【作者】 张蕊；

【导师】 郭瑞臣；

【作者基本信息】 山东大学 ， 药理学， 2009， 博士

【摘要】 目的:1.合成去甲斑蝥素钠作为实验药物;2.建立灵敏度高、特异性强的HPLC-MS/MS方法测定小鼠血浆和组织中去甲斑蝥素钠含量;3.进行去甲斑蝥素钠小鼠体内药代动力学及组织分布研究;4.对去甲斑蝥素钠体内、体外代谢物及可能代谢途径进行研究分析;5.采用逆相蒸发法制备去甲斑蝥素钠脂质体并进行理化性质考察;6.进行去甲斑蝥素钠脂质体小鼠体内药代动力学特征、组织分布研究,评价其缓释性和靶向性。方法:1.采用酸酐碱性条件下水解反应制备,经乙醇重结晶纯化得到去甲斑蝥素二钠盐,并通过红外吸收光谱和质谱对其结构进行确证。2.生物样本在酸性条件下经丙酮沉淀蛋白后浓缩,采用C₁₈色谱柱（150×4.6mm,5μm）,以乙腈-水（20:80,2mM乙酸铵,含0.1%甲酸）为流动相,利巴韦林为内标,选用正离子模式,以多反应检测（MRM）方式检测m/z 169.1→123.1（去甲斑蝥素钠）及267.1→135.1（利巴韦林）,从方法专一性、基质效应、绝对回收率、相对回收率、精密度与准确度及稳定性等方面进行方法学考察确证。3.健康昆明小鼠102只,随机分为12组,每组6只（其中0.25,1,2,4,8h组12只,雌雄各半）,按20mg·kg^-1剂量灌胃给予1mg·mL^-1去甲斑蝥素钠水溶液,按设计时间点分别于给药前和给药后经摘眼球取血,离心取血浆;取血后脱颈处死小鼠,取出心、肝、脾、肺、肾、脑、肠、胃、胆囊、睾丸/子宫,生理盐水冲洗干净后滤纸吸干。样本经处理后采用已建立的测定方法测定药物含量,采用DAS软件计算药代动力学参数,对去甲斑蝥素钠在小鼠体内的药代动力学特征及组织分布特点进行详细分析。4.采用体内代谢和体外代谢两种方法对去甲斑蝥素钠代谢物和代谢途径进行初步分析。体内收集给药后大鼠尿液、粪便等生物样本;体外采用肝微粒体温孵方法,通过钙沉淀法制备大鼠肝微粒体,辅以氧化还原辅酶,模拟体内Ⅰ相和Ⅱ相代谢反应。上述样本经处理后,利用液相色谱-质谱联用技术对去甲斑蝥素钠动物体内外代谢物及代谢过程进行研究。5.采用逆相蒸发法制备去甲斑蝥素钠脂质体。以包封率为评价指标,通过正交设计优化脂质体的最佳处方和制备工艺。电子透射显微镜观察脂质体的外观形态,激光散射粒度分析仪测定脂质体的粒径及其粒度分布,电势显微电泳仪测定脂质体表面Zata电位;葡聚糖凝胶色谱法分离脂质体和游离药物,HPLC法测定脂质体包封率和载药量;动态膜透析法测定脂质体体外释药特性。6.进行去甲斑蝥素钠脂质体小鼠体内药代动力学和组织分布研究,计算主要药代动力学参数和靶向评价参数,评价其靶向性和缓释作用。结果:1.合成纯化后计算去甲斑蝥素钠的产率约为94%,经红外吸收光谱和质谱确证其结构为去甲斑蝥素二钠盐。2.经方法学考察,本文建立的HPLC-MS/MS方法在0.01～5.00μg·mL^-1线性范围内,在血浆、肝脏、肺、肾脏、胃、小肠、子宫和睾丸样本中线性关系良好,最低定量限为0.01μg·mL^-1;在0.005～0.50μg·mL^-1线性范围内,心脏、脾脏和脑中线性关系良好,最低定量限为0.005μg·mL^-1;相关系数（r²）均在0.9918～0.9976之间;日内、日间精密度均小于15%,血浆和组织中绝对回收率均大于50%,相对回收率均在91%-107%之间,室温放置4h,-20℃条件下反复冻融2次、存放24h和一周后均稳定,RSD均小于15%,能满足生物样本中去甲斑蝥素钠含量测定的要求。3.去甲斑蝥素钠在小鼠体内的吸收半衰期t_1/2α为0.196±0.117h,消除半衰期t_1/2β为6.345±3.321h,单位体重表观分布容积V1/F为0.089±0.039L·kg^-1,单位体重血浆清除率CL/F为0.088±0.021L·h^-1·kg^-1,药时曲线下面积AUC_0-24和AUC_0-∞分别为11.22±2.614mg·L^-1·h和11.894±2.805mg·L^-1·h,平均滞留时间MRT_0-24和MRT_0-∞分别为5.392±1.12h和8.285±4.941h,达峰时间Tmax为0.3754±0.137h,峰浓度为3.9734±0.656mg·L^-1。给药后各组织药时曲线下面积依次为:小肠＞胃＞子宫＞肾＞睾丸＞肝＞肺＞脾＞心＞脑。给药后各组织体内平均滞留时间依次为:肺＞肝＞睾丸＞脑＞脾＞肾＞子宫＞心＞胃＞小肠。4.体内代谢研究结果显示,给药后大鼠尿液、粪便样本经HPLC-MS/MS方法系统分析,尿液中检测到分子量为169和205的分子离子峰,分别为去甲斑蝥素（M0）和Ⅰ相环氧化物水合反应产物（M1）;粪便检测到分子量为169和539的分子离子峰,分别为去甲斑蝥素（M0）和结合两分子葡萄糖醛酸的Ⅱ相结合产物;体外代谢研究结果显示,去甲斑蝥素钠主要葡萄糖醛酸结合产物形式代谢,未见Ⅰ相代谢产物。体内外代谢结果初步阐明,除部分以去甲斑蝥素原型药物排出体外,可能在环氧化物水合酶的作用下代谢为Ⅰ相水合产物,在葡醛酸转移酶作用下与2分子葡萄糖醛酸发生Ⅱ相结合反应结合,形成Ⅱ相代谢结合产物。5.HPLC法测定去甲斑蝥素钠脂质体种药物含量,去甲斑蝥素钠在50.0～350.0μg·ml^-1浓度范围内,线性关系良好,相关系数r=0.9998;批内、批间精密度均＜2%,回收率在90%～110%之间。按优化处方工艺制备的空白脂质体和含药脂质体均多为单层封闭的囊状或球状。空白脂质体和去甲斑蝥素钠脂质体平均粒径分别为209.6nm和243.1nm,Zeta电位分别为-14.05mv和-22.94mv,PH值7.54±0.13;包封率为（34.34±1.21）%。初步稳定性试验表明,去甲斑蝥素钠脂质体4℃放置3个月包封率未见明显变化。6.去甲斑蝥素钠在小鼠体内的吸收半衰期t_1/2α为0.763±0.101h,消除半衰期t_1/2β为12.235±5.279h,单位体重表观分布容积V1/F为0.169±0.138L·kg^-1,单位体重血浆清除率CL/F为0.057±0.012L·h^-1·kg^-1,药时曲线下面积AUC_0-24和AUC_0-∞分别为17.555±2.941mg·L^-1·h和18.231±3.209mg·L^-1·h,平均滞留时间MRT_0-24和MRT_0-∞分别为11.631±1.282h和12.395±1.4331h,达峰时间Tmax为0.708±0.459h,峰浓度为3.286±1.114mg·L^-1。