WO2018090266A1 - 口服结肠靶向的递送系统及其制备方法和应用 - Google Patents

口服结肠靶向的递送系统及其制备方法和应用 Download PDF

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WO2018090266A1
WO2018090266A1 PCT/CN2016/106175 CN2016106175W WO2018090266A1 WO 2018090266 A1 WO2018090266 A1 WO 2018090266A1 CN 2016106175 W CN2016106175 W CN 2016106175W WO 2018090266 A1 WO2018090266 A1 WO 2018090266A1
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delivery system
colon
targeted delivery
drug
oral colon
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PCT/CN2016/106175
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English (en)
French (fr)
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肖泽宇
陆蕾
陈高贤
蔺淼
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上海交通大学医学院
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Priority to PCT/CN2016/106175 priority Critical patent/WO2018090266A1/zh
Priority to CN201680001612.XA priority patent/CN108401418B/zh
Priority to US16/461,564 priority patent/US11771656B2/en
Publication of WO2018090266A1 publication Critical patent/WO2018090266A1/zh

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    • 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
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Definitions

  • the invention relates to the technical field of medicine, in particular to a novel oral colon-targeted delivery system and a preparation method and application thereof, which can obtain ideal colon selective targeting and have good diagnosis and treatment for colon diseases. Application value.
  • colon diseases such as colon cancer and inflammatory bowel disease are threatening human health.
  • the cancer death rate statistics in 2015 the number of colorectal cancer cases in the world is 1.02 million, and the incidence rate is the third highest among all lethal cancers.
  • the incidence rate is the third highest among all lethal cancers.
  • the distribution of the incidence of inflammatory bowel disease shows that there is a high incidence in Western countries, and the incidence of Chinese people is increasing with the changes in the environment and living habits, and its incidence is on the global scale. The speed has increased year by year. This will pose a huge threat to the health of all human beings, so it can be of great significance to design a relevant way of treatment.
  • PH-dependent targeted drug delivery system is a relatively common type of targeted drug delivery system. It utilizes a pH-sensitive polymer as a coating-coated drug by utilizing the pH of the colon site higher than that of the small intestine. The coating is dissolved under the pH condition of the colon and the drug is released in large quantities.
  • a pH-sensitive polymer such as: acrylic resin copolymer, cellulose phthalate, etc.
  • S100 is an anionic copolymer based on methacrylic acid and methyl methacrylate.
  • Time-dependent colon-targeted drug delivery system refers to the time difference caused by the swelling property of the biological material, and releases the drug after a longer period of activity in the gastrointestinal tract to achieve the purpose of intestinal targeting.
  • the regulation of time lag mainly reaches the colon site by particle size, thickness and permeability of the coating, but this system is greatly affected by individual differences, food types, eating time, pathological state and other factors.
  • the bacterial/enzyme-triggered colon-targeted drug delivery system utilizes the colon site to be more acute than the stomach and small intestine.
  • the flora can secrete many specific reductases and hydrolases, such as ⁇ -glucuronide.
  • Enzyme, ⁇ -glucosidase, cellulase, nitroreductase, azoreductase, ⁇ -dehydroxylase, cholesterol dehydrogenase, etc. while azo polymer and polysaccharides, etc., can be colon-specific enzymes Degraded to design a flora-triggered colon-targeted formulation.
  • chitosan is a natural polysaccharide, in which the 1,4 glycosidic bond can not be degraded and absorbed in the stomach and small intestine, but it can be degraded under the action of enzymes produced by bacteria unique to the colon. It is highly targeted and safe. Stable, non-toxic and biodegradable, it is a good carrier material for colon-targeted drug delivery systems.
  • the gastrointestinal environment is complex, the pH varies widely, the length of the gastrointestinal tract is different, and there are individual differences.
  • the current reported oral colon delivery system has the disadvantage that a single pH-dependent or time-dependent delivery of colon drugs cannot be due to individual differences.
  • the drug is released in advance, so that the drug is absorbed or decomposed in advance, resulting in less dose reaching the colon, which requires a higher initial dose or repeated administration, resulting in serious systemic side effects.
  • the materials used in the single microbial sensitivity mechanism are mostly azo-containing compounds and polysaccharides.
  • the azo-containing compounds are more prominent in colon targeting, but the azo-based small molecule compounds are a strong carcinogen, while polysaccharides Due to its good water absorption, it is easy to absorb water and swell and release the drug in advance.
  • the technical problem to be solved by the present invention is to provide an oral colon-targeted delivery system which combines pH-sensitive and microflora-sensitive mechanisms to enhance selective release and release of the colon site by using double-layer protection, and enhances the treatment. Or diagnose imaging effects while reducing toxic side effects.
  • an oral colon targeted delivery system comprising:
  • a microbial sensitive layer comprising a polysaccharide and coated on the outside of the active ingredient
  • a pH sensitive layer comprising any polymer material composition dissolved at pH ⁇ 7 and coated on the outside of the sensitive layer of the flora.
  • the polysaccharide material includes a ⁇ -cyclodextrin derivative, and/or a plant extract and a derivative thereof.
  • the polymer material composition of the pH sensitive layer comprises the following components by weight: 10% to 43% of methacrylic acid and methyl methacrylate copolymer, 5% to 36% of plasticizer, 5% of talc. twenty two%.
  • coating includes coatings for solid carriers and encapsulating fluids and/or solids of capsules or microcapsules.
  • active ingredient means any compound or biological material useful for the diagnosis and/or treatment of a disease.
  • the outer layer is a pH sensitive layer material, since it dissolves only at pH ⁇ 7, it can resist the acidic environment of the upper end of the stomach and small intestine; the microbial sensitive material of the middle layer can only be unique to the colon site.
