去甲斑蝥素钠体内处置过程及其脂质体制剂的研制
|
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C_(18)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(r~2)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。
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 C_(18) 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-MS~n method after disposition.In vitro,rat liver microsome was prepared with Ca~(2+) 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-MS~n 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(r~2) 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%o
Conclusion:
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.
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C_(18)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(r~2)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。
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 C_(18) 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-MS~n method after disposition.In vitro,rat liver microsome was prepared with Ca~(2+) 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-MS~n 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(r~2) 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%o
Conclusion:
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.
引文
[1]杨敏一,梁宝英,余清平,等.B超导向瘤体中心注射去甲斑蝥素治疗中晚期肝癌4l例[J].人民军医,1993,406(9):44-46.
[2]刘安,单德琴.肝复乐与去甲斑蝥素联用治疗中晚期肝癌[J].内蒙古中医药,1998,4:6.
[3]郑英兰,史春雷,贺延新.去甲斑蝥素治疗肝癌的临床研究[J].河北医学,2005,11(9):820-821.
[4]董丽华,曲雅勤,刘锋等.复方去甲斑蝥素合并放射治疗中晚期食管癌近期疗效的临床观察[J].吉林医学,2002,(5):301.
[5]孙震晓,赵天德,魏育林,等.去甲斑蝥素诱导人肝癌BEL-7402细胞凋亡的研究[J].解剖学报,1999,30(1):65-67.
[6]戴书静,王继峰,莫日根等.去甲斑蝥素增强BEL7402中IκBα表达和抑制细胞增殖的研究[J].解剖学报,2001,32(2):155-158.
[7]FAN Y Z,FU J Y,ZHAO Z M,et al.Effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells[J].World J Gastroenterol,2005,11(16):2431-2437.
[8]赵泽明,范跃祖,傅锦业,等.去甲斑蝥素对荷瘤裸鼠胆囊癌移植瘤nm23、基质金属蛋白酶-2和基质金属蛋白酶组织抑制因子-2的影响[J].中华实验外科杂志,2005,22(3):307-309.
[9]范跃祖,傅锦业,赵泽明,等.去甲斑蝥素对原发性胆囊癌GBC-SD细胞系增殖和凋亡的影响[J].上海医学,2003,26(增刊):1-4.
[10]傅锦业,范跃祖,赵泽明,等.去甲斑蝥素对人胆囊癌GBC-SD细胞系增殖相关基因蛋白PCNA、Ki-67的影响[J].同济大学学报(医学版),2003,24(5):374-376.
[11]Fan YZ,Zhang JT,Yang HC,et al.Expression of MMP2,TIMP2 protein and the ratio of MMP2/TIMP2 in gallbladder carcinoma and their significance[J].World J Gastroenterol,2002,8:1138-1143.
[12]范跃祖,傅锦业,赵泽明,等.去甲斑蝥素对人胆囊癌GBC-SD细胞系增殖及侵袭的影响[J].中华肿瘤杂志,2004,26(5):272-274.
[13]戎煜,梁福佑,陈莉,等.去甲斑蝥素对人乳腺癌细胞系的凋亡诱导作用及bcl-2基因的表达[J].癌症,2000,19(12):1077-1081.
[14]黄松音,袁广卿,姚燕丹等.去甲斑蝥素抑制乳腺癌SKBR3细胞增殖及侵袭转移的体外研究[J].中国病理生理杂志,2007,23(6):1062-1067.
[15]唐龙英,王昭梅,宓鹤鸣,等.去甲基斑蝥素治疗卵巢癌的实验研究[J].肿瘤,2000,20(2):96-99.
[16]S.H.Kok,C.Y.Hong,M.Y.P.Kuo,et al.Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes[J].Oral Oncology,2003,39:19-26.
[17]黄雪强,凌昌全,张登海,等.去甲斑蝥素诱导人T淋巴细胞白血病细胞株凋亡[J].基础医学与临床,2002,22(1):36-39.
[18]刘晓兰,陈家旭.去甲斑蝥素诱导HL60细胞凋亡的研究[J].北京中医药大学学报,2000,23(4):35-38.
[19]孙震晓,魏育林,赵天德,等.斑蝥素及去甲斑蝥素诱导人红白血病K562细胞凋亡的细胞学研究[J].解剖学报,2000,31(1):56-60.
[20]Dorn DC,Kou CA,Png KJ,Moore MA.The effect of cantharidins on leukemic stem cells[J].Int J Cancer.2009,124(9):2186-2199.
