【作者】 吕志华；

【导师】 刘昌孝；

【作者基本信息】 中国海洋大学 ， 水产品加工与贮藏工程， 2008， 博士

【摘要】 海洋硫酸多糖PS916是以来源于海洋的甲壳质为基础原料,经多步分子修饰而得到的一种硫酸氨基多糖,化学名为β-(1,4)-3-氧-硫酸基-6-氧-硫酸基-6′-氧-羧甲基醚钠盐,英文名为β-(1,4) polyglycosamine-3-O-sulfate-6-O-sulfate-6′-O- carboxylmethyl ether sodium,药效学研究结果表明PS916具有调血脂、抗脂质过氧化、抑制血管平滑肌细胞增殖等作用,具有多途径抗动脉粥样硬化作用,系正在进行系统研究的I类新药。本文对PS916在大鼠体内的药代动力学、组织分布及排泄进行了研究,初步阐明了PS916在大鼠体内的吸收,分布及排泄过程,为新药开发和临床用药提供了一定的理论依据。本论文主要研究工作如下:一、PS916的荧光标记研究PS916为多糖类物质,在分子结构中缺乏发色团和荧光团,不能直接用紫外或荧光法进行检测,质谱法也不适合多糖的定量分析。因此,我们以异硫氰酸荧光素(FITC)为荧光标记试剂,对PS916进行了荧光标记,标记后的PS916的荧光最大激发波长Ex为480nm,最大发射波长Em为515nm;荧光标记的PS916中含FITC 0.74%;以高效凝胶色谱法测定PS916分子量及分子量分布,测定结果表明,标记后的PS916平均分子量及分子量分布宽度与标记前比较均未发生显著变化。二、PS916的体内外稳定性研究以荧光标记的PS916为研究对象,采用高效凝胶色谱法考察PS916在磷酸盐缓冲液(PBS)及大鼠血浆中的稳定性。PS916体外在PBS、大鼠血浆及大鼠尿液中37℃孵育1h、20h,其分子量及分子量分布宽度均未发生变化,荧光强度也未发生变化,表明PS916体外在PBS、大鼠血浆及大鼠尿液中稳定。同时,对大鼠以口服和静脉注射两种给药方式给药后PS916在血浆、尿液和粪便中的稳定性进行了研究,体内大鼠口服PS916后血浆和粪便中PS916均以原型形式存在;PS916静脉注射给药后尿液中以原型形式从尿液中排泄,说明大鼠口服和静脉注射PS916后,主要以原型的形式吸收代谢。三、PS916在生物样品中的定量分析方法研究建立了生物样品中PS916的荧光定量分析方法,PS916的血浆浓度在0.2-20μg/mL的浓度范围内PS916的荧光强度与浓度具有良好的线性关系,线性方程为:y =22.951x– 3.3561,相关系数r2=0.9991,血浆中PS916最低定量限为0.2μg/mL;低、中、高(0.2、2.0、20.0μg/mL) 3种浓度的日内精密度在1.52–7.83%之间,日间精密度在2.14–8.42%之间,组内回收率在84.3-108.0%之间,组间回收率在88.7-100.1%之间;血浆样品稳定性考察显示PS916在-70℃条件下放置4周相对稳定。PS916在组织、尿液及粪便中的线性、精密度、准确度及稳定性分析均符合生物样品体内定量分析的要求。证明本实验方法适用于PS916在大鼠血浆、组织等生物样品中含量的测定。四、PS916在大鼠体内的药代动力学研究1、大鼠口服和静脉给药PS916后,血药浓度-时间曲线数据采用非房室模型计算药代动力学参数,通过Cmax和AUC与给药剂量的相关性分析结果表明,大鼠口服和静脉给药后,PS916在大鼠体内呈线性动力学特征。2、大鼠口服PS916后,三种剂量的平均Tmax为3.28±0.58h,可知PS916在大鼠体内吸收较慢,达峰迟缓。大鼠口服三种剂量下平均消除半衰期t1/2为7.84±0.12h,大鼠静脉注射三种剂量下平均消除半衰期t1/2为8.74±1.01h,口服和静脉给药后的t1/2无统计学差异(P>0.05),PS916在大鼠体内的半衰期较长,3、大鼠口服和静脉给药后,用AUC0-∞计算PS916绝对生物利用度为9.06%,显示大鼠口服PS916吸收较差,生物利用度较低。4、在大鼠连续给药实验中,口服50mg/kg剂量PS916多次给药与单次给药相比,药-时曲线基本吻合,首、末次给药后的t1/2和AUC0-∞值均无统计学差异,提示PS916多次给药体内不易蓄积,且对其代谢酶无诱导抑制作用。5、大鼠单次口服PS916后,组织分布实验表明,给药后1h,药物能分布到大部分组织中,在肠和胃中分布最高,另外主要分布在血流充足的肝和肺组织;给药6h后,肠和胃组织中药物浓度明显下降,肾脏中药物浓度明显升高,表明PS916吸收入血后的药物主要经肾脏随尿液排出;给药24h后,大部分组织中药物浓度均明显降低。给药后脑组织中可检测到药物,说明PS916能透过血脑屏障,向脑内分布。6、大鼠单次静脉给予PS916后,组织分布实验表明,PS916静脉给药后在大鼠各主要组织中广泛分布。给药后1h,PS916在大鼠肾脏、肝脏、血浆中的浓度较高,在肾脏中浓度最高,在肠中亦有相对较高浓度,在肾脏中含量高说明该药物主要经肾排泄。PS916静脉注射6h后,肾脏中药物浓度在3个时间点中最高,在肾脏、肝脏、肠、心、肺、胃、脾等组织中的浓度均大于其血浆浓度,说明PS916的组织分布比例较大。给药后PS916在脑中亦可检出,说明PS916能透过血脑屏障,向脑内分布。7、大鼠口服给予PS916后,主要以原型从粪便和尿液中排出体外,给药72h后,尿中PS916的累积排泄量占给药剂量百分比为1.288±0.958%;粪中PS916的累积排泄量占给药剂量百分比为78.974±5.498%;灌胃给药后72h内,有相当于给药量80.26%的药物以原型的形式随尿粪排出体外,灌胃给药后PS916主要是从粪便中排出。8、大鼠静脉注射给予PS916后,主要以原型从粪便和尿液中排出体外,给药72h后,尿中PS916的累积排泄量占剂量百分比为54.388±11.832%;粪中PS916的累积排泄量占剂量百分比为6.286±1.938%;静脉注射给药后72h内,有相当于给药量60.67%的药物以原型的形式随尿粪排出体外,静脉给药后PS916主要是从尿液中排出体外。更多还原

【Abstract】 PS916 is a new form of sulfated amino polysaccharide that is derived from marine chitin and by ways of molecular modifications. It is characterized by 1, 4-linkedβ-D-glucosamine with an average of 1.0 sulfate and 0.5 carboxylmethyl groups per unit monosaccharide. The average molecular weight of PS916 is 7000 Da and the distribution width of molecular weight is less than 1.5. Systematic pharmacodynamic research shows that PS916 has good antiatherosclerotic activity and was authorized to enter clinical trial in China. This paper