title: Cellular pharmacology of the anti-hepatitis B virus agent beta-L-2',3'-didehydro-2',3'-dideoxy-N4-hydroxycytidine: relevance for activation in HepG2 cells.
abstract: Beta-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED(50)] = 0.03 microM; 50% cytotoxic dose [CD(50)] = 2,500 microM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[(3)H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5'-mono-, 5'-di-, and 5'-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ss-d-N(4)-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5'-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 microM l-[(3)H]Hyd4C for 24 h, the 5'-triphosphate accumulated to 19.4 +/- 2.7 pmol/10(6) cells. The predominant peak belonged to 5-diphosphate, with 43.5 +/- 4.3 pmol/10(6) cells. The intracellular half-life of the 5'-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5'-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5'-diphosphate during a drug-free incubation. The high 5'-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.
authors.affiliation: Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13092 Berlin, Germany. emat@mdc-berlin.de
authors.forename: E
authors.initials: E
authors.lastname: Matthes
authors.forename: H
authors.initials: H
authors.lastname: Bünger
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D001711:Biotransformation; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003564:Cytidine Deaminase / Q000494:pharmacology; D003841:Deoxycytidine / Q000378:metabolism; D006207:Half-Life; D006515:Hepatitis B virus / Q000187:drug effects*; D006801:Humans; D008099:Liver / Q000378:metabolism; D010766:Phosphorylation; D016047:Zalcitabine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
chemical_list: C547756:2',3'-didehydro-2',3'-dideoxy-N4-hydroxycytidine; D000998:Antiviral Agents; D003841:Deoxycytidine; D016047:Zalcitabine; D003564:Cytidine Deaminase