG-DOX 2.5 Hekou 5.0 wt%) and a-CD (6.0 Hekou 8.0 wt%) 0 in the mixed system
  • time lapse rheology analysis was carried out in an oscillating mode using an advanced galvanometer extensometer (ARES, TA).
  • the reaction conditions were: 25 °C, parallel
  • the plate (diameter 20 mm, gap 0.5 mm) was placed in a parallel plate immediately after mixing and measured after 1 min.
  • the relationship between the viscoelastic force of the sample and the time was measured in the linear region of the viscoelastic force determined by the previous tension scanning.
  • a dynamic frequency sweep test (0.1 to 100 rad/s) was applied to the hydrogel.
  • the hydrogel sample needed to be aged for 12 h before testing.
  • the gelation time was reduced from 20 min to 12.5 min when the MPEG-DOX concentration was increased from 2.5 to 5.0 wt%.
  • the MPEG-DOX concentration was 5 wt% and the a-CD concentration was increased from 6.0 to 8.0 ⁇ 1%, the gelation time was reduced from 12.5 min to 4 min. This result indicates that any increase in MPEG-DOX or a-CD concentration favors the formation of supramolecular hydrogels, probably because a-CD contains a mechanism for the gelation system.
  • the gum system contains a composite of a-CD and PEO segments to form a neck-ring structure containing composite, and self-assembly as a physical cross-linking presents the initial driving force for sol-gel conversion. Therefore, a high concentration of solution can increase the probability of inclusion body formation.
  • the relationship between the elastic modulus (G') of the formed supramolecular hydrogel and the concentrations of MPEG-DOX and ⁇ -CD was measured using a rheometer. As shown in Fig. 5, in different concentrations of the MPEG-DOX/a-CD mixed system, G' changes with the change in its concentration.
  • the G' value increases.
  • the MPEG-DOX concentration is increased from 2.5 to 5.0 wt% at a frequency of 1.0 rad/s
  • the G' value increases from approximately 8.3 kPa to ⁇ 800 kPa.
  • the concentration of ⁇ -CD increased from 6.0 to 8.0 wt% G' value increased from about 98 kPa to about 800 kPa.
  • all G' values have little to do with frequency.
  • a total of 0.1 ml of the mixture (5 wt% MPEG-DOX/8 wt% a-CD) was injected into a 2 ml tube for each test sample and allowed to stand overnight to form a hydrogel.
  • the tubes were incubated in oscillated water (85 rpm, 37 °C). According to the preset time point, take 0.5 ml from the supernatant of 1 ml of the tube, then add 0.5 ml of pre-warmed buffer to maintain its volume of 1 ml. The time of each sampling is determined to be about 30 S. , the mixture will not reach equilibrium during this time.
  • the solution was taken at different time points and the characteristic absorption wavelength of DOX in solution was measured at 480 nm. Each sample was tested 3 times.
  • the MTT kit was used to detect the inhibition rate of hydrogel on MCF-7 cell line.
  • Human breast cancer cells (MFC-7) were supplemented with 10% fetal bovine serum (FBS, GIBCO l.Ox lO 5 U/l penicillin (Sigma 100 mg/1 streptomycin (Sigma) in DMEM medium (DMEM, GIBCO) Medium, 37°C 5% C0 2
  • MCF-7 cells were seeded at a density of 8000 cells/well in a 96-well plate, cultured at 37 ° C 5% CO 2 , and after 24 hours, about 20 ⁇ l of growth medium was taken and injected into a roughly equal amount of hydrogel, and allowed to stand. 1 h;
  • the medium was then replaced with fresh DMEM.
  • the collected release solution and the control solution (without DOX) were added to the well plate (6 holes per sample) for 24 h, then ⁇ MTT solution was added to the well, and the culture was continued for 4 h;
  • the culture medium in the well plate was removed and 200 L of DMSO was added to each well, which was blown several times to dissolve the formazan therein; the absorbance of each well was measured using an ELISA plate analyzer at a measurement wavelength of 570 nm, and the reference wavelength was The method for calculating the cell inhibition rate in the 630 nm sample is as follows:
  • I and I represent the absorbance of different test samples and control cells, respectively.
  • the hydrogel showed a lower cell inhibition rate than pH 6.0 at a pH of 7.4.