给药后各组织药时曲线下面积依次为:子宫＞胃＞小肠＞肾＞肝＞肺＞心＞脾＞睾丸＞脑。给药后各组织体内平均滞留时间依次为:脾＞肝＞小肠＞睾丸＞心＞脑＞肺＞子宫＞肾＞胃。去甲斑蝥素钠脂质体血浆中消除半衰期t_1/2β由6.354h增加到12.235h,平均滞留时间MRT_0-∞由8.285h增加至12.395h,表明延长了药物在血中的循环时间。表观分布容积V1/F由0.089L·kg^-1增加至0.169L·kg^-1,表明脂质体载体脂溶性增加了药物在体内组织和器官的药物浓度。血浆清除率CL/F由0.088L·h^-1·kg^-1降低至0.057L·h^-1·kg^-1,表明单位时间内被清除体外的药物量减少。药-时曲线下面积AUC_0-∞分别为11.894mg·L^-1·h和18.231mg·L^-1·h,相对于去甲斑蝥素钠溶液的生物利用度F为153.3%。与去甲斑蝥素钠溶液相比,除睾丸外,其它组织的相对摄取率r_e均大于1,表明脂质体明显提高药物在这些组织器官中的分布,对脑、心脏和肺的相对摄取率大于4,对肝脏、肾脏、胃和子宫的相对摄取率大于2;脑、心脏、肺、子宫和胃的靶向效率Te分别从0.42%,2.58%,2.88%,18.38%和18.40%提高到1.13%,5.58%,6.00%,22.44%和20.73%,肝脏、肾脏的靶向效率无变化,而小肠、睾丸的靶向效率由21.40%,8.61%降低到12.12%,3.81%。结论:1.本文建立的HPLC-MS/MS方法快速、灵敏,能满足生物样本中去甲斑蝥素钠含量测定的要求。将药代动力学结果与同位素标记方法进行比较,提示该方法更能准确描述去甲斑蝥素钠体内处置过程;2.小鼠组织分布研究结果显示子宫为去甲斑蝥素钠作用的优势靶向器官,提示其在子宫癌治疗方面的潜在优势,为临床应用提供新的选择;3.体内外代谢研究初步阐明去甲斑蝥素钠在体内除去甲斑蝥素原型外,可能以Ⅰ相水合产物和结合两分子葡萄糖醛酸的Ⅱ相结合产物消除;4.逆相蒸发法制备的脂质体包封率较高,初步稳定性良好,体外释放结果表明起到了长效缓释的作用;5.去甲斑蝥素钠脂质体在体内各组织的分布均明显增加,并具有明显的缓释作用,可降低给药剂量减轻不良反应。更多还原

【Abstract】 Objectives:1.To synthesize disodium norcantharidate（DSNC） as the test drug; 2.To establish and validate a sensitive and high specificity HPLC-MS/MS method for determination of DSNC in mice plsma and tissues;3.To study the pharmacokinetic and tissue distriburion of DSNC solution（DSNC-sol） in mice;4. To study the metabolite and metabolic pathway of DSNC in vivo and in vitro;5.To prepare DSNC liposomes by reverse phase evaporation method and evaluate its pharmaceutical characterater;6.To study the pharmacokinetics and tissu distribution of DSNC liposomes（DSNC-lip） to evaluate its delayed releasing and targeting character.Methods:1.Disodium norcantharidate was synthesized by acid-base neutralization reaction and purified by recrystallization in ethanol.Its structure was identified by infrared absorption（IR） spectrum and mass spectrum.2.Biological samples were extracted by protein precipitation with acetone from 0.3mL plasma or tissue homogenates under acid condition.The samples were analysized on a C₁₈ column with a mobile phase of acetonitrile-ammonium acetate （2mM,0.1%formic acid）（20:80,v/v） and ribavirin was used as internal standard （IS）.DSNC was quantified using a triple quadrupole mass spectrometer operating in positive electrospray ionization（ESI） in multiple reaction monitoring（MRM） mode, ion transitions of m/z 169.0→123.0 for DSNC and m/z 267.1→135.1 for I.S.were selected.The pecificity,matrix effect,absolute recovery,calibration curve,LLOQ, precision,accuracy and stability were studied to validate the method.3.102 Kunming mice were divided into 12 groups randomly,one was blank group and others were experiment groups.The DSNC solution(1mg·mL^-1) was administered to experiment groups via intragastric administration at a dose of 20mg·kg^-1.Blood samples were obtained