摘要
【目的】研究莱柯霉素在Caco-2细胞单层模型中的转运特性,为进一步深入了解莱柯霉素在体内的吸收过程提供试验依据。【方法】建立Caco-2细胞单层模型,通过测定跨膜电阻(TEER)值、低渗透性药物荧光素钠通透性试验的表观渗透率Papp(AP-BL)来验证Caco-2细胞单层模型的致密性并进行模型筛选。考察在Caco-2细胞单层模型中莱柯霉素从顶侧(apical side,AP)到基底侧(basolateral side,BL)及基底侧到顶侧的转运特性,分析药物浓度、pH及外排转运蛋白(P-gp、MRP2和BCRP)抑制剂3种因素对莱柯霉素转运的影响。用超高效液相色谱串联质谱(UPLC-MS/MS)法检测药物浓度,计算其表观渗透率和外排率。【结果】连续20 d的跨膜电阻值检测及荧光素钠通透性试验显示,随培养时间增加,Caco-2细胞单层模型的跨膜电阻值逐渐增加,在20 d时达到400Ω·cm 2左右;荧光素钠在0.1~6μg/mL内线性关系良好(R^(2)=0.991),计算荧光素钠AP侧向BL侧的表现渗透率(Papp(AP-BL))为(1.57×10^(-7)±9.92×10^(-9))cm/s,小于试验规定的0.5×10^(-6) cm/s,表明Caco-2细胞单层模型建立成功,可用于莱柯霉素的转运特性研究。莱柯霉素Papp(AP-BL)值随时间无明显变化且水平较低,而BL侧向AP侧的表现渗透率(Papp(BL-AP))值随时间递增,在孵育120 min时达到最高水平(2.0350×10^(-6) cm/s),表明肠道外排莱柯霉素的能力较强。随莱柯霉素浓度递增,Papp(BL-AP)值呈上升趋势,具有浓度依赖性。当溶液pH为8.0时,Papp(BL-AP)值(39.3871×10^(-6) cm/s)明显高于pH为6.5和7.4的溶液,表明碱性环境可加速莱柯霉素外排。维拉帕米、丙磺舒和GF1209183种转运蛋白抑制剂均能显著抑制莱柯霉素由BL侧向AP侧的转运,外排率均>1.5,可能有外排蛋白参与转运。【结论】莱柯霉素在体外Caco-2细胞单层模型上的转运较差,吸收率低,表现为低渗透性化合物;随着浓度和pH的升高,莱柯霉素外排增强;外排转运�
【Objective】This experiment was aimed to investigate the transport characteristics of lekethromycin across Caco-2 cell monolayer model,and to provide further experimental evidence for further understanding of the absorption process of lekethromycin in vivo.【Method】A Caco-2 cell monolayer model was established to verify the compactness of the Caco-2 cell monolayer model and to screen the model by measuring the transmembrane resistance(TEER)value and the apparent permeability Papp(AP-BL)of the low permeability drug fluorescein sodium permeability test.The transport characteristics of lekethromycin from the apical side(AP)to the basolateral side(BL)and from the basolateral side to the apical side in the Caco-2 cell monolayer model were investigated,and the effects of three factors,drug concentration,pH and efflux transporter(P-gp,MRP2 and BCRP)inhibitors on the transportation of lekethromycin in Caco-2 monolayer model were analyzed.The drug concentrations were measured by UPLC-MS/MS,and its apparent permeability and efflux ratio were calculated.【Result】The resistance value test and sodium fluorescein leakage test for 20 days showed that with the increase of culture time,the transmembrane resistance value of Caco-2 cell monolayer model gradually increased and reached about 400Ω·cm 2 at the 20th day.The linear relationship of fluorescein sodium was good within 0.1-6μg/mL(R^(2)=0.991),and the calculated fluorescein sodium Papp(AP-BL)was(1.57×10^(-7)±9.92×10^(-9))cm/s,which was less than 0.5×10^(-6) cm/s specified in the test.The Caco-2 cell monolayer model was successfully established and could be used to study the transport characteristics of lekethromycin.Lekethromycin Papp(AP-BL)values did not change appreciably over time and were low,while Papp(BL-AP)values increased over time,reaching a maximum level of 2.0350×10^(-6) cm/s at 120 min of incubation,and intestinal efflux of lekethromycin was strong.Papp(BL-AP)values increased with increasing concentrations of lekethromycin in a concentration-depen
作者
温泽宇
肖红芝
孙攀
刘羽
杨宇欣
王悦
李靖
张璐
龚晓会
周德刚
曹兴元
WEN Zeyu;XIAO Hongzhi;SUN Pan;LIU Yu;YANG Yuxin;WANG Yue;LI Jing;ZHANG Lu;GONG Xiaohui;ZHOU Degang;CAO Xingyuan(College of Veterinary Medicine,China Agricultural University,Beijing 100193,China;National Center for Veterinary Drug Safety Evaluation GCP Laboratory,Beijing 100193,China;Luoyang Huizhong Animal Medicine Co.,Ltd.,Luoyang 471022,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2023年第12期5094-5103,共10页
China Animal Husbandry & Veterinary Medicine
基金
抗菌药物代谢转归及环境微生态风险评估(2018YFD0500301)
LKMS在比格犬和大鼠临床前药动学研究(202005510510087)。
