【作者】 孙明辉；

【导师】 杨祥良；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2011， 博士

【摘要】 西罗莫司(Sirolimus, SRL)是一种新型大环内酯类免疫抑制剂,其与FK506蛋白(FKBP-12)结合形成西罗莫司-FKBP-12复合物,后者再与哺乳类动物的西罗莫司靶点(Mammalian target of rapamycin, mTOR)结合,阻断T淋巴细胞及其他细胞由G1期至S期的进程,从而发挥免疫抑制效应。进一步的研究发现SRL独特的作用机制还可使其成为一种化疗药物用于抗肿瘤,如乳腺癌、结肠癌、前列腺癌、肾细胞瘤等。虽然SRL显示出较好的药理活性,然而由于其水中溶解度低及对胃酸敏感,加之其在肠壁和肝中被CYP3A4同功酶广泛代谢,且又是P-gp的底物导致其口服剂型生物利用度较低,如临床常用口服液及纳米结晶片的平均生物利用度均低于20%。本文研制了西罗莫司自微乳化药物递送系统(SMEDDS),并以市售口服液制剂为对照,对SRL-SMEDDS的处方工艺、理化性质、体外分散、肠吸收机理以及大鼠体内药物动力学进行了研究。首先建立了溶媒及SMEDDS制剂中西罗莫司含量的HPLC分析方法、分散介质中和肠灌流液中药物浓度的HPLC测定方法以及大鼠体内全血样品中药物浓度的HPLC /MS/MS测定方法。结果表明采用选定的色谱或质谱条件测定西罗莫司的体内外样品含量,峰形较好,空白基质对药物测定无影响；主成分峰分离较好,灵敏度高,专一性强,测定结果稳定准确。以溶解度实验、相溶性和自乳化效率实验、伪三元相图绘制以及药物的稳定性考察等确定了SRL-SMEDDS的处方成分,即MCT为油相,Cremophor RH40为表面活性剂,丙二醇为助溶剂。采用二因素五水平星点设计-效应面优化法对西罗莫司SMEDDS处方进行了优化,所建数学模型取得了良好的预测效果,确定SRL-SMEDDS处方的油相质量百分比(X1)和表面活性剂与助溶剂质量比(X2)的优选范围分别为25～30%和2.0～3.0。优选处方体外快速分散后生成粒径小于50nm的微乳；与原料药相比,微乳可使药物在pH 1.2 HCl溶液中的半衰期延长近4倍,在pH6.8的磷酸盐缓冲液中的半衰期延长近9倍。此外,助溶剂在制剂中不仅发挥着增溶药物的作用,而且还可显著提高了SMEDDS的分散速率。采用单向灌流法考察了西罗莫司微乳在大鼠体内的肠吸收特征,并对西罗莫司微乳的最佳吸收部位和吸收机制进行了初步探讨。西罗莫司自微乳化制剂吸收速率常数不受药物质量浓度的影响而与灌流速度和大鼠肠段不同吸收部位有关；胆汁分泌和排泄在本实验条件下不影响药物肠道吸收；药物在大鼠小肠主要通过被动扩散方式吸收；西罗莫司微乳在整个肠段均有吸收,其中以回肠吸收最好,且全肠吸收效果优于市售西罗莫司口服液(Rapamune(?))。考察了大鼠灌胃给予市售Rapamune(?)口服液以及SRL-SMEDDS后的体内药动学行为。相对于市售口服液制剂,SMEDDS的达峰时间短、达峰浓度高、相对生物利用度可达217.59%；经统计学分析,二者Cmax、tmax和AUC0→48h均具有显著性差异(P<0.05)；可见将SRL制成自微乳化制剂后可显著提高药物的口服吸收。通过联用乳糜微粒流阻塞技术和在体单向肠灌流技术评价西罗莫司SMEDDS的药物肠道淋巴转运,同时还考察了淋巴转运对大鼠口服生物利用度的影响。研究结果显示淋巴转运对西罗莫司自微乳化制剂口服生物利用度的影响显著。在各SMEDDS成分中,油相显著影响药物经淋巴转运的肠道吸收,并呈现一定的剂量依赖性；对于不含油相的对照制剂,药物吸收基本不通过淋巴转运；口服药物淋巴转运的启动需要一定量脂质的支持。此外,虽然较小的微乳粒径和较高表面活性剂含量也能一定程度上提高药物的吸收,但其较油相含量通过淋巴转运途径对药物吸收的影响要弱得多。通常认为药物的淋巴转运是伴随着脂质的消化吸收而进行的,药物经过跨细胞途径进行淋巴转运是药物淋巴吸收的最主要途径。而本文单向肠灌流研究的结果提示M细胞和肠道相关淋巴组织也是微乳中药物淋巴转运的重要途径。更多还原

