Metabolism of the Anti-Hepatitis C Virus Nucleoside β- d - N ⁴ -Hydroxycytidine in Different Liver Cells

Abstract

␤-D-N4 -Hydroxycytidine (NHC) was found to have selective anti-hepatitis C virus (HCV) activity in the HCV replicon system (clone A). The intracellular metabolism of tritiated NHC was investigated in the HCV replicon system, Huh-7 cells, HepG2 cells, and primary human hepatocytes. Incubation of cells with 10 M radiolabeled NHC demonstrated extensive and rapid phosphorylation in all liver cells. Besides the 5-mono, -di-, and -triphosphate metabolites of NHC, other metabolites were characterized. These included cytidine and uridine mono-, di-, and triphosphates. UTP was the predominant early metabolite in Huh-7 cells and primary human hepatocytes, suggesting deamination of NHC as the primary catabolic pathway. The intracellular half-lives of radiolabeled NHC-triphosphate and of CTP and UTP derived from NHC incubation in Huh-7 cells were calculated to be 3.0 ؎ 1.3, 10.4 ؎ 3.3, and 13.2 ؎ 3.5 h (means ؎ standard deviations), respectively. Studies using monkey and human whole blood demonstrated more-rapid deamination and oxidation in monkey cells than in human cells, suggesting that NHC may not persist long enough in plasma to be delivered to liver cells.Hepatitis C virus (HCV) causes liver disease and is spread primarily by contact with the blood of an infected person. Globally, an estimated 170 million persons are chronically infected with HCV (20). An estimated 3.9 million Americans (1.8%) have been infected with HCV, and cirrhosis will eventually develop in at least 15 to 20 percent of them (1, 12). HCV is one of the most important causes of chronic liver disease and seems to progress more rapidly to liver damage in human immunodeficiency virus-infected persons than in noninfected persons (13). Interferon, alone and in combination with ribavirin, is approved for the treatment of persons with chronic hepatitis C. Treatment with interferon for genotype 1-infected individuals is effective in about 15 to 20% of patients (6, 16), but when combined with ribavirin, its effectiveness increases to almost 42%; ribavirin alone, however, is ineffective (6). The combination of ribavirin and interferon is 80% effective against genotypes 2 and 3 (12, 16). Therefore, we need to develop new antiviral agents active against all genotypes of HCV, particularly genotype 1, which is commonly found in the United States and China (11).␤-D-N 4 -Hydroxycytidine (NHC), a base-modified ribonucleoside analogue, was identified as a potent and selective anti-HCV candidate (19). In a bovine viral diarrhea virus infection system and in HCV replicon RNA in Huh-7 cells, NHC had 90% effective concentrations (EC 90 ) of 2 and 5 M, respectively (19). NHC was nontoxic (50% inhibitory concentration, Ͼ100 M) in the HCV replicon system (clone A cells, genotype 1b), Huh-7 cells, HepG2 cells, and human peripheral blood mononuclear cells (19). Levels of mitochondrial DNA and RNA or lactic acid in HepG2 cells treated with NHC did not change, suggesting no delayed toxicity (19). Although the antiviral mechanism of action of NHC is not completely understood, it...

Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

“…Although 1,5-and N,N-DMHA are more volatile than TBA and phosphate, many presented methods still require flushing of the ESI source to prevent clogging and a loss of signal (Hernandez-Santiago et al, 2004;Pruvost et al, 2008). Pollution could be repressed by reducing the column diameter and flow.…”

Synthesis of β-Enantiomers of N4-Hydroxy-3′-Deoxy-Pyrimidine Nucleosides and Their Evaluation against Bovine Viral Diarrhoea Virus and Hepatitis C Virus in Cell Culture

“…Based on this observation, and on the fact that 3′-β-D-deoxynucleosides have shown some interesting biological activity (Walker & Hong, 2002), we report the synthesis of 3′-deoxy-pyrimidine-NHC analogues modified at the 5 position of the base and their biological activity against BVDV and HCV. Since NHC was converted in cells to the natural substrates cytidine and uridine (Stuyver et al, 2003;Hernandez-Santiago B et al, 2003), or their respective triphosphates, it is anticipated that the 3′-deoxy-NHC analogues could undergo a similar metabolic conversion. Therefore, the biological activity and the chemical syntheses of 3′-deoxyuridine and 3′-deoxycytidine analogues are also reported.…”

Characterization of β- d - N 4 -Hydroxycytidine as a Novel Inhibitor of Chikungunya Virus

“…In this study, we identify and characterize a nucleoside analog, ␤-D-N 4 -hydroxycytidine (NHC), which has been reported previously by our group to affect hepatitis C virus (16). Herein, we examine the impact of NHC on CHIKV replication in terms of antiviral activity, toxicity, intracellular metabolism, and mechanism of action.…”