Clevudine is efficiently phosphorylated to the active triphosphate form in primary human hepatocytes

Clevudine (CLV) is a nucleoside analogue of the unnatural L-configuration that has demonstrated potent activity against hepatitis B virus (HBV) in vitro and in Phase III clinical studies. Human hepatoma cell lines are the only liver-derived cells in which CLV metabolism has been investigated. Here, we examine CLV metabolism in primary human hepatocytes, which better represent CLV metabolism in the liver.

HPLC analysis of primary human hepatocyte extracts incubated with radiolabelled CLV was used to determine the time course of CLV phosphorylation. Effects of the exogenous cell concentration of CLV on phosphorylation were assessed and the half-life of the CLV phosphorylated forms was determined.

The major CLV metabolite formed in human primary hepatocytes was 5'-monophosphate, whereas in the hepatoma cell lines the major metabolite was 5'-triphosphate. The level of CLV 5'-triphosphate was similar in both cell types. In primary hepatocytes the conversion of CLV 5'-monophosphate to the corresponding 5'-diphosphate was the rate-limiting step in CLV phosphorylation; the level of CLV phosphorylation was dependent upon exogenous drug concentration and exposure time. CLV 5'-triphosphate accumulated rapidly with peak levels observed after approximately 8 h. When cells were incubated with 1 microM CLV, the approximate maximal plasma concentration achieved in individuals receiving the 30 mg dose, the average intracellular concentration of CLV 5'-triphosphate was 41.3 +/- 8.4 pmols/10(6) cells (approximately 10 microM). The initial half-life of CLV triphosphate was approximately 11 h.

These results are consistent with once daily CLV dosing currently being used in Phase III clinical studies.