[21]安巍巍,龚显峰,王敏伟,等.去甲斑蝥素诱导人黑色素瘤A375-S2细胞凋亡[J].中国医院药学杂志,2005,25(6):501-504.
[22]安巍巍,薛莲,王敏伟,等.去甲斑蝥素诱导小鼠肺纤维瘤L929细胞凋亡[J].中国癌症杂志,2005,15(1):22-25.
[23]谭知显,李聪贤,王广生.去甲斑蝥素治疗乙型肝炎表面抗原携带者的疗效观察[J].药学通报,1998,23(1):46.
[24]孙伟生,赵慧荣.去甲斑蝥素片治疗银屑病24例疗效观察[J].临床皮肤科杂志,1990,(4):209.
[25]徐跃林,康玉薇,矫雪萍.去甲斑蝥素凝胶治疗尖锐湿疣58例报告[J].青岛医药卫生,1997,29(8):44-45.
[26]王广生,董成,张贺仲.去甲斑蝥素升高白细胞作用的研究[J].药学通报,1987,22(9):517-519.
[27]周爱如,邓戈,王广生.去甲斑蝥素与去甲去氢斑蝥素对小鼠骨髓细胞DNA 合成的影响[J].药学通报,1987,22(7):427.
[28]Chen Y J,Tsai YM,Kuo CD,et al.Norcantharidin is a small-molecule synthetic compound with anti-angiogenesis effect[J].Life Sci.2009,85(17-18):642-651.
[29]钟大放.药物代谢.北京:中国医药科技出版社,1996:1-14
[30]宋振玉.药物代谢研究意义、方法、应用.北京:北京人民卫生出版社,1990:4-8.
[31]钟大放.对我国药物代谢及药物动力学基础研究发展战略的思考.见吴镭主编.药学科学前沿与发展方向.北京:中国医药科技出版社,1994:54-59
[32]Lee MS,Kerms EH.LC/MS application in drug development.Mass Spectrum Rev.1999,18:187-205.
[33]胡蓓,江骥.串联质谱在药品及其代谢产物分析中的应用.张均田主编.现代药理实验方法.北京:北京医科大学中国协和医科大学联合出版社,1998:1637-1642.
[34]Oliveira EJ,Watson DG.Liquid chromatography-mass spectrometry in the study of the metabolism of drugs and other xenobiotics[J].Biomed Chromatogr,2000,14:351-372.
[35]李惠义,罗淑荣,周同惠.液相色谱-质谱联用技术及其在药物代谢研究中的应用[J].国外医学药学分册,1997,24:257-263.
[36]Ermer J,Vogel M.Application of hyphenated LC-MS techniques in pharmaceutical analysis[J].Biomed Chromatogr.,2000,14:373-383.
[37]Wu Y.The use of liquid chromatography-mass spectrometry for the identification of drug degradation products in pharmaceutical formulations[J].Biomed Chromatogr.,2000,14:384-396.
[38]Jemal M.High-throughput quantitative bioanalysis by LC/MS/MS[J].Biomed Chromatogr.,2000,14:422-429.
[39]Wang T.A new ultra-high throughput method for characterizating combinatorial libraries incorporating a multiple probe autosampler coupled with flow injection mass spectrometry analysis[J].Rapid Commun Mass Spectrom,1998,12:1123-1129.
[40]陈笑艳,钟大放.液相色谱-串联质谱在药物动力学研究中的应用.第七届全国药物与化学异物代谢会议论文集(南京),2003:12-13.
[41]钟大放,陈笑艳.采用液相色谱-离子阱质谱联用法鉴定药物代谢产物.第七届全国药物与化学异物代谢会议论文集(南京),2003:14.
[42]Tong XC,Ita IE,Wang JY,Pivnichny JV.Characterization of a technique for rapid pharmacokinetic studies of mutiple co-eluting compounds by LC/MS/MS[J].J Pharm Biomed Anal.,1999,20:773-784.
[43]Olah TV,McLoughlin DA,Gilbert JD.The simultaneous determination of mixtures of drug candidates by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry as an in vivo drug screening procedure[J].Rapid Commun Mass Spectrom,1997,11:17-23.
[44]Berman J,Halm K,Adkison K,Shaffer J.Simultaneous pharmacokinetic screening of a mixture of compounds in the dog using API LC/MS/MS analysis for increased throughput[J].J Med Chem.,1997,40:827-829.
