Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β- l -2′,3′-Didehydro-2′,3′-Dideoxy-N 4 -Hydroxycytidine: Relevance for Activation in HepG2 Cells

• Published in: Antimicrobial agents and chemotherapy Vol. 54; no. 1; pp. 341 - 345
• Main Authors: Matthes, E., Bünger, H.
• Format: Journal Article
• Language: English
• Published: 01.01.2010
• ISSN: 0066-4804; 1098-6596
• DOI: 10.1128/AAC.01176-09

ABSTRACT ß- 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 μM; 50% cytotoxic dose [CD 50 ] = 2,500 μM). 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 μM 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.