依托泊苷论文：依托泊苷白蛋白纳米混悬剂的研究.doc

依托泊苷论文：依托泊苷白蛋白纳米混悬剂的研究【中文摘要】肺癌是一种严重威胁人类健康的疾病,近20多年来发病率呈逐年上升趋势。依托泊苷(Etoposide,EPEG)是一种细胞周期特异性抗肿瘤药物,为治疗肺癌的一线用药,现有上市剂型为注射液和软胶囊剂。依托泊苷水溶性差,口服剂型生物利用度较低,个体差异较大；注射液采用了大量的表面活性剂增溶,刺激性大,使用过程中药物易析出,患者顺应性差；另外,依托泊苷本身常见的不良反应为骨髓抑制,可使患者白细胞及血小板减少等。上述弊端在一定程度上制约了其在临床上的广泛应用。近年来,以脂质体、纳米粒等为载体的纳米给药系统在解决难溶性药物溶解度方面发挥了巨大的作用。通过制剂手段将难溶性药物制成纳米给药系统可以增加难溶性药物的溶解度、提高药物特别是抗肿瘤药物在靶向部位的浓度,从而能以最小的药量达到令人满意的治疗效果、减少毒副作用的发生,因此受到了广大科研工作者的青睐。纳米混悬剂是一种纯药物纳米颗粒的亚微细粒胶态分散体,近年来,针对此领域的研究十分活跃,其旨在增加药物的溶解度、生物利用度和给药的靶向性,目前药物纳米混悬剂正引领纳米释药系统向前发展。制备纳米混悬剂使那些溶解性差的抗生素类药物、抗肿瘤药物和一些治疗窗窄的药物有了重现其自身价值的机会,使其能够发挥出更好的作用。本课题以白蛋白为载体材料,构建了依托泊苷纳米混悬剂给药系统,控制其粒径在100-300nm,利用EPR效应及白蛋白的主动靶向作用将依托泊苷靶向于肿瘤部位并缓慢释放,减少了其在非肿瘤部位的分布,有利于降低其副作用,使其更好的发挥疗效。方法与结果：本课题采用高效液相色谱法测定依托泊苷的含量并对其方法学进行考察；采用高压乳匀-溶剂沉淀法制备依托泊苷纳米混悬剂,以粒径、包封率、载药量为评价指标,在单因素考察基础上,采用正交设计对处方和工艺进行优化；采用电子透射显微镜观察纳米混悬剂的外观形态,激光散射粒度分析仪测定粒径及其粒度分布,电势显微电泳仪测定表面Zeta电位,动态膜透析法测定体外释药特性,以零级动力学方程、一级动力学方程Higuchi方程和Weibull方程等数学模型拟合其体外释药动力学,并对纳米混悬剂冻干粉初步稳定性进行考察；以家兔和小鼠为研究对象,对依托泊苷纳米混悬剂进行了注射部位局部刺激性、小鼠骨髓抑制等体外评价；采用HPLC法测定家兔血浆、小鼠组织中的依托泊苷的含量,并建立其方法学；以依托泊苷注射剂为对照,分别以家兔和小鼠为研究对象,对依托泊苷纳米混悬剂进行了体内药动学和组织分布动力学的研究。实验结果表明,采用HPLC法测定依托泊苷纳米混悬剂中依托泊苷的含量,辅料不干扰测定,专属性强,方法简便、灵敏、可行。采用高压乳匀-溶剂沉淀法制备依托泊苷纳米混悬剂,可成功控制该系统的粒径,优化的最佳处方组成为：三氯甲烷与乙醇的比例为8：1(V/V),白蛋白/药物的比例为10：1(W/W),油相与水相的比例为10：1(V/V),同时控制水相pH值为5.4；最佳的制备工艺为：乳匀压力1500bar,乳匀次数15次。冷冻干燥法制备的依托泊苷白蛋白纳米混悬剂冻干粉为外观饱满、无塌陷的疏松多孔块状固体,用生理盐水复溶后为具有乳光的胶态溶液；冻干前的平均粒径为(190.27.3)nm,多分散系数为0.290.08,Zeta电位为(-23.051.78)mV；冻干后的平均粒径为(182.38.91)nm,多分散系数为0.270.11,Zeta电位为(-9)mV；依托泊苷白蛋白纳米混悬剂的pH为5.460.24,符合注射剂的要求。与市售注射剂相比,自制纳米混悬剂的体外释放表现出一定的缓释性,释药曲线符合Higuchi方程。DSC图谱显示,依托泊苷制成纳米混悬剂冻干粉后在白蛋白中以无定形状态存在。稳定性结果表明,在4条件下,依托泊苷纳米混悬剂冻干粉在3个月的时间内保持稳定。注射部位局部刺激性实验结果表明,受试样品无明显刺激性；小鼠骨髓抑制试验表明,与注射液相比,白蛋白纳米混悬剂能明显降低依托泊苷对小鼠的骨髓抑制。采用HPLC测定家兔血浆、小鼠体内组织中的依托泊苷的含量,在所选色谱条件下,内源性物质不干扰测定,标准曲线、精密度、回收率均符合测定要求。家兔体内动力学试验结果表明,静脉注射给药后,纳米混悬剂组和注射液组分布半衰期(t1/2)分别为10.296h和5.225h,前者比后者延长了1.97倍；平均滞留时间(MRT0-)分别为2.707h和1.170h,前者比后者延长了2.31倍；最大血药浓度(Cmax)分别为48.05 mgL和61.08mgL,纳米混悬剂组是注射液组的0.787倍,血药浓度-时间曲线下面积(AUC0-)分别为116.87mgL*h和111.16 mgL*h,这表明制备成纳米混悬剂后最大血药浓度得到降低,但并不影响治疗效果。主要以Ce(Ce=(Cmax)nanosuspensions/(Cmax)injection)作为靶向性参数,对纳米混悬剂靶向性进行了评价。其中,肺中的Ce值是1.93,肝和脾分别是1.79和2.52,而在心、脑、肾中的Ce值分别是0.94、0.95和0.88,该结果表明纳米混悬剂能使依托泊苷的肝、脾、肺靶向性大大提高,相反的在心、肾中的分布有所下降,有利于降低其在这些部位的副作用。结论：本课题成功研制了依托泊苷白蛋白纳米混悬剂,所采用的制备工艺简便可行,重现性好,适合工业化生产,成品对注射部位无刺激性、且降低了主药的骨髓抑制。将依托泊苷制备成纳米混悬剂后,明显改变了药物的体内配置,与市售注射剂相比,纳米混悬剂避免了大量表面活性剂的使用,能使药物浓积于肝、脾、肺部,尤其是增加了肺局部的药物浓度,药物的缓慢释放可以长时间维持肺部肿瘤组织的有效药物浓度,并且可以减少非靶部位如心、肾、脑的蓄积,从而减轻毒副作用,提高患者用药的顺应性和用药水平,具有良好的社会和经济效益。【英文摘要】:Lung cancer is a serious threat to human health, and the incidence increased year by year.Etoposide (EPEG) is a cell cycle specific anticancer drug which is on the first line treatment in lung cancer, and the primary cellular target for etoposide is topoisomerase. The existing etoposide dosage forms in market are injections and soft capsules. Because of its poor water-solubility, the bioavailability of oral dosage form is low and individual differences are apparent; the injection of etoposide utilizes a large number of surfactant as solubilizers, resulting in irritation, poor patient compliance. The most serious adverse event associated with etoposide is myelosuppression, and the number of WBC and platelets of patients can be reduced. The above drawbacks to some extent restricted the widespread clinical application of etoposide. In recent years, scientific researchers focus their work on new drug delivery systems such as liposomes, nanoparticles to increase the solubility of insoluble drugs and the local concentration of drugs at the specific site, thereby