[45]De Anel J,Vickery WE,Diamond FX.Simultaneous analysis of 14non-steroidal anti-inflammatory drugs in human serum by electrospray ionization-tandem mass spectrometry with chromatography[J].J Am Soc Mass Spectrom,1998,9:255-257.
[46]梁福佑,张胜华,潘颖,等.去甲斑蝥素用于单克隆抗体为载体的肿瘤导向治疗实验研究[J].首都医科大学学报,1999,20(1):5-8.
[47]南昌弘益科技有限公司.去甲斑蝥素滴丸及其制备方法:中国,03159066.7[P].2004-05-05.
[48]沈阳药科大学.去甲斑蝥素软胶囊及其制备方法:中国,200410021279.8[P].2005-01-12
[49]凌昌全,陈坚,陈哲,等.去甲斑蝥素.泊洛沙姆407缓释剂瘤内注射治疗肝癌的临床研究[J].第二军医大学学报,2000,21(11):1074-1076.
[50]袁敏,田建明,苏国强,等.去甲斑蝥素明胶微球肝节段性栓塞的犬实验研究[J].中国中西医结合影像学杂志,2003,1(1):13-16.
[51]徐连敏,陈改清.正交设计优化去甲斑蝥素微囊的制备工艺[J].中国医院药学杂志,2005,25(3):234-236.
[52]匡长春,何文,罗顺德,等.去甲斑蝥素壳聚糖纳米粒在小鼠体内分布及肝靶向作用[J].中国医院药学杂志,2005,25(6):527-530.
[53]张莉,向东,洪诤,等.去甲斑蝥素肝靶向微乳的研究[J].药学学报,2004,39(8):650-655.
[54]王登之.治疗恶性肿瘤的去甲斑蝥素脂质体及其制备方法:中国,02156853.7[P].2003-05-21.
[55]陆彬主编.药物新剂型与新技术.第3版.北京:人民卫生出版社,1998:108.
[56]王洪武主编.肿瘤药物靶向治疗技术[A].现代肿瘤靶向治疗技术[M].第1版.北京:中国医药科技出版社,2005:367-390.
[57]程宇慧,廖工铁,侯世祥,等.去甲斑蝥素钠白蛋白微球的研究[J].药学学报,1993,28(5):384-388.
[58]斑蝥素类似物及衍生物的合成.青岛海洋大学硕士论文.
[59]Li Qimin and Dong Shannian.Gas chromatographic analysis of norcantharidin and related compounds using derivatization to imides[J].Journal of Pharmaceutical & Biomedical Analysis,1989,7(12):1635-1639.
[60]张莉,向东,洪诤等.去甲斑蝥素肝靶向微乳的研究[J].药学学报,2004,39(8):650-655.
[61]匡长春,何文,罗顺德,等.去甲斑蝥素壳聚糖纳米粒在小鼠体内分布及肝靶向作用[J].中国医院药学杂志,2005,25(6):527-530.
[62]邵瑞莹,唐星.高效液相-质谱法联用测定大鼠体内去甲基斑蝥素血药浓度[J].药物分析杂志,2005,25(6):607-609.
[63]徐幸民,魏科达,刘建宇,赵绪元.HPLC-MS法测定大鼠血浆中去甲斑蝥素的浓度[J].中国医药导报,2009,6(12):50-52.
[64]Chunmin Wei,Rui Zhang,Benjie Wang,Guiyan Yuan,Ruichen Guo.Determination and pharmacokinetic study of norcantharidin in human serum by HPLC-MS/MS method[J].Biomed.Chromatogr.,2008,22:44-49.
[65]董成.~3H-去甲基斑蝥素在小鼠体内分布的研究[J].江西医学院学报,1984,(1):24-25.
[66]魏春敏,王本杰,马娅等.~3H-去甲斑蝥素小鼠体内药代动力学与组织分布[J].药学学报,2007,42(5):516-519.
[67]王广生,李荣昌,窦培延.去甲基斑蝥素在胃肠液中的存在状态[J].药学通报,1984,19(8):24.
[68]化学药物临床药代动力学技术指导原则.2005年3月.
[69]B.K.Matuszewski,M.L.Constanzer and C.M.Chavez-Eng.Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS[5].Anal.Chem.,2003,75:3019-3030.
[70]钟大放.药物代谢.北京:中国医药科技出版社,1996:1-14
[71]Portenoy PK,Thaler HT,Inturrisi CE,Foley KM.The metabolite morphine-6-glucuronide contributes to the analgesia produced by morphine infusion in patients with pain normal renal function[J].Clin Pharmacol Ther.,1992,51:422-431.
