一种新型MCC-478衍生物抗乙型肝炎病毒活性及体外毒性实验

Anti-hepatitis B virus activity and toxicity of a novel nucleoside analogues in vitro

目的:研究一种具有新结构类型的MCC-478衍生物030705的抗乙肝病毒活性和体外毒性。方法:实验组以不同浓度受试化合物030705(0.01、0.03、0.10、0.30和1.0μmol.L-1)、对照组以不同浓度阿德福韦酯(0.10、0.30、1.0、3.0和10.0μmol.L-1),分别作用于HepG2.2.15细胞,采用Southern blotting杂交法测定其对HBV DNA的抑制率,计算其50%抑制浓度(IC50)和90%抑制浓度(IC90)值,采用ELISA法测定不同药物浓度受试化合物030705对HBeAg分泌的抑制率。采用MTT法测定不同浓度受试化合物030705(10、30、100、300和1 000μmol.L-1)对HepG2细胞毒性,计算其50%致死浓度(CC50)值。采用Dotblotting法分别测定不同浓度受试化合物030705(0.10、1.0和10.0μmol.L-1)对HepG2细胞线粒体含量的抑制率;同时设相应浓度双脱氧胞苷(ddC)阳性药物对照组和仅加入培养基的阴性对照组。结果:受试化合物030705抑制HepG2.2.15细胞HBV DNA作用与阿德福韦酯对照组比较差异无显著性(P>0.05),显示其具有与阿德福韦酯相近的抗乙肝病毒活性。不同浓度受试化合物030705(0.01、0.03、0.10、0.30和1.0μmol.L-1)对HBeAg分泌的抑制率分别为5.94%、6.08%、6.32%、10.31%和12.49%,与阴性对照组比较差异均无显著性(P>0.05)。受试化合物030705对HepG2细胞毒性的CC50值为2 014μmol.L-1(>1 000μmol.L-1),属于低细胞毒性药物。阳性对照药物ddC在不同浓度下(0.10、1.0和10.0μmol.L-1),对HepG2细胞线粒体含量的抑制率分别为38.43%、46.51%、56.51%,与阴性对照组比较差异均有显著性(P<0.05)。相同浓度下受试化合物030705抑制率分别为7.00%、5.81%、5.78%,与阴性对照组比较差异均无显著性(P>0.05)。结论:受试化合物030705对HepG2.2.15细胞HBV DNA的抑制作用与阿德福韦酯的作用相近,对HepG2细胞毒性的CC50值2 014μmol.L-1,无明显线粒体毒性,是一种低毒、高效的新型核苷类抗乙肝病毒药物。

Objective To study the anti-HBV activity and toxicity of a novel MCC-478 devivative 030705 with specific structure in vitro. Methods Anti-HBV activities in HepG2.2.15 cells were analyzed by Southern blotting in experimental groups with different doses of 030705 （0.01, 0.03, 0.10, 0.30 and 1.0μmo·L^-1） and Adefovir Dipioxil positive control groups （0.10, 0.30, 1.0, 3.0 and 10.0μmo·L^-1 ）. Concentration of 50% inhibitation （IC50） and concentration of 90% inhibitation （IC90） were obtained. The inhibitory effect of HBeAg was analyzed in HepG2.2.15 cells by ELISA method. The cytotoxicities were tested by MTT method in the experimental groups （10, 30, 100, 300 and 1 000 μmo·L^-1） in HepG2 cells. Concentration of 50% extinction （CC50） was obtained. The inhibitory effects of mitochondrial DNA contents in experimental groups （0.10, 1.0 and 10μmo·L^-1） were tested by Dot blotting in HepG2 cells. While didenoxyeytidine （ddC） groups were as positive control groups and untreated groups were as negative groups. Results Compared with Adefovir Dipioxil positive control groups, the inhibitory effects of the test compound 030705 on HBV DNA had no obvious difference （P〉0. 05）. Anti-HBV activity of the test compound 030705 was similar to that of Adefovir Dipioxil in HepG2.2.15 cells. The inhibitory rates of HBeAg in experimental groups （0.01, 0.03, 0.10, 0.30 and 1.0 μmo·L^-1 030705） were 5.94%, 6.08%, 6.32%, 10.3% and 12.49%, respectively in HepG2.2.15 cells; compared with negative control group, there was no obvious difference （P 〉0.05）. CC50 value of the test compound was 2 014 μmo·L^-1（〉1 000μmo·L^-1） in HepG2 cells. The inhibitory rates of mitochondrial DNA content from positive drug ddC （0.10, 1.0 and 10 μmo·L^-1） were 38. 43%, 46.51%, 56.51%, respectively in HepG2 cells; compared with negative control group, there was obvious difference （P〈0.05）. The inhibitory rates of mitochondrial DNA content from the test compound 030705 with the same concentrations were 7.00 %, 5.81%, 5.78%, respectively in HepG2 cells; compared with negative control group, there was no obvious difference （P〉0.05）. Conclusion Anti-HBV activity of the test compound 030705 was similar to that of Adefovir Dipioxil in HepG2.2.15 cells. CC50 value of the test compound is 2014 μmo·L^-1 （〉1000μmo·L^-1） in HepG2 cells. No apparent reductions of mitochondrial DNA content are observed in HepG2 cells. The test compound 030705 with specific structure may be a new promising anti-HBV agent.