  • the flora triggers and dissolves, which can effectively enhance the selective delivery ability of the delivery system to the colon site;
  • the inner layer is an active ingredient, including drugs or diagnostic reagents, wherein the drug includes antibody drugs, protein drugs, gene drugs, nano drugs, small molecules Drugs, etc.; diagnostic reagents include contrast agents, imaging probes and the like for imaging inflammatory bowel disease or colon cancer diagnosis.
  • the drug is a targeted nanoparticle dosage form.
  • the target molecules modified by the surface of the nanoparticles bind to cell surface specific receptors, and enter the cells under the action of cell uptake to achieve safe and effective targeted drug therapy.
  • the targeted nanoparticles are CD-98 antibody modified PLGA-PEG nanoparticles.
  • the targeted nanoparticle modified by the CD-98 antibody of the invention can specifically recognize and enrich in the inflammatory part of the colon, and long-term stay, slow release of the drug, prolong the action time of the drug, and improve the therapeutic effect of the drug, thereby effectively Overcoming the treatment of diffuse colitis, because the small molecule drug reaches the colon site, it is quickly taken out of the body with the sputum or feces, resulting in a technical problem that the drug can not stay in the inflammation site.
  • the ⁇ -cyclodextrin derivative of the microbial sensitive layer material of the present invention is preferably an epichlorohydrin crosslinked ⁇ -cyclodextrin polymer.
  • the flora sensitive layer material may also be selected from natural plant extracts such as celery extract, konjac gum, or pectin, and derivatives of these natural plant extracts may also be used.
  • the bacterial sensitive layer material of the present invention may also be a mixture of a ⁇ -cyclodextrin derivative and a plant extract and a derivative thereof.
  • the ratio of methacrylic acid and methyl methacrylate copolymer in the pH sensitive layer material of the invention preferably methacrylic acid and methyl methacrylate, is 1:2.
  • the plasticizer in the pH sensitive layer material of the present invention may be selected from methyl or ethyl esters, organic acid fatty esters or inorganic acid esters (such as lactic acid, citric acid, succinic acid, adipic acid, azelaic acid, Phthalic acid, glutaric acid or phosphoric acid), or an acetate or fatty ester selected from the group consisting of monohydric, dihydric, trihydric or polyhydric alcohols. Specifically selected may be selected from the group consisting of diacetin (diacetin), triacetin (triacetin), or triethyl citrate, and any mixture of these products.
  • the plasticizer is preferably triethyl citrate.
  • the oral colon-targeted delivery system of the present invention can be formulated into any dosage form, including capsules, microcapsules, tablets, pills, or granules.
  • the preparation method of the oral colon-targeted delivery system of the capsule dosage form comprises the following steps:
  • the polymer material composition dissolved under the condition of pH ⁇ 7 is mixed into a coating liquid of the pH sensitive layer, sprayed on the outer surface of the capsule shell containing the sensitive layer of the bacteria, and dried to obtain a double sensitivity of the pH and the bacteria group.
  • Colon-targeted delivery system Colon-targeted delivery system.
  • the colonic diseases include colon cancer, inflammatory colitis, amebic bowel disease, or intestinal tuberculosis.
  • the oral colon-targeted delivery system of the present invention combined with the pH-sensitive and microflora-sensitive mechanism, effectively enhances the selective dissolution release of the colon site by using double-layer protection, so that the inner layer of the drug reaches the colon site and is released, thereby reducing
  • the number of administrations reduces toxic and side effects, achieves better therapeutic effects, and has broad application prospects.
  • Example 1 is a graph showing capsule release curves in vitro simulated gastric juice, small intestinal juice and colonic fluid according to Example 1 of the present invention
  • Figure 2 is a graph showing the release profile of the susceptible layer of the flora of the present invention in the bacterium and the sterile simulated colonic liquid;
  • Figure 3 is a schematic view showing the structure of an oral colon-targeted delivery system of the present invention.
  • Example 4 is a graph showing the characterization of the targeted nanoparticles and the drug release profile in vitro according to Example 2 of the present invention.
  • Figure 5 is a graph showing the results of nanoparticle targeting experiments in Example 3 of the present invention.
  • Figure 6 is a diagram showing the distribution of pH and microbiocidal capsules in vivo in Example 4 of the present invention.
  • Figure 7 is a columnar comparison diagram of the capsules of Example 4 of the present invention distributed in the body;
  • Fig. 9 is a graph showing the results of treatment of a DSS-induced rat disease model by a conventional capsule, a pH-sensitive capsule containing free drug, and an encapsulated NP-CD98PH and a bacterial double sensitive capsule according to Example 6 of the present invention.
  • an oral colon-targeted delivery system comprising: a bacterial sensitive layer comprising a polysaccharide and coated On the outside of the active ingredient; a pH sensitive layer comprising any polymer material composition dissolved at pH ⁇ 7 and coated on the outside of the sensitive layer of the flora.
  • the active ingredient includes a drug or a diagnostic imaging reagent.
  • the polysaccharide material includes a ⁇ -cyclodextrin derivative, and/or a plant extract and a derivative thereof.
  • the polymer material composition of the pH sensitive layer comprises the following components by weight: 10% to 43% of methacrylic acid and methyl methacrylate copolymer, 5% to 36% of plasticizer, 5% of talc. twenty two%.
  • the drug is a targeted nanoparticle dosage form.
  • the targeted nanoparticles are CD-98 antibody modified PLGA-PEG nanoparticles.
  • the CD-98 antibody-modified targeting nanoparticle can specifically recognize and enrich the inflammatory site of the colon, and long-term stay, slow release of the drug, prolong the action time of the drug, and improve the therapeutic effect of the drug. This is of great significance for the current treatment of diffuse colitis, because the existing common dosage form of small molecule drugs quickly come out of the body with the sputum or feces after reaching the colon site, so that the drug can not stay in the inflammation site to play its proper role.
