【作者】 赵春顺；

【导师】 张汝华； 何仲贵；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2003， 博士

【摘要】 3-正丁基苯酞（3-n-butylphthalide，简称NBP）是从芹菜籽挥发油中分离得到的抗脑缺血的有效成分，经化学合成后用于临床试验的新药。本文旨在研究NBP在体内的药动学过程及组织分布情况，考察其在肠粘膜的吸收性质及肝脏的首过效应，从而阐明影响其口服吸收的因素；研究NBP向靶器官—脑组织转运的机制，为合理给药途径的提出及剂型的设计提供理论依据；通过考察NBP在鼻粘膜的吸收性质及对鼻纤毛的毒性，论证NBP鼻腔给药的可行性；并通过研究NBP脂质体经鼻给药的生物利用度及向脑内转运的途径对实验设想予以证实。 首先，建立了NBP在家兔和大鼠体内的测定方法，采用HPLC法以荧光检测器进行检测。色谱条件为：以0.05 mol/L醋酸钠（用醋酸调pH4.5）-乙腈（40：60）为流动相，采用Selectosil C₁₈色谱柱，检测波长为λ_ex=280 nm，λ_em=304 nm，以布洛芬为内标，样品经乙醚提取处理测定。该法专属性强，灵敏度高，适于NBP体内药动学研究。 分别以家兔和大鼠为模型，考察了NBP在不同动物体内的药动学过程：NBP在家兔和大鼠体内均呈线性动力学过程，符合双隔室开放模型。静注给药后从体内消除迅速：不同剂量组NBP在家兔体内的T_1/2α为10.3～11.3 min，T_1/2β为96～109min：在大鼠体内的T_1/2α为4.7～9min，T_1/2β为68～125 min。NBP分别以羟丙基-β-环糊精（Hydroxypropyl-β-cyclodextrin，简称HPCD）包合物和脂质体静注给药，并不影响NBP在大鼠体内的动力学过程。NBP口服给药后，吸收迅速，在家兔和大鼠体内达峰时间(T_max)约为30 min。家兔口服生物利用度为4.7±1.7％；于大鼠口服给药后，NBP脂质体的生物利用度（30.3±9.6％）与NBP溶液（50％1，2-丙二醇溶液）相比（26.3±10.3％）无显著性差异（P＞0.05）；NBP-HPCD包合物的生物利用度显著降低（15.0±3.7％）。 采用同位素示踪技术考察了[³H]NBP在大鼠体内的分布，NBP主要分布于肝、肾、肺、脑、脾等组织，各组织的浓度均呈一定的剂量依赖性，NBP在大脑、嗅球、小脑、脑室、脑干的浓度间无显著性差异（P＞0.05）。 以大鼠在体肠灌流技术（In-situ single-pass perfusion）考察了NBP在肠粘膜的吸收性质，NBP在大鼠十二指肠、空肠、回肠和结肠的表观吸收系数分别为0.0381±0.0103，0.0314±0.0041，0.0277±0.0060和0.0574±0.0050 ml/min/cm（灌流液中NBP浓度为10 μg/ml），NBP浓度对肠段的表观吸收系数无明显影响（P＞0.05）。表明NBP在各肠段的吸收均呈被动扩散机制，也说明肠粘膜吸收过程非NBP口服生物利用度低的主要影响因素。灌流液中HPCD的加入对NBP在大鼠肠粘膜的吸收具有较强的抑制作用，其抑制程度随HPCD用量的增加而显著增强。而NBP包沈阳药科大学博士学位论文摘要封于脂质体中后对NBP在大鼠肠粘膜的吸收无显著性影响。说明NBP一HPCD包合物于大鼠口服生物利用度低是由HPCD的抑制作用所引起石 采用离体大鼠肝灌流技术测得不同浓度NBP（2，5和20林g/inl）在肝脏的清除率分别为6.31士0.45，3.14士0.15，2.71士0.48 ml/m in;HPCD对NBP在离体大鼠肝脏的消除有抑制作用;NBP脂质体对NBP在大鼠离体肝脏的消除无显著性影响;尼莫地平对NBP的消除无显著性影响。原代大鼠肝细胞对护H]NBP的摄取实验表明，NBP能迅速进入离体肝细胞中并达到稳态，且细胞中NBP浓度比培养介质中高约4倍，细胞的摄取过程无自身浓度抑制作用，呈能量非依赖性过程，说明NBP进入肝细胞为被动扩散机制。由以上实验结果表明NBP在大鼠肝脏具有较强的首过效应，由此可解释NBP口服生物利用度低和NBP在肝脏中浓度高的现象。 离体脉络丛摄取实验表明，脉络丛对NBP的摄取无自身浓度抑制作用，呈能量非依赖性转运过程，且NBP的T/M值均较高（1 4.巧士1 .18）。说明NBP在大鼠脉络丛中的转运主要为被动扩散机制，NBP易于透过脑屏障。尼莫地平和桂利嗦对NBP在离体大鼠脉络丛的转运方式无显著性影响（P>0.05）。 NBP在大鼠鼻粘膜吸收迅速，用药后5 min即可吸收55%以上;但对大鼠鼻粘膜具有较强的纤毛毒性，NBP经HPCD包合后可显著降低NBP的纤毛毒性，但对NBP在鼻粘膜的吸收有一定抑制作用。NBP包封于脂质体中后不但显著降低了纤毛毒性，且对NBP在鼻粘膜的吸收无显著性影响。说明脂质体可作为NBP鼻腔给药的剂型之一。 采用超声一高压乳匀法制备了NBP脂质体。磷脂的种类和用量、载药量以及胆固醇用量对脂质体的包封率均有一定的影响。以合成DPPC为磷脂膜的脂质体包封率较高且稳定;随载药量的增大包封率显著降低;制备工艺对脂质体粒径有显著性影响。以超声一高压乳匀法制得的脂质体有效粒径为88.snln，聚分散度为0 .1 84。 NBP脂质体于家兔经鼻给药后，迅速吸收进入体循环，2一5 min即可达最大血药浓度，体内药时过程与静注给药相似，其绝对生物利用度为88.7%。于大鼠经鼻给药后，2 min即可在脑组织中达到较高的浓度，尤其以脑组织的嗅球部位浓度最高。30一60 min NBP在脑组织中的浓度显著高于静注给药。说明NBP经鼻给药后进入脑组织的途径有两条:a)嗅粘膜上皮细胞途更多还原