[72]Kroemer HK,Klotz U.Glucuronidation of drugs:a re-evaluation of the pharmacological significance of the conjugates and modulating factors[J].Clin Pharmacokinet.,1992,23(4):292-231.
[73]曾苏主编.药物代谢学.杭州:浙江大学出版社,2004:
[74]丁黎,张正行,倪沛洲,等.LC/DAD/MSD技术研究大鼠胆汁中盐酸非洛普的Ⅱ相代谢产物[J].药学学报,2001,36(6):440-444.
[75]车庆明,黄新立,李艳梅,等.黄芩苷的药物代谢产物研究[J].中国中药杂志,2001,26(11):768-769.
[76]阳长明,侯世祥,张志荣.中药代谢化学研究新进展[J].中草药,2000,31(9):641-643.
[77]姚庆强,王琰,杨树民.大鼠肝微粒体温孵体系中(+)-,(-)-黄皮酰胺及其代谢产物的LC-MS分析[J].药物分析杂志,2000,20(1)3-7.
[78]诸敏,姚彤伟,曾苏.银杏黄酮的体外葡醛酸反应及其药物相互作用[J].浙江大学学报(医学版),2004,33(1):15-20.
[79]国家食品药品监督管理局标准:注射用去甲斑蝥素钠.
[80]陆彬主编.药物新剂型与新技术[M].第2版.北京:人民卫生出版社,1998:123.
[81]高晓黎,季兴梅.葡聚糖凝胶色谱法测定脂质体包封率得条件筛选[J].中国药学杂志,2003,38(7):515-517.
[82]张俊红,朱家壁,王虹,等.阿米卡星脂质体包封率的测定[J].中国药学杂志,1999,34(12):818-820.
[83]仵文英,薛红安,席枝侠,等.黄芩苷脂质体的制备及在小鼠体内的分布[J].中国生化药物杂志,2004,25(5):282-283.
[84]赵蓉,朱家壁.全反式维甲酸立体稳定脂质体得制备[J].药学学报,2000,35(12):921-923.
[85]严文伟,肖超菊,齐宪荣,王文浩.水溶性药物8-氯腺苷多囊脂质体的制备[J].北京大学学报,2002,34(4):399-400.
[86]李琴,平其能,SE秋娟,郭建新.前列地尔脂质体的制备及刺激性考察[J].中国新药与临床杂志,2004,23(7):423-426.
[87]杨莉,齐宪荣,石靖,等.8-氯腺苷长循环脂质体的制备及其在大鼠体内的药代动力学[J].药学学报,2005,40(4):382-384.
[88]蔡钦生,黄海,周佩.阿昔洛韦脂质体的制备及其体外抗乙肝病毒的研究[J].中国药学杂志,2005,40(18):1396-1398.
[89]Szoka FJR,Vapahadjopoulos D.Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation[J].Proc Natl Acad Sci USA.1978,75(9):4194-4198.
[90]陆彬主编.药物新剂型与新技术[M].第2版.北京:人民卫生出版社,2002:116-117,121.
[91]Yoshiyuki Hagiwara,Hidetoshi Arima,et al.Preparation and Pharmaceutical Evaluation of Liposmes Entrapping Salicylic Acid/γ-Cyclodextrin Conjugate[J].Chem.Pharm.Bull.,2006,54(1):26-32.
[92]Ryo Yamauchi,Hiromi Mizuno,and Koji Kato.Preparation and Characterization of 8a-(Phaosphatidylcholine-dioxy)-α-tocopherones and their Formation during the Peroxidation of Phosphatidylcholine in Liposomes[J].Biosci.Biotechnol.Biochem.,1998,62(7):1293-1300.
[93]Townsend RW,Zutshi A,Bekersky I.Biodistribution of 4-[~(14)C]Cholesterol-AmBisome following a Single Intravenous Administration to Rats[J].Drug Metab Dispos,2001,29(5):681-685.
[94]Krishna R,Webb MS,Onge GS,et al.Mayer Liposomal and Nonliposomal Drug Pharmacokinetics after Administration of Liposome-Encapsulated Vincristine and Their Contribution to Drug Tissue Distribution Properties[J].J Pharmacol Exp Ther,2001,298(3):1206-1212.
[95]Jin F,Krzyzanski W.Pharmacokinetic Model of target- mediated disposition of thrombopoietin[J].AAPS Pharm Sci,2004,6(1):1-8.
