【作者】 沙先谊；

【导师】 方晓玲；

【作者基本信息】 复旦大学 ， 药剂学， 2005， 博士

【摘要】 9-硝基喜树碱（9-nitrocamptothecin,9-NC）是一种新型的喜树碱水不溶性衍生物,动物实验显示对多种癌细胞具有强抑瘤活性,尤其是对胰腺癌有着独特的疗效。本文旨在研究9-硝基喜树碱的基本理化性质和小肠吸收机理,阐明影响药物口服吸收的因素,从而为制备9-硝基喜树碱的新型口服制剂提供合理依据;制备9-硝基喜树碱自微乳化给药系统,考察影响制剂中药物细胞摄取和转运的因素;研究自微乳化给药系统的分子药剂学性质,并对其体内药动学和组织分布进行了研究。 首先,建立了9-硝基喜树碱内酯和羧酸盐形式的HPLC分析方法,考察药物的基本理化性质和水解动力学。9-硝基喜树碱是一种水难溶性药物,并且随着介质pH的升高,其油水分配系数逐渐降低,说明药物的脂溶性降低。9-NC在水溶液中的水解为假一级动力学反应;在水溶液中受OH^-催化,药物的水解是一种pH依赖性的水解反应,在酸性条件下稳定,在中性偏碱性条件下,水解速度加快;药物的水解反应速度与温度成正相关性。在pH为4.5、6.5、7、7.4、8与9.06缓冲液中的反应活化能分别为5.039,12.476,10.094,10.021,9.647,11.35 KJ/mol。 以外翻肠囊法考察了9-硝基喜树碱在肠黏膜的吸收性质。结果表明9-硝基喜树碱在十二指肠、空肠、回肠的吸收没有显著性部位差异性（P>0.05）,均主要以被动扩散的方式吸收。P-糖蛋白抑制剂维拉帕米能显著增加各段小肠对9-NC的吸收（P<0.05）,在小肠下端更加明显。随着维拉帕米浓度的增加,作用逐渐增强,在50μg/ml的维拉帕米作用下,药物90min小肠累积透过量增加了大约2倍。 建立了Caco-2细胞摄取和转运模型,首次考察了9-硝基喜树碱的Caco-2细胞摄取、跨膜转运及外排动力学研究,评价了表面活性剂Cremophor EL、Labrasol和Transcutol P对药物转运的影响。结果表明:（1） 9-硝基喜树碱主要以被动扩散的方式被细胞摄取。药物的摄取与时间呈正相关,与温度、pH呈负相关。P-gp抑制剂环孢菌素和维拉帕米显著增加9-NC细胞摄取（P<0.05）。（2） 9-硝基喜树碱的吸收转运具有pH依赖性,在弱酸性条件下有助于药物的吸收。9-硝基喜树碱主要以被动扩散的方式吸收转运,而药物的分泌具有浓度依赖性和饱和性的非线性动力学特征,其米氏常数Km为49.8±1.2μM,V_max为38.28±0.8ng/cm²/min。（3） 9-硝基喜树碱在Caco-2细胞中的转运呈现较强的方向性,从Basolateml侧（BL,基底面）到Apical侧（AP,肠腔面）跨膜转运速度大于AP到BL转运（2.6-6.9倍）。当加入P糖蛋白抑制剂环孢菌素和维拉帕米后,增加了药物吸收,同时降低了药物的外排,其表观渗透系数P_{app BL-AP}/P_{app AP-BL}的比值从3.4分别降更多还原