  • DOX was about 1.5 ⁇ ⁇ / ⁇ 1, about 6 g / ml at ⁇ 7.4.
  • the viability of the cells in a pure MPEG/a-CD hydrogel was also examined by the MTT method, and the results are shown in FIG. As expected, the results show that at low concentrations (PEG ⁇ 100 ⁇ ⁇ / ⁇ 1 ), the hydrogel is not toxic to MCF-7 cells, or even (1 mg / PEG ml) is at very high concentrations, the cells pH7. In the case of 4, the vitality is still around 70%.
  • MCF-7 cells were co-cultured with free DOX released or MPEG-DOX released for 3 h, 24 h and 48 h, respectively, and then the uptake and drug distribution of DOX were studied using confocal laser scanning microscopy (CLSM).
  • CLSM confocal laser scanning microscopy
  • MCF-7 cells were seeded in a petri dish (3.5 cm in diameter) with a cover glass, seeded at a density of 2> ⁇ 10 5 cells/dish for 24 h, and then the gel was injected onto the wall of the dish. ;
  • the coverslips were rinsed 3 times with PBS, and the treated cells were incubated with 4% paraformaldehyde for 15 min.
  • the fixed cells were immersed in PBS containing 0.1% Triton X-100 for 10 min to increase. Permeability of the cell membrane, followed by rinsing 3 times with PBS;
  • FIG. 9 The experimental results are shown in Fig. 9.
  • A, C and E are micrographs of drug release culture at pH 7.4 for 3 h, 24 h and 48 h, respectively.
  • B, D and F release drug culture at pH 5.0, respectively.
  • the drug released from the gel at pH 5.0 and 7.4 was incubated for 3 h, and DOX fluorescence was concentrated in and around the nucleus. Due to the reported intracellular and lysosomal pH values of 5.0 to 7.0, the MPEG-DOX conjugate can be rapidly degraded and effectively release doxorubicin into the cytoplasm. After exposure to the released drug for 24 h, most of the DOX fluorescence was distributed in the nucleus of PCF-7 cells. However, compared with the drug released at pH 7.4, after 24 hours of exposure to the drug at pH 5.0, the cell nucleus expanded and the cytoplasm contracted. This is because the difference in DOX release at different pH is significant.
  • the MPEG-DOX hydrogel of the present invention can effectively increase the efficiency of cellular uptake of DOX.
  • the nucleus of DOX released by exposure to pH 7.4 became larger and cytoplasm decreased after 48 h of release of DOX.
  • prodrugs of pH 7.4 exhibited higher effective cell inhibition rates than drugs released under acidic conditions.
  • the drug release behavior of supramolecular gels the drug release of cells and gels under co-culture of P H 7.4, 6.8 and 6.0 was compared. The pH of the culture broth was adjusted to about 6.0 and 6.8, respectively, by the addition of a 1 M HCl solution.
  • the highest concentration of DOX contained in each petri dish gel was set to 10 ⁇ ⁇ / ⁇ 1.
  • the experimental results are shown in Fig. 10.
  • A, B, and C were co-focused microscopic images after co-culture for 1 h at pH 6.0, 6.8, and 7.4, respectively.
  • the upper graph is an overlay of cells and DOX fluorescence, and the text is separate. DOX fluorescence map.