[96]杨凯,温玉明,王昌美,等.葫芦素BE聚乳酸纳米微粒对口腔癌颈淋巴结转移灶的靶向性评价[J].华西口腔医学杂志.2003,21(6):477-480.
[97]王韶宁,邓意辉,吴红兵,等.半乳糖化脂质体在小鼠体内肝靶向性评价[J].中国药学杂志,2006,41(14):1076-1079.
[2]刘安,单德琴.肝复乐与去甲斑蝥素联用治疗中晚期肝癌[J].内蒙古中医药,1998,4:6.
[3]郑英兰,史春雷,贺延新.去甲斑蝥素治疗肝癌的临床研究[J].河北医学,2005,11(9):820-821.
[4]董丽华,曲雅勤,刘锋等.复方去甲斑蝥素合并放射治疗中晚期食管癌近期疗效的临床观察[J].吉林医学,2002,(5):301.
[5]孙震晓,赵天德,魏育林,等.去甲斑蝥素诱导人肝癌BEL-7402细胞凋亡的研究[J].解剖学报,1999,30(1):65-67.
[6]戴书静,王继峰,莫日根等.去甲斑蝥素增强BEL7402中IκBα表达和抑制细胞增殖的研究[J].解剖学报,2001,32(2):155-158.
[7]FAN Y Z,FU J Y,ZHAO Z M,et al.Effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells[J].World J Gastroenterol,2005,11(16):2431-2437.
[8]赵泽明,范跃祖,傅锦业,等.去甲斑蝥素对荷瘤裸鼠胆囊癌移植瘤nm23、基质金属蛋白酶-2和基质金属蛋白酶组织抑制因子-2的影响[J].中华实验外科杂志,2005,22(3):307-309.
[9]范跃祖,傅锦业,赵泽明,等.去甲斑蝥素对原发性胆囊癌GBC-SD细胞系增殖和凋亡的影响[J].上海医学,2003,26(增刊):1-4.
[10]傅锦业,范跃祖,赵泽明,等.去甲斑蝥素对人胆囊癌GBC-SD细胞系增殖相关基因蛋白PCNA、Ki-67的影响[J].同济大学学报(医学版),2003,24(5):374-376.
[11]Fan YZ,Zhang JT,Yang HC,et al.Expression of MMP2,TIMP2 protein and the ratio of MMP2/TIMP2 in gallbladder carcinoma and their significance[J].World J Gastroenterol,2002,8:1138-1143.
[12]范跃祖,傅锦业,赵泽明,等.去甲斑蝥素对人胆囊癌GBC-SD细胞系增殖及侵袭的影响[J].中华肿瘤杂志,2004,26(5):272-274.
[13]戎煜,梁福佑,陈莉,等.去甲斑蝥素对人乳腺癌细胞系的凋亡诱导作用及bcl-2基因的表达[J].癌症,2000,19(12):1077-1081.
[14]黄松音,袁广卿,姚燕丹等.去甲斑蝥素抑制乳腺癌SKBR3细胞增殖及侵袭转移的体外研究[J].中国病理生理杂志,2007,23(6):1062-1067.
[15]唐龙英,王昭梅,宓鹤鸣,等.去甲基斑蝥素治疗卵巢癌的实验研究[J].肿瘤,2000,20(2):96-99.
[16]S.H.Kok,C.Y.Hong,M.Y.P.Kuo,et al.Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes[J].Oral Oncology,2003,39:19-26.
[17]黄雪强,凌昌全,张登海,等.去甲斑蝥素诱导人T淋巴细胞白血病细胞株凋亡[J].基础医学与临床,2002,22(1):36-39.
[18]刘晓兰,陈家旭.去甲斑蝥素诱导HL60细胞凋亡的研究[J].北京中医药大学学报,2000,23(4):35-38.
[19]孙震晓,魏育林,赵天德,等.斑蝥素及去甲斑蝥素诱导人红白血病K562细胞凋亡的细胞学研究[J].解剖学报,2000,31(1):56-60.
[20]Dorn DC,Kou CA,Png KJ,Moore MA.The effect of cantharidins on leukemic stem cells[J].Int J Cancer.2009,124(9):2186-2199.
[21]安巍巍,龚显峰,王敏伟,等.去甲斑蝥素诱导人黑色素瘤A375-S2细胞凋亡[J].中国医院药学杂志,2005,25(6):501-504.
[22]安巍巍,薛莲,王敏伟,等.去甲斑蝥素诱导小鼠肺纤维瘤L929细胞凋亡[J].中国癌症杂志,2005,15(1):22-25.
