新的核苷类似物β-L-D4A的细胞内代谢与生物转化  

Intracellular metabolism and bioconversion of β-L-D4A

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 [2]华中科技大学同济医学院同济医院肝病研究所

出　　处：《中国临床药理学与治疗学》2005年第6期709-712,共4页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：国家自然科学基金资助项目(№39970858)

摘　　要：目的:研究β-L-D4A在2.2.15细胞内的代谢情况,以便为进一步明确其抗HBV作用机制和动物体内实验及人体内试验研究其抗HBV作用与药代动力学奠定基础。方法:以2μmol·L-1[3H]βLD4A或[3H]βDD4A处理2.2.15细胞2、4、8、12、24h,再以0.4mol·L-1预冷的高氯酸(含0.08mol·L-1磷酸三乙胺)抽提,离心后,溶解于酸的上清液,直接进行HPLC分离、相连的紫外检测器检测,然后分析各个峰值。结果:两化合物均出现4峰,且出现时间一致,保留时间依次在6、10、19、28min,24h处理后,β-L-D4A细胞内代谢峰明显高于βDD4A的代谢峰;β-L-D4A于2.2.15细胞内代谢进行了动态的检测,发现细胞内代谢物的含量于加药后迅速增加,以三磷酸形式增加最快,至8h时,达到最大值,以后开始缓慢降低;β-L-D4A处理2.2.15细胞24h后,换用不含药培养基继续培养,停药后3种磷酸化代谢物含量于前8h迅速减少,继后16h降低缓慢,于24h时,仍有8h时35.6%(0.320.9)的三磷酸化合物存在。结论:β-L-D4A较之β-D-D4A更易被磷酸化代谢;一磷酸化过程可能为限速步骤;β-L-D4A三磷酸化物在2.2.15细胞内降解缓慢。AIM: To investigate the metabolism of β-L-D4A in 2.2.15 cells for further clarifying its anti-HBV mechanism and establishing a background on the studies of its anti-HBV effect and pharmacokinetics in animal model and human body. METHODS: 2.2.15 cells were treated with [ 3H]β-L-D4A or [ 3H] β-D-D4A at 2 μmol·L -1 concentrations for 2,4,8,12 and 24 hours, then the cells were extracted by adding 0.4 mol·L -1 perchloric acid containing 0.08 mol·L -1 triethylammonium phosphate. Then it centrifuged at 1 000×g for 5 min, the acid-soluble supernatant were directly isolated immediately by HPLC and monitored by connected ultraviolet detector, then the peaks were analyzed. RESULTS: Both of the two compounds present 4 peaks of metabolites, and the emergence time of each corresponding metabolite peak were similar. The retention times for β-L-D4A, mono-, di and triphosphates were 6,10,19 and 28 min, respectively. Peaks for β-L-D4A metabolites were significantly higher than that for β-D -D4A metabolites in 2.2.15 cells after treated with each of the compounds for 24 h. Rapid conversion of β-L-D4A to its phosphorylated forms could be seen, especially for triphosphorylated form. With the concentrations used, maximal metabolite formation was observed at 8 h, then the metabolites began to reduce gradually. After 24 h treatment, when β-L-D4A was withdrawn, the levels of mono-, di, and triphosphates dropped rapidly in the first 8 h. In the subsequent 16 h, the triphosphate was removed at a lower rate, with 35.6% of the triphosphate still present at 24 h in comparison with the amounts at 8 h. CONCLUSION:β-L-D4A can be more easily phosphated metabolism than β-D-D4A in 2.2.15 cells. Monophosphorylation may be the rate-limiting step. Triphosphate of β-L-D4A is degraded at a lower rate in 2.2.15 cells, and it may have a longer half-life.

关 键 词：核苷类似物 Β-L-D4A 乙型肝炎病毒 2.2.15细胞代谢 生物转化 

分 类 号：R969.1[医药卫生—药理学]