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Abstract

一种阿霉素前药及其制备方法和可注射的组合物。所述阿霉素前药由MPEG和DOX通过腙键连接而成，其制备方法简单，对反应条件的要求低，产率高，生产成本更易于控制。该前药的免疫性低，可以有效提高阿霉素的水溶性，在体内被清除时间长，可以有效地延长药物的作用时间，具有pH敏感性，在酸性pH环境下可以原位释放出阿霉素，同时形成PEG。所述阿霉素前药组合物，可溶解在接近中性的水性溶液中，形成性质稳定的水溶液，在pH变化为酸性时，可以在数分钟内形成超分子水凝胶，可以长效原位释放阿霉素，可以有效避免阿霉素扩散，影响正常细胞的功能，减少药物的毒副作用。

Description

阿霉素前药及其制备方法和可注射的组合物 技术领域
本发明涉及一种前药及使用该前药制备得到的新型药剂， 具体涉及阿霉素前药及其可注 射的 pH敏感水凝胶。
背景技术
癌症是一高发及致命性疾病， 虽然目前化疗药物能杀死癌细胞， 但他们常区分不了正常 细胞与癌细胞而有相应的副作用， 致正常组织受到药物毒性损伤并增加病人痛苦。 而增加药 物疗效的方法主要包括提高药物靶向选择性和提高局部活性药物的浓度。 目前静脉给药是药 物达到实体瘤的主要途径。 而循环系统情况及肿瘤动脉血运供应情况决定了给药效果。 而局 部注射药物水凝胶的方法与静脉给药相比有以下优越性：
1、 它能有效地释放治疗因子于瘤体局部间隙内， 而避免了复杂的血管系统；
2、 水凝胶能让药物缓释， 作用时间长；
3、 水凝胶能据周围环境对载体物的剌激而达到想要的释放效果；
4、 生物可降解性可注射水凝胶减少了纳米药物合成物的原发或继发的毒性及免疫 反应风险；
5、 应用可注射水凝胶能应用非损伤的方式达到治疗效果， 而减少了手术的风险；
6、 应用可注射水凝胶局部注射药物是病人情况欠佳而不能耐受手术时另一合适的 治疗手段。
超分子水凝胶是通过聚合物成分与大量水通过物理相互交联作用后，形成的交联聚合物。 过去的 10年里， 它们作为载荷分子或细胞潜在的有效系统而被广泛研究。 由于交联的非共价 属性， 在剪力的作用下， 超分子凝胶显示出凝胶-溶胶的可逆性转变。 超分子水凝胶 "摇溶 "的 特性使得其适用于局部、 非侵袭性的药物注射。 被注射的溶胶在体内局部变成凝胶， 同时在 局部使其载荷的药物缓释。 许多可注射水凝胶用来载荷亲水治疗药物等， 如小的亲水药物分 子、 蛋白、 多肽及寡核苷酸。 但只有小部分用来制作缓释的亲水抗癌药物， 包括 5-氟尿嘧啶， 阿霉素及顺铂等为基础的药物。
在这些凝胶中， 近年来， 由 a-CD与其他聚合物络合形成的超分子凝胶引起了研究者们 广泛的兴趣。 Li等应用环氧乙烯聚合物与 α-CD形成超分子凝胶。 Zhu等应用顺铂被包在纳 米粒内作为复合结构与 a-CD结合， 这种复合的在癌症的治疗中具有程序性缓释性能。
药物前体是药物分子与亲水性复合物如 (聚乙二醇 PEG、 多糖或丙烯酸聚酰胺)结合的产 物。 当 PEG与那些溶解性差的药物共价结合后， 药物前体被赋予 PEG的被清除时间长、 低 免疫性及增加药物的水溶性等性能。 PEG与药物的复合物与 α-CD相互作用而形成具有缓释 功能的超分子凝胶。凝胶分解时间的延长是由于低溶性药物间的疏水键的相互作用而形成的。 发明内容
本发明的目的在于提供一种阿霉素前药。
本发明的另一个目的在于提供上述阿霉素前药的制备方法。
本发明的再一个目的在于提供一种使用了上述阿霉素前药的可注射 pH敏感的水凝胶。 本发明所采取的技术方案是：
阿霉素前药， 其结构式如式 （ I ) 所示:
Figure imgf000003_0001
式 （ I )
式中， n为 10〜100， 优选为 30〜50之间的整数。
上述阿霉素前药的制备方法， 包括如下步骤：