  • the therapeutic effect while the pH and micro-sensitive dual-delivery delivery system of the invention is combined with the targeted nanoparticles, can achieve long-term retention of the colonic site, slow release of the drug, and full effect, because the pH and the flora are both sensitive.
  • the delivery system ensures complete delivery of the targeted nanoparticles to the site of the colon, and the targeted nanoparticles specifically recognize the cells at the site of the colon and are endocytosed into the cells without squirming with the sputum or feces. It was quickly excreted.
  • the outer layer of the capsule is coated with a pH sensitive layer according to the method of the above 1, and the colon-targeted acid-base and microbial dual-sensitive oral administration capsule is obtained. .
  • CD-98 antibody modified PLGA-PEG nanoparticles were prepared by the double emulsion method.
  • the drug or imaging reagent was dissolved in the aqueous phase, and 20 ⁇ 10 mg of PLGA-PEG was dissolved in dichloromethane (DCM) (Belling, China), and ultrasonically added to the emulsion and added to the water containing 1.5 ⁇ 1% Tween-80.
  • DCM dichloromethane
  • the second phacoemulsification was carried out in the phase. After stirring for 2.5 ⁇ 1h, use ultracentrifuge tube for super Filtered, washed twice with two steamed water to obtain nanoparticles, and lyophilized for use.
  • the epichlorohydrin crosslinked ⁇ -cyclodextrin polymer or plant fiber extract is dissolved in dimethyl sulfoxide. A suitable amount of the solution is taken up into a mold gelatin capsule shell prepared by 3D printing, and then dried at a high temperature. After cooling to room temperature, an intermediate layer of capsules sensitive to the colonic flora is obtained.
  • Eudragit S100, triethyl citrate and talc are mixed to form a coating liquid of the pH sensitive layer, and stirred and uniformly mixed.
  • the coating liquid is uniformly sprayed on the outside of the capsule by a coating pan, and after drying, the outer layer is a pH sensitive layer, and the middle layer is a double sensitive oral capsule which is a sensitive layer of the flora.
  • the drug-loaded targeting nanoparticle prepared in the above 3 is added into the double sensitive oral capsule to prepare the oral colon-targeted delivery system of the present invention, and the structure thereof is shown in FIG. 3, and the outer layer is a pH sensitive layer, and the layer is only in the layer. When dissolved at pH ⁇ 8, it can resist the acidic environment in the upper part of the stomach and small intestine; the middle layer is the sensitive layer of the flora, which can only be triggered by the flora unique to the colon site, which can effectively enhance the selection of the colon site by the delivery system.
  • the active ingredient in the inner layer is a drug-loaded targeting nanoparticle, which can specifically recognize and enrich in the inflammatory site, long-term stay, slow release of the drug, reduce the number of administrations, and enhance the therapeutic effect of the colon lesion .
  • the particle size and zeta potential of the nanoparticles and the targeted nanoparticles were determined by dynamic light scattering method. 1 mL of the solution sample was taken and added to the sample cell to determine the absence of bubbles in the container. The British Malvern Nanoparticle Size Analyzer (DLC) was used. The particle size and zeta potential of the sample were measured, and each sample was repeatedly measured three times to obtain a particle diameter and a potential. It was observed using an electron microscope (TEM).
  • DLC British Malvern Nanoparticle Size Analyzer
  • NP-CD98 exhibited a slow release profile with more than 80% release after 48 h incubation. This allows the drug to achieve sustained release at the site of inflammation of the IBD.
  • DAPI 6-Dimercapto-2-phenyl dihydrochloride
  • RAW 264.7 macrophages were seeded in 6-well plates. After incubation with NP and NP-CD98 for 2 hours, in order to remove free nanoparticles, wash 3 times with PBS, disperse the cells, centrifuge (1000 rpm, 10 minutes), and redisperse up to 1 ⁇ 10 6 cells / 1 ml concentration . The fluorescence uptake of the cells was measured using an Accuri C6 flow cytometer (Becton Dickinson, Calif.).
  • the results are shown in Figure 5.
  • the red-fluorescent nanoparticles can be endocytosed.
  • the black line in Fig. 5B represents the nanoparticle, and the red line represents the targeted nanoparticle. It can be observed that the red line is significantly shifted to the right of the black line, indicating that the fluorescent signal generated by the targeted nanoparticle is significantly higher than that of the nanoparticle, that is, the target nanoparticle. Targeting is obvious.
  • A Nanoparticle endocytosis laser confocal map
  • B NP and NP-CD98 endocytic flow diagram.
  • Example 4 Distribution of capsules in the gastrointestinal tract
  • Fig. 6 As can be seen from Fig. 6, after oral administration for 3 hours, capsule-shaped fluorescence appeared in the stomach, and after 6 hours, appeared in the middle of the small intestine. After 12 hours, the intact capsule appeared at the end of the small intestine. The location near the cecum indicates that the pH and microbio-sensitive capsules of the present invention maintain integrity as they undergo the gastrointestinal tract.
  • Fig. 7 The results are shown in Fig. 7.
  • the ordinary capsules showed obvious red fluorescence in the five segments, indicating that they did not play any protective role. While the pH-sensitive layer alone shows fluorescence only at the lower end of the small intestine and the colon, the pH of the present invention and the double-layer sensitive capsule of the flora show only significant fluorescence in the colon, which is consistent with the statistical data, fully demonstrating that in vivo, the invention The double-layer sensitive capsule has a good colon targeting effect.
  • the free dye, NP and NP-CD98 were placed in pH+bacteria capsules and administered to rats, and the colon site was sacrificed after 18H, 24H, 30H.
  • the nuclei were stained with DAPI. More details on Texas Red can be observed by laser confocal.