they could reduce the amount of drugs, side effects occurrence and improve drug treatment.Nanosuspensions are sub-micron colloidal dispersions of pure drug particles in an outer liquid phase. The nanosuspensions can be used to formulate compounds that are insoluble in both water and oils and to reformulate existing drugs to remove toxicologically less favourable excipients. By preparing nanosuspension makes it possible for those drugs including antibiotics, anticancer drugs and drugs possessing a narrow therapeutic window to play a better role. We constructed the nanosuspensions of etoposide,in which albumin was utilized as carrier material. The particle size range of etoposide nanosuspensions was 100-300nm, so EPR effect and the active target of albumin will make etoposide concentrate at the tumor site. The release of etoposide can be slowed by existing in particle forms, which improve the drug availability at the tumor site while reducing harmful side effects.Methods and results:HPLC method was established to determine the etoposide concentration in vitro and its methodology was examined. EPEG-BSA-Nanos were prepared by high pressure homogenizer-Solvent precipitation method. The evaluating indexes included drug content, entrapment efficiency and particle size. The formulation and technological parameters were optimized based on the results of the single factor study and the orthogonal design. The obtained nanosuspensions were dried using freeze-drying method. The properties of EPEG-BSA-Nanos such as diameter and size distribution were observed by transmission electric microscope (TEM) and laser dispersive analyzing apparatus for granularity, and Zeta potential was measured by micro-electrophoresis apparatus. In vitro releases of EPEG-BSA-Nanos were performed by dialysis method.The stability of EPEG-BSA-Nanos at 4was evaluated. The in vitro reliability evaluation such as local irritation, hemolysis and bone marrow suppression of EPEG-BSA-Nanos in rabbits and mice were investigated. HPLC method was established to investigate etoposide concentrations of plasma and tissue in rabbits and mice. The pharmacokinetics and tissue distribution of the EPEG-BSA-Nanos in rabbits and mice were investigated and EPEG injection was used as control.The results showed that using HPLC method to determine the etoposide concentration in vitro was easy, convenient and correct. EPEG-BSA-Nanos with appropriate particle size were obtained by high pressure homogenizer-Solvent precipitation method. The optimized prescription was as follows:trichlormethane/ dehydrated alcohol (8:1,v/v), BSA/drug (10:1,g/g), oil phase/water phase (10:1, v/v),and the pH of water phase is 5.4.The optimized procedure was as follows: homogenization pressure was 1500bar and homogenization cycles were 15. Lyophilized EPEG-BSA-Nanos were prepared to improve the stability and the obtained lyophilized nanosuspensions appeared as full, no collapse of the porous solid block. The lyophilized nanosuspensions showed good redispersibility and the reconstituted nanosuspensions with saline were opalescent, colloidal solution. The mean diameters of EPEG-BSA-Nanos changed to (182.38.91) nm from (190.27.3) nm after lyophilization, and the polydispersity was from0.290.08 to 0.270.11, zeta potential was from (-23.051.78) mV to (-9) mV, respectively. The pH of EPEG-BSA-Nanos was 5.460.24, which is consistent with the requirements of injection.Compared with etoposide injection, EPEG-BSA-Nanos presented controlled release properties and the etoposide