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

Abstract: 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-LD4A, 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 After 24 h treatment, when β-L-D4A was withdrawn, the levels of mono-, di, and triphosphates dropped rapidly in the first 8 h. In the subsequent 16 h, the triphosphate was removed at a lower rate, with 35.6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-LD4A can be more easily phosphated metabolism than β-DD4A 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.

Key words: nucleoside, analogue, β-L-D4A, hepatitis B virus, 2.2.15 cells, metabolism, bioconversion