  • Tissue samples were homogenized in PBS and subsequently extracted with an ethanol/DMSO mixture (1:1 volume/volume). The fluorescence content of the sample was analyzed using a fluorescent plate reader.
  • Fig. 8 The results are shown in Fig. 8.
  • the fluorescence intensity decreased significantly during the 18 to 30 hours, indicating that the residence time was short.
  • the targeted nano-groups were significantly more intense and less variable than the nano-sets, indicating that the targeted nanoparticles entered the colon tissue more and sustained release, prolonging the time.
  • Rats (about 150 g) were divided into 5 groups of 10 each. Except the first group was the negative control group, the other groups provided free drinking sodium sulfate dextran (DSS) (6.5%) for 10 days, The second group was used as a positive control, and the other groups were given a daily capsule filled with free drug, free drug with pH layer protection, pH+bacteria double sensitive layer to protect capsules carrying NP-CD98-encapsulated drug, and weighed every day. Rat body weight and number of dead mice were counted. After 10 days, all rats were sacrificed and the colon and spleen were removed. The length of the colon was measured and the spleen was weighed.
  • DSS free drinking sodium sulfate dextran
  • MPO activity is expressed as one unit per milligram of protein unit, defined as an amount of 1 millimolar which degrades to hydrogen peroxide per minute at 25 °C.

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Abstract

一种口服结肠靶向递送系统、其制备方法和在制备治疗或诊断结肠疾病的药物或试剂中的应用，所述递送系统包括菌群敏感层和pH敏感层，其中所述菌群敏感层包含多糖类，并包覆于活性成分外面；所述pH敏感层包含任何在pH≥7条件下溶解的高分子材料组合物，并包覆于所述菌群敏感层外面。

Description

口服结肠靶向的递送系统及其制备方法和应用 技术领域
本发明涉及医药技术领域，具体涉及一种新型口服结肠靶向的递送系统及其制备方法和应用，该递送系统可获得理想的结肠选择性靶向，对结肠类疾病的诊断和治疗具有很好的应用价值。
背景技术
目前，结肠类疾病，比如：结肠癌、炎症型肠炎正在威胁着人类的健康。2015年的癌症致死率统计结果显示：世界结直肠癌的发病人数是102万，发病率高居所有致死癌症的第三位。而其中中国患者17.7万，排所有国家第四位。而炎症型肠炎发病率的分布统计显示，在西方国家有较高的发病率，在我们中国发病人口随着环境的变化及生活习惯的改变日趋增涨，并且其发病率正在全球范围内以惊人的速度逐年上升。这将给全人类的健康带来巨大的威胁，因此能设计出相关的方式治疗，将会有巨大的意义。
目前针对结肠部位疾病的治疗主要是单一pH依赖型结肠靶向给药系统、时间依赖型结肠靶向给药系统或菌群/酶触发型结肠靶向给药系统三种给药系统，详述如下:(1)PH依赖的靶向给药系统是一类比较常见的靶向给药系统，它通过利用结肠部位pH值高于小肠的特点，采用一定pH敏感型聚合物作为包衣包裹药物，使包衣在结肠部位的pH条件下溶解并大量释放药物。如：丙烯酸树脂共聚物、邻苯二甲醋酸纤维素等，其中
Figure PCTCN2016106175-appb-000001