from the fossa orbitalis vein at 0.083,0.25, 0.5,1,1.5,2,4,6,8,10,24h after administration.After centrifuged,the plasma was separated and stored at -20℃for analysis.Heart,liver,spleen,lung,kidney,brain, stomach,small intestine,testis,uterus were rapidly collected at 0.25,1,2,4,8h following blood collection,respectively.The tissue samples were washed with 0.9% NaCl and blotted on filter paper,then weighted accurately and homogenized in 0.9% NaCl(2mL·g^-1 tissue),and the homogenates were stored at -20℃for analysis.The samples were analysized by etablished HPLC-MS/MS method,and pharmacokinetic parameters were calculated by DAS 2.0 software.4.The metabolite and metabolic pathway of DSNC in vivo and in vitro were studied.In vivo,rat urine and feces were collected after administration of DSNC-sol and were analysized by HPLC-MSⁿ method after disposition.In vitro,rat liver microsome was prepared with Ca²⁺ precipitation method,assisted with oxidoreduction coenzyme to simulate the conditiong of phaseⅠand phaseⅡmetabolism in vivo.The reaction was terminated with acetonitrile and products were analysized by HPLC-MSⁿ method after disposition.5.The reverse phase evaporation method was used to prepare disodium norcantharidate liposome（DSNC-Lip）.The orthogonal experimental design was applied to optimize the prescription and the technology of preparation.The diameter and size distribution properties of disodium norcantharidate liposome were observed by transmission electric microscop and laser dispersive analyzing apparatus for granularity,and Zeta potential was measured by micro-electrophoresis apparatus. Liposome containing drug was separated from free drug by sephadex gel column method.The encapsulation efficiency and drug loading capacity were assayed by HPLC method.In vitro releases of liposome were performed by dialysis method.6.The pharmacokinetics and tissue distribution of DSNC in mice after administration of DSNC-lip were investigated.The main pharmacokinetic parameters and targeting parameters were calculated to evaluate the delayed releasing and targeting characters.Results:1.The average yield of synthesis was about 94%.The compoud was validated to be DSNC by IR spectrum and Mass spectrum.2.Good linearity of DSNC in plasma,liver,lung,kidney,stamoch,small intestine,uterus and testis were observed in the range of 0.01-5.0μg·mL^-1,the LLOQ was 0.01μg·mL^-1,and good linearity was observed in the range of 0.005-0.5μg·mL^-1 in other tissus（heart,spleen and brain） too,the LLOQ was 0.005μg·mL^-1,the correlation coefficients（r²） were between 0.9918 and 0.9976.The intra-day and inter-day RSD were less than 15%.All absolute recoveries exceeded 50%and relative recoveries were between 91%and 107%.DSNC in plasma and all