新的核苷类化合物β-L-D4A及其异构体体外抗HBV作用  

Inhibition of β-L-D4A and its related analogues on replication of hepatitis B virus in vitro

作　　者：吴金明[1] 林菊生[2] 章金艳[2] 梁扩寰[2] 

机构地区：[1]温州医学院附属第一医院消化内科,浙江温州325000 [2]华中科技大学同济医学院同济医院肝病研究所,湖北武汉430030

出　　处：《中国药学杂志》2005年第16期1270-1273,共4页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金资助项目(39970858)

摘　　要：目的探索比较β-L-D4A(L-D4A)及其异构体体外抗HBV作用,以期获得高效、高治疗指数(TI)的新一类抗HBV化合物。方法L-D4A及其异构体干预2.2.15细胞(Hep G2细胞进行HBV基因组转染后所得)培养,ELISA法检测上清HBs Ag, HBe Ag;32p标记HBV DNA为探针,Southern印迹法检测上清液及细胞内HBV DNA,再以计算机图像处理进行定量分析,求出50%抑制的药物浓度(ED50)。洗去HBV探针后,以32P标记细胞色素氧化酶ⅢDNA为探针再次进行杂交,以探索线粒体的毒性。以MTT法检测不同浓度药物的细胞毒性,求出50%细胞抑制的药物浓度(ID50)。结果上清液病毒DNA的Southern印迹结果显示,L型异构体较之D型异构体具有更强的HBV DNA抑制作用,L-D4A最为突出,并呈显明显的浓度依赖性,计算出ED50为0.2μmol·L-1;D-D4A具有明显的线粒体毒性,L-D4A则不明显;L-D4A细胞毒性实验显示ID50为200μmol·L-1;低浓度L-D4A对上清HBs Ag,HBe Ag无明显的影响,高浓度时有显著的抑制作用。结论体外实验显示L-D4A具有明显的抑制病毒DNA复制作用,且无明显的细胞毒性和线粒体毒性,TI(ID50/ED50)值为1 000,高于拉米夫定组(TI值为750),将有望进一步得到开发。the substitution and modification of groups in 2′, 3′-dideoxy-adenine. To explore the inhibition of β-L-D4A against hepatitis B virus （HBV） in 2.2.15 cells derived from Hep G2 cells transfected with HBV genome. METHODS 2.2.15 cells were cultured, and treated with various concentrations of β-L-D4A and its related analogues. HBs Ag and HBe Ag were determined by ELISA. Intracellular DNA was extracted and digested with Hind Ⅲ , and then were subjected to Southern blot, hybridized with a ^32p-labeled HBV probe and autoradiographed. The intensitv of the autoradiographic bands was quantitated by densitometric scans of computer and ED50 was calculated. Then Hybond-N membrane was washed and rehybridized with a ^32p- labeled mtDNA-speeifie probe, and the effect of β-L-D4A on mitoehondrial DNA was studied, Cytotoxicity with different concentrations was examined by MTT method. ID50 was calculated. RESULTS Autoradiographie bands showed β-L-D4A was the most potent anti-HBV candidate compared with other analogues. Episomal HBV DNA was inhibited in a dose-dependent manner. ED50 was 0.2 μmol·L^-1. HBs Ag or HBe Ag was not apparently decreased. The Inhibition of β-L-D4A on mitochondrial DNA was not obvious.The experiment of cytotoxieity gained ID50 at 200 μmol·L^-1. CONCLUSION β-L-D4A possessed potent inhibitory effect on the replication of HBV in vitro with little eytotoxicity and mitocondrial toxicity, TI value was 1000. It is expected to be developed clinically as a new anti-HBV drug.

关 键 词：核苷类化合物 β-L-双脱氧双脱氢腺苷 乙肝病毒 

分 类 号：R978.7[医药卫生—药品]