S100为基于甲基丙烯酸和甲基丙烯酸甲酯的阴离子共聚物，在pH≥7时溶解，使药物在胃部得到保护，免受酸性环境破坏，直到到达肠道才释放其有效成分。(2)时间依赖型结肠靶向给药系统指生物材料的溶胀性产生的时间差，在胃肠道进行更长时间的活动后释放药物，以达到肠道靶向的目的。时滞的调控主要通过颗粒大小、涂层的厚度及渗透性到达结肠部位，但此系统受个体差异、食物种类、进食时间、病理状态等因素影响较大。(3)菌群/酶触发型结肠靶向给药系统是利用结肠部位较胃和小肠，菌群浓度急剧上升，这些菌群可以分泌许多特异的还原酶和水解酶，如β-葡萄苷酸酶、β-葡萄苷酶、纤维素酶、硝基还原酶、偶氮还原酶、α-脱羟酶、胆固醇脱氢酶等，而偶氮聚合物和多糖等材料，可被结肠特异的酶所降解，以此设计菌群触发型结肠靶向制剂。如壳聚糖为天然多糖，其中的1,4糖苷键使其在胃和小肠中不能降解和吸收，却可在结肠处所特有细菌产生的酶的作用下降解，靶向性强，并且安全、稳定、无毒及可生物降解，故其是一类良好的结肠靶向给药系统的载体材料。
但是胃肠道环境复杂，pH变化范围广，胃肠道长度不一，存在个体差异，当前报道的口服结肠递送系统存在的缺点是：单一的pH依赖或时间依赖型递送结肠药物由于个体差异不能 保证完全到达结肠部位，通常在小肠中下端就提前将药物释放，使药物提前吸收或分解，导致到达结肠部位剂量较少，从而需要给予较高的初始剂量或者重复给药，造成严重的全身副作用。而单一的菌群敏感性机制所用材料多为含偶氮结构化合物及多糖类，含偶氮结构化合物结肠靶向较显著，但偶氮类小分子化合物是一种强致癌物质，而多糖类材料由于吸水性好，极易吸水溶胀，提前将药物释放。
发明内容
本发明要解决的技术问题是提供一种口服结肠靶向的递送系统，该递送系统结合pH敏感及菌群敏感机制，通过采用双层保护有效增强了结肠部位的选择性溶解释放，增强了治疗或诊断成像效果，同时降低了毒副作用。
此外，还需要提供一种上述口服结肠靶向的递送系统的制备方法及应用。
为了解决上述技术问题，本发明通过如下技术方案实现:
在本发明的一个方面，提供了一种口服结肠靶向的递送系统，包括：
菌群敏感层，其包含多糖类物质，并包覆于活性成分外面；
pH敏感层，其包含任何在pH≥7条件下溶解的高分子材料组合物，并包覆于所述菌群敏感层外面。
所述多糖类物质包括β-环糊精衍生物、和/或植物提取物及其衍生物。
所述pH敏感层的高分子材料组合物包含以下重量百分比的组分：甲基丙烯酸和甲基丙烯酸甲酯共聚物10％～43％、增塑剂5％～36％、滑石粉5％～22％。
在本发明中，术语“包覆”包括用于固体载体的包衣及封套了流体和/或固体的胶囊或微型胶囊。
在本发明中，术语“活性成分”是指对疾病的诊断和/或治疗有用的任何化合物或生物材料。
本发明的递送系统，外层是pH敏感层材料，由于其只在pH≥7时溶解，可抵制胃及小肠中上端的酸性环境；中层的菌群敏感材料，其只能被结肠部位特有的菌群触发而溶解，能有效增强该递送系统对结肠部位的选择性递送能力；内层为活性成分，包括药物或诊断试剂，其中药物包括抗体药物、蛋白药物、基因药物、纳米药物、小分子药物等；诊断试剂包括造影剂、影像探针等用于炎性肠病或结肠癌诊断的影像试剂。
优选的，所述药物为靶向纳米粒剂型。
采用靶向纳米粒的药物，通过纳米粒表面修饰的靶向分子与细胞表面特异性受体结合，在细胞摄取作用下进入胞内，实现安全有效的靶向性药物治疗。
更优选的，所述靶向纳米粒为CD-98抗体修饰的PLGA-PEG纳米粒。
本发明CD-98抗体修饰的靶向纳米粒，可以特异性识别和富集在结肠炎性部位，并长效停留、缓慢释放药物，延长了药物作用时间，提高了药物的治疗效果，从而有效克服了弥散型结肠炎治疗时，由于小分子药物到达结肠部位后很快随食糜或粪便蠕动带出体外，致使药物不能在炎症部位停留起不到治疗效果的技术问题。
本发明的菌群敏感层材料β-环糊精衍生物，优选为环氧氯丙烷交联的β-环糊精聚合物。该菌群敏感层材料也可选用天然植物提取物，如芹菜提取物、魔芋胶、或果胶等，也可选用这些天然植物提取物的衍生物。本发明的菌群敏感层材料还可选用β-环糊精衍生物和植物提取物及其衍生物的混合物。
本发明pH敏感层材料中的甲基丙烯酸和甲基丙烯酸甲酯共聚物，优选甲基丙烯酸和甲基丙烯酸甲酯的比例为1：2。
本发明pH敏感层材料中的增塑剂可以选自甲基或乙基酯类、有机酸脂肪酯类或无机酸酯类(如乳酸、柠檬酸、琥珀酸、己二酸、癸二酸、邻苯二甲酸、戊二酸或磷酸)，或选自一元醇、二元醇、三元醇或多元醇的乙酸酯类或脂肪酯类。具体可选自甘油二乙酸酯(二乙酸甘油酯)、甘油三乙酸酯(三乙酸甘油酯)、或柠檬酸三乙酯，以及这些产品的任何混合物。增塑剂优选为柠檬酸三乙酯。
本发明口服结肠靶向的递送系统可以制成任意剂型，包括胶囊、微囊、片剂、丸剂、或颗粒剂。
所述胶囊剂型的口服结肠靶向递送系统的制备方法，包括以下步骤：
将多糖类物质溶解在有机溶剂中，吸取适量该溶液加到胶囊壳内，在45～65℃的温度下干燥5～15小时，然后冷却至室温，得菌群敏感层；
将pH≥7条件下溶解的高分子材料组合物混合制成pH敏感层的包衣液，喷涂在所述含菌群敏感层胶囊壳的外面，干燥后即得pH和菌群双敏感的口服结肠靶向的递送系统。
在本发明的另一方面，还提供了上述口服结肠靶向的递送系统在制备治疗结肠疾病的药物中的应用。
在本发明的另一方面，还提供了上述口服结肠靶向的递送系统在制备诊断结肠疾病的试剂中的应用。