【Abstract】 9-nitrocamptothecin is a novel analog of camptothecin, which had indicated that the antitumor activity for many cancer cells, especially for advanced pancreatic cancer. The main aims of this research were to study the physicochemical properties and the intestinal absorption mechanisms of 9-nitrocamptothecin, and investigate the factors that affect the absorption of 9-nitrocamptothecin for preparing a novel oral drug delivery system. Self-microemulsifying drug delivery systems (SMEDDS) of 9-nitrocampthecin were developed. The uptake and transport of 9-nitrocamptothecin SMEDDS in Caco-2 cells was investigated. The molecular pharmaceutical properties of SMEDDS were evaluated. Finally, the studies on pharmacokinetics and tissue distribution of different SMEDDSs of 9-nitrocamptothecin were determined.Resolution of the lactone and carboxylate forms of 9-nitrocamptothecin was accomplished by reverse-phase HPLC. 9-nitrocamptothecin is sparingly solubility in water. The oil-water partition coefficient of 9-nitrocamptothecin was the pH-dependent. With increasing of pH, the coefficient became decreasing. The hydrolysis and lactonization reactions followed pseudo-first-order kinetics in the approach to equilibrium. The hydrolysis rate was dependent on pH, with the closed form (lactone) dominant at low pH and the open form dominant at physiological pH or above. The hydrolysis rate of 9-nitrocamptothecin was affected by temperature. The Ea at different pH were 5.039, 12.476, 10.094, 10.021, 9.647, 11.35KJ/mol, respectively.The intestinal transport of 9-nitrocamptothecin was performed by applying everted intestinal sacs method. The absorption of 9-nitrocamptothecin was passive diffusion and had no significant difference in different gut regions (P>0.05). Adding verapamil in the everted gut sacs, the absorption of 9-nitrocamptothecin was significantly enhanced (P<0.05), especially more markedly in lower small intestine. The accumulative transport flux of 9-nitrocamptothecin was increasing by two-fold in 90min added 50 μg/ml verapamil.The Caco-2 cell model was used to study the uptake, transport and efflux kinetics of 9-nitrocamptothecin. The effect of surfactants, such as Cremophor EL, Labrasol and Transcutol P, on the transport of drug was investigated. The results indicted: (1) The uptake of 9-nitrocamptothecin is passive diffusion as the dominating process. The uptake of 9-nitrocamptothecin is positively correlated to uptake time, and negatively correlated to pH and temperature. The inhibitors, cyclosporine A and verapamil,significantly enhanced the uptake amount of 9-nitrocamptothecin (.P<0.05) .(2) The absorptive transport of 9-nitrocamptothecin was pH dependent and the transport was enhanced at weakly acidic pH on the apical side. No concentration dependence and saturation were observed for the absorptive transport of 9-nitrocamptothecin, while secretory transport were concentration dependent and saturable process, Km was (49.8±1.2) uM, Vmaxwas (38.28±0.8) ng/cm2/min, respectively. (3) The transport of 9-nitrocamptothecin across Caco-2 cell monolayers was directional. Papp of Basolateral to Apical was much more than that of Apical to Basolateral (2.6-6.9 folds). In the presence of verapamil and CsA, potent inhibitors of P-glycoprotein or MRP2, the Papp BL-Ap/Papp ap-bl ratio was decreased from 3.4 to 1.4 and 1.3, respectively. (4) The efflux of 9-nitrocampothecin was fitted to apparent two-order process. The m0 [(148.0±2.18)] pmol-cm’2) and the efflux rate (41.1 pmol-cm2-min*’) on Apical side were higher than the m0 [(121.3±6.9) pmol-cm’2] (PO.05) and the efflux rate (29.2 pmol-cm2-min"1) on Basolateral side (PO.01). (5) Surfactants could enhance the absorption of 9-nitrocampthothecin, and decrease the efflux. Overall, the current study suggests that pH and efflux transporters are capable of mediating the absorption and efflux of 9-nitrocamptothecin, and they may play significant roles in limiting the oral absorption of 9-nitrocamptothecin.In the study of preformulation, the equilibrium solubility of 9-nitrocampothecin in different oil, surfactants and cosurfactants was investigated and pseudo-ternary phase diagrams were constructed to evaluate the formulation effect. An optimized formulation consisted Maisine 35-1: Miglycol 812 N (1:1) as oil phase, Cremopher EL: Labrasol (4:1) as surfactants, and Transcutol P as co-solvent. With the amount of oil and Km as the independent variables, the optimized formulation was determined by the central composite design-response surface methodology. The response variables were particle size, Zeta potential, solubility, and cloud point, the cumulative percentage of 9-nitrocamptothecin in 5 min or 60 min. The optimized formulation of 9-nitrocamptothecin was quickly and conveniently obtained by the central composite design-response surface methodology, i.e. oil: surfactants: cosolvent=30%:47%:23%. For the positively charged SMEDDS of 9-nitrocamptothecin, oleylamine (1%, w/w) was additionally added. For the super-saturable SMEDDS of 9-nitrocamptothecin, HPMC (0.5% E5LV, w/w) was additionally added.The effect of dilution on the particle size and zeta potential of 9-nitrocamptothecin in Caco-2 cells was investigated. The results indicated that dilution had hardly effecton the particle size of microemulsion, while the zeta potential were increased with dilution. When the negatively charged, positively charged and HPMC SMEDDS were diluted with water in the ratio of 1:10, the size of microemulsions were (24.7±7.9), (25.0±8.3) and (32.8±7.5) nm, respectively. The SMEDDS of 9-nitrocamptothecin could decrease the hydrolysis rate to a certain extent, increase the stability of 9-nitrocamptothecin in buffer solution.The results of the uptake and transport of 9-nitrocamptothecin SMEDDS in Caco-2 cells indicated: (1) Self-microemulsifying drug formulations of 9-nitrocamptothecin could significantly improve the uptake of 9-nitrocamptothecin (PO.05), except for the positively charged SMEDDS at the dilution of 1/50.(2) SMEDDS of 9-nitrocamptothecin could enhance the transport across Caco-2 cell monolayers. (3) The effect of pH on the transport of 9-nitrocamptothecin SMEDDS formulations was markedly decreased compared with the control. The ratio (2.19-3.68) of Papp of SMEDDS formulation between different pH was lower than that (6.02) of control.Effect of the negatively charged and positively charged self-microemulsifying drug delivery systems on the P-glycoprotein and cellular tight junction complex was to been investigated at molecular cell level. The results indicated: negatively charged or positively charged SMEDDS at different dilution could significantly affect the integrity of Caco-2 cell monolayers (PO.05), which depended on the dilution times. The cell accumulation of RH123, a substrate of P-glycoprotein, were significantly enhanced by two SMEDDSs in different dilutions (1.6-6.4 folds).The improvement of positively charged SMEDDS on the accumulation was higher than that of negatively charged SMEDDS in the same dilution. Immunoflurescent and Western-blotting results indicated the mechanism of opening of tight junctions by SMEDDS was changed the distribution of ZO-1 and actin. In addition, to change the cell membrane fluidity inhibited the efflux activity of P-glycoprotein. MTT results showed the positively charged SMEDDS in lower dilution presented certain cytotoxicity.The pharmacokinetic parameters of 9-nitrocamptothecin solution, micelle, negatively charged, positively charged and HPMC SMEDDS were investigated after oral administration to rats at a single dose of 6mg/kg. The results were as follows: Cmax was (93.83±72.79), (143.25±29.91), (176.42±93.43), ( 167.38±52.12 ) and (333.23±96.26) ng/ml, respectively; AUC was (127.62±51.6), (239.78±137.60), (331.91±161.04), (595.04±42.00) and (412.21±136.04) ng-mr’-h, respectively; MRT was (1.6±0.9 ), (1.3±0.4), (1.2±0.6), (4.3±2.6) and (2.6±0.6) h, respectively.更多还原