【作者】 张聪；

【导师】 杨祥良； 徐辉碧；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2014， 博士

【摘要】 雷公藤甲素(Triptolide, TP)是传统中药雷公藤(Tripterygium wilfordii Hook F, TWHF)中最具代表性的、集活性和毒性于一身的化合物。TP具有广泛的生物活性,包括抗炎、免疫抑制、抗生育及抗肿瘤等。然而治疗窗窄和对肝、肾、消化道、生殖系统、血液系统等的严重毒性限制了TP的临床应用。TP口服给药后被机体迅速吸收并被快速消除：在大鼠中,达峰时间Tmax在10.0～19.5min之间,消除半衰期t1/2在16.8～50.6min之间；在小鼠中,Tmax、t1/2分别约为5.0和13.7min。TP的药物动力学行为被认为是良好的胃肠道渗透性、肝脏细胞色素P4503A酶的代谢和广泛的组织分布三个因素综合作用的结果。TP快速吸收入血和短暂停留导致血药浓度波动较大,促进了TP毒性的产生。因此,通过缓慢释放药物降低血药浓度波动从而有效提高TP的安全性具有重要意义。大量研究结果表明,脂质纳米粒在实现药物缓释和减毒方面具有独特的优势,本论文首先制备并优化了雷公藤甲素固体脂质纳米粒,继而制备了包封率和稳定性更佳的雷公藤甲素纳米结构脂质载体,研究并比较了二者的药物动力学、急性和亚急性毒性,为开发优良的雷公藤甲素脂质纳米粒新制剂提供实验依据,也可为研究雷公藤甲素药物动力学改变与其毒性降低之间的关系提供参考。本论文完成的主要研究工作有：(1)研究了雷公藤甲素固体脂质纳米粒(Triptolide-loaded solid lipid nanoparticles, TP-SLN)两种制备方法(高压均质法和微乳法),并进行了初步的处方筛选。结果表明TP在SLN制备过程中会向水相分配而不易包封,微乳法比高压均质法更适合制备具有较高包封率的TP-SLN;空白SLN的处方组成为：固体脂质Compritol888ATO,表面活性剂Cremophor RH40,助表面活性剂卵磷脂的TranscutolHP溶液(质量比1：1)；在绘制伪三元相图的基础上,通过单因素实验确定了影响TP-SLN包封率的三个关键因素及取值范围：脂质百分含量(40%～60%)、表面活性剂／助表面活性剂质量比(2：1～6：1)、脂质／药物质量比(50：1～100：1)(2)优化了TP-SLN处方并研究了最优TP-SLN的性能。采用三因素五水平中心复合设计(Central composite design, CCD),评价了脂质百分含量(X1)、表面活性剂／助表面活性剂质量比(X2)、脂质／药物质量比(X3)三个因素对TP-SLN粒径(Y1)、包封率(Y2)和载药量(Y3)的影响并优化了TP-SLN。结果表明,Y1、Y2、Y3最优的回归模型均为二次多项式模型,用拟合方程预测的最优处方为：X1=49.73%,X2=3.25,X3=55.27；最优TP-SLN的各指标分别为：粒径179.8±5.7nm,包封率(56.5±0.18)%,载药量(1.02±0.003)%,各预测值与实测值接近,预测效果良好；TP-SLN表现出明显的体外缓释特性；TP-SLN在人工胃液中温孵3h后具有较好的稳定性,粒径未见明显改变,且大部分的药物(91%)仍然被保留；SLN能够降低TP口服诱导的大鼠胃刺激毒性,该保护作用与降低胃粘膜氧化应激水平和减轻局部炎症细胞浸润有关。(3)为改善TP-SLN的包封率和稳定性,在最优TP-SLN基础上,采用微乳法制备了雷公藤甲素纳米结构脂质载体(Triptolide-loaded nanostructured lipid carriers, TP-NLC)并进行了表征。TP在液体脂质Capryol90中的溶解度最大；Capryol90在固-液脂质二元混合物中的最优比例为20%；电镜下观察发现TP-NLC和TP-SLN均近似于球形,TP-NLC平均粒径较TP-SLN大(231.8±4.3nm vs.179.8±5.7nm,p<0.001),而PDI却明显较小(0.143±0.012vs.0.283±0.012,p<0.001)；TP-NLC包封率明显提高[(71.6±0.6)%vs.(56.5±0.2)%,p<0.001]；TP-NLC粒径在一个月内基本没有变化(p>0.05),而TP-SLN粒径从179.8±5.7nm增加到200.2±7.4nm(p<0.05); TP-NLC贮存一个月后包封率略减(从71.6%降到69.1%,p>0.05),相比之下,TP-SLN包封率显著降低(从56.5%降到48.3%,p<0.01)；TP-NLC的体外缓释效果较TP-SLN好,48h的累积释放度为71.2%,显著小于TP-SLN的91.3%(p<0.001)。(4)研究了大鼠口服给予雷公藤甲素脂质纳米粒(TP-NLC和TP-SLN)后体内药物动力学。建立了测定血浆中TP含量的HPLC-MS方法学；相对于TP混悬液,TP-NLC和TP-SLN的血药浓度-时间曲线均明显平缓；TP的Tmax和tl/2z分别为0.200±0.075h和0.706±0.087h；与TP比较,TP-NLC和TP-SLN的Tmax均明显推迟,分别为0.717±0.240h(p<0.01)和0.450±0.183h(p>0.05),tl/2z显著延长,分别为1.509±0.216h(p<0.01)和1.544±0.574h (p<0.01); TP-NLC的Cmax从TP的8.656±2.077μg/L下降到3.361±0.666μg/L (p<0.01), TP-SLN的Cmax下降到5.794±1.747μg/L(p<0.05)：与TP比较,TP-NLC和TP-SLN的CLz/F也显著降低(p<0.01, p<0.05); TP-NLC和TP-SLN的AUC0-t分别为9.012±1.576μg h/L,7.318±1.628μg h/L,分别是TP的AUC0-t的1.54倍和1.25倍；与TP-SLN比较,TP-NLC的MRT0-t和Tmax显著延长(p<0.001,p<0.05),Cmax显著降低(p<0.05),显示出更加理想的药物动力学行为。(5)研究了雷公藤甲素脂质纳米粒(TP-NLC和TP-SLN)的小鼠急性毒性和大鼠亚急性毒性。TP-NLC, TP-SLN和TP小鼠口服的LD50分别为1.87mg/kg,1.50mg/kg,1.08mg/kg(以雷公藤甲素计)；亚急性毒性试验结果表明,NLC能够显著缓解大鼠长期口服TP所引起的体重增长抑制,避免诱导大鼠心、肺、肝、脾和肾等脏器相对重量的变化,效果优于SLN；在TP低剂量水平(500μg/kg), NLC和SLN都能有效降低TP的肝毒性,剂量升高时(650μg/kg), SLN对肝和肾的减毒效果不明显,但TP-NLC仍表现出良好的安全性,提示NLC减毒效果明显好于SLN；在高剂量水平(650μg/kg),与TP组相比,TP-NLC组大鼠表现出显著降低的血清MDA含量(p<0.05)和显著升高的SOD活力(p<0.05),提示NLC减毒机理与降低TP诱导的脂质过氧化损伤有关；高剂量TP-SLN组发现肝细胞脂肪变性,肾小管扩张和脾巨噬细胞死亡,而TP-NLC组未见明显病理改变。本论文的实验结果表明NLC和SLN均能显著改善TP口服后的药物动力学参数,并能有效提高TP的安全性,且TP-NLC优于TP-SLN。这对于开发优良的雷公藤甲素脂质纳米粒新制剂具有重要意义,也为研究雷公藤甲素药物动力学改变与其毒性降低之间的关系提供了参考。更多还原