在本发明的另一方面，还提供了上述口服结肠靶向的递送系统在制备诊断和治疗结肠疾病的药物中的应用。
在本发明的另一方面，还提供了上述口服结肠靶向的递送系统在制备口服结肠吸收的药物中的应用。
所述结肠疾病包括结肠癌、炎症型结肠炎、阿米巴肠病、或肠结核。
本发明的口服结肠靶向的递送系统，结合pH敏感及菌群敏感机制，通过采用双层保护有效增强了结肠部位的选择性溶解释放，使内层的药物达到结肠部位才释放出，减少了给药次数，降低了毒副作用，实现了更好的治疗效果，具有广阔的应用前景。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1是本发明实施例1的体外模拟胃液、小肠液及结肠液中的胶囊释放曲线图；
图2是本发明实施例1的菌群敏感层在有菌和无菌模拟结肠液中的释放曲线图；
图3是本发明口服结肠靶向的递送系统的结构示意图；
图4是本发明实施例2的靶向纳米粒表征及其体外药物释放曲线图；
图5是本发明实施例3的纳米粒靶向性实验结果图；
图6是本发明实施例4的pH和菌群双敏感胶囊在体内的分布情况图；
图7是本发明实施例4的胶囊在体内分布的柱状比较图；
图8是本发明实施例5的纳米粒在结肠部位累积18h、24h、30h时结肠部位荧光统计数据柱状图；
图9是本发明实施例6的普通胶囊、包封游离药物的pH敏感胶囊及包封NP-CD98PH和菌群双敏感胶囊对DSS诱导的大鼠疾病模型的治疗结果图。
具体实施方式
为了克服目前单一pH机制递送结肠药物，常导致到达结肠部位剂量太少，而单一的菌群触发型结肠靶向给药由于使菌群敏感层材料高亲水性容易在经过胃和小肠的长时间作用后出现溶胀、不利于在结肠部位正常起效的技术问题，本发明研制出了一种口服结肠靶向的递送系统，包括：菌群敏感层，其包含多糖类物质，并包覆于活性成分外面；pH敏感层，其包含任何在pH≥7条件下溶解的高分子材料组合物，并包覆于所述菌群敏感层外面。其中，活性成分包括药物或诊断影像试剂。
所述多糖类物质包括β-环糊精衍生物、和/或植物提取物及其衍生物。
所述pH敏感层的高分子材料组合物包含以下重量百分比的组分：甲基丙烯酸和甲基丙烯酸甲酯共聚物10％～43％、增塑剂5％～36％、滑石粉5％～22％。
优选的，所述药物为靶向纳米粒剂型。在本发明下述一个优选实施例中，所述靶向纳米粒为CD-98抗体修饰的PLGA-PEG纳米粒。该CD-98抗体修饰的靶向纳米粒，可以特异性识别和富集在结肠炎性部位，并长效停留、缓慢释放药物，延长了药物作用时间，提高了药物的治疗效果。这对于目前的弥散型结肠炎治疗具有重要意义，因为现有的普通剂型小分子药物到达结肠部位后很快随食糜或粪便蠕动带出体外，致使药物不能在炎症部位停留起到应有的 治疗效果，而本发明pH及菌群双敏感的递送系统再结合靶向纳米粒，能实现结肠发病部位长效停留、缓慢释放药物、充分发挥药效，这是因为pH及菌群双敏感的递送系统能确保将靶向纳米粒完整递送到结肠发病部位，而该靶向纳米粒在结肠发病部位可特异性识别细胞，并由细胞内吞作用进入细胞里面，不会随食糜或粪便蠕动很快被排出体外。
下面通过具体实施例阐述本发明。
实施例1 制备口服结肠靶向的递送系统
1.pH敏感层制备
20％±10％的尤特奇S100(德国罗姆公司)，15％±10％柠檬酸三乙酯(美国西格玛奥德里奇)被用作增塑剂以及5％±10％的滑石粉(美国西格玛奥德里奇)作为防黏剂，三组份加入到乙醇中组成pH敏感层的包衣液，搅拌(宁波Scientz生物技术有限公司，中国)混合均匀。通过BY-300A型小型包衣锅(上海黄海药检仪器有限公司)，在45±10℃下均匀喷涂在胶囊上，干燥后即为胶囊外层的pH敏感层膜，其只在pH≥7时溶解，可使胶囊抵制胃及小肠中上端的酸性环境。
2.菌群敏感层制备
1.8±1g环氧氯丙烷交联的β-环糊精聚合物(β-CDP)(滨州智源生物技术,中国)或植物纤维提取物溶解在8±5毫升二甲基亚砜DMSO(国药控股,中国)中。吸取8±5ul该溶液添加到3D打印制作的模具明胶胶囊壳内，然后在55±10℃的温度下干燥10±5h。在冷却到室温后，即得到对结肠菌群敏感的胶囊中间层，其只能被结肠部位特有的菌群触发而溶解，可有效增强对结肠部位的选择性。在将该结肠菌群敏感的胶囊壳上下卡紧后，再按上述1的方法对胶囊外层进行pH敏感层的包衣，得结肠靶向的酸碱和菌群双敏感的口服给药胶囊。
利用模拟胃肠液对双敏感胶囊的溶解释放情况进行体外考察，结果如图1所示，由图1中可以看出，普通的胶囊加染料在pH＝1.2环境下就完全溶解释放，单独的pH敏感层经受住了酸性条件后在pH＝6.8的模拟小肠液里有部分释放，而双敏感层胶囊完全经受住模拟胃液和小肠液达到结肠液后才进行释放。随后，对菌群敏感的特异性进行了考察，胶囊在有菌和无菌的模拟结肠液中的释放结果如图2所示，在有菌条件下进行了快速的释放，而在无菌的条件则表现不完全的释放，因此证明了菌群敏感层的特异性。
3.靶向纳米粒制备
CD-98抗体修饰的PLGA-PEG纳米粒由双乳液法制备。药物或影像试剂溶解在水相中，把20±10mg PLGA-PEG溶解在二氯甲烷(DCM)(百灵威，中国)中，超声使成乳液后加入到含有1.5±1％吐温-80的水相中进行第二次超声乳化。搅拌挥发2.5±1h后用超速离心管进行超 滤，二蒸水洗三次，得纳米粒，冻干备用。
加入NHS及EDC活化PEG末端-COOH，加入CD-98抗体孵育得修饰CD-98抗体的纳米粒，冻干备用。
4.口服结肠靶向的递送系统制备