【Abstract】 Zhao Chunshun (Major: Pharmaceutics) Advisors: Prof. Zhang Ruhua and Associate Prof. He Zhonggui3-n-Butylphthalide (NBP), a novel cerebral antiischemic agent, was isolated and identified from several plants including celery oil. At present, NBP was synthesized and approved for Phase IV trials in treatment of stroke by the State Drug Administration of China.The main objectives of this research were to study the pharmacokinetics, absolute bioavailability, disposition, intestinal absorption and first-pass effect of NBP in rabbits and rats, to investigate the mechanism of transport of NBP in the blood-cerebrospinal fluid barrier, to research nasal mucosa absorption and nasal ciliotoxicity of NBP in rats, and to develop reasonable delivery system of NBP used in intranasal administration. We also determined the uptake of NBP into the brain after intravenous and intranasal administration in rats.A rapid, sensitive and specific RP-HPLC method was developed for the determination of NBP in rabbits or rats plasma in combination with fluorescence detection at an excitation wavelength of 280 nm and an emission wavelength of 304 nm. Ibuprofen was used as internal standard. Plasma samples were extracted with diethyl ether under acidic conditions. After evaporation of the organic phase, the extract was dissolved in mobile phase and injected into the chromatograph with C18 column and a mobile phase of 0.05mol/L sodium acetate buffer (pH 4.5)-acetonitrile (400:600). The achieved limit of quantification of 0.0212 ug/ml is sufficient to study the pharmacokinetics of NBP in rabbits or rats.The pharmacokinetic investigation were performed on rabbits and rats. NBP exhibited linear pharmacokinetics over the dose range tested (l-10mg/kg) in rabbits and rats after intravenous administration. NBP was rapidly eliminated from the plasma. The mean elimination (T1/2a,10.3-11.3 min and T1/2B, 96-109 min in rabbits; T1/2a, 4.7-9 min and T1/2B, 68-125 min in rats), total plasma clearance, and apparent volume values were independent of doses. The dosage forms had no effect on the pharmacokinetic of NBP in rats. After oral administration to rabbits and rats, NBP reached the peak plasma concentration at a time ranging between 10 to 45 min. Oral bioavailability was lower inrabbits (4.7±1.7%) than in rats (26.3±10.3%) for NBP solution. NBP-HPCD inclusion reduced the bioavailability (15.0±3.7%) of NBP in rats.The distribution of [3H]NBP in tissues was determined at 10, 30, 60, and 90 min after intravenous administration of a single 1, 5, and 10 mg/kg dose to rats by liquid scintillation counter. The highest concentration was found in the liver, and the concentration decreased in the order of liver, kidney, lung, brain, heart and spleen. NBP in tissues is dose-dependent. NBP concentration was not significantly different among cerebrum, cerebellum, olfactory bulb, cerebral ventricle and brain stem.The intestinal transport of NBP was performed by applying single-pass perfusion techniques in situ rats. NBP is a high permeability drug in rat intestine and colon. The apparent permeability, Papp, of NBP was independent of both intestinal region and concentration of NBP present in the perfusion solution. The Papp for NBP in the colon is higher than that in various intestinal regions. The Papp for NBP decreased when HPCD is co-perfused in the intestine and colon. The absorption inhibitor effect of HPCD was concentration dependent and it was supposed that the concentration of free NBP is too low in the perfusion solution when HPCD was added in.The hepatic clearance of NBP during isolated rat liver perfusion were 6.31±0.45, 3.14±0.15, and 2.71±0.48 ml/min for 2, 5, and 10 ug/ml, respectively. The elimination of NBP was inhibited when HPCD is co-perfused in the isolated liver. Liposomes didn’t influence the elimination of NBP from the isolated rat liver. Uptake of [3H]NBP by rat primary hepatocytes was rapid and a steady-state was achieved in 15 seconds. The accumulation of [3H]NBP in hepatocytes wasn’t inhibited by unlabeled NBP,更多还原