[23]谭知显,李聪贤,王广生.去甲斑蝥素治疗乙型肝炎表面抗原携带者的疗效观察[J].药学通报,1998,23(1):46.
[24]孙伟生,赵慧荣.去甲斑蝥素片治疗银屑病24例疗效观察[J].临床皮肤科杂志,1990,(4):209.
[25]徐跃林,康玉薇,矫雪萍.去甲斑蝥素凝胶治疗尖锐湿疣58例报告[J].青岛医药卫生,1997,29(8):44-45.
[26]王广生,董成,张贺仲.去甲斑蝥素升高白细胞作用的研究[J].药学通报,1987,22(9):517-519.
[27]周爱如,邓戈,王广生.去甲斑蝥素与去甲去氢斑蝥素对小鼠骨髓细胞DNA 合成的影响[J].药学通报,1987,22(7):427.
[28]Chen Y J,Tsai YM,Kuo CD,et al.Norcantharidin is a small-molecule synthetic compound with anti-angiogenesis effect[J].Life Sci.2009,85(17-18):642-651.
[29]钟大放.药物代谢.北京:中国医药科技出版社,1996:1-14
[30]宋振玉.药物代谢研究意义、方法、应用.北京:北京人民卫生出版社,1990:4-8.
[31]钟大放.对我国药物代谢及药物动力学基础研究发展战略的思考.见吴镭主编.药学科学前沿与发展方向.北京:中国医药科技出版社,1994:54-59
[32]Lee MS,Kerms EH.LC/MS application in drug development.Mass Spectrum Rev.1999,18:187-205.
[33]胡蓓,江骥.串联质谱在药品及其代谢产物分析中的应用.张均田主编.现代药理实验方法.北京:北京医科大学中国协和医科大学联合出版社,1998:1637-1642.
[34]Oliveira EJ,Watson DG.Liquid chromatography-mass spectrometry in the study of the metabolism of drugs and other xenobiotics[J].Biomed Chromatogr,2000,14:351-372.
[35]李惠义,罗淑荣,周同惠.液相色谱-质谱联用技术及其在药物代谢研究中的应用[J].国外医学药学分册,1997,24:257-263.
[36]Ermer J,Vogel M.Application of hyphenated LC-MS techniques in pharmaceutical analysis[J].Biomed Chromatogr.,2000,14:373-383.
[37]Wu Y.The use of liquid chromatography-mass spectrometry for the identification of drug degradation products in pharmaceutical formulations[J].Biomed Chromatogr.,2000,14:384-396.
[38]Jemal M.High-throughput quantitative bioanalysis by LC/MS/MS[J].Biomed Chromatogr.,2000,14:422-429.
[39]Wang T.A new ultra-high throughput method for characterizating combinatorial libraries incorporating a multiple probe autosampler coupled with flow injection mass spectrometry analysis[J].Rapid Commun Mass Spectrom,1998,12:1123-1129.
[40]陈笑艳,钟大放.液相色谱-串联质谱在药物动力学研究中的应用.第七届全国药物与化学异物代谢会议论文集(南京),2003:12-13.
[41]钟大放,陈笑艳.采用液相色谱-离子阱质谱联用法鉴定药物代谢产物.第七届全国药物与化学异物代谢会议论文集(南京),2003:14.
[42]Tong XC,Ita IE,Wang JY,Pivnichny JV.Characterization of a technique for rapid pharmacokinetic studies of mutiple co-eluting compounds by LC/MS/MS[J].J Pharm Biomed Anal.,1999,20:773-784.
[43]Olah TV,McLoughlin DA,Gilbert JD.The simultaneous determination of mixtures of drug candidates by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry as an in vivo drug screening procedure[J].Rapid Commun Mass Spectrom,1997,11:17-23.
[44]Berman J,Halm K,Adkison K,Shaffer J.Simultaneous pharmacokinetic screening of a mixture of compounds in the dog using API LC/MS/MS analysis for increased throughput[J].J Med Chem.,1997,40:827-829.
[45]De Anel J,Vickery WE,Diamond FX.Simultaneous analysis of 14non-steroidal anti-inflammatory drugs in human serum by electrospray ionization-tandem mass spectrometry with chromatography[J].J Am Soc Mass Spectrom,1998,9:255-257.
[46]梁福佑,张胜华,潘颖,等.去甲斑蝥素用于单克隆抗体为载体的肿瘤导向治疗实验研究[J].首都医科大学学报,1999,20(1):5-8.