将环氧氯丙烷交联的β-环糊精聚合物或植物纤维提取物溶解在二甲基亚砜中。吸取适量该溶液加到3D打印制作的模具明胶胶囊壳内，然后高温干燥。在冷却到室温后，即得到对结肠菌群敏感的胶囊中间层。
在将结肠菌群敏感的胶囊壳上下卡紧后，将尤特奇S100、柠檬酸三乙酯以及滑石粉混合组成pH敏感层的包衣液，搅拌混合均匀。利用包衣锅将包衣液均匀喷涂在胶囊外，干燥后得外层为pH敏感层、中层为菌群敏感层的双敏感口服胶囊。
将上述3制得的载药靶向纳米粒加入双敏感口服胶囊中，制得本发明口服结肠靶向的递送系统，其结构如图3所示，外层为pH敏感层，该层只在pH≥8时溶解，可抵制胃及小肠中上端的酸性环境；中层为菌群敏感层，其只能被结肠部位特有的菌群触发而溶解，能有效增强该递药系统对结肠部位的选择性递送能力；内层的活性成分为载药靶向纳米粒，其可以特异性识别并富集在炎性部位,长效停留、缓慢释放药物，减少给药次数，增强结肠病变部位的治疗效果。
实施例2 靶向纳米粒的表征及体外药物释放
1.靶向纳米粒粒径及表面zeta电位测定
采用动态光散射法测定纳米粒及靶向纳米粒的粒径和zeta电位，包取溶液样品1mL，加入样品池中，确定容器内无气泡产生，利用英国马尔文公司纳米粒径测定仪(DLC)测定样品的粒径和zeta电位，每个样品重复测量三次，得到粒径及电位。并使用电镜(TEM)对其观察。
动态光散射的结果表明(见表1)，纳米颗粒尺寸约为143.1nm，其ζ电势约-27.9mv。正如图4所示，对NP-CD98的分析显示通过TEM评价平均粒径为图4A中约200nm的，而由DLC评价是在图4B约为224.9nm。因为TEM技术需要真空下干燥颗粒，这会导致颗粒收缩。图4C示出了纳米粒子的ζ电位为-16.8±0.452mV。大小增加80nm左右,ζ电势也产生了较大的变化，这都表明NP和CD98抗体结合成功。
表1 纳米粒及靶向纳米粒的尺寸及电荷分布
  PDI SIZE(nm) ZETA(mv)
NP 0.219±0.030 143.1±4.073 -27.9±0.379
NP-CD98 0.245±0.067 224.9±8.965 -16.8±0.452
2.药物释放
靶向纳米粒的体外释放在磷酸盐缓冲液(PBS，pH6.8)中进行。0.02毫克/毫升浓度纳米颗粒悬浮在微量透析器中(赛默飞，美国)，然后在摇床中以100rpm速度37℃孵育。在每个指定的时间点(0，1，2，3，4，...0.12，24和48小时)，从三个管中分别取出100ul并加入100ul的PBS(pH＝7.4，0.01M)(Gibio，USA)补足体积，平衡系统。用NanoDrop 2000UV-Vis紫外可见分光光度计测量PBS中的5-ASA药物含量，并拟合成曲线。
结果如图4D显示，通过使用pH 7.4的PBS作为释放介质，以提供一个良好的漏槽条件，NP-CD98呈现一个缓慢的释放曲线，48h孵育后才有80％以上的释放。这样可以使药物在IBD的炎症部位实现持续释放。
实施例3 纳米粒靶向性实验
1.细胞摄取
RAW 264.7巨噬细胞在培养皿中培养过夜。将培养基更换为含有NP和NP-CD98无血清培养基。孵育3小时后，将RAW264.7巨噬细胞用PBS(pH值＝7.4)彻底漂洗干净，来清除过量的纳米颗粒，然后用4％多聚甲醛固定20分钟后。6-二脒基-2-苯基二盐酸盐(DAPI)稀释10,000倍后染色细胞核5分钟，在激光共聚焦下观察纳米粒子的细胞摄取。
2.纳米粒子的定量细胞摄取
RAW 264.7巨噬细胞在6孔板中接种。与NP和NP-CD98孵育2小时后，为了除去游离的纳米粒，用PBS洗涤3次，分散细胞，离心(1000转，10分钟)，并再分散达1×106个细胞/1毫升浓度。细胞的荧光摄取量用Accuri C6流式细胞仪(Becton Dickinson公司，加利福尼亚州)测量。
结果如图5所示，从图5A可以看出，带红色荧光的纳米粒是可以被内吞。图5B黑色线代表纳米粒，红色线代表靶向纳米粒，可以观察到红色线比黑色线明显的右移，说明靶向纳米粒产生的荧光信号明显比纳米粒高，即靶向纳米粒的靶向性明显。图5中，A.纳米粒内吞激光共聚焦图；B.NP及NP-CD98内吞流式图。
实施例4 胶囊在胃肠道的分布
1.体内成像
用罗丹明B(30毫克/毫升)放入pH和菌群双敏感(pH+bacteria)胶囊中制备荧光胶囊， 然后给大鼠灌胃，把大鼠分为三组。3小时，6小时，12小时后处死，取整个胃肠道，包括胃、小肠(SI-1，SI-2，SI-3)、结肠部分，由Kodak IS ex vivo FX成像系统对成像进行观察和分析。EM＝600nm处，Ex＝535nm。
结果如图6所示，从图6可以看出，口服灌胃3小时后，胃部出现了胶囊形状的荧光，6小时后，在小肠中部出现，12小时后，完整的胶囊出现在小肠末端靠近盲肠的部位，说明本发明pH和菌群双敏感胶囊在经历胃肠道的时候，保持了完整性。
2.生物分布情况
将普通胶囊、pH敏感胶囊、含TEXAS RED pH+bacteria胶囊这三种分别对大鼠灌胃，18小时后处死。取整个胃肠道并分为四个部分，包括胃、SI-1、SI-2、SI-3、结肠。然后由冰冻切片机进行切片，在激光共聚焦扫描显微镜下观察荧光的具体分布情况。组织样品在PBS中匀浆，随后用乙醇/DMSO混合物萃取(1:1体积/体积)。用荧光板读数器对样品中的荧光含量进行分析。
