【作者】 徐凌云；

【导师】 杨祥良； 徐辉碧；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 雷公藤内酯（triptolide,TP）具有显著的抗炎、免疫抑制、抗肿瘤和抗生育作用,在临床上已用于治疗类风湿性关节炎、皮肤病、肾炎等疾病。近年来,TP因抗瘤谱广、抗瘤活性强而引起了人们极大的关注。TP难溶于水,对多种器官系统,如消化系统、泌尿系统、生殖系统、心血管系统、免疫系统、骨髓和血液系统具有严重的毒性。本论文制备了载雷公藤内酯的聚合物胶束（triptolide loaded polymeric micelles,TP-PM）,并对TP-PM的抗肿瘤作用、对荷瘤小鼠免疫功能的影响、急性毒性、亚急性毒性进行了研究,为制备一种治疗实体瘤的雷公藤内酯新制剂提供实验依据。此外,我们采用微乳作为载体,制备了包载雷公藤内酯的微乳凝胶（triptolide-loadedhydrogel-thickened microemulsion,TP-MTH）,以期通过皮肤给药降低TP的毒性或提高疗效,本论文对TP-MTH治疗类风湿关节炎的药效学进行了研究。全文完成的工作包括以下几方面:1.以单甲醚聚乙二醇（MePEG）、D,L—丙交酯为原料,以辛酸亚锡为催化剂,合成了单甲醚聚乙二醇—聚乳酸（MePEG-PLA）两亲性两嵌段共聚物。用¹H-NMR、FT-IR、GPC和芘荧光探针对其结构和性质进行了表征。结果显示MePEG和丙交酯发生了酯化反应,共聚物的分子量为2.8×10⁴,临界胶束浓度为8.9×10^-7 mol/L,表明合成的嵌段共聚物在水溶液中能够形成稳定的胶束。2.以合成的MePEG-PLA嵌段共聚物为载体材料,采用溶剂蒸发法制备载雷公藤内酯的聚合物胶束。用激光粒度仪、透射电镜、RP-HPLC对聚合物胶束进行表征。结果所制备的聚合物胶束粒径为80 nm左右,包封率＞65%,多分散指数＜0.3,聚合物胶束呈核壳结构的球形粒子,分散均匀,表面光滑,形态规整,没有粘附和聚集现象。TP-PM体外释放缓慢,24 h累计释放百分率为67%,在4℃下储存4月,无明显聚集与分层现象,聚合物胶束的粒径、Zeta电势、包封率无明显变化,稳定性好。3.以人卵巢癌细胞株A2780为体外细胞模型、以小鼠荷S180移植瘤为体内动物模型,考察了TP-PM的抗肿瘤作用及对荷瘤小鼠免疫功能的影响。TP-PM和TP在1～50μg/L的浓度范围内,在作用48 h或72 h后对A2780细胞的增殖均具有明显的抑制作用,抑制强度具有时间依赖性和浓度依赖性,TP-PM和TP的作用强度相似。用Hochest33258染色在荧光显微镜下观察了药物在10μg/L浓度时作用72 h的细胞形态,可见肿瘤细胞出现核固缩、碎裂、边聚等细胞凋亡表现。TP-PM低、中、高剂量组（0.0375,0.075,0.15 mg/kg,尾静脉注射,6次,隔日一次）对荷瘤小鼠肿瘤生长的抑制率分别为42.5%、46.0%、49.9%,高于同剂量TP组。肿瘤组织病理学检查显示TP-PM组和TP组肿瘤呈现坏死性改变。与模型组相比,TP明显抑制胸腺指数、血清IL-2和TNF-α水平、ConA诱导的淋巴细胞转化率,脾脏异常增大,表现出明显的免疫抑制作用,而TP-PM对上述指标无明显影响。上述结果表明,聚合物胶束能够保持或增强TP的抗肿瘤作用,并且能够降低TP免疫抑制的副作用。4.研究了TP-PM对小鼠的急性毒性和对大鼠的亚急性毒性,并与TP进行比较。小鼠静脉注射TP-PM的LD₅₀值为1.06 mg/kg,TP的LD₅₀值为0.83 mg/kg。雄性大鼠静脉注射TP-PM或TP（0.1 mg/kh/d、0.3 mg/kg/d,×14 d）,与生理盐水组相比,TP-PM对血清ALT、AST、BUN、Cr和GSH-Px的水平无明显影响,明显降低MDA水平、提高SOD活性,TP组的血清AST水平和睾丸组织ACP活性显著降低、胸腺系数、睾丸系数降低、脾系数显著增大。TP组的脾脏、睾丸呈现明显病理性改变,肾和肝呈现轻微病理性改变,TP-PM组中上述器官的病理损伤较TP组轻。与同剂量TP比较,TP-PM能够明显增加睾丸ACP活性、明显降低血清MDA水平。结果表明,TP有明显的免疫毒性和生殖毒性,轻度的肝、肾毒性。而TP-PM对上述器官的毒性较TP明显降低,降低脂质过氧化损伤可能是TP-PM毒性较低的原因之一。5.观察雷公藤内酯微乳凝胶（TP-MTH）皮肤给药的抗炎和镇痛作用。与空白凝胶组相比,雷公藤内酯微乳凝胶在20～120μg/kg剂量范围内（以雷公藤内酯计）能抑制二甲苯致小鼠的耳廓肿胀,低、中、高剂量组的抑制率分别为46.7%、47.8%和49.5%,TP-MTH低、中、高剂量组对棉球所致大鼠肉芽组织增生的抑制率分别为17.9%、28.5%和40.9%,能显著抑制角叉菜胶致大鼠的足趾肿胀,对佐剂性关节炎大鼠的继发性病变具有明显的治疗作用,能抑制醋酸所致小鼠扭体反应,延长热板致痛的潜伏期。结果表明TP-MTH具有良好的抗炎和镇痛作用。本论文实验结果表明聚合物胶束保持或增强了雷公藤内酯的抗肿瘤作用,并且降低了雷公藤内酯免疫抑制的副作用以及对生殖系统、肝、肾等器官的毒性,有望成为雷公藤内酯静脉给药治疗实体瘤的有效载体。雷公藤内酯微乳凝胶透皮给药系统具有良好的抗炎、镇痛作用,经湖北省药物安全性评价中心的实验证实具有良好的安全性,提示该纳米载药系统有望成为治疗类风湿关节炎的新型制剂。更多还原

【Abstract】 Triptolide （TP） shows multiple pharmacological activities, such as anti-inflammatory, immuno-suppressive, anti-tumour and male anti-fertility activities. It has been usually used to treat rheumatoid arthritis （RA）, leprosy and nephritis. Recently, there has been growing interest in anti-tumour researches and applications of TP due to its broad anti-tumour spectrum and potent anti-tumour activity. However, the clinical use of TP is restricted due to its poor water solubility and some toxic effects on the gastrointestinal, urogenital, cardiovascular, blood circulatory system, immuno-system, bone marrow and so on. In order to develop an intravenous formulation for clinical use to treat solid tumours, TP-PM （triptolide loaded polymeric micelles） was constructed and the anti-tumour activities of TP-PM, the influence of TP-PM on the immunity of tumour-bearing mice, its toxicities were evaluated in the present paper. Besides, a novel TP loaded transdermal delivery system, named TP loaded hydrogel-thickened microemulsion （TP-MTH） was prepared previously to enhance the efficiency of TP and decrease the toxicity of TP. The anti-inflammatory and analgesic effects of TP-PM were also investigated in this paper. The main contents of this paper are as follows:（1） A diblock copolymer （MePEG-PLA） containing one block