SGLT2选择性抑制剂筛选及药效评价方法建立  

Development of a method to study the activity and selectivity of SGLT2 inhibitors

作　　者：张晓琳 王亚男 叶菲 

机构地区：[1]中国医学科学院、北京协和医学院药物研究所,新药作用机制研究与药效评价北京市重点实验室,天然药物活性物质与功能国家重点实验室,北京100050

出　　处：《药学学报》2017年第6期897-903,共7页Acta Pharmaceutica Sinica

基　　金：中国医学科学院医学与健康科技创新工程团队项目(CIFMS-2016-I2M-3-012);天然药物活性物质与功能国家重点实验室开放基金(GTZK201512)

摘　　要：本研究的目的是建立SGLT2选择性抑制剂筛选及药效学评价体系。分别将人全长SGLT2和SGLT1c DNA连接至p MSCVpuro载体并转染HEK293细胞,嘌呤霉素筛选单克隆细胞,RT-PCR、Western blot或细胞免疫荧光染色检测目的基因、蛋白在细胞中的表达。采用荧光标记的1-脱氧葡萄糖(1-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-1-deoxy-D-glucose,1-NBDG)作为底物进行葡萄糖转运实验,检测模型细胞的Na+依赖的葡萄糖摄取功能,并对阳性药达格列净、根皮苷选择性进行评价。最后,进行整体药效评价,观察达格列净对正常小鼠及四氧嘧啶诱导的T1DM小鼠血糖的影响。结果显示p MSCVpuro-SGLT1转染组细胞SGLT1基因及蛋白水平均显著升高,p MSCVpuro-SGLT2转染组SGLT2蛋白高表达于细胞膜及细胞质,说明SGLT1/SGLT2稳定过表达细胞构建成功。葡萄糖转运实验结果显示,与p MSCVpuro-null转染组相比,p MSCVpuro-SGLT1、p MSCVpuroSGLT2转染组细胞Na^+依赖性1-NBDG摄取明显增高。达格列净具有良好的选择性,对SGLT1的半数抑制浓度(IC50,6.20×10^(-7) mol·L^(-1))远高于对SGLT2抑制的IC50值(2.24×10^(-10) mol·L^(-1)),优于非选择性抑制剂根皮苷。达格列净单次给药即可改善正常小鼠葡萄糖耐量。对T1DM小鼠,达格列净从给药后1 h血糖水平即显著降低,能够剂量依赖地降低0~24 h血糖-时间曲线下面积。连续给药20天,能够较平稳地降低小鼠餐后血糖。本研究建立了较完善的SGLT2选择性抑制剂的体外筛选方法和整体药效学评价方法。该方法具有无同位素污染,高转运效率、良好稳定性等优点,将为新的SGLT2高选择性抑制剂研发提供良好的技术平台。The purpose of this study was to develop a screening method to determine the activity and selectivity of SGLT2 inhibitor. Human SGLT1/SGLT2 cDNA was inserted into the pMSCVpuro mammalian expression vector and the plasmid was transfected into HEK293 cells. Stably transfeeted clones were selected in puromycin containing medium. To evaluate the expression of human SGLT1 and SGLT2 in stable transfected cells, RT-PCR, Western blot and immunofluorescence analysis were performed. 1-[N-（7-Nitrobenz-2-oxa-l,3- diazol-4-yl）amino]-l-deoxy-D-glucose （1-NBDG） was used as a substrate in the uptake assay to evaluate the Na＋ dependent glucose transport activities of SGLT1/2. The inhibitory activity and selectivity of dapagliflozin/ phloridzin were also determined, respectively. The hypoglycemic efficacy of dapagliflozin was evaluated in mice with normal blood glucose and mice with alloxan-induced T1DM. The result showed that SGLT1 was overexpressed in pMSCVpuro-SGLT1 transfected HEK293 ceils. SGLT2 protein was overexpressed in pMSCVpuro-SGLT2 transfected HEK293 cells and located in both cytoplasm and membrane. The Na＋ dependent 1-NBDG uptake was significantly increased in pMSCVpuro-SGLT1/SGLT2 transfected cells compared to that in pMSCVpuro-null transfected cells. The selectivity of dapagliflozin, whose half maximal inhibitory concentration （IC50） for SGLT2 （2.24× 10-10 mol.L-1） was far lower than that for SGLT1 （6.20×10-7 mol L-1）, was better than that of phloridzin. The oral glucose tolerance was elevated after a single dose of dapagliflozin in normal mice. In T1DM mice, compared with model group, no-fasting glucose level was decreased at 1 h after administration and maintained at a lower level for 24 h in a dose-dependent manner. A 20-day administration with dapagliflozin dose-dependently improved the hyperglycemia status. Taken together, a system to evaluate the activity and selectivity of SGLT2 inhibitors was established using 1-NBDG in vitro and the hypoglycemic efficacy in vivo in this study. The

关 键 词：Na+依赖性葡萄糖摄取 钠-葡萄糖协同转运蛋白2 钠-葡萄糖协同转运蛋白1 荧光标记1-脱氧葡萄糖 抑制剂筛选 

分 类 号：R965.1[医药卫生—药理学]