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Antimicrobial Agents and Chemotherapy, June 2002, p. 1728-1733, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1728-1733.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Pharmacology of ß-L-Thymidine and ß-L-2'-Deoxycytidine in HepG2 Cells and Primary Human Hepatocytes: Relevance to Chemotherapeutic Efficacy against Hepatitis B Virus

B. Hernandez-Santiago,^1,2 L. Placidi,³ E. Cretton-Scott,³ A. Faraj,⁴ E. G. Bridges,³ M. L. Bryant,³ J. Rodriguez-Orengo,² J. L. Imbach,⁵ G. Gosselin,⁵ C. Pierra,⁴ D. Dukhan,⁴ and J. P. Sommadossi^3*

Department of Pharmacology, University of Alabama at Birmingham, Birmingham, Alabama,¹ Novirio Pharmaceuticals, Inc., Cambridge, Massachusetts,³ Novirio Pharmaceuticals, SARL,⁴ Laboratorie de Chimie Organique Biomoleculaire de Synthese, UMR CNRS-UM II 5625, Université Montpellier II, Montpellier, France,⁵ Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico²

Received 17 August 2001/ Returned for modification 19 November 2001/ Accepted 11 March 2002

ß-L-Thymidine (L-dT) and ß-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 µM 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/10⁶ 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/10⁶ 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/10⁶ cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of ß-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/10⁶ 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/10⁶ 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.

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* Corresponding author. Mailing address: Novirio Pharmaceuticals, Inc., 125 Cambridge Park Dr., Cambridge, MA 02140. Phone: (617) 250-3100. Fax: (617) 250-3101. E-mail: sommadossi.jean-pierre{at}novirio.com.

Dedicated to the memory of Martin L. Bryant.

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Antimicrobial Agents and Chemotherapy, June 2002, p. 1728-1733, Vol. 46, No. 6
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.6.1728-1733.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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