将 PEG-酰肼与阿霉素溶解于无水的极性溶剂中， 搅拌使其反应完全， 加入过量三乙胺继 续反应完全， 纯化得到阿霉素前药， 命名为 MPEG-DOX。
作为本发明的进一步改进， MPEG-酰肼的合成方法包括如下步骤：
1) 取过量硫酸肼盐溶于水中， 调节其 PH为碱性， 得到反应液 A;
2) 将甲氧基聚乙二醇琥珀酰亚胺乙酸酯加入反应液 A 中， 搅拌反应完全， 透析去除未 反应的小分子， 冻干得到纯化的 MPEG-酰肼。
作为本发明的进一步改进， 加入 NaOH溶液调节硫酸肼盐溶液的 pH至 8.5〜10 作为本发明的进一步改进， 甲氧基聚乙二醇琥珀酰亚胺乙酸酯与硫酸肼盐的摩尔比为 1 :
0.5〜10， 优选为 1： 3〜5。
作为本发明的进一步改进， MPEG-酰肼与阿霉素的摩尔比为 1 : 0.2-5, 优选为 1 : 1〜3。 可注射的 pH敏感阿霉素前药组合物，由阿霉素前药、α-环糊精及可接受的药用辅料组成， 其中， 阿霉素前药如上所述。 阿霉素前药与 α -环糊精的摩尔比为 1 : 0.5-5, 优选为 1 : 1.5〜2.0。
本发明的有益效果是：
本发明的阿霉素前药， 制备方法简单， 产率高， 生产成本更易于控制。 该前药的免疫性 低， 可以有效提高阿霉素的水溶性； 此外， 本发明的阿霉素前药在体内被清除时间长， 可以 有效地延长药物的作用时间， 具有 pH敏感性， 在酸性 pH环境下可以原位释放出阿霉素， 同 时形成 PEG。
本发明的制备方法， 对反应条件的要求低， 室温下搅拌即可完成反应； 反应过程易于控 制， 安全性高， 产率高， 产品易于纯化。
本发明的阿霉素前药组合物， 可溶解在接近中性的水性溶液中， 形成性质稳定的水溶液， 在 pH变化为酸性时， 可以在数分钟内形成超分子水凝胶， 可以长效原位释放阿霉素， 可以 有效避免阿霉素扩散， 影响正常细胞的功能， 减少药物的毒副作用。
附图说明
图 1是本发明 MPEG-DOX的核磁共振图谱；
图 2是 DOX、 PEG和 MPEG-DOX的 FT-IR图；
图 3是本发明阿霉素前药组合物的溶胶-凝胶转化图；
图 4是 MPEG-DOX与 α-CD超分子聚合物凝胶的流变动力学测试图；
图 5是 MPEG-DOX 与 a-CD浓度与凝胶模量的变化关系图；
图 6是不同 pH下， 5 wt% MPEG-DOX/8 wt% α-CD体外 DOX释放情况图；
图 7是不同 DOX浓度时超分子水凝胶内药物释放的细胞活力情况图；
图 8是不同 PEG浓度时 MPEG/a-CD水凝胶细胞毒性情况图；
图 9是不同时间点人类 MCF-7细胞与药物释放的样品共培养时的共聚焦显微镜图； 图 10是不同 pH下人类 MCF-7细胞在超分子水凝胶中培养时的共聚焦显微镜图。
具体实 ¾ ^式
阿霉素前药， 其结构 （ I ) 所示：
Figure imgf000004_0001
式 （ I )
式中， n为 10〜100， 优选为 30〜50之间的整数。
PEG单元在于提供足够的亲水性，保证其可以很好的被溶于水，而当单元重复数过高时， 因为高分子量 PEG本身的溶解度降低， 反而会影响前药的水溶性。 因此， 上述结构式中， n 为 10〜100， 优选为 30〜50之间的整数。
上述阿霉素前药的制备方法， 包括如下步骤：
将 MPEG-酰肼与阿霉素溶解于无水的极性溶剂中， 搅拌使其反应完全， 加入过量三乙胺 继续反应完全， 纯化得到阿霉素前药， 命名为 MPEG-DOX。
作为本发明的进一步改进， MPEG-酰肼的合成方法包括如下步骤：
1) 取过量硫酸肼盐溶于水中， 调节其 PH为碱性， 得到反应液 A;
2) 将甲氧基聚乙二醇琥珀酰亚胺乙酸酯加入反应液 A 中， 搅拌反应完全， 透析去除未 反应的小分子， 冻干得到纯化的 MPEG-酰肼。