of methoxypolyethylene glycol （MePEG） and one block of either poly （D,L-lactide） （PLA） was synthesized by a ring opening bulk polymerization by changing the monomer weight ratios in the presence of stannous octoate. The copolymer was characterized by ¹H-NMR, FT-IR, GPC and pyrene. The spectrum showed that the reaction between lactide and MePEG had occurred. The weight-average molecular weight （M_W） of these copolymers were 2.8×10⁴. The critical micelle concentrations （CMC） of the copolymer was was determined to be 8.9×10^-7 mol/L, which was much lower than that of common low-molecular weight surfactants.（2） A novel polymeric micelle system containing TP （TP-PM） was constructed by the solvent evaporation method using methoxypolyethylene glycol-poly （D,L-lactic acid）-block copolymer as the carrier and characterised using photon correlation spectroscopy, transmission electron microscopy and high-performance liquid chromatography. Results demonstrated that TP-PM had an average diameter of 78.9nm, encapsulation efficiency of 66.7%, core-shell morphology and a long-term stability.（3） The anti-tumour and immuno-modulation effects of TP-PM were evaluated in sarcoma 180-bearing mice and A2780 cells. TP-PM could significantly inhibit tumour growth via intravenous injections at dose levels of 0.0375, 0.075 and 0.15 mg/kg, and their inhibition rates were 42.5%, 46.0% and 49.9%, respectively; they showed similar cytotoxicity against A2780 cells compared to that of TP. Simultaneously, TP-PM had no effect on the thymus index, spleen index, spleen lymphocyte proliferation and the TNF-αand IL-2 levels in serum as compared with TP. Therefore, TP encapsulated in polymeric micelles does not demonstrate immuno-suppressive activity but does not lose its anti-tumour effect. These results show that polymeric micelles are a promising carrier for cancer therapy using TP.（4） The acute toxicity in mice and sub-acute toxicity in rat were studied for TP-PM. Results demonstrated that the LD₅₀ for TP-PM and TP administered intravenously were 1.06 mg/kg and 0.83 mg/kg in mice, respectively. In toxicity study in rat, TP-PM and TP were administered intravenously at the dose levels of 0.1 mg/kg and 0.3 mg/kg for 14 d. Compared to the control, there was a significant difference （P<0.05） about the serum AST, ACP levels of the testis, thymus index and spleen index in rats treated with TP, however, TP-PM had no effect on above parameters. Compared to TP, TP-PM significantly increase the ACP activity of the testis and decrease the MDA level in serum. So, TP-PM Showed lower toxicity than TP.（5） The anti-inflammatory and analgesic effects of TP-MTH were evaluated. TP-MTH （0.003%） was administered on the skin at the 0.02～0.12 mg/kg dose levels in rats and mice. TP-MTH could significantly inhibit the adjuvant-induced rat paw edema, xylene-induced mouse ear edema, carrageenan-induced rat paw edema, cotton pellet implantation in rats, acetic acid-induced writhes in mice and the hot plate-induced pain in mice. Results demonstrated that TP-MTH possessed good anti-inflammatory and analgesic activities.This study demonstrated that TP encapsulated in polymeric micelles does not act as an immuno-suppressant without losing its anti-tumour effect and demonstrated that polymeric micelles are a stable and effective drug delivery carrier of TP for the therapy of solid tumours. Good anti-inflammatory and analgesic activities of TP-MTH showed in this study, in combination with lower toxicity described by Hubei center for safety evaluation of drugs, make it a valuable system for the delivery of TP for treating rheumatoid arthritis.更多还原