[47]南昌弘益科技有限公司.去甲斑蝥素滴丸及其制备方法:中国,03159066.7[P].2004-05-05.
[48]沈阳药科大学.去甲斑蝥素软胶囊及其制备方法:中国,200410021279.8[P].2005-01-12
[49]凌昌全,陈坚,陈哲,等.去甲斑蝥素.泊洛沙姆407缓释剂瘤内注射治疗肝癌的临床研究[J].第二军医大学学报,2000,21(11):1074-1076.
[50]袁敏,田建明,苏国强,等.去甲斑蝥素明胶微球肝节段性栓塞的犬实验研究[J].中国中西医结合影像学杂志,2003,1(1):13-16.
[51]徐连敏,陈改清.正交设计优化去甲斑蝥素微囊的制备工艺[J].中国医院药学杂志,2005,25(3):234-236.
[52]匡长春,何文,罗顺德,等.去甲斑蝥素壳聚糖纳米粒在小鼠体内分布及肝靶向作用[J].中国医院药学杂志,2005,25(6):527-530.
[53]张莉,向东,洪诤,等.去甲斑蝥素肝靶向微乳的研究[J].药学学报,2004,39(8):650-655.
[54]王登之.治疗恶性肿瘤的去甲斑蝥素脂质体及其制备方法:中国,02156853.7[P].2003-05-21.
[55]陆彬主编.药物新剂型与新技术.第3版.北京:人民卫生出版社,1998:108.
[56]王洪武主编.肿瘤药物靶向治疗技术[A].现代肿瘤靶向治疗技术[M].第1版.北京:中国医药科技出版社,2005:367-390.
[57]程宇慧,廖工铁,侯世祥,等.去甲斑蝥素钠白蛋白微球的研究[J].药学学报,1993,28(5):384-388.
[58]斑蝥素类似物及衍生物的合成.青岛海洋大学硕士论文.
[59]Li Qimin and Dong Shannian.Gas chromatographic analysis of norcantharidin and related compounds using derivatization to imides[J].Journal of Pharmaceutical & Biomedical Analysis,1989,7(12):1635-1639.
[60]张莉,向东,洪诤等.去甲斑蝥素肝靶向微乳的研究[J].药学学报,2004,39(8):650-655.
[61]匡长春,何文,罗顺德,等.去甲斑蝥素壳聚糖纳米粒在小鼠体内分布及肝靶向作用[J].中国医院药学杂志,2005,25(6):527-530.
[62]邵瑞莹,唐星.高效液相-质谱法联用测定大鼠体内去甲基斑蝥素血药浓度[J].药物分析杂志,2005,25(6):607-609.
[63]徐幸民,魏科达,刘建宇,赵绪元.HPLC-MS法测定大鼠血浆中去甲斑蝥素的浓度[J].中国医药导报,2009,6(12):50-52.
[64]Chunmin Wei,Rui Zhang,Benjie Wang,Guiyan Yuan,Ruichen Guo.Determination and pharmacokinetic study of norcantharidin in human serum by HPLC-MS/MS method[J].Biomed.Chromatogr.,2008,22:44-49.
[65]董成.~3H-去甲基斑蝥素在小鼠体内分布的研究[J].江西医学院学报,1984,(1):24-25.
[66]魏春敏,王本杰,马娅等.~3H-去甲斑蝥素小鼠体内药代动力学与组织分布[J].药学学报,2007,42(5):516-519.
[67]王广生,李荣昌,窦培延.去甲基斑蝥素在胃肠液中的存在状态[J].药学通报,1984,19(8):24.
[68]化学药物临床药代动力学技术指导原则.2005年3月.
[69]B.K.Matuszewski,M.L.Constanzer and C.M.Chavez-Eng.Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS[5].Anal.Chem.,2003,75:3019-3030.
[70]钟大放.药物代谢.北京:中国医药科技出版社,1996:1-14
[71]Portenoy PK,Thaler HT,Inturrisi CE,Foley KM.The metabolite morphine-6-glucuronide contributes to the analgesia produced by morphine infusion in patients with pain normal renal function[J].Clin Pharmacol Ther.,1992,51:422-431.
[72]Kroemer HK,Klotz U.Glucuronidation of drugs:a re-evaluation of the pharmacological significance of the conjugates and modulating factors[J].Clin Pharmacokinet.,1992,23(4):292-231.