【Abstract】 Triptolide (TP), a diterpenoid triepoxide, is the major active and toxic component of the extracts derived from the traditional Chinese medicine Tripterygium wilfordii Hook F (TWHF). TP has been demonstrated to possess a unique bioactive spectrum of anti-inflammatory, immunosuppressive, anti-fertility and anticancer activities. However, the clinical application of TP has been limited by its narrow therapeutic window and high toxicities on the hepatic, renal, digestive, reproductive and hematological systems. After oral administration in rats, TP is absorbed rapidly. The time of maximum plasma concentration (Tmax) ranges from10.0to19.5min, with an elimination half-life (ti/2) from16.8to50.6min. Similarly, the reported Tmax and t1/2of TP in mice are5.0and13.7min, respectively. These pharmacokinetic behaviors of TP indicate that it gains fast access into the blood circulation with very short stay. This phenomenon can be explained by the combined effects of efficient gastrointestinal permeability, rapid metabolism by cytochrome P4503A system in the liver, and extensive tissue distribution. The significant and rapid fluctuations of TP in plasma likely contribute to the toxicity of TP following oral administration. Therefore, with the aim of prolonging TP release and improving its safety, triptolide-loaded solid lipid nanoparticles (TP-SLN) and triptolide-loaded nanostructured lipid carriers (TP-NLC) were developed. In addition, pharmacokinetics and toxicology profiles of TP-SLN and TP-NLC were investigated and compared with each other. The main contents of this thesis are as follows:(1) Triptolide was found to tend to partition toward the aqueous phase during the production process of TP-SLN. TP-SLN prepared by microemulsion technique had higher encapsulation efficiency (EE) than that prepared by high pressure homogenization method. Compritol888ATO, Cremophor RH40and the Lipoid E80in Transcutol HP (with a ratio of1:1, w/w) were selected as solid lipid, surfactant and co-surfactant, respectively. The critical variables including fraction of lipid (X1), surfactant to co-surfactant ratio (X2) and lipid to drug ratio (X3) that influenced the EE of TP-SLN were adjusted, and results of single-factor experiments showed that the appropriate ranges of X1, X2and X3were40%～60%,2:1～6:1and50:1～100:1, respectively. (2) The optimized TP-SLN were prepared for further evaluation. The microemulsion technique was used to formulate TP-SLN employing a five-level central composite design (CCD) that was developed for exploring the optimum levels of three independent variables on particle size, EE and drug loading (DL). Quadratic polynomial models were generated to predict and evaluate the three independent variables with respect to the three responses. The optimized TP-SLN were predicted to comprise fraction of lipid of49.73%, surfactant to co-surfactant ratio of3.25, and lipid to drug ratio of55.27, which showed particle size of179.8±5.7nm, EE of56.5±0.18%and DL of1.02±0.003%that were in good agreement with predicted values. In addition, the optimized nanoparticles manifested a sustained-release pattern in vitro and were stable during3h of incubation in simulated gastric fluids without significant size change and the majority (91%) of the drug was protected. Furthermore, SLN had a potential of preventing gastric mucosa irritation caused by oral administration of TP in rats, this could be attributed to reduced lipid peroxidation levels and inflammation of the stomach mucosa.(3) TP-NLC were developed from the optimized TP-SLN. A binary mixture consisting of20%(w/w) Capryol90and80%(w/w) Compritol888ATO would be the most suitable combination of liquid and solid lipid for the formulation of TP-NLC. TEM photographs showed that the particle shape appeared close to spherical. The size of the TP-NLC (231.8±4.3nm) was significantly larger (p<0.001) than that of the TP-SLN (179.8±5.7nm). TP-NLC had a smaller PDI (0.143±0.012vs.0.283±0.012,p<0.001). Moreover, the presence of Capryol90in TP-NLC was useful to increase the EE from56.5to71.6%in comparison with TP-SLN. The particle growth of TP-NLC was negligible (p>0.05), but the size of TP-SLN significantly increased from179.