title: Pharmacology of beta-L-thymidine and beta-L-2'-deoxycytidine in HepG2 cells and primary human hepatocytes: relevance to chemotherapeutic efficacy against hepatitis B virus.
abstract: beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 microM in 2.2.15 cells). The intracellular metabolisms of L-dT and L-dC were investigated in HepG2 cells and primary cultured human hepatocytes. L-dT and L-dC were extensively phosphorylated in both cell types, with the 5'-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5'-triphosphate levels were 27.7 +/- 12.1 and 72.4 +/- 1.8 pmol/10(6) cells for L-dT and L-dC, respectively. In primary human hepatocytes, the 5'-triphosphate levels were 16.5 +/- 9.8 and 90.1 +/- 36.4 pmol/10(6) cells for L-dT and L-dC, respectively. Furthermore, a choline derivative of L-dCDP was detected at concentrations of 15.8 +/- 1.8 and 25.6 +/- 0.1 pmol/10(6) cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of beta-L-2'-deoxyuridine (L-dUMP and L-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 +/- 0.4 and 18.2 +/- 1.0 pmol/10(6) cells, respectively. In human hepatocytes, L-dUMP and L-dUTP were detected at concentrations of 5.7 +/- 2.4 and 43.5 +/- 26.8 pmol/10(6) cells, respectively. It is likely that deamination of L-dCMP by deoxycytidylate deaminase leads to the formation of L-dUMP, as the parent compound, L-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of L-dTTP, L-dCTP, and L-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to L-dT in combination with L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of L-dT and L-dC are associated with their extensive phosphorylation.
authors.affiliation: Department of Pharmacology, University of Alabama at Birmingham, USA.
authors.forename: B
authors.initials: B
authors.lastname: Hernandez-Santiago
authors.forename: L
authors.initials: L
authors.lastname: Placidi
authors.forename: E
authors.initials: E
authors.lastname: Cretton-Scott
authors.forename: A
authors.initials: A
authors.lastname: Faraj
authors.forename: E G
authors.initials: EG
authors.lastname: Bridges
authors.forename: M L
authors.initials: ML
authors.lastname: Bryant
authors.forename: J
authors.initials: J
authors.lastname: Rodriguez-Orengo
authors.forename: J L
authors.initials: JL
authors.lastname: Imbach
authors.forename: G
authors.initials: G
authors.lastname: Gosselin
authors.forename: C
authors.initials: C
authors.lastname: Pierra
authors.forename: D
authors.initials: D
authors.lastname: Dukhan
authors.forename: J P
authors.initials: JP
authors.lastname: Sommadossi
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003841:Deoxycytidine / Q000378:metabolism / Q000494:pharmacology*; D006207:Half-Life; D006509:Hepatitis B / Q000188:drug therapy* / Q000821:virology; D006515:Hepatitis B virus / Q000187:drug effects*; D022781:Hepatocytes / Q000187:drug effects*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D010766:Phosphorylation; D013936:Thymidine / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D000998:Antiviral Agents; D003841:Deoxycytidine; D013936:Thymidine