release behavior from nanosuspensions in vitro was in accord with Higuchi model. The formation of EPEG-BSA-Nanos was validated by DSC, which indicated that EPEG was encapsuled into BSA successfully. Results of the preliminary stability experiments indicated that the lyophilized EPEG-BSA-Nanos were almost intact at 4for 3 months.The results of local irritation test and hemolysis test showed that EPEG-BSA-Nanos had no stimulatory effects for ear veins of rabbits and hemolysis and coagulation were not observed in hemolysis test. The results of myelosuppression test showed that EPEG-BSA-Nanos could distinctly reduce myelosuppression of etoposide to mice.HPLC method was established to investigate etoposide concentrations of plasma and tissue in rabbits and mice and the results showed that endogenous substances did not interfere with etoposide determination in the selected chromatographic conditions. The extraction recovery, method recovery of drugs in blood of rabbits and tissues in mice, the linearity, RSDs of intra-day and inter-day and the accuracy were all satisfied with the need of analysis for biological detection. After tail vein injection of EPEG-BSA-Nanos and EPEG injection, the plasma drug concentration of two kinds of preparation were compared. Pharmacokinetic parameters were calculated by statistical moment method. The t1/2of nanosuspensions group and injection group were 10.296h and 5.225h respectively, showing of 1.97 folds increasing. MRT0-were 2.707h and 1.170h respectively, showing of 2.31 folds enhancement. Cmax were 48.05 mg/L and 61.08mg/L respectively, however AUC0-of the EPEG-BSA-Nanos was similar with that of EPEG injection group (116.87mg/L*h and 111.16 mg/L*h respectively). It indicated that after encapsulated in the nanosuspensions, the drug concentration was reduced but the therapy effect was not affected.According to Ce (Ce=(Cmax) nanosuspensions/(Cmax)injection), the targeting efficiency of EPEG-BSA-Nanos was evaluated. The results showed that the Ce of lung, spleen and liver was 1.93,2.52 and 1.79 respectively. Meanwhile, the Ce of heart,brain and kindey was 0.94,0.95and0.88 respectively. It demonstrated that nanosuspensions formulations presented accumulative activity in RES sites such as lung, spleen and liver. Conversely the biodistribution of nanosuspensions formulations in non-RES sites such as kidney, brain and heart decreased with descending Ce compared with etoposide injection, which potentially resulted in the reduction of side effects of free drug.Conclusion:In this study, EPEG-BSA-Nanos were successfully prepared with simple, feasible preparation technology and ease of scale-up. EPEG-BSA-Nanos had no stimulatory and hemolysis effects, as well as distinctly reduced myelosuppression of etoposide to mice. Compared with the control group, the distribution of etoposide significantly changed because of the formulation of nanosuspensions. Etoposide encapsulated in BSA could improve drug accumulation in lung, extend retention time of drug in the lung, enhance local etoposide concentration in the lung and improve the therapeutic efficiency. In addition, the side effects in non-target tissues could be avoided and patientscompliance and the medication effects could be improved which accompany by good social and valuable economic benefits.【关键词】依托泊苷 白蛋白 纳米混悬剂 药物动力学 组织分布动力学【英文关键词】etoposide albumin nanosuspensions pharmacokinetics tissue distribution【目录】依托泊苷白蛋白纳米混悬剂的研究中文摘要10-13ABSTRACT13-16符号说明18-19前言19-27第一章 依托泊苷白蛋白纳米混悬剂的处方筛选及工艺研究27-471 仪器与材料27-281.