【作者】 张庆颢；

【导师】 刘克辛；

【作者基本信息】 大连医科大学 ， 药理学， 2008， 硕士

【摘要】 目的:头孢妥仑( cefditoren, CDTR)是前体药物头孢妥仑匹酯(CDTR-PI)经口服吸收在肠壁酯酶的作用下水解生成,从而发挥抗菌作用。本实验主要研究大鼠体内CDTR的主要排泄途径以及体内体外实验研究其跨膜转运吸收及其机制,从而考察CDTR是否为PEPTs的底物。方法:采用高效液相色谱法(HPLC)测定血浆、尿液、胆汁、Krebs-Ringer缓冲液(KRB)、细胞裂解液样品中CDTR的药物浓度。流动相为1‰醋酸胺:甲醇(67:33),内标为4-二甲氨基安替比林,流速为1mL/min,UV检测波长295nm。体内实验中,麻醉大鼠分别行胆管插管和膀胱插管手术,颈静脉给药,分别于各时间点收集胆汁和尿液,经处理后HPLC测定CDTR药物浓度,计算胆汁中和尿液中经时累积药物排泄量。大鼠静脉同时给予PEPTs抑制剂甘氨酰肌氨酸(glycylsarcosine,Gly-Sar)测定其对大鼠体内CDTR排泄的影响。应用大鼠空肠灌流模型,考察Gly-Sar以及　2受体激动剂可乐定(clonidine)在该模型中对CDTR经肠道转运吸收入血的影响。制备大鼠翻转肠样品,测定离体肠管对药物浓度依赖性摄取以及Gly-Sar对离体肠管摄取CDTR的影响。将于24孔板培养15天的Caco-2细胞用于药物摄取实验,分别考察pH值、时间、温度、浓度以及Gly-Sar对CDTR在该模型中摄取的影响。结果:HPLC方法学验证表明血浆、胆汁、尿液、KRB缓冲液、细胞裂解液样品中干扰峰对CDTR峰无干扰,CDTR保留时间为21.2min,内标保留时间为22.0min,二者分离良好。稳定性实验测得CDTR在HBSS和KRB缓冲液中37℃放置至少60min内稳定。大鼠静脉给予0.5mg/kg的CDTR,6小时内大约34%给药量的CDTR经胆汁排泄,约4%给药量的CDTR经尿液排泄。当静脉同时给予CDTR和Gly-Sar,与单独给予CDTR相比,胆汁中CDTR累积排泄量无显著性差异,但是尿液中药物累积排泄量则明显增加从3.64%增加到10.28% ,肾脏清除率从1.34mL/min增加到4.16mL/min。在大鼠空肠灌流实验中,50mM Gly-Sar竞争性的抑制了0.1 mM CDTR经小肠的转运吸收。灌流15min,30 min,60 min后,加入Gly-Sar组的门脉血浆药物浓度(0.19μg/mL,0.28μg/mL,0.46μg/mL)与对照组(0.52μg/mL,0.98μg/mL,1.56μg/mL)相比显著性降低。当于灌流前15min静脉推注400μg/kg的可乐定,可以显著的增加1mM CDTR经空肠灌流模型转运入血的量,从而提高门脉的血浆药物浓度,灌流60min的门脉血药浓度与对照组相比,从1.03μg/mL增加到1.50μg/mL。当于灌流液中同时加入50mM Gly-Sar后,可抵消可乐定诱导的CDTR在空肠灌流模型中转运吸收增加的作用。在外翻肠囊模型实验中,CDTR的浓度依赖性摄取存在饱和现象,当CDTR的浓度达到5mM时浆膜侧药物浓度达到饱和(28.93nmoL/mL)。与空肠灌流实验相似,200mM Gly-Sar显著抑制了CDTR在翻转肠模型中的摄取。在Caco-2细胞模型摄取实验中,Caco-2细胞对CDTR的摄取在60min内随摄取时间推移近似线性增加,60min内未见摄取饱和现象,Gly-Sar显著的抑制各时间点Caco-2细胞对CDTR的摄取。浓度依赖性特异性摄取部分是一条饱和曲线,计算Km=1.095mM, Vmax=1.115nmol/mg protein/30min。4°C条件下CDTR的摄取与37°C条件下的摄取相比显著降低。另外pH值也显著的影响Caco-2细胞对CDTR的摄取,在pH=6.0摄取量最大,在pH=8.5时摄取量最小。结论:CDTR主要经过胆汁排泄,少量经肾脏排泄,静脉给药6小时内约40%给药剂量的药物经胆汁及肾脏排泄。体内实验以及大鼠空肠灌流模型,外翻肠囊模型,Caco-2细胞模型实验中,PEPTs的特异性抑制剂Gly-Sar以及PEPTs的调节剂可乐定显著的影响CDTR经肠道的转运吸收以及肾脏的排泄,因此CDTR是H+协同肽转运蛋白PEPTs的底物之一。更多还原

【Abstract】 Objective: H+/oligopeptide cotransporter PEPT1, mainly located at the brush border membrane of intestinal epithelium cell, transport dipeptide/ tripeptide which is the degradation products of protein in digestive tract. Peptide-like drugs such asβ-lactam antibiotics,angiotensin-converting enzyme inhibitor (ACEI) and non-peptide drugs valaciclovir also can be transported via PEPT1. PEPT1 is important for maintaining the homeostasis and the absorption of drugs in gastrointestinal tract. In this study, the effects of glycylsarcosine and clonidine on uptake and transepithelial transport of cefditoren were investigated in vivo and in vitro, to clarify whether cefditoren is a potential substract of PEPT1/ PEPT2.Methods: Rats were administered cefditoren(10mg/kg) by intravenous administration, in the absence and presence of glycylsarcosine. Serial urine and bile samples were obtained in the period of 60 minuts. The effects of Gly-Sar and clonidine on transport activities was measured by everted small intestinal preparations or in situ intestinal loop technique. Uptake of cefditoren in Caco-2 cells was examined with or without Gly-Sar. Effects of pH and temperature on CDTR transport was also examined in Caco-2 cells. The concentration of cefditoren was determined by HPLC.Results: In vivo Gly-Sar coadministration increased the renal clearance of cefditoren by 200% as compared to cefditoren alone. The intestinal cefditoren absorption rate was decreased when Gly-Sar was dissolved in the perfusate at a concentration of 50mM. Before intrajejunal perfusion , intravenous infusing clonidine(400μg/kg) induced a 50% increase of cefditoren absorption across the intestinal mucosa. Saturable intestinal uptake of cefditoren was found using the everted jejunum of rats. The competitively inhibitive effect of Gly-Sar(200mM) on transport of cefditoren was consistant with that of in situ intrajujunal perfusion. Uptake of cefditoren was decreased by Gly-Sar in caco-2 cells as compared to cefditoren alone. pH and temperature significantly influence cefditoren uptake in Caco-2 cells, Km=1.095mM、Vmax=1.115nmol/mg protein/30min. The maximal uptake of cefditoren was found at pH= 6.0.Conclusion: This study provides the first evidence, under in vivo and in vitro conditions, that Gly-Sar and clonidine affect the transepithelial transport of cefditoren. The results demonstrate that cefditoren is the potential substract of the H+/oligopeptide cotransporter PEPTs.更多还原