±5.7nm to200.2±7.4nm after1month’s storage (p<0.05). TP-NLC exhibited good ability to reduce drug expulsion during storage, the EE only reduced from71.6to69.1%(p>0.05, about2.5%TP was expulsed). In contrast,8.2%TP was expulsed from TP-SLN under the same storage condition (p<0.01). The accumulated drug release at48h of TP-NLC and TP-SLN were71.2and91.3%respectively (p<0.001), indicating that TP incorporated in NLC exhibits lower release behavior compared to SLN.(4) A rapid HPLC-MS method was developed for the detection of TP in rat plasma and in vivo pharmacokinetic studies of TP-NLC, TP-SLN and TP in male rats were performed. The Tmax and t1/2z of free TP were0.200±0.075h and0.706±0.087h, respectively. The time to achieve maximum concentration of TP was delayed to0.717±0.240h and0.450±0.183h in the case of TP-NLC (p<0.01) and TP-SLN (p>0.05), respectively. MRTo-t and t1/2Z were markedly longer (p<0.001,p<0.01) for both TP-NLC and TP-SLN compared to free TP. Interestingly, a significant decrease was found in the Cmax from8.656±2.077μg/L for free TP to3.361±0.666μg/L for TP-NLC (p<0.01) and to5.794±1.747μg/L for TP-SLN (p<0.05). CLz/F was also found to be reduced in TP-NLC (p<0.01) and TP-SLN (p<0.05) compared to free TP. In addition, TP-NLC and TP-SLN gave mean values of AUC0-t9.012±1.576μg h/L and7.318±1.628μg h/L, which were1.54-and1.25-fold higher, than that of free TP, respectively. These results showed the superiority of TP-NLC over TP-SLN, as also supported by prominent difference on MRT0-t(p<0.001), Tmax(p<0.05) and Cmax (p<0.05).(5) The acute toxicity in mice and sub-acute toxicity in rat were studied for TP-SLN and TP-NLC. Results demonstrated that the LD50for TP, TP-SLN and TP-NLC administered orally were1.08mg/kg,1.50mg/kg,1.87mg/kg in mice, respectively. In toxicity study in rat, TP, TP-SLN and TP-NLC were administered orally at the dose levels of500μg/kg and650μg/kg for28days. NLC could significantly inhibit the decrease of weight gain caused by TP in rats, showing better performance than SLN, especially at high dose. Relative weights of heart, lungs, liver, spleen and kidney in TP-NLC group had also been significantly improved at high dose. NLC and SLN could also reduce the liver toxicity of TP (500μg/kg) in male rats. However, at high dose (650μg/kg), only NLC had a significant protective effect on the liver and kidney. Compared to free TP, TP-NLC exhibited significantly reduced oxidative stress, which was verified by decreased MDA and improved SOD activity in rats serum at high dose, indicating a better safety than TP-SLN. Microscopically, histopathological changes in liver, spleen and kidney were discovered in high dose of TP and TP-SLN groups (650μg/kg). Fatty degeneration in the hepatocytes, dead cells in the macrophages as a "starry sky" appearance in spleen, and obvious kidney proximal tubular dilation were seen, However, in TP-NLC group at the same dose, no apparent changes were found.Results in this thesis demonstrated that both NLC and SLN could significantly improve the pharmacokinetic parameters of TP after oral administration and enhance the safety of TP. TP-NLC were superior to TP-SLN. These results would be of great importance for the development of new nano-formulation of TP and also provide suggestions to clarify the association between altered pharmacokinetics and reduced toxicity of TP.更多还原