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Antimicrobial Agents and Chemotherapy, January 2010, p. 341-345, Vol. 54, No. 1
0066-4804/10/$12.00+0     doi:10.1128/AAC.01176-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β-L-2',3'-Didehydro-2',3'-Dideoxy-N4-Hydroxycytidine: Relevance for Activation in HepG2 Cells {triangledown}

E. Matthes* and H. Bünger

Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13092 Berlin, Germany

Received 19 August 2009/ Returned for modification 13 October 2009/ Accepted 4 November 2009

ß-L-2',3'-Didehydro-2',3'-dideoxy-N4-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 [ED50] = 0.03 µM; 50% cytotoxic dose [CD50] = 2,500 µM). In the present study, we investigated the intracellular pharmacology of tritiated L-Hyd4C in HepG2 cells. L-[3H]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-N4-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-[3H]Hyd4C for 24 h, the 5'-triphosphate accumulated to 19.4 ± 2.7 pmol/106 cells. The predominant peak belonged to 5-diphosphate, with 43.5 ± 4.3 pmol/106 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.

* Corresponding author. Mailing address: Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13092 Berlin, Germany. Phone: 49 30 9406 3409. Fax: 49 30 9406 3138. E-mail: emat{at}mdc-berlin.de

{triangledown} Published ahead of print on 16 November 2009.

Antimicrobial Agents and Chemotherapy, January 2010, p. 341-345, Vol. 54, No. 1
0066-4804/10/$12.00+0     doi:10.1128/AAC.01176-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.





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