[73]曾苏主编.药物代谢学.杭州:浙江大学出版社,2004:
[74]丁黎,张正行,倪沛洲,等.LC/DAD/MSD技术研究大鼠胆汁中盐酸非洛普的Ⅱ相代谢产物[J].药学学报,2001,36(6):440-444.
[75]车庆明,黄新立,李艳梅,等.黄芩苷的药物代谢产物研究[J].中国中药杂志,2001,26(11):768-769.
[76]阳长明,侯世祥,张志荣.中药代谢化学研究新进展[J].中草药,2000,31(9):641-643.
[77]姚庆强,王琰,杨树民.大鼠肝微粒体温孵体系中(+)-,(-)-黄皮酰胺及其代谢产物的LC-MS分析[J].药物分析杂志,2000,20(1)3-7.
[78]诸敏,姚彤伟,曾苏.银杏黄酮的体外葡醛酸反应及其药物相互作用[J].浙江大学学报(医学版),2004,33(1):15-20.
[79]国家食品药品监督管理局标准:注射用去甲斑蝥素钠.
[80]陆彬主编.药物新剂型与新技术[M].第2版.北京:人民卫生出版社,1998:123.
[81]高晓黎,季兴梅.葡聚糖凝胶色谱法测定脂质体包封率得条件筛选[J].中国药学杂志,2003,38(7):515-517.
[82]张俊红,朱家壁,王虹,等.阿米卡星脂质体包封率的测定[J].中国药学杂志,1999,34(12):818-820.
[83]仵文英,薛红安,席枝侠,等.黄芩苷脂质体的制备及在小鼠体内的分布[J].中国生化药物杂志,2004,25(5):282-283.
[84]赵蓉,朱家壁.全反式维甲酸立体稳定脂质体得制备[J].药学学报,2000,35(12):921-923.
[85]严文伟,肖超菊,齐宪荣,王文浩.水溶性药物8-氯腺苷多囊脂质体的制备[J].北京大学学报,2002,34(4):399-400.
[86]李琴,平其能,SE秋娟,郭建新.前列地尔脂质体的制备及刺激性考察[J].中国新药与临床杂志,2004,23(7):423-426.
[87]杨莉,齐宪荣,石靖,等.8-氯腺苷长循环脂质体的制备及其在大鼠体内的药代动力学[J].药学学报,2005,40(4):382-384.
[88]蔡钦生,黄海,周佩.阿昔洛韦脂质体的制备及其体外抗乙肝病毒的研究[J].中国药学杂志,2005,40(18):1396-1398.
[89]Szoka FJR,Vapahadjopoulos D.Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation[J].Proc Natl Acad Sci USA.1978,75(9):4194-4198.
[90]陆彬主编.药物新剂型与新技术[M].第2版.北京:人民卫生出版社,2002:116-117,121.
[91]Yoshiyuki Hagiwara,Hidetoshi Arima,et al.Preparation and Pharmaceutical Evaluation of Liposmes Entrapping Salicylic Acid/γ-Cyclodextrin Conjugate[J].Chem.Pharm.Bull.,2006,54(1):26-32.
[92]Ryo Yamauchi,Hiromi Mizuno,and Koji Kato.Preparation and Characterization of 8a-(Phaosphatidylcholine-dioxy)-α-tocopherones and their Formation during the Peroxidation of Phosphatidylcholine in Liposomes[J].Biosci.Biotechnol.Biochem.,1998,62(7):1293-1300.
[93]Townsend RW,Zutshi A,Bekersky I.Biodistribution of 4-[~(14)C]Cholesterol-AmBisome following a Single Intravenous Administration to Rats[J].Drug Metab Dispos,2001,29(5):681-685.
[94]Krishna R,Webb MS,Onge GS,et al.Mayer Liposomal and Nonliposomal Drug Pharmacokinetics after Administration of Liposome-Encapsulated Vincristine and Their Contribution to Drug Tissue Distribution Properties[J].J Pharmacol Exp Ther,2001,298(3):1206-1212.
[95]Jin F,Krzyzanski W.Pharmacokinetic Model of target- mediated disposition of thrombopoietin[J].AAPS Pharm Sci,2004,6(1):1-8.
[96]杨凯,温玉明,王昌美,等.葫芦素BE聚乳酸纳米微粒对口腔癌颈淋巴结转移灶的靶向性评价[J].华西口腔医学杂志.2003,21(6):477-480.
[97]王韶宁,邓意辉,吴红兵,等.半乳糖化脂质体在小鼠体内肝靶向性评价[J].中国药学杂志,2006,41(14):1076-1079.