作为本发明的进一步改进， 加入 NaOH溶液调节硫酸肼盐溶液的 pH至 8.5〜10 硫酸肼盐的分子量较小， 易于通过半透膜， 为了便于后续的纯化操作， 在反应过程中， 硫酸肼盐的量宜过量， 以使甲氧基聚乙二醇琥珀酰亚胺乙酸酯尽可能地反应完全。 因此， 作 为本发明的进一步改进， 甲氧基聚乙二醇琥珀酰亚胺乙酸酯与硫酸肼盐的摩尔比为 1 : 0.5〜 10, 优选为 1： 3〜5。
类似的， 为了尽可能地利用合成得到的 MPEG-酰肼， 在偶联反应中， 要使 DOX过量， 因此， 作为本发明的进一步改进， MPEG-酰肼与阿霉素的摩尔比为 1 : 0.2-5, 优选为 1 : 1〜
3。
可注射的 pH敏感阿霉素前药组合物，由阿霉素前药、α-环糊精及可接受的药用辅料组成， 其中， 阿霉素前药如上所述。
阿霉素前药与 α -环糊精的摩尔比为 1 : 0.5-5, 优选为 1 : 1.5〜2.0。
下面结合实施例， 进一步说明本发明。
MPEG-酰肼的合成
取硫酸肼盐 (1 mmol, 130 mg)溶解于蒸熘水中， 加入浓度为 lmmol/L的 NaOH溶液调整 其 pH为 9.0， 得到反应液 A;
将甲氧基聚乙二醇琥珀酰亚胺乙酸酯（0.1 mmol, 500 mg)加入反应液 A中， 室温下搅拌 反应 24h， 透析去除未反应的小分子， 冻干得到纯化的 MPEG-酰肼。
经计算， 反应得率为 90%。
MPEG-DOX的合成 将 MPEG-酰肼 (0.1 mmol, 550 mg)与阿霉素 DOX(0.2 mmol, 118 mg)溶解在 10ml无水的
DMSO, 室温下搅拌 3天， 之后加入过量三乙胺；
将产物在乙醚中沉淀、纯化，真空干燥得粉红色粉末，得到阿霉素前药，记为 MPEG-DOX。 经计算， 反应得率为 95%。
整个合成反应的原理如下所示：
Figure imgf000006_0001
产物的确认：
对得到的产物 MPEG-DOX进行核磁共振分析（300 MHz),所使用的核磁共振仪为 Bruker Avance 300, 溶剂为 d6-DMSO, 其 1H-NMR图如图 1所示。 1.18 ppm, 7.60 - 7.90 ppm处的 峰显示了偶联物的 DOX中的甲基及芳基所产生的特征质子峰； 4.20 ppm和 3.55 ppm处的峰 分别显示了与羰基连接的亚甲基和偶联物的 MPEG 中重复亚甲基的特征质子峰。 证明 MPEG-DOX的结构式如下 ：
Figure imgf000006_0002
PEG通过腙键与 DOX相连接。
根据偶联物中由来自 MPEG亚甲基的质子峰 (δ=4.20)和来自 DOX甲基的质子峰 (δ=1.18) 的积分值计算偶联物的连接率。 结果显示， 每 100个 MPEG分子与约 95分子 DOX相偶联。
采用 FT-IR技术对 DOX、 PEG和 MPEG-DOX进行分析， 其 FT-IR图如图 2所示。 结果 同样显示 MPEG与 DOX偶联了。MPEG-DOX的红外图谱具有典型的来自 PEG的醚键在 1108 cm"1的伸缩振荡峰， 来自 DOX中羰基在 1730 cm"1的伸缩振荡峰。
如图 3所示， MPEG-DOX可以溶解在水中， 形成均一的水溶液。 特别的， 当引入 a-CD ( α-环糊精） 后， MPEG-DOX 水溶液可以转化为可注射的水凝胶。 凝胶的形成取决 MPEG-DOX和 α-CD之间的比例， 在温和条件下即可生成， 无需高温、 乳化剂或交联剂。 这 是因为 MPEG-DOX和 a-CD可以混合体系中形成包含复合体。
凝胶化及超分子水凝胶的特点
为了形成超分子水凝胶， MPEG-DOX与 a-CD ( a-环糊精）分别使用 pH 7.4的 PBS缓冲 液溶解得到其水溶液。 取决于 MPEG-DOX或 a-CD使用的量， 室温下 MPEG-DOX和 a-CD 在混合体系中会通过主客交互作用发生凝胶化。 在本发明中， 在混合体系中使用了两种浓度 的 MPEG-DOX ( 2.5 禾口 5.0 wt % ) 和 a-CD ( 6.0禾口 8.0 wt % ) 0