tissues placed at room temprature for 4h and frozen at -20℃for 24 hours and seven days and after two freeze-thawing cycles was stable,the RSD were less than 15%. This method was suitable for DSNC pharmacokinetic and tissue distribution studies.3.After fitting,the pharmacokinetic process of DSNC in mice after administration of DSNC-sol was coincidence with double compartment model with weight of 1.The main pharmacokinetic parameters of DSNC after administration of DSNC-sol were as follows:t_1/2α 0.196±0.117h;t_1/2β 6.345±3.321h;Tmax 0.375±0.137h;Cmax 3.973±0.656mg·L^-1;AUC_（0-24） 11.22±2.614mg·L^-1·h;AUC_（0-∞） 11.894±2.805mg·L^-1·h;MRT_（0-24） 5.392±1.12h;MRT_（0-∞） 8.285±4.941h;Vl/F 0.089±0.039L·kg^-1;CL/F 0.088±0.021L·h^-1·kg^-1.The AUC of DSNC in tissues was in the order as follows:small intestine＞stomach＞uterus＞kidney＞testis＞liver＞lung＞spleen＞heart＞brain.The MRT of DSNC in tissues were lung＞liver＞testis＞brain＞spleen＞kidney＞uterus＞heart＞stomach＞small intestine. 4.The rat urine and feces were analysized by HPLC-MS/MS method after disposition.The molecular ion peak of molecule weight 169 and 205 were detected in rat urine,which were norcantharidin（M0） and the product of epoxide hydration reaction of phaseⅠmetabolism（M1）.The molecular ion peak of molecule weight 169 and 539 were detected in rat feces,which were norcantharidin（M0） and the glucuronate conjugation metabolite of phaseⅡmetabolism（M2）.The metablic reaction products in vitro were analysized by HPLC-MS/MS after filteration with 0.45μm millipore filter.Norcantharidin and glucuronate conjugation metabolite were detected in phaseⅡmetabolism product.Only norcantharidin was detected in phaseⅠmetabolism product,no metabolite of phase metabolism was found.5.HPLC method was established for the determination of DSNC in liposome, there was a good linearity of the drug concentration within the range of 50.0～350.0μg·ml^-1,the correlation coefficients was 0.9998.The intra-day and inter-day RSD were less than 2%.The recovery rate was between 90%and 110%. The liposome was all spherically shaped with uniform size.The mean diameter of DSNC liposome and blank liposome was 243.1nm and 209.6nm;Zeta potential was -22.94mv and -14.05mv,respectively.PH was 7.54±0.13.The incorporation efficiency was（34.34±1.21）%and the incorporation efficiency didn’t change much during the storage period at 4℃for three month.6.After fitting,the pharmacokinetic process of DSNC in mice after administration of DSNC-lip was coincidence with double compartment model with weight of 1.The main pharmacokinetic parameters of DSNC after administration of DSNC-sol were as follows:t_1/2α 0.763±0.101h;t_1/2β 12.235±5.279h;Tmax 0.708±0.459h;Cmax 3.286±1.114mg·L^-1;AUC_（0-48）17.555±2.941mg·L^-1·h;AUC_（0-∞） 