【Abstract】 Sirolimus (SRL, rapamycin) is a carbocyclic lactone-lactam macrolide antibiotic, which has a molecular weight of 913.6 Da. The drug displays a unique immunosuppressive mechanism of action through its binding with FKBP12 and the inhibition of mammalian target of rapamycin (mTOR), which induces cell-cycle arrest in the G1 phase. These pharmacological properties allow sirolimus not only to be a promising immunosuppressant with the absence of nephrotoxicity but also to be a possible chemotherapeutic agent against many types of solid tumor. Even though sirolimus offers promising pharmacological activities, it has low oral bioavailability (<20%) in current formulations, such as oral solution and tablet. The low bioavailability was reported to be attributed to its sensitivity to gastric acid, partial intestinal absorption, and first-pass hepatic metabolism. Sirolimus has poorly water solubility (2.6μg/ml) and high liposolubility (log Po/w=5.77), and is the substrate of CYP450 3A isozymes and permeability-glycoprotein (P-gp) existing in the small intestinal enterocytes. These factors have been considered as significant contributors to low intestinal absorption.In this paper, self-microemulsifying drug delivery system of sirolimus was developed. Compared to commercially available oral solution (Rapamune(?)), the compositions, in vitro dispersion profile, in vivo bioavailability and mechanisms of intestinal absorption were investigated.The concentrations of SRL in vitro were determined by HPLC/UV and the whole blood concentrations of SRL were determined by HPLC/MS/MS. The HPLC method and HPLC/MS/MS method have been proved to be selective, rapid and suitable for the determination of sirolimus in vitro and in vivo, respectively.The compositions of SRL-SMEDDS were investigated by solubility assay, compatibility test, pseudo-ternary phase diagram analysis and drug stability study. MCT, Cremophor RH40 and propylene glycol were selected as oil, surfactant and co-solvent, respectively. The SMEDDS formulation was optimized using central composite design/response surface methodology. Optimized formulation of SMEDDS for bioavailability assessment was 25% MCT,50% Cremophcr EL and 25% propylene glycol. 1.0g of mixture contained 4.0mg of SRL. The SRL-SMEDDS dispersed rapidly (>90% within 30 min) to form microemulsion with emulsion droplet sizes <50nm and maintained sirolimus in a solubilised state at the end of the 60min dispersion. The resulting microemulsions from SMEDDS increased the drug stability significantly in both pH 1.2 HC1 solution and pH 6.8 phosphate buffer (P<0.05). Compared with the stability of free drug, the drug stability presented an approximate 4-fold improvement in pH 1.2 aqueous media and about 9-fold enhancement in pH 6.8 phosphate buffer. Besides, the co-solvent not only plays a role of drug solubilization but also promotes the dispersion rate of SMEDDS formulation.Rat single-pass intestinal perfusion technique (SPIP) was employed to assay the effects of flow rate, concentrations of sirolimus in perfusion solution, intestinal segments and bile duct ligation on the absorption percentage of drug (P%)、the absorption rate constant (Ka) and the intestinal apparent permeability coefficient (Papp). The absorption rate of sirolimus was not affected by drug concentration but was significantly affected by the flow rate and intestinal segments. The bile secretion had no significant impact on drug intestinal absorption. The absorption of sirolimus showed the passive diffusion process. Sirolimus could be absorbed in whole intestinal segments and the absorption rate showed ileum> duodenum≈jejunum > colon. Furthermore, the sirolimus microemulsion presented the higher absorption rate than that of the conventional oral solution in rat intestine. The pharmacokinetic behavior of SRL-SMEDDS in rats were evaluated comparing to Rapamune(?). Significant differences in Cmax, Tmax and AUC values were observed. The relative bioavailability of SRL-SMEDDS to the conventional oral solution was 217.59%. These results illustrate the potential use of SMEDDS for the delivery of sirolimus by the oral route.Under the precondition of efficient dispersion and the formation of microemulsion (droplet size<100nm), the influence of different amount of oil or surfactant in SMEDDS formulations on the intestinal lymphatic transport of sirolimus were investigated using single-pass intestinal perfusion technique and a chylomicron flow blocking approach. The data obtained highlight the importance of lymphatic transport on the enhanced oral bioavailability of sirolimus. For the SMEDDS formulations containing≥25% oil, the lymphatic transport of sirolimus is a major contributor to oral bioavailability. Among the components included in SMEDDS formulations, oil can significantly affect the intestinal absorption of drug via the lymphatic pathway in a concentration dependent manner. A lipid threshold is necessary to trigger the biochemical cascades responsible for the lymphatic transport. Besides, even though the smaller droplet size of resultant microemulsions and more surfactant content also can positively influence the intestinal absorption of drug, their influences on the drug intestinal lymphatic transport were relatively weaker than that of oil content. Potentially drugs absorbed via the intestinal lymph seem to gain their access into the lymphatic system by three ways:via the paracellular route with the aid of absorption enhancers, via the M cells and gut-associated lymphoid tissues (GALT), and via a transcellular route in association with the triglyceride core of the chylomicrons. Although the precise mechanism of lymphatic transport has yet to be fully elucidated, the third route is historically thought to be the major mechanism of lymphatic delivery of lipophilic drugs when formulated with lipid-based vehicles. However, the present results indicated that the route via the M cells and gut-associated lymphoid tissues (GALT) may play a significant role in lymphatic transport of drug.更多还原