结果如图7所示，普通胶囊在五段均出现明显红色荧光，说明其并未起到任何保护作用。而单独pH敏感层只在小肠下端及结肠部位出现荧光，本发明pH和菌群双层敏感的胶囊则只在结肠部位出现明显荧光，这与统计数据一致，充分说明了在体内，本发明的双层敏感胶囊具有很好的结肠靶向效果。
实施例5 纳米粒在结肠部位的停留时间考察
将游离染料、NP和NP-CD98分别放置在pH+bacteria胶囊中灌胃给大鼠，并在18H，24H，30H后处死取结肠部位。用DAPI染色细胞核。Texas Red在组织更多的细节可以通过激光共聚焦观察。组织样品在PBS中匀浆，随后用乙醇/DMSO混合物萃取(1:1体积/体积)。用荧光板读数器对样品中的荧光含量进行分析。
结果如图8所示，游离染料组，18到30小时过程中，荧光强度明显下降，说明停留时间较短。靶向纳米组明显比纳米组荧光强度高且变化小，表明靶向纳米粒更多的进入到了结肠组织内且进行持续释放，延长了时间。
实施例6 体内药效实验
1.灌胃包封游离药物的普通胶囊、包封游离药物的pH敏感胶囊及包封载药NP-CD98的PH+bacteria敏感的胶囊
利用DSS诱导的急性肠炎模型考察体内的药效。大鼠(约150g)分成5组，每组10只。除了第一组是阴性对照组，其他组分别提供自由饮用硫酸钠葡聚糖(DSS)(6.5％)10天， 第二组作为阳性对照，其他组则每隔一天分别灌胃普通胶囊包裹的游离药物、具有pH层保护的游离药物、pH+bacteria双敏感层保护携带NP-CD98包裹药物的胶囊，每天称量大鼠体重和计算死亡老鼠数量。10天后，杀死所有大鼠，取出结肠和脾脏。量取结肠部位的长度并对脾脏进行称重。
2.MPO活性
中性粒细胞浸润情况通过测量MPO活性定量。用Sigma公司的髓过氧化物酶试剂盒进行测量。MPO活性表示为每毫克蛋白单元，一个单位定义为在25℃降解为每分钟的过氧化氢的1毫摩尔的量。
3.结果
如图9所示(图9中，A.结肠图，B.大鼠的体重变化情况，C.MPO值，D.结肠长度，E.脾脏重量)，DSS处理的大鼠体重在第4天开始有差别，在第10天差别最大。三组中，包封NP-CD98PH+bacteria敏感的胶囊体重减少的最少(图9B所示)。从结肠的长度的减少情况(图9D所示)及脾脏的增重(图9E所示)均是表现出更好的疗效。图9C结果显示了MPO活性和结肠长度及脾脏重量的结果一致。这些结果反映包封NP-CD98PH+bacteria敏感的胶囊组达到了最好的效果，明显的降低了结肠的炎性。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (16)

  1. 一种口服结肠靶向的递送系统，包括：
    菌群敏感层，其包含多糖类物质，并包覆于活性成分外面；
    pH敏感层，其包含任何在pH≥7条件下溶解的高分子材料组合物，并包覆于所述菌群敏感层外面。
  2. 根据权利要求1所述的口服结肠靶向的递送系统，其特征在于，所述多糖类物质包括β-环糊精衍生物、和/或植物提取物及其衍生物。
  3. 根据权利要求1所述的口服结肠靶向的递送系统，其特征在于，所述pH敏感层的高分子材料组合物包含以下重量百分比的组分：甲基丙烯酸和甲基丙烯酸甲酯共聚物10％～43％、增塑剂5％～36％、滑石粉5％～22％。
  4. 根据权利要求1所述的口服结肠靶向的递送系统，其特征在于，所述递送系统的剂型包括胶囊、微囊、片剂、丸剂、或颗粒剂。
  5. 根据权利要求1所述的口服结肠靶向的递送系统，其特征在于，所述活性成分包括药物和/或诊断试剂。
  6. 根据权利要求5所述的口服结肠靶向的递送系统，其特征在于，所述药物包括蛋白药物、抗体药物、基因药物、化学药物、或纳米药物。
  7. 根据权利要求5所述的口服结肠靶向的递送系统，其特征在于，所述诊断试剂包括影像试剂。
  8. 根据权利要求5所述的口服结肠靶向的递送系统，其特征在于，所述药物为靶向纳米粒剂型。
  9. 根据权利要求8所述的口服结肠靶向的递送系统，其特征在于，所述靶向纳米粒为CD-98抗体修饰的PLGA-PEG纳米粒。
  10. 根据权利要求2所述的口服结肠靶向的递送系统，其特征在于，所述植物提取物包括芹菜提取物、魔芋胶、或果胶。
  11. 根据权利要求2所述的口服结肠靶向的递送系统，其特征在于，所述β-环糊精衍生物为环氧氯丙烷交联的β-环糊精聚合物。
  12. 根据权利要求4所述的口服结肠靶向的递送系统，其特征在于，所述胶囊剂型的递送系统的制备方法包括以下步骤：
    将多糖类物质溶解在有机溶剂中，吸取适量该溶液加到胶囊壳内，在45～65℃的温度下干燥5～15小时，然后冷却至室温，得菌群敏感层；
    将pH≥7条件下溶解的高分子材料组合物混合制成pH敏感层的包衣液，喷涂在所述含菌群敏感层胶囊壳的外面，干燥后即得pH和菌群双敏感的口服结肠靶向的递送系统。
  13. 权利要求1所述口服结肠靶向的递送系统在制备治疗结肠疾病的药物中的应用。
  14. 权利要求1所述口服结肠靶向的递送系统在制备诊断结肠疾病的试剂中的应用。
  15. 权利要求1所述口服结肠靶向的递送系统在制备诊断和治疗结肠疾病的药物中的应用。
  16. 权利要求1所述口服结肠靶向的递送系统在制备口服结肠吸收的药物中的应用。
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