18.231±3.209mg·L^-1·h;MRT_（0-24） 11.631±1.282h;MRT_（0-∞） 12.395±1.4331h; Vl/F 0.169±0.138L·kg^-1;CL/F 0.057±0.012L·h^-1·kg^-1.The AUC of DSNC in tissues was in the order as follows:uterus＞stomach＞small intestine＞kidney＞liver＞lung＞heart＞spleen＞testis＞brain.The MRT of DSNC in tissues were spleen＞liver＞small intestine＞testis＞heart＞brain＞lung＞uterus＞kidney＞stomach. The elimination half life(t_1/2β) and the mean residence time（MRT） in plasma of DSNC-lip increased from 6.354h to 12.235h and 8.285h to 12.395h,respectly, which indicated that the time of drug in plasma was prolonged distinguishly.The apparent volume of distribution（Vl/F） increased from 0.089L·kg^-1 to 0.169L·kg^-1, which implied the liposolubility was enhanced than DSNC-sol.The plasma clearance rate（CL/F） decreased from 0.088L·h^-1·kg^-1 to 0.057L·h^-1·kg^-1,which indicated that the elimination of DSNC-lip was more slow than DSNC-sol.The AUC_0-∞ of DSNC-sol and DSNC-lip were 11.894mg·L^-1·h and 18.231mg·L^-1·h, respectly,the bioavailability（F） was 153.3%.DSNC-lip changed the distribution of DSNC in mice obviously.Except testis, the relative uptake efficency（r_e） of DSNC-lip in other tissues were＞1,which indicated that the liposolubility of DSNC-lip was enhanced than DSNC-sol.The targeting efficency（T_e） of DSNC-lip in brain,heart,lung,uterus and stomach were increased from 0.42%,2.58%,2.88%,18.38%and 18.40%to 1.13%,5.58%,6.00%, 22.44%and 20.73%,respectly.The highest distribution was in uterus.The T_e in liver and kidney were no change.The T_e in small intestin,testis decreased from 21.40%and 8.61%to 12.12%and 3.81%oConclusion:1.The HPLC-MS/MS method was simple,rapid and sensitive enough to meet the requirements for the quantitation of DSNC in biology samples.The comparision between HPLC-MS/MS method and isotope labelling method indicated that the HPLC-MS/MS method can describe the disposition of DSNC in vivo more exactly.2.In this study,the results of tissue distribution indicated that uterus was a targeting tissue of DSNC.It hinted us that the further researches were worth to carry out and supplied evidences for clinical applying in future.3.The results of metabolism study in vivo and in vitro demonstrated that DSNC was excrected in the form of norcantharidin,the product of epoxide hydration reaction of phaseⅠmetabolism and the glucuronate conjugation metabolite of phaseⅡmetabolism. 4.DSNC-lip was prepared successfully by reverse phase evaporation method with higher entrapment efficiency.The initial stability and the delayed releasing character were observed to be good in vitro.5.DSNC-lip can increase distributions in most tissues and can prolong the action time,which can decrease the administration dose and reduce adverse effect.更多还原