为研究 MPEG-DOX/a-CD 水溶液混合体系的凝胶化动力学， 应用先进电流计延伸仪 (ARES,TA)以振荡模式进行时间扫描流变学分析， 反应条件为： 25 °C， 平行板 （直径 20 mm， gap 0.5mm) 样品混匀后立即置于平行板内， l min之后进行测量。在之前通张力扫描确定的 粘弹力线性区， 测量样品的粘弹力与时间的关系。 为研究形成的水凝胶的机械性能， 对水凝 胶进行动态频率扫描测试 （dynamic frequency sweep test) (0.1〜100 rad/s)， 在测试前， 水凝 胶样品需要陈化 12h。
为了研究 MPEG-DOX和 α-CD是如何影响超分子凝胶化的，对样品进行时间扫描测量以 确定凝胶的粘弹性，记录其在不同时间点的储存模量 (G')及损耗模量 (G")。测试条件为： 25 °C ; 频率， 6.0 rad/s; (A) 2.5 wt% MPEG-DOX禾 P 8 wt% α-CD; (B) 5 wt% MPEG-DOX禾 P 6 wt% a-CD; (C) 5 wt% MPEG-DOX禾 P 8 wt% a-CD。
如图 4所示， 不同的 MPEG-DOX/a-CD水溶液混合体系具有不同的 G'和 G"-时间曲线。 每个体系中的 G'和 G'-时间曲线均存在一个交点， 显示该处反应了溶胶 -凝胶转化。 在交点之 后， G'值越来越大于 G"值， 表明体系变的更类似于固体。相应的， 从体系由粘性表现至弹性 反应的时间可被认为是 MPEG-DOX与 a-CD成胶的时间。 从图 4可知， 成胶时间的长短与 MPEG-DOX或 a-CD 的浓度有关， 浓度高时其成胶时间缩短。 当 a-CD浓度为 8 wt%时， MPEG-DOX浓度从 2.5升至 5.0 wt %时， 成胶时间从 20min降至 12.5min。 当 MPEG-DOX浓 度为 5wt%， a-CD浓度从 6.0升至 8.0 \¥1 %时， 成胶时间从 12.5min减少至 4 min。 此结果显 示 MPEG-DOX或 a-CD其中任一浓度的提高都有利于超分子水凝胶的形成，可能是因为 a-CD 包含成胶系统的机制。 胶体系中含有由 a-CD与 PEO片断交织而形成颈环结构包含复合物， 自组装作为一种物理交联提交了溶胶 -凝胶转化的初始驱动力。 因此， 高浓度的溶液可以提高 包含体形成的机率。 使用流变仪测量形成的超分子水凝胶的弹性模量（G' )与 MPEG-DOX和 α-CD浓度之间 的关系。如图 5示，在不同浓度的 MPEG-DOX/a-CD混合体系中， G'随其浓度的变化而变化。
MPEG-DOX与 α-CD的浓度升高时， G'值随之升高。 当 MPEG-DOX浓度从 2.5 升高至 5.0 wt % ， 频率 1.0 rad/s, G'值从 约 8.3 kPa升高到 ~800 kPa 。 当 MPEG-DOX浓度维持为 5.0 \¥1%时， α-CD 的浓度由 6.0升高到 8.0 wt% G'值从约 98 kPa增至约 800 kPa。 同时， 所有的 G'值与频率关系不大。 这些结果显示水凝胶很好地交联了。
MPEG-DOX/a-CD混合体系的体外 DOX释放能力
每份测试样品均将总计 0.1 ml的混合液 （5 wt% MPEG-DOX/8 wt% a-CD) 注射至 2 ml 的管内， 放置过夜以形成水凝胶。 分别在管内加入 PBS溶液 （l ml pH=7.4) 或醋酸缓冲液 (1.0 ml, 0.1 mo I, pH=6.0 或 5.0)作为释放介质。测试过程中，管置于振荡的水（85 rpm, 37 °C ) 中保温。 根据预先设定好的时间点， 从管内 l ml的上清液中取出 0.5 ml， 然后加入 0.5 ml预 热的缓冲液， 以维持其体积为 l ml， 每次取样的时间确定约为 30 S， 在该时间内混合物不会 达到平衡。 在不同的时间点取出溶液， 480 nm下检测 DOX在溶液中的特征吸收波长。 每个 样品重复测试 3次。