was designed to evaluate the pharmacokinetics features of PS916 in rats to provid scientific data for the new drug development and clinic use.1. The flurocescent labeling of PS916PS916, similar to other polysaccharides such as glucosan, possesses few chromophoric or fluorophoric structurally, so derect detection is impossible with violet-visible (UV-Vis) or flurocescence method, Mass spectra method is unsuitable to quantification analysis of polysaccharides either. Fluorescein-isothiocyanate (FITC) is a fluroscent labeling regent usually used to label protein, so we chose it to label PS916 because just like protein there is amino group in its structure. UV-Vis and flurocescence spectroscopy demonstrated the labeling of FITC to PS916. The maxium excition wavelength of labeled PS916 is 480nm, and the maxium emission wavelength is 515nm. Furthermore, labeled PS916 was determined spectrophotometrically by visible absorption at 490nm with reference to FITC, the content of FITC in labeled PS916 was 0.71% (w/w). The molecular weight of labeled PS916 was examined by high performance gel permeation chromatography (HPGPC). The average molecular weight and the distribution width of molecular weight of labeled PS916 were not signifficent different from that of unlabeled PS916.2. The stability of PS916 in vitro and in vivoThe stability of PS916 in vitro and in vivo was investigated using HPGPC method with fluorescesent labeled PS916. After 1, 20h incubation in phosphate buffer, rat plasma and rat urine at 37℃, the molecular weight and the width of molecular weight distribution were not changed, that showed PS916 was stable in vitro. After oral administration of PS916 to rats, PS916 was in its original form in rat plasma and rat feces, meanwhile after an intravenous administration of PS916 to rats, PS916 was also excreted in its original form in rat urine, that demonstrated PS916 metabolized in its original form.3. The establishment of analytical method of PS916 in biological matrixA rapid, sensitive fluroescence method was developed to determine PS916 in biological matrix. The linear calibration curve was obtained in the range of 0.2-20μg/mL in rat plasma. The lower limit of quantitation (LLOQ) was 0.2μg/mL. The inter-day CV% at 0.2, 2.0 and 20μg/mL of PS916 was 6.35%, 3.81% and 1.68%, respecttively. The intra-day CV% at the above concentrations was 8.42%, 4.38% and 2.14%, respectively. One-way analysis of variance (ANOVA) was carried out with grouping variable“day”to assess precision at 95% level. The result was non-significant when data for each day was compared with other days and within the same day. The calibration curves were also linear in other biological sample such as tiusse, urine and feces, range with correlation coefficient all above 0.99. The established method is suitable for PS916 pharmacokinetics studies.4. The pharmacokinetics study of PS916Individual plasma-concentration data were analyzed by non-compartmental in this paper.After intravenous and oral administration of PS916 to rats, the proportionality between dosage and Cmax or AUC was calculated,A good