如图 6所示， 48小时内， 在 pH 7.4的 PBS溶液中， MPEG-DOX中偶联的 DOX缓慢释 放了约 30%， 而 pH 5.0和 6.0溶液中的水凝胶在最初的 6小时内就释放了近 30%的 DOX 48 小时后，释放率高达 70%。保温 5天后，酸性 pH下，几乎所有的 DOX都已经释放，而 pH 7.4 下的类似固体的凝胶仅释放了 60%
MPEG-DOX/a-CD水凝胶的 MTT实验
应用 MTT试剂盒来检测水凝胶对 MCF-7细胞系抑制率情况。人类乳腺癌细胞（MFC-7 ) 添加了 10%胎牛血清 (FBS, GIBCO l.Ox lO5 U/l青霉素 （Sigma 100 mg/1链霉素 （Sigma) 的 DMEM培养液 （DMEM, GIBCO)中， 37°C 5% C02
MCF-7细胞以 8000个 /孔的密度接种在 96孔板内， 37°C 5% C02培养， 24小时后， 取 出约 20μ1生长培养基并注射加入大致等量的水凝胶， 静置 1 h;
然后培养液使用新鲜的 DMEM替换。 将收集的释放液和对照液 （无 DOX) 加入孔板中 (每份样品 6孔） 培养 24 h后， 在孔内加入 ΙΟμί MTT溶液， 继续培养 4 h;
将孔板中的培养液移除并在每孔中加入 200 L DMSO, 吹打数次以溶解其中的甲瓒； 使用 ELISA板分析仪测定每个孔的吸光度， 测量波长为 570 nm, 参照波长为 630 nm 样本中的细胞抑制率的计算方法如下：
细胞抑制率 = (I I 1 1 X 100 %
其中， I 和 I 分别代表不同测试样本和对照细胞的吸光度。 如图 7示，水凝胶在 pH7.4值时，显示比 pH6.0时更低的细胞抑制率。水凝胶的 DOX在 H 6.0的 IC 50截流率约为 1.5 μβ/ηι1, 在 ρΗ7.4下约为 6 g/ml。 同样用 MTT法检测了细胞 在单纯的 MPEG/a-CD水凝胶中的活力，结果如图 8所示。不出所料，结果显示在低浓度时 (PEG < 100 μβ/ηι1), 水凝胶对 MCF-7细胞没有毒性， 甚至在极高浓度时 (1 mg/ml 的 PEG), 细胞在 pH7.4的情况下的活力仍在 70%左右。 这些结果表明， MPEG-DOX/a-CD水凝胶可以有效地 抑制细胞， 抑制率与 pH值相关。
细胞对释放药物的摄取
将 MCF-7细胞与释放的自由 DOX或释放的 MPEG-DOX分别共培养 3 h、 24 h和 48 h， 然后使用共聚焦激光扫描显微镜（CLSM)研究 DOX的摄取和药物分布情况。 为了评价细胞 在不同 pH下的摄取率， 通过添加 1 M HC1溶液调节基质的 pH值至约 6.0。
首先， MCF-7细胞接种在设有盖玻片的培养皿（直径 3.5 cm)内，接种密度为 2>< 105 cells/ 皿， 培养 24 h， 然后将凝胶注射至培养皿的壁上；
3 h后， 另外加入 l ml的培养液以使浸没凝胶， 这样细胞就暴露在样本中了；
培养至预定时间后， 使用 PBS冲洗盖玻片 3次， 使用 4%的多聚甲醛保温 15 min固定处 理过的细胞， 固定后的细胞在含有 0.1% Triton X-100的 PBS浸泡 10 min以增加细胞膜的通 透性， 之后使用 PBS冲洗 3次；
使用 10 nM类鬼笔环肽 /1 % (w/v) BSA溶液染色处理 20 min, 之后使用 PBS冲洗 3次使 细胞骨架可视； 使用 10 μΜ Topro-3染色 20 min, 之后使用 PBS冲洗 3次使细胞核可视； 最后将盖玻片置于滴加有无色油脂的显微载玻片上， 用 CLSM观察。
实验结果如图 9所示， 图中 A、 C、 E分别为 pH 7.4下释放药物培养 3 h、 24 h、 48 h的 显微照片， B、 D、 F分别为 pH 5.0下释放药物培养 3 h、 24 h、 48 h的显微图像。 每一小幅 图中， 左侧为叠加了 DOX荧光的细胞染色图， 右侧为单纯的 DOX荧光， 标尺为 50 μηι。
收集保温 3 h后， pH 5.0和 7.4下的凝胶液释放的药物的并孵育 3 h后， DOX荧光集中在 细胞核中和其周围。 由于报导的胞内和溶酶体的 pH值为 5.0〜7.0， MPEG-DOX偶联体可以 很快的被降解并有效释放出阿霉素至细胞质中。 在暴露于释放的药物 24 h后， 大部分 DOX 荧光分布在 PCF-7细胞的细胞核内。 但是， 与暴露于 pH 7.4下释放的药物相比， 暴露于 pH 5.0下释放药物下 24 h后， 细胞的核膨大， 而细胞质收缩。 这是因为不同 pH下的 DOX释放 量差异显著。 这一结果进本发明的 MPEG-DOX水凝胶可以有效提高细胞摄取 DOX的效率。 与暴露 24 h的细胞形态相比， 释放的 DOX培养 48 h后， 暴露于 pH 7.4下释放的 DOX的细 胞核变得更大， 细胞质减少。 结合体外药物释放结果， 与酸性条件下释放的药物相比， pH 7.4 的前药表现出更高的有效细胞抑制率。 为进一步研究超分子凝胶的药物释放行为， 分别比较细胞和凝胶在 PH 7.4、 6.8和 6.0共 培养下的药物释放情况。 通过添加 1 M HC1溶液调节培养液的 pH分别至约 6.0和 6.8。 每个 培养皿凝胶中所含的 DOX的最高浓度设为 10μ§/ηι1。 实验结果如图 10所示， 图中 A、 B、 C 分别在 pH 6.0、 6.8和 7.4共培养 l h后共聚焦显微图像， 其中， 上方的图为细胞与 DOX荧光 的叠加图， 正文为单独的 DOX荧光图。

Claims

权
Figure imgf000011_0001
式 （ I )
式中， n为 10〜100。
2. 根据权利要求 1所述的阿霉素前药， 其特征在于： n为 30〜50之间的整数
3. 权利要求 1或 2所述阿霉素前药的制备方法， 包括如下步骤：
将 MPEG-酰肼与阿霉素溶解于无水的极性溶剂中， 搅拌使其反应完全， 加入过量三乙胺 继续反应完全， 纯化得到阿霉素前药， 命名为 MPEG-DOX。
4. 根据权利要求 3所述的制备方法， 其特征在于： MPEG-酰肼的合成方法包括如下步骤：
1) 取过量硫酸肼盐溶于水中， 调节其 pH为碱性， 得到反应液 A;
2) 将甲氧基聚乙二醇琥珀酰亚胺乙酸酯加入反应液 A中， 搅拌反应完全， 透析去除 未反应的小分子， 冻干得到纯化的 MPEG-酰肼。
5. 根据权利要求 4所述的制备方法， 其特征在于： 加入 NaOH溶液调节硫酸肼盐溶液的 pH 至 8.5〜10。
6. 根据权利要求 4或 5任意一项所述的制备方法， 其特征在于： 甲氧基聚乙二醇琥珀酰亚 胺乙酸酯与硫酸肼盐的摩尔比为 1 : 0.5〜10。
7. 根据权利要求 2或 3所述的制备方法， 其特征在于： MPEG-酰肼与阿霉素的摩尔比为 1 :
0.2〜5。
8. 可注射的 pH敏感阿霉素前药组合物，由阿霉素前药、α-环糊精及可接受的药用辅料组成， 其中， 阿霉素前药如权利要求 1所述。
9. 根据权利要求 5所述的可注射的 pH敏感阿霉素组合物， 其特征在于： 阿霉素前药与 α- 环糊精的摩尔比为 1 : 0.5〜5。
10. 根据权利要求 9所述的可注射的 pH敏感阿霉素组合物， 其特征在于： 阿霉素前药与 α- 环糊精的摩尔比为 1 : 1.5〜2.0。
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