correlation was found between the dosage and AUC and Cmax, PS916 exhibited dose-proportional pharmacokinetics with linear kinetic character.Following oral administration, the average Tmax of three dosages was 3.28±0.58 h, that means the absorption of PS916 was slowly. After intravenous and oral administration, the average t1/2 were 7.84±0.12h and 8.74±1.01h,respectively, PS916 had a long terminal half-life.The absolute oral bioavailability of PS916 was 9.06%,which was calculated by compared the AUC0-∞of oral administration to the AUC0-∞of intravenous administration at the dose of 25mg/kg. The bioavailability of PS916 was low.After multiple dosage of PS916 at 50mg/kg, the plasma concentration–time profile was basically the same as that of signal dosage, t1/2 and AUC0-∞were non-significant between the first and the last dosage, that mean PS916 was not accumulated in rats. PS916 was widely distributed to tissues of most organs in rats 1h after oral administration of PS916, such as stomach, intestine, liver, kidneys, lung and heart. The concentration in stomach and intestine was the most highest in all tissues, the concentration in lung and liver was higher than other tissues. The concentration in stomach and intestine degraded rapidly, and increased rapidly in kidney 6h after oral administration of PS916, that show PS916 was excreted mainly by kidney after absorption. For all tissues, PS916 concentrations had significantly decreased at the time point of 24 h after oral administration. PS916 was also be detected in brain, that mean PS916 could also across blood-brain barrier.PS916 was widely distributed to tissues of most organs in rats 1h after intravenous administration of PS916, such as liver, kidneys, lung, heart and intestine. The concentration in kidney, liver and plasma was higher, the most highest in kidney, that show PS916 was excreted mainly by kidney. The concentration in most tissues such as liver, kidney, lung, heart, spleen and intestine was higher than that of plasma, that mean PS916 was widely distributed to tissues. PS916 was also be detected in brain, that mean PS916 could also across blood-brain barrier. PS916 was mainly excreted in its original form from feces and urine after oral administration. Cumulative amounts percentage of PS916 in feces at 72 h after an oral dose was 78.974±5.498%. Cumulative amounts percentage of PS916 in urine at 72 h after an oral dose was 1.288±0.958%. Drug which was excreted totally in urine and feces in 72 h after an oral administration of PS916 was equal to 80.26% of given dose. PS916 was mainly excreted from feces after oral administration.PS916 was mainly excreted in its original form from feces and urine after oral administration. Cumulative amounts percentage of PS916 in urine at 72 h after an oral dose was 54.388±11.832%. Cumulative amounts percentage of PS916 in feces at 72 h after an oral dose was 6.286±1.938%%. Drug which was excreted totally in urine and feces in 72 h after an oral administration of PS916 was equal to 60.67% of given dose. PS916 was mainly excreted from urine after oral administration.更多还原